[CONTRIBUTION FROM TEE DEPARTMENT OF CHEMISTRY
O F COLUMBIA UNIVERSITY ]
ALLOSTROPHANTHIDIN EDITH BLOCH*
AND
ROBERT C. ELDERFIELD
Received March 28, 1959
During recent years the chemical structures of a large number of cardiac drugs of both plant and animal origin have been elucidated. The former occur as glycosides and the latter as conjugations of an “aglycone” with suberylarginine. The majority of aglycones of plant origin have been shown to contain the cyclopentanophenanthrene ring system, hydroxylated in varying degrees and carrying a characteristic Ab-wnsaturated lactone group as the side-chain in position 17. Glycosides of such aglycones, containing one to three sugar residues have been iso1ated.l It is well known that digitalis preparations lose part of their cardiac activity on storage. While no exact information concerning the cause or nature of this loss of activity is available, various empirical stabilizing measures have been employed, such as buffering of aqueous tinctures or storage of the carefully dried leaves in vacuo. A possible cause for such inactivation may be found in two observations. Jacobs, in a study of the action of enzymes present in Strophanthus Lombt? seeds on the glycosides of the seeds found that a t least three enzymes are present. The first two2 act as glucoside-splitting enzymes and remove one or more of the glucose residues from the original glycoside. The third enzyme3 accomplishes a stereochemical isomerization of the aglycone portion of the glycoside molecule with resultant marked loss of physiological activity. Thus, 4 mg. of isomerized cymarin (allocymarin) failed to kill a 25-g. frog, whereas 0.015 mg. of cymarin is lethal to such an animal. Jacobs clearly showed that isomerization involved the aglycone portion of the cymarin molecule, since the same sugar, cymarose, was obtained from both the active and the inactive glycosides. Further, this change in the aglycone is purely stereochemical, as the same functional groups are present in both aglycones. In r% later study on the glycosides of Strophanthus eminii, Jacobs and
* This work was carried out during tenure of the Hernheim fellowship by one of us (E. B.). 1 ELDERFIELD, Chem. Rev., 17, 187 (1935); TSCHESCHE, Erg. Physiol., 38, 31 (1936). 2 JACOBS AND HOFFMANN, J . Biol. Chem., 67, 569 (1923);67, 609 (1926); 69, 153 (1926); STOLL,RENZ,AND KREIS, Helu. Chim. Acta, 20, 1484 (1937). JACOBS, J . Biol. Chem., 88, 519 (1930). 289
290
EDITH BLOCH AND ROBERT C. ELDERFIELD
Bigelow4 found that a similar isomerization occurred. An aglycone, presumably isomeric with periplogenin, was isolated as its trianhydro derivative from the hydrolysis products of a comparatively inactive bioside. This trianhydro derivative was different from the corresponding substance prepared from active periplogenin. Lamb and Smith5 confirmed these observations, and succeeded in isolating several additional glycosides. Among these were the above allocymarin and a substance called by them alloemicymarin. The latter name is unfortunate, since the glycoside presumably is one of alloperiplogenin and digitalose. Inasmuch as both of these inactive all0 derivatives were found either after digestion of the seeds in aqueous suspension or after prolonged storage of the seeds themselves, it is obvious that a further study of the changes in the aglycone molecule which take place on allomerieation would throw considerable light on the changes occurring during the deterioration of pharmaceutical preparations of similar drugs. Since the change to allostrophanthidin is purely one involving the stereochemistry of the aglycone, eight possible centers of asymmetry can be considered as being involved, namely carbon atoms 3, 5, 8, 9, 10, 13, 14, and 17 (I). However it seems reasonable to assume that those bearing no functional group, namely carbon atoms, 8, 9, and 13 may be eliminated
I from consideration in this respect on the basis of current theories of enzyme action. Furthermore, by adopting the conventional practice of regarding the substituent on carbon atom 10 as projecting out from the plane of the molecule, this center may also be eliminated by being chosen as a fixed point of reference. Moreover, since a similar change occurs both in strophanthidin, which carries an aldehyde group in this position, and in periplogenin, which carries a similarly located methyl group, inversion a t carbon atom 10 seems excluded. It is known from the observations of Tschesche and Bohlea and Chen, Chen, and Anderson7, that changes in the relative configurations of carbon JACOBS AND BIGELOW, ibid., 99, 521 (1933). LAMBAND SMITH, J . Chem. SOC., 1936, 442. * TSCHESCHE AND BOHLE,Ber., 69, 2368, 2443 (1936). 7 CHEN,CHEN,AND ANDERSON, J . Am. Pharm. ASSOC., 26, 579 (1936). 4
291
ALLOSTROPHANTHIDIN
atoms 3, 5, and 10 in certain cardiac aglycones produce marked changes in physiological activity (Table I). From this it is obvious, that uzarin at least approaches allocymarin in lack of activity, and further, that the asymmetric centers a t carbon atoms 3 and 5 become distinct possibilities as sources of the allomerization process. Information a t present available indicates a cis arrangement of the aldehyde group on carbon atom 10 and the hydroxyl group on carbon atom 3 in st:rophanthidin. This conclusion is based on the ready formation of oxidilc bridges between these two groups. In addition, deductions of Tschlesche and Bohles from observations of Jacobs and Elderfield* on the cyanohydrins from dihydrostrophanthidin indicate a cis linkage of rings I and 11. A possible inversion a t carbon atom 5 in allocymarin would then bring its physiological properties in line with those of uzarin. Neither strophanthidin nor allostrophanthidin give precipitates with digitonin, either in 90 per cent. alcoholic or 50 per cent. methyl alcoholic TABLE I CONFIGURATION AND PHYSIOLOGICAL ACTIVITY
I
I
BTEREOMER
Digitoxin.. . . . . . . . . . . . . Thevetin., . . . . . . . . . . . . Uzarin., . . . . . . . . . . . . . .
STEREOCHEMICAL ARRANQEMENT RELATIVE TO
1
MIN. SYBTOLIC DOSE
clo
Frog
0.33 mg./kg. 0.92 mg./kg. 5.08 mg./kg.
trans 3, cis 5
cis 3,
cis 5
0.0080 mg./g. 0.0045 mg./g. 1.5OOO mg./g.
solution. However, too much weight cannot be placed on this observat.ion, since the influence of the aldehyde group on the digitonin reaction is unknown. Therefore successive destruction of the asymmetric centers on carbon atoms 3 and 5 was carried out, starting from both strophanthidinic acid methyl ester and the corresponding allo derivative. If either of these two centers is exclusively involved in the allomerization, identical compounds should be obtained a t some stage of this series of reactions. CH3 0
0 *JACOBS AND
(YY
// //\/
//
I1
CHS 0 0
0
I11
ELDERFIELD, J . Biol. Chem., 113, 625 (1936).
IV
292
EDITH BLOCH AND ROBERT C. ELDERFIELD
Strophanthidinic acid methyl ester (11) has already been prepared by Jacobsg and has been converted to strophanthidonic acid methyl ester (111) and anhydrostrophanthidonic acid methyl ester (IV) by Jacobs and Gustus’O. We have carried out similar transformations, starting with allostrophanthidin. Neither allostrophanthidonic acid methyl ester, nor its anhydro derivative is identical with the corresponding compound from strophanthidin. Therefore carbon atoms 3 and 5 may be definitely eliminated from further discussion. JacobsS observed that allostrophanthidin failed to undergo isomerization under the influence of alkali with the formation of an isoaglycone-a reaction highly characteristic of the physiologically active aglycones1 as well as of the comparatively inactive uearin11 and adynerinI2. Tschesche and BohleI2 also observed a similar failure of Lamb and Smith’s alloemicymarigenin to undergo iso-compound formation. If it be assumed, as a study of the atomic model indicates, that a cis configuration of the hydroxyl group on carbon atom 14 relative to the side-chain on carbon atom 17 is necessary for the formation of the oxidic bridge in the is0 compound (V)13, then the failure of the alloaglycones to form is0 compounds must be attributable to a trans-configuration of these two groups. Such an
arrangement would then be due either to inversion at carbon atom 14 or 17 during the allomerization. Tschesche and Bohle12 prefer the latter, although no new experimental evidence is offered. In order to make a decision between these two remaining alternatives it was planned to attempt the conversion of allostrophanthidin into its trianhydro derivative, the side-chain of which could then be degraded in a manner similar to that described by Jacobs and Gustus14 with eventual destruction of the asymmetric center at carbon atom 17. Jacobs3 reported JACOBS,ibid., 67, 553 (1923). JACOBSAND GUSTUS,ibid., 74, 795 (1927). l1 TSCHESCHE AND BOHLE, Ber., 68, 2252 (1935). 12 TSCHESCHE AND BOHLE,ibid., 71, 654, 1927 (1938). 18 JACOBS AND ELDERFIELD, J . Biol. Chem., 108, 497 (1935). 1‘ JACOBSAND GUSTUS,ibid., 74, 805 (1927). 10
ALLOSTROPHANTHIDIN
293
that, when the preparation of trianhydroallostrophanthidinfrom the dianhydro compound was attempted a chlorine-containing substance was formed, for which no satisfactory analytical figures could be obtained. We have checked this. When dianhydroallostrophanthidin is dissolved in concentrated hydrochloric acid, rapid crystallization of the chloro derivative occurs. However, on recrystallization, or on prolonged exposure to the air, the substance loses chlorine to yield the original dianhydroallostrophanthidin. Likewise all attempts to obtain the desired trianhydro derivative by the use of hot hydrochloric acid or saturated alcoholic hydrogen chloride failed. However, a study of the action of concentrated hydrochloric acid on allostrophanthidin itself furnished information that is significant. Jacobs and Collins16obtained pseudostrophanthidin (VI) in 10 to 15 per cent. yield on similar treatment of strophanthidin. This substance did not
VI form an oxime, and gave a non-crystalline benzoate, which was not further chara,cterized. Hence it was assigned a stable oxidic ring structure, which is possible only if the hydroxyl group on carbon atom 14 and the aldehyde group on carbon atom 10 bear a cis relationship to each other. When allostrophanthidin is dissolved in concentrated hydrochloric acid, a nearly quantitative yield of a chloro derivative is obtained. Like the chloro derivative from dianhydroallostrophanthidin this is unstable, but in contrast to the latter, the present compound yields an anhydro derivative of allostxophanthidin on recrystallization from dilute acetone or on treatment with dilute ammonia. The anhydro derivative forms both on oxime and a monobenzoate, indicating retention of both the aldehyde group on carbon atom 10 and the secondary hydroxyl group on carbon atom 3. No trace of a pseudoallostrophanthidin was found in the products of the above reaction. Therefore, it seems probable that in allostrophanthidin the hydroxyl group on carbon atom 14 has undergone a reversal, so that it is now trans to the aldehyde group on carbon atom 10 and to the side-chain JACOBSAND COLLINB, ibid., 63, 123 (1925); 66, 491 (1926); JACOBSAND ELDER108, 693 (1935).
FIELD, ibid.,
294
EDITE BLOCH AND ROBERT C. ELDERFIELD
on carbon atom 17, an arrangement which would not permit the formation of the stable oxidic bridge which is present in pseudostrophanthidin. Finally, we have reexamined the action of alkali on allostrophanthidin and, in common with Jacobs3, have found that, although the substance is changed, no crystalline products could be isolated even after using a variety of procedures. Likewise, oxidation of saponified allostrophanthidin with hypobromite failed to yield crystalline derivatives. From the above discussion, it can be concluded that the isomerization involved in the allomerization of cymarin consists in an inversion of one of the asymmetric centers of the strophanthidin molecule, located a t carbon atom 14 or 17. We are inclined to favor carbon atom 14 as the seat of this inversion, although carbon atom 17 cannot be excluded. A parallel for such an isomerization on carbon atom 14 is found in the observations of Hirschmann and Wintersteinerl* on isoequilin. Against this view must be cited the more numerous cases of stereoisomerism in natural steroids in which carbon atom 17 is involved.’’ The inability of substances containing a trans configuration of carbon
VI1
VI11
I IX CH3
f
CH-CHZ
\ /
/\ \/ X l6 17
0
XI
HIRSCHMANN AND WINTERSTEINER, ibid., 126, 735 (1938). REICHSTEIN AND GAETZI, HeEv. Chim. Acta, 21, 1185 (1938).
ALLOSTROPEL~NTHIDIN
295
atoms 14 and 17 to form is0 derivatives is difficult to understand, if one accepts the mechanism for iso-compound formation suggested by Jacobs and Elderfield13(VII, VIII, XI). It would be expected that the formation of is0 compounds would proceed at least to a certain extent, irrespective of the configuration originally present. However the failure of aglycones of the all0 series to undergo iso-compound formation can be rationalized on the basis of the mechanism for the formation of such derivatives originally put forward by Jacobs and Gustus'* (VII, IX, X, XI). This was subsequently withdrawn on the basis of the observation that preliminary saponification of the lactone group is presumably not necessary for isocompound formationlg. It is possible that a change in one asymmetric center of the aglycone molecule may profoundly affect the chemical behavior of substituents on some other asymmetric center in a manner at present obscure. We wish to express our appreciation to S. B. Penick and Company, New York City, who generously contributed the strophanthus seeds used in this investigation. EXPERIMENTAL
AlZostrophanthidinic acid.-Two and one-half grams of finely-powdered allostrophanthidin was suspended in 100 cc. of dry acetone (distilled from potassium permanganate), the mixture was chilled to about 5", and 1.25 g. of finely-powdered potassium permanganate was added. The mixture was stirred mechanically and kept at 5' for 8 hrs. with strict exclusion of moisture. The precipitated salts were filtered, washed with acetone, and thoroughly extracted with water. The aqueous extract was concentrated to about 20 cc. and then carefully acidified with acetic acid. On long standing in the refrigerator the acid slowly crystallized. The yield varied from 0.3 to 0.5 g. After recrystallization by careful dilution of a concentrated solution in acetone, the acid formed fine white needles which melted a t 247"; [a]:39.2" (c = 0.976 in methanol). A n d . Calc'd for CnsHszO,: C, 65.7; H, 7.7. Found: C, 65.7; H, 7.8. From the mother liquors, on addition of about 4 volumes of saturated ammonium sulfate solution, a gummy precipitate appeared. By recrystallization from acetone, a small additional amount of acid could be obtained. The original acetone filtrate was concentrated to dryness i n vacuo. The residue was extracted several times with hot chloroform from which about 800 mg. of allostrophanthidin could be recovered. Allostrophanthidinic acid methyl ester.-The above acid was esterified in acetone solution with diazomethane. After recrystallization by careful dilution of its acetone solution with absolute ether, the ester melted at 263-265'. A n d . Calc'd for C2rH,rOl: C, 66.3; H, 7.9. Found: C, 66.4; H 7.9. 18
l9
JACOBSA N D GUSTUS,J . Biol. Chem., 74, 811 (1927). J.ACOBS A N D COLLINS, ibid. 01, 387 (1924).
Allostrophanthidonic acid methyl ester.-Two hundred fifty milligrams of allostzsphanthidinic methyl ester was dissolved in 3 cc. of glacial acetic acid, and, after chilling to about 15", 0.9 cc. of Kiliani's chromic acid solution was added. After standing for 30 minutes at room temperature, the mixture was diluted to about 50 cc. and then saturated with ammonium sulfate. On rubbing, the keto acid gradually crystallized. After several recrystallizations by careful dilution of its solution in methyl alcohol, it melted at 255-260". A somewhat purer product was obtained by extraction of the oxidation products with chloroform. The extracts were washed with sodium carbonate solution and with water. The residue, after removal of the chloroform, was taken up in methyl alcohol, and the hot solution was diluted with an equal volume of water. On slow cooling, the substance crystallized in stout prisms, which melted at 258"; [a]: 20.1" ( c = 0.749 in pyridine). Strophanthidonic acid methyl ester melts at 161-162" and shows [aID26" in pyridine*O. Anal. Calc'd for C a 4 H ~ s OC, ~ : 66.6;H, 7.5. Found: C, 66.2;H, 7.5. Monoanhydroallostrophanthidonic Acid Methyl Ester.-One hundred fifty milligrams of crude allostrophanthidonic methyl ester was refluxed for 15 minutes with 2.5 cc. of a mixture of 20 cc. of methyl alcohol and 5 cc. of 10 per cent hydrochloric acid. No crystallization occurred on dilution and saturation with ammonium sulfate. The mixture was therefore extracted exhaustively with chloroform, the extracts were washed and dried, and the solvent was evaporated in vacuo. The residue was taken up in acetone, the solution was concentrated in vacuo to about 1 cc., and carefully diluted with 5 volumes of anhydrous ether. On rubbing and standing in the refrigerator, the substance slowly crystallized; yield 90 mg. After recrystallization from acetone-ether, the substance formed long rectangular prisms which melted at 138-145". It is easily soluble in alcohol, acetone, and chloroform; less so in ether and petroleum ether; [a]: 118' ( c = 0.693 in pyridine). Monoanhydrostrophanthidonic methyl ester melts between 203" and 213" and 123" in pyridine1O. showsDI.[ Anal. Calc'd for ClrHsoOs: C, 69.5; H, 7.3. Found: C, 69.3;H, 7.3. Dianhydroa1lostrophanthidin.-One and four tenths gram of the oxidoethylal of dianhydroallostrophanthidinSwas refluxed for 30 minutes with 75 cc. of alcoholic hydrochloric acid (5 cc. of concentrated hydrochloric acid in 45 cc. of water and 50 cc. of ethyl alcohol). Solution occurred at once. After cooling, the solution was diluted with 75 cc. of ice water. A crystalline precipitate appeared and was collected by filtration after standing overnight in the refrigerator. The yield was 1.2 g. of fine needles of m.p. 170". After recrystallization from 95 per cent alcohol, the substance formed clusters of heavy prisms which melted at 172-175"; [a]: -123.1" (c = 1.202 in pyridine). Anal. Calc'd for ClsHtsO4: C, 75.0;H, 7.7. Found: C, 75.0;H, 7.8. When 1 g. of this substance was treated with 10 cc. of concentrated hydrochloric acid, solution occurred at once, but after 15 sec. crystallization started. After 30 minutes, the substance was filtered through a sintered glass funnel and washed several times with ice water; yield about 1 g. The substance contained chlorine and formed fine white needles which melted a t 165". One hundred milligrams of the above chloro derivative was refluxed for 10 minutes with 2 cc. of alcohol containing 2 drops of 10 per cent ammonia. On cooling, a mass of heavy prisms separated. After 2 recrystallizations from dilute alcohol, the
ALLOSTROPHANTHIDIN
297
product was free from chlorine and melted at 173". The melting point of a mixture with dianhydroallostrophanthidin was 172-175". Anal. Calc'd for CzaHneO8: C, 78.8; H, 7.5. Calc'd for C Z J H ~ ~C, O 75.0; ~ : H, 7.7. Found: C, 74.8; H , 8.1. Anhydroal1ostrophanthidin.-Four and three-tenths grams of allostrophanthidin was dissolved in 20 cc. of hydrochloric acid (sp. gr. 1.19) at 0". The substance dissolved a t once, forming a light-yellow solution. After standing for about 30 min. in the refrigerator, crystallization started. After 2 hours, the crystals were collected on a sintered glass funnel and washed twice with ice water. The yield was 4 g. After recrystallization from dilute acetone, the substance formed fine needles and plates which melted a t 175". It contained chlorine. Anal. Calc'd for C23H31C106: C, 63.4; H, 7.4. Found: C, 64.5; H, 7.5. Apparently partial decomposition of the chloro derivative had taken place during recrystallization. After dilution of the hydrochloric acid mother liquors, an amorphous precipitate slowly deposited. This material, anhydroallostrophanthidin, was recrystallized several times from dilute acetone and from methanol. It formed shining plates and melted a t 209"; [a]: 119' (c = 0.630 in methanol). Anal. Calc'd for C Z J H ~ ~C, O ~68.3; : H, 7.9. Calc'd for C23H3006: C, 71.5; H, 7.8. Found: C, 71.5; H, 8.2. The chlorine-containing product was warmed for 10 minutes in alcohol containing a few drops of ammonia. On cooling and dilution, anhydroallostrophanthidin crystallized in thin plates which melted at 205"; [a]:119" (c=0.257 in alcohol). Anal. Found: C, 71.8; H, 8.0. 3'-Benzoylanhydroal1ostrophanthidin.-A solution of 0.1 g. of anhydroallostrophanthidin and 0.1 cc. of benzoyl chloride in 2 cc. of pyridine was allowed to stand 24 hours a t room temperature, and was then poured onto cracked ice and dilute sulfuric acid. After extraction with chloroform, the benzoate was recrystallized from methanol, and formed thin prisms which melted a t 252". Anal!. Calc'd for C30HJ40e:C, 73.4; H, 7.0. Found: C, 73.8; H, 7.3. Oxime of anhydroal1ostrophanthidin.-This was prepared by refluxing anhydroallostrophanthidin with a slight excess of hydroxylamine in alcohol for 4 hours. ,4fter concentration and dilution the oxime crystallized. It melted at 182" after recrystallization from alcohol. Anal. Calc'd for C23H&06: N, 3.5. Found: N, 3.4. The analyses here reported were made by Mr. Saul Gottlieb of these laboratories.
