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Helvetica Chimica Acta – Vol. 96 (2013) 159 REVIEW Organic Stereochemistry Part 2 1) Stereoisomerism Resulting from One or Several Stereogenic Centers by Bernard Testa Department of Pharmacy, Lausanne University Hospital (CHUV), Rue du Bugnon, CH-1011 Lausanne (e-mail: [email protected]) With this second review, our Work on organic stereochemistry continues with that most important of stereogenic elements, namely the stereogenic center, as found in a majority of stereoisomers. The presentation is restricted to chiral tetrahedral structures, which contain the most important stereogenic centers in molecules of biological relevance. These are either tetracoordinate or tricoordinate centers where an electron lone pair plays the role of the fourth substituent. The nature of the central element in such structures, e.g., carbon, nitrogen, or sulfur, obviously plays an essential role in their geometry. Our main focus in this review are the two rule-based conventions used to encode with adequate descriptors the absolute three-dimensional (3D) geometry of stereogenic centers. By absolute 3D geometry, stereochemists understand an unambiguous description of the sense of chirality (stereogenicity) of a center with reference to the observer and based on the universal left-hand/right-hand discrimination. With single stereogenic centers and the general principle of enantioselective processes explained and illustrated, the fundament will be laid to consider the stereochemistry of molecules containing two or more stereogenic centers. When there is a single stereogenic center, the compound will exist as enantiomers, namely a pair of structures related to each other as non-superimposable mirror images. When two or more stereogenic centers are present, the result may be another steric relation between molecules known as diastereoisomerism. The discrimination between enantiomeric and diastereoisomeric relationships is a critical one in the chemical and related sciences, and care will be taken to illustrate this difference. This Part 2 will be followed by a presentation, in Part 3, of other stereogenic elements, namely axes and planes of chirality, helicity, and (E,Z)-diastereoisomerism, followed in turn by a presentation of isomerisms about single bonds and in cyclic systems in Part 4. Only then, with the principles of stereoisomerism discussed, will we be able to consider their impact on pharmacology, biochemistry, and xenobiotic metabolism. This will still leave us with just one last facet of stereochemistry to consider, the all important concept of prostereoisomerism and its key role in endogenous and exogenous metabolism (Part 8). 1) For the other Parts, see Helv. Chim. Acta 2013, 96, 1 – 3. 2013 Verlag Helvetica Chimica Acta AG, Zrich 160 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.1. This second review in the series begins by looking at compounds characterized by a single stereogenic center and hence having a non-superimposable mirror image with which they form a pair of enantiomers [1 – 16]. The focus will be on chiral tetrahedral structures, namely a) with tetracoordinate centers, and b) with tricoordinate centers where an electron lone pair plays the role of the fourth substituent. Following an overview of the main tetrahedral structures of interest, the review goes on to explain the two dominant convention systems, namely the d,l- and the (R,S)-conventions, the latter being known as the CIP (CahnIngoldPrelog) convention. These are systems based on rigorous rules and allow an absolute descriptions of molecular structures in 3D space, i.e., description that yields an identical outcome for all observers. As we will see, both conventions have their advantages, although the CIP convention is much broader, far more complete, and devoid of ambiguity. The review continues with the case of compounds with two or more stereogenic centers, which result in the emergence of diastereoisomeric relationships. As a result, we will encounter compounds whose structures allow the simultaneous occurrence of enantiomeric and diastereoisomeric relationships. This review will end with a qualitative discussion of the comparative configurational stability of C-centered chiral tetrahedral structures, which, as we will see, range from the extremely stable to the very labile. Helvetica Chimica Acta – Vol. 96 (2013) 161 Fig. 2.2. Tetrahedral chemical structures contain a central atom X at the geometrical center of a tetrahedron, to which are attached four atoms or substituents (A, B, C, and D) each occupying one of the vertices of the tetrahedron. If the four atoms or substituents are different from each other as in 2.1, the generic structure is chiral. Such a tetracoordinate assembly is indeed asymmetric (point group C1) and occurs in two stereoisomeric forms which form an enantiomeric pair. A comparable situation is depicted with the generic structures 2.2. Here, there are only three atoms or substituents attached to X, making it a tricoordinate assembly. However, if the central atom (e.g., Satom, N-atom) possesses an electron lone pair, this will function as a virtual ligand pointing to the fourth vertex of the tetrahedron [17]. With three different atoms or substituents A, B, and C, and provided the chemical nature of X allows sufficient stability of such an arrangement, two enantiomers can indeed be characterized and isolated. Should the four atoms or substituents fail to occupy the vertices of a tetrahedron and be, for example, coplanar with the central atom, a non-dissymmetric structure is obtained [18]. This is illustrated here with the well-known anticancer drug cisplatin (2.3) which shares a diastereoisomeric relationship with transplatin (2.4). Note also that we will not extend the current presentation to more complex structures such as pentacoordinate centers as exemplified with the trigonal-bipyramidal structure of the generic phosphorus(V) compound 2.5. With five different substituents, such a structure can conceivably exist as ten diastereoisomeric pairs of enantiomers [19]! 162 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.3. This Figure illustrates some chiral generic structures having as their central atom a first- or second-row element of columns IVA and VIA in the periodic table of chemical elements. But first, we present an extraordinary chiral molecule, namely (R)[2H1 ,2H2 ,2H3 ]neopentane (2.6). This chemically inert compound is chiral as a result of a dissymmetric mass distribution, the smallest possible one in fact, and its synthesis and absolute-configuration assignment proved to be at the very limit of what was theoretically and experimentally possible at the time [20] [21]. The rest of the Figure instructs us that carbanions with a lone pair, 2.7, racemize readily, whereas carbon radicals, 2.8, and carbonium ions, 2.9, which both lack a virtual fourth ligand, are usually close to planarity and tend to be achiral independently of their substituents [22]. In column IVA, silanes, 2.10, are configurationally stable. As for column VIA, oxonium salts, 2.11, show very rapid inversion. In contrast, sulfonium salts, 2.12, and sulfoxides, 2.13, have rather stable trisubstituted centers [23]. The higher configurational stability of second-row atoms compared to first-row atoms is clear from these generic examples. Helvetica Chimica Acta – Vol. 96 (2013) 163 Fig. 2.4. Moving to column VA of the periodic table, we encounter the elements nitrogen and phosphorus which again demonstrate the difference in configurational stability between first- and second-row atoms. Indeed, fast inversion is the rule for amines, 2.14, [24 – 26], but the molecular environment also plays a role. Fast inversion is also the case when the N-atom is included in a monocyclic system as in 2.15. In contrast, a N-atom incorporated as bridgehead in a bicyclic system will be configurationally frozen. This was established for the first time when Prelog and Wieland succeeded in resolving Trçgers base whose dextrorotatory (S,S)-enantiomer 2.16 is shown here [27] [28]. In contrast to tertiary amines such as 2.14, N-oxides (tertiary amine oxides), 2.17, and quaternary ammonium species, 2.18, are configurationally stable. The same is true for phosphines, 2.19, and for phosphonium salts, 2.20. 164 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.5 – 2.7. A need for consistency in stereochemical designation prompted Emil Fischer to use C(5) of the dextrorotatory enantiomer of glucose (2.21) as a starting point. First, the molecule was drawn based on the following conventions: a) the longest C-chain is vertical; b) the most highly oxidized end of the chain is at the top; c) at each center along the main chain, the vertical bonds point backwards and the horizontal bonds point toward the observer; and d) the central tetrahedral C-atom(s) can be omitted, an option not followed here for better clarity. (þ)-Glucose was degraded by Fischer to the aldotriose (þ)-glyceraldehyde in which the only stereogenic center originates from C(5) of the parent molecule. Arbitrarily, Fischer assigned the depicted configuration to (þ)-glyceraldehyde, which became (þ)-d-glyceraldehyde (2.22) due to the right-hand-side position of the substituent at the stereogenic center (and not due to its optical rotation as still believed by some!). As a result, ( )-l-glyceraldehyde was assigned the configuration shown in 2.23. All molecules that could be chemically related to d-glyceraldehyde were assigned the d-configuration, as illustrated in Figs. 2.6 and 2.7, while molecules related to ()-l-glyceraldehyde (2.23) were assigned to the l-series. This assignment is based on chemical transformations that do not break any bond to the highest-numbered stereogenic C-atom [5 – 15] [29]. Thus, the aldoses related to d-glyceraldehyde include the two aldotetroses d-erythrose (2.28) and dthreose (2.29), the four aldopentoses d-ribose (2.30), d-arabinose (2.31), d-xylose (2.32), and d-lyxose (2.33), and the eight aldohexoses d-allose (2.34), d-altrose (2.35), d-glucose (2.21), d-mannose (2.36), d-gulose (2.37), d-idose (2.38), d-galactose (2.39), and d-talose (2.40) [30] [31]. Helvetica Chimica Acta – Vol. 96 (2013) 165 Whereas this convention proved useful for carbohydrates, conflicting results arose with other chemical classes. Thus, (þ)-d-glyceraldehyde (2.22) can be chemically related to ()-lactic acid (2.24) via carbonyl reduction, followed by alcohol oxidation, and to (þ)lactic acid (2.25) via oxidation of the carbonyl, followed by bromination of the alcohol and finally reductive debromination. To partly overcome such difficulties, the projection convention came into use. This convention distinguishes itself from the convention discussed above in that no reference is made to the chemical affiliation of the compound under examination. The latter is simply drawn in the Fischer projection and is designated as d or l depending on the right-hand or left-hand position, respectively, of the substituent at the highest-numbered stereogenic C-atom. The projection convention is thus restricted to those molecules that can be unambiguously drawn in the Fischer projection, and which can also obey all relevant rules. Further difficulties arose for distinct chemical classes such as phenylethylamines which are conventionally drawn upside down. Another problem arose with the amino acids, the natural configuration of which is l as shown with the generic structure 2.26 when R=H (R ¼ H is glycine, the only non-chiral natural amino acid). The majority of natural amino acids contain only one stereogenic center and are easily designated according to the carbohydrate rules, for example, l-serine (2.26, R ¼ CH2OH). However, the case of threonine (2.27) is inconsistent, because its highest-numbered stereogenic C-atom center has the d-configuration, and designating it as d-threonine created a discrepancy with serine. The compound is thus designated as l s-threonine (2.27) where the subscript s indicates the serine series; the subscript g is then used for the glyceraldehyde series. The most important point to be made here is that the d,l-convention as originally conceived and proposed by Fischer described relative configurations. In other words, there was no way to decide if all stereochemical representations reflected reality or were objects in a mirror-universe. It was not until 1951 that publications based on the Xray-analysis of sodium rubidium (þ)-tartrate tetrahydrate afforded an experimental basis for absolute configurations and showed Fischers gamble to have hit the correct answer. A further problem exists, especially in the medical literature where non-chemist authors use the low-case letters d and l to indicate the sense of optical rotation (dextro- and levorotatory, resp.), thereby creating a confusion with the d,l-convention. No serious publication should tolerate such uninformed practice. As is clear from the above, the rapid experimental and conceptual progress in stereochemistry in the first half of the 20th Century made it urgent to rely on an (ideally) universal and unambiguous description of the absolute configuration at stereogenic centers and other stereogenic elements. 166 Fig. 2.6. Fig. 2.7. Helvetica Chimica Acta – Vol. 96 (2013) Helvetica Chimica Acta – Vol. 96 (2013) 167 Fig. 2.8. The foundation of a new convention was laid in 1951 by Cahn and Ingold, and expanded and clarified by Cahn, Ingold, and Prelog [5 – 15] [32 – 34]. The convention is often referred to as the CIP System and consists in two parts, namely a) the sequence rule, which defines the ranking of the four vertices of a tetrahedron (namely the substituents A > B > C > D) according to a set of arbitrary but consistent subrules, and b) a rule which specifies that rotation is from A to B to C, when these point toward the viewer while D points away. The two possible arrangements are illustrated in the upper part of the Figure, where a clockwise course is shown to translate into the (R)configuration (2.41; Latin rectus, right), and a counterclockwise (anticlockwise) course into the (S)-configuration (2.42; Latin sinister, left). The comparison with a spinning wheel can help explain the circular path from A to B to C. The sequence rule begins by considering the four atoms linked to the stereogenic center. These are ranked in an order of preference which decreases with decreasing atomic number; isotopes are ranked according to decreasing mass number, while a lone electron pair is considered a phantom atom with atomic number zero. The two structures at the bottom of the Figure provide partial applications. In particular, we can now understand how the configuration of the deuterated neopentane 2.6 in Fig. 2.3 was defined as (R). The case of (S)-ethyl methyl sulfoxide (2.43) involves an additional subrule to explain why ethyl > methyl. 168 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.9. The example of (R)-2-bromo-2-chloro-3-methylbutan-1-ol (2.44) is used to answer the above question. Indeed, the case of Br and Cl is clear, but what about the two other adjacent atoms, both C-atoms? Here, we are instructed to consider the onceremoved atoms; on the left, we find H, C, and C, and on the right H, O, and H. Each triplet is arranged according to atomic number (A > B > C), yielding (C C > H) and (O > H H), respectively. Comparing A with A’, B with B’, and C with C’, and stopping at the first difference yields (O,H,H) > (C,C,H), as shown. The case of compound 2.45 is more complex, since the once-removed atoms C(C,C,H) and C(C,C,H) show no difference. In such a case, exploration is continued further. The two subbranches of the left branch are arranged in the order C(Cl,H,H) and C(H,H,H), while, for the right branch, we find C(O,C,C) and C(O,H,H). Comparing the senior subbranches and stopping at the first difference yields C(Cl,H,H) > C(O,C,C), and the junior subbranches need not be compared. In summary, the left branch has preference over the right branch, and the structure has the (S)-configuration. Helvetica Chimica Acta – Vol. 96 (2013) 169 Fig. 2.10. To avoid discussions on the nature of bonds, Sequence Rule III splits double and triple bonds into two and three single bonds, respectively. This is done by duplicating or triplicating all doubly or triply bonded atoms, respectively, but not other groups or atoms attached to them. The duplicated or triplicated atoms are drawn in brackets and are considered as carrying phantom atoms of atomic number zero. This is illustrated here for generic groups, namely a carbonyl (2.46), a cyano (2.47), and an ethenyl group (2.48). d-Glyceraldehyde (2.22) is used as a specific example; its CHO group is treated as C(O,(O),H) and is thus preferred to the CH2OH group treated as C(O,H,H); hence, the absolute configuration of d-glyceraldehyde is (R) [13]. 170 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.11. Aromatic rings deserve a special explanation. They are treated as Kekulé structures (i.e., with alternating single and double bonds). For aryl groups such as phenyl (2.49) or naphthyl, it does not matter which resonance form is used, because splitting the double bonds gives the same results in all cases. For aromatic heterocycles, however, each resonance form is used in turn, as shown here for the two forms of pyridin-2-yl (2.50) where the atomic numbers (6 or 7) are written instead of the actual atoms (C or N). Each duplicate atom is then given an atomic number that is the mean of what it would have if the double bond were located at each of the possible positions. Thus, we see that in two duplications the mean is 6.5 [13]. Helvetica Chimica Acta – Vol. 96 (2013) 171 Fig. 2.12. Compounds with two or more stereogenic centers (n 2) and an unsymmetrical constitution exist in more than two stereoisomeric forms. In Part 1, we encountered the norephedrines (1.17 in Fig. 1.11) which are natural compounds having two stereogenic centers (n ¼ 2), allowing for the existence of four stereoisomers (i.e., 2n ). Each of these, as explained, has one enantiomer and shares a diastereoisomeric relationship with the two others. As a further example, we look at linalool (2.51), a volatile compound found in many plant tissues, a floral fragrance and a component of perfumery substances. Its two enantiomers are oxidized in plants to a number of linalool oxides, in particular the furanoid linalool oxide (2.52). The biochemical reaction generates a second stereogenic center in the metabolites and yields the four stereoisomers shown, namely a pair of trans-configured enantiomers, and a pair of cisconfigured enantiomers [35]. In Fig. 2.7, we saw the eight aldohexose stereoisomers derived from d-glyceraldehyde where n ¼ 4. It was implicit that l-glyceraldehyde would lead to eight other stereoisomers, making it a total of 16 stereoisomers (i.e., 24 ). Aldohexoses in their open form exist as eight pairs of enantiomers. This implies that each of the 16 stereoisomers has one enantiomer and shares a diastereoisomeric relationship with the 14 others. An important subgroup of diastereoisomers called epimers are those that differ in the configuration of only one stereogenic center. Thus, d-allose (2.34 in Fig. 2.7) has four epimers, namely d-altrose (2.35), d-glucose (2.21), d-gulose (2.37), and l-talose. 172 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.13. A further example of constitutionally unsymmetrical molecules is provided here: menthol (n ¼ 3) and its eight stereoisomers (23 ) grouped in four pairs of enantiomers (2(3–1)). In agreement with the above examples, each stereoisomer has one enantiomer (opposite configuration at all stereogenic centers) and six diastereoisomers (23 – 2), of which three (n) are epimers. Note that, for historical reasons, each pair of enantiomers bears a distinct yet related chemical name. Thus, we have (þ)- and ( )menthol (2.53), (þ)- and ()-isomenthol (2.54), (þ)- and ( )-neomenthol (2.55), and (þ)- and ( )-neoisomenthol (2.56), with their absolute configurations shown and described according to the (R,S)-convention [31] [36]. Helvetica Chimica Acta – Vol. 96 (2013) 173 Fig. 2.14. Acyclic molecules having n stereogenic centers are called constitutionally symmetrical when those centers equidistant from the geometrical center of the molecule are identically substituted. Such molecules have (2(n–1) þ 2(n–2)/2 ) stereoisomers, when n is even, and 2(n–1) when n is odd. Tartaric acid (2.57) is a classical example for n even. Its two stereogenic C-centers are identically substituted. The dextrorotatory form has the (2R,3R)-configuration, and the levorotatory form is (2S,3S)-configured [31]. The expected pair of (2R,3S)- and (2S,3R)-enantiomers, however, does not exist since the molecule is bisected by a plane of symmetry (s) and is thus achiral overall. This achiral (and hence optically inactive) stereoisomer is termed the meso-form, and it shares a diastereoisomeric relationship with the other two stereoisomers. In accordance with the above rule, tartaric acid exists as 2 þ 1 stereoisomers. Similarly, a constitutionally symmetrical molecule with n ¼ 4 exists as ten stereoisomers, namely two meso-forms and four pairs of enantiomers. For clarity, let us mention that the compound called racemic tartaric acid is not itself a stereoisomer but a 50 : 50 mixture of the two enantiomers. This is indeed the definition of a racemate. The issue is considered further in Fig. 2.16. As shown in the lower part of the Figure, some cyclic molecules have structural properties similar to those of acyclic molecules, as exemplified with 1,2-dihydroxycyclohexane (2.58). A more systematic stereochemical treatment of cyclic molecules will be presented in Part 4. 174 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.15. When n is odd as in trihydroxyglutaric acid (2.59), the 2(n–1) rule predicts four stereoisomers. The two stereoisomers (S,S) and (R,R) differ in their absolute configurations at C(2) and C(4), but their central C(3)-atom carries two identical glycolyl substituents and is thus of the type X(A,B,B,C), namely achiral and more exactly prochiral (see Part 8). In other words, the (S,S)- and (R,R)-stereoisomers have only two stereogenic centers, and since, they have opposite configurations at both centers, they are enantiomers. A specific contradiction occurs with the two stereoisomers having opposite configuration at C(2) and C(4). Now C(3) carries four different substituents, namely H, OH, (R)-glycolyl, and (S)-glycolyl (two enantiomorphic substituents; see Part 1); but the molecule is also achiral, because it has a plane of symmetry (s) perpendicular to its main axis and cutting C(3). Due to its two enantiomorphic substituents, C(3) may have either of two different configurations, allowing two physically distinct, but optically inactive stereoisomers both called mesoforms. Stated differently, we now face the situation of a C-atom having four different substituents but lying in a plane of symmetry of the molecule. Such a C-atom is called a pseudostereogenic center (also called as pseudoasymmetric center) [11 – 13] [37 – 40]. A subrule of the Sequence Rule states that (R) > (S) and allows pseudostereogenic centers to be treated like stereogenic centers. But because the molecule is achiral, these centers are given the lower-case symbols (r) and (s). A more general representation of a pseudostereogenic center is given at the bottom of the Figure, where the two mesoforms are depicted. Helvetica Chimica Acta – Vol. 96 (2013) 175 Fig. 2.16. With molecules having several stereogenic centers, the chemist may face the problem of identifying one pure enantiomer of known relative configuration but unknown absolute configuration. The Sequence Rule prescribes to use the stereodescriptors (R*) and (S*) (R-star and S-star) to describe relative configurations [13] [41]. In the example of 1-bromo-3-chlorocyclohexane (2.60), the chemist may know that the two stereogenic centers have opposite absolute configurations but ignore which is (R) and which is (S). The subrule states that the stereogenic center with the lowest locant, i.e., C(1) here, is arbitrarily assigned (R*), while the other center(s) become(s) (R*) or (S*) depending on its/their configuration relative to that at C(1). A more complex example is provided [13] by 1-bromo-3-chloro-5-nitrocyclohexane (2.61) whose relative configuration is (1R*,3R*,5R*), a cumbersome formulation when many stereogenic centers exist, as, e.g., in steroids. In this case and as shown, it is acceptable to use the simplified notation of rel-(1R,3R,5R). The (R*,S*)-convention has been sometimes used to indicate the relative configuration of racemates. This is an infelicitous and rejected practice, since the IUPAC rules [13] recommend to use (RS) for racemates containing a single stereogenic center. When there is more than one center, that with the lowest locant is arbitrarily labeled (RS), and the others (RS) or (SR), depending whether they are (R)- or (S)configured, when the first is considered to have the (R)-configuration. Coming back to tartaric acid (2.57) represented here differently from Fig. 2.14, we see that for its racemate, the prefixes (2RS,3RS) or more simply as () are used. The prefix rac has also been proposed [42]. 176 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.17. While asymmetrically substituted C-atoms are considered as configurationally stable, this may not always be the case, depending on the functional groups on and close to the C-atom. Similar arguments apply to asymmetric arrangements around other atoms (see, e.g., Figs. 2.3 and 2.4) [43 – 48]. First, we reflect on the configurational instability of compounds containing a single stereogenic center, i.e., enantiomers. The term of racemization describes the macroscopic and statistical process by which one optically active compound (be it enantiomerically pure or impure) is irreversibly transformed to the racemic mixture, which by definition, contains equal amounts of the two enantiomers. When starting with a pure optically active compound, this equilibrium state is reached when one half of the molecules have changed configuration. The rate constant of the macroscopic reaction is known as krac , and it is identical for (R) ! rac and for (S) ! rac. The process of enantiomerization describes the process occurring at the level of individual molecules and is defined as the reversible conversion of one enantiomer into the other. This configurational inversion, be it from (R) to (S), or from (S) to (R), occurs by passing through a transition state or an intermediate product. The corresponding rate constant, kenant , is again the same in both directions. Interestingly, the rate constants and kinetic activation parameters (e.g., activation energy, free energy barrier) are experimentally accessible, most notably by dynamic chromatographic techniques [49 – 51]. Since the conversion of one molecule reduces the enantiomer excess by two molecules, it is clear that the rate of enantiomerization is by definition half that of racemization. Helvetica Chimica Acta – Vol. 96 (2013) 177 Fig. 2.18. Racemization involving stereogenic C-centers is illustrated here with the examples of the anorectic drug amfepramone (2.62; R ¼ Et) and its N,N-didemethylated metabolite 2.63 (R ¼ H) whose (S)-enantiomer is an amphetamine-like stimulant found in khat (Catha edulis) and known as cathinone [52] [53]. Their rate of racemization in aqueous solutions were found to be highly pH-dependent and to increase ca. 1000-fold between pH 2.3 and 7.5, pointing to a base-catalyzed reaction with Hþ abstraction. What is more, the rate increased at constant pH and ionic strength, when the concentration of the phosphate buffer was increased. A few results at physiological pH and 378 are presented here as half-lifes (t1/2 ) of racemization; these were in the order of one to a few hours, demonstrating that the reactions were rather fast and could have pharmacological implications. Taken globally, the results indicated a general base-catalyzed SE1 mechanism as shown in the Figure [54]. 178 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.19. Here, we present two extreme examples of racemization, one quite slow but not slow enough to avoid a stability and activity problem, and the other so fast that the separation and pharmacological testing of the separate enantiomers would be pointless and impossible, respectively. Atropine (2.65) is a racemic mixture of the active alkaloid ( )-(S)-hyoscyamine ((S)-2.64) and its weakly active enantiomer (R)-hyoscyamine ((R)-2.64). What is found in plants is not atropine but an unequal mixture of (S)- and (R)-hyoscyamine whose proportions range from 100 : 0 (in young plants) to 51 : 49 [55]. Yet what is used in medicine is the configurationally stable atropine, since (S)-hyoscyamine solutions undergo a slow racemization with progressive halving in therapeutic activity [45]. Atropine solutions are used extensively in ophthalmology, for example, but much of it is stored as autoinjectors for troop protection against poisoning by organophosphate nerve gases. Our example of a very fast racemization is provided by the tranquillizer and hypnotic oxazepam (2.66) [56 – 59]. The drug contains an stereogenic center at C(3) which undergoes rapid inversion via ringchain tautomerism, namely the reversible cleavage of the NC(OH) bond. The kinetics of racemization of oxazepam is interesting and relevant from a pharmacological viewpoint, since it racemizes at ambient temperature and in the neutral pH range with a pseudo-first-order rate constant of 0.1 0.05 min1, suggesting a half-life (t1/2 ) of racemization of 1 – 4 min at 378. This rate of racemization is extremely fast compared to the duration of action of the drug, indicating that oxazepam is correctly viewed as a single compound existing in two very rapidly interconverting configurations. Helvetica Chimica Acta – Vol. 96 (2013) 179 Fig. 2.20. Its sad history made thalidomide (2.67) one of the most infamous chiral drugs, to become synonymous with tragedy following the discovery of its catastrophic teratogenic effects in the early 1960s [48] [60] [61]. Although the mechanisms of action of thalidomide are poorly understood, some of its activities may be related to its capacity to inhibit the production of Tumor Necrosis Factor a (TNF-a). Considering the fact that enantiomers can have very different pharmacological activities (see Part 5), the question arises whether the teratogenic activity of thalidomide (a racemate) was associated with either one or both of its enantiomers. Unfortunately, findings in the literature are confusing. The problem is complicated by the fact that the enantiomers of thalidomide are subject to rapid racemization, as discussed below [47] [62 – 65]. Using a stereoselective HPLC assay, the racemization of both (R)- and (S)thalidomide was found to be pH-dependent, being practically nil at acidic pH. As with amfepramone and cathinone (Fig. 2.18), the reaction was linearly dependent on phosphate concentration, indicating a general base catalysis. Simultaneously with its racemization, thalidomide was hydrolyzed to ring-opened products [66]. At pH 7.4 in 0.1m phosphate buffer at 378, the half-lives of racemization and hydrolysis were ca. 3 and 2 h, respectively. Human serum albumin, presumably acting as a base, also catalyzed racemization. Because the reaction was markedly medium-dependent, care is required when drawing conclusions on in vivo racemization from in vitro studies. Twelve products of (non-enzymatic) hydrolysis have been reported in human urine, only three of them retaining the intact phthalimido moiety, namely 2-phthalimidoglutaramic acid (2.68), 4-phthalimidoglutaramic acid (2.69), and 2-phthalimidoglutaric 180 Helvetica Chimica Acta – Vol. 96 (2013) acid (2.70). These metabolites also showed teratogenic activity [67]. An investigation of the teratogenic potency of the enantiomers of 2-phthalimidoglutaric acid (2.70) in pregnant mice showed that the (S)-enantiomer caused dose-dependent teratogenicity, whereas the (R)-enantiomer was devoid of this effect, even at four-times higher doses [68]. The teratogenic activity of the ring-opened products of hydrolysis, 2.68, 2.69, and 2.70, called for an investigation of their configurational stability. This question was addressed by an indirect method which revealed a complete configurational stability over a period of 1 week at neutral pH and 378 [62]. This finding is in agreement with the fact that a carboxylic group is known to stabilize stereogenically substituted C-atoms of the type R’’R’RCH, as outlined in Fig. 2.22. From a toxicological viewpoint, it indicates that the teratogenicity testing of the separated enantiomers of 2-phthalimidoglutaric acid (2.70) in pregnant mice produced reliable results. Further, the configurational stability of the three teratogenic metabolites of thalidomide invites reflection on a possible enantioselectivity in the teratogenicity of thalidomide. Given the configurational stability of the three teratogenic metabolites, inversion of the configuration at the stereogenic center must stop with the hydrolysis of thalidomide. Thus, after administration of (S)-thalidomide the concentration of teratogenic metabolites with (S)-configuration is postulated to be higher than after application of (R)-thalidomide. Assuming that the teratogenic potency of the metabolites with (S)configuration is markedly greater than that of the metabolites with (R)-configuration, as shown with 2-phthalimidoglutaric acid (2.70), it is conceivable that (R)-thalidomide might be somewhat less teratogenic than its enantiomer. Helvetica Chimica Acta – Vol. 96 (2013) 181 Fig. 2.21. The examples above dealt with the configurational stability of compounds having one stereogenic center and undergoing racemization. Compounds having two or more stereogenic centers can also undergo interconversion in a process called epimerization if and when one of these centers is configurationally labile [47] [48]. As shown in the Figure, the term epimerization defines the reversible interconversion of one stereoisomer into another, as does the term enantiomerization. This type of reaction is documented for a number of drugs and is thus of relevance in pharmaceutical research. Since two epimers, being diastereoisomers, obligatorily differ in their internal energy (see Part 1), it follows that, when the interconversion of two epimers is left to proceed to equilibrium, an exact 50 : 50 ratio cannot be reached. As a result, the rate constant of the conversion of epimer A to epimer B must be different from that of the reverse reaction, the magnitude of the difference depending on the thermodynamic profile of the two reactions. The two rate constants of epimerization are designated here as kepim[(R,R)!(R,S)] and kepim[(R,S)!(R,R)] , and they are perforce different. Note that some diastereoisomers other than epimers are also known to undergo interconversion, in which cases the process is called diastereoisomerization. Here, we take the alkaloid drug pilocarpine (2.71), used in ophthalmology to dilate the pupil, as a pharmaceutically relevant example. Its absolute configuration is (2S,3R)cis and it epimerizes to (2R,3R)-trans-isopilocarpine (2.72) by inversion of the configuration at C(2). Both compounds are dextrorotatory, and only pilocarpine is pharmacologically active. In addition, pilocarpine undergoes reversible hydrolytic 182 Helvetica Chimica Acta – Vol. 96 (2013) lactone ring opening to pilocarpic acid (2.73), whereas isopilocarpic acid (2.74) is produced reversibly from isopilocarpine. Detailed mechanistic and kinetic studies have revealed HO-ion-catalyzed epimerization and hydrolytic ring opening [69] [70]. From the data, one can estimate a pseudo-first-order rate constant of pilocarpine disappearance (epimerization to isopilocarpine plus hydrolysis to pilocarpic acid) corresponding to a half-life (t1/2 ) of ca. 36 days at pH 7.4 and 358. Of mechanistic importance is the fact that neither pilocarpic acid nor isopilocarpic acid can epimerize, due to the presence of the free COO group which acts as a strong configurational stabilizer. The percent of pilocarpine at or near equilibrium having undergone epimerization to isopilocarpine was found to be ca. 20% at room temperature. Activation energies of ca. 120 and 105 kJ/mol were calculated for the reactions of pilocarpine epimerization and hydrolysis, respectively, indicating a higher energy barrier for epimerization than for hydrolysis. The trend for an increasing proportion of isopilocarpine (2.72) being formed relative to pilocarpic acid (2.73) as temperature increases is verified among others by the respective half-lives of epimerization and hydrolysis of pilocarpine at pH 5.7, which were 630 and 80 days (ratio 7.9) at 408 compared to 8 and 2.4 days (ratio 3.3) at 808 [70]. This trend was also confirmed by the finding that, during sterilization, epimerization predominated over hydrolysis, whereas storage at room temperature favored hydrolysis [71]. From a practical and pharmaceutical viewpoint, the above data imply that both reactions can contribute to a decreased activity of pilocarpine solutions following sterilization and inadequate storage. As for the back-epimerization of isopilocarpine (2.72), it was detectable but too slow to be measurable with good precision, ca. 1% of pilocarpine being formed from isopilocarpine at pH 5.9 and 608 [70]. This was due to trans-configured isopilocarpine being intrinsically of lower internal energy than cis-configurated pilocarpine (see Part 4). Helvetica Chimica Acta – Vol. 96 (2013) 183 Fig. 2.22. The above examples and others in the literature allow a preliminary generalization of the structural factors causing configurational instability at a stereogenically substituted C-atom of the type R’’’R’’R’CH. Configurational inversion at such centers is catalyzed by the HO anion (specific base catalysis) and in several cases also by other bases (general base catalysis), and it involves the deprotonated form (i.e., the carbanion R’’’R’’R’C ) as intermediate. It thus appears convenient to distinguish between acid-strengthening, neutral, and acid-weakening substituents [45] [72 – 74]. Substituents in the former group usually act by stabilizing the carbanion, but stereoelectronic effects on the substrate or transition state should not be neglected. Two types of acid-strengthening groups are frequently involved in labilizing a stereogenically substituted C-atom by favoring its deprotonation, namely a carbonyl function and an aryl group, often potentiating each other as seen with amfepramone, cathinone, and hyoscyamine (Figs. 2.18 and 2.19). An amino or amido group may also be found, as seen in Figs. 2.18 – 2.20. To be of relevance (see next Fig.), configurational instability appears to require the presence of at least one strong carbanion-stabilizing substituent at the stereogenic center, and the absence of any acid-weakening group. A preliminary list of such groups based on available evidence is presented here in tabular form [45] [48]; question marks indicate the absence of enough examples. Much additional quantitative work would be needed to confirm some of the above results, to complete the table and arrive at a ranking of the listed groups, to offer rules for quantitative prediction, and, from a mechanistic viewpoint, to assess the relative importance of mesomeric and inductive contributions. 184 Helvetica Chimica Acta – Vol. 96 (2013) Fig. 2.23. An important aspect to be taken into account when considering the configurational lability of stereoisomers is its significance in drug research and development. First, one should always bear in mind that configurational stability and lability are relative phenomena. Under the appropriate conditions of temperature, pH etc., no stereoisomer is configurationally stable. However, only two time scales (and their related sets of conditions) are of relevance as far as drugs are concerned [47] [48]. As schematized in the Figure, the pharmacological time scale applies to the time of residence of a drug in the body and under physiological conditions (378, pH 7.4). A half-life of isomerization of several months is no longer of importance in pharmacology and therapy, while very fast rates of isomerization (in the order of minutes, e.g., oxazepam) are of interest only for drugreceptor or drugenzyme interactions. Halflives of isomerization in the order of weeks, months, or a few years are important in a pharmaceutical and formulation perspective, i.e., compared to the duration of the manufacturing process and the shelf-life of drugs. This is the pharmaceutical time scale, which applies to the manufacturing process and to the shelf-life of medicines, as illustrated above with hyoscyamine and pilocarpine. The author is indebted to his former colleague Dr. Antoine Daina, University of Geneva, for help with the bibliography. Helvetica Chimica Acta – Vol. 96 (2013) 185 REFERENCES [1] J. P. 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