Edited by
`
`
`
`
`
`
`jar.-os Fischer and C. Rob.-"n Car.-eflin
`
`
`
`
`Analogue-based Drug Discovery ll
`
`
`
`
`
`WILEY-
`
`VCH
`
`
`
`
`
`
`
`WILEY-VCH Verlag C.mbH 8; Co. KGaA
`
`Page 1 of 28
`
`Astraleneca Exhibit 2262
`
`Mylan v. Astraleneca
`IPR2015-01340
`
`Page 1 of 28
`
`AstraZeneca Exhibit 2262
`Mylan v. AstraZeneca
`IPR2015-01340
`
`

`
`Al] books publisllcd by Wilt-_\'-\«'(IH are carefillly
`
`
`
`
`
`
`
`
`]'.|10dl.lI.'1’d. !~:ewr1!Jc'h-15. illlthols rdiioréa and
`
`
`
`
`
`publisher dn nu! w:m:mI tlw In|i)n11.1Iiun LrJul.nnrI.l
`
`
`
`
`
`
`
`in thesr |xHJks.1IIt.|IIdi|:g!|:n-. hn-at as be frer nl
`
`
`
`
`
`
`
`
`
`
`errors. Rt".ldl'l"'- an‘ .In1\-used to kw.-p 111 mind that
`
`
`
`
`
`
`
`
`
`sl3'It'3'lli.'1'11..~'. dam. i1|Il!iIfJ!itIl:5. pmcodural dclailu or
`
`
`
`
`
`
`ulhcr |1¢*n1-5 111:3‘ lI1.'|d\‘I.'1"tt"III|_\' be inatulralr.-.
`
`
`
`
`
`
`
`
`
`Library ofcongress Clfd No.:
`
`
`
`
`
`
`
`.11.'IphL-d ior
`
`
`
`
`British Library Cataloguing-in-Puhiicalinn Data
`
`
`
`
`A ('.1.I:l|0QtI\' rt-mrd In: this houk us available Irum tllc
`
`
`
`
`
`
`
`
`
`British iihrary.
`
`
`
`BiIrliograp|1iciIIfotn1al.inn published by
`
`
`
`
`Ihe Delllsclle NI‘IioIIi||I'HiotI|elr
`
`
`
`The Dnllsdlv Natlanulllilaliolhn-k heats this
`
`
`
`
`publicmluul In tllru Dvulr-dw Naiaonnlbibling-nfie;
`
`
`
`
`
`dmaill-d |:i|1!1ugr.':phIc data .'m- available an the’.
`
`
`
`
`
`
`
`
`lIl|l‘lIl(‘1 at I1!l|r,rfdiIlI.d-Illmdx-.
`
`
`
`I
`20111 Wll FY-V('.|'l Vi-rlag CmbH 8: Co. KCaA.
`
`
`
`
`
`
`
`
`
`Wvmhnrl m
`
`
`
`
`MI rIg}1tsrv.\t'rvrd lIm'lm!:'ng Lllum: oftrallslntion inln
`
`
`
`
`
`nthrl iullpgxxugu-sq. Nu pan nfthu bank may be
`
`
`
`
`
`
`
`
`
`r\'f|rDdlIu’d In ulur Ilmn — by phcnzuprinfing.
`
`
`
`
`
`
`nlmafthu. or am nth:-r rm-ans — nor tmnsmilled ur
`
`
`
`
`
`
`tr.'ilI-.~i|Il1l'.'l.l ink) u r!'IilthiIIl' language without wrlttcrl
`
`
`
`
`
`
`prmnssmn from Hlr puhllslu-rs. Registered |1.1m(-ea.
`
`
`
`
`
`
`Iradvnurks. ("IL LL-wd Ill this back. even when nnl
`
`
`
`
`
`
`
`
`
`~:p¢-c:fiual|_\' maslu-d as mth. am not to be c'at1s:du-mi
`
`
`
`
`
`
`
`
`unpro!PI,'I¢-d In" law.
`
`
`
`
`
`
`Cover Design AsJ'ar:1 Drslgn. \l."uinhemI
`
`
`
`
`
`
`Typesathirlg T'huI'n:¢on Dxgilal. Haida. India
`
`
`
`
`
`Printing and Binding
`Strauss {]mbH. .\1'c1r1rnIJ:u'|:
`
`
`
`
`
`
`
`
`Frmlt-d III the F:'(h'r.1l Rcpubltc :1-fficrmaluy
`
`
`
`
`
`
`
`Pruitt-d on atid-[kw paper
`
`
`
`
`ISIN: ‘il?8- 1-32 73-32 1'}-W»3
`
`
`
`T|IeEditots
`
`
`
`Prof Dnjdna-s Fimchtr
`
`
`
`
`Iii-(I119: Plc
`
`
`Gyfimnii: ul NI
`
`
`1105 Budapest
`
`Hungary
`
`
`
`
`
`Pro)’. Dr. C. Rabin Gum!!!»
`
`
`
`
`
`Unive-rsily College lnrldcm
`
`
`Dl.‘[}IlTIl'Tll.’I1IDrcllfllltifilfy
`
`
`20 Cufdflll Slrcel
`
`
`
`Lilndon VICE H CAI
`
`
`
`
`Llnilrd Kingdom
`
`
`
`
`
`
`Suppanzd by
`
`
`
`Thr Imemauonai Lmon cu!" Pure and Applied
`
`
`
`
`Chem isrry {I U PACJ
`
`
`Chflni5lnr' and Human Hralth Dc\'i5io1I
`
`
`
`
`
`PO Box 1335?’
`
`
`
`Research Triangle Park. NC £??TI‘J-H5?
`
`
`
`
`
`USA
`
`
`
`
`
`
`Page 2 of 28
`
`Page 2 of 28
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`ipeptidyl Peptidase IV Inhibitors for the Treatment of Type 2
`
`Diabetes
`
`5 D
`
`
`
`
`
`
`jens-Uwe Peters and Patrizio Mattsi
`
`5.1
`
`Introduction
`
`
`
`
`
`
`
`
`
`
`
`
`The symptoms of diabetes mellitus, a metabolic disorder characterized by hyper~
`
`
`
`
`
`
`
`
`
`
`
`
`glycemia {abnormally high blood glucose} due to inadequate irlsuliil levels. have been
`
`
`
`
`
`
`
`
`
`
`described since antiquity. The intrnductimi of insulin replacement therapy for
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`diabetes in 1922 was :1 major feat in the history of medicine and was awarded with
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`the Nobel Prize in medicine in the following year. Later in the 19205. the firs! oral
`
`
`
`
`
`
`
`
`
`
`
`antidiabetic drugs [OADs} were introduced. Although they were imperfect and later
`
`
`
`
`
`
`
`
`
`
`
`
`
`withdrawn, they led to the recognition that two types 0fdi3l3€‘l'lC‘5 exist — the invenile
`
`
`
`
`
`
`
`
`
`
`
`
`
`type. 1'equi_ring insulin therapy. and the late-onset type, which also benefits from OAD
`
`
`
`
`
`
`
`
`
`
`
`
`
`treatment [1, 2]. The late—onset form. today known as type 2 diabetes, accounts for
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`more than 90% of all diabetic patients and affects about 4% of the world
`
`
`population [3].
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The treatment oftype 2 diabetes aims to normalize blood glucose levels by diet.
`
`
`
`
`
`
`
`
`
`
`exercise. and medication, and is monitorecl by measuring glycosyiated hemoglobin
`
`
`
`
`
`
`
`
`
`
`
`
`{HbA.L.} as a long~term marker of elevated blood glucose. The amount of Hl)A.,
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`refiects the average glucose level over the last 120 days {the life span ofred blood cells]
`
`
`
`
`
`
`
`
`
`
`
`
`
`and should be maintained below 7% {4]. Each percentage reduction in HbA ._ leads to
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`a 21% reduction of the risk for any diabetes-related end point {S}. Poorly controlled.
`
`
`
`
`
`
`
`
`
`chronic h)'perglyceInia causes microvascular damage. which affects organs with
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`delicate capillary systems such as the eyes and kidneys, and can lead to blindness and
`
`
`
`
`
`
`
`
`
`
`
`
`
`renal failure.
`[:1 addition. hyperglycemia leads to atherosclerosis of larger vessels.
`
`
`
`
`
`
`
`
`
`
`
`which increases the risk of myocardial infarction and stroke. An important compli-
`
`
`
`
`
`
`
`
`
`
`
`cation resulting frmn micro— and niacroangiopatliy are lesions of the lower limbs
`
`
`
`
`
`
`
`
`
`
`("diabetic foot") that may ultimately require amputation. Utiiortilriately. the tnaiurity
`
`
`
`
`
`
`
`
`
`
`
`
`
`ofdiabetic patterns do not reach recommended HbAh. levels and are therefore at risk
`
`
`
`
`
`
`
`
`
`
`
`
`of" developing, these disabling comorbidities. Furthermore. the prevalence of type 2
`
`
`
`
`
`
`
`
`
`
`
`diabetes has increased over recent years. mainly due to higher life expectancies and
`
`
`
`
`
`
`
`
`
`
`
`
`
`an increasing prevulerice ofobesity [3]. Several classes oi‘OADs have been introduced
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`into clinical practice since Lhe 1950s and are widely pI‘esr.‘I‘ibecl. However‘, they all
`
`
`
`
`
`
`
`
`
`
`
`
`
`come along with side effects such as hypoglvceniia, weight gain. or gastroiritestinal
`
`.|ntl (,'. ROIIIII C-.m('ilIII
`l'):s.a.-wt} H Edited lJ\' lfultrrs I-'I.t-rlwl
`-\r1;tli~pm'-i.Ictm.l Dni-3:
`
`
`
`
`
`
`
`
`
`
`tfurptrigiit
`\\-‘\'IItit1'!nt
`'' 1tIlt'|\.\'|l.i-"I"\r'CH Verlag Cmbtl 52 Cu KGJA.
`
`
`
`
`ISRN '}'."R-1-527-129-fllvfl
`
`
`Page 3 of 28
`
`
`
`Page 3 of 28
`
`

`
`110
`
`
`
`5 Dipeptidyl Peptidase lV lnl1ibitor5 for the Treatment of Type 2 Diabetes
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`problems. Moreover. they often fail to achieve sustained glycemic control. Thus. there
`is a critical unmet need for 0.405 with novel modes of action.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`In the late 1980s. several research groups could Show that the peptidic hormone
`
`
`
`
`
`
`
`
`
`
`
`
`
`GLP-l (glucagon~|ii(e peptide 1]. vvhicli
`is secreted by the I-cells of the intestinal
`
`
`
`
`
`
`
`
`
`
`
`epithelium in response to loud ingestion. is a potent stiniulator oufglucose-dependent
`
`
`
`
`
`
`
`
`
`
`
`
`
`insulin release. This finding raised hopes that exogenous (3Ll"—] might be used to
`
`
`
`
`
`
`
`
`
`
`
`stimulate the impaired insulin secretion in type 2 diabetic patients. Disappointingiy.
`
`
`
`
`
`
`
`
`
`
`single subcutaneous iniections of GLP-I were ineffective in normalizing blood
`
`
`
`
`
`
`
`
`
`
`
`
`
`glucose [6]. A few years later.
`it was discovered that DPP-IV [-dipeptidyl peptidase
`
`
`
`
`
`
`
`
`
`
`
`
`
`I V}. a serine protease first isolated in 1966. rapidly cleaves and inactivates GLP-1 [7].
`
`
`
`
`
`
`
`
`
`Several research groups recognized the implications ofthis finding:
`
`
`
`
`
`
`
`
`
`
`
`
`Inhibition of DPP-IV should prevent the rapid degradation of GLP-1 and should
`
`
`
`
`
`
`thus increase circulating G LP-I levels.
`
`
`
`
`
`
`
`Increased GLP-1 levels should enhance glucose-depetiderlt insulin secretion.
`
`
`
`
`
`
`leading to lower blood glucose levels.
`
`
`
`
`
`
`
`
`Consequently. DPP~lV inhibitors should have an antidia|)el:iL' effect.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The glucose-loweringfantidialietic effect of DPP-IV inhibitors was soon demon-
`
`
`
`
`
`
`
`
`
`
`strated in animals and httrnans and triggered enormous researc h activities tl'LrOugh-
`
`
`
`
`
`
`
`
`
`
`
`
`
`out the pharniaceutical industry in the first decade of the new millennium [8].
`
`5.2
`
`in Vitro Assays and Animal Models for the Assessment of DPP-IV Inhibitors
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The discovery of DPI’-[V inhibitors was facilitated by the availability of robust and
`
`
`
`
`
`
`
`
`
`
`
`
`high-throughput in uitro assays. which often rely on a simple chromogenic or
`
`
`
`
`
`
`
`
`
`fluorogenic readout. For instance. DPP-IV cleaves Ala-Pro-AFC. a peptidyl derivative
`
`
`
`
`
`
`
`
`
`of 7-amirio-4-trifluorometliylcoumarin [AFC]. and the green fluorescence of the
`
`
`
`
`
`
`
`
`
`
`
`
`cleavage product. AFC. can be distinguished from the violet-blue Fluorescence of the
`
`
`
`
`
`
`
`
`
`
`
`
`
`substrate {Figure 5.1). The cleavage of Ala~Pro-AFC serves as a measure of DP P-[V
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`activity in an in vitro assay. in which the candidate inhibitor is evaluated by its ability to
`
`
`
`
`
`
`
`
`
`
`
`suppress the formation of fluorescent AFC. Furfitermora. animal models with high
`
`
`
`
`
`
`
`
`
`
`
`
`
`relevance to the human disease state were available. For instance, the oral glucose
`
`
`
`
`
`
`
`
`
`
`
`
`
`tolerance test (0G'I'T} in diabetic rats measures the glucose excursion. or the insulin
`
`
`
`
`
`
`
`
`
`
`
`
`
`response. after an oral ingestion ofa standardized at-noun t ofglucose. and is equivalent
`
`
`
`
`
`
`
`
`
`
`
`
`to the OGTTused in the diagnosis ofcliabetes in liumans. The eilicaciousness ofDPP-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`[V inhibitors can be evaluated in such an animal model by their ability to reduce the
`
`
`
`
`
`
`
`
`
`
`glucose excursion aller their adtni.nistrat1'ori prior to the glucose chailenge.
`
`
`
`5.3
`
`SI.Il:IslIate—Based DPP-N Inhibitors
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Speculations about the relevance of DPP-IV in the processing of bioactive peptides.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`and its potential role .in diseases such as cancer and AI D8. might have provided much
`
`Page 4 of 28
`
`Page 4 of 28
`
`

`
`5.3 Substrate-Based DPP-W tiihibitors
`
`
`
`
`
`111
`
`
`
`
`HgNJfi-(lg / lalffip-.
`
`
`N
`
`
`H
`0
`Ala-Pro-AFC
`
`GF3
`
`o
`
`0
`
`
`
`
`
`CF3
`
`+ m
`
`
`
`H251
`
`o
`
`o
`
`o
`
`H2~J\r”?
`
`OH
`
`0 0
`Ala-Pro—0H
`
`
`
`AFC
`
`green fluorescent
`
`
`
`
`Figure 5.1 DPP-IV liberates AFC from its dipeptidyl derivative, Ala-Pro-AFC. The green
`
`
`
`
`
`
`
`
`
`
`
`
`fluorescence of the product is used as a readout in a DPP-IV inhibition assay.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ofthe impetus for DPP~lV inhibitor research in the 19805 [9]. At this time. the ACE
`
`
`
`
`
`
`
`
`
`
`
`
`inhibitor success story had just proven that substratebased design is a viable
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`approach to drug discovery. and it seems natural that this concept was also pursued
`
`
`
`
`
`
`
`
`
`
`in DPP-IV research. DPP-W is an endopeptidase that releases dipeptides from the N-
`
`
`
`
`
`
`
`
`
`
`
`
`
`terminus ofa wide variety ofpeptidic hormones. with a preference for prolinue at the
`
`
`
`
`
`
`
`
`
`
`penultimate position. This proline preference is pronounced in small substrates
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`[such as Ala-Pro-AFC. Figure S.1),e-ven ifthe1argerpeptideGLP-1 {3(iamino acids} is
`
`
`
`
`
`
`cleaved after an aianine [Figure 5.2}.
`
`
`
`
`
`
`
`
`
`
`In the early 1990s, several academic research groups disclosed dipeptide~|ike
`
`
`
`
`
`
`
`
`
`
`
`
`DPP-[V inhibitors. in which a pyrrolidine or a thiazolidine replaces the proliirie, and
`
`
`
`
`
`
`
`
`
`
`
`
`
`an attached amino acid with a free amino group mimics the N-tE'l‘l1‘1lTlt_l5 ofa
`
`
`
`
`
`
`
`
`
`
`
`
`
`substrate peptide [Figure 5.3}. The scissile peptide bond was either omitted. as in
`
`
`
`
`
`
`
`
`
`
`
`the prototypical DPP-IV inhibitor P32,v’98 [10]. or replaced by a functional group
`
`
`
`
`
`
`
`
`
`
`
`
`
`designed to mimic the proteolytic transition state or to covalently bind to the
`
`
`
`
`
`
`
`
`
`
`
`
`
`enzymes active site serine. For instance, prolineboronic acids such as 1 have been
`
`
`
`
`
`
`
`
`
`designed as transition-state analogues and are reversible. slow-binding inhibitors
`
`
`
`
`
`
`
`
`
`
`
`
`
`with activities in the low nanomolar range 111]. Phosphonates such as 2 are
`
`
`
`
`
`
`
`
`
`
`ir1'eversibleinhibitors. which form stable esters with DPPvlV's catalytically active
`
`
`
`
`
`
`
`
`
`
`
`serine. However, these early types ofserine-interacting inhibitors did initially not
`
`
`
`
`
`
`
`
`
`
`
`
`provide clear advantages over the noncovalent inhibitors, as they were too unstable,
`
`
`
`
`
`
`
`
`
`
`
`too unseiective. or did not show a substantially improved activity. Nevertheless.
`
`
`
`
`
`
`
`
`
`
`
`the boronic acid dutogliptiu. a DP?-[V inhibitor discovered by Phenomix, has
`
`Haw‘ ® ®®@
`
`
`cleavage
`
`by DPF’-IV
`
`
`
`
`
`
`Figure 5.2 DPP-IV cleaves GLP-"I at the penultimate position from the N—terminus.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Page 5 of 28
`
`Page 5 of 28
`
`

`
`112
`
`
`5 Dipeptidyl Peptidase W inl1i'bi£ors_fi3r the Trentirierit of Type 2 Drab.-ztcs
`
`
`
`
`
`
`
`
`
`
`
`
`Cs
`
`HQN
`
`0
`
`0
`
`B{0H)__.
`
`O
`
`PO(OPh)2
`
`F’32f98
`
`
`Kl: 123 nM
`
`
`
`Demuth at al.. 1991
`
`
`
`
`
`
`1
`
`
`
`IC:_.,[, = 20 nM
`
`Flentke et al., 1991
`
`
`
`
`
`2
`
`irreversible
`
`Powers et al., 1995
`
`
`
`
`
`
`5:
`
`F"
`N
`nfir ‘r
`0
`atom?
`
`>
`
`Dutogliptin
`
`
`IO“, : 25 nM
`
`
`Phenomix. 2005
`
`
`
`
`H N
`
`N
`
`0
`
`N
`
`
`
`3
`
`K: 2.2 nM
`
`
`
`Ferring. 1996
`
`
`
`
`Figure 5.3
`
`
`
`Early substrate-based E:-PP~|V inhibitors and dutogliptin.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`apparently overcome these lirniitations and entered phase 3 clinical development in
`20os[i2.13i
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`In 1994. a publication demonstrated that nitriles could be used as serine-inter-
`
`
`
`
`
`
`
`
`
`
`
`
`acting motifs in inhibitors: ofpirolyl endopepticlase {PEP}. a serine protease related to
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`DPP—lV [14]. So far, nitrilea had only been known to he cysteine protease il‘|l1ll)'li()l‘!-i.
`
`
`
`
`
`
`
`
`
`
`
`but were regarded unreactive to typical serine protease.-;. This surprising i'lt'ILli1‘lg
`
`
`
`
`
`
`
`
`
`
`
`
`
`prompted Sherwin Wilk's reSi?aI‘cl1 group at the City University of New York, and
`
`
`
`
`
`
`
`
`
`
`
`researchers working with Paul D. Jenkins at Ferring Pliarmaceuticals. to introduce
`
`
`
`
`
`
`
`
`
`
`nitriles into their Sl.1l)Stl‘alI’;’-lJ£I:S€'d DPP-IV inhibitors [15-17]. These new Cyanop}-‘r-
`
`
`
`
`
`
`
`
`
`
`
`
`
`rolidine-type DPP-IV inl'lll'Jit0T'S. For example, 3 [Figure 5.3]. turned out to have an
`
`
`
`
`
`
`
`
`
`
`approxiiiiately I00-Fold improved inhibitoiy potency. and additionally both a good
`
`
`
`
`
`
`
`selectivity profile and an acceptable chemical stability.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Up to this point.
`the role of DPP-[V in glucose homeostasis was not fully
`
`
`
`
`
`
`
`
`
`
`
`
`recognized. Rolfhzientleiii et ill. from the University ofKiel had alreacly demonstrated
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`in 1993 that Gl.P-1 is a sulastmte of DPP-[V in vitro {7]. but this did not nec.e5sai‘ily
`
`mean that DPP-IV would be the main metabolic enzyme ol‘C LP-I in vi1ro.Ach.lal1y,3
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`was proposed as a potential i1‘.nmunomodulator. as DPP—lV is identical to CD26, a
`
`
`
`
`
`
`
`
`
`
`
`
`
`component of the T-cell receptor complex. In 1995. lens Holst and CUWOl'l~ZE’t'S from
`
`
`
`
`
`
`
`
`
`
`
`
`the Univer.~_'-ity oi'CoperIhagen concluded from their studies that DPP-IV is respon-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`sible. at least in part. For the obsewed rapid degradation OFGLP-I in humans and
`
`
`
`
`
`
`
`
`
`
`
`
`
`proposed that inhibition of‘DP'P-IV could be a useful adjunct in the management of
`
`
`
`
`
`
`
`
`
`
`
`type 2 diabetes [19]. Shortly tliereaiter. a collaborating team oi5ciei1I_ists workingwitli
`
`
`
`
`
`
`
`
`
`
`
`l-lans;—Ul:'ich Demuth irorn the University oilialle and Ci1|'lS'(Opl‘tE't' H.S. Mclntosh
`
`
`
`
`
`
`
`
`
`
`
`
`and Ray A. Pederson from the University of British Columbia patented DPP-IV
`
`Page6of28
`
`Page 6 of 28
`
`

`
`5.3 SubstraIr—Bas£d DPPJV inhibitors
`
`
`
`
`
`‘H3
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`inhibition as a method to tower blood glucose [£01. The patcnt applit::ttiI:'m was
`
`
`
`
`
`
`
`
`
`
`
`
`
`disclosed in 1997 and demonstrated that DPP-IV inhibition with l’32,t‘J8 did indeed
`
`
`
`
`
`
`
`
`
`
`
`
`
`improve glucose tolerance in rats. Demuth. wlto had spent most of his academic
`
`
`
`
`
`
`
`
`
`
`
`
`career working on DPP-W. would later start the biotech company. Prohioclrug. to
`
`
`
`
`
`
`
`
`
`
`
`
`
`exploit this invention and to bring P32,-'98 into the clinic. The imp1'0ven'tent in
`
`
`
`
`
`
`
`
`
`
`
`
`;__=,Iucosu.* tolerance by P32f98 was then reproduced in human Iioaltlty volttnteeI'-_-: and
`
`
`
`
`
`
`
`
`
`
`diabetic patients. P32,-‘98 and the epimcric allo-isoieucvl-tlriazolidiclc were licensed to
`
`
`
`
`
`
`
`
`
`
`
`
`Merck in late 2000. However. development ofboth cornpouncls was Lliscorltinued in
`
`
`
`
`
`
`
`
`
`
`
`February 2001. after Merck had identified tmaccoptable toxicity profiles for both
`
`
`
`
`
`
`
`
`
`
`
`compounds. Later. insufficient sc-lectivity over the related dipeptidases D P P-8 and for
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`DFP-9 was postulated to be the reason for the observed toxicities [ll ]. At that time.
`
`
`
`
`
`
`
`
`
`
`Merck had already identified Huoropyrrolidinte 4 [Figure 5.4} as 3 potential devel-
`
`
`
`
`
`
`
`
`
`
`
`
`
`opment compound. Because tho rationale for subtype selectivity was compelling. 4
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`was reiectecl on the basis of a selectivity of only S0-Fold over DPP-8 and DPP-‘J. and
`
`
`
`
`
`
`
`
`
`medicinal chemistry focused on l-ITS-based DPP-[V inhibitors. which culminated in
`
`
`
`
`
`
`
`
`
`
`
`
`
`the discovery of sitagliptin {see Section 5.4). Furtlter exploration of the substrate
`
`
`
`
`
`
`
`
`
`
`
`
`
`analogue series provided 5. with a selectivity of ‘>10 000-fold over DI’?-819 [22]. This
`
`
`
`
`
`
`
`
`
`
`compourtd was brought forward as a backup for sitagliptin [23].
`
`
`
`
`
`
`
`
`
`Another potent and selective difluoropyrroiicline derivative. PF-00734200. has
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`been discovered by Pfizer. This compound was reported to be in phase 2 clinical
`
`
`
`
`
`
`studies in September 2008 [.24. 15}.
`
`
`
`
`
`
`
`
`
`
`
`Dtlri rig this time, the cyanopyrrol id ines originally discovered by Sherwin Will: and
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`the group at Ferring had become the most popular class of DP!’-W inhibitors. as
`
`
`
`
`
`
`
`
`
`
`
`
`
`ittdgecl by the number of patent applications [18]. While the SAR around the
`
`
`
`
`
`
`
`
`
`
`
`
`cyanopyrroiidinc ring was ratlier limited. a wide variety of attached amirzto pcids
`
`
`
`
`
`
`
`
`
`
`
`
`with lipophilic or polar. negatively or positively charged. side chains were tolerated.
`
`
`
`
`
`
`
`which provided ample room For proprietary structures.
`
`
`
`
`
`\
`N
`
`, N
`
`o
`
`N
`
`a
`
`O
`
`F
`
`F
`
`6
`N
`
`5
`
`
`
`K: 8.8 nM
`
`
`Merck. 2004
`
`
`
`
`PF-00734200
`
`
`
`
`ICW = 13 nM
`Pfizer. 2005
`
`
`
`
`
`OCFJ.
`
`o=_s.
`
`
`H
`H?“
`
`F
`
`(3
`N
`
`O
`
`
`
`4
`
`
`
`
`|C_¢.., = 35 NM
`
`Merck. 2004
`
`
`
`Figure 5.4
`
`
`
`Pyrrolidides without a serim.--interacting motif
`
`
`
`
`
`Page 7 of 28
`
`
`Page 7 of 28
`
`

`
`11-1
`
`
`
`5 Dipepridyl Peptidase IV lnlnbitors for the Treatment of Type 2 Diabetes
`
`
`
`
`
`
`
`
`
`
`
`
`
`:> 0:1“?
`
`N
`
`7
`
`
`low = 54 nm
`
`
`
`
`0
`
`t
`
`
`
`5
`
`ICE, = 2 nM
`
`
`
`
`
`::>
`
`N\ x/''\
`JCS
`
`N
`
`NW
`
`0
`
`no
`
`1N
`
`NVP-DPP728
`
`
`I05“ = 22 nM
`
`
`
`Novarti-5. 1998
`
`
`
`
`Scaffold change leading to N-alkylglycine DPP-IV inhibitors: NVP-DPW23 was
`Figure 5.5
`
`
`
`
`
`
`
`
`
`
`
`efficacious in a proof-oF—concepL trial.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`An important extension of this SAR was made already in 1996 by scientists at
`
`
`
`
`
`
`
`
`
`
`
`Novartis. Edwin B. Villhauer. a cthemistwith a long-standing interest in diabetes. was
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`looking for a new project when lens Holst‘s paper was published in 1995. Within a
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`few days. he and his colleagues had a DPP-IV project running. Cells that happened to
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`express DPP—lV were just available and provided an in 1"2l.lJ"O assay. A paper fron1‘|988.
`
`
`
`
`
`
`
`
`
`describing a DPP—[V substrate with sarcosine {N-methylglycinel as an N-terminal
`
`
`
`
`
`
`
`
`
`
`
`
`amino acid [26]. caught Villhauter's attention and led him to explore N-alkylglvcine
`
`
`
`
`
`
`
`
`
`
`
`
`Cyanopyrrolidines. in which the side chain ofthe pyrrolidine-attached amino acid is.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`forrnally. shified to the nitrogen atom (e.g.. 6 —~ 7, Figure 5.5} [27]. The novel N-
`
`
`
`
`
`
`
`
`
`alkylglycine cyanopyrrolidines were amenable to resin-based chemistry. which was a
`
`
`
`
`
`
`
`
`
`
`
`
`very popular technology in those years. enabling the preparation of 1300 diverse
`
`
`
`
`
`
`
`
`
`
`
`compounds within 7 months. Only a few inhibitors with low nanomolar activities
`
`
`
`
`
`
`
`
`
`
`were identified in this campaign. one of them carrying a {S-nitro-pyridin-2-yl}-
`
`
`
`
`
`
`
`
`
`
`
`aminoethyl substituent. Replacement ofthe nitro functionality by a nitrile then led
`
`
`
`
`
`
`
`
`
`
`
`
`to NVP~Dl’P728 {Figure 5.5) with an improved selectivity over DP?-ll and PPCE
`
`
`
`
`
`
`
`
`
`
`lpostproline cleaving enzyme}. which were then standard enzymes in DPP-W
`
`
`
`
`
`
`
`
`
`
`
`
`selectivity studies. Within only 9 months. the Novartis proiect team had identified
`
`
`
`
`
`
`
`
`
`
`
`
`a development compound. Clinical trials with NVP-DPP728 began in 1998. A First
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`phase 2 trial based on the then widely held paradigm that any type 2 diabetes patient
`
`
`
`
`
`
`
`
`
`
`
`treated with a DPP-IV inhibitor should experience an immediate benefit. gave
`
`
`
`
`
`
`
`
`
`
`
`disappointing results and almost stopped the p1'oiect. A detailed data analysis
`
`
`
`
`
`
`
`
`
`
`
`
`
`suggested that patients with a certain level of pancreatic beta cell activity might
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`beriefit over a longer time frame. A second trial designed with the hindsight from this
`
`
`
`
`
`
`
`
`
`
`
`
`analysis was a huge success: after 4 weeks of treatment. NVP-DPP728 reduced
`
`Page 8 of 28
`
`Page 8 of 28
`
`

`
`5.3 Substrate» Based DPP-W lmhilnutors
`
`
`
`
`
`‘H5
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`postmeal glucose excursion. fasting glucose. and 24 h mean giitcrm‘. For the First
`
`
`
`
`
`
`
`
`
`
`
`time. it was shown that chronic DPP-IV inhibition in diabetic patients was safe and
`
`
`
`
`
`
`
`
`
`
`also led to a reduction in Him“ levels [28].
`
`
`
`
`
`
`
`
`
`
`
`
`NVI’-DPP728's relatively short half-life of 0.85 h was initially not seen as a
`
`
`
`
`
`
`
`
`
`
`
`disadvantage. On the contrary.
`the many possible physiological roles. of DP!’-IV
`
`
`
`
`
`
`
`
`
`
`made it desirable for a proof-of-concept compound that any potential adverse effects
`
`
`
`
`
`
`
`
`
`
`
`would abate quickly after a discontinuation ofarlrninistration. DPP-IV cl»;-ave-s. at least
`
`
`
`
`
`
`
`
`
`
`
`
`
`in iritro. not only GLP-1 but also several peptidic hormones. neL1rot1'attsrnitters.and
`
`
`
`
`
`
`
`
`
`
`
`
`
`cheniokiues. Of particular concern was initially the fact that DPP-[V is identical to
`
`
`
`
`
`
`
`
`
`
`
`CD26. a surface protein on activated T-cells. which mediates stimulatory signals:
`
`
`
`
`
`
`
`
`
`
`
`lorttt nalely. it was found that NV P- D PP’/'28 had no immunosuppressant eliect. { Later
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`on it was shown that the enzymatic activity of DPP-IV is not required for T-cell
`
`
`
`
`
`
`
`
`
`
`
`
`Function.) It might have been envisioned that NV P—D PP728 could be a sltorl:—acting,
`
`
`
`
`
`
`
`
`meal-dependently administered drug to reduce postprandial glucose excursiotrt. Such
`
`
`
`
`
`
`
`
`
`
`
`
`
`a tretttment would allow an intermittent recovery of DP P—IV activity. and the normal
`
`
`
`
`
`
`
`
`
`regttlation of other potential DPP-W substrates.
`thus minimizing side eflects.
`
`
`
`
`
`
`
`
`
`
`
`
`
`However, a team of Novo Nordisk researclters, collaborating with the Miami School
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`of Medicine. demonstrated in 200] that :1 24 h infusion ofG1.P~1 over 7 days: gave a
`
`
`
`
`
`
`
`
`
`
`
`
`
`much better outcome for diabetic patients than a 16 h infusion. indicating that a 24-h
`
`
`
`
`
`
`
`
`
`
`
`
`
`blockade of DPP-IV was needed to maxitnize the therapeutic eilect I291. In 2002.
`
`
`
`
`
`
`
`
`
`
`
`Farting researchers published their results with the long—acting DPP-IV inltilrtitor FE
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`99901] {Figure 5.6), which dearly showed that full inhibition of DPP-IV over 24 it
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`gave the best resu.lts in animal models ofdialraetes [30|. In the following years, most
`
`
`
`
`
`
`
`
`
`
`
`
`companies therefore focused on inhibitors with high metabolic stability. and today all
`
`
`
`
`
`
`
`
`
`
`
`clinically proven inhibitors show '.~5tl% plasma DPP-[V inhibition over 24h.
`
`
`
`
`
`
`
`
`
`
`
`
`Apart from the demonstrated clinical efficacy and the facile synthetic access. there
`
`
`
`
`
`
`
`
`
`
`
`
`might be yet another reason wily the N-alkylglycine inhibitors became very popular
`
`
`
`
`
`
`
`
`
`
`
`
`tliroughout the industry in the Following years: it was generally perceived that they
`
`
`
`
`
`
`
`
`
`
`had a superior chemical stability. As already mentioned. cyanopyrrolidine lDPP-IV
`
`
`
`
`
`
`
`
`inltibitors. and other substrate-based inhibitors with an electropltilic serine-inter
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`acting motif. are chemically unstable in solution. This solution insetability is due to an
`
`
`
`
`
`
`
`
`
`
`
`intramolecular reaction between the amino function and the electrophilic motif. as
`
`
`
`
`
`
`
`
`
`
`
`
`
`depicted in Scheme 5.1. The short soiution half-life typically of a few hours was
`
`
`
`lN
`
`
`
`H-‘I0
`
`
`
`FE 999011
`
`
`
`
`K, = 3.8 nht
`
`
`Farting. 1996
`
`
`
`
`
`Figure 5.6 Studies with FE 999011 showed that sustained Inhibition oi'DFPvIV leads to best results
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`in animal models of diabetes.
`
`
`
`
`
`
`Page 9 of 28
`
`
`Page 9 of 28
`
`

`
`
`‘I16 |
`
`5 Di'pep:i'dvl .°epti::l'ase lV inhibitorsfor the Treatment ofType 2 Diabetes
`
`
`
`
`
`
`
`
`
`
`
`Ft
`
`
`
`HZN
`
`N
`
`0
`
`l l
`N
`
`cyelization
`0
`
`—a-in j\:
`H
`
`N
`
`M
`H
`
`NH
`
`Scheme 5.1 The limited solution stability of cyanopyrrolicline DPP-IV inl-nibitors 15 due to an
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`mtrarnolecular reaction between the mandatory amino and cvano rIJI"ICt|Gt12lI[tES.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`causing problems for Formulation and was n1adeI'espuII.silJle For the short in uivol1a|l'-
`
`
`
`
`life of some compounds.
`
`
`
`
`
`
`
`
`
`
`To ove:‘c.oIr1e Lhis limitation. many research groups explored Nalkyiglycines with
`
`
`
`
`
`
`
`
`
`
`
`sterically hindered amines. which would undergo cyclization less readily. Early on.
`
`
`
`
`
`
`
`
`
`
`
`
`Novartis scientists had identified an adamantyl derivative 8 [Figure 5.7}. which was
`
`
`
`
`
`
`
`
`
`
`
`
`
`one of" the I‘I't0S‘-I potent inhibitors discovered in their program. Also. the primary
`
`
`
`
`
`
`
`
`
`
`
`
`
`metabolites of‘ lhi.'~: compound were found to be highly active. Already in 1998.
`
`
`
`
`
`
`
`
`
`
`
`
`Villhauer syiitliesixed one ofthe putative metabolites. LAF-237. wllich turned out to
`
`
`
`
`
`
`
`
`
`
`
`
`have an excellent solution stability, potent lI‘Il‘tli'}il0I')' activity. and good selectivity over
`
`
`
`
`
`
`
`
`
`
`related enzymes [31]. The improved pharmacokinetic profile and longer lasting
`
`
`
`
`
`
`
`
`
`
`
`pharmacoclynamic eiiect oi‘ LAF-237 led to a replacement of Novartis frrnit-runner
`
`
`
`
`
`
`
`
`
`
`
`NVPDPP728. LA F-237 was later narnecl vildagliptin. in reference to Villhaut-r. its
`
`
`
`
`
`
`
`
`
`
`
`
`inventor [32]. Viidagliptin has heen. after sitagliptin. the second compound to obtain
`
`
`
`
`
`
`
`
`
`
`
`
`
`market approval in the European Union and other countries. In the United States.
`
`
`
`
`
`
`
`
`
`
`
`
`
`Novartis; has paused its eliorts to seek regulator}-’ approval after the FDA had
`
`
`
`
`
`
`
`
`
`
`
`
`requested additional data to address concerns about the tolerahility in patients with
`
`
`
`
`
`
`
`
`
`
`
`
`renal iinpairmenl and skin lesions in nonhuman primates ]3El| {although no skin
`
`H
`
`0
`
`on
`
`8
`
`lcm = 3 nM
`
`
`
`
`
`OH
`
`0
`
`cm
`
`LAF-237. vildagliptin
`
`
`lC_.,n = 3.5 nM
`
`
`Novartis. 2000
`
`
`
`
`
`
`
`
`
`
`cm
`
`HEN
`
`N
`
`0
`
`
`9
`
`K|=7nM
`
`
`
`
`:>
`
`
`
`HEN
`
`N
`
`0
`
`
`
`
`:{> “O3
`H9N'
`
`CN
`
`
`
`
`CN
`
`
`
`
`
`N
`
`
`
`o
`
`
`10
`
`K.:0.9 nN'|
`
`
`
`
`
`saxagliptin
`
`
`
`
`K,=0.6 nM
`B-MS. 2001
`
`
`
`Figure 5.? Discovery of vlldagliptin and Sattagllptln.
`
`
`
`
`
`
`
`
`
`Page 10 of 28
`
`Page 10 of 28
`
`

`
`Table 5.1 Chemical stabilities of primary amine inhibitors.
`
`
`
`
`
`
`
`
`
`
`
` Compound Halt’-life"
`
`
`
`5.3 Substrate-Based DPP-N inhibitors
`
`
`
`
`
`
`‘I1?
`
`
`
`3 (lllj._',tlI'I.’ 3.3:
`
`
`
`
`I"-ii ‘}‘J‘.iUl l {Figure 5.6]
`
`
`
`
`
`
`9 iFii.J.uI':'- 9.7}
`
`
`
`
`a}
`
`
`in ;1<|ut-nus butTer at pH ?_2: 39.3 (I.
`
`
`
`
`
`
`
`
`
`3 li
`
`)7 ll
`
`4.2 It
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`lesions have been observed in humans during clinical trials [67]]. Vilclagliptini is only
`
`
`
`
`
`
`
`
`
`
`
`moderately selective over DPP-8 and DPP-‘J. Following Ll1e highly publicized Merck
`
`
`
`
`
`
`
`
`
`
`study on the potential toxicities associated with DPP-8}‘) inhibition [21]. Novartis
`
`
`
`
`
`
`
`
`
`
`
`undertook long-term rodent toxicity studies with \.-ildagliptin at exposures that are
`
`
`
`
`
`
`
`
`
`
`
`
`high enough for complete inhibition of DPP-IV. DPP-8. and DPP-‘J. As vildagliptin
`
`
`
`
`
`
`
`
`
`
`
`
`
`did not display any of the toxicities observed with P32f98 and structurally related
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`molecules. the toxicity ofthe compounds studied by Merck is more likely the result of
`
`
`
`
`
`
`
`
`
`
`
`
`unidentified oft"-target effects that are independent ol‘DPl’-3,39. and the relevance of
`
`
`
`
`
`isolbrm selectivity remains unclear [34].
`
`
`
`
`
`
`
`
`
`
`
`Researchers at Bristol-Myers Squilih found that converting a tertiary (3. Figure 5.3}
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`to a quaternary alpha-carbon {I-‘E 9991)! 1. Figure 5.6] improves the solution halif-life by
`
`
`
`
`
`
`
`
`
`
`
`
`fivefold (Table 5.1 1. The long~|asting pltarrnacodynamic effect or FE 99901 I might. at
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`least in part. be attributed to this improved solution stability. Also. the i ntroduction ofa
`
`
`
`
`
`
`
`
`
`
`
`
`methylene bridge into the cyanopyrrolidine ring leads to steric bull: that similarly
`
`
`
`
`
`
`
`
`
`
`
`
`improves the chemical stability [compare FE 999011 and 9. Table 5.1}. Molecular
`
`
`
`
`
`
`
`
`
`
`
`
`modeling demonstrated that these effects a re, in both cases. due to in tratnolec ular van
`der Waals interactions. These interactions disfavor a cis conformation of the amide.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`which is a prerequisite for cyclizatioti, and thereby increase stability [35]. These
`
`
`
`
`
`
`
`
`
`
`
`
`
`findings led the Bristol-Myers Squibb scientists. in striking analogy to the ellbrts at
`
`
`
`
`
`
`
`
`
`
`
`
`Novartis. to 10 with an adarnantyl substituent. This compound showed an excellent
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`plasma-DPP-IV inhibition after oral dosing in rats. despite a low liioavailability [2%l.
`This seemed to indicate that It] is converted into an active metabolite in vim. which
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`prompted the synthesis ofa l'I)'drt)K)' analogueas a putative metabolite. Quite si milnr to
`
`
`
`
`
`
`
`
`
`
`
`
`the vildagliptin story.
`it was found that this metabolite. later named saxagliptiri
`
`
`
`
`
`
`
`
`
`
`
`
`
`[Figure 5.7}. was highly potent and had an excellent solution stability llfii. Tlliis high
`
`
`
`
`
`
`
`
`
`
`solution stability. together with a relatively high distribution volume. tnrtkes s:ax:iglip-
`
`
`
`
`
`
`
`
`
`tinalong-actingDPP-IVinhibitor. Bflfilol-M)-'ErSSqLl‘ll.‘)lJ3]'1£lAStt‘3Z('t'll2CE1l'l£t\'nt‘5ilti1t'Erl
`
`
`
`
`
`
`
`
`
`
`
`the clinical development and filed a New Drug Application in 2008 [37].
`
`
`
`
`
`
`
`
`
`
`
`
`
`Other companies also came up quickly with N-alkylglyciites with a wide variety of
`
`
`
`
`
`
`
`
`
`
`
`
`quaternary N-suhstituents. T5-02.1. 11. and ABT-279 [Figure 5.8} are exam pies o