Functional Receptor Mapping for Modified Cardenolides: Use of the

12 Functional Receptor Mapping for Modified Cardenolides: Use of the PROPHET System DOUGLAS C. ROHRER—Medical Foundation of Buffalo, Inc., Buffalo, NY 14203 DWIGHT S. FULLERTON and KOUICHI YOSHIOKA—School of Pharmacy, Oregon State University, Corvallis, OR 97331

Downloaded by SUFFOLK UNIV on January 17, 2018 | http://pubs.acs.org Publication Date: November 28, 1979 | doi: 10.1021/bk-1979-0112.ch012

ARTHUR H. L. FROM—Cardiovascular Division, Department of Medicine, Veterans Administration Medical Center, Minneapolis, MN 55417, and University of Minnesota, Minneapolis, MN 55455 KHALIL AHMED—Toxicology Research Laboratory, Veterans Administration Medical Center, Minneapolis, MN 55417, and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455

Digitalis preparations have been used therapeutically in the management of cardiovascular disease for almost 200 years (1). Their ability to increase the contractility of the heart muscle (the inotropic activity) combined with the slowing of the heart rate (the chronotropic activity) resulting in a generally improved heart efficiency keep these drugs among the top ten prescribed even today (2). Unfortunately, these drugs are also extremely toxic, with cardenolide toxicity accounting for up to half of drug induced in-hospital deaths (3). When the dose level required to attain the desired therapeutic response has been administered, 60% of the toxic dosage has also been given (4). Clearly, the demonstrated importance of these types of drugs combined with the need for improvement in the therapeutic-toxic ration provides a strong incentive for their study and for reaching a better understanding of their mode of action. The pharmacological effects of the digitalis glycosides, [such as digitoxin (Ia) or digoxin (IIa)] and their genins [Ib or IIb] appear to be the result of the inhibition of the membrane-bound Na ,K -ATPase (5,6,7). Thus it has been suggested that the Na ,K -ATPase enzyme is the digitalis receptor or certainly very closely related to i t (7). This enzyme system is found in the plasma membranes of nearly a l l mammalian cells and is involved in the active transport of Na and K+ across the cell membrane. This transport process is particularly important in cardiac muscle cells where the transport process must occur prior to each heart contraction. A number of often conflicting models (8,9) have been proposed to describe the chemical and structural characteristics of a genin which govern its ability to inhibit the Na ,K -ATPase. Our earlier studies (10-18) have shown that none of these models has been able to consistently explain the activities of a number of the modified genins. A multi-disciplinary approach, including X-ray crystallography, +

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0-8412-0521-3/79/47-112-259$05.25/0 © 1979 American Chemical Society

Olson and Christoffersen; Computer-Assisted Drug Design ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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conformational energy calculations, organic synthesis and Na^K"*"ATPase inhibition studies has been used to find the direct rela tionship between genin structure and activity reported here.


I.

Cardenolide Structural Studies

The structural and electronic characteristics of a molecule determine i t s a b i l i t y to bind to and/or cause a physiological response with a given receptor. As described i n the previous section, the old models for cardiac glycoside action did not account for the a c t i v i t y of the modified genins. To delineate the true nature of the structure-activity relationship of cardenolide genins, a thorough study of cardenolide structure was begun using X-ray crystallography and the graphic analytical features of the NIH PROPHET computer system. Digitoxigenin, l b , i s generally considered to be the car denolide prototype. Its detailed structural features were there fore of particular interest. Figure 1 shows i t s crystallographi c a l l y determined structure (19). The curved shape of the ster oid backbone caused by ois fusions between the A and Β rings and the C and D rings and the axial methyl substituents are common features of most genins. Most synthetic modifications of d i g i toxigenin involve the 173-substituent. This substituent has generally been recognized to be a major contributor to receptor binding of digitoxigenin analogues. A l l of the natural genins including digitoxigenin, lb, have a 173-lactone substituent (see Table I: T., i i , and VIII) . The 38- and 143-hydroxyl substitu ents are also common structural features. Until recently, the 146-hydroxyl substituent has been considered necessary for binding (13). The molecular structures of 11 other cardenolides were determined crystallographically [Table I: l i b (14), III (18), IVa (16), V (18), VI (18), VII (18), VIII (20), IX (17)> X (18), XI (18) and XII (21)] and compared with the structure of digitoxigenin, lb (19), on the NIH PROPHET computer system. A l l of the genins i n this study were synthesized i n our laboratories (10,11,12,16^) from digitoxin, l a , - except for XII, a g i f t from Dr. Mitsuru Yoshioka, Associate Director of Research, Shionogi Research Laboratories, Osaka, Japan; IVa, a g i f t from Dr. Romano Deghenghi,Director of Research, Ayerst Laboratories, Montreal, Canada; and VIII and l i b which were obtained commercially. The synthesis of the methyl ester, VI, and the n i t r i l e , VII, follow the procedures reported previously (8). These structures to gether with the Na ,K -ATPase inhibition data were used to for mulate the structural and activity correlations to be presented. +

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Major Structural Classification Based on Ring D A l l genins examined i n this study can be divided into classes with; (1) a f u l l y saturated steroid backbone (I -> VIII), (2) a 14-ene-steroid backbone ( IX + XI) or (3) an 8(14)-enesteroid backbone (XII). Crystallographic analysis of these genins revealed that the A and Β rings and most of the C rings are nearly identical. It therefore seems reasonable to assume that this constant portion of the molecule must f i t into the


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(a)

261

(b)


Figure 1. PROPHET (a) top and (b) side views of the molecular structure of digitoxigenin lb from cristallographie coordinates



262

same location at the receptor site. Any structural (as l i s t e d above) or conformational variation i n the D-ring portion w i l l have a marked effect on the position of the 178-side group r e l a tive to the constant portion and also influence the Na ,K -ATPase inhibitory potency.


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Saturated D-Ring Structures. Normally, saturated cardeno lide backbones are thought to be r i g i d structures with very l i t t l e conformational v a r i a b i l i t y . Analysis of the 10 crystal structures representing 8 different molecules, however, indicates that there i s unexpected conformational freedom i n the D rings of these molecules. Furthermore, there are conformational trends which can be linked to the nature of the 178-side group. There are three types of 178-side groups represented; the normal lac tone ring (structures, I II, and VIII), and the modified lactone rings with a substituent on a carbon adjacent to the bond to the backbone (structures III and IV) and the acyclic side chains (structures, V, VI and VII). The major classes of D-ring conformations are a C148-envelope, Figure 2a, where C14 i s displaced i n the 8 direction from the remaining four coplanar atoms and a C148/C15a-half chair, Figure 2b, where C14 and C15 are displaced i n the 8 and α direc tions respectively from the three atom plane formed by C13, C16 and C17. Since the symmetry of these conformational forms (a mirror plane in the envelope and a two-fold rotation axis in the half chair) i s manifested i n the intra ring torsion angles, the torsion angles can be used to evaluate the deviation of a given conformer from the ideal form (22). A plot of the asymmetry parameters representing the deviation from ideal C148-envelope symmetry, AC (C14), versus the deviation from ideal C148/C15ahalf chair symmetry, AC2(C17), i s given i n Figure 3. The asymmetry parameter i s zero when the conformer has the ideal symmetric conformation. From this plot, i t i s apparent that the D-ring conformations of the normal lactone structures are clus tered i n the C148-envelope region, while the modified lactone structures have D-ring conformations i n the C148/C15a-half chair region. The acyclic structures show a large amount of D-ring conformational freedom which probably results from the decreased strain i n the 178-side group. The D-ring conformation of V i s a C13a-envelope, VI i s a distorted C148/C15a-half chair, and VII i s a C13a/C148-half chair. It appears from these results that the D-ring of the modified lactone group has a highly preferred conformation as a result of the location of the substituent on the lactone ring. The normal lactone places some limitations on the D-ring conformation, but not as great as the modified lac tones. Finally, the acyclic side groups place few restrictions on the D-ring conformations. The effect these D-ring conformational differences have on the orientation of the 178-side group relative to the constant portion of the molecular are shown i n Figure 4. It i s apparent 9

g



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ROHRER ET AL.

Ο

4

8

12 16 AC (C14)

20

24

S

Figure 3. Correlated variation in ring D principle asymmetry parameters. The two-fold rotational asymmetry parameter &C (C ) is plotted vs. the mirror plane asymmetry parameter ^Cs(C ). 2

17

1!t



264


Ο ET a

Figure 4. PROPHET/FITMOL overlay of the D-ring regions in structures lib, IVa, and V showing the shift in location of the Πβ-bond associated with different saturated ring D conformations

(a)

(b)

Figure 5. PROPHET/FITMOL overlays of (a) lb and IX and (b) lb and XI showing the shift in the location of the ΙΊβ-side group associated with the pres ence of a C -C double bond in the D ring n

(a)

î5

(b)

Figure 6. (a) Top and (b) side views of the PROPHET/FITMOL overlays of lb and XII clearly showing the change in the overall backbone structure in XII associated with the presence of a Cs-C^ double bond


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that the exocyclic C178 bond orientation changes dramatically with change in conformation. This change w i l l be amplified at each successive location out on the chain causing a substantial change i n the relative location of the functional oxygen, independent of differences in the nature of the side group. 14-Ene D-Ring Structures. The C14-C15 double bond i n these molecules restricts the D-ring conformation to a C17a-envelope. This observation i s consistent with the structures of other noncardenolide steroid molecules with a C14-C15 double bond (23,24). There are 3 structures with this type of D ring (IX, X> and XI). Figure 5 shows the marked effect of this D-ring conformation on the orientation of the 178-side group relative to the constant portion. 8(14)-Ene D-Ring Structure. Here the double bond i s i n the C ring, but i t s effect extends into the D ring. There i s only one cardenolide structure of this type available for comparison XII. The D-ring conformation i s a C17a-envelope, but the C8-C14 double bond i n ring C also affects the directionality of the 178-side group relative to the constant portion of the molecule (Figure 6). 178-Side Group Orientation The 178-side group also has rotational freedom about the C17-C20 bond. In order to explore this conformational freedom, relative conformational energies were calculated using a version of the CAMSEQ (25) program which was specially modified to be used in conjunction with the NIH PROPHET computer system (26). Starting with the crystallographic coordinates, the energy was evaluted at 10° rotation steps of the 178-side group while minimizing the nonbonded interaction with the C13 angular methyl by also allowing i t to rotate. No differences were found i n the locations of minima or shape of the curve when solvent ( i . e . water) interactions were included i n these calculations. In every case, the crystallographically observed conformation was at a potential energy minimum. Saturated D-Ring Structures. These calculations agree with earlier reported observations (17,27) that there are two energy minimum conformations for the normal lactone side group related by a rotation of approximately 180°. Figure 7 shows the resulting energy curves shifted so that the lowest energy i s zero kcal/mole. Since the differences i n the three energy curves result from relatively small differences i n the overall structures of the molecules, a curve representing both structural and conformational f l e x i b i l i t y can be generated by using the minimum energy at each side group orientation, Figure 7d. The very large energy barrier at 180° results from the steric interactions be-


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Figure 7. Potential energy diagrams for the rotations of the normal lactone 17 βside group of (a) lb, (b) lib, (c) VIII, and (d) a combined diagram using the minimum energy at each conformational point. The arrows indicate the confor mations of the crystal structure.


12. ROHRER ET AL.


267

tween the hydrogens on C18 and C21, w h i l e the lower b a r r i e r at 0° i n v o l v e s the hydrogens of C22 with those on C18. The energy diagrams, F i g u r e 8, f o r the modified lactone r i n g s t r u c t u r e s i n d i c a t e much l e s s r o t a t i o n a l freedom than the normal lactone s t r u c t u r e s . T h i s i s a r e s u l t of the e x o c y c l i c s u b s t i t u e n t adjacent to the r o t a t i n g bond i n t e r a c t i n g with the s t e r o i d backbone. The t i l t of the lactone r i n g r e l a t i v e to the C17-C20 bond i n I I I caused by C20 being a t e t r a h e d r a l sp atom r a t h e r than planar s p a l s o e f f e c t s i t s energy p l o t . In each case, the conformations i n the c r y s t a l s t r u c t u r e (two independent s t r u c t u r e s f o r I I I and one f o r IV) a r e l o c a t e d i n the lowest and widest energy minimum r e g i o n o f the p l o t which should represent the l a r g e s t conformational populations. The a c y c l i c s t r u c t u r e s represent a somewhat more d i f f i c u l t problem s i n c e there i s an added degree o f freedom a s s o c i a t e d with the C21-C22 bond. However i n each of these s t r u c t u r e s the s i d e group atoms are n e a r l y coplanar. T h i s means that only the e i s o i d and t r a n s o i d o r i e n t a t i o n s f o r the C21-C22 bond need to be considered. The r e s u l t i n g c a l c u l a t i o n s , however, show no e f f e c t on the r o t a t i o n energy of the C17-C20 bond a s s o c i a t e d with changes i n the s i d e group C21-C22 conformation. Figure 9 shows the r e s u l t i n g energy diagrams f o r V, VI and VII. While the r e s u l t i n g curves have a number of minima, i n each case the X-ray conformation represents the p r e f e r r e d o r i e n t a t i o n - i . e . , i n the deepest and broadest minimum. 3


2

14-Ene D-ring S t r u c t u r e s . The energy curves f o r IX and X are i n t e r e s t i n g f o r s e v e r a l reasons. F i r s t , C20 i s an s p carbon r a t h e r than the s p carbon of a normal l a c t o n e . This means that the lactone r i n g i s not coplanar with the C17-C20 bond. Second, these C20 epimeric molecules have remarkably d i f f e r e n t Na+,K -ATPase i n h i b i t i o n a c t i v i t i e s which w i l l be discussed l a t e r . Figure 10 shows the energy diagram. In these molecules there i s only one p r i n c i p a l energy minimum conformat i o n , with one or more a d d i t i o n a l very narrow minima. Again the c r y s t a l s t r u c t u r e o r i e n t a t i o n s are i n the p r i n c i p l e energy minima, one s t r u c t u r e of _IX and two independent s t r u c t u r e s f o r X. The energy p l o t f o r XI, F i g u r e 10c, i s very s i m i l a r to the p l o t s obtained f o r the normal lactone s t r u c t u r e s ( l b , l i b , and VIII) discussed above. The C14-C15 double bond i n the D r i n g e v i d e n t l y does not g r e a t l y change the r o t a t i o n o f the s i d e groups. T h i s s t r u c t u r e , l i k e s t r u c t u r e s l i b and V I I I , has a d i s o r d e r e d 17$-side group. The two s i d e group o r i e n t a t i o n s of the c r y s t a l s t r u c t u r e represent two separate minima rather than a s i n g l e wide minimum on the energy p l o t . 3

2

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8(14)-Ene D-Ring S t r u c t u r e . The change i n the s t e r o i d backbone and a s s o c i a t e d D-ring conformation g r e a t l y reduces the s t e r i c i n t e r a c t i o n s between the l a c t o n e r i n g hydrogens and the



268


Figure 8. Potential energy diagrams for the rotation of the modified lactone Πβ-side group of (a) III and (b) IVa. The arrows indicate the conformations of the crystal structures. Compound III has two independent structural observations.

Figure 9. Potential energy diagrams for the rotation of the acyclic Πβ-side groups of (a)V, (b) VI, and (c) VII. The arrows indicate the conformations ob served in the crystal structure.



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Figure 10. Potential energy diagrams for the rotation of the ΙΊβ-side group of the C -C double bond D-ring structures: (a) IX, (b) X, and (c) XI. The arrows indicate the locations of the crystal structure conformations. Compound X has two independent conformations and XI has two alternate orientations. lk

ls



270

angular methyl group, see Figure 11. This means that the lactone ring has much greater orientational freedom than any of the other molecules studied. II.

Na^K^-ATPase Inhibition Studies

The I (molar concentration for 50% inhibition) in vitro Na ,K -ATPase inhibitory a c t i v i t i e s of the genins were determined using rat brain Na ,K -ATPase (E.C. 3.6.1.3), the preparation and assay of which have been reported previously by our laboratories (28,29). Rat brain enzyme was used i n these studies for two reasons: (1) the ease of preparation of high activity enzyme; and (2) the comparable sensitivity of most heart and brain enzyme preparations (29). I50 ranges for the less water soluble genins X and XII were extrapolated from their doseresponse curves at concentrations where they were completely soluble. Each genin was preincubated with the enzyme ten minutes, i.e. mixing steroid, enzyme, and media lacking K before adding KC£ to maximize inhibitory effects. Each genin was also tested without preincubation, however, there was l i t t l e or no difference i n I50 with or without preincubation for any of the genins except digoxigenin (lib) and strophanthidin (VIII). The added hydroxyl group of these genins decreased l i p o p h i l i c i t y with the result that binding to the enzyme i s concomitantly slower. 5 0

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Structure/Activity Correlation The results of the potential energy calculations show clearly that i n every case the crystallographically observed orientation of the 178-side group i s i n the energetically preferred conformation or conformations represented by the widest and deepest energy minima when calculated both with and without including a contribution for solvent water. Therefore, the crystallographically determined structure i s a suitable model for use in the biological a c t i v i t y and structural comparisons to follow. The PROPHET procedure FITMOL (30) was used to analytically superimpose the constant portion of structures l i b through XII upon the corresponding portion of the most active genin i n the series digitoxigenin, l b , using a least squares method. This permits a quantitative measure of the effects the modification of the 178-side group structure and variation i n the D-ring conformation have upon the functional groups, i n particular the position of the carbonyl oxygen. Comparison of the relative carbonyl oxygen separations obtained from FITMOL with the Na ,K -ATPase inhibition a c t i v i ties given in Table II revealed a striking correspondence. A simple linear regressior model was used to test the relationship between oxygen separation and the I50 data for Na ,K -ATPase +

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12.

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Ο

Figure 11.

60

120 180 -120 -60 TORSION ANGLE (dcg.) C13- C17-C20-C22

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Ο

The potential energy diagram for compound XII. The arrow indi cates the x-ray crystal structure conformation.



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Q

VI

7.0 x 10"

IVb

2.2 χ 10

1.2 χ 10

1.0 x 10

IVa

-6

7.0 x 10

III

III

7,0 x 10"

lib

1

7.0 x 10

-8

1,30

1,43

(C13-C17-C20-C21) -107.6 (C13-C17-C20-C21) -129.0

5.22

4.90

175.9 (C13-C17-C22-C23) -106.9

5,06

0.42 (2,31)

0,00

Carbonyl Oxygen Separations (%) obtained from FITMOL.

180,0

70.4 (alternate: -99.6)

76.3

•8 4.0 χ 10 -7 3,5 x 10

Ha

lb

la

Compound

X-Ray Structure Side Group Torsion Angle (°) C13-C17-C20-C22.

150 00 with 10 min. Preincubation,

Table II. Na ,K -ATPase Inhibition Data (I- ,M) and Side Group Structural Data for Compounds I to XII.

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XII

XI

5

J

(3*6) x 10~ [extrapolated from

1.6 x 10~

data]

100.3

-102.4 (alternate:

-76.9)

4.61

3.76 (5.51)

5.67

-178.8

[extrapolated from I^g data]

(1-K3) x 10"

X

X

5.75

173.5

4

2.0 x 10"

IX

f

4.08

-144.2

0.88 (2.60)

5

-111.0)

7

85,0 (alternate :

(C13-C17-C20-C21) -129,9

Carbonyl Oxygen Separations (X) obtained from FITMOL.

7.5 χ 10"

VII

X

ο

to


274 with p r e i n c u b a t i o n . shown i n Figure 12:

Eleven p o i n t s were found to f i t the

log I

5

0

= 0.459 D -

6.471

where D i s the r e l a t i v e carbonyl separations i n X. The r e s u l t i n g r i s 0.993 and f o r t e s t of the r e g r e s s i o n r e l a t i o n s h i p , ρ = 0.0001. T h i s i n d i c a t e d that f o r each 2.28 the oxygen was d i s p l a c e d from that of d i g i t o x i g e n i n , a c t i v i t y drops by one order of magnitude [An e a r l i e r r e p o r t (15) with nine analogues showed n e a r l y the same r e l a t i o n s h i p , r = 0.994.] In genins l i b , V I I I and _XI where two 178-side group o r i e n t a t i o n s were observed i n the c r y s t a l s t r u c t u r e , i t i s c l e a r that (as one would p r e d i c t ) only one i s p r e f e r r e d by the receptor. As shown i n Figure 13, the a l t e r n a t e conformations of these genins are more a c t i v e than can be explained by t h e i r carbonyl oxygen s e p a r a t i o n s . However, a n e a r l y p e r f e c t c o r r e l a t i o n Figure 12, e x i s t s f o r t h e i r other 178-side group conformations. The carbonyl oxygen separations f o r both s t r u c t u r e s of I I I and X are n e a r l y equal, F i g u r e 12, so e i t h e r conformation would be expected to have equivalent a b i l i t y to i n h i b i t Na ,K -ATPase. One s t r u c t u r e , e s t e r VI, i s more a c t i v e than i t s carbonyl oxygen s e p a r a t i o n would suggest. However, even i f t h i s p o i n t i s included i n the l i n e a r r e g r e s s i o n model, the r value i s s t i l l e x c e l l e n t (0.946). S e v e r a l p o s s i b i l i t i e s may e x p l a i n t h i s apparent anomaly and f u r t h e r s t u d i e s on VI are i n progress. This simple l i n e a r r e l a t i o n s h i p provides a q u a n t i t a t i v e explanation f o r a number of genins where other models have failed. In p a r t i c u l a r : 2

2


line

+

+

2

(A) The p r e d i c t e d a c t i v i t y of aldehyde, V, was up to 123 times higher than d i g i t o x i g e n i n based on previous models, but i t was found to be s l i g h t l y l e s s a c t i v e (10). F i g u r e 12 c l e a r l y shows that V s a c t i v i t y i s a d i r e c t r e s u l t of i t s carbonyl oxygen p o s i t i o n . The r i n g D con f o r m a t i o n a l d i f f e r e n c e and the s t r u c t u r a l d i f ferences of the a c y c l i c 178-side group r e s u l t i n t h i s displacement. T

(B) I t has been g e n e r a l l y b e l i e v e d that the C20-C22 double bond i n the l a c t o n e r i n g has an a c t i v e r o l e i n i n h i b i t i n g the Na+,K+-ATPase (31,32). A lactone r i n g with an e x o c y c l i c double bond would be p r e d i c t e d to be even more a c t i v e than one with an e n d o c y c l i c double bond (32) and c e r t a i n l y more a c t i v e than one w i t h no double bond. However, IX and X, which should there f o r e be more a c t i v e than d i g i t o x i g e n i n , are much l e s s a c t i v e (11). Furthermore, the 20,22-


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3:

REL.CARBONYL OXYGEN SEPARATIONS [A]

Figure 12. Correction between the carboni/l oxygen separations relative to digitoxigenin, lb, and the log of the Na\K*-ATPase inhibition activity: (χ) mea sured 1 data; (+) I so data extrapolated from lower concentrations in which the analog was completely soluble 50

-3. η

Ο.

1.

2.

3.

4.

5.

6.

REL.CARBONYL OXYGEN SEPARATIONS[Â]

Figure 13. Correlation between relative carbonyl oxygen separations and the log of I so showing the oxygen separations associated with the alternate Ιΐβ-lactone orientations (A); carbonyl separation for VI (O)



276


dihydro analogues with no double bond though 100 times less active than digitoxigenin are more active than _IX and X (13). These activ i t y differences are readily explained by the conformational and structural effects on the position of the carbonyl oxygen; that i s , the C20-C22 double bond i s primarily serving a passive role i n Na+jK^-ATPase inhibition keeping the carbonyl oxygen i n the "right" position for maximal inhibitory effect (see Figure 12). (C) It has also been generally believed that the 148-hydroxyl greatly enhances the inhibition of Na ,K -ATPase (_5,6,1,8,9,33) . However, IX without the 148-hydroxy i s more active than the corresponding 148-hydroxy analogues III and i t s 20R stereoisomer (11). Figure 12 suggests that changes i n carbonyl oxygen position may account for these differences in activity. +

+

(D) The activity of genin IVb i s remarkably low (70 times lower than i t s 8-D-glucose analogue (IVa)). The effect of the glucose on the a c t i v i t y was not predicted by a recently proposed binding model (34). The structural correlation shown i n Figure 12, however, accurately f i t s the activity of genin IVb based on i t s modified lactone structure. Conclusions The data presented here are consistent with the following structural correlations of d i g i t a l i s genin-Na ,K+-ATPase inhib ition: +

(A) Rings A and Β with a ois fusion, most of the C ring, plus the axial methyl group form a structurally r i g i d CONSTANT PORTION of the molecule. (B) The saturated D rings have a great deal of conformational f l e x i b i l i t y with preferences influenced by the nature of the 178-side group. (C) There are substantial barriers to rotation of the 178-side group which provide orientational preferences modeled by the crystal


12.

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277

structures. (D) The acyclic α,8-unsaturated 178-side groups form acoplanar unit and are not as flexible as may have been thought. +

+

The relationship of these structural features to Na ,K ATPase inhibition and receptor modeling are:


(A) The CONSTANT PORTION of the genin molecules must a l l f i t onto a specific position on the receptor surface. (B) The 148-hydroxyl and C20-C22 double bonds do not seem to play as direct a role i n the i n hibition as was previously thought. (C) The minimum energy conformation of the mole cule i s preferred at the receptor s i t e . (D) There i s a single carbonyl oxygen position relative to the CONSTANT PORTION which d i rectly controls the activity (a 2.28 shift decreases the activity ten fold). Acknowledgement The authors are grateful to Miss Melda Tugac, Miss Gloria Del Bel and Mrs. Q. E. Bright for preparation of i l l u s t r a t i o n s and to Miss Kathleen Castiglione, Mrs. Brenda Giacchi and Miss Deanna Hefner for manuscript preparation. This work was sup ported i n part by Grant Number HL-21457 from the National Heart, Lung and Blood Institute and LM-02353 from the National Library of Medicine, the Oregon and Minnesota Heart Associations, and the Veterans Administration Medical Research Fund. The organi zation and analysis of the data base associated with this i n vestigation were carried out i n part using the PROPHET system, a unique national resource sponsored by the NIH. Information about PROPHET including how to apply for access can be obtained from the Director, Chemical/Biological Information-Handling Program, Division of Research Resources, NIH, Bethesda, Maryland 20205.

Literature Cited 1. 2.

Withering, W., "An Account for the Fox Glove and Its Medical Uses"; (1785), in "Readings in Pharmacology"; Shuster, L. Ed.; Little Brown: Boston, 1962; p. 109. Pharmacy Times, "1977: Top 200 Drugs", 1978, April, p. 41.


278 3. 4. 5. 6. 7. 8.


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Oglivie, R. I.; Ready, J . Can Med. Assoc. J., 1967, 97, 1450. Dreyfus, L. S.; Watanabe, Y. Seminars in Drug Treatment, 1972, 2, 179. Schwartz, Α.: Lindenmayer, G. E . ; Allen, J . C. Pharmacol. Rev., 1975, 27, 3. Flasch, H.; Heinz, N. Naunyn-Schmiedeberg's Arch. Pharma col., 1978, 304, 37. Akera, T. Science, 1977, 198, 569. Thomas, R.; Boutagy, J.; Gelbart, J . J . Pharm. Sci., 1974, 63, 1649. Guntert, T. W.; Linde, H. H. Experimentia, 1977, 33, 697. Fullerton, D. S.; Pankaskie, M. C.; Ahmed, K.; From, A. H. L. J . Med. Chem., 1976, 19, 1330. Fullerton, D. S.; Gilman, D. M.; Pankaskie, M. C.; Ahmed, K.; From, A. H. L.; Duax, W. L.; Rohrer, D. C. J. Med. Chem., 1977, 20, 841. Yoshioka, K.; Fullerton, D. S.; Rohrer, D. C. Steroids, 1978, 32, 511. Fullerton, D. S.; Yoshioka, K.; Rohrer, D. C.; From, A. H. L . ; Ahmed, K. J . Med. Chem., 1979, 22, in press. Rohrer, D. C.; Fullerton, D. S. Acta Crystallogr., Sect. B, submitted. Fullerton, D. S.; Yoshioka, K.; Rohrer, D. C.; From, A. H. L . ; Ahmed, Κ., Science, submitted. Fullerton, D. S.; Yoshioka, K.; Rohrer, D. C.; From, A. H. L . ; Ahmed, K. Mol. Pharmacol., submitted. Rohrer, D. C.; Duax, W. L.; Fullerton, D. S. Acta Crystal logr., Sect. B, 1976, 32, 2893. Rohrer, D. C.; Fullerton, D. S. Unpublished results. Karle, I. L . ; Karle, J . Acta Crystallogr Sect. Β 1969, 25, 434. Gilardi, R. D.; Flippen, J . L. Acta Crystallogr., Sect. B, 1973, 29, 1842. Gilardi, R. D.; Karle, I. L. Acta Crystallogr., Sect. B, 1970, 26, 207. Duax, W. L.; Weeks, C. M.; Rohrer, D. C. "Topics in Stereo chemistry", Vol. 9, Allinger, N. L. and E l i e l , E. L., Eds., Interscience: New York, NY, 1976; p. 271. Rohrer, D. C.; Strong, P. D.; Duax, W. L.; Segaloff, A. Acta Crystallogr., Sect. B, 1978, 34, 2913. Rohrer, D. C.; Duax, W. L.; Segaloff, A. Acta Crystallogr., Sect. B, 1978, 34, 2915. Weintraub, H. J. R.; Hopfinger, A. J . Int. J . Quantum Chem., Quantum Biol. Symp., 1975, 2, 203. Rohrer, D. C.; Lavin, M. "EDITMODEL"; in "Public Proce dures: A Program Exchange for PROPHET Users"; Wood, J. J., Ed., Bolt, Beranek and Newman, Inc.: Cambridge, MA, 1978.


12. 27. 28. 29. 30. 31.


32. 33. 34.

ROHRER ET AL.


279

Rohrer, D. C.; Fullerton, D. S. American Crystallographic Association Meeting Abstracts, 1978, Norman, Oklahoma, Abstract: J1. Quarforth, G.; Ahmed, K.; Foster, D. Biochem. Biophys. Acta, 1978, 526, 580. Ahmed, K.; Thomas, B. S. J . Biol. Chem., 1971, 246, 103. Rohrer, D. C.; Perry, H. "FITMOL"; in "Public Procedures: A Program Exchange for PROPHET Users"; Wood, J. J., Ed.; Bolt, Beranek and Newman, Inc.: Cambridge, MA, 1978. Thomas, R.; Boutagy, J.; Gelbart, A. J . Pharmacol. Exptl. Ther., 1974, 191, 219. Kupchan, S. M.; Ognyanov, I.; Moniot, J . L. Bioorg. Chem., 1971, 1, 24. Witty, T. R.; Remers, W. Α.; Besch, J r . , H. R. J . Pharm. Sci., 1975, 64, 1248. Thomas, R.; Allen, J.; Pitts, B. J . R.; Schwartz, A. Eur. J . Pharm., 1979, 53, 227.

Received June 8, 1979.


Functional Receptor Mapping for Modified Cardenolides: Use of the

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