Dipeptidyl peptidase-4 inhibitor with β-amino amide scaffold: Synthesis, SAR and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2012.07.019

Inhibitors of dipeptidyl peptidase-4 (DPP4) have been shown to be effective treatments for type 2 diabetes. Several series of β-amino amide containing piperazine derivatives have been prepared and evaluated as a inhibitor of DPP4. Finally compound 5m was selected for further evaluation.

Full text of DOI:10.1016/j.bmcl.2012.07.019

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5545–5549
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dipeptidyl peptidase-4 inhibitor with b-amino amide scaffold: Synthesis,
SAR and biological evaluation
Heung Jae Kim a,b, Woo Young Kwak a, Jong Pil Min a, Si Young Sung a, Ha Dong Kim a, Mi Kyung Kim a,
Hae Sun Kim a, Kyung Jin Park a, Moon Ho Son a, Soon Hoe Kim 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, 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:
Inhibitors of dipeptidyl peptidase-4 (DPP4) have been shown to be effective treatments for type 2 diabe-
tes. Several series of b-amino amide containing piperazine derivatives have been prepared and evaluated
as a inhibitor of DPP4. Finally compound 5m was selected for further evaluation.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 15 April 2012
Revised 1 July 2012
Accepted 6 July 2012
Available online 14 July 2012
Keywords:
Diabetes
DPP4
GLP-1
b-Amino amide
Inhibition of the serine protease dipeptidyl peptidase 4 (DPP4)
has proven to be an effective treatment for improving glycemic
control in type 2 diabetic patients.1 Recovery of glucose homeosta-
sis by DPP4 inhibition results from the prolonged action of the
incretins glucagon-like peptide 1 (GLP-1) that are normally rapidly
inactivated by the N-terminal cleavage of the peptide by the ami-
nopeptidase dipeptidyl peptidase-4 (DPP4, CD26, EC 3.4.14.5), a se-
quence-specific non-classical serine protease.2 Glucagon-like
peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion,
inhibits hepatic glucose production, lowers blood glucose levels,
and promotes the growth and differentiation of b-cells. GLP-1
may also play a role in suppressing appetite in humans and in
modulating peripheral glucose uptake.3 Inhibition of DPP4 leads
to increased levels of endogenous GLP-1 by prolonging its half-life
and consequently enhances the beneficial effects of GLP-1 in glu-
cose dependent insulin secretion and b-cell restoration.4 Since
Type 2 diabetes is a disease characterized by elevated blood glu-
cose levels and a relative insufficiency of insulin, a therapeutic
agent that extends the duration of GLP-1 action may aid in control-
ling glucose homeostasis, by enhancing b-cell glucose-stimulated
insulin release and promoting insulin gene expression and its bio-
synthesis.5 Several DPP4 inhibitors have been developed for the
treatment of type 2 diabetes including Sitagliptin6 (Fig. 1: Vildag-
From previous report,11 it is well known that b-amino acid
substituted piperazines showed desirable inhibition activity for
DPP4. We started our initial derivatization from substituted piper-
azine, compound 4.
As shown in Scheme 1, the target compound 4 was prepared
from commercial Boc protected b-amino acid 1 and N-substituted
piperazine 2 using standard peptide coupling conditions followed
by deprotection of amine.
Table 1 summarizes the DPP4 inhibitory properties of these b-
amino amide piperazines. Several phenyl derivatives of this kind
of piperazine showed inhibitory activity in the range of
liptin,7 Saxagliptin,8 Alogliptin9 and Linagliptin10
)
⇑
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. DPP4 inhibitors in market.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.019

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H. J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5545–5549
Scheme 1. Overall design of b-amino amide containing substitutied piperazine derivatives. Reagents: (a) EDC, HOBt, DIPEA/DMF or CH2Cl2, (b) HCl in ether/MeOH.
Table 1
DPP4 inhibition activities of various substituted compound 4
F
F
NH2
O
N
Scheme 2. Reagents: (a) R0CO2H, PyBOP, Et3N, CH2Cl2 or R0CO2Cl, Et3N, CH2Cl2; (b)
HCl in ether/MeOH.
N
F
R1
12
Compound
R1
DPP4 IC50 (nM)
trile (4f, 4g) showed 10-fold activity difference. Additional substi-
tution to compound 4i with bulky phenyl (4j) led to decrease of the
inhibitory activity.
4a
85.7
194
NO2
In case of compound 4b, from the results of mouse OGTT (oral
glucose tolerance test) study,13 it has a glucose lowering effect of
37.0%, 44.6% at the dose of 3, 10 mg/kg, respectively. It also has
good mouse pharmacokinetic profile (t1/2 = 7.06 h, BA ꢀ100%),
however the evaluation was discontinued due to the limitation of
in vivo efficacy. In mouse DPP4 inhibition assay, compound 4c
has an IC50 of 94.4 nM as well as human DPP4 inhibition study
(IC50 of 291 nM). And the compound showed excellent OGTT study
result (66% reduction of glucose AUC, at the dose of 10 mg/kg) so
dose dependent in vivo DPP4 inhibition study14 was performed.
The dose for 50% plasma DPP4 inhibition (ED50) of compound 4c
was calculated to 0.87 mg/kg (For the dose of 0.3, 1, 3, 10 mg/kg,
DPP4 inhibition % were 33.8, 52.2, 68.6 and 85.9, respectively).
But the compound 4c doesn’t have good selectivity over DPP9
(IC50 = 1763 nM, 18-fold selectivity) and has strong CYP3A4 inhibi-
tion (81.5, 94.0, 100% inhibition at the concentration of 1, 3,
4b
4c
F
F
NO2
F
291
4d
4e
264
OH
O
99.7
4f
569
10
l
M), so we stopped the assessment of the compound 4c.
Compound 4 series has some limitation with in vivo efficacy,
CN
selectivity and undesirable CYP inhibition, we designed another
series of b-amino amide which has moiety of direct carbonyl
extension at the position of -N of piperazine. This design is based
on the concept that through the carbonyl linker, direction to the
S2 pocket of DPP4 active site will be more desirable for the inhibi-
tion activity and the peptidomimetic modification will be more
susceptible to the enzymatic recognition.
The compound N-substituted piperazine with carbonyl function
group was prepared as shown in Scheme 2.
Compound 6, Commercial Boc-protected piperazine, was cou-
pled with acid or acid chloride to give compound 7. Deprotection
of Boc derivatives 6 by treatment with hydrogen chloride in ether
provided compound 8.
4g
4h
50.4
101
CN
OMe
4i
4j
103
212
OMe
F
F
The crystallographic data (PBD id: 1X70) showed that S2 pocket
in active site was composed of key amino acids with phe357,
Arg358, Ser209. And according to following molecular modeling
studies (Fig. 2), simple phenyl ring was introduced after carbonyl
unit and modification of para-position was followed for efficient
targeting on interaction with S2 pocket. Compounds in previous
SAR have little chance to make hydrophobic interaction with
Phe3576,15 and more chance to have steric hindrance with
0.05–0.5 lM. The fluoro substituted compounds 4b was less potent
than compound 4a. In compound 4c, displacement of nitro to flu-
oro resulted in further decrease of inhibitory activity. From the re-
sults of compound 4d and 4e, carbonyl group was more potent
than hydroxyl group. ortho-Substitution with phenyl and carboni-

H. J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5545–5549
5547
Figure 2. Molecular modeling of compound 5m with DPP4 active site. Substituted pyridine ring of 5 m interacts with the side chain of Phe357 and forms pi–pi interaction.
Carbonyl linker enabled to detour steric hindrance with Arg358 and H-bond was formed between carbonyl group and Ser209.
Table 2
Arg358. Compound 5 series which have carbonyl moiety get the
opposite character to the previous compound 4 series. In Table 2,
most compounds showed excellent DPP4 inhibitory activities
around 50 nM. But there was similar problem in CYP3A4 inhibition
DPP4 inhibition activities of various substituted compound 5
F
F
(IC50; 1–5 lM) as well as in compound 4 series.
NH2
O
Replacement phenyl ring with heteroaromatic ring resulted in
very similar DPP4 inhibitory activity to phenyl ring derivatives.
But we could get the compound that has more than 50-fold selec-
tivity over DPP9. In addition to selectivity index, through in vivo
OGTT screening and solubility test, compound 5m was selected
as a representative analog in this series for further assessment.
Table 3 summarizes the results of DPP4, DPP9 inhibition and OGTT
studies.
In vivo DPP4 inhibition study (in Sprague–Dawley rats, Fig. 3)
of compound 5m, maximum inhibition was reached within
30 min. Duration time of inhibition after single oral administration
of the dose of 10 mg/kg were measured. Until 6 h DPP4 inhibition
was maintained up to 60% and after 24 h, DPP4 inhibition in plas-
ma was around 30%. Compound 5m showed dose dependency in
OGTT study. At the dose of 0.3 mg/kg, glucose AUC reduction
was 29.2% compared to control group. The glucose AUC reduction
showed 35.8%, 40.9% at the dose of 1, 3 mg/kg (Fig. 4). We calcu-
lated the dose which reduce glucose AUC to 30% of control group
glucose AUC (ED30), 0.38 mg/kg. In separate experiment, plasma
DPP4 inhibition were measured in C57BL/6 mice at 1 h after oral
administration (five doses were used; 0.1, 0.3, 1.0, 3.0, 10 mg/
kg). At the dose of 1, 3, 10 mg/kg, in vivo DPP4 inhibition were
32.5%, 59.1% and 84.6%, respectively (Fig. 4). Compound 5m was
submitted to the advanced test including pharmacokinetic studies
in rats. Table 4 summarizes the preliminary ADME data of com-
pound 5m.
N
R2
F
N
O
Compound
R2
DPP4 IC50 (nM)
52.6
5a
F
5b
5c
5d
45.3
39.0
64.6
OH
O
CF3
CF3
5e
56.4
SO2NH2
CO2H
5f
45.3
53.4
5g
5h
5i
56.9
47.7
CO2H
In summary, we have shown that the series of b-amino amide
containing the piperazine-phenyl and piperazine carbonyl linker
scaffold can have excellent DPP4 inhibitory activity through the
efficient targeting to S2 pocket in active site and showed desirable
in vivo efficacy with results of oral glucose tolerance test in C57BL/
N
N

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H. J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5545–5549
Table 3
DPP4, DPP9 inhibition activities and OGTT study results of various substituted
compound 5
F
F
NH2
O
N
R2
F
N
O
Compound R2
DPP4 IC50
(nM)
DPP9 IC50
(nM)
OGTT %
(3 mg/kg)
Figure 3. Inhibition of DPP4 activity in plasma obtained from Sprague–Dawley rats
after single oral administration of compound 5m at the dose of 10 mg/kg.
5j
67.7
24.9
2609
1368
40.3
25.9
O
N
5k
5l
65.4
318
21.2
OMe
O
5m
5n
5o
57.6
74.4
91.5
4862
4746
2740
44.2
43.6
15.7
N
N
N
N
OH
OH
N
5p
31.2
471
30.4
OH
Figure 4. Effects of compound 5m on the change of blood glucose levels in C57BL/6
mice after oral glucose loading.
5q
5r
50.6
33.9
1013
427
17.3
28.6
S
N
Table 4
Preliminary ADME data of compound 5m
Liver microsomal stability
(remaining % at 1 h)
Rat(85), mice(79), dog(88),
human(89)
5s
5t
76.2
36.7
885
29.4
38.5
CYP3A4 induction (at 50
l
M)
10% <
O
Rat PK (po, 10 mg/kg): t1/2, Cmax, BA 4.74 h, 2031 ng/mL, 65.3%
Permeability [Papp(ꢁ10ꢂ6 cm/s)]
2.11
1449
Plasma stability (remaining % at 1 h) Mouse (>97), Rat (>97), Human (97)
N
Cl
N
Protein binding (%)
pH solubility (pH 1–9)
Mouse (44.3), Rat (27.1), Human (50.3)
>10 mg/mL
5u
42.2
30.5
4814
658
29.1
11.6
N
N
stability, permeability and solubility), so further evaluation will
be conducted.
5v
F
References and notes
5w
113.9
57.4
4226
2096
20.2
48.1
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N
N
F
F
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F
5x
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Madsen, J. L. Lancet 2002, 359, 824.
6 mice. Among the compound 5 series, compound 5m showed a
very promising activity profile including excellent in vivo efficacy
and other ADME parameters (liver microsomal stability, plasma
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bated with 20 ng/ml rhDPP-4 enzyme and 50 mM H-Gly-Pro-AMC for 1 h at
25 °C. The fluorescence (360 nm excitation/465 nm emission) was measured
after 1 h.
13. OGTT study (in the C57BL/6J mice): Compound or vehicle (0.5% methylcellu-
lose) 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.
14. Compound or vehicle (0.5% methylcellulose) was treated orally. After 1 h,
plasma sample were collected and DPP-4 inhibition assay (Ref. 13) followed.
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