Synthesis and structure-activity relationships of potent 3- or 4-substituted-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.09.010

A series of 3- or 4-substituted-2-cyanopyrrolidines were synthesized and evaluated as dipeptidyl peptidase IV inhibitors. Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treatment of type 2 diabetes because they prevent degradation of glucagon-like peptide-1 (GLP-1) and extend its duration of action. A series of 2-cyanopyrrolidines are among the most potent of DPP-IV inhibitors. We focused our attention on substitutions at the 3- or 4-position of 2-cyanopyrrolidines and synthesized and evaluated various derivatives. Among them, the 4-fluoro derivative was found to exhibit better DPP-IV inhibitory activity and higher plasma drug concentrations after oral administration to rats than the 4-unsubstituted derivative. We report here on the synthesis and biological data of the aforementioned derivatives.

Full text of DOI:10.1016/j.bmc.2004.09.010

Bioorganic & Medicinal Chemistry 12 (2004) 6053–6061
Synthesis and structure–activity relationships of
potent 3- or 4-substituted-2-cyanopyrrolidine
dipeptidyl peptidase IV inhibitors
Hiroshi Fukushima,* Akira Hiratate, Masato Takahashi, Masako Saito, Eiji Munetomo,
Kiyokazu Kitano, Hidetaka Saito, Yuji Takaoka and Koji Yamamoto
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
Received 30 June 2004; revised 9 September 2004; accepted 9 September 2004
Available online 29 September 2004
Abstract—Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treatment of type 2
diabetes because they prevent degradation of glucagon-like peptide-1 (GLP-1) and extend its duration of action. A series of 2-cyano-
pyrrolidines are among the most potent of DPP-IV inhibitors. We focused our attention on substitutions at the 3- or 4-position of
2-cyanopyrrolidines and synthesized and evaluated various derivatives. Among them, the 4-fluoro derivative was found to exhibit
better DPP-IV inhibitory activity and higher plasma drug concentrations after oral administration to rats than the 4-unsubstituted
derivative. We report here on the synthesis and biological data of the aforementioned derivatives.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Many DPP-IV inhibitors are shaped like dipeptides,
which contain a basic nitrogen and a proline mimic.
Among proline mimics, 2-cyanopyrrolidine is one of
the most potent moieties.7
Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5, CD26)1
is a highly specific serine protease that cleaves N-termi-
nal dipeptides from polypeptides with L-proline or L-ala-
nine at the penultimate position.2 The natural substrates
of DPP-IV have been described, and it is recognized that
inhibition of DPP-IV is of potential therapeutic use in
modulation of glucagon-like peptide processing and in
attenuation of the immune response.3 The role of
DPP-IV in glucagon-like peptide (GLP) processing is
well known.4 Given the importance of the regulation
of this process, DPP-IV inhibition has an obvious appli-
cation to the treatment of diabetes.5 Since the blood glu-
cose-lowering effects of GLP-1 are dependent on
elevated blood glucose and abate as glucose levels return
to normal, the incidence of hypoglycemia during treat-
ment with a DPP-IV inhibitor is expected to be very
low.6
We focused on substitutions of 2-cyanopyrrolidine,
because the affinity between DPP-IV and its inhibitors
appeared to be increased by hydrogen bonding or
hydrophobic interaction or by change in the conforma-
tion around the pyrrolidine ring of the inhibitor. Since
DPP-IV specifically recognizes a proline residue at the
P1 site, we suspected that there would be affinity be-
tween DPP-IV and the pyrrolidine ring. We therefore
attempted to introduce substitutions at the 3- or 4-posi-
tion on the pyrrolidine ring to obtain more potent
inhibition.
2. Chemistry
Carboxylic acids 2a–h, which were commercially availa-
ble or prepared by methylation8 or fluorination9 of
hydroxyprolines and difluorination of 4-oxoproline,
were coupled with ammonia using 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDC) and
1-hydroxybenztriazole (HOBt) as coupling reagents
Keywords: Dipeptidyl peptidase IV; Inhibitor; Fluoropyrrolidine;
Diabetes.
*
Corresponding author. Tel.: +81 (0)48 669 3029; fax: +81 (0)48 652
7254; e-mail: hiroshi.fukushima@po.rd.taisho.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.010

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H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
R1
R1
a
b
N
N
Boc
Boc
CO2H
CONH2
a;R1=(4R)-OH b;R1=(4S)-OH c;R1=(3S)-OH
2
3a-h
d;R1=(4R)-OMe e;R1=(4S)-OMe
f;R1=(4R)-F g;R1=(4S)-F h;4,4-diF
H
H
R1
R1
d
N
N
Fmoc N
H
N
H
e
Fmoc
H
c
R1
R1
O
O
CONH2
CN
HN
( HX )
N
5a-g
6a-g
H2N
( HX )
CONH2
f
O
CN
H
H
h
4a-h
R1
R1
1a-h
g
N
N
Boc N
H
N
Boc
H
O
O
CONH2
CN
8h
7h
Scheme 1. Reagents: (a) EDC, HOBt, NH3, DMF or CH3CN; (b) 4M HCl/AcOEt or 4M HCl/1,4-dioxane; (c) Fmoc-L-isoleucine, EDC, HOBt,
N,N-diisopropylethylamine, DMF or CH3CN; (d) (CF3CO)2O, THF; (e) Et2NH, 1,2-dichloroethane; then HCl; (f) Boc-L-isoleucine, EDC, HOBt,
N,N-diisopropylethylamine, THF–DMF; (g) cyanuric chloride, DMF; (h) 2M HCl.
(Scheme 1). Acid-catalyzed cleavage of the N-Boc-pro-
tecting group in 3a–h then yielded the amines 4a–h.
coupling reagents to yield the dipeptides 5a–g or 7h
quantitatively.
Isoleucine was selected for use at the P2 site because it
is one of the most potent P2 site residues among
natural amino acids.7 It was possible to use either the
fluorenylmethoxycarbonyl (Fmoc) group or the t-but-
oxycarbonyl (Boc) group as the N-protecting group of
isoleucine. Compounds 4a–h were coupled with Fmoc-
L-isoleucine or Boc-L-isoleucine using EDC/HOBt as
Fmoc-protected prolinamides 5a–g were treated with tri-
fluoroacetic anhydride to obtain 2-cyanopyrrolidines
6a–g and Boc derivative 7h was dehydrated with cyan-
uric chloride in dimethylformamide10 to yield cyano
derivative 8h. The dehydrating reaction was performed
in mild acidic conditions in order to avoid deprotection
of the N-Boc-protecting group. Removal of the N-Fmoc
H
H
H
a
b
N
N
N
OH
F
F
Boc N
H
Boc N
H
H2N
CF3CO2H
O
O
O
CN
CN
CN
8i
1i
8c
H
H
H
Cl
Cl
OH
c
d
N
N
N
Fmoc N
H
H2N
HCl
Fmoc N
H
O
O
CN
CN
O
CN
6a
6j
1j
H
H
H
H
O
O
O
OH
b
e
f
N
N
N
N
Boc N
H
Boc N
H
Boc N
H
H2N
CF3CO2H
O
O
O
CONH2
CN
CONH2
O
CN
7k
8k
7a
1k
Scheme 2. Reagents: (a) DAST, CH2Cl2; (b) TFA; (c) Ph3P, CCl4; (d) Et2NH, 1,2-dichloroethane; then HCl; (e) PCC, MS4A, AcOH, CH2Cl2; (f)
(CF3CO)2O, N,N-diisopropylethylamine, THF.

H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
6055
Table 1. DPP-IV inhibitory activity
group with diethylamine or the N-Boc group with acids,
and in some cases producing salts with hydrochloric
acid, yielded 1a–h.
H
R
H2N
( HX )
(3R)-Fluoro derivative 8i was obtained via fluorination
of (3S)-hydroxy derivative 8c with (diethylamino)sulfur
trifluoride (DAST) and the N-Boc group of 8i was
deprotected using trifluoroacetic acid (TFA) to obtain
1i (Scheme 2). (4R)-Hydroxy derivatives 6a and 7a were
converted to the corresponding 4(S)-chloride 6j using
Ph3P/CCl4 and to 4-oxo derivative 7k using pyridinium
chlorochromate (PCC). Compound 6j was deprotected
using diethylamine and treated with hydrochloric acid
to yield hydrochloric acid salt 1j. Compound 7k was
treated with trifluoroacetic anhydride and N,N-diisoprop-
ylethylamine to yield 8k quantitatively without produc-
ing a Boc-deprotection product. The N-Boc group of 8k
was then deprotected using TFA to obtain 1k.
O
Compd
R
IC50 (nM)
OH
N
1a
5700
370
CN
OH
N
1b
CN
N
1c
1d
>10,000
>30,000
OH
CN
OMe
It is well known that N-acylproline derivatives exist as
mixtures of cis- and trans-amide rotamers in solution.
It has been reported that N-acyl-2-cyanopyrrolidines ex-
ist as mixtures of rotamers as well.11 NMR revealed that
some compounds among 1a–k occurred as mixtures of
conformers in solution. The protons at the 2- and 4-
position of the pyrrolidine ring of 1e and 1g tended to
appear as separate peaks on the NMR spectra. The pro-
tons of the compounds for which the minor conformers
were clearly observed are described in the NMR data.
N
CN
OMe
N
1e
1f
850
290
CN
F
(R)
N
CN
F
(S)
N
1g
0.6
3. Results and discussion
CN
The hydroxy compounds 1a–c and the methoxy com-
pounds 1d and 1e were all several orders of magnitude
less potent than the non-substituted analogue 9.7 Stereo-
chemically, the 4S derivatives 1b and 1e were more
potent than the 4R derivatives 1a and 1d (Table 1).
Introduction of a ketone to the 4-position slightly de-
creased inhibitory potency (1k, IC50 = 21nM).
F
F
N
1h
0.8
CN
N
1i
1j
65
F
CN
Cl
The (4S)-fluoride analogue 1g had a very potent inhibi-
tory effect (IC50 = 0.6nM) and was one of the most
potent DPP-IV inhibitors found. The 4,4-difluoride
analogue 1h also had a potent inhibitory effect
(IC50 = 0.8nM). However, the (4R)-fluoride analogue
1f had 480-fold less potency (IC50 = 290nM) than 1g.
N
23
21
CN
O
N
1k
9
CN
The (3R)-fluoride analogue 1i and the (4S)-chloride ana-
logue 1j demonstrated slightly decreases in inhibitory
potency.
N
1.5
CN
The introduction of substituents to the 3- or 4-position
on the pyrrolidine ring did not lead to increased potency
with the exception of the incorporation of (4S)-fluoro
substitution. The narrow space of the pyrrolidine-bind-
ing site of DPP-IV may not permit substituents greater
in size than fluorine. It is unclear why the introduction
of (4S)-fluorine augmented DPP-IV inhibitory activity.
However, the 4S position is surrounded by hydrogen
atoms from Tyr and Trp that have slight positive
charges, while the 4R position is adjacent to the p-cloud
of another Tyr that has a slight negative charge, which
might have caused the variation in potency seen with
incorporation of fluorine at the 4-position. Reports on
X-ray crystallographic analysis of DPP-IV12 were used
to aid in speculation regarding bonding patterns.
Introduction of a fluorine atom affected the drug plasma
concentration present after oral administration. Follow-
ing oral administration of 1mg/kg to rats, the Cmax for
1g (372ng/mL at 10min) was 2.5-fold that for 9
(147ng/mL at 10min) (Fig. 1). The reason for the

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H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
level might stimulate insulin secretion by acting upon
b-cells in the pancreas, resulting in suppression of hyper-
glycemia after glucose loading.
400
300
200
100
0
1g
9
4. Conclusion
We have designed and synthesized a series of 3- or 4-
substituted-2-cyanopyrrolidines as DPP-IV inhibitors.
Among these compounds, the (4S)-fluoro derivative 1g
exhibited greater DPP-IV inhibitory activity and higher
plasma drug concentrations after oral administration to
rats than the 4-unsubstituted derivative 9. Based on
these pharmacology efficacy data, the 4-fluoro derivative
of 2-cyanopyrrolidine and potent DPP-IV inhibitor 1g is
expected to have potential as a therapeutic agent for use
in lowering postprandial hyperglycemia and treatment
of type 2 diabetes mellitus. Subsequent reports will de-
scribe the results of further investigation of 4-fluoro-2-
cyanopyrrolidines.
0
60
Time after administration (min)
120
Figure 1. Plasma drug concentrations after oral administration of 1g
or 9 at a dose of 1mg/kg to Wistar rats.
increase in Cmax with the introduction of fluorine was
not clear, but may have been the increase in absorption
and reduction of distribution volume.
The effect of 1g on glucose tolerance was examined in
Zucker fatty rats, a model of obesity and impaired glu-
cose tolerance. Oral administration of 1g at the doses of
1 and 4mg/kg reduced the increase in plasma glucose
beginning 30min after glucose loading (Fig. 2A) and
at 4mg/kg significantly suppressed hyperglycemia (Fig.
2B). DPP-IV activity was almost completely inhibited
at 15min after glucose loading at both doses, with the
inhibitory effect gradually declining beginning 90min
after glucose loading in the 1mg/kg dose group (Fig.
2D). These results indicate that the efficacy of 1g on
hyperglycemia is based on its inhibitory effect on plasma
DPP-IV activity. Insulin secretion was significantly en-
hanced in the 1 and 4mg/kg dose groups (Fig. 2C). This
finding provides support for the proposed mechanism
that 1g might prevent the inactivation of active GLP-1
via DPP-IV inhibition and an increased active GLP-1
5. Experimental
5.1. Chemistry
1H NMR spectroscopy was performed using a Varian
VXR-300 or JEOL GX500 spectrometer. Chemical
shifts are reported in parts per million relative to tetra-
methylsilane as an internal standard (in NMR descrip-
tions s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, and br = broad peak). 13C NMR spec-
troscopy was performed using a JEOL GX500 spectro-
meter. 19F NMR spectroscopy was performed
using a Varian VXR-300 spectrometer. ESI Mass
350
20000
300
250
*
15000
##
200
10000
150
100
5000
0
50
0
0
30
60
90
120
Vehicle
1 mg/kg 4 mg/kg
Lean
Time after glucose administration (min)
(A)
(B)
Vehicle
1 mg/kg
4 mg/kg
Lean
***
***
30
25
20
0.25
0.20
15
10
5
0.15
0.10
0.05
0
0
0
30
60
90
120
Vehicle
1 mg/kg
4 mg/kg
(C)
(D)
Time after glucose administration (min)
Figure 2. Effects of oral administration of 1g (1 or 4mg/kg) on plasma glucose, plasma insulin and plasma DPP-IV activity during OGTT in Zucker
fatty rats. Each point represents the mean SE (n = 6). *p < 0.05, ***p < 0.001 versus Vehicle, Dunnettꢀs test. ##p < 0.01 versus Vehicle, Studentꢀs t-
test.

H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
6057
spectra were recorded with a Shimadzu/Kratos HV-300.
Melting points were measured with a Buchi 535 melting
point apparatus without correction. Infrared spectra
were recorded with a Perkin–Elmer 1760spectrometer.
Elemental analyses were performed using a Perkin–
Elmer 240C (for carbon, hydrogen, and nitrogen) or
Yokokawa–Denki IC7000P (for halogens and sulfur).
F, 11.26; N, 16.46. IR (KBr) 3319, 3174, 3063, 2902,
2778, 2731, 1707, 1626, 1583, 1399, 1340, 1322, 1207,
26
1162, 1055, 1026, 965, 863, 635cmꢀ1. ½aꢁ ꢀ48.8 (c
D
0.5, MeOH).
5.1.3. N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-isoleucyl-
(4S)-4-fluoro-L-prolinamide (5g). In THF (40mL)–DMF
(10mL), 4g (2.4g, 14.2mmol), and N-[(9H-fluoren-9-
ylmethoxy)carbonyl]-L-isoleucine (5.1g, 14.4mmol)
were dissolved, and then 1-hydroxybenzotriazole
monohydrate (2.6g, 17.0mmol), 1-(3,3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (3.3g,
17.2mmol), and N,N-diisopropylethylamine (2.5mL)
were added with ice-cooling, and the temperature was
gradually increased, followed by stirring at room tem-
perature overnight. The solution was concentrated in
vacuo, and water was added to the resulting residue.
The resulting powder was collected by filtration and
purified by silica gel column chromatography (develop-
ing solvent; hexane–EtOAc = 4:1–1:4) to yield the
Analytical thin-layer chromatography was conducted on
precoated silica gel 60F254 plates (Merck). Chromato-
graphy was performed on 100–200mesh silica gel C-200
(Wako Pure Chemical) using the solvent systems (vol-
ume ratios) indicated below.
5.1.1. tert-Butyl (2S,4S)-2-(aminocarbonyl)-4-fluoropyrr-
olidine-1-carboxylate (3g). In acetonitrile (50mL), (4S)-
1-(tert-butoxycarbonyl)-4-fluoro-L-proline 2g9 (4.5g,
19.3mmol) was dissolved, and 1-hydroxybenzotriazole
monohydrate (3.6g, 23.5mmol) and 1-(3,3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (4.5g,
23.5mmol) were then added with ice-cooling. The tem-
perature was gradually increased, and the mixture was
then stirred at room temperature overnight. The reac-
tion solution was again ice-cooled, 25% aqueous ammo-
nia (5mL) was added, and stirring was continued with
ice-cooling for 30min and then at room temperature
for 30min. Acetonitrile (50mL) was added to the reac-
tion mixture, and an insoluble substance was removed
by filtration. The filtrate was concentrated in vacuo
and purified by silica gel column chromatography
(developing solvent; hexane–EtOAc = 4:1–1:5). Hexane
was added to the resulting residue to yield the desired
product (4.2g, 94%) as a colorless powder. Mp 169–
desired product (6.9g, quant) as
a pale yellow
amorphous substance. 1H NMR (300MHz, DMSO-
d6): d 7.89 (2H, d, J = 7.5Hz, aromatic H), 7.72 (2H,
d, J = 7.5Hz, aromatic H), 7.41 (2H, t like, J = 7.5Hz,
aromatic H), 7.32 (2H, t like, J = 7.5Hz, aromatic H),
7.05 (1H, br s, NH), 6.91 (1H, br s, NH), 5.31 (1H, br
d, J = 52.5Hz, H-4), 4.45 (1H, dd, J = 8.8, 2.2Hz, H-
2), 4.32–3.96 (5H, m), 3.89–3.74 (1H, dd, J = 25.6,
12.6Hz, H-5), 2.45–2.12 (2H, m, H-3), 1.88–1.76 (1H,
m), 1.61–1.49 (1H, m), 1.20–1.10 (1H, m), 0.95 (3H, d,
J = 6.7Hz, Me), 0.84 (3H, dd, J = 7.5, 7.3Hz, Me).
5.1.4. 9H-Fluoren-9-ylmethyl ((1S,2S)-1-{[(2S,4S)-2-cya-
no-4-fluoropyrrolidin-1-yl]carbonyl}-2-methylbutyl)carba-
mate (6g). In THF (70mL), 5g (6.9g, 14.8mmol) was
dissolved, and trifluoroacetic anhydride (4.0mL,
28.3mmol) was added with ice-cooling, followed by stir-
ring with ice-cooling for 1.5h. The reaction solution was
concentrated in vacuo, and the residue was purified by
silica gel column chromatography (developing solvent;
hexane–EtOAc = 8:1–3:2) to yield the desired product
1
170°C. H NMR (300MHz, DMSO-d6): d 7.21, 7.15,
and 6.95 (2H, each br, CONH2), 5.21 (1H, br d,
J = 54.0Hz, H-4), 4.13 (1H, d, J = 9.6Hz, H-2), 3.47–
3.68 (2H, m, H-5), 2.26–2.55 (1H, m, H-3), 2.09–2.22
(1H, m, H-3), 1.41 and 1.36 (9H, each s, (CH3)3CO).
MS (ESI pos.) m/z 255 ([M+Na]+). Anal. Calcd for
C10H17FN2O3: C, 51.71; H, 7.38; F, 8.18; N, 12.06.
Found: C, 51.72; H, 7.46; F, 8.14; N, 11.90. IR (KBr)
1
3474, 3283, 3218, 3162, 2990, 1696, 1649, 1397, 1378,
(6.2g, 97%) as a pale yellow amorphous substance. H
22
D
1360, 1223, 1167, 1119, 1072, 848cmꢀ1. ½aꢁ ꢀ56.3 (c
NMR (300MHz, DMSO-d6): d 7.91 (1H, d, J = 7.9Hz,
NH), 7.89 (2H, d, J = 7.3Hz, aromatic H), 7.72 (2H,
d, J = 7.5Hz, aromatic H), 7.42 (2H, t like, J = 7.3Hz,
aromatic H), 7.35–7.29 (2H, m, aromatic H), 5.49 (1H,
br d, J = 50.5Hz, H-4), 5.05–4.98 (1H, m, H-2), 4.34–
3.80 (6H, m), 2.60–2.30 (2H, m, H-3), 1.90–1.74 (1H,
m), 1.65–1.48 (1H, m), 1.20–1.13 (1H, m), 0.87 (3H, d,
J = 6.8Hz, Me), 0.86 (3H, t, J = 7.3Hz, Me). MS (ESI
pos.) m/z 472 ([M+Na]+).
0.5, MeOH).
5.1.2. (4S)-4-Fluoro-L-prolinamide hydrochloride (4g). In
4M HCl/dioxane (45mL), 3g (4.2g, 18.1mmol) was sus-
pended, and after stirring at room temperature for 2h,
the reaction solution was concentrated in vacuo. Tolu-
ene (50mL) was added to the residue, followed by fur-
ther concentration in vacuo. This was repeated three
times to obtain the desired product (3.1g, 100%) as a
colorless powder. This intermediate was used for the
next reaction without purification. Mp 230–231°C (de-
5.1.5. (2S,4S)-4-Fluoro-1-L-isoleucylpyrrolidine-2-carbo-
nitrile hydrochloride (1g). In 1,2-dichloroethane (90mL),
6g (6. 2g, 13.8mmol) was dissolved, and after addition
of diethylamine (10mL) with ice-cooling, stirring was
continued with ice-cooling for 30min and then at room
temperature for 5h. The solution was concentrated in
vacuo, the residue was dissolved in a mixture of diethyl
ether (100mL), THF (50mL) and CHCl3 (50mL), and
then 4M HCl/dioxane (4.0mL) was added with ice-cool-
ing. The resulting salt was collected by filtration and
washed with diethyl ether. The resulting powder was
1
comp.). H NMR (300MHz, DMSO-d6): d 9.72 (2H,
br s, H2N+), 8.10(1H, br s, CONH ), 7.70(1H, br s,
2
CONH2), 5.38 (1H, dt like, J = 52.5, 3.6Hz, H-4), 4.31
(1H, dd, J = 10.4, 3.7Hz, H-2), 3.56 (1H, ddd,
J = 19.9, 13.4, 1.7Hz, H-5), 3.39 (1H, ddd, J = 36.5,
13.4, 3.6Hz, H-5), 2.48–2.72 (1H, m, H-3), 2.24–2.40
(1H, m, H-3). MS (ESI pos.) m/z 155 ([M+Na]+). Anal.
Calcd for C5H9FN2OÆHCl: C, 35.62; H, 5.98; Cl, 21.03;
F, 11.27; N, 16.62. Found: C, 35.57; H, 5.95; Cl, 21.03;

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H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
purified by silica gel column chromatography (develop-
ing solvent; CHCl3–MeOH–25% aqueous ammo-
nia = 40:1:0.1–25:1:0.1). The resulting residue was
dissolved in CHCl3 and, after addition of 4M HCl/diox-
ane (4.0mL) with ice-cooling, the resulting salt was col-
lected by filtration, washed with CHCl3, and dried in
vacuo to yield the desired product (2.9g, 80%) as a
1H NMR (300MHz, CDCl3): d 4.76 (1H, br s, H-2),
4.60(1H, br d, J = 3.7Hz, H-3), 3.83–3.67 (2H, m, H-
5), 3.33 (1H, d, J = 6.4Hz, NCHCO), 2.40–2.26 (1H,
m, H-3), 2.19–2.09 (1H, m, H-3), 1.70–1.54 (2H, m),
1.27–1.05 (1H, m), 0.96 (3H, d, J = 6.8Hz, Me), 0.92
(3H, t, J = 7.3Hz, Me). MS (ESI pos.) m/z 248
([M+Na]+). HRMS calcd for C11H20N3O2 [M+H]+
226.1556, found (m/z) 226.1545.
1
colorless powder. Mp 245–248°C (decomp.). H NMR
(500MHz, DMSO-d6): d 8.59 (3H, br s, H3N+), 5.54
(0.9H, br d, J = 52.1Hz, H-4 of the major conformer),
5.45 (0.1H, br d, J = 53.3Hz, H-4 of the minor con-
former), 5.39 (0.1H, d, J = 6.7Hz, H-2 of the minor con-
former), 5.06 (0.9H, d, J = 9.4Hz, H-2 of the major
conformer), 4.07–3.77 (3H, m, H-5 and NCHCO),
2.55–2.34 (2H, m, H-3), 1.94–1.83 (1H, m), 1.66–1.53
(1H, m), 1.22–1.13 (1H, m), 0.94 (3H, d, J = 6.7Hz,
Me), 0.88 (3H, t, J = 7.3Hz, Me); 13C NMR
(125.4MHz, DMSO-d6): d 168.2, 118.3, 92.9 (d, JC–F
= 175.7Hz), 54.4, 53.6 (d, JC–F = 21.7Hz), 44.7, 36.3,
35.4 (d, JC–F = 20.7Hz), 23.7, 13.9, 10.9; 19F NMR
(282.2MHz, DMSO-d6): d ꢀ175.1. MS (ESI pos.) m/z
250([M+Na] +); (ESI neg.) m/z 262 ([M+Cl]ꢀ). HRMS
calcd for C11H19FN3O [M+H]+ 228.1512, found (m/z)
228.1508. Anal. Calcd for C11H18FN3OÆHClÆ0.5H2O:
5.1.9. (2S,4R)-1-L-Isoleucyl-4-methoxypyrrolidine-2-car-
bonitrile hydrochloride (1d). The title compound was
obtained as a colorless amorphous powder in a manner
similar to the preparation of 1g. H NMR (500MHz,
DMSO-d6): d 8.37 (3H, br s, H3N+), 4.70(1H, dd,
1
J = 9.1, 7.9Hz, H-2), 4.20(1H, br d,
J = 4.3Hz,
NCHCO), 4.07 (1H, br s, H-4), 3.99 (1H, d,
J = 11.5Hz, H-5), 3.63 (1H, dd, J = 11.5, 3.6Hz, H-5),
3.25 (3H, s, Ome), 2.54–2.47 (1H, m, H-3), 2.34–
2.27 (1H, m, H-3), 1.95–1.86 (1H, m), 1.53–1.44
(1H, m), 1.26–1.12 (1H, m), 0.98 (3H, d, J = 7.0Hz,
Me), 0.88 (3H, t, J = 7.3Hz, Me). MS (ESI pos.) m/z
262 ([M+Na]+), 240([M+H] +); (ESI neg.) m/z 274
([M+Cl]ꢀ). HRMS calcd for C12H22N3O2 [M+H]+
240.1712, found (m/z) 240.1723.
C, 48.44; H, 7.39; N, 15.41. Found: C, 48.20; H, 7.37;
25
D
N, 15.23. ½aꢁ ꢀ62.7 (c 0.3, MeOH).
5.1.10.
(2S,4S)-1-L-Isoleucyl-4-methoxypyrrolidine-2-
carbonitrile hydrochloride (1e). The title compound was
obtained as a colorless powder in a manner similar to
the preparation of 1g. Mp 175–178°C. 1H NMR
(500MHz, DMSO-d6): d 8.46 (3H, br s, H3N+), 5.23
(0.05H, d, J = 8.5Hz, H-2 of the minor conformer),
4.91 (0.95H, dd, J = 9.2, 1.6Hz, H-2 of the major
conformer), 4.14 (0.95H, br s, H-4 of the major con-
former), 4.09 (0.05H, m, H-4 of the minor conformer),
3.87 (1H, d, NCHCO), 3.80–3.73 (2H, m, H-5), 3.29
(3H, s, Ome), 2.37 (1H, d, J = 14.0Hz, H-3), 2.22 (1H,
ddd, J = 13.7, 9.5, 4.0Hz, H-3), 1.91–1.80 (1H, m),
1.64–1.53 (1H, m), 1.23–1.12 (1H, m), 0.94 (3H, d,
J = 6.7Hz, Me), 0.88 (3H, t, J = 7.3Hz, Me). MS (ESI
5.1.6. (2S,4R)-4-Hydroxy-1-L-isoleucylpyrrolidine-2-car-
bonitrile hydrochloride (1a). The title compound was
obtained as a colorless powder in a manner similar to
the preparation of 1g. Mp 173–176°C (decomp.). 1H
NMR (300MHz, DMSO-d6): d 8.32 (3H, br s, H3N+),
5.45 (1H, d, J = 3.7Hz, OH), 4.75 (1H, t like, J = 9Hz,
H-2), 4.38 (1H, br s, H-4), 4.08 (1H, d, J = 5.3Hz,
NCHCO), 3.74 (1H, d, J = 10.8Hz, H-5), 3.64 (1H,
dd, J = 10.8, 3.8Hz, H-5), 2.37–2.21 (2H, m, H-3),
1.95–1.81 (1H, m), 1.55–1.40(1H, m), 1.26–1.05 (1H,
m), 0.97 (3H, d, J = 7.0Hz, Me), 0.88 (3H, t,
J = 7.3Hz, Me). MS (ESI pos.) m/z 248 ([M+Na]+),
226 ([M+H]+); (ESI neg.) m/z 260([M+Cl] ꢀ). HRMS
calcd for C11H20N3O2 [M+H]+ 226.1556, found (m/z)
226.1570.
+
pos.) m/z 262 ([M+Na]+), 240([M+H] ); (ESI neg.) m/
z
274 ([M+Cl]ꢀ). HRMS calcd for C12H22N3O2
[M+H]+ 240.1712, found (m/z) 240.1717.
5.1.7. (2S,4S)-4-Hydroxy-1-L-isoleucylpyrrolidine-2-car-
bonitrile hydrochloride (1b). The title compound was
obtained as a colorless amorphous powder in a manner
5.1.11. (2S,4R)-4-Fluoro-1-L-isoleucylpyrrolidine-2-car-
bonitrile hydrochloride (1f). The title compound was
obtained as a colorless powder in a manner similar to
1
1
similar to the preparation of 1g. H NMR (300MHz,
the preparation of 1g. Mp 222–225°C (decomp.). H
DMSO-d6): 8.40(3H, br s, H 3N+), 5.44 (1H, br s,
OH), 4.88 (1H, dd, J = 9.0, 2.5Hz, H-2), 4.45–4.38
(1H, m, H-4), 3.94–3.82 (1H, m, NCHCO), 3.75 (1H,
dd, J = 10.8, 4.3Hz, H-5), 3.53 (1H, d, J = 10.8Hz, H-
5), 2.25 (1H, ddd, J = 13.2, 9.2, 4.1Hz, H-3), 2.10(1H,
br d, J = 13.2Hz, H-3), 2.01–1.80 (1H, m), 1.65–1.47
(1H, m), 1.26–1.08 (1H, m), 0.96 (3H, d, J = 7.0Hz,
Me), 0.88 (3H, t, J = 7.4Hz, Me). MS (ESI pos.) m/z
248 ([M+Na]+), 226 ([M+H]+); (ESI neg.) m/z 260
([M+Cl]ꢀ). HRMS calcd for C11H20N3O2 [M+H]+
226.1556, found (m/z) 226.1565.
NMR (500MHz, DMSO-d6): d 8.38 (3H, br s, H3N+),
5.42 (1H, br d, J = 51.9Hz, H-4), 4.87 (1H, dd,
J = 10.1, 8.0Hz, H-2), 4.28–4.20 (2H, m, H-5 and
NCHCO), 3.81 (1H, ddd, J = 39.0, 12.6, 2.5Hz, H-5),
2.77–2.67 (1H, m, H-3), 2.60–2.45 (1H, m, H-3), 1.95–
1.84 (1H, m), 1.52–1.43 (1H, m), 1.23–1.13 (1H, m),
0.98 (3H, d, J = 6.7Hz, Me), 0.88 (3H, t, J = 7.3Hz,
Me). MS (ESI pos.) m/z 250([M+Na] +), 228
([M+H]+); (ESI neg.) m/z 262 ([M+Cl]ꢀ). HRMS calcd
for C11H19FN3O [M+H]+ 228.1512, found (m/z)
228.1518.
5.1.8. (2R,3S)-3-Hydroxy-1-L-isoleucylpyrrolidine-2-car-
bonitrile (1c). In a manner similar to the preparation of
1g, except for hydrochloride salt formation, the title
compound was obtained as a colorless oily substance.
5.1.12. tert-Butyl ((1S,2S)-1-{[(2S)-2-cyano-4,4-difluoro-
pyrrolidin-1-yl]carbonyl}-2-methylbutyl)carbamate (8h).
In DMF (2.5mL), tert-butyl ((1S,2S)-1-{[(2S)-2-(amino-
carbonyl)-4,4-difluoropyrrolidin-1-yl]carbonyl}-2-meth-

H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
6059
ylbutyl)carbamate 7h (900mg, 2.48mmol) was dissol-
ved, and then cyanuric chloride (280mg, 1.49mmol)
was added, followed by stirring at room temperature
for 1h. The reaction solution was taken up in water,
and extracted with EtOAc. The organic phase was
washed with a saturated aqueous NaHCO3 solution
and a saturated aqueous NaCl solution, successively,
and dried over Na2SO4. The drying agent was removed
by filtration, and the filtrate was concentrated under re-
duced pressure. The residue was purified by silica gel
column chromatography (developing solvent; hexane–
EtOAc = 20:1–4:1) to yield the desired product
(760mg, 89%) as a colorless amorphous substance. 1H
NMR (300MHz, DMSO-d6): d 7.29 (1H, d, J = 7.9Hz,
NH), 5.08 (1H, dd, J = 9.1, 4.1Hz, H-2), 4.34–4.10
(2H, m, H-5), 3.90(1H, t like, J = 8.7Hz, NCHCO),
3.03–2.73 (2H, m, H-3), 1.83–1.67 (1H, m), 1.59–1.43
(1H, m), 1.36 (9H, s, Boc), 1.26–1.04 (1H, m), 0.87–
0.78 (6H, m, 2Me). MS (ESI pos.) m/z 368 ([M+Na]+);
(ESI neg.) m/z 344 ([MꢀH]ꢀ).
reaction mixture was taken up in a saturated aqueous
NaHCO3 solution and extracted with EtOAc. The
organic phase was washed with a saturated aqueous
NaHCO3 solution and a saturated aqueous NaCl solu-
tion, successively, and dried over MgSO4. After removal
of the drying agent by filtration, the filtrate was concen-
trated under reduced pressure. The residue was purified
by silica gel column chromatography (developing
solvent; hexane–EtOAc = 3:2) to yield the desired prod-
1
uct (65mg, 38%) as a colorless gummy substance. H
NMR (300MHz, CDCl3): d 5.32 (1H, br d, J =
51.8Hz, H-3), 5.09 (1H, br d, J = 9.3Hz, NH), 4.84
(1H, dd, J = 21.8, 4.4Hz, H-2), 4.27–4.08 (2H, m, H-5
and NCHCO), 3.81 (1H, td like, J = 10.2, 6.2Hz, H-
5), 2.52–2.36 (1H, m, H-4), 2.31–2.05 (1H, m, H-4),
1.83–1.71 (1H, m), 1.66–1.52 (1H, m), 1.42 (9H, s,
Boc), 1.24–1.08 (1H, m), 0.98 (3H, d, J = 6.8Hz, Me),
0.92 (3H, t, J = 7.4Hz, Me). MS (ESI pos.) m/z 350
([M+Na]+).
5.1.15. (2R,3R)-3-Fluoro-1-L-isoleucylpyrrolidine-2-car-
bonitrile trifluoroacetate (1i). In ice-cooled trifluoroace-
tic acid (1.0mL), 8i (62mg, 0.18mmol) was dissolved,
and after stirring at room temperature for 30min, the
solvent was evaporated under reduced pressure. To
the residue, diisopropyl ether (10mL) was added, and
the supernatant was removed. To the residue, diisopro-
pyl ether (10mL) was added again, and an insoluble
substance was collected by filtration to yield the title
5.1.13. (2S)-4,4-Difluoro-1-L-isoleucylpyrrolidine-2-car-
bonitrile hydrochloride (1h). To 8h (560mg, 1.62mmol)
was added 2M aqueous HCl solution (12mL), followed
by stirring at room temperature overnight. An addi-
tional 2M aqueous HCl solution (6mL) was added to
the solution, and after stirring at room temperature
overnight the aqueous solution was washed with EtOAc.
To the aqueous phase, 1M aqueous NaOH solution
(35mL) and an excess amount of NaCl were added,
and after stirring the mixture was taken up in a satu-
rated aqueous NaHCO3 solution and extracted with
EtOAc. The organic phase was washed with a saturated
aqueous NaCl solution and dried over Na2SO4. After
removal of the drying agent by filtration, the filtrate
was concentrated under reduced pressure to obtain
(2S)-4,4-difluoro-1-L-isoleucylpyrrolidine-2-carbonitrile,
which was then dissolved in diethyl ether (20mL), fol-
lowed by addition of 4M HCl/EtOAc (0.50mL) with
ice-cooling. The precipitated insoluble substance was
collected by filtration to yield the desired product
(370mg, 81%) as a colorless powder. Mp 235–243 °C
(decomp.). 1H NMR (300MHz, DMSO-d6): d 8.38
(3H, br s, H3N+), 5.14 (1H, t like, J = 7.1Hz, H-2),
4.46–4.12 (2H, m, H-5), 4.00 (1H, d, J = 6.2Hz,
NCHCO), 2.98–2.81 (2H, m, H-3), 1.96–1.78 (1H, m),
1.60–1.44 (1H, m), 1.28–1.06 (1H, m), 0.95 (3H, d,
J = 6.8Hz, Me), 0.88 (3H, t, J = 7.3Hz, Me). MS (ESI
pos.) m/z 268 ([M+Na]+), 246 ([M+H]+); (ESI neg.) m/
z 280([M+Cl] ꢀ). HRMS calcd for C11H17F2N3O [M]+
245.1340, found (m/z) 245.1353.
1
compound (32mg, 51%) as a yellow gummy solid. H
NMR (300MHz, DMSO-d6): d 8.22 (3H, br s, H3N+),
5.50(1H, br d,
J = 52.8Hz, H-3), 5.08 (1H, dd,
J = 26.1, 4.0Hz, H-2), 4.19 (1H, d, J = 5.4Hz,
NCHCO), 4.05 (1H, t like, J = 8.9Hz, H-5), 3.64–3.54
(1H, m, H-5), 2.42–2.00 (2H, m, H-4), 1.99–1.84 (1H,
m), 1.54–1.38 (1H, m), 1.26–1.08 (1H, m), 0.96 (3H, d,
J = 6.8Hz, Me), 0.89 (3H, t, J = 7.3Hz, Me). MS (ESI
pos.) m/z 250([M+Na] +), 228 ([M+H]+); (ESI neg.)
m/z 340([M+C 2F3O2]ꢀ). HRMS calcd for C11H19FN3O
[M+H]+ 228.1512, found (m/z) 228.1525.
5.1.16. 9H-Fluoren-9-ylmethyl ((1S,2S)-1-{[(2S,4S)-4-
chloro-2-cyanopyrrolidin-1-yl]carbonyl}-2-methylbutyl)-
carbamate (6j). In CH2Cl2 (2mL)–carbon tetrachloride
(2mL), 9H-fluoren-9-ylmethyl ((1S,2S)-1-{[(2S,4R)-2-
cyano-4-hydroxypyrrolidin-1-yl]carbonyl}-2-methylbut-
yl)carbamate 6a (200mg, 0.45mmol) obtained from
(4R)-1-(tert-butoxycarbonyl)-4-hydroxy-L-proline by a
synthetic route similar to that for 6g was dissolved,
and then triphenylphosphine (234mg, 0.89mmol) was
added, followed by stirring at room temperature over-
night. To the reaction solution, EtOH (0.5mL) was
added, followed by stirring at room temperature for
4h. The reaction solution was concentrated in vacuo,
and the resulting residue was purified by silica gel col-
umn chromatography (developing solvent; hexane–
EtOAc = 1:1–2:3) to yield the desired product (126mg,
60%) as a colorless amorphous substance. 1H NMR
(300MHz, CDCl3): d 7.76 (2H, d, J = 7.5Hz, aromatic
H), 7.57 (2H, d, J = 7.5Hz, aromatic H), 7.40(2H, t,
J = 7.5Hz, aromatic H), 7.30(2H, t, J = 7.5Hz, aro-
matic H), 5.42 (1H, d, J = 9.2Hz, NH), 4.88 (1H,
dd, J = 8.3, 3.5Hz, H-2), 4.63–4.56 (1H, m, H-4),
5.1.14. tert-Butyl ((1S,2S)-1-{[(2R,3R)-2-cyano-3-fluoro-
pyrrolidin-1-yl]carbonyl}-2-methylbutyl)carbamate (8i).
In CH2Cl2 (5mL), tert-butyl ((1S,2S)-1-{[(2R,3S)-2-cya-
no-3-hydroxypyrrolidin-1-yl]carbonyl}-2-methylbutyl)-
carbamate 8c (168mg, 0.516mmol) obtained from
(3S)-1-(tert-butoxycarbonyl)-3-hydroxy-L-proline by a
synthetic route similar to that for 6g or 8h was dissolved,
and diethylaminosulfur trifluoride (0.206mL) was added
dropwise with cooling on a dry ice–acetone bath. The
temperature was gradually increased to room tempera-
ture, and the mixture was then stirred overnight. The

6060
H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
4.44–4.08 (5H, m), 3.96 (1H, dd, J = 11.5, 1.9Hz, H-5),
2.73–2.56 (2H, m, H-3), 1.93–1.79 (1H, m), 1.72–1.56
(1H, m), 1.30–1.13 (1H, m), 1.12 (3H, d, J = 6.7Hz,
Me), 0.96 (3H, t, J = 7.4Hz, Me). MS (ESI pos.) m/z
488 ([M+Na]+).
0.92 (3H, t, J = 7.3Hz, Me). MS (ESI pos.) m/z 378
([M+MeOH+Na]+), 346 ([M+Na]+); (ESI neg.) m/z
322 ([MꢀH]ꢀ).
5.1.20. (2S)-1-L-Isoleucyl-4-oxopyrrolidine-2-carbonitrile
trifluoroacetate (1k). The title compound (76mg, 80%)
was obtained as a colorless powder from 8k (91mg,
0.28mmol) in a manner similar to the preparation of
1i. Mp 121–124°C (decomp.). 1H NMR (300MHz,
DMSO-d6): d 8.21 (3H, br s, H3N+), 5.30(1H, dd,
J = 9.8, 5.6Hz, H-2), 4.29 (1H, d, J = 17.7Hz, H-5),
4.18 (1H, d, J = 17.7Hz, H-5), 4.09–3.98 (1H, m,
NCHCO), 3.11 (1H, dd, J = 19.0, 9.8Hz, H-3), 3.01
(1H, dd, J = 19.0, 5.6Hz, H-3), 2.00–1.90 (1H, m),
1.54–1.38 (1H, m), 1.34–1.10(1H, m), .098 (3H, d,
J = 7.0Hz, Me), 0.88 (3H, t, J = 7.3Hz, Me). MS (ESI
pos.) m/z 278 ([M+MeOH+Na]+), 246 ([M+Na]+).
HRMS calcd for C11H18N3O2 [M+H]+ 224.1399, found
(m/z) 224.1381.
5.1.17. (2S,4S)-4-Chloro-1-L-isoleucylpyrrolidine-2-car-
bonitrile hydrochloride (1j). The title compound (32mg,
53%) was obtained as a colorless powder from 6j
(100mg, 0.22mmol) in a manner similar to the prepara-
tion of 1g. Mp 189–192°C. 1H NMR (300MHz,
DMSO-d6): d 8.46 (3H, br s, H3N+), 5.01–4.92 (2H, m,
H-2 and H-4), 4.11 (1H, dd, J = 12.0, 4.3Hz, H-5),
3.98 (1H, br d, J = 12.0Hz, H-5), 3.90 (1H, d,
J = 6.8Hz, NCHCO), 2.73 (1H, ddd, J = 14.6, 9.4,
4.8Hz, H-3), 2.54–2.43 (1H, m, H-3), 2.00–1.80 (1H,
m), 1.66–1.48 (1H, m), 1.27–1.10(1H, m), 0.97 (3H, d,
J = 6.8Hz, Me), 0.88 (3H, t, J = 7.4Hz, Me). MS (ESI
pos.) m/z 266 ([M+Na]+), 244 ([M+H]+); (ESI neg.) m/
z 278 ([M+Cl]ꢀ), 242 ([MꢀH]ꢀ). HRMS calcd for
C11H19ClN3O [M+H]+ 244.1217, found (m/z) 244.1231.
5.2. Biological methods
5.1.18. tert-Butyl ((1S,2S)-1-{[(2S)-2-(aminocarbonyl)-
4-oxopyrrolidin-1-yl]carbonyl}-2-methylbutyl)carbamate
(7k). In CH2Cl2 (10mL), tert-butyl ((1S,2S)-1-{[(2S,4R)-
2-(aminocarbonyl)-4-hydroxypyrrolidin-1-yl]carbonyl}-
2-methylbutyl)carbamate 7a (250mg, 0.73mmol) was
dissolved, and then molecular sieves 4A (1.5g), pyridi-
nium chlorochromate (235mg, 1.09mmol), and acetic
acid (0.07mL) were added, followed by stirring at room
temperature for 2h. The reaction solution was directly
purified by silica gel column chromatography (develop-
ing solvent; hexane–EtOAc = 1:1–1:3) to yield the de-
sired product (180mg, 72%) as a brown amorphous
substance. 1H NMR (300MHz, CDCl3): d 6.70and
5.2.1. DPP-IV inhibitory activity. Activity inhibition
testing of DPP-IV was carried out according to the
method by Deacon et al.13 Plasma including DPP-IV
was prepared via centrifugation of blood collected from
healthy human volunteers. Enzyme reactions were car-
ried out using 96-flat-bottom-well plates in buffer solu-
tion of pH7.8 containing 25mM HEPES, 140mM
NaCl, and 1% BSA. To a mixture of 25lL of 10 0lM
Gly-Pro-4-methylcoumaryl-7-amide solution (manufac-
tured by Peptide Institute, Inc.), 7.5lL of 133mM
MgCl2 solution, and 5lL of the test compound,
12.5lL of plasma diluted to 1/100 with the above buffer
solution was added. The solution was allowed to react at
room temperature for 2h and 50lL of 25% aqueous
acetic acid solution was added to stop the reaction.
The fluorescence intensity of the liberated 7-amino-4-
5.50(1H each, br s each, CONH ), 5.25–5.05 (2H, m,
2
BocNH and H-2), 4.41 (1H, d, J = 17.3Hz, H-5),
4.17–4.07 (1H, m, NCHCO), 3.92 (1H, d, J = 17.3Hz),
3.05 (1H, br d, J = 18.2Hz, H-3), 2.76–2.60(1H, m,
H-3), 1.80–1.50 (2H, m), 1.43 (9H, s), 1.27–1.07 (1H,
m), 0.98–0.85 (6H, m, 2Me). MS (ESI pos.) m/z 364
([M+Na]+); (ESI neg.) m/z 340([M ꢀH]ꢀ).
methylcoumarin was determined using a fluorescence
TM
plate reader (1420ARVO
Multilabel Counter manu-
factured by Wallac Oy; excitation: 390nm; emission:
460nm).
5.1.19. tert-Butyl ((1S,2S)-1-{[(2S)-2-cyano-4-oxopyrrol-
idin-1-yl]carbonyl}-2-methylbutyl)carbamate (8k). In
THF (10mL), 7k (168mg, 0.49mmol) was dissolved,
and after ice-cooling, trifluoroacetic anhydride
(0.21mL) and N,N-diisopropylethylamine (0.51mL)
were added, followed by stirring with ice-cooling for
1h. The reaction solution was diluted with EtOAc
(100mL), and washed with water, 10% aqueous KHSO4
solution, water, a saturated aqueous NaHCO3 solution
and a saturated aqueous NaCl solution, successively,
and the organic phase was dried over MgSO4. After
removal of the drying agent by filtration, the solvent
was evaporated in vacuo to yield the desired product
5.2.2. Drug concentration measurement of 1g and 9 in
blood following oral administration to rats. Male Wistar
rats (eight weeks of age) fasted overnight were used.
Aqueous solution of 1g or 9 was orally administered
at a dose of 1mg/kg. At 5, 10, 15, 30, 60, and 120min
after administration, 200lL of blood was collected from
the jugular vein. After centrifugation, 50lL of the
resulting plasma was added to 200lL of acetonitrile.
The supernatant was injected into a liquid chromatogra-
phy system with a CAPCEL PAK C18, UG1205 lm
(150mm long, 2mm diameter) column using a mixture
of 10mM aqueous ammonium acetate solution and
90% aqueous acetonitrile solution (1:9) as an eluent,
the Sciex API3000 LC/MS/MS System (Perkin Elmer
Sciex) for MS/MS, ESI as an ionization method. The
indicators of compound 1g were m/z 228.0and m/z 86.0.
1
(174mg, quant) as a brown amorphous substance. H
NMR (300MHz, CDCl3): d 5.38 (1H, br d, J = 7.6Hz,
H-2), 5.07 (1H, br d, J = 8.1Hz, NH), 4.52 (1H, d,
J = 18.0Hz, H-5), 4.03–3.95 (1H, m, NCHCO), 4.00
(1H, d, J = 18.0Hz, H-5), 3.10–2.86 (2H, m, H-3),
1.90–1.72 (1H, m), 1.70–1.54 (1H, m), 1.41 (9H, s,
Boc), 1.34–1.10(1H, m), 0.98 (3H, d, J = 6.5Hz, Me),
5.2.3. Oral glucose tolerance test (OGTT) in Zucker fatty
rats. An OGTT in Zucker fatty rats was carried out
based on the method of Balkan et al.14 Male Zucker

H. Fukushima et al. / Bioorg. Med. Chem. 12 (2004) 6053–6061
6061
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