Spectral characterization of coumarins and cinnamic acids

MICROCHEMICAL

Spectral

JOURNAL

14, 567-572

Characterization

(1969)

of Coumarins

and Cinnamic

Acids

J. MBNDEZ AND M. I. LOJO Department of Plant Biochemistry, Santiago de Compostela, Received

Jurle

C.S.I.C. Spain

12, 1969

INTRODUCTION In continuing the spectral analysis of coumarins and cnmamic acids present in plants, five coumarins, petunic (3,4-dihydroxy,5-methoxycinnamic) , o-ferulic (2-hydroxy, 3-methoxycinnamic) acids and methyl 3,4,5-trimethoxycinnamate, not included before (3-5) and reported as naturally occurring (I, 6) were studied. Work on synthetic 6-hydroxy and 4-methyl, 6,7-dihydroxycoumarins is also reported here for comparative purposes. Attention to 2,5-dihydroxycinnamic acid was paid because gentisic (2,5dihydroxybenzoic) acid is present in plants and it is well established that C&C, compounds are formed by p-oxidation of the analogous cinnamic acids (II). Microorganisms can accomplish P-oxidation of phenoxyalkylcarboxylic acids (IO). Whether 2,4- and 3,5-dihydroxybenzoic acids could be derived by p-oxidation, either from shikimic acid or a closely related compound, remains unsolved. However, 2,4- and 3,5-dihydroxycinnamic acids are included since the corresponding substituted benzoic acids were recently identified as metabolites of the fungus Epicoccum nigrum (2) and of Azotobacter chroococcum (7).

The occurrence of a mechanism for the formation of hydroxybenzoic ethers by O-methylation of C,-C, precursors (6) accounts for the study of 2,3- and 2,4-dimethoxycinnamic acids. MATERIALS

AND METHODS

The compounds were dissolved in methanol and analyzed in presence of sodium acetate (NaOAc), boric acid (H,BO,), aluminum chloride (AlCI,), sodium hydroxide (NaOH) and sodium borohydride (NaBH,). The addition of NaOH to caffeyl derivatives was discontinued since they are unstable in alkaline media. Instead, NaBH, was used and no further alkali was added because the final solution had already a basic pH. The spectra were recorded in an Unicam SP.800 spectrophotometer with l-cm silica cells in the range 220-450 nm. 567

6-Methoxy,

6-Methoxy,

7, 8-Dihydroxycoumarin

IV.

V.

VI.

(daphnetin)

7-0-primeverosecoumarin

7-O-glucosecoumarin

7-0-glucosccoumarin

6-Hydroxy,

III.

6, 7-dihydroxycoumarin

4-Methyl,

II.

Coumarins I. 6-Hydroxycoumarin

Compound

DATA

268 274 329

226-8 29&3 338

225-8 26Oa 287-97 344

221 276 339

290-3 338

234 273a 295a 372 214 335

228 273 336

245 27Oa 300-02 379

234 265a 298 360

242 288 384

NaBH4

237 288-92 335 275 336

240 260” 291-8 344

236-7 28oa 392 234

226 254-E 288 343

231 253 287-90 343

225 277 346

AlC13

(nm)

247 281 295 351

227 253 287-90 343

x,,,,,

COMPOUNDS

240 27CP 3104 374

277 346

229

HzB03

CINNAMIC

239 265” 305a 370

239 2658 309” 382

230a 299 364

NaOAc

RELATED

228 247 278 346

AND

1

226 271 346

MeOH

OF COUMARINS

(fabiatrin)

(scopolin)

(cichoriin)

SPECTRAL

TABLE

278 343

-

246-7 275 303 390

242-5 275” 3158 394

244 290 387

___NaOH

2-Hydroxy, 3-methoxycinnamic (o-ferulic acid)

Methyl 3, 4, Strimethoxycinnamate

3, 4-Dihydroxy, (petunic acid)

XIII.

XIV.

XV.

aShoulder.

2, 4-Dimethoxycinnamic

XII.

b(dec.) decomposition.

5-methoxycinnamic

acid

acid

acid

(daphnin)

acid

2, 3-Dimethoxycinnamic

XI.

acid

3, 5-Dihydroxycinnamic

X.

acid

2, 5-Dihydroxycinnamic

IX.

acid

7-o-glucose, 8-hydroxycoumarin

.Cinnamic acids -VIII. 2, 4-Dihydroxycinnamic

.VII.

-

223 245a 2600 293 324 -

221 273

225 281

225 280

225 279

228 285

227 290

248a 25ga 349

255 322

285" -

283" 31oa

-

251

240 273

235 280

236 275

235 274

235 279

237 293

2480 257a 341

227 255 324

250-3 -

240 213

233 279

236 273

233 274

234 278

236 293

392

24ga

234 273 370

265a 298 335 380a

223

283

225 289

225 290

228 293

226 293

248,= 25ga 362

255 322

34oa

223 25@~ 286-90

224 273

233 278

224 274

227 272

224 274

225 302

237 289 383

268 366

dec.

dec.

dec.

dec.

dec.

dec.

decb

273 370

VI $

570

MfiNDEZ

AND

LOJO

Spectra in sodium acetate. To 3 ml of the methanolic solutions an excess of powdered NaOAc was added. After shaking and allowing to settle, the spectra of the solutions were determined. Spectra in boric acid. To the above solution, saturated with NaOAc, powdered H,BO, was added. The spectrum was determined after shaking and allowing to stand for 10 minutes. Spectra in aluminum choloride. Three drops of ethanolic 5% AlCl, were added to the cell with the methanolic solution, and the spectrum was determined after 5 minutes. Spectra in sodium borohydride. An excess of powdered NaBH, was added to the solution of the compound and the spectrum was recorded after 24 hours. Spectra in sodium hydroxide. Two drops of 5% NaOH were added to the neutral methanolic solutions. In all cases the blank was treated in an identical manner as the solutions of the compounds. Except for the spectra in NaOAc, the observations were confined to the long wavelength bands because the short wavelength proved to be less sensitive. RESULTS

AND

DISCUSSION

Coumarins

Spectral shifts were noted (3, 5) on adding to coumarins the diagnostic reagents used in the structural identification of flavonoids. Similarly to the bathochromic shifts reported for 7-hydroxy derivatives (3) in the presence of NaOAc, coumarins with a free 8-hydroxyl (VI, VII. Table 1) showed shifts which were more prominent when the hydroxyl group was adjacent to a protected 7-hydroxyl (VII) than to a free 7-hydroxyl (VI). On the other hand, a derivative lacking 6- or 7-hydroxyl groups (IV) undergoes anomalously large bathochromic shift, and 6-hydroxycoumarins exhibited a small shift (III) or no shifts at all [I, 6-hydroxy, 7-methoxycoumarin (3)] as already established (3). Glucosilation, but not methylation, of the 7-hydroxyl seems to enhance the reactivity of hydroxyls in both adjacent positions (Table 1). Thus, whereas the spectrum of a 7-methoxy derivative of 6-hydroxycoumarin was not altered (3) that of its 7-0-glucoside (III) was shifted 8 nm. The effect is, however, much larger between daphnetin (VI) and daphnin (VII) where the difference is 38 nm. Complexation with glucose (9) could be connected with the phenomenon. Coumarins with a catechol grouping, not only in 6 and 7 positions [II, aesculetin (3), 4-methyl aesculetin (S)] but also in 7 and 8 [VI, fraxetin (5)], shift in the presence of AICI:, and/or H,,BO,, with more

COUMARINS

AND

CINNAMIC

ACIDS

571

SPECTRA

remarkable bathochromic effects with AlCl, in general (unpublished results). No anomalous responses were observed (Table 1). With hydroxycoumarins, NaOH proved to be more effective than NaBH,. Bathochromic shifts are more noticeable and hydrolysis does not seem to occur, probably because relactonization is favored in very alkaline conditions (I). With compounds having o-dihydroxyls, NaBH, showed small hypsochromic (II) or bathochromic shifts (VI, aesculetin Ll3). Cinnamic Acids The application of NaOAc and AlCl, can be extended to cinnamic acids as sources of supplementary data but their use do not make it possible to establish structural features. So, the addition of NaOAc causes, at the short wavelength, anomalous bathochromic shifts with o-ferulic acid and cinnamyl derivatives with all their hydroxyls blocked (XI-XIV. Table 1). Lactonization could account for the large and small shifts observed, respectively, for 2,4- and 2,5-dihydrcxycinnamic acids. The former behave as the related 7-hydroxycoumarin, and the latter as 6-hydroxycoumarins. On the other hand, the spectral shifts on adding AlCl, to compounds either with free or protected hydroxyls fail to provide any correlation on a systematic basis. In the presence of HaBOa, petunic, o-ferulic, 2,%dihydroxycinnamic acids and methyl 3,4,5-trimethoxycinnamate behave as might be expected, but the long wavelength band of other compounds (VIII, X-XII. Table 1) undergoes hypsochromic shifts. The instability reported for the caffeyl moiety in basic media (8) is extended in the present study to m-dihydroxy, dimethoxy, trimethoxy, and methoxyhydroxycinnamyl structures, a fact that reinforces the opinion that olefinic bonds but not catechol groupings bring about the instability, since the addition of NaBH, appears to stabilize these compounds. SUMMARY Sodium acetate, boric acid, aluminum chloride, sodium borohydride, and sodium hydroxide were used as diagnostic reagents in the spectral characterization of five coumarins, naturally occurring in plants, and eight related cinnamic derivatives. A structural correlation for the former but not for the latter was found, The spectral data of cinnamic compounds, however, provided useful supplementary information. REFERENCES

1. Dean, F. M., “Naturally Occurring Oxygen Ring Compounds,” worth, London, 1963.

661 pp.

Butter-

2. Haider, K. and Martin, J. P., Synthesis and transformation of phenolic cornnounds by Epicoccum ni~runz in relation to humic acid formation. soi1 Sci Sot. Amer. Proc. 31, 766-772 (1967).

572

MkNDEZ

AND LOJO

3. Horowitz, R. M. and Gentili, B., Flavonoids of Citrus. IV. Isolation of some aglycones from the lemon (Citrus Zimon). I. Org. Chem. 25, 2183-2187 (1960). 4. Mtndez, J. and Lojo, M. I. Spectral behavior of some cinnamic acids. Microthem. J. 13, 232-235 (1968). 5. Mendez, J. and Lojo, M. I., Spectral analysis of coumarins. Microchem. J. 13, 506-512 (1968). 6. Pridham, J. B., Low molecular weight phenols in higher plants. Ann. Rev. Plant Physiol. 16, 13-36 (1965). 7. Robert-G&o, M., Vidal, G., Hard&on, C., Le Borgne, L., and Pochon, J., Etude biogtnttique des polymeres humiques. Relation entre polymeres humiques naturels, d’origine microbienne et l&nine. Ann. inst. Pasteur 113, 911-921 (1967). 8. Schroeder, H. A., Stabilization of caffeyl compounds in alkaline media. Phytochemistry 6, 1.589-1592 (1967). 9. Swain, T., The identification of coumarins and related compounds by filter-paper chromatography. Biochem. J. 53,200-208 (1953). 10. Towers, G. H. N., Metabolism of phenolics in higher plants and micro-organisms. Irz “Biochemistry of Phenolic Compounds” (J. B. Harbome, ed.), pp. 249-294. Academic Press, New York, 1964. 11. Zenk, M. H., Biosynthesis of C,-C, compounds. In “Biosynthesis of Aromatic Compounds” (G. Billek, ed.), pp. 45-60. Macmillan (Pergamon), New York, 1966.