Zeitschrift fiir Krebsforschung 67, 192--204 (1965)

From Deutsches Krebsforschungszentrum, Heidelberg, Biochemisches Institut (Direktor: Prof. Dr. E. HECK]m) and Max:Planck-Institut fiir Biochemie, Miinchen

(Direktor: Prof. Dr. A. BV~ENAI~I)T)

On the Active Principles of Croton 0il

u Purification and Characterisation of Further Irritant and Cocarcinogenic Compounds of the B-Group*

By

ERIC CLARKE** and ~ERIcH HECKER

With 7 Figures in the Text

(Received May 5, 1965)

The sepa ra t ion of the biological ly ac t ive cons t i tuen ts of Croton 0 i l in to two groups, A and B, has been descr ibed (H:EGKER, JARCZYK, MEY~I~, BI~ESCI~ and BI~ACI~aANN). Subsequent work on these groups resul ted in the i so la t ion and chemical , as well as biological , charac te r i sa t ion of the i r r i t an t and cocarcino- genic compounds A1 (HEcKEI~, BI~ESC~ and v. SZOZEPANSI(I; I-IEcKER and BR~SCH), B1 and B 2 toge the r wi th a s imi la r ly ac t ive fract ion, B x (HECKER, KUBINYI and BlZ~SOH; I-IEcKER and K v ~ I ~ u of unknown composi t ion (see also Fig. 1).

The compounds A1, B1 and B 2 are de r iva t ives of a Di t e rpene alcohol, Phorbo l C20H~s06, which contains one carbonyl - and five hydroxy- func t ions as well as 2 C = C double bonds (HECKER, BRESCH and Mwvn~). I n the compounds i so la ted two of the h y d r o x y groups are esterif ied, one wi th a long chain and the o ther wi th a shor t cha in s a t u r a t e d f a t t y acid. The presen t s t u d y describes the resolu t ion of two fract ions, B x and B 2 ~- Bx , in to compounds B 3 - - B 7 b y means of Cra ig-d i s t r ibu t ion (C.d., Fig. 1), and es tabl ishes the close chemical re la t ionsh ip of these new compounds to the cons t i tuen ts A 1, B 1 and B 2 of Croton Off.

Substances and Methods

Melting points were determined on a Leitz heating microscope and are uncorrected. Ultramicroanalyses were carried out by the microanalytical laboratories of the European Research Associates, Brussels 1 and of the Organic Chemistry Department, University of Saarbriicken 1. UV absorption spectra were measured in ethanol, using a Beckmann Spectro- photometer Model DK-2. IR absorption spectra were determined in KBr discs with a Perkin- Elmer Spectrophotomcter Model 21. NMR spectra were measured on a Varian Associates A 60 Model using tetramethylsilane as internal standard, "Elektronenanlagerung"-mass- spectra on the special mass-spectrometer devised by v. A R n ~ . Gas-liquid chromato- graphy was carried out on a Perkin-Elmer gas chromatograph Model 116 E, using packed 6' columns, helium as carrier gas and a detector of the thermobalance type. Optical rotations were measured in dioxane using the Kreis Polarimeter Zeiss-Winkel (•

* Dedicated to Prof. Dr. K. H. BAITER on his 75th birthday. ** Research chemist on secondment from present address: Imperial Chemical Industries

Ltd., Petrochemical and Polymer Lab., P.O. Box 11, The Heath, Runcorn, Cheshire, England. 1 We are gratefully indebted to Dr. It. REIMLI~Gm% Brussels, and Dr. W. W~nlSCl%

Saarbriicken, for ultra-microanalyses.

On ~he Active Principles of Croton Oil 193

Unless otherwise specified thin-layer chromatography was on Kieselgel G (Merck) in the following systems:

A: tetrachlormethane-benzene (2:1); B: benzene-ethyl acetate (10:1) ; C: acetone-ethyl acetate (2 : 1) ; D: benzene-ethyl acetate (3 : 1).

For column chromatography Merck Kieselgel (0.05--0.20 ram), deactivated by the addition of 13% water, was used.

Commercial nitromethane was distilled from phosphorus pentoxide, and then redistilled. Di-n-butyl ether was washed with ferrous sulphate solution, dried over magnesium sulphate and distilled from sodium, n-Heptane and cyclohexane were fractionally distilled from sodium. Commercial samples of rac. e-methylbutyrie acid (Th. Schuehardt, Munich, Germany), tiglic acid (Fluka, Buehs, Switzerland) and angelica acid (EGA Chemic, Steinheim, Krs. Heidenheim, Germany) were converted with diazomethane into the corresponding methyl esters. These esters without isolation were reduced by LiA1Ha and converted into their 3,5-dinitrobenzoates in the usual way. Their m.p.'s and R/-vaines on Kieselgel G in system A (all three compounds on the same plate) are as follows: tiglic alcohol-DNB m.p. 96 ~ R~ 0.22; angelica alcohol-DNB m.p. 105--106 ~ R/0.26; (--)-S-2-methylbutanol-DNB m.p. 82.5--83 ~ (HECKER and KUBINYI), R/ 0.26.

Craig.distributions o/ B Compounds. Automatic batteries were used: an apparatus with z = 2 0 0 elements, using 25 ml stationary phase (v. METZSCE), and a z = 1 0 2 0 element Craig battery, using 3 ml stationary phase (CE~G and Kr~G). The conditions used in the various distributions are summarized in the table below.

Table 1. Specification o/ the Craig-distributions 1--5

No. of complete Settling No. of tubes

Craig- Battery type V n period receiving distribution shaking sample

movements rain

1 * v. Metzsch 25/~ 450 20 3 1 2 * Craig 3/3 2000 10 2 5 3 ** Craig ~3/3 2500 20 1.5 6 4 ** Craig a/3 3020 20 1.5 3 5 ** Craig /3 4085 20 1.5 3

�9 System: n-heptane (15), di-n-butyl ether (5), nitromethane (20); single phase with- drawal procedure.

�9 * System and procedure: see legends Fig. 2, 3, 4 and 5 respectively.

The usual Craig-distribution procedures (H~cxE~ 1955, Ct~G, CII4I~- and SC~EZB~L) were followed, the sample being introduced at the beginning of the battery using as few elements as possible (Table 1). Normally longer settling periods than stated in Table 1 were used at the beginning of the distribution. I t was convenient, in that it removed most of the high-boiling ether from the important fractions, to finish distributions with a complement of double-phase withdrawal. The distribution curves are constructed by the weighed-sample method, the samples consisting of between 2 and 5 neighbouring fractions, and the average fraction-weight being plotted against the middle fraction number. The shape of the dis- tribntion curve governed the subsequent combination of fractions for the long-chain fat ty acid content analyses.

Quantitative determination o] long-chain ]arty acid content. Saponification, and subsequent methylation of the various fractions with diazomethane, was carried out by the method described in a preceding paper (HECKER and KUBr~YI). Gas*liquid Chromatography (15% polyestersuecinate on Celite 54:5) at 1400 permitted quantitative analysis of the resultant methyl ester mixtures. The results are summarized in Table 2 as weight % of long chain fat ty acids.

194 E. CLARKE and E. HECKER:

Table 2. Long chain fatty acid content, Rf values and some physical data o/the more signi/ican~ fractions from Craig-distributions 1--5

Com- pound

B2 B3 B4 B5 B6 B7

Source $

C.d. 3 Fr. 961--1080 C.d. 3 Fr. 861--900 C.d. 3 Fr. 781--800 C.d. 5 Fr. 926--98G C.d. 5 Fr. 711--780 C.d. 4 Fr. 676--720

Fat ty acid analysis

Cs C~o C~2

0 98 1 0 98 2 0 3 96

94 6 0 95 5 0

2 98 0

[a]D 28.50

+ 3 3 ~ -~45~

+ 4 8 ~

~inax mtt

irLfl., 335 232, 336 229,334

infl., 333 232,333

- - , 80 5360,70 5330,80

- - , 90 5300, 110

* For softening region see Table 5. ** See HECKER and KUBINYI.

*** Not measured because of scarcity of compounds and requirements for assays�9

2,4-Dinitriphenylhydrazone derivative of B2. By the procedure previously described (HEC~ER and B~ESC~; ttECKE~ and KVBINYI) B2 was oxidised and converted into the 2,4- dinitrophenylhydrazone derivative, which separated from methanol, m.p. 120--121 ~ With B 2 - D N P H (m.p. 119,5--120,5 ~ the mixed m.p. is undepressed. Both compounds show the same R! value (thin-layer-chromatography, System B), and their IR-, UV- and l~MR-speetra are identical.

Preparation of 4-(d-nitrophenylazo)-benzoate derivatives. By the usual procedure (HECKE~ and BRESCH) 4-(4-nitrophenylazo)-benzoates were prepared from the B-compounds. Chro- matography on Silica Gel in benzene-ethyl acetate afforded the NPAB-derivatievs, which separated from methanol as follows (Table 3).

Table 3. Characterisation o

NPAB- ester of

B3

B4

B5

B6

B7

Type of crystals

pnsms

needlc~ r

prlsm

ueedlef

m.p. oC

90--91

150--152

94--98

148--150

the N PA B-derivatives ]rom compounds B 3--B 7

~[olecular formula

C4sH59NsOn requires found

C47tI59NaOn requires found

C4eHs~Ns0 n requires found

C45H55N30n requires found

67.5 6.96 4.9 67.4 7.06 4.8

67.1 7.06 5.0 66.7 6.91 4.9

66.9 6.71 5.1 66.7 6.63 5.2

66.4 6.81 5.2 66.6 6.73 4.9

* Oily NPAB-ester.

20.6 20.5

20.9 20.9

21.3

21.6 21.4

Lithium Aluminium Hydride Cleavage o /B compounds�9 The procedure with substance B4, typical of that used with the other compounds, is described. I t is an advantageous modifica- tion of the procedure previously described (I-IECKER and B~ESC~I, I-IEC]~mr and Ku]~INu

A solution of B4 (203 rag) in ether (20 ml) was added slowly (30 rain) to a stirred slurry of lithium aluminium hydride (150 rag) in ether (20 ml). After further 1.5 hrs the reaction mixture was added, with stirring, to a phosphate buffer solution (pH 6.2) at 0 ~ which was covered with a layer of light petroleum (b.p. 40--60 ~ to minimise losses of volatile products. Filtration of the mixture and separation of the organic layer, followed by a further extrac- tion with ether gave an aqueous solution, which was evaporated almost to dryness at 350 . Extraction of the residual gum with acetone and evaporation of the combined acetone extracts

On the Active Principles of Croton Off 195

afforded a colourless solid (76 mg, 60% ), m.p. 180--184 ~ separating from acetone in prisms, m.p. 197--198 ~ Thin-layer chromatography in System C gave a single spot, R/0.50, identical with t h a t of Phorbolol.

The ether extracts were dried over magnesium sulphate, filtered, and the fi l trate was refluxed with an excess of 3,5-dinitrobenzoyl chloride. The normal work-up procedure gave a resinous residue which was chromatographed on Silica Gel. Elut ion with l ight petroleum (b.p. 40--60~ (2: I) and evaporat ion of the fractions gave the dodeeyl 3,5-dinitro- benzoate, separating from methanol in prisms (63 rag, 47 %), m.p. and mixed m.p. 58--59 ~ Fur ther elution afforded the ethyl derivative (49 rag, 59 % ), separating from aqueous ethanol in prisms, m.p. and mixed m.p. 90--91 ~ Both fractions gave single spots on thin-layer chro- matography, corresponding to those of authent ic specimens. The I R spectra were identical wi th those of the authent ic samples, and the ~ M R spectra were compatible in all eases wi th the proposed structures. Da ta of the DNB-derivat ives obtained from the ethereal layer of the reductive cleavage of the B-compounds are summarized in Table 4.

Table 4. Characterisation o] the DNB-derivatives ]rom the reductive cleavage o] B2- -B7 RFvalues have been obtained in the paper chromatographic system described by M ~ H

using heptane sa turated wi th methanol as the mobile phase.

Com- pound

B2

B3

B4

B5

B6

B7

Alcohols

(--)- S-2-Methylbutanol . Decanol . . . . . . .

Tiglic . . . . . . . . Decanol . . . . . . .

E thanol . . . . . . . Dodeeanol . . . . . . .

( - - ) - S -2-Methylbutanol . Octanol . . . . . . .

Tiglie . . . . . . . . Oetanol . . . . . . .

E thanol . . . . . . . Deeanol . . . . . . .

3,5-dinitro- benzoate

(m.p.)

750 570 _ _ * *

57 o

92 930 590

74--75 o 60--61 o

_ _ * *

60--610

92--930 56--57 o

Bf value on paper

0.69 0.88

0.69 0.88

0.50 0.89

0.69 0.84

0.69 0.84

0.50 0.88

Identity shown by *

mixed m.p., t.1. c., p.c., IR, NMR mixed m.p. , t . l .c . , IR,

t . l .c . , p e., IR, NMR mixed m.p. , t.1. e., IR, NMR

mixed m.p. , t . l .c . , p.e., I R mixed m.p. , t . l .c. , IR, NMR

mixed m.p., t . l .e . , p.c., I R mixed m.p. , t . l .c . , I R

t . l .c . , p.c., I R mixed m.p. , t . l .c. , I R

mixed m.p. , t . l .e . , p.e., I R mixed m.p. , t . l .e. , IR, NMR

* ;.1. c. = th in layer chromatography; p.c. = paper chromatography. ** Shown to contain also 2-methylbntanol, see text .

In each case the alcohol obtained lh'om the aqueous phase of tile l i th ium alumininm hydride t r ea tment was converted into the te t raacctate derivative, by the method previously described (HEcKE~ and BR~SC~). This product was shown, in each case, to be identical with Phorbolol te t ra-acetate by m.p., mixed m.p., t.l.c, in System D (R! 0.51), and by infrared spectroscopy (IR). Where substance quant i ty sufficed (B3, B4, and B7) the ident i ty was fur ther confirmed by NMR spectroscopy.

Biological assays. For inf lammation assays SI(Sandoz-Inzucht)-mice have been used in a modification of the s tandard procedure and the results are being expressed as " inf lamma- t ion u n i t " I.U. in /~g/mouse ear ( "Entz i indungse inhe i t " EE. ; H r c x ~ 1963). In this modified procedure compounds A 1 ( H ~ c K ~ and BR~SCH) and B 2 (g~CX~l~ and KUBII~YI) show an I.U. of 0,1 and 0,3/~g/ear respectively. The I.U. of compounds B 3 - - B 7 are summarized in table 6. For est imation of cocarcinogenie act ivi ty groups of 14 male and 14 female RLYIRI-miee were t rea ted in the s tandard manner (HEc~I~ 1963, H~CK~R and B ~ s o ~ ) with a sub-threshold amount of DMBA (0.1 or 1 #MoI) in 0.1 ml acetone and sub- sequently twice weekly wi th 0.1 ml of an 0.01% acetone solution of the cocarcinogens. Turnout counts were carried out after 12 weeks of application of cocareinogens.

196 E. CLARKE and E. HECKE~:

Results and Discussion

Separation and Puri]ieation o] the new compounds. I n this work solvent systems consist ing of cyclohexane (or n-heptane) , d i -n-butyle ther , and n i t romethane in different proport ions have been used. Suitable par t i t ion coefficients K 1 were obta ined by a d j u s t m e n t of the vo lume ratio V 1 of the two phases or of the hydro- carbon: ether ratio of the system. As hydrocarbon cons t i tuen t cyelohexane is preferable, because, compared with n-heptane, a smaller propor t ion of ether is necessary to obta in a suitable K-v~lue. Details of solvent systems and procedures are included in Substances and Methods (Table 1). Al though the effectiveness

Croton Oil D A B 6 1958

I A Fraction B _Fraction

B1 B2 B 2 + B x (7.12g)

C.d. 1 n = 450

Fr 36--160 (6.54 g)

C.d. 2 n ~ 2000

Fr 601--680 (1.07 g)

B2

Fr 961--1080 (0.45 g)

B2

Fr 101--600 (5.04 g)

C.d. 3 n~2500

Fr 861--900 (1.23 g)

B3

Fr 781--800 (0.29 g)

B4

,

B x (3.63 g)

C.d. 4 n ~ 3020

Fr 476--610 (0.29 g)

Fr 761--1270 (1.49 g)

Cid. 5 n =4085

Fr 926--980 Fr 711--780 (0.24 g) (0.36 g)

B5 B6

Fr 676--720 (0.48 g)

B7

Fig. 1. Flow scheme for the isolation of biologically active constitutents of Croton 0il and the further resolution of the fractions Bx an4 B 2 + B x by Craig-distribution (C.d.), n number of transfers

1 As usual the following abbreviations are being used (HECK~ 1955): partition eoeffi- Vu

eient K, partition number G, volume ratio of the two phases V ~ VI-I' Vu = volume of upper,

V 1 ~ volume of lower phase, n = number of ~ransfers, z = number of elements, r = fraction number.

On the Active Principles of Croton OiJ 197

of the separation is indicated by the appearance of the distribution curves, they are insufficient as a criterion of puri ty because the partition isotherms of the mixture in the systems and concentration ranges used are not linear. This has been also experienced with compounds A1, B 1 and B2 in the systems used previously (see also Fig. 2). Therefore a gas-liquid chromatographic method (H~c~:n~ and KuBINYI) was used as a criterion of puri ty and for the identifica- tion and analysis of the long chain fa t ty acid residues, as their methyl esters.

This method assumes that each compound contains one long chain f~tty acid and it does not distinguish between compounds differing only in the nature of the short chain fatty acid. I-Iowever, as a criterion of purity, it has been found satisfactory, as confirmed by the subsequent desTadative work.

The progress of the separation is outlined in the flow scheme, Fig. 1. Dis- tribution 1 using the system n-heptane (15), di-n-butylether (5), nitromethane (20) with n = 450 transfers achieved no resolution of the fraction B 2 ~F B x. The material accumulated main- ly in fractions 36--160 and was used for Distribu- tion 2 in the same solvent system applying n = 2000 transfers. Now, in the region of fractions 101--680, three distinct but overlapping bands were apparent, the peaks corresponding to partition numbers G ~- 0,13, 0,31, and 0,47.

30

G 0

- l z~176

/

7o z o 3o 4o Eo BO ~g/~tl 8o

- - ~ - C/owe r

Fig. 2. Par t i t ion isotherms of ]~2-F B x in different solvent systems. Q CC1, (2), methanol (1), water (0.15) (HECKER and B~SCH); [] n- heptane (15), di-n-butylether (5), ni tromethane (20); A cyelohexane

(15), di-n-butylether (5), ni tromethane (16), methanol (4)

The substance in fractions 601--680 (G=0,47) , containing decanoic acid as acyl-residue as revealed by the gas-liquid chromatographic method, was shown to be identical with the compound B2 (HwcK]~ and Kv]~I~u by comparison of partition coefficients and of UV-, IR- and 57MR-spectra, by chemical degradation, and by preparation of the corresponding aldehyde char- acterized by its crystalline 2,4-dinitrophenylhydrazone. The bands G =0,31 and G ~--0,13 occupying fractions 101--600 are rather broad in appearance and gas liquid chromatographic analyses of the fractions indicate that they consist of at least three unresolved compounds, containing the acyl-residues of octanoic-, decanoic-, and dodecanoic-acids.

In order to achieve better resolution the material in fractions 101--600 was subjected to a third Craig-distribution, the solvent system being modified by using cyclohexanc and by addition of a small portion of methanol in order to improve linearity of the partition isotherm (Fig. 2). A corresponding distribu- tion (Fig. 3) resulted not only in the isolation of more compound B2 (fractions 961--1080, C10), but also in a partial resolution of the other components. Maximum fractions 861--900 gave compound B3 (Clo) and maximum fractions 781--800 compound B4 (C12) with a separation factor of fl----1,13. Material containing octanoic acid accumulated in the fractions 476--610 (Fig. 3). On gas-liquid

198 E. C~A~K~ and E. H~cx~:

Z8

7B l" 72

8 i I #

2

787~ --~8on -- -~ 8#7.ooo~ ~ 9#7-7080 -----

V"

zoOc

i

,ze-elu . ~ i I

B 2 "-~

~ ~ I . . I I 1 ) ) !

son 600 ?no 8on ono 1000 Z 100

- - ~ f ruc t ion No, r

Ftg. 3. Craig-dis t r ibut ion 8, single phase wi thdrawal procedure, z = 1020, n = 2500, u = 5]8. Sys t em: Cyclo- hexanc (15), d i -n-butyle ther (5), n i t rome thane (16), me thano l (4). F rac t ions r = 0- -1020 represent the ave rage weight oi two ne ighbour ing fr~ctions r and ~-1 p lo t ted aga ins t r - l , l r~eti0ns 10~1--1080 represent the ave rage

we igh t of three ne ighbour ing f ract ions

z z--...~?~..~ 9 7 8 7 - 7 Z 7 0 = ~zn

76 u ~ : ~

,L A

. Z

Z 7 '~

0 } I I I t .I t I [ r ) l 500 #00 ?00 800 000 7000 7700 7200 7300 7~00 7#00

-- fpaction no.

Fig . 4. Cra ig-dis t r ibut ion 4, z ~ 1020, single wi thdrawal procedure for n ~ 2000 transfers, double wi thdrawal procedure ~or fu r the r n = 1020 transfers , V ~ 3i3. Sys t em: cyclohexane (13), d i -n-butyle ther (7), n i t rometbane(20) . F rac t ions r ~ 0- -1020 represent the ave r age we igh t of three, f ract ions r ~ 1021--1500 of f ive ne ighbour iug

f rac t ions

chromatographic analysis fractions in between the maximum fractions show intermediate compositions of the corresponding fatty acids.

Attention was then turned to the fraction Bx, and a preliminary separation (Fig. 4) afforded the major component B7 (fractions 676--720, C10 ) of adequate purity. However, the separation of this and the neighbouring compound B6 was incomplete (separation factor/7 ~ 1,19). The unresolved material in fractions

On the Active Principles of Croton 0il 199

777- 82S-

78D geO

74

mg

72"

2'

6=o,21

A 2ooc

5 . 1 1 ~. a/ ;l *

/ I e< , l

o

7oo 2gO 30o ooo WOo Soo 20g 8gO 900 7000

�9 fraction no.

Fig. 5. Craig-distribution 5, z = 1020, single withdrawal procedure for n = 3065 transfers, double withdrawal procedure for further n ~ 1020 transfers, V = 3]3. System: cyclohexane (15), di-n-butylether (5), nitromethanc (20).

Fraction weights represent average of three neighbouring fractions

761--1270, containing B 6 and a minor component B5, and the tail fraction f rom Distr ibution 3 (fractions 476--610) - - which was shown to have a similar K-vMue to tha t of B 6 - - were combined and used in Distribution 5 (Fig. 5). This f i na l separation afforded, besides a shoulder f rom compound BT, the compound B 6 (fractions 711--780, Cs) and compound B 5 (fractions 926--980, Cs) as well separated peaks (fl = 1,48), containing sufficiently pure fractions for subsequent chemical charaeterisation.

Chemical characterisation o / t h e new compounds. The compounds B2- - -B7 are optically active resins which could not be crystallized. They are easily soluble in mos t organic solvents and can be converted into brittle foams which give light powders softening below 100~ (see Table 5). They show almost the same R! values in thin layer ch romatography on silica gel. The spectra (IR, UV and NMR) of these compounds are similar in type, bu t ra ther unspecific. However, in the eases of B 4 and B7, an aeetyl group is shown to be present by NMI~

Table 5. Chemical characterisation and structural elucidation of pure compounds B2 B 7

Chloroform- Ethyl acetate

3:2**

0,32 0,31 0,30 0,32 0,31 0,28

ttegion of Corn- softening *

pound

oC

B2 60--62 B3 72--77 B4 71--74 B5 73--75 B 6 62--'65 B 7 75--78

Parent alcohol

C20H2sO~ Phorbol,

Esterified with acids ~5olecular formula long chain short chain -

decanoic decanoic

dodecanoic octanoic octanoic deeanoic

(~-)-S-2-methylbutanoic ~igiic acetic

(~) - S-2-methylbutanoic tiglic acetic

CssH~4Os C~5H5208 C34H520s Cs3t~50Os C~sH~sOs C32H4sO8

* 1Vfaterial has especially treated to remove solvents. ** On Kieselgel H (Merck), artificial mixture of B compounds R/0.34.

200 E. CLAgKE and E.H~exEg:

(d =2 ,0 , singlet). Also in case of the NMg-speet ra of compounds B3, B4 and B7 it has been ascertained tha t the singlet at d =3 ,95 ppm characteristic of a free - - C H 2 0 H group in allylie alcohols is present (H~o~Ex and K~rBI~YI). Crystalline derivatives of B3, B4, B6 and B7 were obtained by preparation of the 4-(4-nitrophenylazo)-benzoates (NPAB), the melting-points of which are given in Table 3. As experienced with compounds B1 and B2 (I-IEoK]~R and KgBI~YI) the NPAB-derivat ive of compound B5 did not crystallize.

Chemical degradation of the new compounds was carried out by reduction with lithium aluminium hydride. This procedure leads, in each ease, to the isolation of a water-soluble, erystailine alcohol and to a mixture of two ether- soluble alcohols.

The water-soluble alcohol was further characterized by its R/-value anti by conversion into a tetra-acetate. This alcohol is identical with Phorbolol C20Ha00s which can also be produced by a similar reduction of Phorbol C20It2sO s (I-IECKER, BRESCI~ and M~YE~; H1gCKEI~, KUBINYI, V. SZCZEPANSKI, HXI~LE and B~Esc~). Therefore, the parent-alcohol for the compounds B 2 - - B 7 is Phorbol, as has been found also for compounds A1, B 1 and B2 (HEcKEI~ and B~ESCH; H~Cs:En and K v ] ~ Y I ) .

The ether-soluble alcohols were isolated as their 3,5-dinitrobenzoates (DNB). The DNB-mixture was separated by column chromatography and the individual compounds were identified by comparison (IR and mixed m.p.) with authentic specimens. Nuclear magnetic resonance spectroscopy was useful for the detection of unsaturation or chain-branching in these derivatives, and also confirmed the identi ty of the long chain aeyl residues as suggested by the gas liquid chromato- graphic data (see Table 4). The short chain aeyl residue of compounds B4 and B7 is tha t of acetic acid, B5 contains 2-methyl-butanoie acid most probably as the (~ ) - S- antipode.

The DNB-derivative of (--)-S-2-methylbutanol prepared from compound B2 shows m.p. 82.5--83 ~ the derivative of the racemic mixture of 2-methylbutanol m,p. 700 (tt]se~:El~ and I~2IrBINYI). The preparations of compounds ]32 and B 5 described here yield DNB-deriva- tives of 2-methylbutanol m.p. 750 and 74--75 ~ respectively, which on admixture with the derivative m.p. 82.5--83 o do not cause depression of the m.p. Therefore it can be concluded that the substances m.p. 75 and 74--75 ~ are DNB-derivatives from (--)-S-2-methylbutanol which is derived from (d-)-S-2-methyl-butanoie acid.

The reductions of the compounds B3 and B6 presented a complication. " N o r m a l " and " reverse" addition procedures yielded a short-chain alcohol derivative fraction which is a mixture. Mass-spectrM data (discussed below) indicate tha t the short chain residue in these compounds is an unsaturated C 5 unit, and the NMI~ spectrum clearly excludes the (CHa)~C=CH-- and CI'I 2 = C < groupings, suggesting tha t the substance is a tiglic or angelica alcohol derivative. However the NM1% spectrum shows resonances at d = l , 1 4 (triplet, J = 7 cps) which can be assigned to the 2-methylbutanol derivative. Authentic samples of tiglic- and angelica-alcohols were prepared from the corresponding methyl esters by a procedure similar to tha t described in detail for the B4 compound. Paper chromatography indicated clearly that the angelica alcohol derivative was absent but did not differentiate between the tiglic alcohol and 2-methylbutanol derivatives. The NMI~ spectra of a synthetic mixture composed of tiglic DNB (65%) and 2-methylbutyl DNB (35%), and of the derivative mixtures from 133

On the Active Principles of Croton Oil 201

and B 6 were identical in all respects. The fragmentation patterns in the mass spectra of B 3 and B 6, the good resolution of them and their saturated analogues B2 and B 5 in the distributions and the preparation of crystalline NPAB deriva- tives, all support the high purity of B3 and B6 claimed. Therefore it is con- cluded that partial reduction of a tiglic acid residue has occurred in the lithium alumirdum hydride reaction.

Mass spectrometry applied as " M o l e k i i l " - or "Elektr0nenanlagerungs"- Massenspektrographie (v. A I ~ D E b l N E ; V. AI~DENNE, STEINFELDER and TOMMLEI~;

700

%

80

GO

4,0

20

500

1

308

zgz

310 328

388

_[ [,,I . . . . l lltZ~It31 e , . I 28O 300 320 3/,0 3B0/,00 1~0 ~bO /-/s

~ rnsss D u m b e r

55x#

5~3

#72 L d I__

080 500 5GO 580

Fig. 6. "Elektronenanlagerungs"-mass spectrum of compound B6. Temperature of vaporisation 1~00 C

6OO -$00-

400

3 2 0

-700

- 7~ - 500-

328

-588-

\- -528-

-388- /

328"

- 574 -

- 7~,b t 702

- 4 7 2 -

- ~30- I

328

-5?2- h -seo-_

/ I-" i l -~,28- i # I 772

-"\,I 328 3 2 8 - -

Go ~sG (3~)-zG~ Fig. 7. Fragmentation patterns of the compounds ]33--]37 from "Elektronenardagerungs"-mass spectrometry.

Temperatures of vaporisation: B3, B4 and B7 200 o C, B5 and B6 140 ~ C

v. A~D~N~, STEI~ELDE~, T~MML~I~ and S c ~ I ~ E ~ ) has proved a powerful and convenient tool in the characterisation of these compounds (see also HECKEI~ and B~sc t t ) . A typical spectrum, that of compound B 6, is illustrated Z. Krebsforsch. Bd. 67 14

202 E. CLAiKE and E. HEOKE~:

in Fig. 6. The spectra of the B compounds are all similar in general appear- ance and m a y be convenient ly considered together. Significantly, in each case there is a peak at mass number 328, which can be assigned to an ent i ty "Phorbol minus 2 t I 2 0 " . The f ragmenta t ion pat terns in the region below 328 are very similar. P resumably they represent the f ragmenta t ion of the parent alcohol which is obviously independent of the nature of the acid residues. The pa t te rn above mass number 328 is the mos t impor tan t for the elucidation of the structures of these residues. The molecular weight or parent peak is visible in all cases and the fragments arising by removal of the acids can be identified as more or less intense lines, thus permit t ing calculation of the molecular weights of the fa t ty-acid constituents. Fig. 7 shows these pat terns for compounds B 3 - - B 7 , and for clarity other f ragments (most arising apparent ly by the removal of water) are omitted. These results confirm those obtained in the chemical degradations.

--O--C---R 1 II 0

--O--C--I~ 2 II

O --OI-I

--OI-I I

>C=C--CH2--OH 1~ i = Alkyl residue of short chain fatty acid

>C:C- -C : 0 I~ : Alkyl residue of long chain fatty acid I I

I n summary compounds B 3 - - B 7 are diesters of Bhorbol with one short chain (acetic-, 2-methylbutanoic- , tiglic acid) and one tong chain f a t t y acid (octanoic-, decanoic-, dodecanoic acid).

The almost identical R fva lues of compounds B 3 - - B 7 together with NMR- da ta and their abil i ty to form mono-NBAB-der ivat ives indicate t h a t of the

Table 6. Biological characterisation o] compounds B 3- - B 7 (Assays see Substances and Methods)

r176176 I

Croton Oil DAB 6 1958

B3

]B4

B5

B6

B7

2,4

0.7

0.2

0.15

0.7

0.3

Carcinogen ~ ~Mol

Cocarc inogenie a c t i v i t y

1 0.1

1 0.1

1 0.1

0.1

0.1

1 0.1

9,10-Dimethyl- 1,2-benzanthracene (DMBA). b Application: twice weekly during 12 weeks. c After 12 weeks.

Carc inogen ~ g / a p p l i c a t i o n b

500 500

10 1O

10 10

i0

10

10 10

% mice w i t h t u m o u r s r

t t tmors/li lOllSe c

90 5 64 4

86 6 61 3

89 6 50 2

32 2

29 1

57 1 57 2

On the Active Principles of Croton 0il 203

three free hydroxyls found, the pr imary allylie hydroxyl is free as has been shown for compounds A1, B1 and B2 (HEcKER and BI~.SOH; HECKER and KUBINu Together with the ~,/~-unsatnrated earbonyl-group all oxygen functions of the compounds are accounted for.

Biological characterisation o/ the new compounds. The biological activities of the compounds isolated are summarized in Table 6 and compared with Croton Oil DAB 6 1958 from which they h ive been isolated. The inflammation unit (I.U.) i.e. the amount in/zg which causes a clearly visible inflammation of the mouse ear (HwcI(m~ 1963), is similar for all five compounds. Also the cocarcinogenic activity as assayed by the standardized procedure (HEcKleR 1963) and expressed as percent mice with tumours and as tumours/mouse is similar for all the compounds. Further- more these biological activities are comparable with the activities of compounds A1 (HwcKV, R and BRESCIa), as well as B1 and B2 (HEcKEI~ and KVBINYI).

The authors are gratefully indebted to Prof. BUTENANDT, Imperial Chemical Industries Ltd. and Deutsche Forschungsgemeinschaft for material support of this work. Measurement of spectra and stimulating discussions by Prof. Dr. M. v. ARDV, NI~E, Dr. J. SOWNENBICI~Lm~ and Dr. T. Fu~" are gratefully acknowledged. Mr. H. BAUMANN assisted in some of the practical work.

Summary

Resolution of fraction B x from Croton Oil by Craig-distribution reveals 5 new irritant and cocarcinogenic compounds B 3 - - B 7. By physical methods such as mass-spectrometry and NMR-spectroscopy combined with chemical degrada- tion these compounds are shown to be diesters of the polyfunctional Diterpen alcohol Phorbol C20H2s06 each with a short chain (acetic-, (+)-S-2-methyl- butanoic-, tiglie acid) and a long chain fa t ty acid (octanoie-, decanoic-, dodecanoie acid). The new compounds are being characterized by their irri tant and cocarcino- genic act ivi ty in mice.

t3ber die Wirkstoffe des Crotoniils V. Reindarstellung und Charakterisierung weiterer entziindlicher

und coearcinogener Wirkstoife der B-Gruppe

Zusammenfassung

Die Auftrennung der frfiher beschriebenen Fraktion B x aus Croton61 dutch Craig-Verteilung ffihrt zu f/inf bisher unbekalmten entzfindlichen und cocarei- nogenen Wirkstoffen B 3- -B7, die rein dargestellt werden. Dttrch physikalisehe Methoden wie Massenspektrometrie und NMR-Spektroskopie kombiniert mit chemischen Abbauverfahren wird gezeigt, dal~ diese Wirkstoffe Diester des polyhmktionellen Diterpenalkohols Phorbol C20H2sO6 mit jeweils einer kurz- kettigen (Essig-, (+)-S-2-Methylbutter-, Tiglinsaure) und einer langkettigen Fettsaure (Capryl-, Caprin-, Laurinsi~ure) sind. Die neuen Wirkstoffe werden dutch ihre entzfindliche und cocarcinogene Wirkung an Mausen charakterisiert.

References

ARDEN~I]~, M. V. : Tabellen zur Angewandten Physik, Bd. I. Berlin: VEB Deutscher Ver]ag tier Wissensch~ften 1962.

- - i . S T EINF ELDER U. R . Tf fMMLER: Elektronenanlagerungs-Massenspektrogramme konden- sierter, aromatiseher Kohlenwasserstoffe. Angew. Chem. 73, 136 (1961).

- - - - u. EL. SCRlCEIBER: Molekiil-Masscnspektrographie yon Naturstoffen. Steroide. Experientia (Basel) 19, 178 (1963).

14

204 E. CLARKE and E. HEOKER: On the Active Principles o~ Croton Oil

CI~AIG, L.C., D. CI~AIG, and G. SCHEIBEL: Laboratory extract ion and countercurrcnt dis- tr ibution. Technique of organic chomistry, edit. by A. W]~ISSBEI~G]~I% 2. ed., vol. I I I , par t I, p. 149. New York: Interscience Publ. 1956.

- - , and T. P. KII~G- Design and use of a 1000-tubo countercurrent dis tr ibut ion apparatus. Fod. Proc. 17, 1126 (1958).

Ha~CKEI~, E. : Vertcflungsverfahren im Laboratorium. Weinheim. Verlag Chemio 1955. - - Uber d i e Wirkstoffe des CrotonSls. I. Biologische Teste zur quant i ta t iven Messung der

ontziindliehen, cocareinogenen und toxisehen W~rkung. Z. Krebsforseh. 65, 325 (1963). - - , u. H. BlzEScg: t ibe r die Wirkstoffe des CrotonSls. I I I . Reindarstel lung und Charakteri-

siorung t ines toxisch, entziindlich und cocarcinogen hochaktivon Wirkstoffos. Z. Natur- forsch. 20b, 216 (1965).

- - - - u. J . G. MEYEt~: lJbcr cocarcinogene Wirkstoffe dos CrotonSls. I. World Fa t Congr., Hamburg, Oct. 12--18, 1964, Abstracts of Papers, p. 176. Fet tc , Scifen, Anstr ichmit te l 67, 78 (1965).

- - - u. CIr. v. SZCZEPANSKI: Cocarcinogen A1 - - der erste reine, hochaktivo Wirkstoff aus Croton51. Angew. Chem. 76, 225 (1964); Angew. Chem. internat , edit. 3, 227 (1964).

- - H. J. JAI~CZYK, J . G. MEYm~, H. B I ~ s c ~ u. I. BI~ACmVtANN: ~Jber die Wirkstoffo des Croton5ls. II . Eine systematisehe Frakt ionierung des Croton5ls. Z. Krobsforsch. 66, 478 (1965).

- - u. H. KI~BINYI: ~ b e r die Wirkstoffe dos CrotonSls. IV. I~eindarstcllm~g und Char~kteri- sierung dcr entziindlichen und cocarcinogcncn Wirkstoffe B 1 und B2. Z. Krebsforsch. 67, 176 (1965).

- - - - u. H. BRESCIa: Eine neue Gruppe yon Cocarcinogenen aus Croton61. Angcw. Chem. 76, 887 (1964); Angew. Chem. internat , odit. ] , 747 (1964).

- - - - Ct t . V. SZGZEPAI~ISKI, E . I-I~RLE U. H . BRESOII : Phorbol - - ein tetracyclisches Diterpen aus Croton5l. Tetrahedron Letters 1965, 1837.

MEmo, D. F. : Separat ion of the 3,5-dinitrobonzoates of volatile alcohols by paper chromato- graphy. Nature (Lond.) 169, 706 (1952).

M~TzscI~, F . A . v . : 200-stufige, vollautomatische Apparatur . Zur fraktionierten Gegen- stromvertcilung. Chem.-Ing.-Tcch. 25, 66 (1953).

Prof. Dr. E. NECKER Biochemisehos Ins t i tu t , Deutsehes Krebsforschungszentrum

69 Heidelberg, Berliner Str. 23, Germany