Lead discovery of quinoxalinediones as an inhibitor of dipeptidyl peptidase-IV (DPP-IV) by high-throughput screening

Article abstract of DOI:10.1016/j.bmcl.2004.02.065

N-Ureido-quinoxalinedione derivatives have been discovered as leads for a novel series of dipeptidyl peptidase-IV (DPP-IV) inhibitors through high-throughput screening of our chemical library. A brief structure-activity relationship of the compounds was investigated. Among them, entry 5 showed the most potent inhibitory activity. The nitro group in quinoxaline moiety and the aromatic sulfonyl substituted ureido functional group seem to be important to increase the potency dramatically.

Full text of DOI:10.1016/j.bmcl.2004.02.065

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2661–2664
Lead discovery of quinoxalinediones as an inhibitor of dipeptidyl
peptidase-IV (DPP-IV) by high-throughput screening
Hyae-Gyeong Cheon,a Chul-Min Lee,b Beom-Tae Kimb and Ki-Jun Hwangb,*
aBiomedicinal Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yusung,
Taejeon 305-706, South Korea
bDepartment of Chemistry and Research Center of Bioactive Materials, College of Natural Science, Chonbuk National University,
Dukjindong 664-14, Chonju 561-756, South Korea
Received 9 December 2003; accepted 12 February 2004
Abstract—N-Ureido-quinoxalinedione derivatives have been discovered as leads for a novel series of dipeptidyl peptidase-IV (DPP-
IV) inhibitors through high-throughput screening of our chemical library. A brief structure–activity relationship of the compounds
was investigated. Among them, entry 5 showed the most potent inhibitory activity. The nitro group in quinoxaline moiety and the
aromatic sulfonyl substituted ureido functional group seem to be important to increase the potency dramatically.
Ó 2004 Published by Elsevier Ltd.
1. Introduction
O
HO
N
Glucagon-like peptide-1 (GLP-1) is an incretin secreted
from the small intestine in response to food ingestion,
and exhibits several biological effects including stimu-
lation of insulin secretion, inhibition of glucagon secre-
tion, slowing gastric emptying and induction of satiety.1
However, GLP-1 action has a very short half life about
1 min due to the degradation by DPP-IV.2 DPP-IV is a
serine protease, which removes the dipeptide from the
N-terminus of substrate proteins by cleaving post pro-
line or alanine residues.3 DPP-IV is expressed in many
tissues and body fluids, and exists as either a membrane-
bound or a soluble enzyme.4
N
N
H
S
NH2
O
CN
LAF-237
P-32/98
Figure 1. DPP-IV inhibitors.
pounds (Korea Chemical Bank) was screened by high
throughput screening (HTS) techniques. DPP-IV HTS
was carried out on 96-well plates with HTS automation
instruments using rat plasma as the enzyme source and
Ala-Pro-7-amino-4-trifluoromethyl-coumarin (AFC) as
the substrate. As a result, we found a novel scaffold of
quinoxalinedione as a DPP-IV inhibitor. Here, we
report structure–activity relationship of quinoxaline-
diones on in vitro DPP-IV inhibitory activity.
Since the discovery that GLP-1 secretion is impaired in
type 2 diabetes, several approaches have been employed
to enhance GLP-1 action, thereby ameliorating type 2
diabetes.5 Among them, DPP-IV inhibition has been
proved to be an effective method for type 2 diabetes
pharmacotherapy.6 Several peptide-like DPP-IV inhibi-
tors including LAF-237 and P-32/98 are under clinical
trials (Fig. 1).7
2. Chemistry
Quinoxalinediones 2 were synthesized by the reaction of
aryldiamines 1 with oxalic acid in 3 N HCl under reflux.8
The yields were generally over 90%. The next amination
step was not trivial. Among several other aminating
agents, hydroxylamine sulfonic acid9 was successfully
utilized to produce the key intermediate 3 by treating
quinoxalinedione 2 with it under basic conditions
In order to discover novel nonpeptide DPP-IV inhibi-
tors, a chemical library of approximately 32,000 com-
* Corresponding author. Tel.: +82-63-270-3577; fax: +82-63-270-4322;
e-mail: kijun@moak.chonbuk.co.kr
0960-894X/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.02.065

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H.-G. Cheon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2661–2664
(10 equiv KOH and K2CO3 in water). The yields were at
the range of 40–62% depending upon the functional
group. With the strong electron withdrawing substitu-
ents like nitro group, the yield was generally low
(Scheme 1). The amino-quinoxalinediones represented
as 3 deserve some mentions due to the unsymmetrical
case such as compound 3 (A ¼ H, B ¼ NO2). It was
envisioned that monoamination of compound 2 (A ¼ H,
B ¼ NO2) gave 1-amino-7-nitro-quinoxalinedione (3;
A ¼ H, B ¼ NO2) rather than 1-amino-8-nitro-quin-
oxalinedione (3; A ¼ NO2, B ¼ H) among the possible
regioisomers, judging from the character of resonance
stabilized anion of 2a by contrast with anion of 2b (Fig.
2) and from chemical shifts comparison of the aromatic
protons between the observed values10 and simulated
ones by ChemDrawâ program.
with appropriate isocyanates and isothiocyanates in
DMF, respectively, in 52–75% yields. However, com-
pounds 5 with nitro group could not be synthesized
due to the low electrophilicity of isothiocyanate (Scheme
2).
Encouraged by the strong biological activity of some
compounds of 4, the synthesis of compounds 7, whose
nitrogen atom of side chain of 4 is replaced with carbon,
were examined next. Quinoxalinediones 2 were treated
with methyl bromoacetate and sodium hydride in DMF
to give compounds 6 in 40–55% yields with the excep-
tion of nitro containing compound. It is assumed that
the nucleophilicity of the anion derived from 2 con-
taining nitro group was not strong enough to react with
methyl bromoacetate. The conversion of 6 to 7 was
accomplished by activating the carboxyl group of 6 with
carbonyldiimidazole (CDI)11 followed by treatment with
appropriate sulfonamides (Scheme 3).
Ureido compounds 4 and 5 were synthesized with no
incidence simply by treating aminoquinoxalinediones 3
NH2
H
N
A
A
B
NH2
NH2
A
O
O
N
O
O
NH2OSO3H
Oxalic acid
3N-HCl
K2CO3 / KOH
H2O
B
N
H
B
N
H
3
1
2
Scheme 1. Synthesis of key intermediates 3.
H
N
O
O
N
O
O
N
O
No resonance
form
O
O
O
N
N
H
N
H
N
O
N
O
N
O
O
2a
2b
Figure 2. Resonance stabilized form of 2a.
O
R
HN
N
N
NH2
N
H
O
A
B
A
O
O
R-N=C=O
DMF
N
H
B
N
H
O
3
4
S
R
HN
N
N
H
O
A
R-N=C=S
DMF
B
N
H
O
5
Scheme 2. Synthesis of ureido compounds 4 and thioureido compounds 5.

H.-G. Cheon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2661–2664
2663
O
O
O
S
N
H
OH
R
H
N
O
A
O
O
O
A
R
A
N
O
N
O
Br
SO2NH2
/ NaH
/ NaH
OCH3
DMF
CDI / DMF
B
N
H
B
N
H
O
B
N
H
O
2
7
6
Scheme 3. Synthesis of compounds 7, carbon analogues of compounds 4.
Table 1. Activity data of ureido compounds 4
3. Results and discussion
DPP-IV enzyme assay was carried out using rat plasma
by measuring 7-amino-4-trifluoromethyl-coumarin
(AFC) liberated from Ala-Pro-AFC in the presence or
absence of a test compound.7;12 Rat plasma preparation
(20 lL) was incubated with Ala-Pro-AFC (40 lM) at
room temperature, pH 7.8 for 1 h in the presence or
absence of test compounds (20 lM). Test compounds
were dissolved in DMSO. DMSO concentration in the
assay mixture was 5%, which did not affect enzyme
activity. After 1 h incubation, the fluorescence of AFC
released by the reaction was measured at 360 nm (exci-
tation wavelength) and at 485 nm (emission wave-
length). P-32/98 was used as a reference compound.
O
R
HN
N
N
H
O
A
B
N
H
O
4
Entry
1
A
H
B
R
% Inhibition
NO2
57.2
SO2
SO2
2
3
79.1
48.0
Analysis of structure–activity relationship of ureido
compounds 4 revealed that nitro group on quinoxaline
ring moiety (entries 1–5) influenced the DPP-IV activity
greatly. Simple substitution of chlorine or hydrogen for
the nitro group (entries 6–16) diminished the potency. In
addition, the sulfonyl group in ureido part appears to be
essential since entries 17–20 exhibited very weak inhi-
bition when compared with the activities of entries 1–5
(Table 1). For the most active compounds (entries 1–5)
their IC50 values were determined,13 although they are
not included in Table 1 for the purpose of table clarity.
H3C
SO2
CH3
Cl
4
5
6
66.8
72.2
17.3
SO2
CO
H
Cl
SO2
SO2
7
26.4
We moved on the structural modification by substituting
sulfur atom for the oxygen of the ureido side chain of
compounds 4 to produce the thioureido compounds 5
(entries 21–26), which deserves the following mention.
Although the compounds 5 containing nitro and sulfo-
nyl group (like entries 1–4 type compounds) are highly
desirable in terms of biological activity, the synthetic
attempts to couple nitro group containing quinoxaline 3
with several isothiocyanates were failed possibly due to
the weak electrophilicity of the isothiocyanates. How-
ever, most of the thioureido compounds 5 showed
moderate to good activities in spite of the absence of the
nitro and sulfonyl groups (Table 2). Next, the strong
biological activity exhibited by the compounds of entries
1–5 of Table 1 prompted us to synthesize the carbon
analogues represented by compounds 7. However, the
biological activity shown by this class of compounds is
too weak to make comments.
H3C
8
20.5
14.8
23.7
SO2
CO
CH3
9
10
Cl
Cl
SO2
SO2
11
10.4
H3C
12
13
21.0
14.1
SO2
SO2
SO2
CH3
H
H
14
15
12.3
12.3
H3C
SO2
In conclusion, we discovered a novel scaffold for DPP-
IV inhibitor containing quinoxalinedione moiety
through high throughput screening of chemical library
of approximately 32,000 compounds (Korean Chemical
CH3
(continued on next page)

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H.-G. Cheon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2661–2664
Table 1 (continued)
to maintain the inhibitory potency of this series of
compounds. The through investigations regarding these
quinoxalinedione compounds toward the DPP-IV inhi-
bition are now underway.
Entry
16
A
B
R
% Inhibition
13.0
CO
17
18
H
NO2
6.6
3.6
Cl
References and notes
F
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19
20
5.1
1.0
F
CH3
Table 2. Activity data of thioureido compounds 5
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S
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A
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B
N
H
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5
Entry
21
A
H
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R
% Inhibition
26.5
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1H, -8H), 8.13 (m, 1H, -6H), 12.06 (br, 1H, –NH–).
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H
22
32.1
Cl
H2C
23
24
25
41.5
40.1
50.0
–CH2CH3
Cl
Cl
O
C
26
20.5
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Bank), and that nitro group in the quinoxaline ring as
well as sulfonyl group in ureido part seems to be critical
13. IC50 values (lm): entry 1, 22.1; entry 2, 18.7; entry 3, 25.8;
entry 4, 18.4; entry 5, 12.7.