(2S,4S)-1-[2-(1,1-Dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl] -4-fluoro-pyrrolidine-2-carbonitrile: A potent, selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV

Article abstract of DOI:10.1016/j.bmcl.2010.04.124

A series of 2-[3-[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3- methyl-1-oxobutyl]-based DPP-IV inhibitors with various monocyclic amines were synthesized. The structure-activity relationships (SAR) led to the discovery of potent DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP-II, DPP8, DPP9 and FAP (IC₅₀ > 20 μM). Of these compounds, the analogues 12a, 12h and 12i exhibited a long-lasting ex vivo DPP-IV inhibition in rats.

Full text of DOI:10.1016/j.bmcl.2010.04.124

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3596–3600
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(2S,4S)-1-[2-(1,1-Dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl]-
4-fluoro-pyrrolidine-2-carbonitrile: A potent, selective, and orally
bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV
Teng-Kuang Yeh , Ting-Yueh Tsai , Tsu Hsu , Jai-Hong Cheng, Xin Chen, Jen-Shin Song, Horng-Shing Shy,
Mei-Chun Chiou, Chia-Hui Chien, Ya-Ju Tseng, Chung-Yu Huang, Kai-Chia Yeh, Yu-Lin Huang,
Chih-Hsiang Huang, Yu-Wen Huang, Min-Hsien Wang, Hung-Kuan Tang, Yu-Sheng Chao,
*
*
Chiung-Tong Chen , Weir-Torn Jiaang
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli Country 350, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-[3-[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]-based DPP-IV
inhibitors with various monocyclic amines were synthesized. The structure–activity relationships (SAR)
led to the discovery of potent DPP-IV inhibitors, having IC50 values of <100 nM with excellent selectivity
Received 20 January 2010
Revised 23 April 2010
Accepted 27 April 2010
Available online 17 May 2010
over the closely related enzymes, DPP-II, DPP8, DPP9 and FAP (IC50 > 20
analogues 12a, 12h and 12i exhibited a long-lasting ex vivo DPP-IV inhibition in rats.
Ó 2010 Elsevier Ltd. All rights reserved.
lM). Of these compounds, the
Keywords:
Dipeptidyl peptidase IV
Inhibitor
F
F
OH
F
Dipeptidyl peptidase IV (DPP-IV, also known as CD26)
(EC 3.4.14.5) is a prolyl dipeptidase involved in the in vivo degra-
dation of two insulin-sensing hormones, glucagon-like peptide-1
(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP),
by cleaving at the peptide bond of the penultimate position.1,2 Glu-
cagon-like peptide-1 (GLP-1) is an incretin hormone secreted by
intestinal L-cells in response to food intake.3 The active form of
GLP-1 is a 30-amino acid peptide, which stimulates insulin release,
inhibits glucagons release, and slows gastric emptying, each a ben-
efit in the control of glucose homeostasis in patients with type II
diabetes.4–7 Thus inhibition of DPP-IV extends the half-life of
endogenously secreted GLP-1, which in turn enhances insulin
secretion and improves the glucose tolerance. DPP-IV inhibitors
offer several potential advantages over existing therapies including
decreased risk of hypoglycemia, potential for weight loss, and the
potential for regeneration and differentiation of pancreatic b-cells.8
Therefore, DPP-IV has become a validated target for the treatment
of type II diabetes, and several inhibitors of DPP-IV are currently
undergoing late-stage clinical trials; the first DPP4 inhibitor,
sitagliptin 1 (Januvia, Merck) was approved by FDA in October
2006 (Fig. 1),9,10 vildagliptin 2 (Glavus, Novartis) was approved
for use in Europe in September 2007.11 Recently, The FDA approved
NH2
O
N
N
N
N
N
H
N
O
CN
CF3
2
1
O
N
HO
N
N
H N
2
N
H
O
CN
O
CN
3
4
O
O
Cl
O
O
F
HN
HN
N
N
N
H
N
H
O
CN
O
CN
* Corresponding author.
E-mail address: wtjiaang@nhri.org.tw (W.-T. Jiaang).
These authors contributed equally to this work.
5
6
Figure 1. Inhibitors of DPP-IV.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.124

T.-K. Yeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3596–3600
3597
saxagliptin 3 (Onglyza, Bristol–Myers Squibb), a once-daily treat-
TFA
NH2
H
H
N
HO
W
N
ment for type 2 diabetes to be taken in combination with diet
a
b, c
Boc
Boc
and exercise.12
O
O
O
With few exceptions, most DPP-IV inhibitors resemble the P2–
P1 dipeptidyl substrate cleavage product. As shown in Figure 1,
compounds 2 and 3 are covalent inhibitors. Often this electro-
philic cyanopyrrolidine is able to bind covalently with the serine
630 in the S1 pocket of DPP-IV, whereas the non-covalent inhib-
itor 1 depends on non-covalent protein–ligand interactions and
the substituted phenyl group occupies very well the hydrophobic
S1 pocket of the enzyme.9 Previous report from our laboratories
described three related series of 2-[3-[[2-[(2S)-2-cyano-1-pyrro-
9
7
8
W=1o or 2o amine
R
W
d
O
N
R
N
H
O
CN
N
Br
11 R = H
12 R = F
O
CN
lidinyl]-2-oxoethyl]amino]-3-methyl-1-oxobutyl]-based
(struc-
10
ture I) DPP-IV inhibitors (Fig. 2), compounds 4–6 are the
representative inhibitors in these three series (Fig. 1). Compound
4 is a potent, selective and orally available inhibitor of DPP-IV. In
the mice ex vivo plasma DPP-IV inhibition assay, 4 provided >80%
inhibition and lasted for at least 4 h after oral administration of
18 mg/kg.13 Compound 5 is less potent than compounds 4 and
6 (Table 1), and furthermore the abilities of 5 in decreasing the
glucose excursion and inhibiting ex vivo plasma DPP-IV activity
are not comparable to compound 6.14 Novel trans-2-arylcyclopro-
pylamine analogue 6 is equipotent with 4 in DPP-IV inhibition
and exhibits good selectivity profile, but this analogue does not
exhibit a long-lasting ex vivo DPP-IV inhibition profile at a dose
of 3 mg/kg in rats (30% inhibition at 8 h after an oral dosing).15
Scheme 1. Reagents: (a) KOCl, 1,4-dioxane/H2O; (b) DCC, HOSu, 1,4-dioxane/
CH2Cl2, various amines; (c) CF3COOH; and (d) K2CO3, THF.
The data shown in Table 1 compare the DPP-IV potency and
selectivity of compounds 4–6 to known DPP-IV inhibitors 1–3.16
Since the purpose of the project was to identify a drug candi-
date, we continued to search for other series of structure I-based
inhibitors that increased the inhibitory activity and duration of
in vivo DPP-IV inhibition. In this letter, we describe our extensive
SAR studies regarding structure I-based derivatives to develop
long-acting DPP-IV inhibitors, we here report on the discovery
of a novel series of monocyclic amine derivatives 11 as DPP-IV
inhibitors (Fig. 2). To further improve the potency of this series
of compounds, introduction of fluoro substitution at the 4-posi-
tion of 2-cyanopyrrolidine led to the discovery of compound
12a (Fig. 2).17 This approach resulted in not only increased po-
tency, but also improved in vivo efficacy. Compound 12a is a po-
tent DPP-IV inhibitor (IC50 = 15 nM) with excellent selectivity
X
R
R1
R2
O
n
N
N
O
N
N
N
H
N
H
O
O
CN
CN
over DPP-II, DPP8, DPP9 and FAP (>50 lM), and 12a demonstrated
in vivo efficacy better than that of inhibitors 5 and 6.
I
11
(2S)-2-Cyanopyrrolidine analogues 11–12 were prepared as de-
scribed in Scheme 1 and are listed in Table 2. 3-Boc-amino-3-meth-
ylbutyric acid 8 was prepared through oxidation of commercially
available N-Boc-diacetonamine 7 using potassium hypochlorite.
Acid 8 was DCC-coupled with various amines followed by Boc-
deprotection by TFA to provide TFA salt 9. 1-Bromoacetyl-2-cya-
no-(S)-pyrrolidine 10 was coupled with free base amine 9 to pro-
vide the desired 2-cyanopyrrolidine analogues 11–12. The
synthesis of bromoacetylated compound 10 was carried out accord-
ing to the literature procedure.13,17a
Generally, this class of inhibitors shown in Table 2 exhibited
potent DPP-IV inhibition (IC50 < 100 nM), therefore improvement
of selectivity against the off-target enzymes, in particular DPP8
and DPP9 was of great concern since inhibition of DPP8 and/or
DPP9 is associated with significant toxicity in preclinical spe-
cies.18,19 These inhibitors were also tested against DPP-II20 and
FAP (Fibroblast activation protein),21 significant inhibition
Improve in vivo efficacy
Improve potency
N
F
O
N
N
H
O
CN
12a
Figure 2. Design of monocyclic amine derivatives as DPP-IV inhibitors.
Table 1
(IC50 < 20 lM) was not observed, thus only DPP-IV, DPP8 and
Inhibitory profiles of compounds 1–6
DPP9 data are reported. As shown in Table 2, the lead compound,
the unsubstituted pyrrolidine 11a, is a potent DPP-IV inhibitor
with an IC50 value of 71 nM. When the polar hydroxyl substituent
was introduced at the 3-position of pyrrolidine, (3R)-pyrrolidin-3-
ol 11b led to a 2.5-fold decrease in DPP-IV inhibitory potency
(IC50 = 180 nM). To further improve the potency of compounds
11a and 11b, introduction of fluoro substituent at the 4-position
of 2-cyanopyrrolidine (S form) at the P1 site provided compounds
12a and 12b, and evaluated fluoro effects on both potency and
selectivity. As expected, 4-fluoro-2-cyanopyrrolidine derivatives
12a and 12b exhibited 5- and 6-fold better DPP-IV inhibitory
activity than 12a and 12b, respectively. In view of this fact, we
Compound
IC50 (l
M)a
DPP-IV
DPP8
DPP9
DPP-II
FAP
1
0.030
0.051
0.0034
0.015
0.116
0.015
>20
>100
1.2
>100
>50
>100
>100
ND
>100
>50
2
14.2
0.24
>100
>50
3b
4
0.10
>100
>20
NDc
>100
>20
>100
5
6
>20
72%d
>20
a
b
c
Means of at least three experiments; standard derivatives are 20%.
Matsuyama-Yokono et al. Ref. 16.
ND, no data.
d
Inhibition at 20 lM.

3598
T.-K. Yeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3596–3600
Table 2
focused our efforts on studying the diverse monocyclic amines
with (4S)-4-fluoro-2-cyanopyrrolidine at the P1 site.
Inhibition of DPP-IV, DPP8 and DPP9 by compounds 11–12
W
As shown in Table 2, the unsubstituted pyrrolidine 12a is a po-
tent DPP-IV inhibitor with an IC50 value of 15 nM. However, substi-
tution at the pyrrolidine ring had little effect on DPP-IV activity
and selectivity; compounds 12b–g exhibited high potency
(IC50 < 50 nM) against DPP-IV, with excellent selectivity versus
W
F
O
O
N
N
N
N
H
H
O
CN
O
CN
11
12
DPP8 and DPP9 (IC50 > 20 lM). Next, we investigated the effects
of six-membered ring derivatives, such as piperidine 12h–j, mor-
pholine 12k and piperazine 12l–n. In comparison with five-mem-
bered ring 12a, six-membered ring 12h–l exhibited a similar
potency and selectivity profile (DPP-IV IC50 = 18–27 nM, DPP8/9
a
Compound
W
IC50
(lM)
DPP-IV
0.071
DPP8
DPP9
>20
11a
11b
12a
12b
N
N
>20
IC50 > 20 lM). As for 1-methylpiperazine 12m (IC50 = 63 nM) and
HO
ethyl 1-piperazinecarboxylate 12n (IC50 = 93 nM), both of them
showed a significant reduction in DPP-IV inhibition compared to
12a (IC50 = 15 nM). The relatively lipophilic azepane analogue
12o and 7-azabicyclo[2.2.1]heptane analogue 12p were 10- and
4-fold, respectively, less potent than 12a as DPP-IV inhibitors. Fur-
ther modification was carried out at bringing the nitrogen out of
the ring system and developing the cyclopentylamine derivative
12q, aniline derivative 12r and pyridine derivatives 12s–t. Cyclo-
pentylamine 12q is still a potent inhibitor of DPP-IV (IC50 = 29 nM).
Aniline 12r (4-fluoro-2-cyanopyrrolidine) is 4-fold more potent
than its closely relative analogue 5 (2-cyanopyrrolidine), but 12r
had weak inhibition against DPP9. When the phenyl ring of 12q
was replaced with a more polar heterocyclic ring, pyridin-2-yla-
mine 12s and 5-methoxypyridin-2-ylamine 12t are equipotent
against DPP-IV (IC50 = 19 nM), but 12t had weak inhibition against
DPP9.
A further evaluation of potency in 50% human (rat) serum and
ex vivo DPP-IV inhibition22 was carried out to provide a finer dis-
crimination between these potent compounds within this struc-
turally related series of inhibitors with respect to
pharmacodynamic effects (serum potency shift) and duration of
action.12 DPP-IV inhibitory activities in 50% human (rat) serum
and ex vivo DPP-IV inhibition for selected structure I-based inhib-
itors are listed in Table 3. For comparison, first-generation DPP-IV
inhibitors 1 and 2 are also included in the table. trans-2-Aryl-
cyclopropylamine 6 and pyrrolidine 12a were all comparable to
1 and 2 in terms of intrinsic potency, but 6 suffered from large
serum potency shift (IC50 = 847 and 210 nM in human and rat
plasma, respectively), resulting in only 66% inhibition of plasma
DPP-IV activity at 30 min after oral administration of 3 mg/kg to
rats. High non-covalent plasma protein binding of 1 might be
one of reasons for its unexpectedly less potent plasma DPP-IV
inhibition (30% at 30 min). Compounds 2 and 12a maintained
excellent DPP-IV potency in both human and rat plasma, afforded
potent plasma DPP-IV inhibition (>80% at 30 min, 70% at 8 h).
Other pyrrolidine analogues 12b, 12f and 12g exhibited a shorter
duration of action than 12a. (3R)-pyrrolidin-3-ol 12b showed less
potent ex vivo DPP-IV inhibition (63% at 30 min) most likely due
to the decreased oral absorption for the increase in hydrophilicity.
As for six-membered ring derivatives, the slightly more lipophilic
piperidine analogues 12h–i exhibited ex vivo potency (>85% at
30 min) and duration of action (>70% at 8 h) similar to that of pyr-
rolidine 12a, the more hydrophilic morpholine analogue 12k
exhibited 89% plasma DPP-IV inhibition with a modest duration
of action, however another hydrophilic analogue, 1-methanesulfo-
nylpiperazine 12l, showed weaker inhibition of plasma DPP-IV
and its efficacy diminished within 8 h. When the nitrogen was
brought out of ring system, cyclopentylamine 12q showed no
improvement in ex vivo potency and duration compared to that
of 12a. Aromatic analogue 12r and heteroaromatic analogues
12s–t reached at least 85% inhibition at 30 min, but none of them
are long-lasting DPP-IV inhibitors in rats.
0.18
>20
>20
>50
>20
N
0.015
0.030
>100
>20
HO
HO
F
N
N
12c
12d
0.050
0.039
>20
>20
>20
>20
N
OH
12e
12f
0.024
0.032
>20
>20
>20
>20
N
N
N
S
12g
12h
0.027
0.027
>20
>20
>20
>20
N
N
12i
12j
12k
0.018
0.026
0.020
>20
>20
>20
50%b
>20
>20
O
O
N
N
O2
S
12l
N
0.024
0.093
>20
>20
>20
N
N
12m
86%b
N
N
O
12n
12o
0.063
0.16
>20
>20
>20
>20
O
N
N
N
12p
12q
0.066
0.029
>20
>20
>20
>20
H
N
O
12r
0.027
>20
73%b
O
NH
N
H
N
12s
12t
0.019
0.019
>20
>20
>20
N
H
61%b
O
N
a
Means of at least three experiments; standard derivatives are 20%.
b
Inhibition at 20 lM.

T.-K. Yeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3596–3600
3599
Table 3
presented above, compound 12a was chosen for further evaluation
in preclinical studies. Preliminary acute toxicity studies in rats and
monkeys demonstrated the compound to be well tolerated.
In summary, we have identified a novel series of 2-[3-[[2-[(2S)-
2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-3-methyl-1-oxobutyl]
-based analogues as potent and selective DPP-IV inhibitors. Nota-
ble among these is compound 12a having monocyclic pyrrolidine
ring in the P2 site. This compound is an IC50 = 15 nM DPP-IV inhib-
itors and displays a more than 3000-fold selectivity over DPP8,
DPP9, FAP and DPP-II. The in vivo effects of compound 12a, includ-
ing inhibition of plasma DPP-IV activity and suppression of blood
glucose elevation, were also demonstrated. The results of these
studies indicate that 12a is a potent, selective, long-acting and safe
DPP-IV inhibitor as a potential treatment of type 2 diabetes
mellitus.
Potency in the presence of 50% human and rat serum and ex vivo plasma DPP-IV
inhibition in rats
Compound 50% HSa IC50 (nM) % plasma DPP-IV inhibition at 3 mg/kg oral
dose, normal rats
30 min
8 h
1
2
6
18 (46)b
17 (16)
847 (210)
5 (10)
21 (23)
NDc
ND
14
ND
13 (14)
15
30
84
66
82
63
75
77
86
85
89
64
81
86
87
88
20
73
30
70
15
48
56
74
70
59
7
12a
12b
12f
12g
12h
12i
12k
12l
12q
12r
12s
12t
ND
ND
19 (24)
18
60
18
31
41
Acknowledgment
National Health Research Institutes, Taiwan, financially
supported the study.
a
b
c
Assay done in the presence of 50% human serum.
Values in parentheses are the IC50 values in the presence of 50% rat serum.
ND, not determined.
References and notes
Compound 12a was chosen for more extensive study in acute
1. Mentlein, R. Regul. Pept. 1999, 85, 9.
efficacy model measurement due to its excellent human plasma
DPP-IV inhibition (IC50 = 5 nM) and pharmacodynamic duration
of action in this preliminary ex vivo assay in rats. In an oral glucose
tolerance test (OGTT), when compound 12a was administered by
oral route at 3 mg/kg to C57BL/6j mice 30 min before glucose
administration (3 g/kg), and then blood samples drawn and ana-
lyzed for glucose levels. The glucose AUC was determined from 0
to 120 min, and OGTT data on 12a were summarized in Figure 3.
Compound 12a significantly suppressed the glucose excursion ob-
served after glucose challenge (67% reduction in the area under the
glucose levels vs. time curve) and provided a comparable efficacy
to both the known DPP-IV inhibitors 1 and 2.
2. Zhu, L.; Tamvakopoulos, C.; Xie, D.; Dragovic, J.; Shen, X.; Fenyk-Melody, J. E.;
Schmidt, K.; Bagchi, A.; Griffin, P. R.; Thornberry, N. A.; Roy, R. S. J. Biol. Chem.
2003, 278, 22418.
3. Holst, J. J.; Orskov, C.; Nielsen, O. V.; Schwartz, T. W. FEBS Lett. 1987, 211, 169.
4. Pospisilik, J. A.; Stafford, S. G.; Demuth, H. U.; McIntosh, C. H.; Pederson, R. A.
Diabetes 2002, 51, 2677.
5. Ahren, B.; Holst, J. J.; Martensson, H.; Balkan, B. Eur. J. Pharmacol. 2000, 404,
239.
6. Deacon, C. F.; Danielsen, P.; Klarskov, L.; Olesen, M.; Holst, J. J. Diabetes 2001,
50, 1588.
7. Sudre, B.; Broqua, P.; White, R. B.; Ashworth, D.; Evans, D. M.; Haigh, R.; Junien,
J. L.; Aubert, M. L. Diabetes 2002, 51, 1461.
8. Deacon, C. F.; Holst, J. J. Int. J. Biochem. Cell Biol. 2006, 38, 831.
9. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher, M. H.; He, H.; Hickey, G. J.;
Kowalchick, J. E.; Leiting, B.; Lyons, 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.; Weber, A. E. J. Med. Chem. 2005, 48, 141.
10. Deacon, C. F. Expert Opin. Investig. Drugs 2007, 16, 533.
11. 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.
12. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson,
J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.;
Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.;
Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med. Chem.
2005, 48, 5025.
Compound 12a is a potent, selective and orally active inhibitor
of DPP-IV, and 12a showed modest and good oral bioavailability in
rats and dogs (F = 19% and 68%, respectively). Besides, 12a also pos-
sesses an excellent safety pharmacology profile: additional in vitro
profiling of 12a in an extensive panel of 8 enzyme assays showed
no significant inhibition at a test concentration of 10
did not block the Potassium Channel hERG, Calcium Channel L-type
and Sodium Channel Site II at a test concentration of 10
l
M,23 and it
l
M (À6%,
13. Tsu, H.; Chen, X.; Chen, C. T.; Lee, S. J.; Chang, C. N.; Kao, K. H.; Coumar, M. S.;
Yeh, Y. T.; Chien, C. H.; Wang, 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.; Chao, Y. S.; Jiaang, W. T. J.
Med. Chem. 2006, 49, 373.
À9% and 1% inhibition, respectively). Based on these results
14. Coumar, M. S.; Chang, C. N.; Chen, C. T.; Chen, X.; Chien, C. H.; Tsai, T. Y.; Cheng,
J. H.; Wu, H. Y.; Han, C. H.; Wu, S. H.; Huang, Y. W.; Hsu, T.; Hsu, L. J.; Chao, Y. S.;
Hsieh, H. P.; Jiaang, W. T. Bioorg. Med. Chem. Lett. 2007, 17, 1274.
15. Tsai, T. Y.; Hsu, T.; Chen, C. T.; Cheng, J. H.; Yeh, T. K.; Chen, X.; Huang, C. Y.;
Chang, C. N.; Yeh, K. C.; Hsieh, S. H.; Chien, C. H.; Chang, Y. W.; Huang, C. H.;
Huang, Y. W.; Huang, C. L.; Wu, S. H.; Wang, M. H.; Lu, C. T.; Chao, Y. S.; Jiaang,
W. T. Bioorg. Med. Chem. 2009, 17, 2388.
16. Matsuyama-Yokono, A.; Tahara, A.; Nakano, R.; Someya, Y.; Nagase, I.;
Hayakawa, M.; Shibasaki, M. Biochem. Pharmacol. 2008, 76, 98.
17. (a) Haffner, C. D.; McDougald, D. L.; Reister, S. M.; Thompson, B. D.; Conlee, C.;
Fang, J.; Bass, J.; Lenhard, J. M.; Croom, D.; Secosky-Chang, M. B.; Tomaszek, T.;
McConn, D.; Wells-Knecht, K.; Johnson, P. R. Bioorg. Med. Chem. Lett. 2005, 15,
5257;
b Melogliptin, Glenmark’s lead DPP-IV inhibitor with a fluorine
substituent on the 4-position of the P1 2-cyanopyrrolidine ring, is a novel,
oral DPP-IV inhibitor in phase III development for type 2 diabetes.
18. Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G. J.; Beconi, M. G.; Biftu, T.; Chan,
C. C.; Edmondson, S.; 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, L.; Weber, A. E.; Thornberry, N. A. Diabetes
2005, 54, 2988.
19. Kim, D.; Kowalchicka, J. E.; Edmondsona, S. D.; Mastracchioa, A.; Xua, J.;
Eiermannb, G. J.; Leitingb, B.; Wub, J. K.; Pryorb, K. D.; Patelb, R. A.; Hea, H.;
Lyonsa, K. A.; Thornberryb, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett. 2007, 17,
3373.
Figure 3. Effects of 1, 2 and 12a on the glucose levels after an oral glucose tolerance
test in C57BL/6j mice (3 mg/kg, po).

3600
T.-K. Yeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3596–3600
20. Chiravuri, M.; Schmitz, T.; Yardley, K.; Underwood, R.; Dayal, Y.; Huber, B. T. J.
Immunol. 1999, 163, 3092.
21. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol. 2003, 7, 496.
22. DPP-IV inhibition in rats: Adult male Wistar rats were orally gavaged with the
test compounds dissolved in 0.5% methyl cellulose at a single dose of 3.0 mg/
assay buffer that was composed of 25 mM tris(hydroxymethyl)aminomethane
HCl (pH 7.4), 140 mM NaCl, 10 mM KCl, and 0.1% bovine serum albumin. The
fluorescence was determined by using a Fluoroskan Ascent FL (excitation at
390 nm and emission at 460 nm) (Thermo LabSystems; Thermo Electron
Corporation). DPP-IV activity in plasma was described as units per milliliter (U/
mL). One unit of activity is defined as the amount of enzyme that produces
kg. Blood samples of 25–50 lL were collected from the tail veins at the time
points indicated in Table 3, and the plasma fraction was kept frozen until DPP-
IV activity measurement. The plasma DPP-IV activity was determined by the
cleavage rate of Gly-Pro-AMC (H-glycyl-prolyl-7-amino-4-methylcoumarin;
1
l
M products per minute.
23. Compound 12a (10 M) inhibition of selected proteases: b-Secretase (3%),
l
Caspase 1 (23%), Caspase 10 (8%), Cathepsin B (À10%), Cathepsin H (0%), Factor
BACHEM). Plasma (10 lL) was mixed with 140 lL of 150 lM Gly-Pro-AMC in
Xa (2%), Thrombin (À7%), Trypsin (0%).