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THE CHEMISTRY OF THE * W. L. FOWLKS, Ph.D.

The psoralens belong to a group of compoundsfled since biological activity of the psoralens and which have been considered as derivatives ofangelicins has been demonstrated. They appear , the furocoumarins. There are twelveto have specific biochemical properties which different ways a ring can be condensed withmay contribute to the survival of certain the coumarin molecule and each of the resultingspecies. Specifically these compounds belong to compounds could be the parent for a family ofthat group of substances which can inhibit certain derivatives. Examples of most of these possibleplant growth without otherwise harming the furocoumarins have been synthesized; but natureplant (2, 3, 5). is more conservative so that all of the naturally It is interesting that it was this property which occurring furocoumarins so far described turnled to the isolation of the only new naturally out to be derivativesof I or angelicinoccuring furocoumarin discovered in the United II (1). States. Bennett and Bonner (2) isolated tharn- nosmin from leaves of the Desert Rue (Tham- n.osma montana) because a crude extract of this 0 0 plant was the best growth inhibitor found among /O\/8/O\/ the extracts of a number of desert sur- i' Ii veyed for this property, although all the extracts showed seedling growth inhibition. One could I II speculate as to the role such growth inhibition plays in the economy of those desert plants These natural derivatives of psoralen and angeli-when survival may depend upon a successful cm have one or more of the following substituentsfight for the little available water. The structure at the 5,8 or 5,6 positions respectively: hydroxy,of thamnosmin was determined by Crosby (4) methoxy, butanyloxy isoainyleneoxy, geranyloxyto be a derivative of namely 5- and substituted isoamyleneoxy derivatives such methoxy-6-(2 ,3-epoxyisopentanlyoxy-)angelicin. as epoxy, dihydroxy or esterified hydroxy. InRodighiero (5) has also shown that psoralen, addition derivatives of both psoralen and angeli-8-methoxypsoralen and angelicin inhibit seed cm are found with isopropyl or substituted iso- germination, root growth and seedling growth. propyl groups at the 5' position and some of Such findings suggest that further investiga- these derivatives have oxygen functions; keto,tion may reveal certain furocoumarins in the hydroxy or methoxy; at the 4' position. Alto-role of natural growth regulator of certain gether about two dozen furocoumarins have beenplants just like another coumarin derivative, isolated from natural sources. , which Goodwin and others (6, 7) Although the first furocoumarin was isolatedhave shown regulates cell division in the root of over a hundred years ago, when in 1834 Kal-Avena sativa. This idea is strengthened if one brunner isolated (5-methoxypsoralen)recalls that the psoralens of , from bergamot oil, the biological importance of8-methoxypsoralen, 5-methoxypsoralen and 8- these compounds has not been known until veryisoamylenoxypsoralen () are found recent times. The fact that these compounds areconcentrated in the pericarp of the fruit (seed). found in various plant materials leads naturallyThe psoralen and angelicin of to teleological speculation as to what biologicalare also found in the pericarp of the fruit (28) function they fulfill. Such speculation was justi-and thus the germ of the seed is surrounded by *Fromthe Division of Dermatology, Uni-a tissue containing a germination inhibitor, versity of Oregon Medical School, Portland,which could regulate the time when sprouting Oregon. Presented at the Brook Lodge Invitationalwill occur by the rate at which it diffuses into Symposium on the Psoralens, sponsored by Thethe surrounding soil. Chakraborty, DasGupta, Upjohn Company, Kalamazoo, Michigan, March 27—28, 1958. and Bose (8) have very recently shown that out 249 250 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY of seventeen natural eoumarin derivatives whichturned to paper chromatography as a means of they tested, the psoralens, including psoralenseparation and identification of furocoumarins. and imperatorin, were the most effective anti-The first effort along these lines was that of fungal agents tried. And thus the economy ofSvendsen (14) who reported the separation of nature would be beautifully demonstrated if thepimpinellin, , bergapten, and iso- same compound acts as germination inhibitorbergapten from Pimpinella magna and P. saxi- and also decontaminates the soil by killing patho-fraga.Hechromatographed on paper the mic- genic fungi as it diffuses away. rosubhmates from the crude plant extracts Musajo (3) has mentioned some hitherto un- using petroleum ether (65—70° C.)——95 % reported results of Dolcher, Rodighiero andmethanol (5:4:2) to develop the ehromatograms. Caporale who described the mutagenic propertiesAt about the same time in 1952 Riedl and of five furocoumarins and found 5-methoxy-Neugcbauer (15) also published a chromato- psoralen and psoralen to be almost as effectivegraphic method for the isolation and identifica- as the most effective mutagcnic agent known,tion of from plant sources. They used trypafiavin, when tested on onion root tips.a paper that had been presprayed with glycol Psoralen at 5.6 x10M. and 5-mcthoxy-(ethylene or propylene) and was developed with psoralen at 5.0 x10M. induced 40% of mitosisbenzene at 10°C. for their separation. Swain (16) with chromosome when incubatedstudied a number of solvent systems for the with the onion roots for 4 hours at 20°C. Atseparation of a wide variety of naturally occurring higher concentrations there was total inhibitioncoumarins including three furocoumarins. Rodig- of mitosis. Chromosome aberrations noted werehicro, Caporale and Ragazzi (17) reported the agglutinization of chromosomes and liquificationidentification of psoralen and bergapten from of the chromosome surface (stickiness effect). ItRuta graveolens using chromatographic separation is not reported if these experiments were per-on paper developed with methanol-- formed in the absence of light which could havewater (18:1 :1) solution. They determined the caused these effects due to photosensitization inRf. of a number of different furocoumarins in the the presence of the furocoumarins (29). Themcthanol-pyridine-water solvent and also in photoscnsitizing properties of several of themethanol-phenol-water (45:4:1). Chakraborty psoralens have been described elsewhere (3, 9). and Bose (18) have made the most extensive study of the paper chromatographic separation 5EPAEATION OF FUEOcOUMAEIN5 of natural coumarins. They studied the separa- With new discoveries of the biological ac-tion of 12 representative coumarin derivatives, tivities of the several furocoumarins which haveincluding furocoumarins, in 21 different solvent appeared in recent years there has also been asystems. Unlike other investigators they also renewed activity concerning the chemistry andattempted the separation of a mixture of their physical properties of these compounds. Incoumarin derivatives and discovered that most particular a number of investigators have soughtspots contained more than one compound and ways of isolating, identifying and quantitativelywhen a single compound was separated from the determining the furocoumarins in general andmixture its Rf. was usually altered sufficiently so certain of them in particular. that positive identification was rarely possible. There is no simple quantitative method for theThe author has confirmed these observations of isolation of the furocoumarins of plant origin.difficulties with the chromatographic separation The usual methods which have been employedof mixtures and also that the best separations on for the isolation of numerous natural productspaper arc obtained with solvent mixtures con- have been used for the furocoumarins namely:taining 85% or more water. It is our observation extraction with lipid solvents or alcohol followedthat the addition of up to 15% of solvents such by partition between immiscible solvents withas acetic acid or methylethyilcetone to the water or without the use of acids or alkalies, thenincreased the capacity of the system and usually further purification by repeated recrystalliza-sharpens the separation somewhat. Paper chro- lions or combinations of sublimation and re-matography alone can not be relied upon for the crystallization. Such methods are not suitablyseparation and identification of furocoumarins quantitative, nor are they technically satisfac-unless a preliminary purification has first been tory. carried out. A number of investigators have therefore The separation of a number of furocoumarins CHEMISTRY OF THE PSORALENS 251 using paper eleetrophoresis (19) on a circular disk apparatus has also been reported. Difficulties are encountered with this method in mixtures. — Psornien Preliminary separations of eoumarins from 5 —Methnsy Psnrnlen 8—Methney Pso,alen natural sources using aluminum oxide column 5,8—Diniethnsy Psornien chromatography has been reported (20—22) but in general pure compounds have not been ob- tained. ANALYTICAL EEACTION5 The analytical determination of the furoeou- marins remains a problem. One might expect from general considerations that furoeoumarins & would be quite reactive or at least undergo reactions typical of activated aromatic com- pounds but in fact they are rather unreaetive in the usual sense. No one for example has reported coupling a diazonium compound with a furo- eouinarin, a reaction which is characteristic of activated aromatic compounds. Likewise efforts to prepare derivatives based upon reactions de- Wave length mp the signed to open the laetone ring, i.e., phenyl- FIG.I. Traces of the individual Cary record- hydrazides or hydroxamie acids, are met withing spectrophotometer records made of 4.0 X failure. Such lack of reactivity has frustrated10 M solutions of each of the furocoumarins: psoralen, 5-methoxypsoralen, 8-methoxypsoralen efforts to find specific reactions which will leadand 5,8-dimethoxypsoralen in 25% . to colored derivatives with somewhat specific absorption spectra. The only reaction known which goes with facility is a reaction with dilutedand natural origin of the eoumarins including nitric acid. In acetic acid this reaction appearsthe furoeoumarins (1, 3, 10—13). These reviews to give a quantitative yield of nitro derivative.have adequately covered the methods of synthesis But the absorption spectrum of the resultingof furoeoumarins and the proofs of structure of compound has little to recommend it over themost of the naturally occurring compounds. Some degradation reactions and photoehemieal reac- spectrum of the parent compound for analyticaltions will be considered here. purposes. The methods used to degrade psoralens for The absorption spectra of psoralen, 5-methoxy-structure determination have been well described, psoralen, 8-methoxypsoralen and 5, 8-dimethoxy- psoralen are reproduced in Figure I. Theseparticularly in papers by Spath and his co- spectra were obtained on a Cary, model 14,workers (for references see 10—13), therefore, recording speetrophotometer. One should noteonly a brief description of three degradation reac- that as with coumarins (31) there are three re-tions will be given here. One of the three reactions gions in which one or more absorption maximagives information as to the presence of a con- occur, these are the less than 225 region, thedensed furan ring which positively identifies an unknown compound as a furoeoumarin if other 230—270 region and the 290—330 region. Distinct minima occur between these regions. Hydro-reactions have identified it as a eoumarin. Furan- 2, 3-dicarboxylie acid is obtained when either genation in the 3,4 position (30) or opening of the laetone ring greatly reduces the absorptionpsoralen or angeliein derivatives are oxidized coefficient in the 295—325 region. Psoralen, 5-with alkaline peroxide. Reaction of an alkoxy methoxypsoralen 8-methoxypsoralen and 5,8-furoeoumariu, other than a methoxy derivative, dimethoxypsoralen obey Beers' law to the limitwith an acetic acid-sulfuric acid mixture results of the of the compounds. in cleavage of the ether. One may then identify the resulting furocoumarin phenol and the alcohol CHEMISTRY OF PSOEALEN5 by other reactions. The third reaction can give Excellent reviews have appeared, some ininformation as to whether the compound is a recent years, which summarize the chemistrypsoralen or angeliein derivative. For this reaction 252 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY the ring is opened in strong sodium hy- droxide solution and the resulting phenolie compound is methylated. Permanganate oxida- o—-- /O\ /O\ —o tion followed by methylation gives derivatives of 4, 6-dimethoxyisophthalic acid from psoralen H3CO\ \/_\//OCH and its derivatives and derivatives of 2, 4-di- OCH, OCH, methoxyisophthalie acid from angelicin and its IV derivatives. This method fails to distinguish 5-methoxypsoralen from 5-methoxyangeliein and

p CR, pimpinellin from isopimpinellin. 0 PHOTOCHEMICAL REACTIONS OF P5ORALENS N/OyON/O\/ Wessely and Dinjaski (23) exposed 5, 6-di- methoxyangelicin, pimpinellin, in a thin layer to /o 0 diffused sunlight for 2.5 months and obtained a CH, dimer which regenerated the parent compound Nc upon heating and gave furan-2 ,3-dicarboxylic V acid upon alkaline oxidation with peroxide. Hydrogenation of the dimer gave agens of phenanthraquinone to give a derivative tetrahydro derivative which was split with heatof furobenzodioxin V. Sehonberg gives no proof to a single compound that melted at 132—3° andof this structure but reasons from analogy. He added a mole of hydrogen. Irradiation of 5,6-found that coumarin does not react with phen- dimethoxyangeliein in acetic acid solution gave aanthraquinone in sunlight while and different dimer that underwent the identicalits derivatives do. reactions of the first dimer. In a later paper (24) In a paper by Lerner et ci. (26) changes in the \\Tessely with Plaiehinger investigated the hydro-absorption spectra of 8-methoxypsoralen were genation of photodimers of eoumarin and herni-reported as a result of irradiation. The change arm and a synthetic dieoumarin and reaffirmedconsisted of a loss of the characteristic peaks and the earlier conclusion that the photodimers ofvalleys of the spectrum and a generalized absorp- pimpinellin have a eyelobutane structure eithertion getting stronger toward shorter wavelengths. III or IV. Upon irradiation of psoralen the author noted this In a paper on photoehemieal reactions in sun-same response and chromatography of the re- sulting solution revealed at least two new fluores- light Sehonberg ci ci. (25) investigated the reac- tion of phenanthraquinone with a number ofcent compounds had been formed. One of these compounds, on the basis of its ehromatographic ethylene derivatives including 8-methoxypsora- len and some benzofuran derivatives. He postU-behavior and by analogy with the results ob- lated the reaction as between the 4', 5' positionstained with 5, 6-dimethoxyangeliein, has been of the furoeoumarin and the orthoquinone oxy-designated a dimer. The other proved to be furocoumarie acid identical with the one re- ported by Stoll (20). Similar results are obtained upon irradiation of 8-methoxypsoralen and 5-methoxypsoralen. The absorption CR, spectra of the three compounds eluted from the H,C 0 OCR, irradiated psoralen chromatograms are given in Figure II. From the differences in the absorption 0 spectrum of the compound designated a dimer as C H, compared with the parent compound it would 0 appear that while the assignment of a eyelo- butane derivative for its structure as made by Wessely (24) might be possible in this ease it is III not probable. Absorption in the 295 m.i to 325 CHEMISTRY OF THE PSORALENS 253

Paul B. Elder Company of Bryan, Ohio for gifts of S-mcthoxypsoralen and Sandoz Ltd. of Basle, Switzerland for generous supplies of psoralen. 0 DIMER These investigations were supported by a grant from the United States Public Health Service, Grant No. C2S37. R 0.52 PSORALEN REFERENCES 0.61 FUROCOUMARIC ACID 1. Grssaraic, T. A. AND HTNREJNEE, E.: Theories of the biogenesis of flavanoid compounds. Botanical Rev., 18: 77, 1952. .0 2. BENNETT, E. L. AND BONNER, J.: Isolation of plant growth inhibitors from Thamnosma k. monlclnc. Am.J., 40: 29, 1953. 3. MusAjo, L.: Interessanti proprieta' dclle furocumarine naturali. II farmaco. Ed. sci., 10: 2, 1955. 4.BONNER, J.: Personal communication. c:. 5. RODIGHIERO, G.: Influenza di furocumarine naturali sulla germinazione dci semi e sullo sviluppo dci germogli e delle radici di lat- 0.5 tuga. Giorn. di biochim., 3: 138, 1954. 6.GOODWIN,R. H. AND POLLOcK, B. M.: Studies on roots. I. Properties and distribution of fluorescent constituents in Avena roots. Am. J. Botany, 41: 516, 1954. 7. POLLOCK, B. M., GOODWIN, R. H. AND GREEN, S.:Studieson roots. II. Effects of coumarin, scopoletin and other substances on growth. Am. J. Botany, 41: 521, 1954. S.CNAKRABORTY,D. P., DA5GUPTA, A.AND 200 300 BOSE, P. K.: On the action of WAVE LENGTH mp some natural coumarins. Annals Biochem. and Exptl. Mcd., 17: 57, 1957. FIG. II. Traces of a Cary recording spectro- 9.FOWLKS,W.L., GRIFFITH,D. G. AND OGINSKY, photometer record of the spectra made of the E.L.: Photosensitization of bacteria by eluates of the three fluorescent bands of a paper furocoumarins and related compounds. chromatograph on which an irradiated psoralen Nature,181: 571, 1958. solution in 40% ethanol had been streaked. The10. DEAN,F.M.: Naturally occurring coumarins. chromatograph wns developed with 15% acetic Progress in the Chem. of Organic Natural acid in water, air dried 24 hours and eluted with Products, 9: 225, 1952. 95% ethanol. The concentrations are unknown. 11.SETHNA, S. M. AND SHAH, N. M.: The chemis- try of coumarins. Chem. Rev., 36: 1, 1945. 12. SPATR, E.: Die naturlichen Cumarine. Ber., mi region for coumarin and its derivatives ap- 70A: 83, 1937. pears to be due principally to the lactone ring13. SPATH, E. AND KUFFNEE, F.: Die natOrlichcn (30) since if this ring is open or the 3,4-double Cumarine und ihrc Wirkung auf Fische. Monatsh., 69: 75, 1936. bond is hydrogenated absorption in this region14. SWEND5EN, A. B.: Papierchromatographischcr is lost. The furano ring also makes a contribution Nachiweis naturlichcr Cumarinc in Pflan- in the 295—325 inz region but it is slight as is zen. Pharm. Acta Helv., 27: 44, 1952. 15.RIIIDL,K. AND NEUGEBATJER, L.: Uber die shown by the spectrum of furocoumaric acid. papierchromatographischcTrennung von The spectrum of the dimcr on this basis suggests Cumarinen. Monatsh., 83: 1083, 1952. that the dimerization involves a bond between 16. SWAIN,T.:The indentification of coumarins and related compoundsby filter-paper chro- 3 or 4 atom of one molecule of psoralen and the matography. Biocbem. J., 53: 200, 1953. 4' or 5' atom of another molecule with saturation 17. RODIGHIERO, G., CAPOLALE, G. AND RAGAzzI, E.: Ricerche sulle cumarine presenti nclla of the double bond of the furano ring. Such Rule grcveolens, nellc foglie di FICNS carica photodimers would be consistent with the find- c nell' essenza di C'ilrus limonNm. Atti Isti- tuto Veneto Sd. Lett. e Arti, 111: 125, 1953. ings of botb Wessely and Sehonberg. More work 18. CHAKEABOETT, D. P. AND BOSE, P. K.: Paper will be done in these laboratories on this problem chromatographic studies of some natural to ascertain the exact structure of these photo- coumarins. J. Indian Chem. Soc., 33: 905, dimers. 1956. 19. BEELINGOzZI, S. AND PAEEINI, V.:Separazione di alcune cumarine per elettroforcsi circo- We would like to thank E. W. Peterson who ran lare su carta. Lo Sperimentale. Sez. Bio- the absorption spectra reported here and the chim., 6: 59,1955. 254 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

20. STOLL, VON A., PEREIRA, A. AND RENTZ, J.: reactions between o-quinones and cthylenes Das Furocumarin und die j3-D-Glucosido- in the dark and in the light. J. Chem. Soc., furocoumarinsiiure aus den Samen von Coro- 1951: 1364, 1951. nilla-Arten. Hclv. Chim. Acta. 33: 1637,26. LERNER, A. B., DENTON, C. R. AND FITZ- 1950. PATRICK, T. B.: Clinical and experimental 21. CALDWELL, A. G. AND JONES, B. R. H.: The studies with 8-methoxypsnralen in vitilign. constituents of expressed West Indian J. Invest. Dermat., 20: 299, 195?. oil. J. Chem. Soc., 1945: 540, 1945. 27. PATZAK, R. AND NEIXGEBAUER, L.: tJber Pnla- 22. Ronioniano, G., CAPORALE, 0. AND ALBIERO, rographische Untersuchungen Von Coumari- 0.: Ricerche sulle cumarine della R'uta nen II. Monatsh, 83: 776, 1952. graveolens. Gazz. chim. Ital., 84: 874, 1954.28. MUKEEJI, B.: Psoralea and other indigenous 23. WEssELv, 0. VON AND DINJA5KI, K.: Action drugs used in leucoderma. J. Sci. and md. of light on substances of the type of furo- Research, iSA: 1, 1956. coumarins. Monatsh., 64: 131, 1934. 29. FowLKs, W. L.: The mechanism of the photo- 24. WE5SELY, F. VON AND PLAICRINOER, I.: dynamic effect. This Symposium. p. 233. Uber die Knnstitution der Photodimerisate30. BROKKE, M. E. AND CIJEI5TENSEN, B. E.: der Cumarine und Furocumarine. Ber. 75B: Psoralene I: Certain reactions of xantho- 971, 1942. toxin. J. Org. Chem., 23: 589, 1958. 25. SCHöNBERG, A., LATIF, N., MOUBASHER, R.31. NAKABATASHI, T., T0K0R0YAMA, T., MIYA- AND SINA, A.: Photochemical reactions in ZAKI, H. AND IsoNo, S.: Studies on coumarin sunlight. Part XVI. (a) Photoreduction of derivatives II. Ultraviolet absorption spec- phenylglyoxylic acid. (b) Photoreactions tra of coumarin derivatives. J. Pharm. Soc. between aldehydes and o-quinones. (c) Japan, 73: 669, 1953.