Monthly Summary:
`
`DP4 Inhibitors
`
`The goal of the program is to discover small molecule inhibitors of dipeptidyl peptidase IV (DP4) for
`use in the treatment and prevention of diabetes.
`Inhibition of DP4 should prevent the degradation of
`GLP-1 and GIP, potentiating their actions in vivo. The remarkable in vivc potency of l":3MS—431285 in
`the acute rat piasma ED50 assay in spite of poor pharmacokinetic properties led us to investigate this
`compound’s metabolism and stability behavior. Results from a combination of studies indicate rapid
`conversion to an inactive species through the cycioisomerization mechanism depicted below, as well
`as conversion to a metabolite with a MW consistent with hydroxylatlon. Efforts are underway to
`identify the metabolite through analytical methods, as well as parallel synthetic pursuit of hydroxylated
`side—chain versions of BlviS—431285, expioring the likely sites of oxidation, postulated to be at either
`carbon of the vinyl moiety.
`
`\\_z
`
`H2N
`
`N T’ ‘-
`
`O
`
`ON
`
`BMS—4312B5
`DP4 K. = 12 nM
`E05,, (30 min) = 0.39 umoukg
`ED50 (4 h) = 3.2 urnollkg
`11,2 = 1.23 h, F = 5.3% (rat)
`
`0
`
`id
`
`N
`H
`
`NH
`
`Competitive Update: A new patent application from Novartis has appeared claiming N-substituted 2-
`cyanopyrroli-dines bearing substituted adamantyl groups as DP4 inhibitors. The best compounds
`show rat and human plasma DP4 |C50’s of 2.3 and 2.7 nl‘v1 respectively, and 64% increase in early
`insulin response to oral glucose chailenge in rats at an oral dose of 10 umollkg. Claims for these N-
`substitutecl glycine derived compounds do not overlap with our existing series. Phase I clinical data
`on the ProBioDrug compound P—32l98 is showing a ciean profile, with no changes in BP, pulse, or
`ECG in normals. Plasma DF'4 inhibition ranged from 90% to 20% of control with increasing close up
`to 240 mg. GLP~1 was eievated 2-3 fold, presumabiy at the highest dose. A single dose of F’-32l'98
`increased insulin over placebo with a concurrent decrease in glucose.
`
`H0
`
`H/fig
`
`CN
`
`2
`
`O
`
`P-2/98 {ProBioDrug)
`
`Detailed Report:
`
`DP4 Inhibitors
`
`P
`
`age
`
`f 4
`
`1
`
`0
`
`Astraleneca Exhibit 2188
`Mylan V. Astraleneca
`IPR2015-013-I0
`
`AstraZeneca Exhibit 2188
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 4
`
`

`
`Page 2
`
`The goal of the program is to discover small molecule inhibitors of dipeptédyl peptidase IV (DP4) for
`use in the treatment and prevention of diabetes.
`Inhibition of DP4 should prevent the degradation of
`GLP-1 and GIP, potentiating their actions in vivo. Previous SAR has revealed an exceedingly limiting
`steric environment surrounding the cyanoproline moiety of these inhibitors, and consequently the vast
`majority of recent analoging efforts has focussed on optimization through modification of the amino
`acid fragment. Furthermore, as a definitive ruling from legal regarding the opportunity for a proprietary
`position on non-methano-bridged cores is contingent upon pending in vivo results, nearly all analoging
`has centered on 3,4— and 4,5—methano-bridged cores, with 4,5-methano favored due to relative ease
`of synthesis.
`
`Exploration of SAR surrounding the amino acid fragment of DP4 inhibitors has revealed a strong
`favorable effect of B—branching. The activity of analogues with amino acid fragments where |3—tertiary
`and [3—quaterriary centers are part of a cycloalkyl ring have shown the greatest potencies of all, with in
`vitro E<i’s in the 3-10 nM range. A series of compounds were prepared exploring ring size and the
`nature of the 4"‘ B-substituent.
`
`H2)‘n
`
`R
`
`H2N
`
`N
`
`0
`
`CN
`
`BMS #
`
`440927
`
`440823
`
`442616
`
`442371
`
`431 289
`
`431285
`
`461 059
`
`461017
`
`395067
`
`429636
`
`430452
`
`440925
`
`459819
`
`‘
`
`n
`
`4
`
`4
`
`5
`
`5
`
`5
`
`5
`
`5
`
`5
`
`6
`
`6
`
`6
`
`6
`
`7'
`
`442378
`
`4—pyrany|
`
`R
`
`Et
`
`vinyl
`
`H
`
`Me
`
`Et
`
`Vinyl
`
`isopropyl
`
`isopropenyl
`
`H
`
`Me
`
`Et
`
`vinyl
`
`vinyl
`
`H
`
`DP4 K. n M
`
`15.2
`
`5.4
`
`4.1
`
`3.3
`
`8.1
`
`11.?
`
`14.6
`
`10.2
`
`6.2
`
`4.5
`
`1 5.7
`
`11.8
`
`13.9
`
`20.3
`
`Page 2 of 4
`
`Page 2 of 4
`
`

`
`Page 3
`
`Though in vivo work had been suspended during biology group relocation, some critical data was
`obtained for these two potent analogues. Pharmacokinetic measurements on BMS-431285 and BMS-
`431289 were obtained in rats. EMS-431285 showed a tug of 1.23 h, with 5.3% bioavailabiiity; BMS—
`431289 showed a 211,2 of 0.99 h, and 31% bioavailability. These compounds were also evaluated for
`cytotoxicily in a human hepatocyte assay and showed no cytotoxicity up to the maximum solubility
`tested (25 pg/mL).
`
`1 I ‘A,
`f.'!2F““ 13"!»
`'
`t-;—
`
`CN
`l \
`/ N
`
`16 f‘
`
`H N
`
`2
`
`N
`
`0
`
`cm
`
`E
`2
`
`H N
`
`0
`
`N
`
`cN
`
`(NH
`H/Y
`
`N
`
`0
`
`N
`
`CN
`
`
`
`I
`
`BMS-431235
`DP4 K, = 12 nM
`ED50 (30 min) = 0.39 umollkg
`ED50 (4 h) = 3.2 umollkg
`
`BMS-431289
`DP4 K, = 3 nM
`ED59 (30 min) = 7.2 umollkg
`E05,, (4 h) = 3.4 umoiikg
`
`NVP—DF'P-728 (Novartis)
`BMS-428245
`DP4 K; = 3 HM
`ED5o (30 min) = 4.7 umoflkg
`ED5o (4 I1) = 31 umollkg
`
`Despite the relatively low bioavailability and short half—life of BMS—-431285, this compound is nearly an
`order of magnitude more potent than any DP4 inhibitor tested to date in the acute rat plasma DP4
`inhibition model, both at the 30 min and 4 h timepoints. These findings suggested possible rapid
`metabolism to a more active compound in vivo and prompted further study.
`Incubation with both
`human and rat liver microsomes, as weii as measurements of compound stability in phosphate buffer
`solution led to two important discoveries. Microsomal incubation results in rapid (-<10“/o of parent
`remaining after 60 min) conversion to both a compound with a MW consistent with hydroxylation, as
`well as to a peak with identical mass. This "latter compound is aiso produced, though more slowly,
`upon standing in phosphate buffer, and is believed to be the product of cyctoisomerization resulting
`from amide bond rotation and subsequent attack of the free amine onto the nitrile. The cyclic amidine
`produced is known from earlier investigations to occur in solution over time upon liberation of the free
`base form of the compound, and has also been shown to be an inactive template.
`
`\/
`
`H2
`
`l\i
`
`O
`
`C
`El!
`N
`
`EMS-431285
`
`c H N o
`MoI.'\?vt.2;12§9.35
`
`0
`
`T'''"
`
`N
`
`N
`H
`
`NH
`
`C ‘HEW O
`M°'-W‘-l 2g935
`
`The current hypothesis is that, since formation of the cyclic amidine constitutes a siphoning off of
`active compound, the remarkable in vivo activity of BMS-431285 is likely attributable to the metabolite.
`It is further postulated that this metabolite either has far superior DP4 inhibitory potency, and/or is far
`less susceptible to cyclization to an inactive compound. and therefore maintains a higher plasma
`concentration of an active species for Eonger duration. Concerted efforts are underway to prepare
`oxygenated side—chains on the cyc|opentyl—containing analogues to attempt to elucidate the putative
`hydroxylated metabolites. Additionally, modeling efforts are focussing on conformational aspects of
`the current and planned DP4 inhibitors with respect to stabilizing a conformation less favorable for
`unwanted cycloisomerization. Heavy emphasis will be placed on gathering additional MAP data and in
`vivo data from a wider set of compounds related to BMS-481285 in the acute rat model.
`
`Page 3 of 4
`
`Page 3 of 4
`
`

`
`Page 4
`
`Competitive Update: A new patent application from Novartis has appeared claiming N-substituted 2-
`cyanopyrrolidines bearing substituted adamantyl groups as DP4 inhibitors for treatment of NIDDM.
`The best compounds show rat and human plasma DP4 |C50's of 2.3 and 2.7 nM respectively, and
`64% increase in early insulin response to oral glucose challenge inrats at an oral dose of 10
`micromol/kg. Claims for these N-substituted glycine derived compounds do not overlap with our
`existing series. Phase I clinical data on the ProBioDrug compound P—32f98 is showing a clean profile,
`with no changes in BP, pulse, or ECG in normals. Plasma DP4 inhibition ranged from 90% to 20% of
`control as the dose was increased (7.5, 15, 30, 60, 120, 240 mg). GLP-'1 was elevated 2-3 fold,
`presumably at the highest dose. A single dose of P-32/98 increased insulin over placebo with a
`concurrent decrease in glucose.
`
`EB i
`HO
`
`0
`
`CN
`
`8
`
`L;
`
`HZN
`
`0
`
`P-2/98 (ProBioDru g)
`
`Page 4 of 4
`
`Page 4 of 4