Discovery of DA-1229: A potent, long acting dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmcl.2011.04.029

A series of β-amino amide containing substituted piperazine-2-one derivatives was synthesized and evaluated as inhibitors of dipeptidyl pepdidase-4 (DPP-4) for the treatment of type 2 diabetes. As results of intensive SAR study of the series, (R)-4-[(R)-3-amino-4-(2,4,5-trifluorophenyl)- butanoyl]-3-(t-butoxymethyl)-piperazin-2-one (DA-1229) displayed potent DPP-4 inhibition pattern in several animal models, was selected for clinical development.

Full text of DOI:10.1016/j.bmcl.2011.04.029

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3809–3812
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of DA-1229: A potent, long acting dipeptidyl peptidase-4 inhibitor
for the treatment of type 2 diabetes
Heung Jae Kim a, Woo Young Kwak a, Jong Pil Min a, Jae Young Lee a, Tae Hyun Yoon a, Ha Dong Kim a,
Chang Yell Shin a, Mi Kyung Kim a, Song Hyen Choi a, Hae Sun Kim a, Eun Kyoung Yang a, Ye Hwang Cheong a,
Yu Na Chae a, Kyung Jin Park a, Ji Myun Jang a, Soo Jung Choi a, Moon Ho Son a, Soon Hoe Kim a, Moohi Yoo a,
Bong Jin Lee b,
⇑
a Dong-A Pharm. Co., Ltd, 47-5, Sanggal-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-905, Republic of Korea
b Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of b-amino amide containing substituted piperazine-2-one derivatives was synthesized and eval-
uated as inhibitors of dipeptidyl pepdidase-4 (DPP-4) for the treatment of type 2 diabetes. As results of
intensive SAR study of the series, (R)-4-[(R)-3-amino-4-(2,4,5-trifluorophenyl)-butanoyl]-3-(t-butoxym-
ethyl)-piperazin-2-one (DA-1229) displayed potent DPP-4 inhibition pattern in several animal models,
was selected for clinical development.
Received 5 July 2010
Revised 31 March 2011
Accepted 7 April 2011
Available online 13 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dipeptidyl peptidase-IV
DPP-IV
DPP-4
GLP-1
DPP-4 inhibitor
DA-1229
Type 2 diabetes mellitus is a chronic disorder characterized by
hyperglycemia coupled with a gradual decline in insulin sensitivity
and insulin secretion. The main goal of the management of type 2
diabetes is to achieve glycemic control as close to the non diabetic
range as practicable, in order to reduce the risk of late-stage com-
plications.1 However, the therapeutic effect provided by existing
medications is often not sustainable, since the multi-organ defects
responsible for the disease are only insufficiently addressed.2 The
incretin hormone glucagon-like peptide 1 (GLP-1) is a potent stim-
ulator of endogenous insulin release. GLP-1 has beneficial effects
on islet b-cell function and insulin sensitivity without induction
of hypoglycemia.3 Further contributing factors are the inhibition
for the treatment of type 2 diabetes (Fig. 1). Long-term studies
with DPP-4 inhibitors in patients are underway in order to confirm
the safety and sustainability of these effects, and, in particular,
their ability to prevent the progressive loss of b-cell function. On
the basis of these background, we discovered various kind of
DPP-4 inhibitors including DA-1229. We also disclose that our ini-
tial design concept came from b-amino acid thiazolidides11 and
some substituted piperazines.12
of glucagon release from pancreatic
a-cells, reduction of food in-
take and retardation of gastric emptying, which are mediated by
GLP-1.4 Unfortunately, GLP-1 is rapidly degraded in vivo by the
serine protease dipeptidyl peptidase-4 (DPP-4);5 therefore DPP-4
inhibitors have emerged as a new therapeutic option to treat type
2 diabetes.6–10 Clinical proof of concept has been established for
several DPP-4 clinical candidates, four of which have become mar-
keted drugs. Januvia™ (sitagliptin), Onglyza™ (Saxagliptin), Gal-
vus™ (vildagliptin) and Nesina™ (alogliptin) have been approved
⇑
Corresponding author. Tel.: +82 2 880 7869; fax: +82 2 872 3632.
E-mail address: lbj@nmr.snu.ac.kr (B.J. Lee).
Figure 1. DPP-4 inhibitors in market.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.029

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H. J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3809–3812
Scheme 1. Overall design of b-amino amide containing substituted piperazine-2-one derivatives. Reagents: (a) EDC, HOBt, DIPEA/DMF or MC; (b) HCl in ether/MeOH.
As shown in Scheme 1, the target compound 4 and 5 were pre-
pared from commercial Boc protected b-amino acid 1 and substi-
So, we tried to study more about benzyl moiety in that position
and then various other substituents were introduced.
tuted heterocyclic amine
conditions followed by deprotection of amine.
2
using standard peptide coupling
As shown in Table 2, there was no effect on activity according to
position and size of halogens (4m, 4n, 4o, 4p; ꢀ30 nM). In the case
of nitro group, ortho- 4h is threefold more potent than para- 4g. We
The substituted heterocyclic amine 8 was prepared as shown in
Scheme 2. Commercial Boc-protected piperazin-2-one (6) was cou-
pled with alkyl or aryl halide to give compound 7, respectively.
Deprotection of Boc derivatives 7 by treatment with hydrogen
chloride in ether provided compound 8.
As shown in Table 1, introduction of the simple methyl 4a re-
sulted in DPP-IV inhibitory activity with an IC50 value of around
100 nM. Substitution of pyridine 4b or non aromatic replacement
4e showed decreased activity compared to methyl substitution.
In case of five-membered hetero fusion ring 4d and bi-phenyl 4c
increased potency by twofold than methyl. We concluded that
there were limitations of activity elevation and the essential moi-
ety might be the benzyl 4f which is possible to make hydrophobic
interaction with side chain of Phe35713,14,16 as shown in Figure 2.
Figure 2. Docking structure of 4f in the active site (from 1X70) of DPP-4.
Trifluorophenyl ring is energetically stabilized in the hydrophobic pocket formed
by Tyr547, Tyr662 and Tyr666. The amine group forms H-bond with side chain of
Glu205, Tyr662. Phenyl ring of 4f interacts with the side chain of Phe357 and forms
edge-phase pi–pi interaction.
Scheme 2. Reagents: (a) R1Br, NaH, or R1Br/DMF, Pd(OAc)2, DPPF, KO-t-Bu, THF; (b)
HCl in ether/MeOH.
Table 1
DPP-4 inhibition activities of various substituted compound 4
Table 2
DPP-4 inhibition activities of various substituted compound 4
F
F
NH2
O
F
N
F
NH2
O
F
N
R1
N
O
F
N
R1
O
R1
DPP-4 IC50 (nM)
15
Compound
4a
4b
92.6
311
Compound
R1
DPP-4 IC50 (nM)
N
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4-Nitro-benzyl
2-Nitro-benzyl
2-Cyano-benzyl
2-Trifluoromethyl-benzyl
4-Ethyl-benzyl
4-Methoxy-benzyl
4-Fluoro-benzyl
4-Iodo-benzyl
38.5
13.7
48.9
50.7
38.9
33.9
33.8
30.8
26.7
29.0
7.40
4c
44.8
42.7
N
N
N
4d
N
4e
4f
116
O
3-Chloro-benzyl
2-Chloro-benzyl
2-Nitro-4,5-dimethoxy-benzyl
54.1

H. J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3809–3812
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Table 3
From our preliminary study for proper chirality, in case of polar
hydroxyl group 5a, (R)-form was threefold more potent than (S)-
form and compound 5a overcame CYP inhibition problem. For
example, compound 4l showed DPP4 IC50 of 33.9 nM and CYP3A4
IC50 of 1075 nM (Selectivity Index CYP3A4 IC50/DPP4 IC50 ꢁ 31.7).
On the other hand, introduction of hydroxylmethyl substituent
raised selectivity index more than 1000-fold. So we set the new
start point to hydroxy derivatives. The DPP-4 inhibitory activities
of hydroxyl derivatives (5b, 5c, 5e, 5d) were directly correlated
to the number of alkyl size (the IC50 value of methyl, ethyl, isopro-
pyl and cyclopentyl were 7.4, 4.3, 2.2 and 1.7 nM, respectively). In
this SAR, we found tert-butyl addition to (R)-hydroxy moiety 5f
was highly potent with an IC50 value of around 1 nM. Replacement
of oxygen in the hydroxyl linker to nitrogen showed similar activ-
ity pattern which were dependent on the bulkiness of direct sub-
stituent to nitrogen (5g, 5h, 5i). Finally incorporating sulfur
instead of the oxygen of hydroxy resulted in highly potent DPP-4
inhibitory activity (5j).
DPP-4 inhibition activities of chiral substituted compound 5
F
F
NH2
O
X
N
F
NH
O
R2
Compound
X
R2
DPP-4 IC50 (nM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
O
O
O
O
O
O
N
N
N
S
H
12
Methyl
Ethyl
Cyclopentyl
Isopropyl
tert-Butyl
Ethylmethyl
Diethyl
–(CH2CH2)2O
tert-Butyl
7.4
4.3
1.7
2.2
0.9
17
11
5.2
1.3
Representative analog in the series, compound 5f was selected
for evaluation of in vivo efficacy. In Sprague Dawley rats, com-
pound 5f dose-dependently inhibited plasma DPP-4 activity within
30 min of administration. At 24 h following oral administration of
compound 5f (1, 3, 10 mg/kg), plasma DPP-4 activity was inhibited
by 72.5 2.4%, 82.9 1.6% and 87.2 0.6%, respectively (Fig. 3).
5j
achieved with an IC50 value of around 10 nM, so further investiga-
tion was conducted with ortho-nitro group. Replacement of nitro
group to electron-withdrawing cyano 4i or trifluoromethyl 4j re-
sulted in 3- to 4-fold decrease of inhibition activity. From these re-
sults, we fixed ortho position substituent to nitro group and
through additional substitution of methoxy to meta and para 4q,
we got the highest inhibitory activity (7.4 nM) in these series.
On the other side, we tried R0 substitution (compound 5) for
more activity enhancing and the results are shown in Table 3.
The substituted heterocyclic amine 18 was prepared as shown in
Scheme 3.
The N-protected aziridine compound 9 was reacted with vari-
ous alcohols (X = O) or amines (X = N) or thiol (X = S) to provide
compound 10, followed by deprotection using Pd/C and hydrogen
gas gave compound 11 which was condensed with Boc-amino
acetaldehyde to provide 12 having a variety of substituents X
and R2. Compound 12 was protected with Cbz, followed by depro-
tection of Boc to provide 13 which was conducted using trimethyl-
aluminium and deprotected to give 15. Additionally, compound 5a
Figure 3. Inhibition of DPP-4 activity in plasma obtained from SD rats after single
oral administration of compound 5f at different doses. Data were presented as
means SEM.
was prepared from D-serine methyl ester (11; X = O, R = H).
Scheme 3. Reagents: (a) R2XH, BF3OEt2/CHCl3; (b) Pd/C, H2(g)/MeOH; (c) Boc-aminoacetaldehyde, NaBH(OAc)3/MC; (d) CbzCl/THF; (e) HCl in ether/MeOH; (f) (CH3)3Al/MC;
(g) Pd/C, H2(g)/MeOH.

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H. J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3809–3812
Figure 4. Effects of compound 5f on the change of blood glucose levels in C57BL/6 mice after oral glucose loading. Data were presented as means SEM.
References
Table 4
1. Nathan, D. M.; Buse, J. B.; Davidson, M. B.; Ferrannini, E.; Holman, R. R.;
Sherwin, R.; Zinman, B. Diabetes Care 2009, 32, 193.
2. DeFronzo, R. A. Diabetes 2009, 58, 773.
Preliminary pharmacological and kinetic data of compound 5f
CYP450, 3A4/2D6/2C9/2C19/1A2
Human liver microsome t1/2
t1/2 (rat)
CL (rat)
Vss (rat)
>50 lM
>90 min
6.10 h
60.5 mL/min/kg
12.0 L/kg
74.8%
3. Knudsen, L. B. J. Med. Chem. 2004, 47, 4128.
4. Murphy, K. G.; Dhillo, W. S.; Bloom, S. R. Endocr. Rev. 2006, 27, 719.
5. For recent reviews, see: (a) Mest, H.-J.; Mentlein, R. Diabetologia 2005, 48, 616;
(b) Weber, A. E. J. Med. Chem. 2004, 47, 4135; (c) Drucker, D. J. Expert Opin.
Invest. Drugs 2003, 12, 87; (d) Augustyns, K.; Van der Veken, P.; Senten, K.;
Haemers, A. Expert Opin. Ther. Patents 2003, 13, 499.
Foral (rat)
hERG, patch clamp IC50
79.6 lM
6. Villhauer, E. B.; Coppola, G. M.; Hughes, T. E. Annu. Rep. Med. Chem. 2001, 36,
191.
7. Drucker, D. J.; Nauck, M. A. Lancet 2006, 368, 1696.
8. Ahren, B. Expert Opin. Emerg. Drugs 2008, 13, 593.
9. Pei, Z. Curr. Opin. Drug Disc. Dev. 2008, 11, 512.
10. Gwaltney, S. L., II Curr. Top. Med. Chem. 2008, 8, 1545.
11. Xu, J. et al Bioorg. Med. Chem. Lett. 2004, 14, 4763.
12. Brockunier, L. L. et al Bioorg. Med. Chem. Lett. 2004, 14, 4759.
13. Oefner, C.; D’Arcy, A.; Sweeney, A. M. Acta Crystallogr., Sect.
1206.
14. Kim, D.; Wang, L.; Beconi, M. J. Med. Chem. 2005, 48, 141.
In a separated experiment, compound 5f was evaluated using
oral glucose tolerance test (OGTT) in the C57BL/6J mice.17 Com-
pound 5f (0.1, 0.3 or 1.0 mg/kg) significantly inhibited the eleva-
tion of blood glucose and reduced the glucose AUC in dosage-
dependent manner achieving maximal efficacy at 1 mg/kg, with
63% inhibition (Fig. 4).
D 2003, 59,
Compound 5f was submitted to the advanced test including
preliminary experiments on pharmacokinetic parameters in rats
(Table 4). Additionally, compound 5f was applied for a panel of
receptors and enzymes and it showed no known liabilities at test
15. DPP-4 inhibition assay: Inhibitors at various concentrations were incubated
with 20 ng/ml rhDPP-4 enzyme and 50 M H-Gly-Pro-AMC for 1 h at 25 °C. The
l
fluorescence (360 nm excitation/465 nm emission) was measured after 1 h.
16. Aertgeerts, K.; Ye, S.; Tennant, M. G. Protein Sci. 2004, 13, 412.
17. Compound 5f or vehicle (0.5% methylcellulose) was treated orally and then
challenged with glucose (2 g/kg) 30 min post-dose. The blood glucose
excursion profile from 0 to 120 min was used to integrate an area under for
curve (AUC) and percent inhibition values for each treatment were generated
from the AUC data normalized to the non-glucose controls.
concentrations of 1 l
M.18
In conclusion, we have shown that variations of b-amino amide
containing the piperazine-2-one scaffold used in the clinical devel-
opment compound 5f (DA-1229), which retains excellent DPP-4
inhibitory activity, in vivo efficacy and preliminary safety profiles,
as well as subtype selectivity.19 DA-1229 is currently undergoing
phase II clinical trials and holds the potential for once-daily treat-
ment of type 2 diabetics.
18. Off target selectivity was screened by MDS pharm service pharmacology
packages: LeadProfilingScreenÒ, EnzymeProfilingScreenÒ
.
19. Kim, H. J.; Kim, H. D.; Kwak, W. Y. Diabetes 2008, 57. 2611-PO.