2-[3-[2-[(25)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl] -1,2,3,4-tetrahydroisoquinoline: A potent, selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV

Article abstract of DOI:10.1021/jm0507781

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2- oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC₅₀ values of <50 nM with excellent selectivity over both DPP8 (IC₅₀ > 100 μM) and DPP-II (IC₅₀ > 30 μM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development. 2006 American Chemical Society.

Full text of DOI:10.1021/jm0507781

J. Med. Chem. 2006, 49, 373-380
373
2-[3-[2-[(2S)-2-Cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]-
1,2,3,4-tetrahydroisoquinoline: A Potent, Selective, and Orally Bioavailable
Dipeptide-Derived Inhibitor of Dipeptidyl Peptidase IV
Hsu Tsu,†,‡ Xin Chen,†,‡ Chiung-Tong Chen,†,‡ Shiow-Ju Lee,† Chung-Nien Chang,† Kuo-His Kao,† Mohane Selvaraj Coumar,†
Yen-Ting Yeh,† Chia-Hui Chien,† Hsin-Sheng Wang,† Ke-Ta Lin,† Ying-Ying Chang,† Ssu-Hui Wu,† Yuan-Shou Chen,†
I-Lin Lu,†,§ Su-Ying Wu,† Ting-Yueh Tsai,† Wei-Cheng Chen,† Hsing-Pang Hsieh,† Yu-Sheng Chao,† and Weir-Torn Jiaang*,†
DiVision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Rd., Zhunan Town,
Miaoli County 350, Taiwan, Republic of China, and Graduate Institute of Life Sciences, National Defense Medical Center,
No.161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan, Republic of China
ReceiVed August 8, 2005
Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most
known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1
site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies,
it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of
2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was syn-
thesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC50 values of <50 nM with excellent
selectivity over both DPP8 (IC50 > 100 µM) and DPP-II (IC50 > 30 µM). Compound 21a suppressed the
blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV
activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo
activities comparable to those of NVP-LAF237 (4), which is in clinical development.
Introduction
Glucagon-like peptide-1 (GLP-1), the most potent insulino-
tropic hormone known,1 is a 30 amino acid hormone produced
by L-cells of the intestinal mucosa that results from tissue-
specific processing of the proglucagon gene.2 The active form
of GLP-1 stimulates insulin secretion, inhibits glucagons
release,3 and slows gastric emptying,4 each a benefit in the
control of glucose homeostasis in patients with type 2 diabetes.5
However, therapeutic use of GLP-1 itself is severely compro-
mised by its lack of oral activity and rapid degradation by
plasma DPP-IV. Thus, a small molecule inhibitor of DPP-IV
could extend the duration of action of GLP-1 and prolong the
beneficial effects of this incretin hormone. Indeed, inhibition
of DPP-IV has been shown to be effective in improving glucose
Figure 1. Reported DPP-IV inhibitors.
tolerance in diabetic patients and healthy volunteers in human
clinical trials and therefore offers a new strategy for treating
type 2 diabetes.6
replacing hydrogen with an electrophilic nitrile group at the
Dipeptidyl peptidase IV (DPP-IV) is a serine protease
2-position of the pyrrolidine resulted in a 1000-fold increase in
potency compared to the unsubstituted pyrrolidines.10 A series
of 2-cyanopyrrolidine derivatives was prepared for optimizing
the N-terminal residue, and the result showed that bulky and
lipophilic R-substituted amino acid gave more potent com-
pounds. Cyclohexylglycine-(2S)-cyanopyrrolidine (2) is one of
the potent and stable representatives of this nitrile class. This
compound has a Ki value of 1.4 nM and an excellent chemical
stability t1/2 ∼ 48 h at pH 7.4.11 Another class of proline-mimetic
inhibitors was synthesized by Novartis with diverse N-substi-
tuted glycines in the P2 site.6a The side chain in this class was
moved from the R-carbon to the terminal nitrogen. Two potent
derivatives in this series were 3 and 4, both of which showed
great efficacy in clinical trials. A monotherapy for 12 weeks
with 4 showed sustained reductions in HbA1c (from a baseline
value of 8 to 7.4% by the end of the study),12 and when given
in combination with metformin, no weight gain was reported
over the study period.13
cleaving the N-terminal dipeptide with a preference for L-proline
or L-alanine at the penultimate position.7 Many DPP-IV inhibi-
tors resemble the P2-P1 dipeptidyl substrate cleavage product.
The simplest inhibitors of DPP-IV are product-like compounds
lacking the carbonyl function of the proline residue, such as
aminoacyl pyrrolidines and thiazolidines, which bind and inhibit
DPP-IV with moderate ability. One of these compounds,
N-isoleucylthiazolidine 1 (Figure 1, also called P32/98 by
Probiodrug),8 has moderate potency toward DPP-IV. In NIDDM
patients, 60 mg of 1 improved insulin release by 70% and, in
combination with acarbose or glibenclamide, suppressed glucose
excursions by 20 and 30%, respectively.9 In some cases,
* Corresponding author. Phone: 886-37-246166, ext 35712. Fax: 886-
37-586456. E-mail: wtjiaang@nhri.org.tw
† National Health Research Institutes.
‡ Contributed equally to the work.
§ National Defense Medical Center.
10.1021/jm0507781 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/07/2005

374 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsu et al.
Table 1. Inhibition of DPP-IV, DPP8, and DPP-II by Compounds 1-4
IC50a (nM)
compd
DPP-IV
DPP8
DPP-II
1
2
3
4
1660 ( 230
12 ( 4
2283 ( 201
27 ( 4
4573 ( 220
14219 ( 579
68985 ( 598
39873 ( 1670
26520 ( 764
53 ( 8
51 ( 10
>100000
a Values are expressed as the mean ( SD of three independent
determinations.
In addition to DPP-IV, the prolyl peptidase family contains
DPP-II, DPP8, DPP9, and FAP (fibroblast activation protein),
which also cleave the peptide bond at the penultimate proline
residue.14 The selectivity of the DPP-IV inhibitors against other
proline-specific dipeptidyl peptidases is one of the key issues
in drug development, since the inhibition of DPP-II15 has been
shown to result in the apoptosis of quiescent T-cells.15b Recent
in vivo studies indicate that selective inhibition of DPP8/916
may be associated with profound toxicities (but it is not known
if both DPP8 and DPP9 inhibition are required and how much
inhibition of each is required for producing the toxicity), whereas
selective inhibition of DPP-IV is not.17 We first assessed the
selectivity of existing compounds 1-4 for DPP-IV, DPP-II, and
DPP8 inhibition. As shown in Table 1,18 R-carbon-substituted
glycine in the P2 site (1 and 2) exhibited DPP8 inhibition with
IC50 values of 2283 and 27 nM, respectively, with no selectivity
for DPP-IV over DPP8. In comparison, N-substituted glycines
in the P2 site combined with a (2S)-cyanopyrrolidine in the P1
site (3 and 4) were weak DPP8 inhibitors with IC50 values of
4.6 and 14.2 µM, respectively, with high selectivity for DPP-
IV over DPP8. Thiazolidine (1) showed equipotent inhibition
against DPP8 and DPP-IV. In addition, compounds 1-3 showed
weak inhibitory activities against DPP-II with IC50 values
ranging from 27 to 69 µM, while there is no inhibitory activity
for compound 4 against DPP-II (Table 1). Compound 4 exhibits
excellent selectivity over other compounds shown in Table 1.
Clinical results show that 4 does not have serious side effects.
In fact, MK-0431 [(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)-
butan-2-amine] developed by Merck is a highly potent and
selective inhibitor of DPP-IV. It does not inhibit DPP-II or
DPP8/9 among other proteases tested and currently it has
successfully gone into phase III clinical trials.6c,e
Figure 2. Design and retrosynthesis of 2-[3-[[2-[(2S)]-2-cyano-1-
pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors.
Scheme 1a
a Reagents: (a) DCC, HOSu 1,4-dioxane/CH2Cl2, various amines; (b)
CF3COOH; (c) K2CO3, THF; (d) KOCl, 1,4-dioxane/H2O.
Starting materials Boc-glycine 5, Boc-â-alanine 6, 4-Boc-amino-
butyric acid 7, Boc-L-â-alanine or Boc-L-â-leucine 8, and 3-
Boc-amino-3-methylbutyric acid 9 were DCC-coupled with
various amines followed by Boc-deprotection by TFA to provide
TFA salts 10, 11, 12, 13, and 14, respectively. Acid 9 was
prepared through oxidation of N-Boc-diacetonamine 15 using
potassium hypochlorite. 1-Bromoacetyl-2-cyano-(S)-pyrrolidine
16 was coupled with free base amines 10-14 to provide the
desired 2-cyanopyrrolidine analogues 17-21. The synthesis of
bromo compound 16 was carried out according to the literature
procedure.19
From the selectivity data of compounds 1-4, it can be
concluded that (2S)-cyanopyrrolidine derivatives with N-sub-
stituted glycine in the P2 site (3 and 4) are more selective for
DPP-IV than R-carbon-substituted glycine (1 and 2). On the
basis of the results, we endeavored to develop a new pharma-
cophore in the P-2 site with N-substituted glycine. We first
designed P-2 site amine extensions using â-alanine as building
block and coupled the C-terminal with various substituted
amines to generate a novel pharmacophore in the P2 site. Then,
the N-terminal of the â-alanine derivative was combined with
the P1 site R-bromoacetyl (2S)-cyanopyrrolidide to construct
2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxo-
propyl]-based (structure I) DPP-IV inhibitors (Figure 2). The
structure-activity relationships of several series of structure
I-based DPP-IV inhibitors were investigated. This study has
led to the discovery of the potent and selective DPP-IV inhibitor
21a and its analogues. This selective DPP-IV inhibitor 21a
exhibits in vivo potency comparable to that of DPP-IV inhibitor
4.
Results and Discussion
Structure I-based inhibitors with various substituted amines
were explored, including the bicyclic ring system (series I),
monocyclic piperazine ring (series II) and phenylalkyl groups
(series III). These N-substituted glycine derivatives 17-21
described above were tested for inhibition of DPP-IV, DPP8,
and DPP-II, and the data is presented in Table 2. Since
significant inhibition of DPP-II (IC50 < 20 µM) was not
observed in all the series of inhibitors shown in Table 2 except
Chemistry. (2S)-Cyanopyrrolidine analogues 17-21 were
prepared as described in Scheme 1 and are listed in Table 2.

Dipeptide-DeriVed Inhibitor of DPP-IV
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 375
Table 2. Inhibitory Properties of Structure I-Based DPP-IV Inhibitors
IC50a (nM)
compd
series
n
m
R1
R2
R3
R4
R5
DPP-IV
DPP8
DPP-II
>100000
17
I
I
I
I
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
H
H
H
H
3236 ( 599
116 ( 11
651 ( 21
83 ( 3
4169 ( 736
3583 ( 281
3340 ( 599
1700 ( 761
2121 ( 483
202 ( 33
3416 ( 3928
2000 ( 141
2117 ( 294
10744 ( 917
2387 ( 151
21961 ( 1638
5532 ( 941
12847 ( 3251
8338 ( 262
2592 ( 891
855 ( 288
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
18o
18p
19
20a
20b
21a
21b
21c
21d
21e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>100000
>100000
>100000
>100000
>100000
>10000
6,7-diOMe
6-F
H
I
132 ( 14
676 ( 75
1418 ( 5
629 ( 78
527 ( 46
452 ( 20
317 ( 39
447 ( 45
369 ( 23
784 ( 54
119 ( 40
564 ( 60
298 ( 11
428 ( 27
54 ( 3
II
II
II
II
III
III
III
III
III
III
III
CO(3,5-F2C6 H3)
SO2C6H4-4-NHCOCH3
nicotinonitrile
benzothiazole
18100 ( 987
6452 ( 756
17400 ( 3865
36003 ( 2573
0
0
0
0
0
0
1
H
4-NO2
H
3,5-F2
H
H
H
H
H
H
H
CH3
H
H
H
ethylb
H
>100000
34444 ( 1683
29315 ( 1749
i-Prb
CH3
H
>100000
H
20720 ( 424
57176 ( 3548
31248 ( 1027
18869 ( 3193
I
I
I
I
I
I
I
III
2
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
H
H
H
H
CH3
CH3
CH3
CH3
CH3
H
H
H
H
6-F
6,8-F2
H
1407 ( 300
5346 ( 291
41859 ( 942
CH3
i-Pr
CH3
CH3
CH3
CH3
CH3
811 ( 60
49 ( 5
>100000
>100000
34201 ( 1661
50146 ( 3858
31165 ( 2355
30 ( 2
>100000
>100000
>100000
>100000
22 ( 3
15 ( 2
>100000
>100000
H
CH3
CH3
1108 ( 82
a Values are expressed as the mean ( SD of three independent determinations. b The stereochemistry at the benzylic carbon is not defined (mixture of
diastereomers).
18h (IC50 ) 6452 nM), discussion of SAR studies is focused
on inhibition of DPP-IV and DPP8. We started initially with
the bicyclic system (series I) using the isoquinoline ring.
Compound 18a inhibited DPP-IV with an IC50 value of 116
nM, had 31-fold selectivity over DPP8, and was inactive toward
DPP-II. A focused structure-activity profiling effort was
initiated by modifying the aromatic ring of isoquinoline.
Introduction of 3,4-dimethoxy substituents in 18b had a moder-
ate decrease in DPP-IV potency by 4-fold of magnitude relative
to 18a, but compound 18b was approximately equipotent to 18a
as DPP8 inhibitor. On the other hand, introduction of an
electron-withdrawing fluorine atom at the 6-position of the
isoquinoline ring20 in 18c led to some increase in potency (IC50
) 83 nM) with a slight drop in selectivity over DPP8 (20-fold).
When the isoquinoline ring system was replaced by isoindoline
in compound 18d, it resulted in a similar level of DPP-IV
inhibition, as seen for 18a, but selectivity was decreased by
half (16-fold). Replacement of the bicyclic ring system (series
I) with monocyclic piperazine derivatives (series II), such as
benzoyl 18e, sulfonamide 18f, nicotinonitrile 18g, and ben-
zothiazole 18h, led to at least a 4.5-fold decrease in DPP-IV
inhibitory potency compared to lead compound 18a. In contrast,
all the four compounds exhibited better inhibition of DPP8 than
18a. The benzoyl derivative 18e even showed 3-fold higher
inhibitory activity for DPP8 (IC50 ) 202 nM) over DPP-IV (IC50
) 676 nM). This compound is the most potent DPP8 inhibitor
among the compounds in our investigation.
Next, we brought the nitrogen out of the ring system and
developed the acyclic benzylamine and phenylethylamine
derivatives (series III). Benzylamine 18i had DPP-IV potency
similar to the phenylethylamine 18o, but benzylamine 18i
showed good selectivity over DPP8 (24-fold) compared to
phenylethylamine analogue 18o (5-fold). Therefore, we focused
our efforts on studying the benzylamine series of compounds
(18j-n). Introduction of an electron-withdrawing group in the
phenyl ring, such as the 4-nitro substituent in 18j and 3,5-
difluoro substituents in 18l, gave compounds with a similar
range of activities against DPP-IV, but a decreased selectivity
(<15-fold) for inhibition of DPP-IV over DPP8 relative to 18i.
Introduction of ethyl group at the carbon 1 of benzylamine gave
the diastereomer 18k, which provided no improvement in DPP-
IV inhibition (IC50 ∼ 447 nM). However, there was a significant
increase in selectivity over DPP8 (49-fold). Replacement of the
ethyl group with isopropyl gave diastereomer 18m, which
showed reduced potency (IC50 ∼ 784 nM) and selectivity over
DPP8 (16-fold). A significant improvement in DPP-IV inhibition
was obtained with 1,1-dimethylbenzylamine 18n, which was
more potent as a DPP-IV inhibitor (IC50 ∼ 119 nM) compared
to the unsubstituted 18i and also showed improved selectivity
(70-fold). Moving the aromatic ring close to the nitrogen yielded
aniline derivative 18p, which was only twice less potent (IC50
∼ 298 nM) compared to 18a at DPP-IV inhibition. However,
it showed a drastic reduction in selectivity for DPP-IV over
DPP8 (3-fold).

376 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsu et al.
Having failed to improve both the potency and selectivity of
the lead compound 18a significantly by the above manipulations
of the N-substituents, we set out to investigate the effect of
varying the length of alkyl chain between the P2 site amine
and carboxylic position. In comparison with 18a, shortening
by one carbon (17) or lengthening by one carbon (19) decreased
the inhibitory potency of DPP-IV by 28- and 4-fold, respectively.
The study of the P2 site N-substituted glycines was continued
with the evaluation of acyclic branching R to the terminal amine.
Introduction of methyl substitution into the â-alanine chain of
18a to give the diastereomeric pair 20a was well-tolerated,
providing both significantly improved potency (IC50 ∼ 54 nM)
and selectivity (99-fold) over DPP8. Increasing the bulk of the
branching by introduction of an isopropyl group gave diaster-
eomers 20b, leading to a 7-fold reduction in DPP-IV potency
(IC50 ∼ 811 nM) but still maintaining good selectivity (51-fold).
Apparently, the alkyl group adjacent to the P2 site amine has
some effect on the selectivity over DPP8. In view of this, we
introduced a gem-dimethyl group adjacent to the P2 site amine.
Compound 21a exhibited an IC50 value of 49 nM and selectivity
index of more than 2000 over DPP8; this compound can be
considered as an equipotent but more selective DPP-IV inhibitor
than 20a. Compound 21a also showed weak inhibitory activity
against DPP-II (IC50 ∼ 34 µM). Having arrived at the optimized
structure in the â-alanine portion of the P2 site for good potency
and selectivity, we went back to optimize the isoquinoline
portion of the inhibitor. As expected, the gem-dimethyl series
of derivatives with the introduction of 6-fluoro (21b) or 6,8-
difluoro (21c) substituents20 in the phenyl ring of 21a, gave
slight improvement in the inhibitory potency of DPP-IV, while
both still maintained excellent selectivity for DPP-IV over DPP8
and DPP-II. Replacement of the isoquinoline ring of 21a with
isoindoline gave a very potent DPP-IV inhibitor 21d, with an
IC50 value of 15 nM and >6700-fold selectivity over DPP8 and
DPP-II. Interestingly, the effect of the gem-dimethyl substituent
was not observed in the acyclic (series III) compound 21e, which
showed 10-fold reduced potency compared to 18n. Nevertheless,
21e was still inactive toward DPP8. Beginning with our lead
compound 18a in bicyclic series I and carrying out a focused
structure-activity optimization, we have demonstrated that
structure I-based DPP-IV inhibitors prefer a bicyclic ring system
(series I) with a gem-dimethyl group adjacent to the P-2 site
amine. Inhibitors with these structure entities, 21a-d, showed
better potencies and selectivities than the other series of
compounds investigated.
Figure 3. Effects of 21a and 4 on the glucose excursion levels of
adult male Wistar rats during oral glucose tolerance test. All rats
received 1.5 g/kg glucose orally at 0 min. Each compound was orally
administered at a 10 mg/kg dose to rats at -30 min. Data are
represented as mean ( SEM (n ) 4/group).
Figure 4. Effects of 21a, 21d, and 4 on the plasma DPP-IV activity
in mice. Each compound was orally administered at a single dose of
18 mg/kg to mice at 0 h. Data are expressed as mean ( SEM (n )
4/group).
were orally dosed with water or with 10 mg/kg inhibitors 21a
and 4, followed by the administration of 1.5 g/kg glucose 0.5 h
after the dosing. Glucose levels were then monitored over a 2
h period. Figure 3 shows the glucose excursion in response to
an oral glucose challenge. Vehicle-treated controls were mark-
edly glucose intolerant with an average peak glucose level of
160 mg/dL at 30 min after glucose challenge, whereas the drug-
treated animals exhibited a 36% decrease in glucose levels
compared to controls (21a-treated animals, 102 mg/dL; 4-treated
animals, 103 mg/dL). In addition, the inhibitory effects of orally
administered compounds 4, 21a, and 21d at a single dose of 18
mg/kg on plasma DPP-IV activity were measured in mice
(Figure 4). A maximum inhibition of plasma DPP-IV activity
was observed approximately 30 min after the oral dosing of
compound 4, 21a, or 21d and the inhibition of >80% lasted
for at least 4 h. The most potent compound 21d showed similar
inhibition profile of plasma DPP-IV activity as 21a, even though
21d was 3-fold more potent than 21a in the in vitro assay.
Interestingly, potent fluoro analogues 21b and 21c inhibited
plasma DPP-IV activity (80-90%) within 30 min, but the
duration of DPP-IV inhibition of fluoro analogues was shorter
than that of 21a when compared at the same dose (data not
shown). The in vivo effects shown in Figures 3 and 4
demonstrate the ability of 21a to both significantly decrease
the glucose excursion and inhibit plasma DPP-IV activity, and
After the discovery that compounds 21a-d were potent and
selective inhibitors of DPP-IV in vitro, the aqueous solubility
and stability of these analogues need to be investigated before
testing the in vivo efficacy of these compounds. The representa-
tive analogue 21a was found to have good solubility (>1000
µg/mL) in distilled water. The aqueous solution stability of 21a
was determined by monitoring the disappearance of 21a on
reverse-phase HPLC in aqueous solution at room temperature.
HPLC mass spectral analysis revealed that the amino group of
21a did not intramolecularly cyclize with the nitrile to form a
cyclic amidine or diketopiperazine19,21 during the time frame
of the stability experiment (72 h). Because of the steric size
imposed by the fully branched carbon adjacent to the P2 site
amine, compound 21a is much more stable than less branched
3 in aqueous solution.19
Compound 21a, with its potent, selective in vitro DPP-IV
inhibition, excellent stability, and water solubility, was chosen
for further evaluation of its ability to improve glucose tolerance
in Wistar rats and DPP-IV inhibition in mice. The fasting rats

Dipeptide-DeriVed Inhibitor of DPP-IV
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 377
(mixture of trans/cis amide rotomers) δ 2.09-2.37 (m, 4H), 2.84-
2.93 (m, 2H), 3.42-3.73 (m, 7H), 3.83 (t, 1H, J ) 6.0 Hz), 4.57
(s, 0.9H, ArCH2N), 4.73-4.78 (m, 1.9H, overlapped singlet at 4.73,
1.1H ArCH2N and 0.8H CHCN), 4.95 (d, 0.2H, J ) 7.5 Hz,
CHCN), 7.09-7.27 (m, 4H); HRMS (EI) m/z calcd for C18H22N4O2
326.1743, found 326.1743.
these pharmacological profiles of 21a are comparable to those
of compound 4, currently in late-stage clinical development.
In summary, we have discovered a novel series of potent and
selective DPP-IV inhibitors.
Notable among these is series I inhibitors with a gem-dimethyl
group adjacent to the P2 site amine (21a-d), which are IC50
<
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-1,2,3,4-tetrahydroisoquinoline (18a): 1H NMR (CD3OD)
δ 2.16-2.28 (m, 4H), 2.87 (t, 1H, J ) 6.0 Hz), 2.94-2.98 (m,
3H), 3.41 (t, 2H, J ) 5.9 Hz), 3.46-3.51 (m, 2H), 3.63-3.68 (m,
1H), 3.73 (t, 1h, J ) 6.0 Hz), 3.81 (t, 1H, J ) 6.0 Hz), 4.11-4.13
(m, 2H), 4.70 (d, 2H, J ) 9.9 Hz, ArCH2N), 4.81 (dd, 1H, J ) 9.9
and 5.1 Hz, CHCN), 7.18-7.19 (m, 4H); HRMS (EI) m/z calcd
for C19H24N4O2 340.1899, found 340.1899; HPLC (condition A)
tR ) 9.85 min, 97.4%, (condition B) tR ) 6.57 min, 97.4%.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (18b): 1H
NMR (CDCl3) (9/1 mixture of trans/cis amide rotomers) δ 2.16-
2.28 (m, 4H), 2.68 (t, 2H, J ) 6.0 Hz), 2.77 (t, 1H, J ) 6.0 Hz),
2.83 (t, 1H, J ) 6.0 Hz), 3.04 (t, 2H, J ) 6.3 Hz), 3.41-3.62 (m,
4H, overlapped singlet at 3.51), 3.67 (t, 1H, J ) 6.0 Hz), 3.79-
3.87 (m, 7H, overlapped singlet of OCH3 at 3.87), 4.56 (s, 0.9H,
ArCH2N), 4.65 (s, 1.1H, ArCH2N), 4.73-4.74 (m, 0.9H, CHCN),
4.83 (d, 0.1H, J ) 6.0 Hz, CHCN), 6.60-6.63 (m, 2H); HRMS
(EI) m/z calcd for C21H28N4O4 400.2111, found 400.2112.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-6-fluoro-1,2,3,4-tetrahydroisoquinoline (18c): 1H NMR
(CDCl3) (4/1 mixture of trans/cis amide rotomers) δ 1.94-2.37
(m, 4H), 2.55-2.70 (m, 2H), 2.83 (t, 1H, J ) 6.0 Hz), 2.90 (t, 1H,
J ) 6.0 Hz), 2.93-3.10 (m, 2H), 3.30-3.72 (m, 5H, overlapped
triplet at 3.68, J ) 6.0 Hz and singlet at 3.47), 3.80 (t, 1H, J ) 6.0
Hz), 4.50-4.80 (m, 2.8H, two overlapped singlet at 4.58 and 4.68,
2H ArCH2N and 0.8H CHCN), 4.85 (d, 0.2H, J ) 7.5 Hz, CHCN),
6.75-6.92 (m, 2H), 7.01-7.11 (m, 1H); MS (ES+) m/z 359.2 (M
+ H)+.
50 nM DPP-IV inhibitors with an excellent selectivity profile
over DPP8 (IC50 > 100 µM) and DPP-II (IC50 > 30 µM). This
series of inhibitors also exhibited excellent aqueous solubility
and stability. The in vivo effects of compound 21a, including
inhibition of plasma DPP-IV activity and suppression of blood
glucose elevation, were also demonstrated. The result of these
pharmacological studies indicates that 21a is a potent, selective,
and orally available inhibitor of DPP-IV.
Experimental Section
All commercial chemicals and solvents are reagent grade and
1
were used without further treatment unless otherwise noted. H
NMR spectra were obtained with a Varian Mercury-300 spectrom-
eter operating at 300 MHz. Chemical shifts were recorded in parts
per million (ppm, δ) and were reported relative to the solvent peak
of TMS. High-resolution mass spectra (HRMS) were measured with
a Finnigan (MAT-95XL) electron impact (EI) mass spectrometer.
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 irradiation (254 nm) or by spraying with phosphomo-
lybdic acid reagent (Aldrich) followed by heating at 80 °C. All
starting materials and amines were commercially available unless
otherwise indicated. The purity of compounds was determined by
an Agilent 1100 series HPLC system. HPLC retention times were
reported using a Chromolith performance RP-18e 4.6 mm × 100
mm column with two elution conditions. Condition A was 40:60
MeOH-H2O and condition B was 70:30 MeOH-H2O. The flow
rate was 0.2 mL/min and the injection volume was 5 µL. The system
operated at 25 °C. Peaks were detected at 210 nm.
General Procedure for DCC Coupling Reaction for Com-
pounds 10-14. A solution of BOC-protected carboxyl compounds
5-9 (1.0 equiv) and HOSu (1.0 equiv) in 20 mL of CH2Cl2/1,4-
dioxane (2/1) was cooled in an ice-water bath. To this solution
was added DCC (1.1 equiv) and the mixture stirred at room
temperature for 1 h. To the resulting solution was added the
appropriate amine (1,2,3,4-tetrahydroisoquinoline derivatives, ben-
zylamine derivatives, phenylethylamine derivatives, piperazine
derivatives, 1.5 equiv). After 4 h of stirring at room temperature,
the precipitate was removed by filtration and washed by ethyl
acetate (EA). The filtrate and washings were combined and washed
sequentially with 10% aqueous citric acid and saturated aqueous
NaHCO3. The organic layer was dried over MgSO4 and concen-
trated in vacuo. Purification by flash column chromatography
(eluted with hexane/CH2Cl2/EA ) 4/5/1) yielded the desired
compounds 10-14 (>70%) as a foam.
General Procedure for the Synthesis of Compounds 17-21.
A solution of compound 10-14 (3 equiv) in cool TFA was stirred
at room temperature for 10 min and concentrated in vacuo for 3 h.
The resultant yellowish oil was dissolved in THF, K2CO3 (9 equiv)
was added, and the mixture stirred for a period of 3 h to liberate
free amine. The solution was diluted with EA and filtered to remove
potassium salts. The filtrate was concentrated in vacuo so as to
obtain about 0.5 M free amine solution. To this solution was added
bromide compound 16 (0.4 equiv), and the reaction mixture was
stirred overnight at room temperature. The mixture was diluted with
CH2Cl2, washed with saturated aqueous NaHCO3, dried over
MgSO4, and concentrated in vacuo. Purification by flash column
chromatography (eluted with CH2Cl2/MeOH ) 97/3-95/5) yielded
desired compounds 17-21 (30-45%) as light yellow gums.
2-[2-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
ethyl]-1,2,3,4-tetrahydroisoquinoline (17): 1H NMR (CDCl3)
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-1,3-dihydroisoindole (18d): 1H NMR (CDCl3) δ 2.05-
2.31 (m, 4H), 2.62-2.66 (m, 2H), 3.04-3.08 (m, 2H), 3.43-3.50
(m, 3H, overlapped singlet at 3.50, ArCH2NCH2, CHCN), 3.59-
3.65 (m, 1H), 4.75-4.83 (m, 5H, two overlapped singlet at 4.79
and 4.83), 7.28-7.30 (m, 4H); HRMS (EI) m/z calcd for C18H22N4O2
326.1743, found 326.1744.
1-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
1
propyl]-4-[3,5-difluorobenzoyl]piperazine (18e): H NMR (CDCl3)
δ 2.10-2.20 (m, 2H), 2.21-2.30 (m, 2H), 2.60 (bs, 2H), 2.85-
2.95 (bm, 2H), 3.36-3.55 (m, 3H), 3.56-3.88 (m, 9H), 4.77-
4.80 (m, 1H, CHCN), 6.88-6.96 (m, 3H); HRMS (EI) m/z calcd
for C21H25F2N5O3 433.1925, found 433.1922.
1-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-4-[4-[1-oxoethylamino]phenylsulfonyl]piperazine
(18f): 1H NMR (CD3OD) δ 2.11-2.23 (m, 7H, overlapped singlet
of NHC(O)CH3 at 2.13), 2.57 (t, 2H, J ) 6.6 Hz), 2.88 (t, 2H, J )
6.6 Hz), 2.96 (bt, 2H, J ) 4.8 Hz), 3.01 (bt, 2H, J ) 4.8 Hz),
3.41-3.67 (m, 8H, overlapped doublet at 3.49, J ) 6.0 Hz), 4.71
(t, 1H, J ) 5.4 Hz), 7.69-7.73 (m, 2H), 7.79-7.83 (m, 2H); MS
(ES+) m/z 491.4 (M + H)+, 513.3 (M + Na)+.
1-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
propyl]-4-[benzothiazol-2-yl]piperazine (18h): 1H NMR (CDCl3)
δ 2.09-2.36 (m, 4H), 2.62 (t, 2H, J ) 6.3 Hz), 2.97-3.04 (m,
2H), 3.37-3.52 (m, 3H, overlapped singlet at 3.47), 3.53-3.81
(m, 9H), 4.74-4.78 (m, 1H), 7.09-7.14 (m, 1H), 7.29-7.35 (m,
1H), 7.56-7.64 (m, 2H); HRMS (EI) m/z calcd for C21H26N6O2S
426.1838, found 426.1841.
N-Benzyl-3-[2-(2-cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-
propionamide (18i): 1H NMR (CDCl3) (4/1 mixture of trans/cis
amide rotomers) δ 2.05-2.32 (m, 4H), 2.45 (t, 2H, J ) 6.0 Hz),
2.95 (t, 2H, J ) 6.0 Hz), 3.31-3.57 (m, 4H, overlapped doublet at
3.38, J ) 3.3 Hz), 4.45 (d, 2H, J ) 5.7 Hz), 4.64 (dd, 1/5H, J )
7.5 and 1.5 Hz, CHCN), 4.70-4.73 (m, 4/5H, CHCN), 7.22-7.36
(m, 5H), 7.56 (bs, 1/5H, ArCH2NH), 7.74 (bs, 4/5H, ArCH2NH);
HRMS (EI) m/z calcd for C17H22N4O2 314.1743, found 314.1741.

378 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsu et al.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-(4-ni-
trobenzyl)propionamide (18j): 1H NMR (CDCl3) (4/1 mixture
of trans/cis amide rotomers) δ 2.05-2.35 (m, 4H), 2.50 (t, 2H, J
) 6.0 Hz), 3.00 (t, 2H, J ) 6.0 Hz), 3.30-3.70 (m, 4H, overlapped
doublet at 3.44, J ) 7.5 Hz), 4.44-4.60 (m, 2.2H, overlapped
doublet at 4.54, J ) 7.5 Hz, 2H ArCH2N and 0.2H CHCN), 4.68-
4.76 (m, 0.8H, CHCN), 7.50 (d, 2H, J ) 12.0 Hz), 8.16 (d, 2H, J
) 12.0 Hz), 8.28 (bt, 1/5H, ArCH2NH), 8.40 (bt, 4/5H, ArCH2NH);
HRMS (EI) m/z calcd for C17H21N5O4 359.1594, found 359.1594.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-(1-
phenylpropyl)propionamide (18k): 1H NMR (CDCl3) (9/1 mix-
ture of trans/cis amide rotomers) δ 0.88 (t, 3H, J ) 7.5 Hz), 1.74-
1.90 (m, 2H), 2.02-2.31 (bm, 4H), 2.37-2.49 (m, 2H), 2.86-
3.01 (m, 2H), 3.32-3.62 (m, 4H), 4.62 (d, 1/10H, J ) 7.5 Hz,
CHCN) 4.73-4.77 (m, 9/10H, CHCN), 4.89 (q, 1H, J ) 7.5 Hz),
7.22-7.40 (m, 5H), 7.81 (bt, 1H); HRMS (EI) m/z calcd for
C19H26N4O2 342.2056, found 342.2060.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-(3,5-
difluorobenzyl)propionamide (18l): 1H NMR (CDCl3) (9/1
mixture of trans/cis amide rotomers) δ 2.14-2.35 (m, 4H), 2.49
(t, 2H, J ) 5.7 Hz), 2.98 (t, 2H, J ) 6.0 Hz), 3.35-3.66 (m, 4H,
overlapped doublet at 3.44, J ) 4.2 Hz), 4.37-4.51 (m, 2H), 4.64
(d, 1/10H, J ) 7.5 Hz, CHCN) 4.73-4.76 (m, 9/10H, CHCN),
6.64-6.71 (m, 1H), 6.81-6.87 (m, 2H), 8.08 (bs, 1/10H, ArCH2NH),
8.19 (bt, 9/10H, ArCH2NH); HRMS (EI) m/z calcd for C17H20F2N4O2
350.1554, found 350.1550.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-(2-
methyl-1-phenylpropyl)propionamide (18m). A solution of iso-
propyl phenyl ketone (296 mg, 2 mmol), ammoniumacetate (1.54
g, 20 mmol), and NaBH3CN (251 mg, 4 mmol) in MeOH (30 mL)
was refluxed for 1 h. The resulting solution was cooled to room
temperature and ice-cold 6 N HCl was added until pH ∼ 2. The
methanol was evaporated in vacuo and the remaining aqueous
solution washed with CH2Cl2. Solid KOH was added to the aqueous
solution until pH ∼ 12, and the aqueous solution was extracted
with CH2Cl2. The organic layer was dried over MgSO4 and
concentrated in vacuo to yield crude amine. The crude 2-methyl-
1-phenyl-1-propylamine was used without further purification in
the next step. DCC coupling with BOC-protected carboxyl acid,
followed by TFA deprotection and then amine coupling with
bromide compound 16, was done according to the described general
procedure to give 18m: 1H NMR (CDCl3) (4/1 mixture of trans/
cis amide rotomers) δ 0.83 (d, 3H, J ) 6.6 Hz), 0.94 (d, 3H, J )
6.6 Hz), 1.99-2.32 (m, 5H), 2.34-2.41 (m, 2H), 2.89-2.96 (m,
2H), 3.32-3.59 (m, 4H), 4.50 (d, 0.2H, J ) 7.5 Hz, CHCN) 4.70-
4.80 (m, 1.8H, 1H ArCH2N and 0.8H CHCN), 7.22-7.34 (m, 5H),
8.14 (bs, 1H); HRMS (EI) m/z calcd for C20H28N4O2 356.2212,
found 356.2210.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-(1-
methyl-1-phenylethyl)propionamide (18n): 1H NMR (CDCl3)
(9/1 mixture of trans/cis amide rotomers) δ 1.68 (s, 6H), 2.05-
2.31 (m, 4H), 2.38-2.46 (m, 2H), 2.92-2.98 (m, 2H), 3.31-3.44
(m, 1H), 3.45-3.65 (m, 3H, overlapped singlet at 3.48), 4.67 (dd,
1/10H, J ) 7.5 and 2.1 Hz, CHCN) 4.73-4.75 (m, 9/10H, CHCN),
7.17-7.45 (m, 6H); HRMS (EI) m/z calcd for C19H26N4O2
342.2056, found 342.2057; HPLC (condition A) tR ) 10.32 min,
98.6%, (condition B) tR ) 6.67 min, 96.7%.
4.74-4.77 (m, 9/10H, CHCN), 7.07 (t, 1H, J ) 7.4 Hz), 7.30 (t,
2H, J ) 8.1 Hz), 7.61 (d, 2H, J ) 7.8 Hz), 9.86 (s, 1/10H, ArNH),
9.99 (s, 9/10H, ArNH); HRMS (EI) m/z calcd for C16H20N4O2
300.1586, found 300.1586.
2-[4-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
butyl]-1,2,3,4-tetrahydroisoquinoline (19): 1H NMR (CDCl3) δ
1.86-1.91 (m, 2H), 2.05-2.32 (m, 4H), 2.48-2.55 (m, 2H), 2.69-
2.75 (m, 2H), 2.85 (t, 1H, J ) 6.0 Hz), 2.91 (t, 1H, J ) 6.0 Hz),
3.37-3.61 (m, 4H, overlapped doublet at 3.39, J ) 11.1 Hz), 3.70
(t, 1H, J ) 6.0 Hz), 3.83 (t, 1H, J ) 6.0 Hz), 4.66-4.77 (m, 3H,
two overlapped singlets at 4.66 and 4.73), 7.10-7.22 (m, 4H);
HRMS (EI) m/z calcd for C20H26N4O2 354.2056, found 354.2059.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-1-oxo-
butyl]-1,2,3,4-tetrahydroisoquinoline (20a): 1H NMR (CDCl3)
δ 1.20 (d, 3H, J ) 8.4 Hz), 2.07-2.32 (m, 4H), 2.41-2.49 (m,
1H), 2.59-2.68 (m, 1H), 2.85 (t, 1H, J ) 6.0 Hz), 2.92 (t, 1H, J
) 6.0 Hz), 3.24-3.31 (m, 1H), 3.36-3.58 (m, 4H), 3.61-3.73
(m, 1H), 3.75-3.86 (m, 1H), 4.65-4.77 (m, 3H, overlapped doublet
at 4.65, J ) 3.9 Hz and a singlet at 4.74, ArCH2N and CHCN),
7.10-7.23 (m, 4H); HRMS (EI) m/z calcd for C20H26N4O2
354.2056, found 354.2057; HPLC (condition A) tR ) 10.07 min,
99.2%, (condition B) tR ) 6.68 min, 98.6%.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-4-meth-
yl-1-oxopentyl]-1,2,3,4-tetrahydroisoquinoline (20b): 1H NMR
(CDCl3) δ 0.93-0.99 (m, 5H), 1.90-2.00 (m, 1H), 2.01-2.27 (m,
4H), 2.40-2.52 (m, 2H), 2.86-3.07 (m, 3H, three overlapped
multiplets), 3.25-3.89 (m, 6H), 4.49-4.86 (m, 3H), 7.14-7.18 (m,
4H); HRMS (EI) m/z calcd for C22H30N4O2 382.2369, found
382.2369.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-3-meth-
yl-1-oxobutyl]-1,2,3,4-tetrahydroisoquinoline (21a): 1H NMR
(CDCl3) (4/1 mixture of trans/cis amide rotomers) δ 1.23 (s, 3H),
1.25 (s, 3H), 2.05-2.33 (m, 4H), 2.53 (s, 2H), 2.85 (t, 1H, J ) 6.0
Hz), 2.91 (t, 1H, J ) 6.0 Hz), 3.38-3.48 (m, 3H, overlapped
doublet at 3.39, J ) 7.5 Hz), 3.53-3.64 (m, 1H) 3.73 (t, 1H, J )
6.0 Hz), 3.83 (t, 1H, J ) 6.0 Hz), 4.66-4.76 (m, 2.8H, two
overlapped singlets at 4.67 and 4.73, 2H ArCH2N and 0.8H CHCN),
5.10 (d, 0.2H, J ) 7.5 Hz, CHCN), 7.09-7.22 (m, 4H); HRMS
(EI) m/z calcd for C21H28N4O2 368.2212, found 368.2215; HPLC
(condition A) tR ) 9.69 min, 99.8%, (condition B) tR ) 6.74 min,
98.2%.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-3-meth-
yl-1-oxobutyl]-6-fluoro-1,2,3,4-tetrahydroisoquinoline (21b). The
synthesis of 6-fluoro-1,2,3,4-tetrahydroisoquinoline was carried out
as described in ref 20: 1H NMR (CDCl3) (4/1 mixture of trans/cis
amide rotomers) δ 1.22 (s, 3H), 1.25 (s, 3H), 2.02-2.28 (m, 4H),
2.53 (s, 2H), 2.83 (t, 1H, J ) 6.0 Hz), 2.89 (t, 1H, J ) 6.0 Hz),
3.40-3.53 (m, 3H, overlapped doublet at 3.41, J ) 5.4 Hz), 3.58-
3.67 (m, 1H), 3.72 (t, 1H, J ) 6.0 Hz), 3.80 (t, 1H, J ) 6.0 Hz),
4.64-4.76 (m, 2.8H, two overlapped singlets at 4.64 and 4.68, 2H
ArCH2N and 0.8H CHCN), 5.07 (d, 0.2H, J ) 7.5 Hz, CHCN),
6.84-6.93 (m, 2H), 7.06-7.12 (m, 1H); HRMS (EI) m/z calcd for
C21H27FN4O2 386.2118, found 386.2116; HPLC (condition A) tR
) 10.40 min, 100.0%, (condition B) tR ) 6.97 min, 99.5%.
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-3-meth-
yl-1-oxobutyl]-6,8-difluoro-1,2,3,4-tetrahydroisoquinoline (21c).
1H NMR (CDCl3) (4/1 mixture of trans/cis amide rotomers) δ 1.23
(s, 3H), 1.26 (s, 3H), 2.05-2.37 (m, 4H), 2.54 (s, 2H), 2.83 (t, 1H,
J ) 6.0 Hz), 2.89 (t, 1H, J ) 5.7 Hz), 3.43-3.55 (m, 3H,
overlapped doublet at 3.43, J ) 3.3 Hz), 3.60-3.64 (m, 1H), 3.73
(t, 1H, J ) 6.0 Hz), 3.83 (t, 1H, J ) 6.0 Hz), 4.61 (s, 1H), 4.70 (s,
1H), 4.74-4.77 (m, 4/5H, CHCN), 5.04-5.09 (m, 1/5H, CHCN),
6.65-6.72 (m, 2H); HRMS (EI) m/z calcd for C21H26F2N4O2
404.2024, found 404.2025; HPLC (condition A) tR ) 11.16 min,
99.4%, (condition B) tR ) 6.67 min, 99.1%.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-phen-
ethylpropionamide (18o): 1H NMR (CDCl3) (4/1 mixture of trans/
cis amide rotomers) δ 2.05-2.35 (m, 6H, overlapped triplet at 2.31,
J ) 6.0 Hz), 2.75-2.83 (m, 4H), 3.17-3.35 (m, 3H, overlapped
doublet at 3.23, J ) 3.0 Hz), 3.42-3.51 (m, 3H), 4.60 (dd, 1/5H,
J ) 7.5 and 1.8 Hz, CHCN), 4.69-4.73 (m, 4/5H, CHCN), 7.16-
7.27 (m, 5H), 7.29 (bs, 1/5H, ArCH2CH2NH), 7.52 (bs, 4/5H,
ArCH2CH2NH); HRMS (EI) m/z calcd for C18H24N4O2 328.1899,
found 328.1904.
3-[2-(2-Cyano-(S)-pyrrolidin-1-yl)-2-oxoethylamino]-N-phen-
ylpropionamide (18p): 1H NMR (CDCl3) (9/1 mixture of trans/
cis amide rotomers) δ 2.03-2.28 (m, 4H), 2.56 (t, 2H, J ) 6.0
Hz), 3.04 (t, 2H, J ) 6.0 Hz), 3.34-3.75 (m, 4H, overlapped
doublet at 3.48, J ) 4.2 Hz), 4.63 (d, 1/10H, J ) 5.4 Hz, CHCN),
2-[3-[2-[2-Cyano-(S)-pyrrolidin-1-yl]-2-oxoethylamino]-3-meth-
yl-1-oxobutyl]-1,3-dihydroisoindole (21d): 1H NMR (CDCl3) (7/1
mixture of trans/cis amide rotomers) δ 1.28 (s, 6H), 2.05-2.35
(m, 4H), 2.51 (s, 2H), 3.44-3.57 (m, 3H, overlapped singlet at
3.45), 3.62-3.68 (m, 1H), 4.76-4.80 (m, 2.7H, overlapped singlet
at 4.80, 2H ArCH2N and 0.7H CHCN), 4.85 (s, 2H, ArCH2N), 5.13

Dipeptide-DeriVed Inhibitor of DPP-IV
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 379
(2) (a) Drucker, D. J. Minireview: The glucagon-like peptides. Endo-
crinology 2001, 142, 521-527. (b) Ross, S. A.; Gulve, E. A.; Wang,
M. Chemistry and biochemistry of type 2 diabetes. Chem. ReV. 2004,
104, 1255-1282.
(dd, 0.3H, J ) 7.8 and 1.5 Hz, CHCN), 7.24-7.31 (m, 4H); HRMS
(EI) m/z calcd for C20H26N4O2 354.2056, found 354.2058; HPLC
(condition A) tR ) 9.67 min, 100.0%, (condition B) tR ) 6.75 min,
97.9%.
3-[2-(2-Cyanopyrrolidin-1-yl)-2-oxo-ethylamino]-3-methyl-N-
(1-methyl-1-phenylethyl)butyramide (21e): 1H NMR (CDCl3)
(10/1 mixture of trans/cis amide rotomers) δ 1.19 (s, 6H), 1.67 (s,
6H), 2.11-2.31 (m, 6H, overlapped singlet at 2.25), 3.37-3.43
(m, 3H, overlapped singlet at 3.37), 3.54-3.60 (m, 1H), 4.72-
4.75 (m, 1H), 7.16-7.21 (m, 1H), 7.26-7.32 (m, 2H), 7.36-7.40
(m, 2H), 8.12 (bs, 1/10H, ArC(CH3)2NH), 8.50 (bs, 9/10H, ArC-
(CH3)2NH); HRMS (EI) m/z calcd for C21H30N4O2 370.2369, found
370.2370.
Inhibition of DPP-IV, DPP8, and DPP-II in Vitro.15a,22 IC50
determination was done as described in the literature with the
modifications described below.15a A sigma plot was used to obtain
the IC50 values. DPP-IV and DPP8 were purified as described in
our previously published method.22 The purification of DPP-II was
carried out as described in ref 15a with modifications. The buffer
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 and 2500 µM for DPP-IV
and DPP8 assays, respectively. DPP-II activity was assayed by 1.5
mM Gly-Pro-pNA in 50 mM potassium phosphate buffer, pH 5.5.
The concentration of DPP-II is 10 nM. Reaction developed at 37
°C and OD405 was monitored.
Oral Glucose Tolerance Test in Wistar Rats. Adult male
Wistar rats were fasted overnight. Blood samples were obtained
from the tail veins with 27G needles, and the blood glucose was
measured with the Accu-Chek Compact System from Roche (Basel,
Switzerland). Animals were grouped according to the glucose levels
and then orally gavaged with the test compounds dissolved in
distilled water at a dose indicated in Figure 3. Thirty minutes after
the oral dosing of test compounds, the animals were orally gavaged
with freshly prepared glucose solution of 400 mg/mL in distilled
water at 1.5 g glucose/kg. Blood glucose levels of these dosed
animals were monitored at 0, 15, 30, 60, and 120 min after the
oral glucose challenge.
DPP-IV Inhibition in Mice.23 Adult male BALB/c mice were
orally gavaged with the test compounds dissolved in 0.5% methyl
cellulose at a single dose of 18 mg/kg. Blood samples of 25-50
µL were collected from the tail veins at the time points indicated
in Figure 4, 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; BACHEM). Plasma (10 µL) was mixed with
140 µL of 150 µM Gly-Pro-AMC in 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 1 µM
products per minute.
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Acknowledgment. The study was financially supported by
National Health Research Institutes, Taiwan.
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Supporting Information Available: 1H NMR spectra and
HPLC purity analysis for 18a, 18n, 20a, 21a, 21b, 21c, and 21d.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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