LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-1
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-2
`IPR2016-00379
`
`

`
`First published in 1988 by
`ELLIS HORWOOD LIMITED
`Market Cross House , Cooper Street,
`Chichester, West Sussex, P019 lEB, England
`The publisher's colophon is reproduced from James Gillison's drawing of the ancient Market Cross,
`Chichester.
`
`Distributors:
`Australia and New Zealand:
`JACARANDA WILEY LIMITED
`GPO Box 859, Brisbane , Queensland 4001, Australia
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`JOHN WILEY & SONS CANADA LIMITED
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`JOHN WILEY & SONS LIMITED
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`Halsted Press: a division of
`JOHN WILEY & SONS
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`JOHN WILEY & SONS (SEA) PTE LIMITED
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`WILEY EASTERN LIMITED
`4835/24 Ansari Road
`Daryaganj, New Delhi 110002, India
`© 1988 S. G. Allenmark/Ellis Horwood Limited
`
`British Library Cataloguing In Publication Data
`Allenmark, S. G . (Stig G.) , 1936-
`Chromatographic enamtioseparation.
`l . Chromatography
`I. Title
`543'.089
`Library of Congress Card No. 88-1092
`ISBN 0-84312-988-6 (Ellis Horwood Limited)
`ISBN 0-470-21080-X (Halsted Press)
`Typeset in Times by Ellis Horwood Limited
`Printed in Great Britain by Hartnolls, Bodmin
`
`COPYRIGHT NOTICE
`All Rights Reserved. No part of this publication may be reproduced , stored in a retrieval system, or
`transmitted , in any form or by any means, electronic, mechanical, photocopying, recording or otherwise,
`without the permission of Elhs Horwood Limited, Market Cross House, Cooper Street, Chichester, West
`Sussex, England.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-3
`IPR2016-00379
`
`

`
`Table of contents
`
`Preface . ... . .......... . ........ . ............... . ....... 9
`
`List of Symbols and Abbreviations ................ . ... . ......... 11
`
`1 Introduction ..... .. .... .. .. .. . ....................... 13
`Bibliography .................... . ............. . ..... . 18
`References ..... . ....................... ... .... . . .. .. 18
`
`2 The development of modern stereochemical concepts
`2.1 Chirality and molecular structure ......................... 19
`2.1.1 Molecules with asymmetric atoms ...................... 19
`2.1.2 Other types of chiral molecular structures ..... . ........... 20
`2.2 Definitions and nomenclature .......... . ................ 23
`Bibliography ........................... . ...... . .. . . .. 26
`References . ....... . ................ .... .......... .. . 26
`
`3 Techniques used for studies of optically active compounds
`3.1 Determination of optical or enantiomeric purity ............... 27
`3.1. l Methods not involving separation ... .. ................. 27
`3.1. l.1 Polarimetry ................................. 27
`3.1.1.2 Nuclear magnetic resonance .. .. . .. .. . ...... . ..... . 29
`3.1.l.3 Isotope dilutio n .................. . ..... .. ..... 31
`3.1.l.4 Calorimetry .... . ... .. . .. ... . ................ 33
`3.1.1.5 Enzyme techniques ................... . ......... 33
`3.1. 2 Methods based on separation ......................... 34
`3.2 Determination of absolute configuration .................... 35
`3.2.l X-Ray crystallography with anomalous scattering ...... . .. ... 36
`3.2.2 Spectroscopic (ORD , CD) and chromatographic methods
`based on comparison ......... .. ..... .. ............ 37
`Bibliography ................... .. .. ...... .... ...... . .40
`References .. . . ... ............... . ................... 40
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-4
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-5
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-6
`IPR2016-00379
`
`

`
`8
`
`Table of contents
`
`Bibliography .................. . • ........ .. ..... . .. . .. 188
`References .. .... ... .. . .. .... . . ... ... .. .. .. . . .. .. .. . 188
`
`9 Preparative scale enantioseparations- need, progress and problems . . .. 192
`Bibliography . . .... . ..... . ............ . ... .. . .... .... 199
`References ... .. . . . . ... .. . ... . .... .. .... . .. . . .. ..... 199
`
`10 Future trends
`10.l New detector systems . .. .. .. .... . .. .. .... . .... . ... . .. 200
`10.2 Column improvements . . . . ... . .... . ... .. .... . ... . .... 203
`10.3 Supercritical fluid chromatography . . . . .... . . .. .. . ........ 206
`Bibliography .. . .... ... . .. .. ... ... .. ....... .. . . . ..... 206
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
`
`11 ~xperimental procedures for the synthesis of chiral sorbents
`11.1 Techniques for the preparation of chiral sorbents by
`derivatization of polysaccharides . .. .... ... .. .. . .. .. . . .. . 208
`11.1.1 Preparation of microcrystalline cellulose triacetate (MCTA) .... 208
`11.1 .2 Preparation of silica coated with cellulose triacetate .. .. . . .. . 209
`11.1.3 Preparation of silica coated with cellulose triphenylcarbamate ... 209
`11.2 Polymerization procedures used to obtain chiral synthetic
`polymer materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
`11.2.1 Preparation of poly[(S)-N-acryloylphenylalanineethyl ester] ... . 209
`11.2.2 Preparation of polycellulose(triphenylmethyl methacrylate) ... . 211
`11.3 Techniques used for the binding of chiral selectors to silica .. .. . ... 211
`11 .3.1 Preparation of 3-glycidoxypropyl-silica ... . ... ........ . . . 212
`11 .3.2 Large-scale preparation of (R)-N-(3,5-dinitrobenzoyl)-
`phenylglycine covalently silica-bound sorbent . .. .. .. . .. . .. 212
`11 .3.3 Preparation of (S)-(- )-~-N-(2-naphthyl)leucine .. .. .. ...... 213
`11 .3.4 Hydrosilylation of (R)-N-(10-undecenoyl)-~-(6,7-dimethyl-
`1-naphthyl) isobutylamine . ... ... . .. . . . . . . .. ... ... . . 213
`11.3.5 Preparation of silica-bonded (S) -1-(~-napththyl)ethylamine .... 213
`11.3.6 Preparation of silica-bound polyacrylamide and
`polymethacrylamide . . . .. . .... . ...... . ......... . . . 214
`Bibliography .... .. . ... ... .. .. . .. . . .. .. .. . .. .. . ... .. . 214
`References .... . ... . ... . ... ... ... . .. . ... . . .. ... . . ... 214
`
`Appendix: Commercial suppliers of chiral columns for GC and LC . . ....... 216
`
`Index . .. .. ....... .. .... . .... .. ...... .. . . ...... .. .. . . . 218
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-7
`IPR2016-00379
`
`

`
`5
`Theory of chiral chromatography for direct
`optical resolution
`
`In the previous chapter it was noted that separation of enantiomers, by the formation
`of diastereomeric derivatives, is associated with a number of disadvantages which
`make its use relatively unattractive. Therefore , direct separations, i.e. by the use of a
`chiral stationary phase, are far more interesting from an analytical , as well as a
`preparative point of view. Particularly in LC, a technique that is very suitable for
`separation without prior derivatization, development of new chiral stationary phases
`has been a rapidly expanding field during the last few years. It is the purpose of this
`chapter to give a general treatment of the theories underlying the various efforts and
`achievements made in this area.
`
`5.1 THE PREREQUISITE FOR ENANTIOSELECTIVE INTERACTION WITH
`THE CHIRAL STATIONARY PHASE
`In all forms of chromatography, separation is based on differences in retention of
`some kind by the stationary phase. We have already seen how the chromatographic
`process can be regarded as a series of equilibria and how the equilibrium constant
`describing the distribution of a compound between the stationary and mobile phases
`is related to the chromatographic capacity ratio . It has been customary to distinguish
`between partition and adsorption chromatography, depending upon whether the
`stationary phase is a liquid or a solid. With the introduction of bonded organic phases
`in LC and immobilized coatings in fused silica capillary GC, this distinction is no
`longer obvious. Nevertheless the consideration of primary importance should be the
`types of molecular interactions with the stationary phase that cause retention.
`It should be helpful for later discussions , however, to consider the fact that there
`are cases in which retention is not due to bonding interactions with a stationary phase
`but rather to differences in the distance the components under separation have to
`travel through the column. In principle, molecular sieve chromatography, of
`particular importance for protein separation, operates essentially by means of steric
`exclusion, i.e. the larger molecules travel a shorter distance because they are unable
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-8
`IPR2016-00379
`
`

`
`Sec. 5.1)
`
`The prerequisite for enantioselective interaction
`
`65
`
`to diffuse into the finer pores of the matrix and are therefore eluted faster than the
`smaller molecules.
`The theory of chiral chromatography (i.e. separation in which a chiral stationary
`phase selectively retains more of one enantiomer than the other) is still rather
`rudimentary. A number of chiral recognition models have been proposed to account
`for optical resolutions by GC and LC, which are often based on the 'three-point
`interaction' theory advanced by Dalgliesh [1] in 1952. According to this postulate,
`three simultaneously operating interactions between an enantiomer and the station(cid:173)
`ary phase are needed for chiral discrimination. The enantioselective situation is
`visualized in Fig. 5.1. It is obvious that this is a sufficient condition for enantioselec-
`
`chiral
`selector
`
`solute
`enantiomers
`
`Fig. 5.1-The three-point interaction model advanced by Dalgliesh.
`
`tion to occur. Now it may be asked whether it is always necessary. As we shall see
`later, there are many instances where this is probably not the case.
`Dalgliesh arrived at his conclusions from studies of certain aromatic amino-acids
`by paper chromatography. He assumed that the hydroxyl groups of the cellulose
`were hydrogen-bonded to the amino and carboxyl groups of the amino-acid. A third
`interaction was caused, according to these views, by the aromatic ring substituents.
`The 'three-points rule' has often been used in a very uncritical way to rationalize
`experimental results. It is therefore important to try to analyse the situation in more
`detail. It is readily understood that in order to determine the configuration of a chiral
`object by matching with some probe , a minimum of three simultaneous, spatially
`significant contacts or interactions must be present. This minimum number of
`contact points, however, does not necessarily mean points of attachment when it
`comes to molecular interactions. In principle, a situation where only steric inter(cid:173)
`actions cause a molecular steric discrimination is quite possible. In adsorption
`chromatography, though , there must always exist some kind of bonding interaction
`with the sorbent. This may arise through any non-covalent attachment possible
`under the prevailing conditions. Thus , hydrogen bonding as well as ionic or dipole
`attraction is enhanced by non-polar solvents, whereas hydrophobic interactions may
`be important in aqueous media, etc.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-9
`IPR2016-00379
`
`

`
`66
`
`Theory of chiral chromatography for direct optical resolution
`
`[Ch.5
`
`5.2 SOME GENERAL ASPECTS REGARDING CHIRAL RECOGNITION
`MODELS AND CHROMATOGRAPHIC ENANTIOSELECTIVITY
`Optical resolution by chromatography is possible through reversible diastereomeric
`association between a chiral environment, introduced into a column , and solute
`enantiomers. The multiplicity of experimental conditions under which direct chro(cid:173)
`matographic optical resolutions have been achieved also tells us that the difference in
`association which is necessary can be obtained by means of many types of molecular
`interactions. The association, which may be expressed quantitatively as an equili(cid:173)
`brium constant, will be a function of the magnitudes of the binding as well as of the
`repulsive interactions involved. The latter are usually steric, although dipole-dipole
`repulsions may also occur, whereas various kinds of binding interactions may
`operate. These include hydrogen bonding, electrostatic and dipole-dipole attrac(cid:173)
`tions, charge-transfer interaction and hydrophobic interaction (in aqueous systems).
`As we shall see in the following chapters, a single type of bonding interaction may be
`sufficient to promote enantiomer differentiation . For example , it appears to be quite
`evident that hydrogen bonding, as the sole source of attraction, is sufficient for
`optical resolution in some GC as well as LC modes of separation. The fact that
`enantiomeric solutes, bearing only one hydrogen bonding substituent, can be
`separated under such conditions, points to the conclusion that only one attractive
`force is necessary for chiral discrimination in this type of chromatography.
`Taking a one-point binding interaction as a model, we may envisage a difference
`in the equilibrium constant of the two enantiomers at the chiral binding site as due to
`effects from the site forcing one of the enantiomers to take an unfavourable
`conformation. This resembles a situation often assumed to be present in enzyme(cid:173)
`substrate interactions to account for substrate specificity.
`Let us follow the reasoning a bit further. Is it possible to base enantiomer
`differentiation entirely on steric fit? In other words , can chiral cavities be constructed
`for the preferential inclusion of only one enantiomer? Although no chiral stationary
`phase (CSP) has yet been prepared that is based entirely on steric exclusion from
`chiral cavities (cf. Section 7.1.3), some recent work with the use of 'molecular
`imprinting' techniques is very interesting in this respect [2). The idea is to create rigid
`chiral cavities in a polymer network is such a way that only one of two enantiomers
`will find the environment acceptable. Other types of CSPs, where steric fit is of
`primary importance, include those based on inclusion phenomena, such as cyclodex(cid:173)
`trin and crown-ether phases. These are described in Chapter 7.
`In the following sections some theoretical aspects of important binding types
`present in enantioselective sorption processes are given.
`
`5.2.1 Co-ordination to transition metals
`The transition metals are characterized by having unfilled inner-shell d-orbitals. A
`transition metal complex is formed by ligands which may donate electrons to these
`unfilled orbitals. Such co-ordination complexes possess a very well-defined geo(cid:173)
`metry, such that the ligands can only occupy certain given positions in space. The
`donor ligand atoms in the complex are thus held at strictly fixed distances from the
`metal atom and in defined orientations. This so-called co-ordination sphere is
`therefore densely packed with the ligands and with the solvent molecules. The latter
`also form a second (outer) highly organized sphere. This , in turn, means that the
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-10
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-11
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-12
`IPR2016-00379
`
`

`
`Sec. 5.2)
`
`Some general aspects regarding chiral recognition models
`
`69
`
`Inclusion phenomena
`5.2.3
`The ability of certain compounds to use their particular structures to include suitable
`guest molecules has long been known. Classical examples are the host properties of
`urea and starch. Crystal analyses have shown that urea molecules form complexes
`with a channel-like interior into which unbranched alkanes fit nicely. Such n-alkane
`-urea complexes therefore form spontaneously. Branched alkanes do not fit into
`such interiors and consequently this phenomenon can be used to separate n-alkanes
`from mixtures of isomers. Starch is well known for its inclusion of iodine. The
`cyclodextrins (Schardinger's dextrins) are crystalline degradation products of starch,
`which are obtained through the action of micro-organisms (see Section 7.1.1.1.).
`Whereas the (¥-form , composed of a ring with six glucose units, is of the correct size to
`include iodine or benzene , it is too small to include bromobenzene. The P-form
`(composed of seven units) , on the other hand, is (contrary to the (¥-form) precipi(cid:173)
`tated by bromobenzene as a consequence of inclusion complex formation.
`The strict steric requirements for the formation of such 'host-guest' complexes
`imply that these phenomena should be highly stereoselective. Thus , by the use of a
`chiral host, enantiomeric guest molecules might be separated. These principles are
`utilized fully or partially in some of the liquid chromatographic techniques described
`in Chapter 7. A short general treatment of the inclusion phenomena present, or
`thought to be present , in these enantiomer-differentiating phases is given below.
`Let us consider two different types of host molecules, one with a hydrophilic
`interior and hydrophobic exterior and the other with the opposite polarity configu(cid:173)
`ration (Fig. 5.4).
`
`f:::,, hydrophilic groups
`
`0 hydrophobic groups
`
`includes hyd r op hilic
`guest
`
`(al
`
`includes hydrophobic
`guest
`
`(bl
`
`Fig. 5.4 - Simplified models to represent different types of host-guest inclusion complex
`formation.
`
`The hydrophilic interior in a means that the cavity contains hetero-atoms such as
`oxygen, where lone-pair electrons are able to participate in bonding to electron
`acceptors such as metal or organic cations. The hydrophobic exterior makes the
`host-guest complex as a whole soluble in organic media , a phenomenon which has
`been exploited in so-called phase-transfer catalysis (cf. Section 7 .2.1 ). One type of
`these host compounds is found in the naturally occurring macrocyclic polyethers,
`which are known to bind alkali-metal cations. Synthetic chiral analogues of these
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-13
`IPR2016-00379
`
`

`
`70
`
`Theory of chiral chromatography for direct optical resolution
`
`(Ch.5
`
`compounds, chiral crown ethers, have been found to exert remarkable enantioselec(cid:173)
`tivity towards organic ammonium ions . In these cases the ammonium ion is held
`within the cavity by hydrogen bonds to the ether oxygen atoms. Thus, in this case, the
`structural and steric requirements of the guest are high.
`The hydrophobic interior of b, on the other hand, means that a cavity suitable for
`inclusion of hydrocarbon-rich parts of a molecule is present. No bonds are involved
`in the complex formation , as the inclusion is merely a result of the hydrophobic effect
`and the structural demands are not as pronounced as in the previous case. This type
`of host is found in the cyclodextrins, which will be treated in Sections 7 .1.1.1 and
`7.3.2. Under reversed-phase conditions (aqueous media) the combination of hydro(cid:173)
`phobic interaction, which generates inclusion , with steric effects from substituents
`present in a chiral structure on the cavity entrance, is assumed to be the cause of
`enantioselection.
`A rather special case of inclusion effects is found in chiral matrices composed of
`swollen, microcrystalline cellulose derivatives. The triacetate, prepared by hetero(cid:173)
`geneous acetylation in order to preserve microcrystallinity , has been shown to act
`partially by steric exclusion effects. Thus, of a series of aromatic hydrocarbons (with
`essentially no bonding properties) , benzene is highly retained, mesitylene (2,3,5-
`trimethylbenzene) is much less retained and 1,3 ,5-tri-tert-butylbenzene is totally
`excluded (no retention) . This phenomenon can be explained by considering the
`lamellar arrangement of the polysaccharide chains. These yield a kind of two(cid:173)
`dimensional molecular sieve, permitting inclusion of flat aromatic compounds in
`particular, but excluding sterically more demanding structures. The higher retention
`of benzene (compared to that of toluene) has further led to the suggestion of a
`secondary effect, viz. an action by pockets in the chain structure.
`
`5.3 SOME THERMODYNAMIC AND KINETIC CONSIDERATIONS
`5.3.1 Temperature effects on C¥
`Recalling our treatment in the previous chapter and the definition of C¥ , we may
`readily arrive at Eq. (5.1) by considering the equilibrium of each enantiomer
`between the mobile and the stationary phase. If the equilibrium constants are
`denoted by KR and Ks, respectively, the expression for the change in free energy,
`!lG0 = -RTinK, will give the free energy difference: llllG0 = !lG0 R- llG0 s, as
`- !l!lG0 = R11nKRIKs , where K = C/Cm and KR > Ks (arbitrary assumption). From
`our definition of k' we know that k' = KV5 /V m which then gives Eq. (5.1):
`
`-!l!lG0 = RTinkRlk$ = RTinC¥
`
`(5.1)
`
`Thus, the free energy difference associated with a given ex-value is easily
`computed from chromatographic data. The figures given in Table 5.1 are quite
`illustrative as they show the very small energy difference that is needed for complete
`optical resolution , provided a reasonable column efficiency is available.
`In the previous chapter, we have also seen that modern column technology can
`provide us with extremely efficient capillary columns, which in good analytical GC
`instruments give baseline resolution of peaks even at ex-values< 1.05. In columns of
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-14
`IPR2016-00379
`
`

`
`Sec. 5.3)
`
`Some thermodynamic and kinetic considerations
`
`71
`
`Table 5.1 - Free energy differences necessary to produce separation factors > 1
`
`1.05
`1.10
`1.50
`2.00
`10.0
`
`D.D.G , cal/mole (J/mole)
`
`29 (121)
`56 (236)
`240 (1005)
`410 (1717)
`1364 (5705)
`
`effective plate numbers around 2x 105 an n:-value of 1.01 means only 2% peak
`overlap (Rs = 1.11), which, in turn , corresponds to an energy difference of only 5.9
`cal/mole (24. 7 J/mole). Such minute energy values are at least an order of magnitude
`lower than those normally associated with conformational changes in a molecule. It
`is obvious therefore, that binding of two enantiomers to a given chiral site may
`involve different amounts of energy simply because one of the enantiomers, for steric
`reasons , might be forced to adopt an energetically less favourable conformation.
`The much lower column efficiency in LC is often more than compensated for by
`the considerably larger n:-values that can be obtained. In certain cases n:-values of 30
`or more have been found, which then correspond to !l6.G0 values in the range of 2
`kcal/mole (8.4 kJ/mole) . Generally, such values are obtained owing to very low
`retention of the first enantiomer eluted. This means that a very enantioselective
`adsorption process is operating in the column, i.e. one of the enantiomers is virtually
`unbound by the CSP for steric reasons. Such phenomena are not easily explained by
`the three-point interaction model, but rather indicate the operation of a sort of
`'chiral steric exclusion' mechanism , more in line with a 'steric fit' concept involving
`only one binding interaction.
`An expansion of Eq. (5.1) to involve the enthalpy and entropy terms (by an
`application of the Gibbs-Helmholtz equation: G = H-TS) yields :
`
`Inn: = - 6.6.H° + 6.6.S°
`RT
`R
`
`(5.2)
`
`Thus, from a study of the dependence of n: on temperature, Inn: may be plotted as a
`function of 1/T. The slope of the line will then be proportional to the ehthalpy
`difference and Inn: = 0 will give the temperature at which the enthalpy and entropy
`contributions cancel each other. Such studies are easily performed by GC (5] . Figure
`5.5 shows the general appearance of such a plot.
`
`5.3.2 Peak coalescence due to enantiomerization phenomena
`There is always a possibility that the enantiomers of a compound may be intercon(cid:173)
`verted by some mechanism which generates an achiral intermediate. In solution suth
`processes are often acid- or base-catalysed and thus related to the stereochemkal
`fate of positively or negatively charged transient intermediates. Common examples
`are enantiomerization reactions through carbonium ion or carbanion formation
`(Scheme 5.1).
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-15
`IPR2016-00379
`
`

`
`72
`
`Theory of chiral chromatography for direct optical resolution
`
`[Ch. 5
`
`020 I Ince
`
`.15
`
`.10
`
`.05
`
`0 -·
`.8
`
`20o•c
`
`100-C
`
`is·c
`
`OQ..NHPFP
`OONH PFP
`
`{}CH<H3
`NHPFP
`
`O<;H-tH2>iHPrP
`OPFP
`o~H~H-0
`OAcOA<
`A o~ HCHiOPFP
`OPFP
`
`, __
`
`I ! l!r-103 K-1
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`Illustration of the decrease in the enantiomeric separation factor with increasing
`Fig. 5.5 -
`temperature for chiral GC separations. The plots permit evaluation of the enthalpy and entropy
`contributions, respectively, to the separation. Different compounds were studied, with the
`same column. (Reprinted, with permission, from B. KoppenhOfer and E. Bayer, Chromato·
`graphia , 1984, 19, 123. Copyright 1984, Fr. Vieweg & Sohn Verlagsgesellschaft mbH).
`
`In these and similar cases, the free energy barrier to enantiomer interconversion
`will be dependent on substituent effects in the transition state leading to formation of
`the achiral intermediate.
`Even simpler mechanisms of enantiomerization are found in those cases where
`chirality is caused predominantly by steric hindrance, as in compounds with axial or
`planar chirality. Thus , if an atropisomeric compound is taken as an example (Scheme
`5.2), the free energy barrier to internal rotation around the central bond may be high
`enough to prevent any significant racemization of an optically active form, in solution
`at room temperature, and thereby permit its isolation. In many cases, however, the
`temperature has to be lowered considerably in order to decrease the rate of internal
`rotation leading to enantiomer interconversion.
`Taking such enantiomerization reactions into consideration in relation to chro(cid:173)
`matographic methods for direct optical resolution, it is obvious that there are many
`cases where the chromatographic conditions used play an important role. In general
`terms , if the chromatographic conditions are such that the rate of enantiomerization
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-16
`IPR2016-00379
`
`

`
`Sec. 5.3)
`
`Some thermodynamic and kinetic considerations
`
`73
`
`A,,, ©
`C- H + HX
`Ar~
`
`H
`R,,, I
`~c\
`Ar
`X
`
`Scheme 5.1 - Examples of (a) an acid-catalysed and (b) a base-catalysed reaction yielding
`enantiomerization due to formation of achiral charged intermediates.
`
`:.>A____,-< I!__
`
`..____/
`
`,
`A'
`'\.,__;D
`,---\
`A
`
`D I
`
`Scheme 5.2-Thermal enantiomerization reactions by changes in molecular conformation by
`internal rotation, (a) in a biaryl system, and (b) in a polarized alkene system.
`
`is significant on the chromatographic time-scale , the elution pattern will deviate from
`the normal one. During passage of the first-eluted enantiomer through the column, it
`will be partially transformed into the last-eluted enantiomer. This process will result
`in tailing of the leading peak. Conversely, the enantiomer eluted last will also be
`transformed into the first-eluted enantiomer at the same rate. This will give rise to a
`'fronting' of the second peak. The net result will be an extended peak overlap , such
`that the baseline will not be reached between the two peaks. If the enantiomerization
`reaction is fast enough in comparison with the chromatographic process , complete
`coalescence of the peaks will take place. The situation is visualized in Fig. 5.6.
`Consequently, enantiomerization phenomena are readily detected by chromatogra(cid:173)
`enantiomerization
`rates may be
`phy on chiral phases. Conversely ,
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1013-17
`IPR2016-00379
`
`

`
`74
`
`Theory of chiral chromatography for direct optical resolution
`
`[Ch. 5
`
`CH~
`
`D<
`
`N
`I
`Cl
`
`tm
`I
`
`I
`
`.
`.-
`/ - ' ..
`
`0
`
`20
`
`40
`
`60
`
`0
`
`20
`
`min
`
`40 -mtn
`
`60
`
`Fig. 5.6 - C hromatographic patte rn a rising fro m on-wlumn enantiomerization . (Reprinted ,
`with pe rmission, from W. Biirkle , H . Ka rfunkel and V . Schurig, J . Chroma1og., 1984, 288, I.
`Copyright 1984, Elsevier Scie nce Publishers, V.V.).
`
`calculated from chromatographic peak coalescence data by use of suitable computer
`programs. However, very little work has yet been done in this field. It should also be
`kept in mind that the rate observed may not always be representative of that in bulk
`solution , because the reaction process may be catalysed by the surfaces with which
`the solute comes into contact during passage through the column [6].
`We will return to the chiral chromatography of enantiomerization-labile com(cid:173)
`pounds in Section 8.5.
`
`BIBLIOGRAPHY
`J. Porath , Explo rations into the Field of Cha rge-Transfer Adsorption, J. Chroma1og., 1978, 159, 13
`(Chromalog. Rev., 1978, 22, 13).
`V . A . Davankov, Resolution of Racemates by Ligand-Exchange Chromatography, Adv. Chromalog.,
`1980, 18, 139.
`J. L. Atwood , J. E . D . Davies and D. D . McNicol (eds.) , Inclusion Compounds, Acade mic Press,
`London , 1984.
`D . Wo rsch a nd F. Vogt le , Sepa ration o f Enantiomers by Clath rate Fo rmation, Top. Curr. Chem., 1987,
`140, 21.
`
`REFERENCES
`[ I) C . Dalgliesh, J . Chem . Soc., 1952, 137.
`(2] G . Wulff, in Polymeric Reagents and Catalysts, W . T . Ford (ed.) , ACS, Washington DC, 1986, p .
`186.
`(3] W . H . Pirkle and T . C. Pochapsky, J. Am. Chem. Soc., 1986, 108, 5627.
`(4] K. B. L ipkowitz, D . A . Demete r, C. A . Parish and T . Darden, Anal. Chem. , 1987, 59, 1731.
`[5] B . Ko ppenhofer and E . Bayer, Chromatographia , 1984, 19, 123.
`[6] G . Blaschke , A ngew. Chem., 1980, 92, 14.
`
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`Exhibit 1013-18
`IPR2016-00379