LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-1
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-2
`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 1015-3
`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 1015-4
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-5
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-6
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-7
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-8
`IPR2016-00379
`
`

`
`Petitioner
`Exhibit 1015 - 009
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-9
`IPR2016-00379
`
`

`
`Petitioner
`Exhibit 1015 - 010
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-10
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-11
`IPR2016-00379
`
`

`
`Petitioner
`Exhibit 1015 - 012
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-12
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-13
`IPR2016-00379
`
`

`
`164
`
`Analytical applications in academic research and industry
`
`[Ch.8
`
`Table 8.4 - Data obtained from optical resolutions of a series of benzodiazepino nes o n N-(3 ,5-
`dinitrobenzoyl)amino-acid sorbents. (Reprinted, with permission, from W. H. Pirkle and A. Tsipo uras ,
`J. Chromatog., 1984, 291, 291. Copyright 1984, Elsevier Science Publishers 8. V .).
`
`Compound
`
`(X=R=H)
`
`R1
`
`CH3
`CH2CH3
`CH2CH2CH3
`CH(CH3)i
`CH2(CH 2)iCH3
`CH2CH(CH3)i
`CH2CH2SCH3
`CH2C6Hs
`CH2C6H 40H-p
`C02CH2CH3
`OH
`OCH3
`OCOCH3
`OCOC6H5
`OCOC (CH3)J
`
`Adsorbent
`
`(R)-DNB-phenylglycine
`
`(S)-DNB-leucine
`
`kj
`
`3.2(-)-(R)
`2.3(-)
`2.2(-)-(R)
`2.7(- )-(R)
`2.7(-)
`3.2(-)-(R)
`4.0(-)
`4.0(- )-(R)
`12.3(-)-(R)
`4.4(-)
`16.9(-)-(R)
`10.0(-)
`7.4( - )
`3.1( - )
`1.0(-)
`
`<X
`
`1.6
`1.46
`1.42
`1.92
`1.92
`1.89
`1.57
`1.93
`1.48
`1.36
`1.20
`1.33
`1.27
`1.87
`2.05
`
`kj
`
`1.8( + )-(S)
`1.9( +)
`2. 7( + )-(S)
`1.8( + )-(S)
`I. 7( +)
`1.9( + )-(S)
`2.2( +)
`2.2( + )-(S)
`6.3( + )-(S)
`4.3( +)
`11. 7( + )-(S)
`11.0(+)
`3.8( +)
`2.3( +)
`0.7( +)
`
`<X
`
`4.11
`2.22
`2.35
`3.53
`4.0
`4.20
`3.0
`4.33
`2.18
`1.42
`1.13
`l.19
`1.62
`2.84
`3.69
`
`In each case the mobile phase was 10% 2-propanol in hexane
`
`enantiomer is adsorbed on the CSP by simultaneous three-site bonding interactions
`as illustrated by Fig. 8.11.
`
`Fig. 8.11 - Chiral recognition model for retention by the stationary phase of the final
`benzodiazepinone enantiomer eluted. (Reprinted, with permission , from W. H . Pirkle and A .
`Tsipouras, J. Chromatog. , 1984, 291, 291. Copyright 1984, Elsevier Science Publishers B.V.).
`
`Note that the most stable conformation of a 3-substituted diazepam is the one
`shown, with the substituent occupying a pseudo-equatorial position. This conforma(cid:173)
`tion tends to direct the amide carbonyl group of the folded ring towards the amide
`hydrogen atom of the bound selector. The bonding interactions suggested are: (a)
`charge-transfer interaction between the 3,5-dinitrobenzoyl group and the benzene
`ring of the analyte , (b) hydrogen bonding between the 3,5-dinitrobenzamide
`hydrogen atom and the amide carbonyl oxygen atom of the analyte and (c) hydrogen
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-14
`IPR2016-00379
`
`

`
`Sec. 8.3]
`
`Pharmaceutical applications
`
`165
`
`bonding between the amino-acid carbonyl oxygen atom of the CSP and the amide
`hydrogen atom of the analyte.
`This model is also consistent with the findings that the length of the 3-substituent
`does not impair the optical resolution, which indicates that it does not contribute to
`steric interaction.
`The large separation factors obtained for many of the compounds on these CSPs
`have encouraged preparative-scale resolutions, which will be treated in Chapter 9.
`This type of pharmaceutical is also well resolvable by reversed-phase LC on
`albumin-silica sorbents. In investigations by Allenmark and Andersson (53 , 54], a
`series of compounds was shown to give separation factors up to about 7. The
`retention is highly dependent on the hydrophobic character of the 3-substituent, and
`the mobile phase has to be selected accordingly to give reasonable k' values. Table
`8.5 gives an idea of the effects caused by the substituents and of the mobile phase
`composition.
`
`Table 8.5 - Data obtained from the separation of benzodiazepinone enantiomers on a BSA-silica
`("Resolvosil" ) column. (Reprinted from S. Allenmark, 1. Liquid Chromatog., 1986, 9, 425, by courtesy
`of Marcel Dekker, Inc.).
`
`Compound
`
`(X=R=H)
`R1
`
`Mobile phase composition
`
`Molarity
`(mM)
`
`pH
`
`7.8
`7.5
`7.8
`7.8
`7.8
`7.5
`7.8
`7.5
`7.8
`6.6
`
`1-propanol
`(%)
`
`6
`I
`6
`6
`6
`I
`6
`1
`6
`2
`
`k j
`
`5.1
`7.25
`11.1
`41.0
`1.5
`3.0
`3.2
`6.5
`14.0
`2.1
`
`ki
`
`IX
`
`5. 1
`14.7
`17.4
`110.0
`1.65
`5.0
`4.7
`10.75
`28.2
`8.6
`
`1.00
`2.03
`1.57
`2.68
`1.09
`1.67
`1.47
`1.65
`2.01
`4.1 0
`
`CH3
`50
`20
`CH3
`50
`CH2(CH2)iCH3
`50
`CH2C6Hs
`50
`CH2CH2CH20H
`20
`CH2CH2CH20H
`50
`02CCH2CH3
`20
`0 2CCH2CH3
`OCH2CH2CH(CH3)i 50
`10
`OH
`
`In conclusion, many chiral LC methods are well suited to a direct monitoring of
`enantiomer composition or enantiomeric purity of this type of pharmaceuticals. This
`should be of importance in connection with future development of optically pure
`products and pharmacokinetic studies of the antipodes.
`
`8.3.1.5 Miscellaneous compounds
`Various other racemic drugs have been resolved by direct chiral chromatographic
`methods thanks mainly to the work of Blaschke and co-workers (51]. Such resolu(cid:173)
`tions include the anti-cancer drug " lfosfamide" (" Holoxan"), 7, and related com(cid:173)
`pounds, which all contain an asymmetric phosphorus atom in a heterocyclic ring
`system (55]. The resolution of 7 was done on a semipreparative scale on poly((S)-N(cid:173)
`acryloylphenylalanine ethyl ester] with toluene/dioxan (1:1) as mobile phase (51].
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-15
`IPR2016-00379
`
`

`
`166
`
`Analytical applications in academic research and industry
`
`[Ch. 8
`
`7
`
`8
`
`The drug methaqualone, 8, has been used for a long time as a hypnotic and
`anticonvulsive [56]. It was not until 1975, however , when it was shown by NMR that
`some methaqualone analogues had high barriers to rotation around the nitrogen(cid:173)
`aryl bond [57] , that the chiral structure and resolvability of 8 was realized. It was
`recently shown by Mannschreck et al. [58] that the barrier to enantiomer intercon(cid:173)
`version (.1.1G*) in methaqualone is as high as 131.6 kJ/mole (at 135°C). This is
`sufficient to prevent any racemization of an enantiomer, which was also proved
`experimentally by semi preparative partial resolution of the racemate on an MCT A
`column [58]. Typically , 300 mg of racemic 8 gave ca. 100 mg of ( + )-8 (optical purity
`70%, first eluted) and ca. 120 mg of (-)-8 (optical purity 60% ). Both enantiomers
`showed anticonvulsive activity, with (-)-8 the more active form.
`Recently, compounds possessing a 1,4-dihydropyridine structure have come into
`focus as drugs influencing ion-channel transport of calcium [59]. Some of these
`compounds have been resolved into enantiomers, which have opposite effects on
`calcium channels [60, 61]. Optical resolutions have been achieved by chiral LC on
`MCT A columns with 95% ethanol as mobile phase (62-64]. Recycling techniques in
`a two-column configuration have also been used to achieve baseline resolution [64].
`Two drugs with the common structure 9 have been optically resolved by LC.
`
`Ar =
`
`Ar
`
`-CNH
`I
`~
`0
`
`9
`
`Baclophen-lactam (Ar=p-G-C6H4-; 9a) shows an extraordinarily large ex value (28)
`on a "Resolvosil" column (50mM phosphate buffer , pH 7, 2% 1-propanol) [54].
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-16
`IPR2016-00379
`
`

`
`Sec. 8.3]
`
`Pharmaceutical applications
`
`167
`
`Rolipram (9b) is completely separated into enantiomers on an MCT A column (96%
`ethanol) [51]. Other racemic drugs recently resolved on MCTA [51], include
`chlormezanon (10) (also on "Resolvosil" [65]), oxapadol (11) , ketamin (12) and
`mianserin (13). Poly[N-(S)-acryloylphenylalanine ethyl ester] has been successfully
`used for optical resolution of the chiral sulphur compound 14 and of chlorthalidone
`(15) [both with toluene/dioxan (1:1) as mobile phase] [51].
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-17
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-18
`IPR2016-00379
`
`

`
`Sec. 8.3]
`
`Pharmaceutical applications
`
`169
`
`w z
`
`~
`<{
`-J
`-J
`~
`z
`w
`L 0..
`
`w
`z
`w
`.....
`(/')
`>-u
`
`D
`
`D
`
`20
`
`10 MIN
`
`I
`
`I i r-
`0
`
`Fig. 8.12 - Optical resolution of D.L·penicillamine and o ,L·cysteine after conversion into the
`thiazolidin-2-one derivatives. (Reprinted, with permission, from W. A. Konig, E. Steinbach
`and K. Ernst, J. Chromatog., 1984, 301, 129. Copyright 1984, Elsevier Science Publishers B.V.).
`
`On this CSP the Q'. values recorded were 1.085 for penicillamine and 1.079 for
`cysteine. In both cases the L-enantiomer was the first eluted.
`(2) Liquid chromatography [70]. Reaction of 16 with formaldehyde yields the
`dimethylthiazolidinecarboxylic acid, 18, (Scheme 8.6). Separation of the enan-
`
`0 HO-kJ
`
`18
`
`Scheme 8.6 - Derivatization of penicillamine for CLEC.
`
`tiomers was effected by CLEC on a column packed with LiChrosorb RP-8 (Merck)
`coated with the Cu(II) complex of (2S,4R,2'RS)-4-hydroxy-1-(2' -hydroxydodecyl)(cid:173)
`proline 19 [71], and a mobile phase composed of methanol-water (12:88 v/v)
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-19
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-20
`IPR2016-00379
`
`

`
`Sec. 8.3]
`
`Pharmaceutical applications
`
`171
`
`B. Dopa and derivatives.L-Dopa , L-3-(3 ,4-dihydroxyphenyl)alanine, is commonly
`used for the treatment of Parkinson's disease and acts as the precursor of dopamine,
`which is deficient in patients suffering from this illness. D-Dopa, however, is toxic
`(73,74) and its presence in the drug has to be monitored because of the rather large
`amounts of the drug that are usually taken in L-dopa therapy.
`An LC method, utilizing the principle of CLEC and mobile phase addition of the
`chiral constituent (cf. Section 7.3 as well as the technique described above), has
`proved to be very useful for this purpose (75). The chromatographic system consists
`of a C18 column (4.6x250mm) which has been equilibrated with a mobile phase
`composed of L-phenylalanine (6mM) and copper(II) sulphate (3mM) in water.
`Eluted species are detected by UV at 280 nm.
`Two chemically related compounds, methyldopa and carbidopa, the L-forms of
`which are used as inhibitors of the decarboxylative enzyme , as well as tryptophan ,
`can also be determined by the same method. As seen from Table 8.6, which also gives
`
`Table 8.6 - Data obtained for the optical resolution of dopa and dopa-analogues by CLEC. (Reprinted,
`with permission, from L. R. Gelber and J. L. Neumeyer, J. Chromatog., 1983 , 257, 317. Copyright 1983,
`Elsevier Science Publishers B.V.)
`
`Compound
`
`Dopa
`Methyldopa
`Carbidopa
`Tryptophan
`
`kb
`
`1.7
`1.9
`7.4
`5.8
`
`ki..
`
`2.4
`2.6
`9 .5
`7.0
`
`CJ(
`
`1.4
`1.2
`1.2
`1.2
`
`R,
`
`2.9
`J.9
`3.4
`2.6
`
`Mobile phase composition
`
`L-Phe(mM)
`
`Cu(II)(mM)
`
`MeOH( %)
`
`6
`12
`6
`8
`
`3
`6
`3
`4
`
`0
`0
`0
`JO
`
`the optimized mobile phase compositions, all D-enantiomers are eluted prior to the
`L-forms . The ex values (1.2-1.4) are sufficient for complete resolution. Standard
`calibration curves give correlation coefficients >0.986 (for dopa 0.996) and the
`relative standard deviations are < 1 % .
`
`8.3.2.2 Basic (cationic) compounds
`Many important drugs contain aliphatic amino groups and are therefore charged at
`physiological pH. Among these are many anticholinergic alkaloid derivatives,
`amino-alcohols with P-receptor blocking or stimulating action, and a variety of chiral
`local anaesthetics.
`The number and diversity of important chiral basic drugs has led to intensive
`research on their direct chromatographic optical resolution. Selected examples of
`useful bioanalytical procedures will be given after some general comments on the
`different merits of the various strategies.
`The different physiological effects of the enantiomers of P-adrenergic blocking
`agents have stimulated intensive research on chromatographic optical resolution of
`such compounds. Most of them possess the general structure 20. It is known that the
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-21
`IPR2016-00379
`
`

`
`172
`
`Analytical applications in academic research and industry
`
`[Ch.8
`
`(S)-form is often 5~500 times more effective than the (R)-enantiomer [76) and that
`the latter may also be toxic.
`
`20
`
`Ar =
`
`o_;= 6- ,,,
`
`CH30~
`
`lal
`
`s-lbl
`0- (c)
`HNJ
`a<dl
`
`These compounds are too polar to be subjected to GC without derivatization ,
`which is also required by some LC methods. The favoured derivatization process is a
`conversion into the oxazolidones by reaction with phosgene [77). In some cases non(cid:173)
`cyclic carbamates, formed by reaction with an isocyanate, have been used. The
`reactions are shown in Scheme 8. 7. Note that the oxazolidones are easily cleaved by
`dilute alkali to regenerate the amino-alcohol without racemization .
`Some experimental procedures, useful for a determination of enantiomer com(cid:173)
`position of ~-blockers, are given below. The first of these , based on GC of the
`oxazolidinone derivative of metoprolol (20a) and two metabolites (20g) and (20h)
`[78) , used a capillary column (0.25mmx18m, Duran glass) coated with the XE-
`6~L-valine-(R)-cx-phenylethylamide polymer described by Konig and co-workers
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-22
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-23
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-24
`IPR2016-00379
`
`

`
`Sec. 8.3]
`
`Pharmaceutical applications
`
`175
`
`able for the pindolol derivative) .
`Other methods used for direct optical resolution of P-blockers include ion-pair
`chromatographic techniques with chiral counter-ions [83-85] and the use of an
`cx1-acid glycoprotein (EnantioPac®) column [86]. Recently , very impressive results
`were obtained by Okamoto et al. [87] with the use of a silica-based cellulose tris(3,5-
`dimethylphenylcarbamate) column. On this column five P-blockers (alprenolol 20d ,
`oxyprenolol 20e, propranolol 20b, pindolol 20c and atenolol 20f) were completely
`resolved without derivatization , by elution with hexane/2-propanol (9:1). Figure
`8.14 shows some of the results obtained. The chromatographic separation can easily
`
`A
`
`(o.
`
`3 . 87)
`
`B (a
`
`6 . 03)
`
`(+)
`
`( + )
`
`( - )
`
`0
`
`10
`
`0
`
`20
`
`40
`
`Fig. 8.14 - Optical resolution of (a) alprenolol and (b) oxyprenolol on a cellulose tris(3,5-
`dimethylphenylcarbamate) (silica supported) column. (Reprinted, with permission , from Y.
`Okamoto, M. Kawashima, R. Aburatani, K. Hatada, T. Nishiyama and M. Masuda, Chem.
`Lett., 1986, 1237. Copyright 1986, Chemical Society of Japan).
`
`be scaled up. Thus, with a preparative column (20x500mm) , lOOmgof 20b, 150mg
`of 20d and 400 mg of 20e, respectively , were completely resolved, each in a single
`run.
`Optical resolution of 20a and of 20b (as the oxazolidinones) has also been
`achieved on a semipreparative scale by LC on MCTA [88].
`The structurally related ephedrines (p-amino-aicohol structure) have also been
`studied. Direct resolution of the enantiomers of ephedrine and its metabolites (as
`N, 0 -PFP derivatives) by GC on "Chirasil-Val" was described in 1978 [89]. Later,
`ephedrine, pseudoephedrine and norephedrine were optically resolved as the
`oxazolidinone derivatives on an XE-60-L-valine-(R)-cx-phenylethylamide CSP [90].
`An interesting LC separation of ephedrine has been described by Wainer et al.
`[91]. By cyciization with 2-naphthaldehyde to form a naphthyl-substituted oxazoli(cid:173)
`dine , an efficient 1T-donating group was introduced (Scheme 8.8) and optical
`resolution could be achieved (although not completely) by the use of an (R)-N-(3 ,5-
`dinitrobenzoyl)phenylglycine column (Pirkle Type 1-A, 5 µm, Regis Co .,
`4.6 x 250mm) . The (1R ,2S)-enantiomer is eluted before the (1S,2R) enantiomer.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-25
`IPR2016-00379
`
`

`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-26
`IPR2016-00379
`
`

`
`Sec. 8.4]
`
`Studies of microbial and enzymatic reactions
`
`177
`
`( d)
`
`~ flurbiprofen
`_,©-1~ ""'"'"''"
`
`F
`
`Cl
`
`(f)
`
`23
`
`By conversion of the free carboxyl group into amide derivatives, good results
`have also been obtained with the use of other types of columns such as those based on
`covalent (R)-N-(3,5-dinitrobenzoyl)phenylglycine (Regis 'Hi-Chrom Reversible')
`as well as cellulose tris(phenylcarbamate )-coated macroporous silica (Daicel 'Chiral(cid:173)
`cel OC') [92,93]. For analytical purposes , however, the latter column was found to be
`less well suited, despite its good selectivity, owing to the low column efficiency. As
`their napthylmethylamide derivatives in particular, ibuprofen (23a) and fturbiprofen
`(23b) were well optically resolved on the Pirkle-type column with the use of 7.5%
`dioxan in hexane, permitting in each case a determination of ca. 0.1 % contamination
`by the other enantiomer.
`In Table 8. 7 the chiral LC techniques used for optical resolution of some common
`acidic drugs are summarized.
`
`8.4 STUDIES OF MICROBIAL AND ENZYMATIC REACTIONS
`With the progress of biotechnology, a growing interest in the use of enzymes and
`micro-organisms as catalysts for organic chemical conversions has emerged. Of
`particular importance in this respect is the possibility of achieving transformations
`with a high degree of stereoselectivity, to produce optically active compounds.
`Although there is already a wealth of empirical knowledge concerning the use of
`enzymes and cells for such purposes, the field and its potential are enormous. In
`particular, the results from microbial reactions are highly unpredictable and the need
`for small-scale screening work seems almost unlimited. Such studies were earlier
`hampered by a lack of really adequate techniques to monitor the progress of a
`stereoselective reaction. With the new developments in chiral chromatography it is
`now possible to determine , in a very simple way, the exact enantiomeric composition
`in a minute sample taken from an enzyme-catalysed reaction. The chromatographic
`peak areas are measured by an electronic integrator interfaced with the detector. By
`such techniques the progress of the reaction and its stereochemistry c<>,n be readily
`monitored, with a very small volume of sample.
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1015-27
`IPR2016-00379