COMPOSITION OF THE
March, 1945
SECRETION OF
Condensation of o - N i t r o p h e n y l a c e t o Ether with p Nitrobenzenedonyl Chloride.-The normal method of condensation of pyridine failed to give clean products.
Primula Denticulata
49 1
in sixty-five minutes. After filtration from catalyst, the acetone was removed at low temperature i n uatuo. The residue had a strong indole-like odor and was soluble in ether. No pure product was isolated and it was clear that the sulfonyl group had been split off.
o-Nitrophenylacetimino ether base (8.4 g.) was melted with +nitrobenzenesulfonyl chloride (4.4 g.). Reaction was immediate and the temperature rose to 80'. Some The author's thanks are due to the Directors ethyl chloride was evolved, as normally occurs.* The melt, after a few minutes a t 90', was extracted successively of Messrs. May & Baker Ltd., England, for perwith ether and water and the residue (4.9 g.) m. p. 115" mission to publish this work, and to Mr. S. Bance (incomplete) was recrystallized from alcohol. The mix- for the semi-micro analyses. ture was taken up in boiling ethyl acetate (30 cc.) and the product which separated on cooling (0.5 g., m. p. 157summary 158') was removed. After a crystallization from ethyl 1. When 2-aminoindole is condensed with acetate this had m. p. 159' and was identified as o-nitrophenylacetamide (Calcd.: N, 15.55. Found: N, 15.4). acetylsulfanilyl chloride, the sulfonyl residue The ethyl acetate liquors were concentrated and successive enters in the 1 position. crops removed. These were crystallized from 1:l2. A method of obtaining the isomeric 2benzenebight petroleum mixture to give the required p nitrobyzenesulfonyl 2 nitrophenylacetimino ether, sulfanilyl derivative by use of appropriate sulfam. p. 123 ; yield 2.55 g. Anal. Calcd. for CLJ~UGNIS:nilyl amidines or imino ethers is outlined. N, 10.7. Found: N, 10.8. 3. It is suggested that this method might find Attempted Reduction of Above.-Two grams dissolved other similar applications in this field. in acetone (50 cc.) was reduced catalytically in the usual DAGENHAM, ENGLAND RECEIVEDAUGUST24, 1944 way (0.1 g. of platinum oxide) giving an uptake of 12 H
-
- -
[CONTRIBUTION FROM THE
DEPARTMENT O F CHEMISTRY,
UNIVERSITY OF CALIFORNIA]
The Composition of the Solid Secretion Produced by Primula Dentidata' BY WALTER C. BLASDALE A distinguishing feature of a large percentage of the species of Primula is the secretion of white or yellow solids commonly designated by botanists as farina. They are produced by minute, twocelled, gland-tipped hairs found on the leaves, scapes, calyces, or more rarely the petals. The chemical nature of the secretion of P . pulverulentu was studied by Hugo Muller2 who found it to consist of flavone associated with small amounts of a wax-like component. Later Brunswick,a by the use of a microchemical test devised by Klein,* found that flavone was present in the secretions of 25 of the species of Primula as well as three of the closely related genus Dionysiu. This test involved the subjection. of fragments of farinabearing tissue or of minute portions of farina scraped from it, to the fumes of concentrated hydrochloric acid, at a temperature of 40°, in a sublimation ring. If flavone was present a microscopic examination of the tissue or the cover glass revealed bundles of fine white or yellow needlelike crystals. Appreciable differences in the color and consistency of the secretions of the different species suggest that additional components, both colorless and colored, should be looked for. Macroscopic study of them is rendered difficult because of difficulty of procuring enough plants upon which they are found. The seed of many species is unobtainable, many are difficult to grow, and the yield of farina-bearing tissue per plant is very (1) Original manuscript received January 1 1 , 1943.
(2) Miiller.J . Chcna. Soc.. lOT, 872 (1915). (3) Brunswick. Bcr. Akad. Wiss. Wicn., [ I ] 131, 221 (1922). (4) Klein, { b i d . . [I]111, 23 (1922).
small. During the past fifteen years I have been able to grow sufficient plants of twenty-one of the farina-bearing species t o make a partial study of their secretions possible. This paper is concerned chiefly with the secretion of P. denticulata. I desire to express my obligation to Mr. Charles Koch of this Laboratory for microcombustions of several of the products separated. Experimental Part Separation of Farina.-Owing to the sticky nature of the secretion and the small amount of farina-bearing tissue available, mechanical separation of i t was not feasible. The procedure finally adopted was to treat the dried and coarsely powdered leaves and flower heads for twenty minutes, a t room temperature, with a three-fold volume of benzene and allow the filtered extract to evaporate spontaneously. The dry residue consisted of white crystals and green amorphous matter, the total equaling about 4% of the tissue used. The same procedure applied to nonfarinate leaves of the same species yielded nearly 2% of a green sticky mass from which no crystals could be obtained even by use of a variety of solvents. It seems probable that none of the crystals in the extract from the farinabearing tissue came from the tissue itself. Some Properties of Flavone.-It was soon apparent that most of the crystalline material present in the benzene extract was flavone. In separating it from the other components constant use was made of a boiling solution of 6 N hydrochloric acid. Although acid addition products of several flavone derivatives have been isolated,b these compounds hydrolyze a t once unless the concentration of the acids used in their preparation is left high. I t was found that constant boiling hydrochloric acid dissolved 2.65 g. of flavone per liter but about 90% separated, on standing a t room temperature, as long fine needles. This may be attributed to its low melting point as compared with those of its derivatives. By treating the benzene extract with large volumes of 6 N acid twice, the flavone was almost completely separated from the large amounts of plant pig(5) Perkin, J . Chcm. Soc., [Z]69, 14.19 (1896).
WALTERC. BLASDALE
492
ments, waxes, etc. The resulting solution, as well as the crystals which separate on cooling, was lemon-yellow. Repeated recrystallization of the yellow crystals from the pure solvent finally gave white crystals of the same form. Small amounts of yellow' coloring matter were recovered from the solutions from which the first crystals separated. The dry, white crystals, even at room temperature, slowly lost weight and became opaque, owing to the liberation of both water and hydrochloric acid. The opaque residue melted at 95". The melted product, purified by crystallization from either hot petroleum ether or 60% alcohol, formed shorter white crystds which melted at 97", the currently accepted melting point of flavone. Further, the fine white crystals from the acid solution, as well as the crystals which melted at 97'. responded to the triiodide test? that is, gave a deep purple color when treated with a drop of a dilute solution of potassium tri-iodide. When the same process of separation and purification was applied to farina-bearing tissue derived from P. malacoides and P.M o o r e a m products were obtained which melted a t ! K O , gave molecular weights of 209, 232 and 240, and the fnllowing analyses. P.
€2.
malucoides malacoidcs
ib)
I a)
Carbou, I,; IHydrogen,
80.91
81.45
4 :$ti
I. Oii
P. Moorcana
Calcd, for
80.X 4 ,93
81.05
CihHw02
4 , 31
Further, wheii the scapes and calyces of 1'. pulaerulenln species studied by Muller) were subjected to the same procedure both thc fine crystals from the acid solution and the purified product could not be distinguished from the corresponding products obtained from 1'. denliculala. The Composition of Flavone Hydrochloride.-The fine white crystals, which were separated from hydrochloric acid solutions of the species studied, after drying, were found to decompose slowly, even a t room temperature, unless kept in closed containers. Complete elimination of the solvent from which they separated without some decomposition was difficult to effect. Samples derived from three species were allowed to dry a t room temperature and weighed at twenty-minute intervals until the rate of loss showed a decided drop. Three portions of each of these ,aniples were weighed out for determination of (a) loss a t 105', (b) percentage of hydrochloric acid, found by adding water and titrating with 0.01 L'V sodium hydroxide, and (c) melting point in a sealed capillary. The results are tabulated. i the
Loss a t 105' Percentage HCI M. p., 'C.
P. denliculala
P. mala-
18.88 13.60 132-134
19.85 12.81 134-135
coidcs
1'. japonica
CirIIioOs. HCI.HaO
17.44 13.03 134-1345
19.70 13.19
These figures indicate that the fine white crystals corrcspond to the forniula ClsH~oO~.HCl.H20. I t is uoteworthy that wheii heated in a small, sealed capillary they Idiave like a one component system. Heated in an open capillary they began to melt as low as 107'. This i-, a compotind to which Klein's test is due. Its long fine needles of uniform diameter are readily distinguished from the coarser, ,plinter-like crystals of flavone. Separation of a Dihydroxyflav0ne.--Hy treating the dry ariiorphorts mass, left after removal of flavone from the benzriie extract, with a large amount of hot carboil tetrachloride and filtering wliilc hot, a green solution was obtained. Evaporation gave some amorphous matter and a few crystals. The latter were purified by washing with petroleuin ether and recrystallizing from either benzene or ethyl ether. The purified product, which I will designate a i Pr. 238, consisted of orange-yellow, 6- or X-sided crystals of short-columnar or tabular habit. Between crossed nicol< the dominant face gave extinction parallel to the longer edge and marked birefringence. The dtiiniriant a n d atljawii:. narrr)n. fari-.. gave brilliant rvti'
1i:grxvr ( n i l > t a r l : n .
i
ii : p , a
\
I
IOT,
11
i
I i i il!ll i,
VOl. 67
flections. 1 am indebted to Mr. Adolph Pabst of the Department of Geology for measurements of the corresponding interfacial angles on two millimeter-long crystals. These results, in addition to the parallel extinction, indicate that the crystals are monoclinic, with a 6 angle of 82'18'. Heated in a sealed capillary Pr. 228 melted a t 22Xo Determination of its molecular weight, by lowering of the melting point of camphor, gave 269, 234, 265 and 246 Analyses of five mg. samples gave the following results. Sample (2, % K 70 .4,by the writer 70.42 4.02 R , by the writer 70.68 3.81 C, by Charles Koch 70.98 4.12 Calcd. for C16Ill00, 70.86 3.93 When treated with 0.5 N sodium hydroxide Pr. 228 slowly forms a deep yellow solution from which i t is reprecipitated unchanged by neutralization. Acetylation of a 13-mg. sample, by means of acetic anhydride and sodium acetate, gave a white, crystalline compound which, after recrystallization from ethyl acetate, melted a t 183-184'. By hydrolysis and distillation this product yielded 1.71 moles of acetic acid per mole. By dissolving Pr. 228 in glacial acetic acid, adding a sniall amount of strong sulfuric acid and allowing to stand for several days a red crystalline product separated. It yielded 24.90% of sulfuric avid: the compound CljH1001.H2SObshould yield 27.844'. The preceding analytical data, associated with the rcsults obtained by treating Pr. 228 with sodium hydroside and with acetic anhydride suggest that it is a dihydroxyflavone. Its orange-yellow color, the forniation of a red addition produet with sulfuric acid, which is a charactrri\tic. of compounds containing a y pyrone nucleus, and it4 insolubility in hot 0 S hydrochloric acid, confirm this suggestion. I t differs from all of the eight dihydroxyflavones listed by Beilstein in its physical properties. Since it constitutes only about 0.15vjC of the selected farina-bearing tissue (about one-ninth that of flavonej further work Icading to t h e determination of the position of the hydroxyl groups must await the accumulation of larger amounts of such tissue. Its occurrence in the farina rather than in the main leaf tissue was proven by allowing a few drops of chloroform to flow over an undried, farina-bearing leaf and allowing the solution to evaporate. The residue, examined under a microscope, showed, in addition t o flavonc, the easily identifiable crystals of Pr. 228. Neither flavone nor Pr. 228 could be separated from the benzene extract of dry rion-fariria-bearing leaves of this species. The Wax-like Components.-The carbon tetrachloride extrart left after removal of Pr. 228 formed a sticky greeii mass composed of plant pigments and the decompositioii products resulting from treatment of them with hydruchloric acid. 'Small amounts of wax-like preparations, were separated by means of dilute mcltiiig from 40 to &io, alcohol but they could not be resolved iiito pure coinpouncls.
Composition of the Farina of Other Species.- -Tlie proccss described above was used, with several modificrttious, i i i htutlying the farina of the twenty o t h e r sprvit., Flavone was separated i n pure form from all of them m1i1 tlic available data indicate that it conztituteb ai k a i i I a$.,& of the secretions of all these species. Small aniouiit. of two additional compounds: were isolated. b'ronl I'. vcrlirilkutu a yellow crystalline substance, which meltcd at 1.i.Y" ant1 gavc a molecular weight a n d (*omposition correqiiondiiig to C ~ S H I Owas O ~ , obtained. From P . ,ffurindoea sccoiid yellow conipound, which begau to deconipox at 18.5' and a t 305" gave a black arnorphoui solid aiitl a fen. white crystals was obtained. Pr. 3% was found i n thc secretion of P.deitticulatn only. Kine of the twenty-one species, namely, P.auriricla, cepitntu, denticulata, frloizdostz, .pponictl, mtdacnides, murginatn, puloerulmdo arid viertirrilnttr arc inclutlctl i n t h e li5t found to give qualitative t
