\’d.
ti
Notes Subsidiary Constituents from .4ntanita
’I’he residual rnushrooni pulp ( 11 4 Lg ) \\as di ieti i n cb curreiit itir at 35” Thr dried material \vas thoroughl\ cx\tractetl at i (ion1 teriiperztiirr v ith ahsolutr methanol Thc. combinrti eltracts n erc’ conceiitrated in a low temperature v:iciiiini -till Frddi(Jn 11 \ \ A S d light hro\rn sirup ( 2 9 kg , plI 7.5) The ‘ riiaw ’ f r o r i i the t n o previcius r\tractions \\as tiirthei ix\tractetl :it roori1 teniperaturr 3 times \rith p\ rogen-freia distillctf n ater T l i t . ailueous filtrates vere concentrated i n .i l(n1 teriiperature vacuum still Fraction IT1 was a (lark bruwri ‘LIYrou5 solutioli ( 3 3 kp , 1‘ {if
.MuscariaI a.17
D e p a r t m e n t ol C h e m i s f !11, .1iontana S t a f r College, Hoze?nan. .liontana
Review articles”-‘ record chroiiological accounts of the chemistry of muscarine, whose structure finally has heen elucidated. However, the chemical basis for the pqychoactive principles of the mushroom, Amanita inuscaria, continues t o baffle investigators. It is generally agreed that intoxication by the mushrooin is due primarily to excitation of the central nervous slsten1 and that muscarine cannot possibly be responsible for the observed activity.s Investigators have labeled the mushroom’s elusive hallocinogen as “Pilzatropine” for ease of reference.6 Hyoscyamine’ and bufotenineY have been reported to be found in Amanita muscaria. These could, if present, account for the activity of the drug. Howm w 7such claims have not been substantiated by other iiivestigators in the field.6 lo Eugster3 reported the muscaria atiselice of hyoscyamine and bufotenine in 9. i n the course of intensive investigations. He detected :t labile substance which had a spectrum similar to that of the 4-hydroxy indole derivative, psilocybin, the psychoactive principle of the Mexican fungus, P d o c y b e me.xicana. l 1 systematic chemical investigation of A . muscaria was initiated in this laboratory in an attempt to identify tlie psychoactive principles. Fractionation was carried out with four successive batches of mushroom,12taking precautions to prevent the formation of artifacts and progressively making changes in procedure based on data from biological tests. Details of this work will be rrported later. A summary of the extraction procedure is given hew. Fresh, whole mushrooms, preserved in methanol, were filtered 1-, draining through muslin cloth. The swollen mushrooms (40 3 kg.) were pulverized in a meat grinder and the pulp was spun L ~ Y dry
as possible in a basket centrifuge. The combined filtrates from draining and centrifuging were concentrated in a lox teniperature vacuum still. The concentrate. fraction I,was a thick, dark broxn sirup ( 3 6 kg., pH 5.1) (1) (a) .4 prciiniinary report of this work \\-as presented before the IUPAC‘ Symposium on the Chemistry of Natural Products, Melbourne. .\ustralia, \ugust, 1960. (b) This investigation was supported i n part by t h e Medical Sciences Research Foundation. Stanford Cniversity . (2) S. Wilkinuon. Quart. Rer. (London), 15, 153 (1961). f.3) C. 11. Eugster. Rev. de Mliycologie., 24, 369 (1959). (,‘4) K. Bowden and G. A. Mogey, J . P h a r m . Pharmocol., 10, 145 (1938). ( 5 ) V. P. Wasson a n d R. G. Wasson, “Mushrooms, Russia and History”, Vol. 1, Pantheon Books, New York, N. Y . 1957. pp. 190-214. (6) 1’. E. Tyler, Jr., A m . J . Pharm. Pharmacol., 10, 145 (1958). fi)H. S. Lewis. S. < l j v . .Wed. J . , 29, 262 (195,5), (81 T. Rleland and W. Motzel. Ann., 581, 10 (1953). (9) v. Iiwasnienski, Deut. A p o t k - Z t g . , 94, 1177 (1954). (10) L. R. Brady and V. E. Tyler, Jr., .I. Am. Pharm. Ass7r. (Sci. Ed), 48, 417 (1959). i l l ) :I. Hofmann, A. Frey, H. O t t , Th. Petrizilka, and F. Troxler, Ezperientia, 14, 397 (1958). 1 1 ?) .4uthentic samples of An~anzta viu,srar7a u e r P supplied hs t i i t s lf:-vcdogicxl Society (if Sari Franrisro.
Hiological tcistiiig (‘Fable I) indicated that aqueou‘ extract 111 n.a\ relatirely free from muscarinic effects ciharacteristir of fractions I and I1 l‘lethanolic ex tracts I a d I1 w r p subjected to extensive solvent fractioiiation u u d chroniatographic separation ‘t‘hc +everal resulting fractions which included minor crystalroduct- i ~ i i da rather large essential oil fractioir uhjectccl to hologicd screeiiing. Xonc cxliibitcd bant “\~~riipatEii)toiiia.”t h r 1ivprrpyrc.uia-iur driasiy drug \viidioiw vharacteiistic of LSD-23 1‘mi I, I 13101 O G I I
b\
%I
IC111 1 l I L S O E F R . \ L T I O \ S ” L I ) - X mi3 /kg I1
rnptoir,\
Ataxia of gait Hypomatilit\ Loss of righting refle\ Prone position MlOSlS
Salivation Lacrimation Hyperp yrexi‘t Sloning of respiratiun Convulsions Death Opisthotono\ Proptosis Hypoton1 I hfydriasis
2 1.) 1
48
1 .36 2 t; IO3 1 04 1 01 1 01 1 01 2 30 6 23 1 ’32 2 l i
0 07
* sc
I 0,i I .io 2 19 0 09 1 3h 0
0 50 0 56
-I rartion I11 i SI
x
0 29 0 d0
1s
I 0% 0 18
2 1 1 0 10 2 0 07 2 0 0; 2
40 40 0 13 1 53 63 0 10 0 ‘3-1 10 0 13 25 0 14 >I) G L: result uf these fiiidings the ultimate natei 3olublc portion of tlir mushroom, freed from alkaloids, proteins, lipids arid inorganic constituents, such as KCl, u - a h considered to be the “active” fraction Small doses of this fraction, caomparable to those employed f o ~ LSD-23, produced in test animals mydriasis acc‘onipanied by hyperpyrexia. Whether the hypothetical “pilzatropinr” or >onw such single constituent is responsible for the activity of tlie water concentrate o ~ ’ whether the hallucinogenic activity of the niushrooin is due t o a combined drug syndrome will have to bc proved by human experimentq in view of the manifestation of various pharmacological effects by the mushroom which are not always easily distinguishable and \yhicli require extreme cautioii in interpretation. It should bo iwiitioned that hyperpvrexia per sc need not iieces.:irily produce nor 1~ accompanied by mydriasis (11’
SOTES
July, 1963 miosis. For example, fumaric acid isolated from A. muscaria produced hyperpyrexia but neither mydriasis nor miosis in experimental animals. Therefore, in the biological screening of various fractions from A . muscuria, a material which produced mydriasis accompanied by hyperpyrexia was considered tentatively to possess LSD-like behavior. Through fractionation of the aqueous concentrate (111) with judicious mixtures of methanol, ethanol, ethyl acetate, and acetone an hygroscopic crystalline material, provisionally designated “z” was isolated (307 mg., m.p. 135-138’ dec). Anal. Calcd. for C15H30K4015: C, 29.7; H, 4.98; K, 25.77. Found: C, 30.17, 30.30; H, 4.83, 4.60; K, 25.23; mol. wt., 615; equiv. wt., 218 (potentiometric titration). The analytical data are consistent with a compound having four COOK and three COOH groups. The n.m.r. spectrum showed only two peaks; one characteristic of methyl-keto functions, the other of labile hydrogens. Integration of these peaks suggests a ratio of three methyl groups to two labile hydrogens. The very small amount of “z” left after pharniacological and analytical experiments was methylated with diazomethane. Its n.m.r. spectrum was complicated and showed no expected relationship to that of the original compound other than to confirm the methylation. “z” was tested intravenously in rabbits a t two-dose levels. A low level dose of “z,” 1.0 mg./kg., resulted in mydriasis, 30 min. after administration, which persisted for 30 min. Pupil dilation \vas 2 mm. above normal. Three hr. after administration, there was a temperature rise of 0.9” which lasted less than 1 hr. At the high level dose of 6.25 mg./kg., mydriasis was immediate, persisting for 30 min. Pupil dilation was initially 1.5 mm. above normal, increasing to 2 mm. later. Thirty min. after administration, temperature rose 0.7” and persisted for 30 min. Fifteen min. after administration, there was rapid respiration persisting for 2 hr. There were no other symptoms. By means of thin-layer chromatography minute quantities of indolic compounds have been isolated from the aqueous concentrate. From fractions I and I1 potassium gluconate, a glucoside m.p. 106’ dec and two minor crystalline substances, m.p. 172174” and 160-165’, respectively, were isolated as hitherto unreported constituents. Mannitol, fumaric acid, a fixed oil fraction, and a considerable amount of KCl also were isolated. The fixed oil was fractionated on alumina columns and subjected to chemical and biological testing. All fractions were found to be mainly esters of oleic acid and devoid of insecticidal properties. 1 3
Acknowledgment.-We wish to thank Dr. Edward Pelikan and his colleagues for carrying out the pharmacological tests, Varian Associates for n.m.r. data and hbbott Laboratories for large scale extractions. (13) A . muscarza is commonly knonn as fly-agaric due t o its reputed propert) as an insecticide.
I. Derivatives of P,P-Bis(1-aziridiny1)phosphinicAmide‘ ORRIEill. FRIEDMAS, ROBERT S.LEVI,
Potential Carcinolytic Agents.
ZINON
B.
PAPAXASTASSIOU,’ AND WILSOX
M. WHALEY
Arthur D . Little, Inc., Acorn Park, Cambridge 40, Massachusetts Received July 1 1 , 1961: reaised manuscript receiued February 1,196s
The alkylating ability of aziridine is known to be enhanced by an elect.ron-withdrawing N-substituent, and there are indications that molecules with several activated aziridine groups are likely to display anti-
449
tumor activity.3 However, a favorable therapeutic index depends on the entire molecular structure rather than simply on the cytotoxic moieties. A degree of selectivity of action has been achieved with certain P,P-bis( 1-aziridiny1)phosphinicderivatives, notably the “dual antagonist^."^ Further work along these lines has led to the preparation of the derivatives (11) reported in the present paper.
Ia, Z Ib, Z
1 = =
7T
11
0
S
IIa, R = C6Hll; Z = 0 IIb, R CeH11; Z = S IIc, R = (CH2)30H; Z = 0 IId, R = (CHz)aOH; Z S IIe, R = (CHz),OCH,; Z = S
The usual procedure (excepting one instance) for preparing P ,P-bis(1-aziridinyl)phosphinic amides has been t’o attach the sensitive aziridine groups in the last steps6 This approach was not used in our work because a number of the intermediates would contain reactive functional groups so situated that unwanted cyclizations could occur prior to the final step. The desired compounds were prepared from the intermediate P,P-bis(1-aziridiny1)phosphinic chloride (Ia) and the thiono analog (Ib), both of which were used directly without isolation. The phosphinic chloride was so unstable that it was used immediately upon preparation, whereas the thiophosphinic chloride could be kept at least overnight in solution at low temperatures. All reactions were conducted in the cold, and the products isolated under the mildest possible conditions because of their inherent instability. Simple amines such as cyclohexylamine, used in excess to serve as condensing agent,s, gave fairly good yields of nearly pure products (generally the yields decreased rapidly with at.tempts a t purification). With other amines where triethylamine was used as the condensing agent the products were less easily purified. The oily products in particular were difficult to purify because they decomposed when subjected to most standard purification techniques, including molecular distillation at. mm. pressure or chromatography. Pure derivatives could not be obtained from 1,3propanediamine, 3-mercapto-l-propylamine,1,3-dimercaptopropane, 3-mercapto-1-propanol, 1,3-propanediol, and ethyl glycinate. The use of sodium or magnesium salts of the alcohols and mercaptans was unsuccessful as was the triet’hylamine t’echnique. I n addition to triethylamine, various other hindered and unhindered amines (X-methylpiperidine, S-methylmorpholine, tri(1) This work was sponsored by the Cancer Chemotherapy National Service Center, National Cancer Institute, National Institutes of Health, Contract No. SA-43-ph-4360. (2) To whom inquiries should be addressed. (3) J. A. Hendry, R. F. Homer. F. L. Rose, a n d -4.L. Walpole, Brit. J. Pharmacol., 6 , 357 (1951); S. &I. Buckley, e t a l . , Proc. Soc. Ezptl. Bid. Med., 78, 299 (1951). (4) 2. B. Papanastassiou and T. J. Bardos, J . M e d . Pharm. Chem., 5, 1000 (1962): D. R. Seeger and 4. 9. Tomcufcik, J. Org. Chem., 26, 3666 (1961). (5) Z. B. Papanastassiou and T. J. Bardos, Belgian Patent 577,883 (1939); journal manuscripts in preparation. (6) E. Kuh and D. R . Seeger, U. S.P a t e n t 2,1370,347 (1954).
