Discovery of potent dipeptidyl peptidase IV inhibitors through pharmacophore hybridization and hit-to-lead optimization

Article abstract of DOI:10.1016/j.bmc.2013.01.062

A novel dipeptidyl peptidase IV inhibitor hit (5, IC₅₀ = 0.86 μM) was structurally derived from our recently disclosed preclinical candidate 4 by replacing the cyanobenzyl with a butynyl based on pharmacophore hybridization. A hit-to-lead optimization effort was then initiated to improve its potency. Most N-substituted analogs exhibited good in vitro activity, and compound 18o (IC₅₀ = 1.55 nM) was identified to be a potent dipeptidyl peptidase IV inhibitor with a significantly improved pharmacokinetic properties (bioavailablity: 41% vs 82.9%; T_1/2: 2 h vs 4.9 h).

Full text of DOI:10.1016/j.bmc.2013.01.062

Bioorganic & Medicinal Chemistry 21 (2013) 1749–1755
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent dipeptidyl peptidase IV inhibitors through
pharmacophore hybridization and hit-to-lead optimization
Shaogao Zeng a, , Hui Xie a, , Li-li Zeng a, Xin Lu a, Xin Zhao a, Gui-cheng Zhang a, Zheng-chao Tu a,
Hong-jiang Xu b, Ling Yang b, Xi-quan Zhang b, Wenhui Hu a,c,
⇑
a Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, 190 Kaiyuan Avenue, Guangzhou Science Park, Guangzhou 510530, China
b Jiangsu Chia-Tai Tianqing Pharmaceutical Co. Ltd, No. 8 Julong North Rd., Xinpu Lianyungang, Jiangsu 222006, China
c State Key Laboratory of Respiratory Disease, Guangzhou 510120, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel dipeptidyl peptidase IV inhibitor hit (5, IC50 = 0.86 lM) was structurally derived from our
Received 22 November 2012
Revised 18 January 2013
Accepted 23 January 2013
Available online 8 February 2013
recently disclosed preclinical candidate 4 by replacing the cyanobenzyl with a butynyl based on pharma-
cophore hybridization. A hit-to-lead optimization effort was then initiated to improve its potency. Most
N-substituted analogs exhibited good in vitro activity, and compound 18o (IC50 = 1.55 nM) was identified
to be a potent dipeptidyl peptidase IV inhibitor with a significantly improved pharmacokinetic properties
(bioavailablity: 41% vs 82.9%; T1/2: 2 h vs 4.9 h).
Keywords:
DPP-IV inhibitor
Type 2 diabetes
Ó 2013 Elsevier Ltd. All rights reserved.
Linagliptin
Pharmacophore hybridization
Hit-to-lead optimization
1. Introduction
glycemia, and less side effect.3 As a result, DPP-IV inhibitors grad-
ually became the major intervention for type 2 diabetics.
Type 2 diabetes (T2D) formerly referred to as non-insulin-
dependent or adult-onset diabetes, results from the body’s ineffec-
tive use of insulin and comprises over 90% of diabetes patients.
With more than 371 million people affected worldwide, diabetes
is a big social burden with more than 471 billion dollars cost on
its healthcare. Developing countries accounts for 80% of diabetic
patients. Especially, China has the most population with diabetics
of 90 million. And this number is predicted to increase to 129.7
million. Thus, more and persistent effort should be paid to fight
against its epidemic damage and the global burden.1
Glucose like peptidase-1 (GLP-1) is an important incretin which
contributes to the increase of insulin secretion and sensitivity, beta
cell mass, and satiety, as well as the reduction of glucagon secre-
tion and gastric emptying, which is helpful for glucose control
for type 2 diabetics. However, in the normal physical condition,
GLP-1 is rapidly truncated by dipeptidyl peptidase IV (DPP-IV)
and lost its function.2 Thus inhibition of DPP-IV could effectively
maintain the GLP-1 function and control glucose level. Compared
to conventional anti-diabetic drugs, dipeptidyl peptidase IV (DPP-
IV) inhibitors have good patient compliance, reduced risks of hypo-
Traditional medicinal strategies for protease inhibitors mostly
rely on the direct tight binding to the target, which leads to cova-
lent compounds.4 However, the emergence of non-covalent com-
pounds to achieve satisfactory binding affinity with lower risk of
selectivity issues has been witnessed in recent years.5 The first
non-covalent DPP-IV inhibitor with good selectivity against DPP-
8 and DPP-9,6 Sitagliptin (1 Fig. 1),7 was marketed in 2006. Aloglip-
tin (2 Fig. 1) was the second non-covalent DPP-IV inhibitor ap-
proved by the EMA, and it exhibited better efficacy than
sitagliptin.8 The most potent and long lasting drug is Linagliptin
(3 Fig. 1).9 The market drug of covalent DPP-IV inhibitors are Vil-
dagliptin,10 and Saxagliptin.11
Our recently disclosed compound 4 was derived from Alogliptin
by keeping its pharmacophore (3-aminopypiperidinyl region, red
box, Fig. 2) while modifying the scaffold.12 It displayed a better
in vivo efficacy than Alogliptin in lean mice and dose-dependent
glucose reduction in T2D model ob/ob mice. Although compound
4 acquires a similar pharmacokinetic profile with Alogliptin in
rat (bioavailability: 40% and half life: 2 h), it still has a lot of space
to improve. Herein, we present how we employed the classic
medicinal chemistry strategy to further improve its drug-like
properties.
⇑
Corresponding author. Tel.: +86 020 32015 211; fax: +86 020 32015 299.
E-mail address: hu_wenhui@gibh.ac.cn (W. Hu).
Close comparison of the pharmacophore of Linagliptin and com-
pound 4 revealed that the cyanobenzyl should be freely changeable
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.062

1750
S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
F
F
O
O
F
CN
NH2
NH2
O
N
N
N
N
N
N
N
N
N
N
N
NH2
N
O
N
CH3
O
N
CF3
1
2 Alogliptin
Sitagliptin (MK-0431)
3 Linagliptin (BI-1356)
Figure 1. Marketed non-covalent DPP-IV inhibitors.
O
NH2
N
N
N
N
N
N
O
N
O
N
CH3
pharmacophore
hybridization
H
N
3 Linagliptin (BI-1356)
N
NH2
N
Br
O
N
CN
5
H
N
N
NH2
N
Br
4
Figure 2. Generation of hit 5.
with butynyl if the 3-(R)-aminopiperidinyl group was still present.
Thus, compound 5 was immediately generated based on pharma-
cophore hybridization. Though its activity was still in the micro
The modification of compound 5 through N-substitution led
to a series of novel DPP-IV inhibitors with significantly increased
activity. The most active compound, 18o (IC50 = 1.55 nM), exhib-
its much better pharmacokinetic profile than compound 4 (bio-
availablity: 41% vs 82.9%; T1/2: 2 h vs 4.9 h), which implies
molar range (IC50 = 0.86
ther optimization.
lM), it could act as a starting point for fur-
O
O
O
O
H
N
O2N
O2N
a
b
c
NH
NH
NH
NH
N
H
O
N
H
O
N
H
O
N
N
H
O
7
8
9
10
O
Cl
N
O
N
Boc
H
N
H
O
g
d
e
f
N
N
NH
Cl
N
Boc
N
NH
Cl
N
N
Cl
N
14
Cl
11
12
13
O
O
O
H
H
H
N
N
i
N
k
h
N
N
N
NH2
N
N
N
Cl
N
Cl
Br
Br
15
5
16
k
j
O
N
O
N
R
N
R
N
N
N
k
NH2
N
Cl
6: R = H
18a-o: R = CH3 and heterocyclic ring
17a-o
Scheme 1. Synthesis of compounds 5, 6 and 18a–o. Reagents and conditions: (a) sulfuric acid, fuming nitric acid, rt; (b) dimethylfomamide–dimethyl acetal, DMF, 80 °C then
140 °C; (c) AcOH, Zn, 80 °C; (d) POCl3, DIEA, toluene, 70–80 °C; (e) 1 N NaOH/H2O, 100 °C; (f) (Boc)2O, DMAP, Et3N, THF, rt; (g) 1-bromo-2-butyne, DIEA, DMF, rt; (h) HCl/H2O,
MeOH, rt; (i) NBS, DCM, rt; (j) RX, NaH, DMF, rt; (k) 3-(R)-aminopiperidine, NaHCO3, 120 °C, ethanol.

S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
1751
potential better and competent in vivo efficacy for glucose
3.2. Hit-to-lead optimization on compound 5
control.
With compound 5 in hand, we immediately initiated a hit-to-
lead optimization effort. First, compound 6, without any substitu-
ent on the pyrolyl ring, was synthesized and found to have better
2. Chemistry
activity with an IC50 of 0.46 lM. By simply adding a methyl group
The synthesis of compounds 5, 6 and 18a–o is outlined in
Scheme 1. Synthesis of compound 13 from 7 was described in
our recent report.9 Alkylation of 13 with 1-bromo-2-butyne
provided precursor14, which was de-protected to give key inter-
mediate 15. The bromination of compound 15 with N-bromosuc-
cinimide (NBS) provided compound 16, which was converted to
5 by amination. The direct amination of 15 with 3-(R)-aminopi-
peridine afforded compound 6. Compounds 18a–o were obtained
by the N-alkylation of 15 followed by the replacement of the chloro
group with a 3-(R)-aminopiperidinyl group.
on the nitrogen (compound 18a), the activity was further in-
creased. This trend suggested that N-substitution might a powerful
way to increase inhibition. Thus, we decided to make a series of N-
substituted analogs (18b–o).
Though the first N-heterocyclic compound (18b) afforded fur-
ther increased activity, it was still relatively inactive compared to
parent compound 4 (IC50 = 44.0 nM). Inspired by the discovery of
Linagliptin, where it was found that the N-5 position could hold
larger steric substituents, a series of compounds with bicyclic rings
at the N-position were made (18c–o). A few of these compounds
displayed activity in the nM range, such as 18f, 18l, and 18m
(<10 nM). Surprisingly, the best compound (18o, IC50 = 1.55 nM)
contained the same N-substituent as Linagliptin (see Table 1)
3. Results and discussion
3.1. Hit identification through pharmacophore hybridization
The similarity of the pharmacophore between Alogliptin, com-
pound 4 and Linagliptin reminded us that the cyanobenzyl group
of 4 could be replaced with a butynyl group without sacrificing
in vitro activity (Fig. 2). Because Linagliptin is the most potent
and longest-lasting DPP-IV inhibitor on the market, we decided
to test our hypothesis with a pharmacophore hybridization ap-
proach.10 However, to our disappointment, compound 5 signifi-
cantly lost activity (IC50 = 0.86 lM). Though this compound did
not achieve the desired inhibition against DPP-IV, it might be a
hit worthy of further optimization.
3.3. Biological evaluation of compound 18o
Compound 18o was picked for preliminary evaluation for its
high DPP-IV inhibitory activity. It had no inhibition against DPP-
8 or 9 up to 10
lM. Compound 18o had no CYP 3A inhibition up
to 30 M, which is likely to decrease the risk of drug–drug interac-
l
tions. The pharmacokinetic study of 18o also displayed a good
drug-like profile and successfully overcame the insufficiency in
the starting compound 4 (Table 2 and Fig. 4). Compared to its par-
ent compound 4, it had a longer half life in rats (approximately 5 h)
Table 1
Modifications at the N-5 position
O
R
N
N
NH2
N
N
No.
R
DPP-IVa
44.0
No.
R
DPP-IVa
14.5
N
N
N
N
∗
18b
18i
∗
∗
F
∗
18c
18d
18e
44.1
27.6
48.2
18j
18k
18l
52.6
11.4
4.67
CH3
N
H
H
N
N
Cl
∗
∗
∗
∗
N
N
F
N
N
N
N
∗
∗
∗
∗
∗
∗
18f
7.05
18m
18n
5.93
15.5
N
N
18g
139.2
Br
Cl
N
N
N
18h
60.1
18o
1.55
N
a
nM. Data represent the mean of at least three independent measurements.

1752
S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
Table 2
Alogliptin, its bioavailability (40%) and half life (2 h) still have some
Pharmacokinetic parameters of 18o in rat
space to improve. Thus we initiated an optimization toward better
PK properties while maintain the necessary in vitro activity. In-
spired by the similarity in pharmacophores between Alogliptin,
compound 4 and Linagliptin, we identified a novel DPP-IV inhibitor
(5) by utilizing a pharmacophore hybridization strategy. Though
Dose IV/po
AUC0–1 po
T1/2 po
Clz IV
Vz IV
MRT0–1 po
F
mg/kg
l
10/30
g h mLꢁ1
25.3 2.6
4.9 2.5v
1.0 0.1
4.0 1.3
8.3 1.6
82.9
h
L hꢁ1 kgꢁ1
L kgꢁ1
the in vitro activity was still low, compound 5 (IC50 = 0.86 lM)
h
%
was a logical hit for optimization and generated a series of DPP-IV
inhibitors with significantly improved activity. Compound 18o
was finally obtained with the desired in vitro activity
(IC50 = 1.55 nM) and much improved pharmacokinetic profile (bio-
availability 82.9%; half life 4.9 h). Thus 18o is highly possible to be
more active than compound 4 and Alogliptin in vivo. This paper is
mainly to disclose the successful optimization by adopting classical
medicinal chemistry strategy and suggests that further optimiza-
tion within this series may generate more active DPP-IV inhibitors.
po: Oral administration. IV: Intravenous injection.
O
H
O
N
H
N
N
N
N
NH2
NH2
N
N
N
Br
5. Experimental section
5.1. Chemistry
6
, IC50 = 456.8 nM
5
, IC50 = 860.0 nM
All commercially available compounds and solvents were of re-
agent grade and used without further treatment unless otherwise
noted. Reactions were monitored by TLC using Qing Dao Hai Yang
GF254 silica gel plates (5 ꢀ 10 cm); zones were detected visually
under ultraviolet irradiation (254 nm) and by spraying with an
ethanol solution of 2,4-DNP or ninhydrin, or by fuming with iodine
steam. Silica gel column chromatography was performed on silica
gel (200–300 mesh) from Qing Dao Hai Yang. NMR spectra were re-
corded on a Bruker NMR AVANCE 400 (400 MHz) or a Bruker NMR
AVANCE 500 (500 MHz). Chemical shifts (d) were recorded in ppm
and coupling constants (J) in hertz (Hz). Splitting patterns describe
apparent multiplicities and are designated as s (singlet), d (dou-
blet), t (triplet), q (quartet), m (multiplet) or br (broad). MS data
were measured on an Agilent MSD-1200 ESI-MS system.
O
N
O
R
N
N
N
NH2
NH2
N
N
N
N
18a, IC50 = 211.9 nM
Figure 3. Hit-to-lead optimization strategy.
18b-18o
5.1.1. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-3H-
pyrrolo[3,2-d]pyrimidin-4(5H)-one (5)
A mixture of 16 (301 mg, 1.0 mmol), 3-(R)-aminopiperidine
dihydrochloride (207 mg, 1.2 mmol) and NaHCO3 (336 mg,
4.0 mmol) in a sealed tube containing 15 mL of ethanol was heated
at 120 °C and stirred overnight. The reaction mixture was subse-
quently cooled to room temperature and filtered. The resulting fil-
trate was concentrated in vacuo and then purified by flash
chromatography to yield compound 5. Yield: 81.8%. 1H NMR
(400 MHz, MeOD) d ppm: 7.37 (1H, s), 4.84–4.82 (2H, m), 3.53–
3.50 (1H, m), 3.47–3.43 (1H, m), 3.06 (1H, s), 2.92–2.98 (1H, m),
2.03–2.00 (1H, m), 1.98–1.96 (1H, m), 1.89–1.87 (1H, m), 1.81
(3H, s), 1.79–1.73 (1H, m), 1.33–1.27 (1H, m); 13C NMR
(500 MHz, MeOD) d ppm: 24.92 (1C), 28.68 (1C), 34.07 (1C),
35.63 (1C), 52.68 (1C), 58.99 (1C), 75.29 (1C), 80.54 (1C), 81.04
(1C), 91.44 (1C), 116.26 (1C), 129.02 (1C), 142.13 (1C), 155.80
(1C), 156.49 (1C); ESI-MS calcd for (C15H19N5O) [M+H]+, 363.07,
365.07, found 364.0, 366.0.
Figure 4. Concentration–time curve of 18o in rat.
and a better oral bioavailability (82.9%) that was comparable with
marketed DPP-IV inhibitors (Alogliptin, 45%; Linagliptin,
50.7%).9,12,13
Since compound 18o acquired better half life and bioavailabil-
ity, it is highly possible to have a better in vivo efficacy than Alog-
liptin. As the structure is similar with Linagliptin, some evaluation
can be done like the peers’ work.14 Currently, there is no more ur-
gent to develop new drugs in DPP-IV inhibitor class, in this paper
we mainly report the optimization strategy started from com-
pound 4 and the improved pharmacokinetic profile of compound
18o.
Compounds 6 and 18a–o were prepared in a manner identical
to that described for 5.
5.1.2. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-3H-
pyrrolo[3,2-d]pyrimidin-4(5H)-one (6)
Yield: 67.8%. 1H NMR (400 MHz, CDCl3) d ppm: 10.87 (1H, s),
7.23 (1H, d, J = 2.8 Hz), 6.36 (1H, d, J = 2.8 Hz), 4.85 (2H, s), 3.49–
3.46 (1H, m), 3.36–3.33 (1H, m), 3.11 (1H, m), 2.92 (1H, m),
2.80–2.78 (1H, m), 2.27 (2H, s), 1.99–1.97 (1H, m), 1.88–1.86
(1H, m), 1.79 (3H, s), 1.73–1.70 (1H, m), 1.37–1.32 (1H, m); ESI-
MS calcd for (C15H19N5O) [M+H]+, 286.16, found 286.1.
4. Conclusion
Though our recently reported novel DPP-IV inhibitor 4 acquires
acceptable drug like properties and has a better in vivo efficacy than

S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
1753
5.1.3. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
5.1.8. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
methyl-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (18a)
(quinolin-4-ylmethyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one
(18e)
Yield: 52.6%. 1H NMR (400 MHz, CDCl3) d ppm: 6.87 (1H, d,
J = 2.8 Hz), 6.17 (1H, d, J = 2.8 Hz), 5.19 (2H, s), 4.77–4.63 (2H, AB
q, J = 34.8 Hz, 16.4 Hz), 3.96 (1H, s), 3.50–3.47 (1H, m), 3.32 (2H,
m), 3.03–2.98 (1H, m), 2.90 (1H, m), 2.05 (1H, m), 1.84 (1H, m),
1.75 (3H, s), 1.65–1.63 (2H, m); 13C NMR (500 MHz, CDCl3) d
ppm: 22.64 (1C), 28.34 (1C), 30.38 (1C), 34.41 (1C), 35.57 (1C),
47.69 (1C), 51.55 (1C), 55.40 (1C), 74.46 (1C), 79.35 (1C), 101.61
(1C), 115.10 (1C), 131.69 (1C), 142.73 (1C), 153.49 (1C), 155.42
(1C); ESI-MS calcd for (C16H21N5O) [M+H]+, 300.17, found 300.1.
Yield: 51.6%. 1H NMR (400 MHz, MeOD) d ppm: 8.67 (1H, d,
J = 8.0 Hz), 8.25–8.23 (1H, d, J = 8.0 Hz), 8.08–8.06 (1H, d,
J = 8.0 Hz), 7.85–7.81 (1H, m), 7.73–7.69 (1H, m), 7.40 (1H, d,
J = 2.8 Hz), 6.62 (1H, d, J = 2.8 Hz), 6.43–6.42 (1H, m), 6.24 (2H, s),
4.79–4.70 (2H, t, J = 19.2 Hz), 3.48–3.45 (1H, m), 3.41–3.38 (1H,
m), 3.03–2.98 (1H, m), 2.93–2.87 (1H, m), 2.76–2.70 (1H, m),
2.01–1.98 (1H, m), 1.90–1.87 (1H, m), 1.79–1.76 (1H, m), 1.74
(3H, s), 1.40–1.30 (1H, m); 13C NMR (500 MHz, MeOD) d ppm:
24.52 (1C), 33.74 (1C), 35.42 (1C), 48.53 (1C), 49.55 (1C), 49.58
(1C), 52.76 (1C), 59.35 (1C), 75.28 (1C), 80.52 (1C), 103.96 (1C),
116.09 (1C), 119.10 (1C), 124.29 (1C), 127.06 (1C), 128.60 (1C),
129.89 (1C), 131.22 (1C), 134.10 (1C), 145.54 (1C), 147.43 (1C),
148.48 (1C), 151.30 (1C), 156.14 (1C), 156.94 (1C); ESI-MS calcd
for (C25H26N6O) [M+H]+, 427.22, found 427.1 (see Fig. 3)
5.1.4. (R)-2-(2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-4-
oxo-3H-pyrrolo[3,2-d]pyrimidin-5(4H)-yl)acetonitrile (18b)
Yield: 54.2%. 1H NMR (400 MHz, CDCl3) d ppm: 7.14 (1H, d,
J = 2.8 Hz), 6.39 (1H, d, J = 2.8 Hz), 5.46 (2H, s), 4.76 (2H, s), 3.49–
3.47 (1H, m), 3.38–3.35 (1H, m), 3.15 (1H, m), 2.96–2.91 (1H, m),
2.80 (2H, s), 2.00–2.96 (1H, m), 1.88–1.84 (1H, m), 1.80 (3H, s),
1.75–1.65 (2H, m), 1.42 (1H, m);13C NMR (500 MHz, CDCl3) d
ppm: 22.95 (1C), 28.35 (1C), 32.54 (1C), 34.80 (1C), 35.47 (1C),
47.58 (1C), 50.39 (1C), 51.26 (1C), 58.04 (1C), 73.97 (1C), 79.86
(1C), 104.73 (1C), 114.60 (1C), 130.28 (1C), 143.98 (1C), 154.66
(1C), 155.57 (1C); ESI-MS calcd for (C17H20N6O) [M+H]+, 325.17,
found 325.1.
5.1.9. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
(quinolin-2-ylmethyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one
(18f)
Yield: 54.8%. 1H NMR (400 MHz, CDCl3) d ppm: 8.20–8.18 (1H,
d, J = 8.0 Hz), 7.98–7.96 (1H, d, J = 8.8 Hz), 7.83–7.81 (1H, d,
J = 8.0 Hz), 7.85–7.81 (1H, m), 7.73–7.70 (1H, m), 7.54–7.51 (1H,
m), 7.40 (1H, d, J = 2.8 Hz), 6.35 (1H, d, J = 2.8 Hz), 5.87 (2H, s),
4.79–4.69 (2H, t, J = 21.6 Hz), 3.44–3.41 (1H, m), 3.36–3.29 (1H,
m), 2.99–2.94 (1H, m), 2.88–2.85 (1H, m), 2.71–2.63 (1H, m),
1.99–1.95 (1H, m), 1.86–1.83 (1H, m), 1.73 (1H, m), 1.73 (3H, s),
1.38–1.26 (1H, m); 13C NMR (500 MHz, MeOD) d ppm: 24.48
(1C), 33.69 (1C), 35.32 (1C), 48.48 (1C), 49.51 (1C), 52.69 (1C),
54.44 (1C), 59.33 (1C), 75.36 (1C), 80.43 (1C), 103.79 (1C), 115.92
(1C), 120.19 (1C), 127.82 (1C), 128.85 (1C), 128.94 (1C), 129.19
(1C), 131.24 (1C), 133.92 (1C), 139.13 (1C), 145.41 (1C), 148.50
(1C), 155.96 (1C), 156.92 (1C), 159.53 (1C); ESI-MS calcd for
(C25H26N6O) [M+H]+, 427.22, found 427.1.
5.1.5. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
(pyrimidin-2-ylmethyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one
(18b)
Yield: 54.5%. 1H NMR (400 MHz, MeOD) d ppm: 8.65–8.63 (2H,
d, J = 4.8 Hz), 7.32 (1H, d, J = 2.8 Hz), 7.31–7.28 (1H, t, J = 4.8 Hz),
6.31 (1H, d, J = 2.8 Hz), 5.81 (2H, s), 4.76–4.67 (2H, q, J = 18.8 Hz),
3.46–3.43 (1H, m), 3.36–3.29 (1H, m), 3.12–3.08 (1H, m), 2.90
(1H, m), 2.81 (1H, m), 2.78–2.76 (1H, m), 2.01–1.98 (1H, m),
1.88–1.86 (1H, m), 1.85–1.75 (1H, m), 1.72 (3H, s), 1.43–1.39
(1H, m); 13C NMR (500 MHz, MeOD) d ppm: 22.34 (1C), 28.87
(1C), 32.60 (1C), 35.16 (1C), 49.51 (1C), 50.51 (1C), 52.77 (1C),
54.39 (1C), 58.06 (1C), 75.36 (1C), 80.41 (1C), 103.23 (1C), 116.32
(1C), 121.15 (1C), 134.41 (1C), 145.10 (1C), 155.54 (1C), 156.79
(1C), 158.70 (1C), 167.68 (1C); ESI-MS calcd for (C20H23N7O)
[M+H]+, 378.20, found 378.1.
5.1.10. (R)-2-(3-Aminopiperidin-1-yl)-5-((6-bromoquinolin-2-
yl)methyl)-3-(but-2-yn-1-yl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18g)
Yield: 43.8%. 1H NMR (400 MHz, MeOD) d ppm: 8.14–8.11 (1H,
d, J = 8.8 Hz), 8.00–7.99 (1H, d, J = 2.0 Hz), 7.90–7.87 (1H, m), 7.80–
7.77 (1H, m), 7.44 (1H, d, J = 2.8 Hz), 7.23–7.21 (1H, d, J = 8.8 Hz),
6.42 (1H, d, J = 2.8 Hz), 5.91 (2H, s), 4.83–4.81 (2H, m), 3.53–3.50
(1H, m), 3.45–3.42 (1H, m), 3.10–3.04 (1H, m), 2.96–2.91 (1H,
m), 2.80–2.75 (1H, m), 2.07–2.03 (1H, m), 1.95–1.90 (1H, m),
1.84 (3H, s), 1.83–1.75 (1H, m), 1.45–1.33 (1H, m); 13C NMR
(500 MHz, CDCl3+MeOD) d ppm: 24.27 (1C), 33.45 (1C), 35.29
(1C), 48.48 (1C), 49.50 (1C), 49.98 (1C), 52.50 (1C), 54.29 (1C),
59.07 (1C), 75.14 (1C), 80.40 (1C), 103.70 (1C), 115.68 (1C),
121.17 (1C), 129.63 (1C), 130.72 (1C), 131.04 (1C), 133.61 (1C),
134.19 (1C), 137.85 (1C), 145.06 (1C), 146.83 (1C), 155.63 (1C),
156.67 (1C), 159.63 (1C); ESI-MS calcd for (C25H25BrN6O) [M+H]+,
505.13, found 505.1.
5.1.6. (R)-5-((1H-Indol-3-yl)methyl)-2-(3-aminopiperidin-1-yl)-
3-(but-2-yn-1-yl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (18c)
Yield: 51.7%. 1H NMR (400 MHz, MeOD) d ppm: 7.46–7.44 (1H,
d, J = 8.0 Hz), 7.30 (1H, s), 7.28 (1H, s), 7.28 (1H, d, J = 2.8 Hz), 7.04–
7.00 (1H, m), 6.92–6.88 (1H, m), 6.13 (1H, d, J = 2.8 Hz), 5.73 (2H, s),
4.81 (2H, s), 3.57 (1H, m), 3.47–3.44 (1H, m), 3.26–3.25 (1H, m),
3.07–3.01 (1H, m), 2.05 (1H, m), 1.89–1.87 (1H, m), 1.78 (1H, m),
1.77 (3H, s), 1.63–1.61 (1H, m), 1.22 (1H, m); 13C NMR (500 MHz,
MeOD) d ppm: 22.47 (1C), 28.92 (1C), 35.44 (1C), 44.25 (1C),
48.49 (1C), 49.51 (1C), 52.92 (1C), 53.85 (1C), 58.06 (1C), 61.44
(1C), 75.36 (1C), 80.41 (1C), 102.73 (1C), 112.44 (1C), 115.73
(1C), 119.23 (1C), 120.29 (1C), 122.77 (1C), 125.81 (1C), 127.80
(1C), 133.07 (1C), 138.14 (1C), 144.66 (1C), 154.56 (1C), 156.82
(1C); ESI-MS calcd for (C24H26N6O) [M+H]+, 415.22, found 415.1.
5.1.11. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((6-
chloroquinolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18h)
5.1.7. (R)-5-((1H-Benzo[d]imidazol-2-yl)methyl)-2-(3-
aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-3H-pyrrolo[3,2-
d]pyrimidin-4(5H)-one (18d)
Yield: 67.8%. 1H NMR (400 MHz, CDCl3) d ppm: 7.96–7.93 (1H,
m), 7.71 (1H, d, J = 2.4 Hz), 7.60–7.57 (1H, dd, J = 2.4 Hz,
J = 9.2 Hz), 7.30–7.26 (1H, m), 7.20 (1H, d, J = 2.8 Hz), 6.34 (1H, d,
J = 2.8 Hz), 5.88 (2H, s), 4.79 (2H, d, J = 2.0 Hz), 3.48–3.43 (1H, m),
3.06–3.00 (1H, m), 2.90–2.85 (1H, m), 2.72–2.67 (1H, m), 1.98–
1.93 (1H, m), 1.89 (2H, s), 1.85–1.82 (1H, m), 1.80 (3H, s), 1.74–
1.65 (1H, m), 1.32–1.24 (1H, m); 13C NMR (500 MHz, CDCl3) d
ppm: 23.41 (1C), 33.45 (1C), 34.49 (1C), 47.83 (1C), 51.36 (1C),
53.67 (1C), 59.26 (1C), 74.47 (1C), 77.00 (1C), 79.34 (1C), 103.12
Yield: 77.1%. 1H NMR (400 MHz, MeOD) d ppm: 7.47 (2H, m),
7.39 (1H, d, J = 2.8 Hz), 7.18–7.15 (2H, m), 6.32 (1H, d, J = 2.8 Hz),
5.84 (1H, s), 4.78 (2H, s), 3.46–3.44 (1H, m), 3.43–3.42 (1H, m),
3.41–3.40 (1H, m), 3.03–2.98 (1H, m), 2.08–2.05 (1H, m), 1.91–
1.88 (1H, m), 1.82–1.79 (1H, m), 1.77–1.75 (1H, m), 1.73 (3H, s),
1.60–1.57 (1H, m); ESI-MS calcd for (C23H25N7O) [M+H]+, 416.21,
found 416.2.

1754
S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
(1C), 114.75 (1C), 120.82 (1C), 126.14 (1C), 127.93 (1C), 130.52
(1C), 130.74 (1C), 131.49 (1C), 132.14 (1C), 136.21 (1C), 143.72
(1C), 145.95 (1C), 154.24 (1C), 155.72 (1C), 158.00 (1C); ESI-MS
calcd for (C25H25ClN6O) [M+H]+, 461.18, found 461.1.
2.76–2.71 (1H, m), 1.98–1.95 (1H, m), 1.88 (1H, m), 1.85 (2H, s),
1.80 (3H, s), 1.76–1.66 (1H, m), 1.33–1.31 (1H, m); 13C NMR
(500 MHz, CDCl3) d ppm: 23.17 (1C), 33.51 (1C), 34.49 (1C),
47.03 (1C), 51.37 (1C), 53.71 (1C), 74.51 (1C), 79.34 (1C), 103.12
(1C), 112.76 (1C), 114.80 (1C), 116.87 (1C), 119.24 (1C), 124.38
(1C), 129.45 (1C), 131.53 (1C), 137.02 (1C), 143.70 (1C), 148.55
(1C), 154.22 (1C), 155.73 (1C), 158.81 (1C), 162.13 (1C), 164.12
(1C); ESI-MS calcd for (C25H25FN6O) [M+H]+, 445.21, found 445.1.
5.1.12. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((6-
fluoroquinolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-
one (18i)
Yield: 62.5%. 1H NMR (400 MHz, CDCl3) d ppm: 8.02–7.97 (2H,
m), 7.46–7.41 (1H, m), 7.36–7.33 (1H, m), 7.31–7.29 (1H, d,
J = 8.4 Hz), 7.20 (1H, d, J = 2.8 Hz), 6.33 (1H, d, J = 2.8 Hz), 5.89
(2H, s), 4.79 (2H, d, J = 2.0 Hz), 3.48–3.43 (1H, m), 3.37–3.34 (1H,
m), 3.06–3.00 (1H, m), 2.90–2.85 (1H, m), 1.98–1.93 (1H, m),
1.87–1.84 (1H, m), 1.83 (2H, s), 1.79 (3H, s), 1.74–1.66 (1H, m),
1.32–1.24 (1H, m); 13C NMR (500 MHz, CDCl3) d ppm: 23.40 (1C),
33.45 (1C), 34.45 (1C), 47.81 (1C), 51.34 (1C), 53.63 (1C), 59.26
(1C), 74.48 (1C), 77.00 (1C), 79.31 (1C), 103.06 (1C), 110.37 (1C),
114.73 (1C), 119.68 (1C), 120.72 (1C), 127.90 (1C), 131.53 (1C),
136.45 (1C), 143.70 (1C), 144.63 (1C), 154.21 (1C), 155.70 (1C),
157.00 (1C), 159.34 (1C), 161.31 (1C); ESI-MS calcd for
(C25H25FN6O) [M+H]+, 445.21, found 445.1.
5.1.16. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
(quinoxalin-2-ylmethyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one
(18m)
Yield: 61.5%. 1H NMR (400 MHz, MeOD) d ppm: 8.67 (1H, s),
7.97–7.90 (2H, m), 7.74–7.69 (2H, m), 7.45 (1H, d, J = 2.8 Hz),
6.35 (1H, d, J = 2.8 Hz), 5.90 (2H, s), 4.78–4.71 (2H, m), 3.44–3.41
(1H, m), 3.36–3.32 (1H, m), 2.99–2.95 (1H, m), 2.86–2.84 (1H,
m), 2.71–2.66 (1H, m), 1.99–1.94 (1H, m), 1.85–1.82 (1H, m),
1.73 (3H, s), 1.69–1.68 (1H, m), 1.37–1.26 (1H, m); 13C NMR
(500 MHz, MeOD) d ppm: 24.46 (1C), 33.68 (1C), 35.33 (1C),
48.48 (1C), 49.98 (1C), 52.64 (1C), 54.40 (1C), 59.33 (1C), 75.35
(1C), 80.43 (1C), 103.71 (1C), 115.75 (1C), 129.67 (1C), 129.98
(1C), 131.16 (1C), 131.58 (1C), 134.10 (1C), 142.64 (1C), 142.93
(1C), 145.16 (1C), 145.45 (1C), 154.23 (1C), 155.90 (1C), 156.81
(1C); ESI-MS calcd for (C24H25N7O) [M+H]+, 428.21, found 428.1.
5.1.13. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((6-
methylquinolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18j)
Yield: 69.4%. 1H NMR (400 MHz, MeOD) d ppm: 8.06–8.04 (1H,
d, J = 8.4 Hz), 7.86–7.84 (1H, d, J = 8.4 Hz), 7.62 (1H, s), 7.61–7.59
(1H, dd, J = 8.8 Hz, J = 1.6 Hz), 7.39 (1H, d, J = 2.8 Hz), 7.30–7.28
(1H, d, J = 8.4 Hz), 6.34 (1H, d, J = 2.8 Hz), 5.73 (2H, s), 4.80–4.74
(2H, m), 3.45–3.43 (1H, m), 3.35–3.34 (1H, m), 3.01–2.95 (1H,
m), 2.87–2.67 (1H, m), 2.65 (3H, s), 2.00–1.96 (1H, m), 1.88–1.84
(1H, m), 1.77 (3H, s), 1.74–1.66 (1H, m), 1.38–1.35 (1H, m); 13C
NMR (500 MHz, MeOD) d ppm: 24.63 (1C), 33.73 (1C), 35.33 (1C),
48.49 (1C), 49.51 (1C), 52.19 (1C), 52.26 (1C), 55.50 (1C), 59.40
(1C), 75.49 (1C), 80.41 (1C), 103.56 (1C), 115.64 (1C), 123.60
(1C), 127.07 (1C), 127.79 (1C), 128.74 (1C), 130.34 (1C), 133.41
(1C), 137.70 (1C), 138.21 (1C), 145.28 (1C), 147.72 (1C), 155.81
(1C), 156.77 (1C), 160.52 (1C); ESI-MS calcd for (C26H28N6O)
[M+H]+, 441.23, found 441.1.
5.1.17. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((3-
methylquinoxalin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18n)
Yield: 53.4%. 1H NMR (400 MHz, CDCl3) d ppm: 7.98–7.92 (2H,
m), 7.69–7.61 (2H, m), 7.07 (1H, d, J = 2.8 Hz), 6.34 (1H, d,
J = 2.8 Hz), 6.04 (2H, s), 4.78 (2H, d, J = 2.0 Hz), 3.49–3.46 (1H, m),
3.38–3.35 (1H, m), 3.06–3.01 (1H, m), 2.91–2.86 (1H, m), 2.72
(3H, s), 2.72 (1H, m), 1.98–1.94 (1H, m), 1.87–1.81 (1H, m), 1.77
(2H, s), 1.77 (3H, s), 1.72–1.68 (1H, m), 1.29–1.27 (1H, m); 13C
NMR (500 MHz, CDCl3) d ppm: 22.13 (1C), 23.44 (1C), 29.63 (1C),
33.46 (1C), 34.48 (1C), 47.84 (1C), 50.93 (1C), 51.37 (1C), 59.32
(1C), 74.46 (1C), 76.78 (1C), 79.35 (1C), 103.08 (1C), 115.00 (1C),
128.29 (1C), 128.98 (1C), 129.81 (1C), 131.41 (1C), 140.66 (1C),
141.61 (1C), 143.54 (1C), 150.83 (1C), 152.80 (1C), 154.17 (1C),
155.84 (1C); ESI-MS calcd for (C25H27N7O) [M+H]+, 442.23, found
442.2.
5.1.14. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((7-
chloroquinolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18k)
5.1.18. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((4-
methylquinazolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-
4(5H)-one (18o)
Yield: 43.4%. 1H NMR (400 MHz, CDCl3) d ppm: 8.03 (1H, d,
J = 1.6 Hz), 8.01 (1H, s), 7.69–7.67 (1H, d, J = 8.8 Hz), 7.45–7.43
(1H, dd, J = 8.8 Hz, J = 2.0 Hz), 7.30–7.28 (1H, d, J = 8.8 Hz), 7.22
(1H, d, J = 2.8 Hz), 6.35 (1H, d, J = 2.8 Hz), 5.90 (2H, s), 4.84–4.75
(2H, m), 3.50–3.47 (1H, m), 3.37–3.34 (1H, m), 3.16–3.12 (1H,
m), 2.97–2.92 (1H, m), 2.84–2.80 (1H, m), 2.43 (2H, s), 1.99–1.96
(1H, m), 1.89–1.85 (1H, m), 1.80 (3H, t, J = 2.4 Hz), 1.76–1.66 (1H,
m), 1.42–1.40 (1H, m); 13C NMR (500 MHz, MeOD) d ppm: 23.07
(1C), 29.65 (1C), 32.65 (1C), 34.53 (1C), 47.70 (1C), 51.40 (1C),
53.67 (1C), 58.27 (1C), 74.45 (1C), 79.46 (1C), 103.11 (1C), 114.78
(1C), 120.14 (1C), 125.74 (1C), 127.53 (1C), 128.21 (1C), 128.71
(1C), 131.64 (1C), 135.49 (1C), 136.98 (1C), 143.59 (1C), 147.96
(1C), 154.12 (1C), 155.67 (1C), 158.74 (1C); ESI-MS calcd for
(C25H25ClN6O) [M+H]+, 461.18, found 461.1.
Yield: 59.0%. 1H NMR (400 MHz, CDCl3) d ppm: 7.97–7.95 (2H,
d, J = 8.4 Hz), 7.77–7.70 (2H, m), 7.51–7.47 (1H, m), 7.13 (1H, d,
J = 2.8 Hz), 6.28 (1H, d, J = 2.8 Hz), 5.86 (2H, s), 4.68–4.58 (2H, dd,
J = 33.6 Hz, J = 4.0 Hz), 3.85 (2H, s), 3.39–3.37 (1H, m), 3.27–3.24
(1H, m), 3.06 (1H, m), 2.85 (1H, m), 2.79 (3H, s), 2.75–2.73 (1H,
m), 1.93–1.90 (1H, m), 1.80–1.76 (1H, m), 1.64 (3H, s), 1.64 (1H,
m), 1.36–1.34 (1H, m); 13C NMR (500 MHz, CDCl3+MeOD) d ppm:
21.24 (1C), 22.30 (1C), 31.24 (1C), 34.09 (1C), 47.11 (1C), 49.02
(1C), 51.32 (1C), 53.40 (1C), 56.65 (1C), 74.01 (1C), 79.28 (1C),
102.17 (1C), 115.14 (1C), 122.87 (1C), 124.82 (1C), 127.31 (1C),
128.35 (1C), 132.27 (1C), 133.72 (1C), 142.95 (1C), 149.42 (1C),
153.76 (1C), 155.34 (1C), 160.96 (1C), 169.08 (1C); ESI-MS calcd
for (C25H27N7O) [M+H]+, 442.23, found 442.2.
5.1.15. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((7-
fluoroquinolin-2-yl)methyl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-
one (18l)
5.2. In vitro inhibition of DPP-IV, DPP-8 and DPP-9
Yield: 45.9%. 1H NMR (400 MHz, CDCl3) d ppm: 8.04–8.02 (1H,
d, J = 8.4 Hz), 7.75–7.64 (1H, m), 7.67–7.64 (1H, dd, J = 5.2 Hz,
J = 2.0 Hz), 7.30–7.28 (1H, m), 7.24 (1H, s), 7.22 (1H, d, J = 2.8 Hz),
6.35 (1H, d, J = 2.8 Hz), 5.90 (2H, s), 4.80 (2H, d, J = 2.0 Hz), 3.49–
3.47 (1H, m), 3.38–3.35 (1H, m), 3.07 (1H, m), 2.93–2.88 (1H, m),
Solutions of test compounds at varying concentrations
(610 mM final concentration) were prepared in dimethyl sulfoxide
(DMSO) and diluted into assay buffer containing 20 mM Tris (pH
7.4), 20 mM KCl, and 0.1 mg/mL BSA. Human DPP-IV (0.1 nM final
concentration) was added to the dilutions and pre-incubated for

S. Zeng et al. / Bioorg. Med. Chem. 21 (2013) 1749–1755
1755
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10 min at ambient temperature before the reaction was initiated
by the addition of Gly-Pro-AMC (H-glycyl-prolyl-7-amino-4-meth-
ylcoumarin, Sigma–Aldrich, 10
lM final concentration). The total
volume of the reaction mixture was 100
lL. The kinetics of the
reaction was monitored (excitation at 400 nm, emission at
505 nm) for 5–10 min, or an endpoint was measured after
10 min. Inhibition constants (IC50) were calculated from enzyme
progress curves using standard mathematical models.
5.3. In vivo pharmacokinetic study
Adult male SD rats (n = 4/group) were administered the test
compounds dissolved in distilled water at a single dose of 20 mg/
kg or 25 mg/kg for oral administration and 5 mg/mL by injection.
Blood samples of 100–200 lL were collected from the orbit at 11
time points within 24 h. The blood concentration of test com-
pounds was determined by LC–MS/MS. The PK parameters were
obtained from the pharmacokinetic software DAS. 2.0.
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Acknowledgments
This research was supported by Grant No. 2009Z1-E871 from
the Bureau of Science and Technology of Guangzhou Municipality
and the Natural Science Foundation of China (NSFC 21102145).
12. Xie, H.; Zeng, L.; Zeng, S.; Lu, X.; Zhang, G.; Zhao, X.; Cheng, N.; Tu, Z.; Li, Z.; Xu,
H.; Yang, L.; Zhang, X.; Huang, M.; Zhao, J.; Hu, W. Eur. J. Med. Chem. 2012, 52,
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