(1,3-diphenyl-1h-pyrazol-4-yl)-methylamine analogues as inhibitors of dipeptidyl peptidases

Article abstract of DOI:10.1002/jccs.200900152

Anumber of pyrazole compounds reported in literatures elicit anti-hyperglycemic effects. By modifying the side chain of the heterocyclic skeleton, a new chemical class of DPP-IV inhibitors structurally derived from the (pyrazol-4-yl)-methylamine scaffold

Full text of DOI:10.1002/jccs.200900152

1048
Journal of the Chinese Chemical Society, 2009, 56, 1048-1055
(1,3-Diphenyl-1H-pyrazol-4-yl)-methylamine Analogues as Inhibitors of
Dipeptidyl Peptidases
Tsu Hsu (
), Chiung-Tong Chen (
), Su-Ying Wu (
), Ting-Yueh Tsai (
), Chung-Nien Chang (
), Yu-Wen Huang (
), Ssu-Hui Wu (
),
Jai-Hong Cheng (
Chia-Hui Chien (
Chen-Lung Huang (
Yi-Kun Chiang (
),
), Kai-Chia Yeh (
), Chung-Yu Huang (
), Min-Hsien Wang (
),
),
),
), Yu-Sheng Chao (
Xin Chen (
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
) and Weir-Torn Jiaang* (
)
No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, R.O.C.
Anumber of pyrazole compounds reported in literatures elicit anti-hyperglycemic effects. By modify-
ing the side chain of the heterocyclic skeleton, a new chemical class of DPP-IV inhibitors structurally de-
rived from the (pyrazol-4-yl)-methylamine scaffold have been discovered and evaluated the biological ac-
tivities of these inhibitors against DPP-IV, DPP8, DPP-II and FAP. The SAR studies showed the (1,3-di-
phenyl-1H-pyrazol-4-yl)-methylamines with 2,4-dichloro substituents at the 3-phenyl ring selectively
preferred as DPP-IV inhibitors, whereas with difluoro substituents at the 3-phenyl ring selectively pre-
ferred as DPP8 inhibitors. The binding mode of representative compound 15h at the active site of DPP-IV
was predicted by computer model. In additional, 15h exhibited the ability to significantly decrease the
glucose excursion in mice.
Keywords: Dipeptidyl peptidase; Inhibitor; Pyrazole; Diabetes.
INTRODUCTION
Dipeptidyl peptidase IV (DPP-IV, also known as
CD26) (EC 3.4.14.5) is a drug target for type II diabetes. It
is a prolyl dipeptidase involved in the in vivo degradation
of two insulin-sensing hormones, glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic polypep-
tide (GIP), by cleaving at the peptide bond of the penulti-
mate position.1,2 DPP-IV knockout mice and rats have con-
sistently displayed healthy phenotypes.3-5 When chal-
lenged with a high concentration of glucose, these animals
show improved glucose tolerance, enhanced insulin secre-
tion and increased circulating active GLP-1 peptide.3-5 By
extending the duration of action of GLP-1, animal studies
demonstrated that DPP-IV inhibitors would stimulate insu-
lin secretion, inhibit glucagon release and slow gastric
emptying; each a benefit in the control of glucose homeo-
stasis.6-10 Antidiabetic efficacy has been demonstrated clin-
ically with DPP-IV inhibitors; MK-0431 (sitagliptin) de-
veloped by Merck was approved by the US Food and Drug
Administration for the treatment of type 2 diabetes,11
Novartis’s LAF237 (vildagliptin) was recently approved
for the European market (Fig. 1).12
Fig. 1. DPP-IV inhibitors.

1,3-Diphenyl-1H-pyrazol-4-yl)-methylamine Analogues
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 1049
Selectivity of the DPP-IV inhibitors for closely re-
lated prolyl peptidase such as DPP8/9, DPP-II and FAP
(Fibroblast activation protein) is an important criterion for
their further development as antidiabetic agents, since inhi-
bition of DPP-II results in the apoptosis of quiescent T-
cells13 and also recent in vivo studies indicate that selective
inhibition of DPP8/9 may be associated with profound tox-
icities.14 Reports suggest that fibroblast activation protein
(FAP) is involved in tumor growth and invasion.15 We are
involved in the design and synthesis of DPP-IV inhibitors
and have reported potent and selective DPP-IV inhibitors
1-3 (Fig. 1).16-18
IV inhibitors published in patents or journals, most of DPP-
IV inhibitors have primary or secondary amines to form
salt bridges with the active site Glu205 and Glu206 of
DPP-IV.19 It is very important interaction; therefore the
4-methylamine group replaced the 4-acetamide group of
SAH 57-749 in order to increase potency against DPP-IV
(Fig. 2). In this paper, we prepared methylamines 8-15 with
substituents at different positions on both the phenyl rings,
the structure-activity relationships of pyrazole-based com-
pounds as selective DPP-IV or selective DPP8 inhibitors
were investigated. The selected compound 15h was further
evaluated in vivo activity, and computer model predicted
the possible binding mode of 15h with DPP-IV protein.
As shown in Fig. 1, vildagliptin and compounds 1-3
are covalent inhibitors. Often this electrophilic cyanopyr-
rolidine is able to bind covalently with the serine 630 in the
S1 pocket of DPP-IV,19 whereas the non-covalent inhibi-
tors sitagliptin and aminomethylpyrimidine derivatives de-
pend on non-covalent protein-ligand interactions and the
substituted phenyl group occupies very well the hydropho-
bic S1 pocket of the enzyme.11,20 We here report on the dis-
covery of a novel series of non-covalent inhibitors. Using
the structure-based design, we speculated that a pyrazole
scaffold could effectively display groups known to interact
with the active site residues of DPP-IV, furthermore a num-
ber of pyrazole compounds have been sited in the literature
that elicit anti-hyperglycemic effects including: 1,3-disub-
RESULT AND DISCUSSION
The general synthetic route for the preparation of
(1,3-diaryl-1H-pyrazol-4-yl)-methylamines 8-15 varying
in 1- and 3-position of pyrazole nucleus is shown in Scheme
I.24 The synthesis commonly began with commercially
available ketones 4 and hydrazines 5 as a starting point.
Condensation between a ketone and hydrazine provided a
hydrazone 6 in high yields in most cases. The Vilsmeier-
Haack reaction using 2.5 equiv of reagent performed a dou-
ble addition of reagent to afford, ultimately after hydroly-
sis, the desired cyclized aldehydes, 7 in 60-75% yield.
Treatment of 7 with at least 10 equiv of ammonium acetate
in dry MeOH/THF co-solvent afforded imines, and subse-
quently the intermediate imines were reduced by NaBH3CN
stituted pyrazoles.21 1,3,5-trisubstituted pyrazoles.22,23
A
putative lead compound, SAH 57-749 (Fig. 2), was shown
to increase [3H] glucose incorporation into lipids in adi-
pocytes in the presence of submaximal concentrations of
insulin, suggesting that the compound might increase the
sensitivity of the adipocyte to insulin.24 For this reason, we
expected to develop structurally new pyrazole-based DPP-
IV inhibitors with dual mechanism of action for reducing
glycemia.
Scheme I The synthetic procedure of compound 8-15
According to the chemical structure of known DPP-
Fig. 2. Lead pyrazole structures.

1050 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Hsu et al.
to provide primary amines 8-15 in 15-20% yield.
Table 1. In vitro DPP-IV inhibitory activity of compounds 8-15a
There is a lack of structural information on the DPP8,
DPP-II and FAP protein. Due to the amino acid homology
between DPP-IV and closely related dipeptidyl peptidases,
we speculate that they may have a similar substrate-binding
pocket. It is likely that some DPP-IV inhibitors may have
an inhibitory effect on DPP8, DPP-II and FAP protein.
Thus, the IC50 values for the inhibition of these enzymes for
compounds 8-15, were determined.16-18 We started initially
to investigate the effects of substitution of the 3-phenyl
ring. As shown in Table 1, lead compound 8 with no substi-
tuent on both the phenyl rings did not showed significant
inhibition against DPP-IV, DPP8, DPP-II and FAP (>20
mM). A single para methoxy substituent (9) had no effect
on activity. Introducing fluorine at the 4-position of phenyl
ring gave a slight of improvement in potency versus DPP8
(10). Introduction of two fluorines at the 2,4-position of
3-phenyl ring (11) had potency versus DPP8 similar to 4-
fluoro analogue 10. The 2,5-difluoro analogue 12 had no
effect on activity. Incorporating two fluorines into the 3,4-
position and 3-5-position of 3-phenyl ring gave 13 and 14,
respectively. Interestingly, both the compounds 13 and 14
significantly increased potency against DPP8 (IC50 = 2.1
mM and 2.5 mM, respectively) with weak inhibition (10 mM
< IC50 < 20 mM) against DPP-II. Compound 13 had IC50
value of 12 mM for DPP-IV inhibition, but compound 14
did not showed significant inhibition against DPP-IV. A
more profound improvement in DPP-IV inhibition was
2,4-dichloro substituted compound 15a, which is a moder-
ately potent 3.5 mM inhibitor of DPP-IV with weak inhibi-
tion against DPP8 and DPP-II.
N
N
R
H2N
IC50 (mM)
Compd
R
DPP-IV DPP8
DPP-II
FAP
> 20
8
> 20
> 20
> 20
OMe
9
> 20
> 20
>20
> 20
10
11
> 20
> 20
15
16
> 20
> 20
> 20
> 20
F
F
F
F
12
> 20
12
>20
2.1
2.5
14
> 20
11
> 20
> 20
> 20
> 20
F
F
13
F
F
F
14
> 20
3.5
11
Cl
15a
17
Cl
Next, we aimed to improve further the potency versus
DPP-IV and selectivity over related enzymes. Having ar-
rived at the optimized structure in the 3-phenyl ring for
moderate DPP-IV inhibition, we went back to optimize the
1-position of pyrazole 15a. As shown in Table 2, replace-
ment with a benzyl group (15b) was found to be detrimen-
tal to activity, indicating that phenyl ring is more suitable
than benzyl group at the 1-position. Attention was next fo-
cused on the modification of 1-phenyl ring of compound
15a. A para fluorine substituent was added to make the
ring less electron rich, compound 15c showed a similar
level of DPP-IV inhibition compared with 15a, had weak
inhibition against DPP8, DPP-II and FAP. A similar po-
tency toward DPP-IV was observed for the introduction of
para methyl (15d) or para trifluoromethyl (15e) substitu-
ents. Moreover, electron-donating methyl group gave 15d
having equipotent against DPP-IV and DPP8, electron-
withdrawing trifluoromethyl group led to 15e, which inhib-
ited DPP-II with an IC50 value of 8.6 mM. The strong elec-
tron-donating para methoxy substituted 15f exhibited a
slight of drop in DPP-IV inhibition relative to 15a, and 15f
had weak DPP-II inhibition with no detectable DPP8 and
FAP inhibition. Introduction of meta methyl (15g) or meta
fluoro (15h) substituent slightly increased DPP-IV inhibi-
tory activity, 15g and 15h both had moderate potency to-
ward DPP8 and DPP-II, respectively. The 2,4-dimethyl
substituted compound 15i gave no improvement in DPP-IV
potency relative to lead 15a, and showed weak DPP8,
DPP-II and FAP inhibitory activities.

1,3-Diphenyl-1H-pyrazol-4-yl)-methylamine Analogues
Table 2. In vitro DPP-IV inhibitory activity of compounds 15a-i
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 1051
arginine residue and the pyrazole core of 15h.20 No interac-
tion with the catalytic Ser630 is observed. Molecular-mod-
eling show that aromatic ring at the 1-position of the pyra-
zole 15h does not interact with the side chain of Tyr547,
which may be one of the reasons that this series of com-
pounds do not act as potent DPP-IV inhibitors.
Cl
R
N
N
Cl
H2N
IC50 (mM)
Compound 15h was chosen for in vivo evaluation.
An oral glucose tolerance test (OGTT) was used to assess
the ability of 15h to improve glucose tolerance in mice. In
lean animals, compound 15h (3 mg/kg) was orally adminis-
tered 30 min prior to dextrose challenge (3 g/kg). Fig. 4
shows the glucose excursion in response to an oral glucose
challenge. Vehicle-treated controls were markedly glucose
intolerant with an average peak glucose level of 245 mg/dL
at 30 min after glucose challenge, whereas the drug treated
animals exhibited a 16% decrease in glucose levels com-
pared to controls (15h-treated animals, 205 mg/dL).
Compd
R
DPP-IV DPP8
DPP-II
FAP
> 20
15a
15b
3.5
18
14
17
> 20
> 20
18
> 20
21
F
15c
15d
15e
15f
2.9
3.4
3.4
4.6
12
Me
3.6
> 20
8.6
20
> 20
> 20
> 20
CONCLUSION
F3C
We have described synthesis and SAR studies of
(pyrazol-4-yl)-methylamine derivatives. Biological data
indicated some of this class of inhibitors showed low mi-
cromolar DPP-IV or DPP8 inhibitory activity. Computer
modeling predicted the 1-position of the pyrazole 15h does
not interact with the side chain of Tyr547 of DPP-IV pro-
tein; the prediction may explain this fact that this class of
inhibitors did not show potent DPP-IV inhibition. Never-
theless, representative compound 15h showed in vivo effi-
> 20
> 20
MeO
Me
15g
15h
2.6
2.6
6.8
> 20
4.7
> 20
> 20
F
> 20
Me
15i
3.3
13
13
16
Me
To explore the mechanism of the inhibition, com-
puter-modeling study was carried out to investigate a possi-
ble interaction between these inhibitors and DPP-IV. The
predicted binding mode of compound 15h at the active site
of DPP-IV is shown in Fig. 3. The 2,4-dichlorophenyl moi-
ety of the inhibitor occupies very well the narrow hydro-
phobic S1 pocket of the enzyme. In the S2 pocket, the pri-
mary amine terminus forms a hydrogen-bonding network
with the side chains of Glu205, Glu206, and Tyr662 of
DPP-IV protein, and a cation-p-interaction between an
Fig. 3. The binding mode of compound 15h is pre-
dicted by computer model.

1052 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Hsu et al.
formed and acetic acid was added to maintain stirring for
15 h. The reaction mixture was diluted with an additional
10 mL of water and subsequently filtered, and the solid was
dried in vacuo to afford crystalline product 6 without fur-
ther purification.
Phosphorus oxychloride (20 mmol, 2 equiv) was added
to 15 mL of DMF at 0 °C and stirred for 30 min. Compound
6 (10 mmol, 1 equiv) was added as a solid slowly to this
mixture and stirred for 15 h. The crude reaction was then
quenched into 100 mL of water and stirred for an additional
15 h. The resulting solid was filtered, dissolved in methy-
lene chloride, and dried over MgSO4, filtered, and evapo-
rated to a crude solid that was crystallized from ether/hex-
ane (5:1) to afford 7 in 60-75% yield. Only one of aldehyde
7 is selected to show its NMR spectrum.
Fig. 4. Effects of 15h on the glucose excursion levels
of adult male BALB/c mice during oral glucose
tolerance test (3 mg/kg, po). Data are expressed
± SEM (n = 6/group, P value < 0.05).
3-(2,5-Difluoro-phenyl)-1-phenyl-1H-pyrazole-4-car-
baldehyde
1H NMR (300 MHz, CDCl3) d 9.92 (d, J = 3.6 Hz,
1H), 8.55 (s, 1H), 7.80-7.76 (m, 2H), 7.56-7.39 (m, 4H),
7.23-7.14 (m, 2H).
cacy in animal model. Administration of single oral dose (3
mg/kg) can suppress the plasma glucose level in an OGTT
in mice.
General procedure for the synthesis of 8-15
To a solution of aldehyde 7 (10 mmol, 1 equiv) in 30
mL of dry THF and 50 mL of dry MeOH was added ammo-
nium acetate (100 mmol, 10 equiv). The mixture was heated
to reflux for 30 min, followed by adding the solid of
NaBH3CN (15 mmol, 1.5 equiv) slowly to the solution. The
resulting mixture was stirred at reflux for 1 hr. The reaction
was cooled to ambient temperature and then quenched into
a cool 6 N solution. The methanol is evaporated in vacuo
and the remaining aqueous was added solid Na2CO3 until
the pH > 9. The solution was extracted twice with CH2Cl2.
Organic layer was dried over MgSO4 and evaporated. The
residue was chromatographed over silica gel (eluted with
CH2Cl2/MeOH = 95/5) to afford 8-15 as white solid in
15-20% yield.
EXPERIMENTAL
All commercial chemicals and solvents are reagent
grade and were used without further treatment unless oth-
erwise noted. 1H NMR spectra were obtained with a Varian
Mercury-300 spectrometer operating at 300 MHz. Chemi-
cal shifts were recorded in parts per million (ppm, d) and
were reported relative to the solvent peak or TMS. High-
resolution mass spectra (HRMS) were measured with a
Finnigan (MAT-95XL) electron impact (EI) mass spec-
trometer. LC/MS data were measured on an Agilent MSD-
1100 ESI-MS/MS System. Flash column chromatography
was done using silica gel (Merck Kieselgel 60, No. 9385,
230-400 mesh ASTM). Reactions were monitored by TLC
using Merck 60 F254 silica gel glass backed plates (5 ´ 10
cm); zones were detected visually under ultraviolet irradia-
tion (254 nm) or by spraying with phosphomolybdic acid
reagent (Aldrich) followed by heating at 80 °C. All starting
materials were commercially available unless otherwise
indicated. Yields were of purified product and were not
optimized.
C-(1,3-Diphenyl-1H-pyrazol-4-yl)-methylamine 8
1H NMR (300 MHz, CD3OD) d 8.29 (s, 1H), 7.79 (dd,
J = 1.2, 8.4 Hz, 2H), 7.69 (dd, J = 1.2, 8.4 Hz, 2H), 7.52-
7.33 (m, 6H), 4.00 (s, 2H). HRMS (EI): calcd for C16H15N3:
249.1266; found: 249.1277.
C-[3-(4-Methoxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 9
General procedure for the synthesis of aldehyde 723
To acetophenone 4 (10 mmol, 1 equiv) in 40 mL of
acetic acid and 1 mL of water was added phenylhydrazine 5
(10 mmol, 1 equiv). Shortly after addition, a solid mass
1H NMR (300 MHz, CD3OD) d 8.22 (s, 1H), 7.76 (d,
J = 7.8 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 7.48 (t, J = 8.1 Hs,
2H), 7.30 (t, J = 7.2 Hs, 1H), 7.02 (d, J = 8.4 H, 2H), 3.92
(s, 2H), 3.84 (s, 3H). HRMS (EI): calcd for C17H17N3O:

1,3-Diphenyl-1H-pyrazol-4-yl)-methylamine Analogues
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 1053
279.1372; found: 279.1406.
7.60 (m, 3H), 7.47-7.45 (m, 2H), 7.30 (d, J = 8.4 Hz, 2H),
3.68 (s, 2H), 2.37 (s, 3H). HRMS (EI): calcd for C17H15Cl2N3:
331.0643; found: 331.0642.
C-[3-(4-fluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 10
1H NMR (300 MHz, CD3OD) d 8.24 (s, 1H), 7.79-
7.70 (m, 4H), 7.50-7.45 (m, 2H), 7.31 (t, J = 7.5 Hz, 1H),
7.22-7.16 (m, 2H), 3.90 (s, 2H). HRMS (EI): calcd for
C16H14FN3: 267.1172; found: 267.1169.
C-[3-(2,4-Dichloro-phenyl)-1-(4-trifluoromethyl-phen-
yl)-1H-pyrazol-4-yl]-methylamine 15e
1H NMR (300 MHz, CD3OD) d 8.43 (s, 1H), 7.99 (d,
J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 1.8
Hz, 1H), 7.51-7.46 (m, 2H), 3.69 (s, 2H). HRMS (EI):
calcd for C17H12Cl2F3N3: 385.0360; found: 385.0357.
C-[3-(2,4-Dichloro-phenyl)-1-(4-methoxy-phenyl)-1H-
pyrazol-4-yl]-methylamine 15f
1H NMR (300 MHz, CD3OD) d 8.23 (s, 1H), 7.67-
7.63 (m, 3H), 7.49-7.46 (m, 2H), 7.04 (d, J = 7.8 Hz, 2H),
3.84 (s, 3H), 3.80 (s, 2H). HRMS (EI): calcd for
C17H15Cl2N3O: 347.0592; found: 347.0598.
C-[3-(2,4-Difluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 11
1H NMR (300 MHz, CD3OD) d 8.29 (s, 1H), 7.76 (d,
J = 8.1 Hz, 2H), 7.66-7.58 (m, 1H), 7.49 (t, J = 7.8 Hz, 2H),
7.35-7.30 (m, 1H), 7.15-7.06 (m, 2H), 3.75 (s, 2H). HRMS
(EI): calcd for C16H13F2N3: 285.1078; found: 285.1071.
C-[3-(2,5-Difluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 12
1H NMR (300 MHz, CD3OD) d 8.48 (s, 1H), 7.82 (d,
J = 7.8 Hz, 2H), 7.56-7.25 (m, 6H), 4.12 (s, 2H). HRMS
(EI): calcd for C16H13F2N3: 285.1078; found: 285.1062.
C-[3-(3,4-Difluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 13
1H NMR (300 MHz, CD3OD) d 8.24 (s, 1H), 7.78 (d,
J = 7.8 Hz, 2H), 7.69-7.62 (m, 1H), 7.53-7.28 (m, 5H), 3.90
(s, 2H). HRMS (EI): calcd for C16H13F2N3: 285.1078;
found: 285.1097.
C-[3-(2,4-Dichloro-phenyl)-1-m-tolyl-1H-pyrazol-4-yl]-
methylaminee 15g
1H NMR (300 MHz, CD3OD) d 8.28 (s, 1H), 7.63-
7.43 (m, 5H), 7.36 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 6.9 Hz,
1H), 3.69 (s, 2H), 2.43 (s, 3H). HRMS (EI): calcd for
C17H15Cl2N3: 331.0643; found: 331.0640.
C-[3-(2,4-Dichloro-phenyl)-1-(3-fluoro-phenyl)-1H-pyr-
azol-4-yl]-methylamine 15h
1H NMR (300 MHz, CD3OD) d 8.34 (s, 1H), 7.63-
7.43 (m, 6H), 7.10-7.04 (m, 1H), 3.67 (s, 2H). HRMS (EI):
calcd for C16H12Cl2FN3: 335.0392; found: 335.0384.
C-[3-(2,4-Dichloro-phenyl)-1-(3,5-dimethyl-phenyl)-1H-
pyrazol-4-yl]-methylamine 15i
1H NMR (300 MHz, CD3OD) d 8.38 (s, 1H), 7.67 (d,
J = 1.8 Hz, 1H), 7.55-7.47 (m, 2H), 7.40 (s, 2H), 7.04 (s,
1H), 4.00 (s, 2H), 2.40 (s, 6H). HRMS (EI): calcd for
C18H17Cl2N3: 345.0800; found: 345.0803.
C-[3-(3,5-Difluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 14
1H NMR (300 MHz, CD3OD) d 8.29 (s, 1H), 7.80 (d,
J = 7.5 Hz, 2H), 7.52-7.47 (m, 2H), 7.37-7.30 (m, 3H),
6.98-6.94 (m, 1H), 3.97 (s, 2H). HRMS (EI): calcd for
C16H13F2N3: 285.1078; found: 285.1097.
C-[3-(2,4-Dichloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-
methylamine 15a
1H NMR (300 MHz, CD3OD) d 8.25 (s, 1H), 7.70 (d,
J = 8.1 Hz, 2H), 7.58 (s, 1H), 7.44-7.41 (m, 4H), 7.32-7.14
(m, 1H), 3.63 (s, 2H). HRMS (EI): calcd for C16H13Cl2N3:
317.0487; found: 317.0479.
Determination the inhibitions of DPP-IV, DPP8,
DPP-II and FAP in Vitro25-27
IC50 determination was done as described in litera-
ture.25 Sigma plot was used to obtain the IC50 values. DPP-
IV and DPP8 were purified as described in our previously
published method.26,27 The Purification of DPP-II was car-
ried out as described in reference 25 with modifications.
The detailed protocol for the cloning and expression of
FAP will be described elsewhere. The buffers for DPP-IV
and DPP8 assays are 2 mM Tris-HCl, pH 8.0 and PBS
buffer, respectively. The substrate used is Gly-Pro-pNA
from Bachem at the concentrations of 500 mM and 2500
mM for DPP-IV and DPP8 assays, respectively. DPP-II ac-
C-[3-(2,4-Dichloro-phenyl)-1-(4-fluoro-phenyl)-1H-pyr-
azol-4-yl]-methylamine 15c
1H NMR (300 MHz, CD3OD) d 8.29 (s, 1H), 7.80-
7.76 (m, 2H), 7.64 (d, J = 1.8 Hz, 1H), 7.51-7.44 (m, 2H),
7.25 (t, J = 8.1 Hz, 2H), 3.75 (s, 2H). HRMS (EI): calcd for
C16H12Cl2FN3: 335.0392; found: 335.0392.
C-[3-(2,4-Dichloro-phenyl)-1-p-tolyl-1H-pyrazol-4-yl]-
methylamine 15d
1H NMR (300 MHz, CD3OD) d 8.25 (s, 1H), 7.63-

1054 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Hsu et al.
N. A.; Zhang, B. B. Proc. Natl. Acad. Sci. USA 2003, 100,
6825.
tivity was assayed by 1.5 mM Gly-Pro-pNA in 50 mM po-
tassium phosphate buffer, pH 5.5. The substrate used for
FAP assay was Gly-Pro-pNA at 6 mM concentration. Reac-
tion developed at 37 °C and OD405 was monitored.
Molecular modeling
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S.; Asano, O.; Tanaka, I. Biochem. Biophys. Res. Commun.
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The compound, 15h was docked into the binding
pocket of DPP-IV by the program GOLD 3.2 (CCDC Soft-
ware Limited, Cambridge, UK) using the X-ray structure of
DPP-IV as a template protein for the docking study.28 The
binding pocket for the docking study was defined as a 12 Å
radius sphere centered on ARG 125 (NH2). Twenty genetic
algorithm (GA) runs were performed for compound 15 h
docking. The scoring function, GoldScore, was employed
to rank the docking positions of compound 15h.
8. Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Dunning, B.
E.; Mangold, B. L.; Mone, M. D.; Russell, M. E.; Weldon, S.
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Oral Glucose Tolerance Test in mice
Adult male BALB/c mice (n = 6) were fasted over-
night. Blood samples were obtained from the tail veins with
needles of 27G and the blood glucose was measured with
the Accu-Chek Compact System from Roche (Basel, Swit-
zerland). The animals were then orally gavaged with the
testing compounds dissolved in distilled water at a dose of
3 mg/kg. Thirty minutes after the oral administration of the
testing compound, the animals were orally gavaged with
freshly prepared glucose solution in distilled water given at
3 g glucose/kg. Blood glucose levels of these dosed ani-
mals were monitored at 0, 15, 30, 60, and 120 min after the
oral glucose challenge.
11. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher, M.
H.; He, H.; Hickey, G. J.; Kowalchick, J. E.; Leiting, B.; Ly-
ons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.; Petrov,
A.; Scapin, G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt,
M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N. A.; Webe, A. E.
J. Med. Chem. 2005, 48, 141.
12. Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey, B.
F.; Dunning, B. E.; Prasad, K.; Mangold, B. L.; Russell, M.
E.; Hughes, T. E. J. Med. Chem. 2003, 46, 2774.
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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,
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ACKNOWLEDGEMENT
National Health Research Institutes, Taiwan, finan-
cially supported the study.
Received March 2, 2009.
15. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.
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H.-S.; Lin, K.-T.; Chang, Y.-Y.; Wu, S.-H.; Chen, Y.-S.; Lu,
I.-L.; Wu, S.-Y.; Tsai, T.-Y.; Chen, W.-C.; Hsieh, H.-P.;
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