Available online at www.sciencedirect.com
`
`Bioorganic & Medicinal Chemistry Letters 17 (2007) 6476–6480
`
`Potent non-nitrile dipeptidic dipeptidyl peptidase IV inhibitors
`
`Ligaya M. Simpkins, Scott Bolton, Zulan Pi, James C. Sutton, Chet Kwon, Guohua Zhao,
`David R. Magnin, David J. Augeri, Timur Gungor, David P. Rotella, Zhong Sun,
`Yajun Liu, William S. Slusarchyk, Jovita Marcinkeviciene, James G. Robertson,
`Aiying Wang, Jeffrey A. Robl, Karnail S. Atwal, Robert L. Zahler,
`Rex A. Parker, Mark S. Kirby and Lawrence G. Hamann*
`
`Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08543-5400, USA
`
`Received 14 September 2007; revised 26 September 2007; accepted 27 September 2007
`Available online 1 October 2007
`
`Abstract—The synthesis and structure–activity relationships of novel dipeptidyl peptidase IV inhibitors replacing the classical
`cyanopyrrolidine P1 group with other small nitrogen heterocycles are described. A unique potency enhancement was achieved with
`b-branched natural and unnatural amino acids, particularly adamantylglycines, linked to a (2S,3R)-2,3-methanopyrrolidine based
`scaffold.
`Ó 2007 Elsevier Ltd. All rights reserved.
`
`Dipeptidyl peptidase IV (DPP-IV) is an exopeptidase
`ubiquitously expressed in mammalian tissues, specifi-
`cally on epithelial and endothelial cells and lympho-
`cytes, which specifically cleaves dipeptides from the
`amino terminus of peptide substrates with proline or
`alanine at the penultimate position.1 DPP-IV is respon-
`sible for the degradation of several important incretin
`hormones, most notably the gut hormone glucagon-like
`peptide-1 (GLP-1) which is released post-prandially
`from the L-cells of the intestine, and acts to potentiate
`glucose-stimulated insulin secretion resulting in the low-
`ering of plasma glucose.2 Due to DPP-IV’s actions, the
`circulating half-life of GLP-1 is <90 s. Several DPP-IV
`inhibitors have reached late stages of clinical develop-
`ment (Fig. 1), including the dipeptidic inhibitors vildag-
`liptin3
`saxagliptin,4
`and
`and
`the
`non-peptidic,
`structurally novel sitagliptin5 and alogliptin.6 Robust
`antidiabetic efficacy has been demonstrated clinically
`with DPP-IV inhibitors, and the most advanced com-
`pound has recently gained FDA approval for treatment
`of type 2 diabetes.7
`
`Although multiple distinct chemical classes of DPP-IV
`inhibitors have been disclosed spanning diverse struc-
`
`Figure 1. Clinically advanced DPP-IV inhibitors.
`
`tural types,8 some of the most potent compounds to date
`are those containing a proline mimetic cyanopyrrolidine
`P1 group.9 This enhanced potency is thought to be due
`in part to a transient covalent trapping of the active site
`Ser630 hydroxyl of DPP-IV by the nitrile group, result-
`ing in delayed dissociation kinetics and slow-tight bind-
`ing of certain inhibitors.10
`
`Keywords: DPP4; Serine protease; Diabetes; Non-nitrile; GLP-1.
`* Corresponding author. Tel.: +1 203 677 6948; fax: +1 203 677
`7884; e-mail: lawrence.hamann@bms.com
`
`Along with this potency enhancement, chemical stability
`issues had been noted with early generations of nitrile-
`based inhibitors. While these issues were largely resolved
`
`0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
`doi:10.1016/j.bmcl.2007.09.090
`
`HO
`
`H2N
`
`N
`
`CN
`O
`saxagliptin
`(BMS) phase III
`O
`
`N
`
`CN
`
`NH
`
`O
`vildagliptin
`(Novartis) NDA
`
`HO
`
`F
`
`F
`
`F
`
`N
`
`N N
`
`O
`
`NH2
`
`CN
`alogliptin
`(Takeda) phase III
`
`N
`
`N
`
`N
`
`CF3
`
`NH2
`
`O
`
`N
`
`sitagliptin
`(Merck) approved
`
`Page 1 of 5
`
`AstraZeneca Exhibit 2014
` Mylan v. AstraZeneca
` IPR2015-01340
`
`

`
`L. M. Simpkins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6476–6480
`
`6477
`
`a
`
`O
`
`O
`
`10
`
`EtO2C
`
`CO2Et
`
`11
`
`d
`
`b, c
`
`O
`
`OO
`
`N
`Bn
`13
`
`O
`
`12
`
`O
`
`9
`
`O
`
`e, f
`
`+
`
`O
`
`NH
`
`6
`
`Cl
`
`Scheme 1. Reagents and conditions: (a) NaH, toluene, rt, 2.5 h, 35 °C,
`1.5 h; (b) NaOH, reflux, 8–10 h; (c) Ac2O, reflux 40 min; (d) BnNH2,
`toluene, 180 °C, 1.0 h, 150 °C, 20 h; (e) Red-Al, Et2O, 0 °C, 70 min,
`reflux 3 h, then rt, o.n.; (f) i—10% Pd/C, CH3OH, HOAc, 40 psi, 4 d,
`ii—4.0 N HCl/dioxane.
`
`diacid which was converted to the methanosuccinic
`anhydride 12 by heating in Ac2O. Reaction of 12 with
`benzylamine in toluene gave the corresponding benzyl
`azabicyclohexane-2,4-dione 13. Finally, reduction of
`the imide with Red-Al followed by catalytic hydrogena-
`tion in the presence of 10% Pd/C effected N-debenzyla-
`tion to give the desired 3,4-methanoproline 8 which
`was obtained as the HCl salt by filtering the methanolic
`solution directly into a solution of 4.0 N HCl in dioxane.
`
`The initial syntheses of enantiomeric 2,3-methanopyrr-
`olidines 7 and 8 utilized a stereorandom construction
`of a racemic 2,3-methanopyrrolidine, which was subse-
`quently coupled to a suitably protected homochiral ami-
`no acid prior to resolution at
`the analogue stage
`(Scheme 2). Commercially available Cbz-protected
`LL-proline (14) was oxidatively decarboxylated by treat-
`ment with iodobenzene diacetate and elemental iodine
`in CH2Cl2, followed by stirring in methanol to provide
`the racemic protected 2-methoxypyrrolidine 16 in 77%
`yield, along with the corresponding hydroxy product
`15 (11%). The hydroxy product could be recycled by
`quantitative conversion to the desired methoxy com-
`pound 16 by treatment with pyridinium p-toluene sulfo-
`nate (PPTS)
`in MeOH. Dehydration of methoxy
`compound 16 was achieved by treatment with Hunig’s
`base and TMSOTf to give protected dihydropyrrole 17
`in 81% yield. Standard cyclopropanation conditions
`
`CO2H
`
`Cbz
`
`N
`
`a, b
`
`Cbz
`
`N
`
`14
`
`HN
`7 (2S,3R)
`8 (2R,3S)
`
`f, g
`79%
`
`Cbz
`
`N
`
`18
`
`c
`quant.
`
`Cbz
`
`N
`
`+
`
`OH
`
`15
`11%
`
`OCH3
`
`16
`77%
`
`e
`78%
`
`d
`81%
`
`N
`
`Cbz
`
`17
`
`Scheme 2. Reagents and conditions: (a) iodobenzene diacetate, I2,
`CH2Cl2, rt; (b) MeOH, rt; (c) PPTS, MeOH, rt, 20 h; (d) TMSOTf,
`N,N-diisopropylethylamine, CH2Cl2, 0 °C; (e) diethylzinc, ClCH2I,
`Et2O, 0 °C to rt; (f) H2, 10% Pd/C, HCl, EtOH; (g) chiral HPLC
`resolution.
`
`with more advanced molecules, we sought to understand
`whether adequate potency might be achieved in a dipep-
`tidic inhibitor without a serine trap in order to obviate
`this potential chemical stability issue altogether. These
`efforts were initially both guided and tempered by two
`fundamental assumptions derived from extensive inter-
`nal and external structure–activity relationship (SAR)
`analysis: (1) that very strict steric constraints exist
`around the pyrrolidine ring of cyanopyrrolidide-based
`inhibitors, with only hydrogen,11 fluoro,12 acetylene,13
`nitrile,14 or methano15 substitution permitted; and (2)
`that presence of a nitrile moiety on the pyrrolidine ring
`is critical to achieving potent activity. The overall strat-
`egy we pursued involved exploration of both P2 and P1
`residues of the dipetide mimics lacking a prolinenitrile
`moiety.
`
`The presumption that the presence of a nitrile group is
`critical to achieving potent DPP-IV inhibition in pyrrol-
`idine-derived P1 containing inhibitors is based on
`known thiazolidine-based inhibitors such as isoleucine
`thiazolidide (P32/98, Ki = 126 nM)16 and simple pyrrol-
`idine-based inhibitors
`such as valine pyrrolidide
`(Ki = 470 nM)17 and fluoropyrrolidides,18 which exhibit
`considerably weaker (typically >10-fold) potency than
`their respective cyanopyrrolidine analogues. Further-
`more, the des-cyano analogue of NVP-DPP728 was re-
`ported to have very weak potency (Ki = 15.6 lM).19
`These inhibitors also lack slow-binding kinetic proper-
`ties, as an additional consequence of their reduced affin-
`ity. Nonetheless, valine pyrrolidide was
`found to
`potentiate plasma levels of active GLP-1 and insulin in
`response to glucose,20 and isoleucine thiazolidide was
`shown to improve glucose tolerance in obese Zucker
`rats.21 Studies with these DPP-IV inhibitor
`series
`showed a preference for b-branched LL-amino acids for
`improved potency. We sought to probe the validity of
`this assumption regarding a requirement for a nitrile
`moiety for potent inhibition, and to evaluate whether
`DPP-IV inhibitors possessing bulkier P2 groups which
`more fully fill the S2 pocket of the enzyme (proven to re-
`sult in potency enhancements in the nitrile series, such as
`with saxagliptin) might experience enhanced potency
`with simple P1 groups. A partial list of those non-nitrile
`containing proline surrogates evaluated in this study is
`shown in Figure 2.
`
`Proline surrogates 1–5 are available commercially. Met-
`hanopyrrolidines 6–8 were prepared as follows. 3,4-Met-
`hanopyrrolidine 6 was prepared according to a known
`procedure (Scheme 1).22 Thus, ethyl chloroacetate (9)
`and ethyl acrylate (10) were reacted in the presence of
`NaH to give the diethyl cyclopropanedicarboxylate 11.
`Saponification with NaOH afforded the corresponding
`
`HN
`
`S HN
`
`5
`
`6
`
`HN
`
`7 (2S,3R)
`8 (2R,3S)
`
`HN
`
`N
`
`34
`
` (Δ2,3)
`
`HN
`
`n
`1 (n = 1)
`2 (n = 2)
`
`Figure 2. P1 Proline surrogates used in synthesis of non-nitrile DPP-IV
`inhibitors.
`
`Page 2 of 5
`
`

`
`6478
`
`L. M. Simpkins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6476–6480
`
`(diethylzinc, chloroiodomethane) to give the methano
`product 18, followed by deprotection of the Cbz group
`under acidic conditions, afforded the racemic 2,3-met-
`hanopyrrolidine 7/8 as the corresponding HCl salt in
`62% overall yield for the two steps.
`
`In a second generation synthesis, the desired 2S,3R-ste-
`reoisomer 7 could be obtained in optically pure form by
`a formal deamidation of a key intermediate used in the
`preparation of saxagliptin (Scheme 3). Beginning with
`L-4,5-methanoprolinamide (19),15 protection of the pro-
`line nitrogen was accomplished using benzyl bromide
`and Hunig’s base in CH2Cl2 to give intermediate 20 in
`90% yield. Dehydration of the amide to the correspond-
`ing nitrile was achieved using TFAA and triethylamine
`in CH2Cl2 to give cyano compound 21 in 67% yield.
`Reductive removal of the cyano group by treatment
`with NaBH4 in aqueous ethanol afforded benzyl pro-
`tected methanopyrrolidine 22 in 60% yield. Removal
`of the benzyl protecting group was accomplished by
`treatment with a-chloroethyl acetyl chloride (ACE-Cl)
`in refluxing CH2Cl2 to give the desired (2S,3R)-2,3-met-
`hanopyrrolidine 7 in optically pure form as the HCl salt
`in 90% yield.
`
`The series of dipeptides in the present study were then
`prepared via standard peptide
`coupling (PyBOP/
`NMO or EDAC/HOBT/DMAP) of the appropriate P1
`proline surrogate with the various Boc-protected P2 LL-
`amino acids. Subsequent removal of the Boc-protecting
`group with TFA in CH2Cl2 or HCl in dioxane afforded
`inhibitors 28–40 as their corresponding TFA or HCl
`salts.23 All compounds were tested in vitro against puri-
`fied human DPP-IV under steady state conditions with
`gly-pro-p-nitroanilide
`as
`substrate
`as
`previously
`described (Table 1).4
`
`We systematically examined the influence of both P1
`and P2 moiety contributions to DPP-IV inhibitory po-
`tency, beginning with a survey of both natural and
`unnatural amino acids in the P2 position, while fixing
`the P1 subunit as the homochiral des-cyano methano-
`pyrrolidine (7) corresponding to saxagliptin. As shown
`previously for nitrile containing inhibitors, P2 amino
`acids with aryl (23–28) or polar (29–36) side-chains
`failed to exhibit any appreciable DPP-IV inhibition
`
`HN
`
`O
`
`NH2
`
`19
`
`Ph
`
`a
`
`90%
`
`N
`
`O
`
`NH2
`
`20
`
`Ph
`
`N
`
`b
`
`67%
`
`CN
`
`21
`
`HN
`
`7
`
`d
`
`90%
`
`Ph
`
`N
`
`22
`
`c
`
`60%
`
`Scheme 3. Reagents and conditions: (a) benzyl bromide, N,N-diiso-
`propylethylamine, CH2Cl2, rt; (b) trifluoroacetic acid anhydride, TEA,
`CH2Cl2, 0 °C; (c) NaBH4, EtOH/H2O, rt; (d) 1-chloroethyl chlorofor-
`mate, CH2Cl2, reflux.
`
`Table 1. Inhibition constants versus human DPP-IV for compounds
`23–60
`
`Compound P1
`
`P2-Xaaa
`
`b (nM)
`DPP4 Ki
`
`Saxagliptin
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`43
`44
`45
`46
`47
`
`48
`49
`50
`51
`52
`53
`54
`55
`56
`57
`58
`
`59
`
`60
`
`2S-CN-7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`8
`3
`7
`7
`
`7
`7
`8
`1
`4
`5
`(±)-2-Me-1
`Isoindole
`Indoline
`6
`7
`
`2
`
`7
`
`3-HO-Ad-Gly
`Ph-Gly
`Phe
`4-Cl-Phe
`His
`Tyr
`Trp
`Asn
`N-Ac-Lys
`Orn
`Ser
`O-Me-Ser
`O-t-Bu-Ser
`homo-Ser
`Thr
`n-Bu-Gly
`Leu
`Neopentyl-Gly
`Val
`Ile
`Allo-Ile
`tert-Leu
`tert -Leu
`tert-Leu
`b,b-di-i-Pr-Ala
`3,3,5,5-tetra-
`Me-Ch-Gly
`3-HO-Ad-Gly
`3,5-di-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3-HO-Ad-Gly
`3,5-di-HO-Ad-Gly
`5,7-di-Me-3-HO-
`Ad-Gly
`5,7-di-Me-3-HO-
`Ad-Gly
`N-(3-HO-Ad)Gly
`
`0.6 ± 0.06
`>10,000
`3653 ± 206
`877 ± 286
`>10,000
`3007 ± 180
`>10,000
`>10,000
`>10,000
`>10,000
`>10,000
`>10,000
`>10,000
`>10,000
`>10,000
`3257 ± 453
`>10,000
`1010 ± 146
`1065 ± 485
`530 ± 36
`731 ± 76
`356 ± 68
`>10,000
`>10,000
`112 ± 9
`152 ± 21
`
`10 ± 3
`14 ± 7
`1944 ± 334
`49 ± 8
`3311 ± 563
`28 ± 3
`>10,000
`1420 ± 196
`>10,000
`270 ± 58
`2.9 ± 0.5
`
`607 ± 27
`
`3081 ± 790
`
`a All P2 amino acids bear
`a-stereocenter.
`b All Ki values are mean ± SD of at least triplicate determinations.
`
`the natural LL-configuration at
`
`the
`
`(Kis P1 lM). Also consistent with findings in the anal-
`ogous nitrile series, a strong potency dependence on
`b-branching in the P2 side-chain was revealed in com-
`parison of those simple alkyl side-chains with and with-
`out b-branching (compare 37–39 vs. 40–43). Increasing
`the steric bulk of the b-branched substituents gave only
`modest incremental enhancement of potency (46, 47). In
`all of these cases, it appeared that potency versus the
`corresponding nitrile series suffered an approximately
`20- to 50-fold loss.
`
`Combining our optimized P2 group present in saxaglip-
`tin (3-hydroxyadamantylglycine) with the 2S,3R-met-
`hanopyrrolidine 7 gave compound 48, with potency
`equivalent to that of some of the most active nitrile-con-
`taining inhibitors in the clinic (Ki = 10 nM); similar
`
`Page 3 of 5
`
`

`
`L. M. Simpkins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6476–6480
`
`6479
`
`potency was observed in the dihydroxy compound 49
`(Ki = 14 nM). As was noted for the nitrile-containing
`series, a strong stereochemical preference was main-
`tained for the methano bridge bearing the 2S,3R-config-
`uration (compare 43 with 44 and 48 with 50). The
`apparently unique potency enhancing properties of this
`bulky P2 unit are further demonstrated with simple pyr-
`rolidine (51) and thiazolidine (53) P1 groups, though the
`3- to 5-fold diminished potency compared with 48 serve
`to validate the role of the methano bridge in this precise
`regio- and stereochemical orientation (compare with 57)
`in favorably contributing to DPP-IV binding affinity. It
`is noteworthy that strict steric constraints exist in the S1
`pocket, such that even simple methyl substitution as in
`54 essentially destroys all activity. The most potent com-
`pound in the series was obtained by packing further
`bulk into the S2 pocket (58, Ki = 2.9 nM), though a
`change as subtle as opening the bicyclic methanopyrroli-
`dine to a piperidine (59) results in a 200-fold drop in po-
`tency. Interestingly, methanopyrrolidine analogues with
`N-linked substitution analogous to vildagliptin (60)
`failed to demonstrate any significant DPP-IV inhibitory
`activity.
`
`The unique structural features imparted to inhibitors by
`the hydroxyadamantylglycine P2 group appear to be
`capable of conferring significant potency to non-nitrile
`compounds, though limited by the same narrow steric
`and stereochemical requirements shown for nitrile-con-
`taining inhibitors. Interestingly, several of the more po-
`tent analogues in this series have retained some slow
`binding kinetic properties, despite the lack of a nitrile
`5/s for saxag-
`(dissociation rate increases from 4.6 · 10
`3/s for compound 48 at 25 °C, unpub-
`liptin to 2.0 · 10
`lished results). Compound 48 maintains potent and fully
`efficacious antihyperglycemic effects in rodent models
`and mirrors the PK and safety profiles of clinical lead
`compound saxagliptin, yet is incapable of undergoing
`degradative cyclization. As previously discussed, this
`inhibitor also shows uniquely potent inhibition relative
`to other non-cyano compounds, suggesting that the
`summation of contributions to the binding energy of
`this compound in the active site is largely dominated
`by the P2 hydroxyadamantylglycine moiety. These stud-
`ies have shown the development of chemically more sta-
`ble and potent DPP-IV inhibitors in the low nM range,
`specifically compounds 48, 49 and 58. Further studies
`examining in vivo pharmacological effects as well as bio-
`chemical and biophysical aspects of the binding interac-
`tions for these potent compounds will be the subject of
`forthcoming disclosures from these laboratories.
`
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`23. Data for compound 48 (HCl salt): HPLC (Phenominex
`Luna 3 l C18 4.6 · 150 mm, 95% A to 95% B (A =
`H2O + 0.05% TFA, B = CH3CN + 0.05% TFA, flow rate
`1 mL/min,
`linear gradient over 42 min) retention time
`
`13.37 min (97.9%); Chiral analytical HPLC (Chiralpak
`AD 10 l 4.6 · 250 mm, 80% heptane + 20% 1:1 EtOH–
`MeOH + 0.1% DEA, flow rate 1 mL/min, isocratic) reten-
`tion time 10.56 min (98.2% ee); LC/MS m/z 291 [M+H]+;
`1H NMR (D2O, 400 MHz) d 4.16 (s, 1H), 3.82 (ddd, 1H,
`J = 13.2, 10.3, 2.9 Hz), 3.48 (td, 1H, J = 6.2, 2.6 Hz), 2.94
`(dt, 1H, J = 13.1, 8.7 Hz), 2.14 (bs, 2H), 1.94–2.05 (m,
`1H), 1.88 (ddd, 1H, J = 12.4, 8.4, 3.3 Hz), 1.74 (ddd, 1H,
`J = 8.8, 11.4, 5.2), 1.3–1.73 (m, 12H), 0.74–0.85 (m, 1H),
`13C NMR (D2O,
`0.65–0.71 (td, 1H, J = 5.7, 2.6);
`100 MHz) d 167.3, 69.1, 59.6, 45.3, 45.1, 43.1, 39.7, 38.2,
`37.1, 36.8, 36.6, 34.5, 30.2, 30.1, 24.4, 18.9, 12.8; Anal.
`Calcd for C18H25N3O3Æ1.64 HClÆ1.33 H20: C, 54.56; H,
`8.16; N, 7.49; Cl, 15.57. Found: C, 54.42; H, 7.86; N, 7.35;
`Cl, 15.57. KF, 6.39.
`
`Page 5 of 5