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5
Chapter • Optical Activity and Biological Eect
. Fig. 5.12 Thiorphan 5.23 inhibits the metabolism of enkephalins
and contains aβ-mercaptopropionic acid, the absolute conguration
of which is analogous to l-phenylalanine. Application of the retro–
inverso concept gives aminothiol 5.24, the absolute conguration of
which corresponds to d-phenylalanine due to incorporation in the
opposite direction. The identical binding mode to the zinc protease
was determined for both thiorphan 5.23 and retro-thiorphan 5.24.
Thermolysin and the neutral endopeptidase 24.11 (NEP 24.11, previously referred to as enkephalinase) are inhibited by both compounds
to the same extent. On the other hand, angiotensin-converting enzyme
(ACE), another zinc protease, discriminates decidedly between these
substances
. Fig. 5.11 Enantiomers have different biological effects. The eud-
ismic ratio of propranolol 5.19 is 100 for β-antagonism, and for unspecic membrane interaction, it is, expectedly, 1. Identical partial
structures can have entirely different eudismic ratios, for instance,
compare the optical center of the alcohol moiety of the cholinergic
compound methacholine 5.20, with the identical center on the anticholinergic compound 5.21. Compound 5.21 also proves that the eudismic ratio of different centers in acompound are independent from
each other. The example butaclamol 5.22 also shows that the same
substance can have different eudismic ratios on different receptors
Some naturally occurring peptide antibiotics contain
d-amino acids. This gives them better metabolic stability.
For the same reason, d-amino acids are incorporated
into many synthetic peptide molecules. In the best cases,
amore potent and longer-acting analogue is obtained.
Synthetic analogues of peptides with aretro–inverso con-
guration are aspecial case. In these molecules, the direction of the peptide chain or part of the peptide chain
is reversed, that is, compared to the original peptide, the
amino and carboxyl groups of individual amino acids are
reversed. To maintain the relative conguration, d-amino
acids or their analogues are used instead of l-amino acids. In this way, it is possible to deceive some enzymes or
receptors; they bind the natural peptide and the retro–inverso peptide in the same way. This is true for thiorphan
5.23 and its retro–inverso analogue 5.24 for two enzymes,
but not for athird (. Fig.5.12). In general, retro–inverso
peptides are metabolically more stable than their original
peptide analogues.
Enantiomers differ not only in the strength of their
potency, but also in their biological qualities. These differences can manifest themselves as undesirable side effects of astereoisomer, such as the chiral barbiturate 5.25
(. Fig.5.13). The most serious drug side effect of the last
70years was the embryonic malformations caused by the
sleeping pill thalidomide 5.26 (Contergan®); these were
caused by one of the two enantiomers (. Fig.5.13). In the
1950s, thalidomide was claimed to be the best tolerated
sleeping pill with the fewest side effects. It was introduced
to the market in 1957 and was available in pharmacies as
an over-the-counter drug. There was no concern that even
women in the rst months of their pregnancy were taking
these sleeping pills. It was withdrawn from the market in
1961 because of its teratogenic effects. If drug testing had
been what it is today, this catastrophe would certainly
have been detected earlier and probably largely avoided.
It could not have been prevented by administering only
one of the two enantiomers. Both enantiomers racemize
in vitro, meaning that one converts into the other even
in the test tube. Accordingly, the effect was conrmed in
vivo, after the administration of the supposedly safe enantiomer had led to teratogenic effects in an animal model.
The “other” enantiomer can also open up new therapeutic possibilities. The enantiomer of asynthetic opiate,
such as propoxyphene 5.27 (. Fig.5.13), has weak an-
. • Dierences in the Activity of Enantiomers
. Fig. 5.13 Enantiomers also differ in their mode of action. The
(R)-(−)-enantiomer of barbiturate 5.25 is a hypnotic agent, whereas the (S)-(+)-enantiomer causes seizures. In rats and mice, only the
(S)-(−)-enantiomer of thalidomide 5.26 (Contergan®) is teratogenic,
that is, it causes embryopathies. Thalidomide 5.26 racemizes in vitro
as well as in rabbits. Therefore, even the (R)-(+)-enantiomer is teratogenic in rabbits. Propoxyphene 5.27 is apotent analgesic, the effect of
which depends on the (2S,3R)-(+)-enantiomer, dextropropoxyphene.
The (2R,3S)-(−)-enantiomer is acough suppressant. The (R)-(+)-en-
antiomer of Bay K8644 5.28 is aweak calcium channel blocker. The
(S)-(−)-enantiomer stabilizes calcium channels in the open form and
is therefore an agonist, that is, acalcium channel opener (Sect.30.4)
. Fig. 5.14 The proportion of achiral, enantiomerically pure, and
racemic drugs approved in the period from 1983–2002. In the meantime, the proportion of newly approved drugs has shifted decidedly in
the direction of enantiomerically pure compounds
algesic and narcotic effects, but good antitussive properties. Enantiomers can also inuence each other in their
effects and even cancel each other out. In the case of the
calcium channel ligand5.28, one enantiomer has achannel-opening effect, the other achannel-closing effect (see
Sect.30.4).
Between 1983 and 2002, 38% of all approved drugs
were achiral, 39% were enantiomerically pure, and 23%
were racemic or diastereomeric mixtures. The fact is that
racemic mixtures of chiral drugs were much more readily accepted in earlier decades than they are today. This
was certainly not due to stereophobia on the part of the
chemical industry. Rather, it was due to alack of understanding of stereospecicity and side effects, and perhaps
also to economic considerations; kinetic resolution and/
or enantiomerically pure syntheses are very expensive.
You can certainly see that the proportion of enantiomerically pure drugs on the market is increasing (. Fig.5.14).
In the 1970s, Ariëns was the rst to argue strongly
against the use of racemic mixtures in therapy. In his
view, racemates are compounds with 50% impurity. The
inactive or less active enantiomer is called isomeric ballast and, despite its lack of effect, places an additional
burden on the metabolism, since any drug-like compound
must be chemically degraded in order to be eliminated
from the body (Sect.27.6). Ariëns used as an example
the diastereomeric mixture labetalol 5.11 (. Fig.5.5),
. Fig. 5.15 Upon metabolism of the monoamine oxidase inhibitor,
selegiline 5.29, which is used to treat Parkinson’s disease, the more potent (R)-(–)-enantiomer is converted to methamphetamine 5.30 and
amphetamine 5.31. The less-active (S)-(+)-selegiline has less severe
side effects because it is not metabolized to CNS-active stimulants
which is not a“mixed α,β-antagonist” but amixture of
four different drugs. The effect of this “combination” is
aresult of the effects of each enantiomer. In most cases,
Ariën’s criticism is fully justied. When designing and
developing new drugs, it is important to ensure that the
biological activity is as specic as possible and that side
effects are minimized. Compound uniformity is usually
easier to achieve for an enantiomer than for aracemate,
which is amixture of two substances, or even for adiastereomeric mixture.
Choosing the correct enantiomer can even reduce or
prevent undesirable side effects of metabolites. Selegiline
5.29, amonoamine oxidase inhibitor, is metabolized to
the CNS active compounds methamphetamine 5.30 and
amphetamine 5.31 (. Fig.5.15). Fortunately, the more
active enantiomer of 5.29 forms the less active of these
two metabolites! Using the correct enantiomer of the racemate will increase the desired effect and decrease the
undesired CNS side effects.
There are also afew counter examples. The (−)-enantiomer of the calcium channel blocker verapamil
(Sects.2.6 and30.4) is more effective than the (+)-enan-
Chapter • Optical Activity and Biological Eect
5
. Fig. 5.16 The (R)-(−)-enantiomer of ibuprofen 5.32 undergoes
ametabolic inversion of its stereocenter to form the (S)-(+)-enantio-
mer. As acyclooxygenase inhibitor in vitro, the (S)-(+)-form is more
tiomer. The therapeutic spectrum of both enantiomers
is practically identical. After oral administration, the
(−)-enantiomer is quickly metabolized. Therefore, the
(+)-enantiomer contributes substantially to the desired
effect. In this case, it would not be economical to try to
separate the racemic mixture.
Ibuprofen 5.32, an anti-inammatory drug of the
arylpropionic acid class (. Fig.5.16 and Sect. 27.9),
is aspecial case. The potency of the enantiomers are
very different in vitro. In vivo, however, the inactive
(R)-(−)-enantiomer is converted to alarge extent to the
(S)-(+)-enantiomer. The reverse reaction does not take
place. Therefore, the racemate and each enantiomer are
therapeutically identical, even at the same dose. Only the
side-effect spectrum is different because the inversion of
the (R)-(−)-enantiomer is not 100% complete.
Sometimes the cost of producing apure enantiomer
is not justied. In such cases, the efcacy and side effects
of the two forms must be compared. Depending on the
results, the continued use of the racemate or the development of an achiral analogue may be considered in special
cases. In any case, these data must be complete before the
drug can receive approval.
5.6 Image and Mirror Image:
Why Is It Different for the Receptor?
Enantiomers and diastereomers have different biological
properties because of the handedness of the proteins to
which they bind. They occur in Nature in only one form.
The amino acids with their chiral centers and the secondary structure elements (Sect.14.2) with their helical
orientation are responsible for these properties. If aprotein is offered aleft- or right-handed ligand, different
binding modes can be expected, just as two right hands
come together to shake hands more easily than aright
hand and aleft hand.
Only afew examples have been reported of protein–ligand complexes with both left- and right-handed ligands.
This will only be possible if both enantiomers have sufcient afnity for the target protein, which means that they
both bind to the protein strongly enough for an X-ray
crystal structure to be determined.
potent than the (R)-(−)-form. The less-active form is converted to the
more-active enantiomer in vivo. Therefore, both compounds exhibit
equally anti-inammatory properties in animal models
The R- and S-enantiomers of the compound BX5633
(5.33) inhibit the serine protease trypsin (Sect.23.3) to
equal extents. They have astereogenic center next to an
acid group. The crystal structure determination explains
this lack of discrimination. The inhibitor’s acid group is
oriented outside of the binding pocket so that no specic
interaction is to be expected (. Fig.5.17). Astereopreference cannot exist.
Both enantiomers 5.34 and 5.35 bind to carbonic anhydraseII, a zinc hydrolase (Sect.25.7). There is adifference of afactor of 100 in their afnities. As the X-ray
structure with both enantiomers shows, they have similar
binding modes (. Fig.5.18). All properties relating to
the solvation of the ligands must be the same for both
enantiomers. The difference in afnity is, therefore, only
caused by differences in the binding mode of the ligands.
The sulfonamide groups of both enantiomeric ligands
bind almost identically to the catalytic zinc. In addition,
the endocyclic SO2 group forms very similar hydrogen
bonds to Gln92. The hydrophobic iso-butyl side chains
are positioned in similar parts of the binding pockets.
However, the six-membered ring must adopt a highly
strained conformation in the case of the weaker binding
enantiomer. The price of this strained conformation is
areduced binding afnity to the enzyme.
The enantiomeric agonists 5.36 and 5.37 bind to the
ligand binding domain of the retinoic acid receptor with
a factor of 1000 difference (Sect.28.2). The receptor
itself adopts the same geometry (. Fig.5.19). The alcohol function in the middle of the molecule is at the
stereogenic center. In both cases, a hydrogen bond is
formed with Met 272. As aresult, the neighboring amide
groups must take on deviating orientations in the binding pocket. On the “right” side, the tetralin moiety for
both stereoisomers is found in similar spatial region. On
the “left” side, the benzoic acid moiety of both enantiomers form ahydrogen-bond network with Arg 278, Ser
289, and Leu 233. The uorine-substituted benzene ring
adopts in both cases a180° ipped orientation. These different orientations, together with the divergently oriented
amide bond are responsible for the marked difference in
the binding afnity of the mirror-image agonists.
. • Image and Mirror Image: Why Is It Dierent for the Receptor?
. Fig. 5.17 The (R)- (gray)
and (S)-enantiomers (beige) of
the inhibitor BX5633 5.33 bind
with the same afnity to trypsin.
Because the protein adopts
practically the same geometry
with both inhibitors, only one
structure of the protein residues
is shown. The crystal structure
shows that both forms of 5.33
adopt almost identical binding
modes. The acid function on the
stereogenic center points out of
the binding pocket and into the
surrounding aqueous medium.
Therefore, no stereochemical
discrimination can take place.
(7 https://sn.pub/iAL3oo)
. Fig. 5.18 The enantiomeric
sulfonamides 5.34 (gray) and 5.35
(yellow) bind in asimilar way to
the enzyme carbonic anhydraseII.
Because the protein adopts practically
the same geometry with both inhibitors, only one structure of the protein
residues is shown. The zinc ion in the
catalytic center (purple sphere) is coordinated to the sulfonamide groups.
The SO2 groups in the six-membered
ring form ahydrogen bond to Gln92
(green). The hydrophobic iso-butylamino moieties on the chiral centers
project into ahydrophobic pocket
and ll this out to the same extent.
In doing this, the six-membered ring
must adopt adeviating conformation
in both enantiomers. In one stereoisomer, this conformation is much more
strained than in the other and causes
aloss in binding afnity.
(7 https://sn.pub/YmklAy)
5
Chapter • Optical Activity and Biological Eect
. Fig. 5.19 Both enantiomers of the agonists 5.36 (beige) and 5.37
(gray) bind the retinoic acid receptor with 1000-fold difference in afnity. Because the protein adopts practically the same geometry with
both ligands, only one structure of the protein residues is shown. Both
ligands form H-bonds with their OH groups to the sulfur atom in Met
272. In doing so, the uorine-substituted aromatic ring of the benzoic
acid moiety on the left with its central amide bond has to adopt adeviating orientation. The tetrahydronaphthalene (tetralin) moiety, on
5.7 An Excursion into the World of
Stereoisomers
Experience has taught us that if an enantiomer crystallizes with aparticular auxiliary base or acid, the other
enantiomer will crystallize with the mirror image of the
auxiliary in the same way if identical reaction conditions
are applied. Polypeptides composed of l-amino acids
form right-handed helices, and polypeptides made of
d-amino acids form left-handed helices.
Some naturally occurring peptides form ion channels
in lipid bilayers. Their synthetic enantiomers can also do
this. The more interesting question is: how does the mirror image of an enzyme behave? In 1992, Stephan Kent
and coworkers prepared HIV protease (Sect.24.3), ahomodimer made up of 2 × 99 amino acids, entirely from
d-amino acids. The naturally occurring protein was also
prepared in parallel. The all-l-enzyme reacts only with
l-peptide substrates and the all-d-enzyme reacts only
the other hand, is positioned in the same way in both enantiomers.
(7 https://sn.pub/l3NCiE)
with the all-d-enantiomer. The same is true for chiral inhibitors that block HIV protease. An achiral inhibitor, on
the other hand, inhibits both enzymes in the same way.
Rubredoxin, an electron transport protein, was prepared as the all-d-protein for the sole purpose of mixing
it with the naturally occurring all-l-protein and to make
the racemate! If the effort involved is considered, this
will certainly be an approach that takes some getting
used to. The reward for the work was very high-quality
crystals. The racemate crystallized in acentrosymmetric
space group (Sect.13.2), which allowed better resolution
of the 3D structure than was possible with the natural,
all-l-enantiomer and the phase determination is reduced
to the assignment of“+” or“−”.
What does avisit to the mirror-image world look like?
Achiral drugs would have an identical potency and mode
of action. On the other hand, many enantiomerically
pure drugs would be useless. We would have to watch
out for chiral barbiturates like 5.25. They would rather
Bibliography and Further Reading
cause aseizure than act as asedative. In cases in which
chiral antibiotics were used to treat bacterial infections,
it would rst have to be established whether the infecting bacteria came from the mirror-image world or the
“normal” world. The administration of trimethoprim
(Sect.27.2) and asulfonamide (both achiral) would help
at any rate.
There would be tremendous nutritional problems.
The carbohydrate and protein metabolism would not
work anymore, nor would the absorption of monomers
from the gastrointestinal tract. We would not be able to
recognize some plants by their smell. (R)-Carvone smells
of caraway seeds; (S)-carvone smells of spearmint. Our
beloved sugar would have lost its sweet taste, and fruit
juices and lemonade would taste sour. Coffee, tea, and
Coca-Cola would retain their stimulatory effects because
caffeine is achiral. Diet drinks would have to be sweetened with saccharine or cyclamate (both achiral) because
aspartame is chiral.
Let us return to the normal world! But rst, let us
have aquick glass of vodka. It could also be cognac,
whisky, or adry red wine. The taste would be the same as
in the normal world, or would it not? Despite the many
hundred avor components of wine, the exchange of
asingle chiral center could have the consequence that
aconnoisseur might no longer recognize the chateau.
The euphoric effects would be the same, though this
would not be the case for the hard, optically active drugs
such as heroin, cocaine, or LSD.
5.8 Synopsis
Compounds with an asymmetric or chiral center give
-
rise to enantiomers, two isomeric forms that relate
to each other like an image and mirror image and
cannot be mutually transferred without breaking and
reforming bonds.
Enantiomers exhibit the same properties as long as
-
they are found in anonchiral environment. If ex-
posed to the asymmetric environment such as apro-
tein-binding site, they will experience different interac-
tions and, thus, result in distinct biological properties.
Chiral centers are mostly found at atoms carrying
-
four different substituents, but also an overall handed
scaffold can give rise to chirality. If nindependent
stereocenters are present, 2n isomers (diastereomers)
are produced occurring as 2
of equally present enantiomers) as long as there is
no internal inversion, mirror, or improper rotation
symmetry present.
Chiral centers are named according to the Cahn–In-
-
gold–Prelog priority rules that bring the substitu-
ents in aunique sequence according to their atomic
numbers. The substituent with lowest priority has to
be oriented to the back and the direction of the re-
n−1
racemic mixtures (pair
maining substituents determine R/S by the sense of
rotation following decreasing priority.
Enantiomers can be separated by fractional crystalli-
-
zation after being converted into diastereomeric salts
with appropriate chiral auxiliaries. Enzymes such as
lipases, esterases, or proteases can also be used for
resolution because they transform one enantiomer
faster than the other for steric and kinetic reasons.
Most natural products are optically active and occur
-
in just one form. Biologically active enantiomers are
called eutomers, inactive ones distomers.
Biological activities of enantiomers and diastereo-
-
mers can vary greatly in strength and quality. Application of racemates has to be examined carefully for
each individual case. Side effects, chemical stability,
and deviating metabolism can have decisive inuence
on the activity prole.
On the molecular level, the afnity discrimination of
-
enantiomers is explained by deviating binding modes
in the binding pocket of the target protein, thus, resulting in differences of the observed interaction pattern or strain of the adopted bound conformation.
Bibliography and Further Reading
General Literature
E. J. Ariëns, W. Soudijn and P. B. M. W. M. Timmermans, Stereo-
chemistry and Biological Activity of Drugs, Blackwell Scientic
Publishers, Oxford (1983)
D. F. Smith, Ed., CRC Handbook of Stereoisomers: Therapeutic
Drugs, CRC Press, Boca Raton, Florida (1989)
B. Holmstedt, H. Frank and B. Testa, Chirality and Biological Activity,
Alan R. Liss, Inc., New York (1990)
C. Brown, Ed., Chirality in Drug Design and Synthesis, Academic
Press, London (1990)
M. Eichelbaum, B. Testa, A. Somogyi, Handbook of Experimental
Pharmacology, Stereochemical Aspects of Drug Action and Disposition, Springer Verlag, Heidelberg (2002)
G. Klebe, Differences in Binding of Stereoisomers to Protein Active
Sites, in Supramolecular Structure and Function 8, Ed. Greta Pifat-Mrzljak, Kluwer Academic/Plenum Pub., New York, pp. 31–53
(2004)
H. Caner, E. Groner and L. Levy, Trends in the Development of Chiral
Drugs, Drug Discov. Today 9, 105–110 (2004)
Special Literature
D. A. Evans, M. D. Ennis, and D. J. Mathre, Asymmetric Alkylation
Reactions of Chiral Imide Enolates. A Practical Approach to the
Enantioselective Synthesis of α-Substituted Carboxylic Acid Derivative, J. Am. Chem. Soc., 104, 1737–1739 (1982)
D. A. Evans, Studies in Asymmetric Synthesis. The Development of
Practical Chiral Enolate Synthons, Aldrichimica Acta, 15, 23–32
(1982)
E. J. Ariëns etal., Stereoselectivity and Afnity in Molecular Pharma-
cology, Fortschritte der Arzneimittelforschung, 20, 101–142 (1976)
E. J. Ariëns, Stereochemistry, a Basis for Sophisticated Nonsense in
Pharmacokinetics and Clinical Pharmacology, Eur. J. Clin. Pharmacol., 26, 663–668 (1984)
5
Chapter • Optical Activity and Biological Eect
M. Bocola, M. T. Stubbs, C. Sotriffer, B. Hauer, T. Friedrich, K.
Dittrich, G. Klebe, Structural and Energetic Determinants for En-
antiopreferences in Kinetic Resolution of Lipases, Protein Eng.,
16, 319–322 (2003)
S. Mason, The Origin of Chirality in Nature, Trends Pharmacol. Sci., 7,
20–23 (1986), and further contributions by additional authors, pp.
60–64, 112–116, 155–158, 200–205, 227–230 and 281–285.
E. J. Ariëns, Nonchiral, Homochiral and Composite Chiral Drugs,
Trends Pharmacol. Sci., 14, 68–75 (1993)
S. C. Stinson, Chiral Drugs, Chemical & Engineering News, 19. Sept
1994, p. 38–72, and Oct. 9, 1995, pp. 44–74
G. Jung, Proteins from the D-chiral world, Angew. Chem. Int. Ed.
Engl., 31, 1457–1459 (1992)
M. T. Stubbs, R. Huber, W. Bode, Crystal structures of Factor Xa spe-
cic Inhibitors in Complex with Trypsin: Structural Grounds for
Inhibition of Factor Xa and Selectivity against Thrombin, FEBS
Lett., 375, 103–107 (1995)
J. Greer, J. W. Erickson, J. J. Baldwin, M. D. Varney, Application of
the Three-dimensional Structures of Protein Target Molecules
in Structure-based Drug Design. J. Med. Chem., 37, 1035–1054
(1994)
B. P. Klaholz, A. Mitschler, M. Belema, C. Zusi, D. Moras, Enantiomer
Discrimination Illustrated by High-resolution Crystal Structures
of the Human Nuclear Receptor hRARγ, Proc. Natl. Acad. Sci.
USA, 97, 6322–6327 (2002)
R. C. Milton, S. C. Milton, S. B. Kent, Total chemical synthesis of
a D-enzyme: the enantiomers of HIV-1 protease show reciprocal
chiral substrate specicity, Science, 256, 1445–1448 (1992)
L. E. Zawadzke, J. M. Berg, The structure of a centrosymmetric protein
crystal, Proteins, 16, 301–305 (1993)
The Search
for the Lead Structure
II
The starting point for the development of anew drug is the search for asuitable
lead structure for atarget protein. First, such atherapeutic target structure must
be identied as adiseaserelevant target in the genome or proteome of acell. Genetic engineering techniques allow the production of this target structure. After
setting up ahighthroughput screening assay, thousands of test molecules are
screened for binding to the target protein. The Xray structure is elucidated and
subsequently used to search for and optimize lead structures. Today, lead search
and optimization is inconceivable without massive support from methods of bio
and chemoinformatics, molecular modeling, and computational chemistry (announcement poster of the author’s group on the occasion of a2003 conference in
Rauischholzhausen, Marburg).
Contents
Chapter 6 The Classical Search for Lead Structures – 85
Chapter 7 Screening Technologies for Lead
Structure Discovery – 95
Chapter 8 Optimization of Lead Structures – 115
Chapter 9 Designing Prodrugs – 127
Chapter 10 Peptidomimetics – 137
The Classical Search for Lead
Structures
Contents
6.1 How It Began: Hits by In Vivo Screening – 86
6.2 Lead Structures from Plants – 86
6.3 Lead Structures from Animal Venoms
and Other Ingredients – 87
6.4 Lead Structures from Microbial Organisms – 88
6.5 Dyes and Intermediates Lead to New Drugs – 89
6.6 Mimicry: How to Copy Endogenous Ligands – 90
6.7 Side Eects Indicate New Therapeutic Options – 91
6.8 From the Traditional Search to the Screening
of Large Compound Libraries – 92
6.9 Synopsis – 93
Bibliography and Further Reading – 93
© The Author(s), under exclusive license to Springer-Verlag GmbH, DE, part of Springer Nature 2024
G. Klebe, Drug Design, https://doi.org/10.1007/978-3-662-68998-1_6