~~~ti~~ers: how valid is feiffer’s rule?

Richard Barlow

in tire 1950s Pfeiffer observed thf trte relative potencies of fire enanfio,ners (mirror-image isot*rers) of sezyeral drugs appeared to be related to fhe dose of the racemate used clinically. The difference was greatest with fhe most active compc~ds and he suggested reasons for what has become known as ‘Pfeiffer’s rule’. ln this shrf arfick Dick Barlow poinfs out that there is a corollary to the rule - fkaf the activity of fhe weaker ena~~fiom~r is defermif~ed by tke acfioify of fke more potent one - which is intzfifiveiy i~nprobable. With more examples and in a simpler sifuafion, fhe picture is more co.mplex and there are marked exceptiofrs. The ideas behirui Pfeiffer’s rule overlook differences iti molecular flexibility and there is a need for a revrsed model that takes entropy differences info account, particalariy as recent developments in fke elucidation of receptor sfrucfure are likely to revive interest in Pfeiffer’s rule.

In a note entitled ‘Optical isomer- ism and pharmacologica action, a generalization’, C. C. Pfeiffer’ observed a striking correlation be- tween the relative potencies of the mirror-image forms (enantiomers) of some clinicafly used drugs and the average human dose of the drug, given as the racemate. With the more active drugs there was a bigger difference between the activities of the enantiomers. For 14 compounds the optical iso- merit ratios (Y) plotted on a loga- rithmic scale against the average human dose (X), also on a logar- ithmic scale (Fig. l), appeared to lie on a straight line described by the equation:

log Y = 1.19 - 0.354 (log X)

The correlation coefficient r ap- peared to be > 0.9.

Pfeiffer’s observation was the more remarkable because no allowance was made for differ- ences in the metabolism of the enantiome~, differences between the receptors involved and differ- ences in the type of action of the drugs (some were agonists, others were antagonists). An additional problem is ensuring that the weaker enantiomer is stereo- chemically pure and does not con-

R. 6. Barlow is Render irr Chenricnl Pbnnnn- cology at the Departrt~ent of Phwnmology, University of Bristol, Tk Medical School, University Walk, Brisfol BSS ITD, UK.

tain traces of the stronger enan- tiomer.

On the basis of the corrc!ations generated by these data Pfeiffer’s rule appears to make sense. In the simplest situation, where activity depends only on binding, as with competitive antagonists, it is plausible that the higher the affin- ity of a compound, the more it matters how groups are arranged about a chiral centre. If mirror- image forms have different affin- ity, binding must involve an interaction at three points (at Ieast); the less active form would perhaps only form an attachmznr at two of them. If the binding is measured as an affinity constant K then log K is directly proportional to the Gibbs free energy of bind- ing [from the van? Hoff relation -AC = R T(ln K), where R is the gas constant and T is the absolute temperature]. The difference be- tween log K for the enantiomers is the logarithm of their relative potency. Various names have been given to the relative potency of mirror-image forms: if it is referred to as the stereospecific index (SSI; Ref. 2) and this is 100, then log SSI is 2 and the difference between the free energy of binding of the two forms is 11.8 kJ mol-r at 37°C.

Further implications There is, however, a corollary to

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Pfeiffer’s rule which it is much more difficult to believe. If 1ogSSI is linearly related to 1ogK for the racemate, it must also be at least approximately linearly related to log K for the stronger enantiomer: this should not be more than twice as active as the racemate so the difference in 1ogK should be less than 0.3 (log 2). Lehmann et aL3 have re-calculated Pfeiffer’s observations in this way and ob- tained a slope of 0.34 with a correlation coefficient, r, of 0.96. If 1ogK for the stronger enantiomer determines logs%, it must also determine 1ogK for the weaker enantiomer. Is this always true?

It does not seem probable, if only because it takes no account of the flexibility of the molecule. In a flexible molecule it would be ex- pected that there would be less difference between enantiomers because the weaker form might adapt its conformation to achieve a degree of fit that would be denied to a more rigid molecule. If flexibility is associated with a smaller difference between enan- tiomers, Pfeiffer’s rule would sug- gest that flexible molecules should have lower affinity, which does not seem likely to be true and is not what is found.

For example, results obtained with several series of antagonists at muscarinic cholinoceptors4,5 are shown in Fig. 2. There is a trend; a least-squares fit to a straight line gives:

log SSI = 0.380 (log K) - 1.906

with the r value of 0.52 (n = 54). The slope is similar to that ob- served with Pfeiffer’s data, indi- cating that SSI roughly doubles for a tenfold increase in affinity: there is, however, a lot of scatter and the correlation could not be used to predict results for new pairs of compounds with any reliability. The highest value of 1ogSSI (3.05, indicating a > lOOO-foid differ- ence between enantiomers) is for an aza-analogue of hyostyamine (IogK 8.36). The calculated value of iog SSZ is 1.19 so the exper- imental value of SSZ is nearly 100 times the calculated value. Al- though the experimental value falls within the 95% confidence limits, these range from -0.82 to 3.20 (i.e. > lOOOO-fold).

Better correlations were ob- tained by Lehmann et ~1.3 who analysed the same data as separate

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_ 2.29

I c

0.5 1.0 5 10 50 100 1000 5000 Average Human Dose

Fig. 7. Pfeiffer’s original observations. The weaker compounds, for which the dose is bigger, lie to the right (Redrawn using data from Ref. 1.)

The corollary to Pfeiffer’s rule - that the affinity of the weaker enantiomer is determined by that of the stronger one - is improb- able. There are many exceptions besides the examples given here. Moreover, as methods for measur- ing affinity become more reliable there is increasing information about the variable effects of

sets of homologues. The results for a particular type of compound often form a pattern (as in Fig. 2) and Lehmann et al. interpreted these and other results in terms of equilibria involving binding at four parts of the molecule to com- plementary sites in a rigid recep- tor. The slopes of the lines differ from one set to another, because the binding groups differ in rela- tive importance from one set to another. The main problem with this approach is in defining a set. The values for hyoscyamine, for instance, are 1ogSSi = 2.5 and 1ogK = 9.38 for the (-)-isomer: should its aza-analogue belong to the same set? If it does, the compounds clearly do not obey Pfeiffer’s rule.

_I temperature on binding; this information gives thermodynamic

the effects of temperature on reasons for exceptions to the rule. binding [dlogKId(lIT) = -AH/R] The entropy of binding is likely to and it is known that these are involve what happens to water in variable-. Usually affinity is the binding process as well as greater at lower temperature but what happens to the conformation

There are thermodynamic rea- sons why there should be differ- ences between sets and even be- tween compounds. The Gibbs free energy, AG, depends on the en- thalpy of binding, AH, and on the entropy of binding, AS (AG = AH - T AS; T is the temperature); there is no reason why AH and AS should be related, though they might be similar in sets of com- pounds that bind to similar areas of the receptor.

Differences in flexibility are likely to be associated with differ- ences in AS. Differences in AH may be detected experimentally because the enthalpy determines

149

for some compounds it is greater at higher temperature.

With the recent developments in the elucidation of the structures of many types of receptor and the possibility of doing molecular modelling in some instances, it is important to re-assess the validity of Pfeiffer’s rule. Because it looks reasonable at first sight there is a danger that it becomes part of established teaching without its implications being explored fully.

z2- cn

5 7 9 11

Log K

Fig. 2. Affinities of some antagonists for muscarinic acetylcholine receptors in guinea- pig ileum. The weaker compounds, with lower affinity, lie to Ihe left. Values of log SSI are plotted against log K for the stronger enantiomer. Mandelic esters (0); phenylcyclo- hexyl acety/ esters (0); phenylcyclohexyl g/yco//ic esters (81; u-methylfropic esters (a); quaternary derivatives of hyoscyamine (m); qualernary derivatives of h.“osclne (0); quaternaty derivatives of homalropine (A). The line is the least-squares fit for aIf54 compounds and has the equation Y = 0.380 (X - 1.906) (X-intercept = 5.02). Results for the aza-analogue of hyoscyamine and its methiodide (0) were nol included in fhe fit (Data from Refs 4 and 5.)

150 TiPS - April 1990 [Vol. 221

of the drug. The effects on water will be particular@ important where hydrophobic binding is in-

which takes account of the reasons 4 Barlow, R. B., Franks, F. M. and Pearson,

why there are exceptions to J. D. M. (1973) /. Med. U&w. 16, 439-446

Pfeiffer’s rule. 5 Barlow; R. B. (1973) /. Med. Clte,,r. 16, 1037-1038

valved. In assessi:lg hydrophobic 6 Barlow, R. 6.. Berry, K. J., Clenl,>n,

effects it is more appropriate to References I’. A. M., Niko!aou, N. M. and Soh, K. S.

think in terms of overlapping I Pfeiffer, C. C. (1956) Scir~r 124. 29-30 (1978) Br. 1. Pltarr~mcol. 58, 613-620

areas, rather than interactions be- 2 Barlow, R. 8. (1971) /. Plrnn~~. Plu~~~ol. 7 Barlow. R. B. and Burston, K. N. (1979)

tween points. There is a need for a 23,9C-97 Br. /. Plu~n~mcol. 66, 581-585

3 Lehmann, P. A., de Miranda, J. F. R. and 8 Barlow, R. B., Birdsall, N. j. M. and revised model of the interactions Ari&s, E. J. (1976) Prog. Dr~rg Rrs. 18, Hulme, E. C. (1979) Br. 1, PJxnr~t~ncol. 66,

between drugs and receptors 101-142 587-590

firnary sequence of cyclic nucleotide phos hodiesterase isozymes and the design of selective inhibitors Joseph A. Beavo and David H. Reifsnyder

Primary seqzle~tce iuforrnntion hns been reported for more than 25 drfferent nmmunlinn cyclic uucleotide phosplzodiesternses. Moreover, recent obser- vntious suggest that mnn!y of these isozyznes me selectively expressed in LI limited number of cell types. The fuct that nearly all these different phosphodiestercses hnzle unique prinmy sequences in their cntnlytic or regulntory dotnnim and thnt they me often selectively expressed itnplies that it tnny be possible to modulate individunl isozynzes using specific drugs. Joe Beavo and David Reifsnyder summrize much of the evidence that hns led to oldr current understanding of multipIe isozyrnes of phosphodiesterczse, with emphasis on aspects thnt my be relezmt to drug design. They also discuss 7ulry ninny previous attempts to isolate isozyme-selective inhibitors may have failed.

Although many texts imply that cyclic nucleotides are degraded by a single enzymatic activity, it is now apparent that inactivation of CAMP and cGMP is catalysed by not one, but rather a large number of different cyclic nucleotide phosphodiesterases. Recent data suggest that at least five different isozyme families exist and more than 20 distinct enzymes are now recognized (see Box). More import- antly, biological reasons for this great diversity are beginning to be appreciated. For example, many of the isozymes are differentially expressed and regulated in dif-

I. A. Benoo is Professor nt tlte Dcparhwn~ of Pharmncolo~y. 51-3U. Utllvcrsity of Wasltiqton, Stvtttc. WA 98195. US& atld D. H. Rrifsnydrr is n Scientist at Geueutech, Sm Fraucisco, CA 94080, USA.

ferent cell types. From a pharma- cological perspective, just as multiple receptors controlling the synthesis of CAMP and cGMP offer opportunity for selective therapeutic intervention., multiple ‘receptors’ for cyclic nucleotide degradation should offer equally good possibilities. Here we dis- cuss the way in which recent data on the structure, regulation and localization of multiple phospho- diesterases provide a conceptual rationale for the design of selec- tive inhibitors and activators of these enzymes (see Refs 1 and 2 for more general reviews on phos- phodiesterases).

Early phosphodiesterase inhibitors

Since the original reports by Butcher and Sutherland3 in 1962

that methylxanthines such as caf- feine and theophylline inhibit CAMP hydrolysis, many studies have been carried out to identify drugs that inhibit cyclic nucleo- tide phosphodiesterase activity. In the first 20 years of this period no new therapeutically important agents were identified. Although drugs like papaverine and theo- phylline were found to be rela- tively potent and effective com- petitive inhibitors of CAMP and later cGMP hydrolysis, most new agents based on these structures were either ineffective or tou toxic tu be of widespread clinical use. We now know that the relatively poor therapeutic index of these agents was due at least in part to the fact that they were not isozyme specific and therefore increased cyclic nucleotide levels in many non-target cells. More- over most had additional modes of action. For example, theophyl- line and its more potent con- gener 3-isobutyl-l-methyl xanthine (IBMX) are also potent antagonists at adenosine receptors. Dipyrid- amole, a classical antithrombotic agent that is now known to be a selective phosphodiesterase inhibi- tor, is also a potent inhibitor of adenosine transport.

It is now evident that the design of most early screening exper- iments was inappropriate. In nearly all of them, whole tissue homogenates or extracts were used as the source of phospho- diesterase activity. Because most tissues are heterogeneous with respect to cell type and many cells contain multiple phospho- diesterases, the screening studies were all conducted with a mixture of isozymes and were therefore unlikely to identify inhibitors selective for an individual iso- zyme. For a few agents it was noted that l&20% of the total phosphodiesterase activity was inhibited by relatively low drug