Discovery of alogliptin: A potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV

Article abstract of DOI:10.1021/jm070104l

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 111 trials in patients with type 2 diabetes.

Full text of DOI:10.1021/jm070104l

J. Med. Chem. 2007, 50, 2297-2300
2297
Discovery of Alogliptin: A Potent, Selective,
Bioavailable, and Efficacious Inhibitor of
Dipeptidyl Peptidase IV†
Jun 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 San Diego, Inc., 10410 Science Center DriVe, San Diego,
California 92121, and Pharmaceutical Research DiVision, Takeda
Pharmaceutical Company Ltd., Osaka, Japan
ReceiVed 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.
Figure 1. Active site of DPP-4.
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 untreated, hyperglycemia may cause nephr-
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 â-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 â-cells.3 Continuous infusion of GLP-1
to patients with T2D results in significant reduction of blood
glucose and hemoglobin A1c 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 of DPP-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-Hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrro-
lidine (LAF-237)6 and (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-
dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluo-
rophenyl)butan-2-amine (MK-0431).7
Figure 2. DPP-4 active site surface.
Figure 3. Structure-based design of compound 1a.
structure-based design, we hypothesized that a quinazolinone
scaffold could effectively display groups known to interact with
the active site residues of DPP-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 S1 pocket (formed by V656,
Y631, Y662, W659, Y666, and V711) and interact with Arg125.
The carbonyl at C-4 was anticipated to provide an important
hydrogen bond to the backbone NH of Tyr631, and the bicyclic
The active site of DPP-4 is shown in Figure 1 with important
residues labeled. Figure 2 shows the surface of the site. Using
† Compound 1a has been deposited into the Protein Data Bank: PDB
code 2ONC.
* To whom correspondence should be addressed. Phone: 858-731-3562.
Fax: 858-550-0526. E-mail: stephen.gwaltney@takedasd.com.
‡ Takeda San Diego, Inc.
§ Takeda Pharmaceutical Company Ltd.
a Abbreviations: DPP-4, dipeptidyl peptidase IV.
10.1021/jm070104l CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/19/2007

2298 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Scheme 1. Synthesis of Compounds 1a-ma
Letters
a Reagents: (a) urea, 200 °C; (b) POCl3, Me2NPh, reflux; (c) NaOH;
(d) NaH, LiBr, 2-CNPhCH2Br; (e) 3-(R)-aminopiperidine, NaHCO3,
150 °C.
Figure 4. Compound 1a in the active site of DPP-4.
Table 1. Selected Data for Quinazolinones 1a-m
DPP-8 RLMb HLMc 3A4d
DPP-4
IC50 (µM)
IC50
t1/2
t1/2
IC50
compd
R
(µM)
(min)
(min) (µM)
1aa
1b
1c
H
5-F
6-F
0.013 ( 0.006
>100
2.5
1.8
31
>200
125
>200
>200
NTe
80
64
113
82
2.0
0.50
2.5
0.005 ( 0.0002 >100
0.004 ( 0.0008 >100
0.005 ( 0.0008 >50
1d
1e
6-Cl
7-Cl
8-Cl
6,8-diCl
6-Br
6-OMe
6-OMe, 7-F
6,7-di-OMe
8-OMe
40
0.25
0.25
0.13
0.08
0.25
0.20
0.25
0.50
0.63
0.79
0.015 ( 0.002
0.029 ( 0.011
0.013 ( 0.003
0.005 ( 0.001
0.008 ( 0.0003 >50
0.004 ( 0.0008 >100
0.019 ( 0.004
0.030 ( 0.005
>100
>100
>100
>100
NTe
3.7
17
1fa
1g
1h
1i
34
10
35
107
7.2
6.1
1j
136
146
61
1k
1la
1m
>100
>100
>50
6-F, 7-morpholinyl 0.018 ( 0.003
33
Figure 5. Compound 1a in the active site of DPP-4 with key
interactions shown.
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 π-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 1a-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-
m. Chlorination with POCl3 followed by selective hydrolysis
gave 4a-m. Selective N-alkylation was performed using
conditions reported by Curran and co-workers.10 Displacement
of the chloride in 6 with 3-(R)-aminopiperidine was performed
in a sealed tube or in a microwave reactor.
Figure 6. Plasma concentrations and DPP-4 inhibition in rats for 1c
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, 1a, 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).
(TFA salt, 10 mpk po).
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 1c was synthesized. This compound
showed a 10-fold improvement in metabolic stability in the rat.
Figure 6 shows the pharmacokinetic (PK) and pharmacodynamic
(PD) profile of 1c in the rat.12 Selected PK parameters are listed
in Table 2.
Compound 1a suffers from a short metabolic half-life in the
rat, making in vivo assessments difficult. Metabolite studies

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2299
Table 2. Selected Rat PK Parameters for Compound 1c (TFA Salt)
species
rat
dose, iv/oral (mg/kg)
1/10
iv t1/2 (h)
oral t1/2 (h)
2.43 ( 0.31
AUCpo (µg h mL-1
7.56 ( 1.80
)
CL (mL kg-1 min-1
)
Vdss (mL kg-1
2865 ( 632
)
F (%)
2.60 ( 1.85
20.61 ( 8.31
85 ( 20
Table 3. Selected PK Parameters for Compound 10
species
salt
doses, iv/oral (mg/kg)
iv t1/2 (h)
oral t1/2 (h)
AUCpo (µg h mL-1
)
CL (mL kg-1 min-1
)
Vdss (mL kg-1
)
F (%)
dog
monkey
HCl
benzoate
1/3
1.1/10
2.93 ( 0.84
5.74 ( 1.70
3.04 ( 0.75
5.66 ( 0.23
1.61 ( 0.51
16.63 ( 2.52
22.96 ( 7.81
8.81 ( 1.07
3508 ( 255
2602 ( 561
68 ( 22
87 ( 13
Scheme 2. Synthesis of 10
As shown in Figure 6, there is a strong correlation between
plasma levels of compound 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 Syrrx106124) 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-
able; however, compound 1c was found to inhibit CYP450 3A4
Figure 7. Plasma concentrations and DPP-4 inhibition in dogs for 10
(HCl salt, 3 mpk po).
Figure 9. Effects of 10 on plasma glucose (A), plasma immunoreactive
insulin (IRI) (B), baseline (0 min)-adjusted AUC0-60min 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 e 0.025 vs control by one-tailed Williams’
test; (#) p e 0.025 vs control by one-tailed Shirley-Williams’ test.
with an IC50 of 2.5 µM and to block the hERG channel at
micromolar concentrations.
In an effort to improve upon 1c, we adopted two strategies.
The first relied on modifications to the quinazolinone substit-
Figure 8. Plasma concentrations and DPP-4 inhibition in monkeys
for 10 (benzoate salt, 10 mpk po).

2300 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Letters
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. Chem.
1999, 34, 189-198.
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-
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 of DPP-4
and exhibits greater than 10,000 fold selectivity over the closely
related serine proteases DPP-8 and DPP-9.14 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
µM. 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
(3) Review: Drucker, D. L. Therapeutic potential of dipeptidyl peptidase
IV inhibitors for the treatment of type 2 diabetes. Expert Opin. InVest.
Drugs 2003, 12, 87-100.
(4) Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. Effect of 6-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. Chem. 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-OR. (b) Ahre´n, 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-OR.
(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. Chem. Lett.
2006, 16, 686-690 and references cited therein.
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.
(10) Liu, H.; Ko, S.-B.; Josien, H.; Curran, D. P. Selective N-function-
alization of 6-substituted-2-pyridones. Tetrahedron Lett. 1995, 36,
8917-8920.
(11) PDB code 2ONC.
(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-
lidine: A potent, selective, and orally bioavailable dipeptidyl
peptidase IV inhibitor with antihyperglycemic properties. J. Med.
Chem. 2003, 46, 2774-2789.
(13) Hansotia, T.; Baggio, L. L.; Delmeire, D.; Hinke, 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 of DPP-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. O.;
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.
Supporting Information Available: X-ray diffraction data,
DPP-4 assay procedure, microsomal stability procedure, general
chemistry procedures, experimental details for synthesis of the target
compounds, and purity data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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1017. (b) Deacon, C. F.; Holst, J. J.; Carr, R. D. Glucagon-like
(15) Alogliptin benzoate clinical data will be the subject of future reports.
JM070104L