LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-1
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-2
`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
`Canada:
`JOHN WILEY & SONS CANADA LIMITED
`22 Worcester Road, Rexdale, Ontario, Canada
`Europe and Africa:
`JOHN WILEY & SONS LIMITED
`Baffins Lane, Chichester, West Sussex, England
`North and South America and the rest of the world:
`Halsted Press: a division of
`JOHN WILEY & SONS
`605 Third Avenue, New York, NY 10158, USA
`South-East Asia
`JOHN WILEY & SONS (SEA) PTE LIMITED
`37 Jalan Pemimpin # 05-04
`Block B , Union Industrial Building, Singapore 2057
`Indian Subcontinent
`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 1014-3
`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 1014-4
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-5
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-6
`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 1014-7
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-8
`IPR2016-00379
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`91
`
`Table 7 .1 - Summary of the main methods used for direct optical resolution by liquid chromatography
`
`Site of chiral
`selector
`
`Stationary phase
`(CSP)
`
`Stationary phase
`(CSP)
`
`Mobile phase
`(CMP)
`
`Basic principle
`
`Capacity
`
`Column efficiency
`
`Use of an intrinsically chiral, polymeric
`stationary phase, either of natural origin
`(polysaccharides and derivatives, pro(cid:173)
`teins) or synthetic (synthetic polymers
`with chiral substituents or 'grafted' chiral
`cavities)
`
`Use of bonded synthetic chiral selectors
`
`Addition of chiral constituents to the
`mobile phase system used. Column
`achiral , usually an alkyl-silica used in
`reversed-phase mode
`
`Analytical to
`preparative
`
`Low to moderate,
`depending on
`whether a support
`material is used or
`not
`
`Analytical to
`preparative
`
`Moderate to fairly
`high
`
`Analytical
`
`Moderate to fairly
`high
`
`7.1 CHIRAL STATIONARY PHASES BASED ON NATURALLY OCCURRING
`AND SYNTHETIC POLYMERS
`Owing to the early recognition of the chiral nature and ready availability of many
`natural products, particularly carbohydrates, such compounds were among the first
`to be tried as sorbents for optical resolution by LC. As early as 1938 a partial
`resolution of a racemic camphor derivative was obtained on a column packed with
`lactose [1]. Lactose remained a column material of interest for some years and was
`used with success in the first nearly complete chromatographic chiral resolution
`described in the literature, which took place in 1944, when Troger's base was
`resolved on a 0.9 m long lactose column [2]. The resolving capacity of a polysacchar(cid:173)
`ide, viz. cellulose, was first realized by the observation that a racemic amino-acid
`could occasionally give two spots in paper chromatography [3-5]. Dalgliesh
`advanced his three-point interaction theory in 1952 on the basis of results from paper
`chromatography of racemic amino-acids [6]. Other early findings on direct optical
`resolution of amino-acids by means of paper chromatography [7] and cellulose thin(cid:173)
`layer chromatography (TLC) (8] were reported. This led to further use of cellulose
`and cellulose derivatives, as well as investigations of starch and cyclodextrins for the
`purpose of chiral LC. At present, derivatives of a large number of natural polysac(cid:173)
`charides are under investigation as potential chiral sorbents.
`The principle of using chiral polymers has also been exploited with many
`different types of totally synthetic materials, by various approaches, and the results
`seem to be very promising.
`Further, the enantioselectivity of proteins, first observed by studies of binding
`equilibria in solution (for reviews see [9,10]), has been successfully utilized for
`analytical chiral LC.
`
`7 .1.1 Polysaccharides and derivatives
`7.1.1.1 Underivatized polysaccharides
`A. Cellulose
`The linear polysaccharide cellulose represents the most common organic compound
`of all. Its chemical constitution is that of a linear poly-~-D-1,4-glucoside (Fig. 7.la).
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-9
`IPR2016-00379
`
`

`
`92
`
`Chiral liquid chromatography
`
`[Ch. 7
`
`(a)
`
`Ii near poly [1- 4-/3-D-glu cos e J
`OH
`Ho·-r--L~~OH H~-r--Cp
`~f ~
`o----
`
`OH
`
`0
`
`0
`
`0
`\
`
`(b)
`
`6-branched po1y[1-4-0'.-0-glu cose J
`
`(some 6 -p ositions are phospho rylated l
`
`Fig. 7.1-The chemical structure of (a) cellulose and (b) amylopectin, the main components of
`starch.
`
`It forms very long chains containing at least 1500 ( + )-D-glucose units per molecule.
`The molecular weight of cellulose ranges from 2.5 x 105 to 1 x 106 or more. In a
`cellulose fibre these long molecules are arranged in parallel bundles and held
`together by numerous hydrogen bonds between the hydroxyl groups . In the native
`state cellulose is therefore built up from partially crystalline regions. These are not
`regenerated on precipitation of cellulose from solution. As seen from Fig. 7 .la the
`( + )-D-glucose repetitive unit contains five chiral centres and three hydroxyl groups.
`All the ring substituents are equatorial.
`It has been found that partial hydrolysis of natural cellulose with dilute mineral
`acid can yield a material with a high degree of crystallinity because hydrolytic
`cleavage will take place preferentially in the amorphous regions. Such a material
`contains ca. 200 glucose units per chain and is usually called 'microcrystalline
`cellulose' [11]. It is marketed as 'Avicel' by several chemical companies.
`Although derivatives of cellulose have been used in most recent research efforts
`and successful resolutions, very good results have also been obtained by the use of
`unmodified cellulose and are therefore worth mentioning. The compounds resolved
`are, without exception, highly polar with multiple sites for hydrogen bond forma(cid:173)
`tion. Some typical results are summarized in Table7.2.
`In a recent work [22], it was found that on treatment with dilute alkali cellulose
`will lose its enantioselective properties owing to a transformation of the native,
`metastable form into a rearranged and stable amorphous form.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-10
`IPR2016-00379
`
`

`
`Sec. 7.1)
`
`Chiral stationary phases
`
`93
`
`Table 7.2- Examples of optical resolutions performed by liquid chromatography on cellulose
`
`Type of compound
`
`Amino-acids, amino-acid
`derivatives
`
`Diaminodicarboxylic acids
`Synthetic alkaloids
`Cathecins
`
`LC mode
`
`Paper
`Thin layer
`Column
`Column
`Column
`Column
`
`References
`
`(3-5,7,12]
`(8,13,14)
`(lS-17]
`(18)
`(19,20)
`(21]
`
`B. Starch
`The other widespread polysaccharide, also built from ( + )-o-glucose units, is starch.
`Its structure is more complex than that of cellulose. It is composed of ca. 20%
`amylase and 80% amylopectin, the latter being an insoluble fraction. Both are
`entirely composed of ( + )-o-glucose units , Jinked by tX-glucoside bonds. Whereas
`amylase is a linear polymer, amylopectin is branched by C1-C6 connections
`(Fig. 7.lb).
`Depending upon the source, there are different particle sizes of starch available.
`The material obtained from potatoes is relatively coarse (60-100 JLm) and has been
`favoured for column chromatography. Despite its ready availability and non(cid:173)
`swellable properties in aqueous media, which give good ftow properties, it has so far
`found very little use.
`As in the case of cellulose, starch appears to be most suitable for polar aromatic
`compounds. Its use for resolution of atropisomers with structures containing polar
`substituents has been particularly well documented [23-27]. These separations show
`a very pronounced dependence on the nature of the mobile phase, and are especially
`inftuenced by the ionic strength. Figure 7 .2 illustrates the chromatographic behav(cid:173)
`iour of a starch column after application of racemic 2,2' -dinitrodiphenic acid and
`elution with lM sodium citrate buffer, pH 7. 7, at 60°C. The separation factors
`obtained are quite satisfactory but the column efficiency is modest .
`
`C. Cyclodextrins
`As early as 1908 it was discovered by Schardinger [28] that new crystalline carbo(cid:173)
`hydrates, so-called dextrins, were formed if starch was subjected to degradation by a
`micro-organism, Bacillus macerans [29]. These compounds were found to be normal
`P-1 ,4-D-glucosides, but cyclized to rings of 6-12 units. Those with the three smallest
`rings (6-8 units) have been called a-, p-, and y-cyclodextrins (CD), respectively, and
`form inclusion complexes with various compounds of the correct size. The diameter
`of a P-CD ring is 8A and its volume is ca. 350A3
`. The stability of the inclusion
`complex is largely dependent on the hydrophobic and steric character of the guest.
`These phenomena make CDs, particularly P-CD, which is easily available, highly
`promising for use in chiral LC.
`The major development in chiral LC with cyclodextrins started with the tech(cid:173)
`nique of using them as mobile phase additives in TLC experiments [30-32]. This
`technique has also been applied with success to column chromatography and will be
`treated in Section 7.3. Earlier some efforts to use cross-linked cyclodextrin gels for
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-11
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-12
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-13
`IPR2016-00379
`
`

`
`Petitioner
`Exhibit 1014 - 014
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-14
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-15
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-16
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-17
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-18
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-19
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-20
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-21
`IPR2016-00379
`
`

`
`104
`
`Chiral liquid chromatography
`
`(Ch. 7
`
`(a) Use of chiral substituent:
`
`polymerization
`cross linking
`
`" - - /
`/-,~
`chiral monomer
`
`chi ral
`polymer network
`
`(b) Use of chiral catalyst:
`
`* c
`
`iso tactic ( stereoregular)
`P.lir.al ~
`
`polymer
`
`~:~~:.,~~
`
`Fig. 7 .6-Two different routes to chiral sorbents based on synthetic polymers. (a) From chiral
`monomer; (b) from achiral monomer.
`
`technique, polymer particles of desired mean diameter and acceptable size homoge(cid:173)
`neity can be obtained. Free-radical initiation is used and the porosity of the gel
`particles is regulated by the relative amount of cross-linking agent added . The
`particles swell in organic solvents and the material is only used in low-pressure LC
`systems.
`The resolving capacity of these sorbents is highly dependent on a variety of
`factors. Apart from the substituents on the polyacrylamide backbone, the degree of
`cross-linking, the nature of the cross-link and the mobile phase compositions are the
`most essential considerations. Systematic investigations of these factors began in
`1973 when partial resolution of doubly radiolabelled (3H and 14C) mandelic acid and
`mandelamide was studied by analysis of the enantiomer composition of eluted
`fractions by liquid scintillation counting (56]. Since the 3H/14C activity ratio is
`proportional to the enantiomer composition this detection technique permits a
`rather precise determination of l¥ even at very low resolution.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-22
`IPR2016-00379
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`105
`
`The method is, of course, limited to those cases where it is possible to radiolabel
`the two enantiomers of a compound with the appropriate radioisotope . Accordingly,
`the elution profiles of both enantiomers are obtained in a single chromatographic
`experiment.
`The preferred route to these polymers was shown to be that given in Scheme 7 .2.
`
`H C=C - COCI
`2
`I
`R
`
`*
`+H N - CH-R -
`2
`1
`I
`R2
`
`0
`*
`II
`H C=C- C- N- CH - R
`2
`H
`I
`I
`1
`R2
`R
`
`suspension - copolymeri zation
`with crosslinker <Cl
`
`R2
`I
`*CH-R 1
`I
`HN-C=O
`I
`
`CH2-?
`R
`
`network
`
`n
`
`R= H, CH3 ; R1 = CH3 , COOR' ; R2= alkyl or aryl groups
`
`Scheme 7 .2 - Synthetic routes to the chiral polyacrylamides and polymethacrylamides.
`
`The great variation of performance with substituents is evident from Table 7.8,
`
`Table 7.8 - Optical resolution ability of a series of variously substituted polyacrylamide sorbents.
`(Reprinted, with permission, from G. Blaschke, Angew. Chem., 1980, 92, 14. Copyright 1980, Verlag
`Chemie GmbH).
`
`R
`
`R,
`
`R1
`
`R'
`
`H
`CH3
`H
`CH3
`CH3
`H
`H
`H
`H
`H
`H
`
`CH3
`CH3
`CH3
`CH3
`CH3
`CH3
`COOR'
`COOR'
`COOR'
`COOR'
`COOR'
`
`C6Hs
`C6Hs
`cyclo-C6H 11
`cyclo-C6H,,
`1-naphthyl
`p-I-C6H4
`CH3
`C6Hs
`C6HsCH2
`p -OH-C6H4CH2
`C6HsCH2
`
`CiHs
`CiHs
`C2Hs
`C2Hs
`tert-C. H9
`
`Optical yield(% )
`
`Mandelic acid
`
`Mandel amide
`
`28
`8
`12
`58
`0
`0
`14
`46
`51
`0
`89
`
`35
`81
`35
`92
`97
`0
`18
`34
`96
`0
`80
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-23
`IPR2016-00379
`
`

`
`106
`
`Chiral liquid chromatography
`
`[Ch. 7
`
`which indicates that two of the most useful polymers are those corresponding to (1)
`R = H, R 1 = C02R ', R2 = C6HsCH2 , R' = C2Hs and (2) R = CH3 , R 1 = CH3 ,
`R2 = cyclo-C6H 11 .
`These substituted polyacrylamides have been found particularly well suited to
`polar compounds with functional groups capable of hydrogen bonding. It is therefore
`logical that relatively non-polar mobile phases have been found most useful.
`Combinations of hydrocarbons, ethers and possibly small amounts of an alcohol are
`typical; examples include toluene-dioxan , hexane-dioxan and toluene-dioxan(cid:173)
`methanol mixtures . Protic co-solvents such as methanol strongly decrease the
`retention and therefore normally form less than 10% of the mobile phase
`composition.
`Although hydrogen bonding is apparently the major binding contribution to
`retention of the solute, the mechanism of enantiomer discrimination appears to be
`quite complex. As in the case of polysaccharide-derived sorbents, enantioselection is
`assumed to be caused by inclusion phenomena , i.e. the binding groups in the
`asymmetric cavities into which the solute enantiomers are thought to diffuse are
`more favourably located for one of the antipodes, which therefore will be preferen(cid:173)
`tially retained.
`Columns packed with sorbents of this kind have mainly been used for semipre(cid:173)
`parative work (sample amounts between ca. 1 and 250 mg) on racemic pharmaceuti(cid:173)
`cals. These applications will be treated in Chapter 9. An interesting recent modifica(cid:173)
`tion of the sorbent consists of a silica-bonded non-cross-linked polyacryloyl (S)(cid:173)
`phenylalanine ethyl ester. This has been used for analytical HPLC and gives
`considerably improved column efficiency [63].
`
`7.1.2.2 Sorbents based on isotactic linear polymethacrylates of helical
`conformation
`A vinyl polymer with chirality caused only by its helicity was first prepared in 1979 by
`Okamoto and co-workers (64,65]. Optically active poly(triphenylmethyl methacry(cid:173)
`late) was then obtained according to Scheme 7 .3 by asymmetric anionic polymeriza(cid:173)
`tion of triphenylmethyl methacrylate under the influence of a chiral initiator in
`toluene at low temperature. The success of the reaction is strongly dependent on the
`initiator used, which is based on a complex between an optically active diamine and
`butyllithium or lithium amide. Both (-)-sparteine-butyllithium, and ( + )-(2S,3S)(cid:173)
`dimethoxy-1 ,4-bis(dimethylamino )butane-lithium amide , were found to give the
`(+)-polymer in good yield [ 66]. At a degree of polymerization greater than ca. 70 the
`polymer is insoluble in most common organic solvents.
`The material can be used as such after grinding and sieving to a particle size
`averaging ca. 30 µ.m [67). However , a more efficient and durable chromatographic
`sorbent is obtained by adsorption of the low molecular-weight soluble fraction of the
`polymer on silanized silica (10 nm, or 1000 or 4000 A) [68].
`It is perhaps not surprising that this CSP turned out to be excellent for optical
`resolution of racemic aromatic hydrocarbons of linear and planar chirality [ 69). The
`compounds in Fig. 7.7. (p. 108) are examples of such hydrocarbons that are all
`resolved with high O'. values. They are characterized by high hydrophobicity and a
`rigid molecular geometry. A difference in the extent to which the enantiomers would
`interact with the helical CSP (in which the triphenylmethyl group is assumed to attain
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-24
`IPR2016-00379
`
`

`
`Sec. 7.1)
`
`Chiral stationary phases
`
`107
`
`Ar
`0
`I
`II
`H C: C- C - 0 -C- Ar
`2
`I
`I
`A
`Ar1
`
`chiral catalyst ( C)
`
`Ar
`I
`Ar-C-Ar
`1
`I
`o-c~o
`CH-C
`2
`I
`A
`
`n
`
`R• CH3; C· (-)-sparteine:
`Ar, Arl - CsHs, C5H4N
`
`linear, isotactic,
`
`single-handed helical
`
`Scheme 7.3 - Asymmetric polymerization reaction used to produce the right-handed helical
`poly(triarylmethyl methacrylates).
`
`a propeller-like conformation) is therefore very reasonable on intuitive grounds.
`This is particularly true in the case of hexahelicene, for which the highest o: value
`(> 13) was reported. The enantiomer most strongly retained on the (+)-CSP is the
`(+)-form, which has P- (right-handed) helicity. Since it was found that the (+)-CSP
`interacts very strongly with itself, but only weakly with the (-)-polymer, it is very
`likely that the (+)-CSP also has P-helicity [70,71). The same P-helicity of the most
`strongly retained enantiomer was also found for all other compounds investigated
`that possessed this type of chirality. The situation is shown in Fig. 7. 7 This correlation
`means that chromatographic retention data can be used for determination of
`absolute configuration of these types of compound.
`Because the structure of this CSP is not cross-linked and the coated-silica version
`of the sorbent is based only on physical adsorption, some limitations are put on the
`choice of mobile phase, for solubility reasons. Thus, aromatic hydrocarbons,
`chloroform and tetrahydrofuran (which dissolves the polymer) should be avoided.
`To date , methanol has been the preferred mobile phase [51) and there is generally a
`tendency towards increase in o: value with increasing polarity of the solvent, but
`retention times may be unacceptably long in many cases.
`The use of these sorbents with protic mobile phase systems is disadvantageous
`owing to the solvolytic instability of the ester bond. Thus in methanol the CSP
`gradually undergoes solvolysis to yield methyl triphenylmethyl ether as a by(cid:173)
`product. (It should be remembered that the trityl group is commonly used for
`protection of carboxyl groups in peptide synthesis and is easily removed under
`weakly acidic conditions). It is therefore advisable to work at low temperature with
`alcohols as eluents.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-25
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-26
`IPR2016-00379
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`109
`
`Ph-CH -Cl
`I
`CH3
`
`Ph- CH-C-0-CH Ph
`2
`I
`11
`OH 0
`
`BzOQ
`
`f3z
`OBz
`OBz
`
`R,
`C=CH -CH-CH-C-0-C Hz
`R,.
`c
`\
`I
`,~
`~
`\
`I
`CH 3 CH 3
`
`0 Cl
`
`OP h
`
`Cr (acac) 3
`Ph- CH-0- C-Ph
`I
`11
`Al (a cac )3
`CH3 0
`1 O
`Cl Cl (XCl
`gQ
`C H -CH-0- C 'I ~
`2 5 I
`CH3
`C H oJ-o ~ cc'-.:: O,~-OCH3
`
`-
`
`Cl Cl
`
`Cl
`
`Cl
`
`Br
`
`Br
`
`2 5
`
`I ~ I
`Ph
`/".
`O
`
`Scheme 7.4- Various compounds optically resolved on ( + )-poly(triphenylmethyl methacry(cid:173)
`late) sorbents.
`
`Basically, the principle rests on an imitation of an enzyme's binding site, which can
`usually be regarded as a chiral cavity or cleft in the protein , often highly specific with
`respect to binding of substrate enantiomers because of the precise steric require(cid:173)
`ments for multiple bond attachment. Because the experimental technique can be
`compared with making a plaster cast from an original template it has also been called
`'molecular imprinting' . Thus, the molecules of a particular compound act as
`templates around which a rigid polymeric network is cast. This procedure, which is
`conceptually straightforward but most delicate to carry out in practice, can be
`summarized in three distinct steps.
`(1) Formation of a complex between the (chiral) compound used as template and
`a polymerizable monomer.
`(2) Polymerization with cross-linking to form a rigid matrix .
`(3) Removal of the template, either simply by some washing technique, or by
`means of a hydrolytic or similar reaction, which has to be used in the case of covalent
`attachment to the template. These steps are visualized in Scheme7.5.
`Extensive research on this technique has been carried out since 1977 when it was
`shown that a polystyrene sorbent, prepared with the use of an optically active
`template, could be used for partial resolution of the corresponding race mate [76-80].
`The preferred method has been to use the rapid and reversible formation of boronic
`acid ester bonds between a carbohydrate structure and a vinyl-substituted phenyl(cid:173)
`boronic acid (Fig. 7 .8). In this way the monomer units are covalently attached to the
`template (cf. Scheme7.5, step 1). Polymerization and cross-linking with a divinyl
`compound, followed by hydrolysis and washing out of the template will then yield the
`chiral sorbent.
`The chromatographic performance of this kind of sorbent is shown in Fig. 7.9.
`The chromatogram illustrates one of the difficulties associated with the technique,
`viz. the necessity of fast mass-transfer to reduce band broadening. Although boronic
`ester formation is a very fast process, it is still somewhat too slow for chromato(cid:173)
`graphic purposes. It is important to keep in mind that chromatographic sorption-de-
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-27
`IPR2016-00379
`
`

`
`110
`
`Chiral liquid chromatography
`
`[Ch. 7
`
`Co mplexa tion wit h monomer units:
`
`Polymer iza tion:
`
`Desorpt ion of templat e:
`
`Scheme 7 .5- The principle of 'molecular imprinting' with formation of a defined arrangement
`of binding groups within the microcavity.
`
`sorption equilibria are always based on non-covalent interactions, except for some
`protein separations by affinity chromatography where thiol-disulphide interchange
`may contribute.
`In accordance with these facts, it was found that the column efficiency could be
`improved by increasing the temperature and also by acid-base catalysis, induced by
`the addition of ammonia to the mobile phase [81].
`The choice of the mobile phase is complicated by the requirement that the
`polymer should be resistant to swelling in the medium, as deformation of the cavities
`will otherwise occur, with loss of selectivity. A mixture of acetonitrile with 4-6% of
`water and 2-8% of concentrated ammonia solution has been found to be very useful.
`The flow-rate has a pronounced influence on the result and very low flow-rates are
`usually necessary.
`Interestingly, the k' values increase with increasing temperature. The increase is
`larger for the most strongly retained enantiomer, leading to an improved a-value.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-28
`IPR2016-00379
`
`

`
`Sec. 7.1]
`
`Chiral stationary phases
`
`111
`
`Fig. 7.8 -
`
`Introduction of polymerizable vinyl groups into phenyl-~-o-mannopyranoside by
`esterification with 4-vinylbenzeneboronic acid.
`
`Start
`
`Fig. 7.9 - Elution profile from the chromatography of phenyl-~-D.L-mannopyranoside on
`macroporous polymer imprinted with the o-enantiomer. (Mobile phase: acetonitrile with 4%
`concentrated ammonia solution and 5% water, flow-rate 0.1 ml/min. Sample amount: 200 µ.g).
`(Reprinted, with permission , from G. Wulff, H .-G. Poll and M. Minarik, }. Liquid Chromato-
`graphy, 1986, 9, 385 by courtesy of Marcel Dekker, Inc.).
`
`7 .1.4 Proteins
`The complicated molecular structure of proteins makes them very interesting for
`binding studies. The technique of affinity chromatography developed from the
`knowledge of the ability of certain protein-ligand pairs to form very strong com(cid:173)
`plexes. Such pairs could be derived from natural systems, such as enzyme-substrate
`analogue, enzyme-cofactor, hormone-receptor protein , etc., but it was soon rea(cid:173)
`lized that many 'unnatural' synthetic compounds could also show very strong binding
`power (high affinity) for proteins.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1014-29
`IPR2016-00379
`
`

`
`112
`
`Chiral liquid chromatography
`
`[Ch . 7
`
`The availability and importance of serum proteins, particularly serum albumins,
`have made them preferred models for binding studies. It was known from Scatchard
`analyses that binding to a protein involves multiple equilibria, i.e. a number of
`binding sites, some of which have different affinity for the ligand. It therefore seemed
`quite probably that the net result, the overall binding constant , could be different for
`the two enantiomers in a racemate. Further, from the knowledge concerning the
`substrate enantioselectivity often shown by enzymes, the presence of high affinity
`sites with enantiomer-differentiating ability would be expected in other proteins as
`well .
`A number of studies of solution equilibria between serum proteins and various
`ligands, particularly pharmacologically active compounds , have been made, which
`demonstrated significantly different binding constants for the respective enan(cid:173)
`tiomers [82,83). Such effects could, however , be demonstrated more clearly by
`chromatographic techniques . Thus, in 1973 the previously known higher affinity of L(cid:173)
`tryptophan for bovine serum albumin (BSA) was used to separate the enantiomers
`on a column packed with a BSA-Sepharose gel [84] . Elution of the o-form was
`performed with a borate buffer (pH 9), then the L-form was desorbed by changing to
`dilute acetic acid. The technique was later used for determination of the enantio(cid:173)
`selectivity of certain drugs for serum albumins [85,86]. In the last few years analytical
`chiral LC-methods based on the use of immobilized proteins as stationary phase
`materials have been rapidly developed and shown to be useful for a variety of
`resolution problems.
`
`Immobilized albumin
`7.1.4.1
`Early work with the use of low-pressure LC-systems and isocratic elution with
`phosphate and borate buffers from columns packed with BSA coupled to Sepharose,
`demonstrated that the optical resolution of charged solutes such as the free amino(cid:173)
`acids tryptophan, kynurenine [3-(2-aminobenzoyl)alanine] and their 5- and 3-
`hydroxy derivatives, respectively, is extremely dependent on the pH of the mobile
`phase [87). It was further shown that compounds with chirality at a sulphur atom,
`such as methyl o-carboxyphenyl sulphoxide, could be resolved and that , in addition
`to pH, the ionic strength of the mobile phase had a great influence on retention and
`r