Journal of Medicinal Chemktry 0 C'op~,.iylit 1070
Cy the A m e r k a ~Chemical ~ Societv
VoLunrE 13, s o . ti
SOVEhlBEIE
1970
Newer Cardiac Glycosides and Aglycones' K. IC. CHEN Department of Pharmacology, Indiana University School of Jf edicine, Indianapolrs, Indiana Received V a r c h 25, 1970
A total of 50 steroidal substances, mostly of plant origin, have been tested pharmacologically, and assayed iii cats for their cardiac activity, using cessation of the heart as the end point. Forty-five of them are glycosides of the cardenolide type, or free aglycones. They are derivatives of digitoxigenin, uzarigenin, periplogenin, sarmentogenin, gitoxigenin, strophanthidin, and strophanthidol with various substituents. Monosides are generally more potent' than biosides, or their corresponding aglycones. The most active glycoside of the entire series is panoside which has a value of 13.14 mean LD's/mg. 3-Epimerization, 17a-orientation, or formation of a double bond from the 56- or 16p-hydroxy group usually resulk in a weak t o an inactive product. Drevogenin *4 ( S I ) is a pregnane derivative; it has no digitalis-like effect. Calactin of the milkweed family is stored in the body of American monarch butterfly or African grasshopper. This is the first example of a potent glycoside of the cardenolide structure that occurs in the animal tissues. The toad synthesizes bufadienolides, but not their glycosides. I n this collection of compounds, arenobufagin (XVI) and +bufarenogin (XV) are derived from t h e toad poison. The results of two new glycosides ( X I 1and XIV) of red squill are reported; their aglycones have the bufadienolide st,ructure.
While the 324th paper2 appeared from the Inqtitute of Organic Chemistry, University of Basel, we have accumulated pharmacological data on 50additional cardiac glycosides and aglycones. As shown in Table I, 49 of them were obtained from natural sources, and one was partially iynthesized. The results afford some structure-activity relationships of variations in the steroid nucleus or side chain. It becomes evident that animals other than the toad are capable of storing glycosides of the carderiolide type from their plant foods. Such example4 are pointed out in the discussion. l l o > t of the 30 substances studied are biosynthesized by plants.
Methods A z in previous investigations3s4the first objective was to demonstrate the presence or absence of digitalis-like action of each new substance, and t o assess its potency in etherized cats if positive evidence was obtained. The cat is the most sensitive laboratory animal, less subject to environmental changes. When sufficient (8-10) animals are used the geometrical mean lethal dose (LD) can be determined and the standard error computed as the limit of reliance. Similar compounds are best compared himultaneously in the same colony of cats in order to minimize any bias. The electrocardiographic changes from start to finish are so characteristic that they are not duplicated by any known drug.
The supply of each rare and valuable material from our collaborators seldom exceeded 30-50 mg. If the mean I D is 0.1 mg/kg, or smaller, a quantity of 5-10 mg is ample for the necessary information. The initial procedure consisted of making a stock solution of 0.1% by weighing 5 mg into a 5-ml volumetric flask, and dissolving the substance in EtOH followed by H,O in equal volumes. dilution of 50,000 was then made up, and injected into an etherized cat from a 50-ml buret a t the rate of 1 nil rnin with the electrocardiograph connected to the animal. The results of this first experiment guided further dilutions so that a %-kg cat was killed within 30-60 min in order to arrive a t the mean LD. The weight of cats varied from 1.6 to 2.9 kg. 1;emale animals in advanced pregnancy were rejected. The mean LD so determined is a n expression of potency, actually the full cardiotoxicity. It can serve as a safe hint for initial clinical trials. -4dose of 3 t o 5 mean LD, injected iv, lowers the ventricular rate of auricular fibrillation. Examples can be found in the first human tests of cinobufaginj and t h e v e t h 6 It must be realized that the cat method, even though by the identical procedure, only gives a figure for comparative purposes, but does not measure the rate of destruction-accumulation tis. rapid disappearance. 120rtunately, none of the substances left the animal body as fast a5 it was injected. Additional investigations mould establish the optimal clinical dose. Thus the digitalization and maintenance doses of digitoxin
( 1 ) This work was supported in part by U. S. Public Health Service Grant HE-0i714 a n d the Indiana Heart Association. (2) -1.Lardon. K. Stockel, a n d T. Reichstein, Helu. Chim. A d a , 53, 167 (5) K. K. Chen a n d A . L. Chen, A r c h . I n t . Pharmacodyn. Ther., 47, 3 0 i (1934). (6) H . L. Arnold, IV. S.hIiddleton and Ii. Ii. Chen, Amer. J . .\fed. Scz., 189, 193 (1935).
(1QiO). (3) F. G . Henderson and K. K. Chen, J. .Wed. Chem., 8 , 577 (1965). (4) K . K . Clien and F. G . Henderson, J . Pharmacal. E x p . Ther., 150, 53 (1963.
1029
C'oronillu glaitca, seeti
17 I l l , I1
2.i
4
/' '1.
S.50
I
I).17:; rL 0 . 0 1 I 0 200 ;t 0 010 0 200 rt- 0 Ill:; I
', il;ll
Journal of Jledicinal ChrtrLislry, IUX, 1'01. 1.1, &Yu. 8 1031
TABLE I (Continued) LD 5 SE, Source
Uryinea maritima, bulb Chan Su, toad poison cake
Compound
Reference
Scillarenin-Gl~~ Scilliruhroside +Buf arenogin
d,d
mglkg, cats
0.111 f 0.006 0.163 f 0.012 Survived 2.65 and 4.92
Partial Synthesis 16-0-Isovalerylgi t,oxin 12 2.27 Abbreviations of sugars: l)yx, digitoxose; Cym, cymarose; Rhs, rhamnose; GIs, glucose. 0.P. ,\Iitt,al, Ch. Tamm, and T. lieischstein, Hela. Chim. A4cta, 45, 907 (1962). c C. H . Hassall and K . Reyle, J . Chem. Soc., 85, (1959). d J . 11. do Nasciemnto, Jr., Ch. Tamm, H. Jager, a n d T . Iteichstein, Hela. Chim. Acta, 47, 1775 (1964). e 11.L. Lewhart, W. Wehrli, and T. Reichstein, ibid., 46, 540 (1963). J 11.L. Lewbart, W. Wehrli, H. Kaufmann, and T. Keichst'eiii,ibid., 46, 517 (1963). 0 W. Schmid, H. P. Uehlinger, Ch.Tamm, and T. lieichstein, ibid., 42, 72 (1959). h L. F. Fieser, T. Golab, H. Jager, and T. Iieichsteiri, ibid., 43, 102 (1960). J. Polonia, A. Kuritzkes, H . Jager, and T. lieichstein, ibid., 42, 1138 (1939). j A. Kuritzkes, J . V. EUR, and T. Reichstein, ibid., 42, 1302 (1959). k 11. Tschesche, W. Freytag, and G. Snatzke, Chem. Ber., 92, 3053 (1959). 1 9 . h1. Kuritzkes, Ch. Tamm, H. Jager, and T . Reichstein, H ~ l o . Chim. Acta, 46, 8 (1963). m E . Schenker, A . Hunger, and T. Heichstein, ibid., 37, 1004 (1954). n R . Berhold, W. Wehrli, and T. lieichstein, ibid., 48, 1634, 1639 (1965). H. H . Sauer, Ek. Weiss, and T . Reichstein, ibid., 48, 837 (1965). P U . P. Geiger, Ek. Weiss, a n d T . lieichst'ein, ibid., 50, 194 (1967). F. Kaiser, E. Haack, AI. Gube, U. Dolberg, and H . Spingler, ~ ~ a t u r ~ i s s e n s c h a f t46, e n ,670 (1959). 11. Zelnik, J. V. Euw,0. Schindler, and T. Reichstein, Hrlv. Chim. Acta, 43, 393 (1960). A . F. Krasso, Ek. Weiss, and T. Iieichst'eiii, ibid., 46, 1961 (1963). t Y t . Hoffmann, Ek. Weiss, and T. Reichst'ein, ibid., 49, 1855 (1966). u P. Hauschild-Rogat, J . V. Euw, 0. Schindler, Ek. Weiss, and T. Iieichst,ein, ibid., 45, 2116 (1962). D P. Hauschild-Rogat, Ek. U'eiss, and T. Reichstein, ibid., 50, 2299, 2322 (1967). H. Kaufmann, W. Wehrli, and T. lieichbtein, ibid., 48, 65 (1965). H. Kaufmann, ibid., 48, 83 (1965). Y A. It. Manzett,i and T. Iteichstein, ibid., 47, 2303, 2320 (1964). 8 K. D. Roberts, Ek. Weiss and T. Iieichstein, ibid., 46, 2886 (1963). a , c . K. L). Roberts, Ek. Weiss, and T. Iieichstein, ibid., 49, 416 (1966); ibid.,50, 1643 (1967). b.h H . hllgeier, Ek. Weiss, and T. Iieichstein, ibid., 50,431, 436 (1967). c , c Z . Kowalewski, H. Jiger, 0. Schindler, and T. lieichstein, ibid., 43, 957 (1960). d , d 4.V. Wartburg, ibid.,47, 1228 (1964); ibid., 49, 30 (1966). e , e K . Huber, H. Linde, and K . Meyer, ibid., 50, 1994 (1967). 1
TABLE I1 STRUCTURE -.4CTIVITY RELATIONSHIP Skeleton Compound
formula
Beaumontoside Wallichoside Vallaroside Mono-0-acetylvallaroside Solanoside Mono-0-acetylsolanoside Uzarigenin 3-Epiuzarigenin do-Uzarigenin Aacleposide Acobioside b
I I I I I I I I I I I1
Acospectoside A
I1
Periplogenin cymarosidodigitoxoside 3-Episarmentogeniri Sarmentogenin biscymaroside Sarmeritogeniii biscymarosidodigitoxoside Sarmentogenin cgmarosidodigitoxoside Opposide
111 IV IV IV IV IV
Malloside
IV
Vallarosolat ioside Moiio-0-acetylacoschimperoside P Beauwalloside Adigoside
v
16-0-Isovalerylgitoxin
V
16-Desacetyl-16-anhydroacoschimperoside P Corotoxigenin rhamnoside Calotropagenin Caiactin
V
V
v V
Varianta
H R
R
= a-L-0ls
OI-L-CYS = a-~-Vls
R
= CU-L-VIS(ac) R = a-~-Acfs R = O-L-Acfs (ac) K = H ; 5a-H R = a-OH; 5,-H R H ; sa-H; 17aIt = P-D-Alms R = a-~-Acvs-p-~-Gb ; It' = H R = CY-L-ACVS-P-D-G~~ ; 11' = AC R = P-D-Dxs-oI-L-C~S; 5p-OH R = 3a-OH; Ila-OH li = ( G - L - C ~ Ylla-OH )~; R = P-D-DXS (-a-L-Cys)~; 11CY-OH R = P-D-Dxs-cz-L-C~S; 11a-OH R = a - ~ - T l s(des); 10-OH, $-OH; 1~ c Y - O H Ii = a-L-RhS; 5a-H; 11P-OH 1i = ~-L-VIS;11' = Ac 11 = a-~-Acfs(ac); It' = Ac 1i = a-L-Cys; Ii' = A4c K. = p-o-I)ns; It' = isovaleryl R = ( p - ~ - D x s ) ~R' ; = isovaleryl R a-L-Acfs; R ' =
LD + SE per mg
5 , 7 7 i 0.38 . L O O i 0.24 5,86 =k 0 . 2 3 3.19 i 0 . 2 3 5 . 7 0 + 0.29 3.16 f 0.12 0.73 f 0.04 0.13 Inactive 1.34 f 0 . 2 0 6 . 5 1 i 0.17 0 , .is
3.27 f 0.23 0.34 i 0.24 2.50 f 0.18 3 . 2 1 i 0.20
,5. 54 & 0 . 35 9.57 f 0.42 6.05 i 0.42 3 . 0 4 f 0.003 3.31 f 0.40 4 . 9 9 f 0.32 Trivial action 2.28 Inactive
A 18-
VI VI VI
R p-D-Rhs R = H ; 2a-OH R = Cncertairi sugar colijugated with 2a-OH and 3p-OH
9.51 f 0.62 0.64 f 0.06 8 , 4 7 =k 0.36
:iw -mailer than thow of digoxin' although the iormrr by iv mcasuremc>iit is thtwreticall? le.< potent than t h r htter. , I few compoiiiid- 111 Table I \yere sparingly soluble i l l tlil EtOH, requiring .iS-fiho/; b: volume for t h e stock solution (uzarige~iin, ~ ~ - ( ~ ~ i u z ~ ~ r iallo-uzarigeiiin. ge~iiri, :ind pachygeniii). Xi1 overn helming- majorit! of the product3 I\ ere -oluhlt. in 50cC EtOH. T h r ultimate dilution- (nith -aIiiir) for the most potrnt member- of t h e group varied from 1 . 100,000 to 1:200,000, and thost. of 1Cssor activit? , from 1 . 10,000 t o 1 .:'5.000. S~vcriilothers so n r u h . or inactive, that their htocli wlutions wpre intermittently injected through :I microburet. X total of 411 cat5 were employed in thiwork . l.'rog\ were used iri a few instance3 for corifirmatorj purpo-e.. The! con-umed much material and. with borderliiie activt. compound-. the) often showed iieg:\tivtk wsults iii contra-t with t h r cat. (1
Results and Discussion r ,
1hc 50 su1)st:mces iirtl rearranged in T n h l r I1 for convviiieiit comp:irison of t,hrir structures. To save space, discussioris were presrrited together with the results. T h c m w n I B ' s of the activo compounds were coriverted into their reciprocals as shown in t,he last, column, n:tmely, the number of LD's per milligram. This ex1)rwses a direct relationship of activity (or toxicit\.) ( T ) C;. I < . 31oe and .\. E . I'aralL "l'liarriiacological Basis of T h e r a p e u t i c 3 ' :ird e(!, 1 . . S. tioudmann arid .\. Gilman, Ed., hIacrnillan Co., Kew \-ark. S.Y.,l!I&?,
thr largcr the. iiumbrr, t h c iiiow potc.rit thc i)rudiict 111 considering the data \ye found the recent r(>view-1)) the authoritir. of the field moat The first (iglycoside< in Table I1 are thoat of digitoxigenin ( I ) . They :ire all monosides arid more active than thc wgl? cone nhich hah ii value of 2.18 f 0.07 mg. \':illaro\ide. beaumoiito-idr, wlarioside, ;ml n allicho-id(, hiivr thr highest activit) . The fuvorablv influenctl of IAeandro*e, L-cjni:Lro-t', ~-vwllnrose,:tiid L-acoiriwe. conjugated with t h e ~econdilr>.OH group at ('-3* i b obvictu-. lIononcet>lation in tht' q + i r residw :Lpparentlj diminishe- the potency :ti indicated 11) mono-O-acrt> lvallaroside arid mono-0-acet! Isolariohidt~. Uzarigeiiiii i. isomeric with digitoxigenin, but has a .Sa-H arid thus an X B-trans configuration In c:itc;, it I\ definitelj :ictive, although \w:il;. l'our aiimpki~ etl, -1ipplied b,~.I'roft+-or 1'. R d i b t t w i t h r w p p i i tvf horn odoroside €3 \I ith i i i c i ~ i iLD'5 11i 1 .i: f '0.1s111 10 cat-, 0 . M f 0.07 in 3 c:it,. :uid 1.39 * 0.16 iii h cat s : anothrr izoiattd from Sysrnalobiccni urir/u/atui,i nilh .L mean J D of 1.44 f 0.14 iiig I\g Sirictl thti +t:mdard errors denoted i i ~ ~ variarict.. o v all thr hgurv- tor cats were combined. ThcJ mean I,D 11 a i computed to he 1 38 i 0.0s mg/kg, and the rcciprocal 0.73 * 0.01 LD/mg (Table 11). During thc -Ion iiit r a v ~ i i o u siiijection in cat
