Discovery of Alogliptin: A Potent, Selective,
`Bioavailable, and Efficacious Inhibitor of
`Dipeptidyl Peptidase IVt
`
`Jrm Feng,+ Zhiyuan Zhang,+ Michael B. Wallace,+
`Jeffrey A Stafford,+ Stephen W. Kaldor,+ Daniel B. Kassel,+
`Marc Navre,+ Lihong Shi,+ Robert J. Skene,+
`Tomoko Asakawa,§ Koji Takeuchi,§ Rongda Xu,+
`David R. Webb,+ and Stephen L. Gwaltney, II*·+
`
`Takeda 3m Diego, Inc., 10410 S::ience Center Drive, 3m Diego,
`California 92121, and Pharmaceutical Rerearch Divison, Takeda
`Pharmaceutical Company Ltd., Osaka, Japan
`
`Recei ved January 26, 2007
`
`Abstract: Alogliptin is a potent, selective inhibitor of the serine
`protease dipeptidyl peptidase IV (DPP-4). Herein, we describe the
`structure-based design and optimization of alogliptin and related
`quinazolinone-based DPP-4 inhibitors. Following an oral dose, these
`noncovalent inhibitors provide sustained reduction of plasma DPP-4
`activity and a lowering of blood glucose in animal models of diabetes.
`Alogliptin is currently undergoing phase III trials in patients with type
`2 diabetes.
`The World Health Organization estimates the number of
`people with diabetes to be approximately 180 million. This
`number is projected to double by 2030. Type 2 diabetes (T2D)
`is a progressive disease characterized by high levels of glucose
`resulting from insulin resistance and impairment of insulin
`secretion. If left rmtreated, hyperglycemia may cause nephr(cid:173)
`opathy, neuropathy, retinopathy, and atherosclerosis. T2D causes
`significant morbidity and mortality and results in considerable
`expense to patients, their families, and society. 1
`Glucagon-like peptide-1 (GLP-1 (7-36 amide or 7-37)), a
`30-amino acid peptide hormone, is secreted by intestinal L-cells
`in response to meal ingestion and stimulates insulin secretion
`from j'J-cells while inhibiting hepatic glucose production. 2
`Furthermore, GLP-1 has been shown in mammals to stimulate
`insulin biosynthesis, inhibit glucagon secretion, slow gastric
`emptying, reduce appetite, and stimulate the regeneration and
`differentiation of islet j'J-cells3 Continuous infusion of GLP-1
`to patients with T2D results in significant reduction of blood
`glucose and hemoglobin A 1c levels 4 However, active GLP-1 is
`rapidly converted to inactive GLP-1 (9-36 amide or 9-37) by
`the serine protease dipeptidyl peptidase IV (DPP-4a), thus
`limiting its therapeutic practicality. Inhibition ofDPP-4 increases
`the levels of endogenous intact GLP-1. Consequently, inhibition
`of DPP-4 is rapidly emerging as a novel therapeutic approach
`for the treatment of type 2 diabetes. 5 Clinical proof of concept
`has already been established with DPP-4 inhibitors such as
`1-[[ (3-H ydroxy-1-adamanty l)amino ]acetyl]-2-cyano-(S)-pyrro(cid:173)
`lidine (LAF-237)6 and (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-
`dihydro[ 1 ,2,4]triazolo[ 4,3-a]pyrazin-7 (SH)-y 1]-1-(2,4,5-trifluo(cid:173)
`rophenyl)butan-2-amine (MK-0431) 7
`The active site ofDPP-4 is shown in Figure 1 with important
`residues labeled. Figure 2 shows the surface of the site. Using
`
`t Compound la has been deposited into the Protein Data Bank: PDB
`code 20NC.
`*To whom correspondence should be addressed. Phone: 858-731-3562.
`Fax: 858-550-0526. E-mail: stephen.gwaltney@takedasd.com.
`t Takeda San Diego, Inc.
`§ Takeda Pharmaceutical Company Ltd.
`a Abbreviations: DPP-4, dipeptidyl peptidase IV.
`
`C0~ NH-Tyr631
`/
`I o
`
`NC Jo
`
`N
`H2N'(rj">N
`
`I
`
`"""
`o
`
`Arg125
`
`Glu205 __
`Glu206 salt
`bridge
`
`J. Mecl. Chern. 2007, 50, 2297-2300
`
`2297
`
`Phe357 Or
`
`Figure 1. Active site of DPP-4.
`
`Figure 2. DPP-4 active site surface.
`
`51 pocket
`
`1a
`
`il 7t-stacking
`
`Tyr547
`Figure 3. Structure-based design of compound la.
`
`structure-based design, we hypothesized that a quinazolinone
`scaffold could effectively display groups known to interact with
`the active site residues ofDPP-4 8 As shown in Figure 3, placing
`the aminopiperidine motif at C-2 was predicted to provide a
`salt bridge to E205/E206 while a cyanobenzyl group at N-3
`was expected to effectively fill the Sl pocket (formed by V656,
`Y631, Y662, W659, Y666, and V711) and interact withArgl25.
`The carbonyl at C-4 was anticipated to provide an important
`hydrogen bond to the backbone NH of Tyr631, and the bicyclic
`
`<GJ 2007 American Chemical Society
`10.1021/jm0701041 CCC: $37.00
`Published on Web 04/19/2007
`
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`Mylan v. Boehringer Ingelheim
`IPR2016-01566
`Page 1
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`

`

`2298 Journal of Medicinal ChemiS:ry, 2007, Vol. 50, No. 10
`
`Letters
`
`b
`
`Scheme 1. Synthesis of Compounds la-ma
`0
`0
`R«OR
`ROCNH
`"'"' NAO
`NH2
`H
`3a-m
`2a-m
`Cl
`ROCN
`"'"' NACI
`4a-m
`
`c
`
`y
`'Q~X" "
`
`0
`ROCNH
`"'"' NACI
`5a-m
`
`y
`'o(J~:,~
`
`1a-m
`Sa-m
`a Reagents: (a) urea, 200 °C; (b) POCh, Me2NPh, reflux; (c) NaOH;
`(d) NaH, LiBr, 2-CNPhCH2Br; (e) 3-(R)-aminopiperidine, NaHC03,
`150 °C.
`
`Figure 4. Compound la in the active site of DPP-4.
`
`Table 1. Selected Data for Quinazolinones la-m y
`
`0
`
`5
`
`CN
`
`R~N
`7"'8 NAO.NH2
`
`DPP-8 RLM" HLM'
`1112
`IC"
`tl/2
`(min)
`(min)
`(uM)
`
`3A4d
`IC"
`(uM)
`
`compd
`
`R
`
`DPP-4
`IC50 (uM)
`0.013 ± 0.006
`la"
`>100
`>200
`H
`2.5
`2.0
`0.005 ± 0.0002
`lb
`>100
`1.8
`5-F
`125
`0.50
`0.004 ± 0.0008
`>100
`>200
`6-F
`31
`2.5
`lc
`0.005 ± 0.0008
`ld
`>50
`>200
`6-Cl
`40
`0.25
`0.015 ± 0.002
`>100
`NT" NT"
`7-Cl
`0.25
`le
`0.029 ± 0.011
`lf"
`>100
`8-Cl
`3.7
`80
`013
`0.013 ± 0.003
`>100
`17
`6,8-diCl
`64
`0.08
`lg
`0.005 ± 0.001
`lh
`>100
`6-Br
`34
`113
`0.25
`0.008 ± 0.0003
`1i
`>50
`6-0Me
`10
`82
`0.20
`0.004 ± 0.0008
`lj
`>100
`6-0Me, 7-F
`35
`136
`0.25
`0.019 ± 0.004
`lk
`>100
`6,7-di-OMe
`107
`146
`0.50
`0.030 ± 0.005
`11"
`>100
`8-0Me
`7.2
`61
`0.63
`0.018 ± 0.003
`lm
`>50
`6-F, 7-morpholinyl
`6.1
`33
`0.79
`a Racemic. b RLM = incubation with rat liver microsomes. c HLM =
`incubation with human liver microsomes. d 3A4 = cytochrome P450 3A4.
`e NT = not tested.
`
`heterocycle was predicted to n-stack with Tyr547. In addition,
`since the quinazolinone scaffold is well represented in bioactive
`natural products and drugs, we surmised that it would impart
`favorable physical properties to our inhibitors 9
`The syntheses of 1 a-m (Scheme 1) began with commercially
`available 2-aminobenzoic acids or esters 2a-m, which were
`heated with urea at 200 °C to generate quinazolinediones 3a(cid:173)
`m . Chlorination with POCh followed by selective hydrolysis
`gave 4a-m . Selective N-alkylation was performed using
`conditions reported by Curran and co-workers10 Displacement
`of the chloride in 6 with 3-(R)-aminopiperidine was performed
`in a sealed tube or in a microwave reactor.
`The compounds shown in Table 1 are potent DPP-4 inhibitors
`and demonstrate excellent selectivity over the related protease,
`DPP-8. Remarkably, the first compound synthesized in the
`quinazolinone series, la, is a 10 nM inhibitor of DPP-4. We
`obtained a cocrystal structure of this compound in the active
`site of DPP-4 (Figure 4)11 The interactions observed in this
`cocrystal structure were consistent with our design (compare
`Figures 5 and 3).
`Compound la suffers from a short metabolic half-life in the
`rat, making in vivo assessments difficult. Metabolite studies
`
`Figure 5. Compound la in the active site of DPP-4 with key
`interactions shown.
`
`100%
`
`80%
`
`60%
`
`c
`~ :;;
`40% ~
`
`20%
`
`0%
`
`10
`
`• % Inhibition
`• [1c]
`
`12
`Time(h)
`Figure 6. Plasma concentrations and DPP-4 inhibition in rats for lc
`(TFA salt, 10 mpk po).
`
`20
`
`24
`
`16
`
`using rat liver microsome preparations revealed that the short
`metabolic half-life in rat was due to oxidation of the A-ring
`phenyl group at position 6 or 7. To address this problem,
`fluorinated derivative lc was synthesized. This compound
`showed a 1 O-f old improvement in metabolic stability in the rat.
`Figure 6 shows the pharmacokinetic (PK) and pharmacodynamic
`(PD) profile of lc in the rat. 12 Selected PK parameters are listed
`in Table 2.
`
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`Mylan v. Boehringer Ingelheim
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`
`

`

`Letters
`
`Journal of Medicinal Chenistry, 2007, Vol. 50, No. 10 2299
`
`Table 2. Selected Rat PK Parameters for Compound lc (TFA Salt)
`dose, iv/oral (mg/kg)
`species
`ivl112(h)
`oral !112 (h)
`2.60 ± 1.85
`2.43 ± 0.31
`rat
`
`l/10
`
`AUCpo (Jlg h mL -1)
`7.56 ± 1.80
`
`20.61 ± 8.31
`
`2865 ± 632
`
`Table 3. Selected PK Parameters for Compound 10
`doses, iv/oral (rng/kg)
`species
`salt
`ivt112(h)
`2.93 ± 0.84
`dog
`HCl
`5.74 ± 1.70
`monkey
`benzoate
`
`1/3
`1.1/10
`
`oral !112 (h)
`3.04 ± 0.75
`5.66 ± 0.23
`
`AUCro (llg h rnL - 1)
`1.61 ± 0.51
`16.63 ± 2.52
`
`22.96 ± 7.81
`8.81 ± 1.07
`
`3508 ± 255
`2602 ± 561
`
`F(%)
`85 ± 20
`
`F(%)
`68 ± 22
`87 ± 13
`
`Scheme 2. Synthesis of 10
`NaH, LiBr
`
`0
`
`£NH
`Cl
`NA,O
`H
`
`9
`
`CN
`
`Br
`
`DMF I DMSO
`54%
`
`JNH
`Cl
`NA,O
`
`cC
`
`CN
`
`8
`
`NaH, CH3 1
`
`THF I DMF
`72%
`
`0
`
`£N,CH 3
`
`HQ·2HCI
`
`0
`
`fN'CH,
`
`Cl
`
`NA,O
`
`cC
`
`CN
`
`NH2
`NaHC0 3
`CH 30H, 100 oc. 2h
`then TFA
`70-76%
`
`H2ND NA,O
`
`• TFA
`
`))
`
`NC
`
`10
`
`(A) 300
`
`~250
`]:200
`~ 150
`
`0 100 J 50
`
`(B)
`
`:::l'
`
`so
`
`70
`liO
`
`30
`
`Time (mml
`
`.---::::::
`
`jso
`5!;40 e 30
`f20
`
`10
`
`(D) 90
`
`~80
`"'70
`~liO
`'6 so
`~
`;;; 40
`~ 30
`~ 20
`£ 10
`0
`
`As shown in Figure 6, there is a strong correlation between
`plasma levels of compmmd 1c and the level of DPP-4 inhibition,
`with a 10 mpk oral dose providing 50% inhibition of DPP-4
`activity after 12 h. Consistent with this effective in vivo
`inhibition, compound 1c (also known as Syrrxl06124) reduced
`glucose excursion following an oral glucose tolerance test
`(OGTT) in mice. 13
`Preliminary safety assessments of compound 1c included an
`Ames test, a safety pharmacology screen, and a 4-day rat
`toxicology study. The results of these assessments were favor(cid:173)
`able; however, compound 1c was found to inhibit CYP450 3A4
`
`6
`'ll
`~
`~"'
`8 E
`n'iio
`~~
`II:
`j
`
`W%
`
`W% ~
`ii
`~
`
`-
`
`Tmo (h)
`Figure 7. Plasma concentrations and DPP-4 inhibition in dogs for 10
`(HCl salt, 3 mpk po ).
`
`10000
`
`1000
`
`-8
`!!!
`~
`8 ~ 100
`~~
`
`.!!l
`II.
`
`120%
`
`- 100%
`
`BO%
`
`60%
`
`40%
`
`" 2 :a
`~
`
`10 -----------------------------------------------
`
`• %1nhlbidon
`• [10]
`------~----~----~----~----~----+0%
`12
`16
`20
`24
`Time (h)
`Figure 8. Plasma concentrations and DPP-4 inhibition in monkeys
`for 10 (benzoate salt, 10 mpk po).
`
`Cootrol
`
`0.01
`
`O.o3
`
`OJ
`
`03
`
`Figure 9. Effects of10 on plasma glucose (A), plasma immunoreactive
`insulin (IRI) (B), baseline (0 min)-adjusted AUC0- 6ornin of plasma
`glucose levels (C), and plasma IRI levels at 10 min after the oral glucose
`load (D) in glucose tolerance test of female Wistar fatty rats. These
`studies were conducted using alogliptin benzoate. Doses are normalized
`to reflect the amount of free base administered. Values are the mean
`and SD (n = 6): (*) p :S 0.025 vs control by one-tailed Williams'
`test; (#) p :S 0.025 vs control by one-tailed Shirley-Williams' test
`
`with an IC50 of 2 .5 ,uM and to block the bERG channel at
`micromolar concentrations.
`In an effort to improve upon 1c, we adopted two strategies.
`The first relied on modifications to the quinazolinone substit-
`
`I
`I
`j;,
`Iff
`
`""-.
`~~
`~~
`~,
`
`30
`
`60
`
`Tm(mn)
`
`l
`
`T
`
`l
`
`T
`
`T
`,...._
`
`f - f -
`
`f - f - -
`f - f - -
`f - f - -
`
`- - - -
`
`Cootrol
`
`O.ol
`
`0.03
`
`.
`
`_l
`
`.
`
`f - ffi
`
`f -
`
`0.1
`
`03
`
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`Mylan v. Boehringer Ingelheim
`IPR2016-01566
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`

`2300 Journal of Medicinal ChemiS:ry, 2007, Vol. 50, No. 10
`
`Letters
`
`uents. The second relied on replacing the quinazolinone with
`other heterocycles. It was the second strategy that yielded
`compounds that were chosen for further development. Interest(cid:173)
`ingly, we found that the phenyl ring of the quinazolinone could
`be eliminated without loss of DPP-4 inhibition.
`Replacing the quinazolinone with a pyrimidinedione resulted
`in 10 (alogliptin, SYR-322), whose synthesis is shown in
`Scheme 2. Selective alkylation,10 methylation and displacement
`of the chloride with 3-(R)-aminopiperidine gave 10.
`Compound 10 is a potent (IC50 < 10 nM) inhibitor ofDPP-4
`and exhibits greater than 10,000 fold selectivity over the closely
`related serine proteases DPP-8 and DPP-914 In addition, in rat
`(data not shown), dog, and monkey treated with 10 (Figures 7
`and 8, respectively, and Table 3), the plasma concentration of
`the compound and the level of DPP-4 inhibition displayed a
`good correlation. Compound 10 also produced dose-dependent
`improvements in glucose tolerance and increased plasma insulin
`levels in female Wistar fatty rats as shown in Figure 9.
`Compound 10 is not an inhibitor of CYP-450 enzymes and
`does not block the hERG channel at concentrations up to 30
`,uM. Further, 10 was profiled in a safety pharmacology screen
`with very favorable results. Based on the data presented above,
`10 was selected for preclinical evaluation. Following scale-up,
`GLP toxicology studies in rat and dog demonstrated the
`compound to be well tolerated. In phase I human trials, 10
`demonstrated human PK-PD suitable for once daily dosing.
`10 has now progressed to phase III testing for the treatment of
`type 2 diabetes. 15
`
`Acknowledgment. The authors thank Michael Tennant and
`Andrew Jennings for technical assistance in computational
`chemistry, Melinda Manuel for technical assistance in analytical
`chemistry, and Gyorgy Snell for technical assistance in structural
`biology. The X-ray crystallography data reported here is based
`on research conducted at the Advanced Light Source (ALS).
`ALS is supported by the Director, Office of Science, Office of
`Basic Energy Sciences, Materials Sciences Division, of the U.S.
`Department of Energy (DOE) under Contract No. DE-AC03-
`76SF00098 at Lawrence Berkeley National Laboratory. We
`thank the staff at ALS for their excellent support in the use of
`the synchrotron beam lines.
`
`Supporting Information Available: X-ray diffraction data,
`DPP-4 assay procedure, microsomal stability procedure, general
`chemistry procedures, experimental details for synthesis ofthe target
`compounds, and purity data. This material is available free of charge
`via the Internet at http:/ /pubs.acs.org.
`
`References
`( 1) http://www. who.int/mediacentre/factsheets/fs312/en/index.html.
`(2) (a) Holst, J. J. Glucagon-like peptide-1, a gastrointestinal hormone
`with a pharmaceutical potential. Curr. Med. Chern 1999, 6, 1005-
`1017. (b) Deacon, C. F.; Holst, J. J.; Carr, R. D. Glucagon-like
`
`peptide-1: a basis for new approaches to the management of diabetes.
`Drugs Today 1999, 35, 159-170. (c) Livingston, J. N.; Schoen, W.
`R. Glucagon and glucagon-like peptide-1. Annu. Rep. Med. Chern.
`1999, 34, 189-198.
`(3) Review: Drucker, D. L. Therapeutic potential of dipeptidyl peptidase
`IV inhibitors for the treatment of type 2 diabetes. Expert Opin. lnveS:.
`Drugs 2003, 12, 87-100.
`(4) Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. Effect of6-week
`course of glucagon-like peptide 1 on glycaemic control, insulin
`sensitivity, and beta-cell function in type 2 diabetes: a parallel-group
`study. Lancet 2002, 359, 824-830.
`(5) Gwaltney, S. L., II; Stafford, J. A. Inhibitors of dipeptidyl peptidase
`4. Annu. Rep. Med. Chern. 2005, 40, 149-165.
`(6) (a) Pratley, R.; Galbreath, E. Twelve-Week Monotherapy with the
`DPP-4 Inhibitor. LAF237 Improves Glycemic Control in patients with
`Type 2 Diabetes (T2DM). Presented at Proceedings of the 64th ADA,
`Orlando, FL, June 2004; Presentation 355-0R. (b) Ahren, B.; Gomis,
`R.; Standl, E.; Mills, D.; Schweizer, A. Twelve- and 52-week efficacy
`of the dipeptidyl peptidase IV inhibitor LAF237 in metformin-treated
`patients with type 2 diabetes. Diabetes Care 2004, 27, 2874-2880.
`(7) Herman, G. A.; Zhao, P.-L.; Dietrich, B.; Golor, G.; Schrodter, A.;
`Keymeulen, B.; Lasseter, K. C.; Kipnes, M. S.; Hilliard, D.; Tanen,
`M.; De Lepeleire,
`I.; Cilissen, C.; Stevens, C.; Tanaka, W.;
`Gottesdiener, K. M.; Wagner, J. A. The DP-IV Inhibitor MK-0431
`Enhances Active GLP-1 and Reduces Glucose Following an OGTT
`in Type 2 Diabetics. Presented at the Proceedings of the 64th ADA,
`Orlando, FL, June, 2004; Presentation 353-0R.
`(8) The aminopiperidine and cyanobenzyl groups have previously been
`used in DPP-4 inhbitors: Kanstrup, A. B.; Sams, C. K.; Lundbeck,
`J. M.; Christiansen, L. B.; Kristiansen, M. World Patent 2003004496,
`2003. Himmelsbach, F.; Mark, M.; Eckhardt, M.; Langkopf, E.;
`Maier, R.; Lotz, R. World Patent 2002068420, 2002.
`(9) See: Liu, J.-F.; Wilson, C. J.; Ye, P.; Sprague, K.; Sargent, K.; Si,
`Y.; Beletsky, G.; Yohannes, D.; Ng, S.-C. Privileged structure-based
`quinazolinone natural product-templated libraries: Identification of
`novel tubulin polymerization inhibitors. Bioorg. Med. Chern. Lett.
`2006, 16, 686-690 and references cited therein.
`(10) Liu, H.; Ko, S.-B.; Josien, H.; Curran, D. P. Selective N-function(cid:173)
`alization of 6-substituted-2-pyridones. Tetrahedron Lett. 1995, 36,
`8917-8920.
`(11) PDB code 20NC.
`(12) For method of determining ex-vivo DPP-4 inhibition in plasma, see:
`Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.;
`Dunning, B. E.; Kapa, P.; Mangold, B. L.; Russell, M. E.; Hughes,
`T. E. 1-[[(3-Hydroxy-1-adamantyl)amino ]acetyl]-2-cyano-(S)-pyrro(cid:173)
`lidine: A potent, selective, and orally bioavailable dipeptidyl
`peptidase IV inhibitor with antihyperglycemic properties. J. Med.
`Chern 2003, 46, 2774-2789.
`(13) Hansotia, T.; Baggio, L. L.; Delmeire, D.; Rinke, S. A.; Yamada,
`Y.; Tsukiyama, K.; Seino, Y.; Holst, J. J.; Schuit, F.; Drucker, D. J.
`Double incretin receptor knockout (DIRKO) mice reveal an essential
`role for the enteroinsular axis in transducing the glucoregulatory
`actions of DPP-IV inhibitors. Diabetes 2004, 53, 1326-1335.
`(14) Inhibition ofDPP-8 and DPP-9 has been associated with toxicity in
`animals. See: Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G.
`J.; Beconi, M. G.; Biftu, T.; Chan, C.-C.; Edmondson, S.; Feeney,
`W. P.; He, H.; Ippolito, D. E.; Kim, D.; Lyons, K. A.; Ok, H. 0.;
`Patel, R. A.; Petrov, A. N.; Pryor, K. A.; Qian, X.; Reigle, L.; Woods,
`A.; Wu, J. K.; Zaller, D.; Zhang, X.; Zhu, L.; Weber, A. E.;
`Thornberry, N. A. Dipeptidyl peptidase IV inhibition for the treatment
`of type 2 diabetes. Diabetes 2005, 54, 2988-2994.
`(15) Alogliptin benzoate clinical data will be the subject of future reports.
`
`JM070104L
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