Synthesis and structure-activity relationships of potent 1-(2-substituted-aminoacetyl)-4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1248/cpb.56.1110

Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treatment of type 2 diabetes because they prevent the degradation of glucagon-like peptide-1 (GLP-1) and extend its duration of action. We previously reported that 2-cyano-4-fluoropyrrolidines act as potent DPP-IV inhibitors and have been modifying the 1-position of pyrrolidine to obtain more useful inhibitors. An L-tert-butylglycine derivative was found to be a stable and potent DPP-IV inhibitor that exhibits a glucose lowering effect in vivo. Here, we report the synthesis of and biological data on the aforementioned derivatives.

Full text of DOI:10.1248/cpb.56.1110

1110
Chem. Pharm. Bull. 56(8) 1110—1117 (2008)
Vol. 56, No. 8
Synthesis and Structure–Activity Relationships of Potent 1-(2-Substituted-
aminoacetyl)-4-fluoro-2-cyanopyrrolidine Dipeptidyl Peptidase IV
Inhibitors
Hiroshi FUKUSHIMA, ,a Akira HIRATATE,a Masato TAKAHASHI,a Masako SAITO-HORI,b Eiji MUNETOMO,b
*
b
Kiyokazu KITANO,b Hidetaka SAITO,c Yuji TAKAOKA,d and Koji YAMAMOTO
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd.; b Molecular Function and Pharmacology
Laboratories, Taisho Pharmaceutical Co., Ltd.; c Non-clinical Quality Assurance Section, Taisho Pharmaceutical Co., Ltd.;
and d Research Computer System, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama, Saitama
331–9530, Japan. Received February 14, 2008; accepted May 8, 2008; published online May 12, 2008
Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treat-
ment of type 2 diabetes because they prevent the degradation of glucagon-like peptide-1 (GLP-1) and extend its
duration of action. We previously reported that 2-cyano-4-fluoropyrrolidines act as potent DPP-IV inhibitors and
have been modifying the 1-position of pyrrolidine to obtain more useful inhibitors. An L-tert-butylglycine deriva-
tive was found to be a stable and potent DPP-IV inhibitor that exhibits a glucose lowering effect in vivo. Here, we
report the synthesis of and biological data on the aforementioned derivatives.
Key words dipeptidyl peptidase IV; inhibitor; fluoropyrrolidine; diabetes
Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5, CD26)1,2) cyanopyrrolidine derivatives have been published. Ashworth
is a highly specific serine protease that cleaves N-terminal synthesized 2-cyanopyrrolidine derivatives using six kinds of
dipeptides from polypeptides with L-proline or L-alanine at amino acids and investigated the DPP-IV inhibitory activity
the penultimate position.3) The natural substrates of DPP-IV and chemical stability.17) Villhauer investigated the SAR of
have been described, and the inhibition of DPP-IV has been N-substituted-glycyl-2-cyanopyrrolidines and determined the
recognized as potentially useful for the modulation of structure of the decomposed compound to be a cyclic ami-
glucagon-like peptide processing. The role of DPP-IV in dine.13) And Magnin reported the synthesis of methanoproli-
glucagon-like peptide (GLP) processing is well-known.4—6) nenitrile derivatives, their DPP-IV inhibitory activity and
Given the importance of the regulation of this process, DPP- their chemical stability and found that the introduction of ei-
IV inhibition has an obvious application for the treatment of ther a sterically bulky amino acid side chain on the N-termi-
diabetes.7—10) Since the blood glucose-lowering effects of nal amino acid or a cis-4,5-methano bridge to the prolineni-
GLP-1 are dependent on an elevated blood glucose level and trile moiety significantly enhanced solution stability.18)
abate as glucose levels return to normal, the incidence of hy-
We thought that the chemical stability of 1a should be fur-
poglycemia during treatment with a DPP-IV inhibitor is ex- ther improved to make it more useful. Consequently, we have
pected to be very low.11) Based on this concept, the clinical been exploring the synthesis of 2-cyano-4-fluoropyrrolidines
development of several DPP-IV inhibitors, including NVP- in the hope of obtaining more effective DPP-IV inhibitors.
DPP728,12) NVP-LAF237 (Vildagliptin),13) BMS-477118
(Saxagliptin),14) and MK-0431 (Sitagliptin),15) has been initi- Chemistry
ated (Fig. 1).
As the cause of the instability of 2-cyano-4-fluoropyrroli-
We previously reported that the introduction of fluorine to dine was undetermined, we investigated the structure of the
the 4-position of 2-cyanopyrrolidine augmented DPP-IV in- decomposed product of 1a (Table 1). Compound 1a was con-
hibitory activity and the drug concentration after oral admin- siderably stable in a pH 1.2 acidic solution, but the residual
istration to rats.16) While investigating the properties of 2- amount decreased in a pH 6.8 neutral solution. The degraded
cyano-4-fluoropyrrolidine 1a, we recognized the presence of compounds derived from 1a were isolated and identified as
decomposed products of 1a in neutral aqueous solution. the anticipated cyclic compounds.
Some previous reports about the chemical instability of 2-
The first step in the degradation process was thought to be
the intramolecular attachment of a basic nitrogen to the
Table 1. Chemical Stability of 1a
Residual amount (%)a)
Compd.
pH 1.2
pH 6.8
1a
95
ꢀ
70
Fig. 1. DPP-IV Inhibitors
a) HPLC determination after 6 h incubation at 37 °C in aqueous solution.
∗ To whom correspondence should be addressed. e-mail: hiroshi.fukushima@po.rd.taisho.co.jp
© 2008 Pharmaceutical Society of Japan

August 2008
1111
cyano group to give cyclic amidine 2, which was subse-
First, 2-aminocarbonyl-4-fluoropyrrolidine 7 was obtained
quently transformed into diketopiperadine 3. Cyclic amidine in three steps from 4-hydroxide 6 and was then converted to
2 (retention timeꢁ4.7 min) was observed as a 67% peak area amine hydrochloride 8 (Chart 2).16,19)
using reverse phase high-performance liquid chromatography
Compound 9 was obtained via the dehydration of com-
(HPLC) after 2 h of the incubation of 1a in a pH 6.8 aqueous pound 7 with cyanuric chloride20) in N,N-dimethylformamide
buffer solution. After 10 h of incubation, compound 2 (DMF) and was treated with hydrochloric acid to obtain in-
changed to diketopiperadine 3 (retention timeꢁ7.4 min), termediate 10. Compound 8 was useful as a common inter-
which was observed as an 86% peak area (Table 2).
mediate, and cyano derivative 10 seemed to be more useful
Cyclic amidine 2 was formed during the decomposition of for the synthesis of a variety of derivatives.
1a in neutral solution, but it was difficult to isolate from
Compound 8 was coupled with N-Boc or N-Fmoc a-
aqueous solution. Since the cyclization of 4, a free base of amino acids using 1-[3-(dimethylamino)propyl]-3-ethylcar-
1a, was accelerated by the presence of carboxylic acid in an bodiimide hydrochloride (EDC) and 1-hydroxybenztriazole
organic solvent, 4 was treated with a 1.1-molar equivalent of (HOBt) as coupling reagents to yield the dipeptides 11a—f,
acetic acid in ethanol–n-hexane to obtain cyclic amidine 5 11k, 11l, 13i and 13j. Either the Fmoc group or the Boc
(acetic acid salt) as a precipitate. Diketopiperadine 3 was ob- group was used as an N-protecting group for of a-amino
tained by heating 5 in a pH 6.8 buffer solution at 80 °C acids. In the same manner, cyanide 10 was converted to
(Chart 1). Spectral data for 5 and 3 supported their struc- dipeptides 12g and 14h (Chart 3).
tures, and the HPLC retention times of 5 and 3 corresponded
Natural amino acids (valine and proline), artificial amino
with those of the degradation products of 1a in buffer solu- acids (alloisoleucine, tert-butylglycine and cyclohexyl-
tion.
Next, conversion at the P2 site was conducted in order to
glycine) and modified amino acids (N-benzyloxycarbonyl-ly-
find a potent, stable and highly efficacious inhibitor. Magnin
reported that the changes in stability caused by the conver-
sion at the P2 site were affected by the substituent of the
pyrrolidine ring.18) Several derivatives converted at the P2
site were then synthesized, as in the case of 2-cyano-4-fluo-
ropyrrolidine.
Reagents: (a) AcOH, EtOH, n-hexane; (b) pH 6.8 buffer.
Table 2. Degradation Peaks of 1a
Chart 1
Retention time
4.7 min
7.4 min
8.7 min
Incubation timea)
0 h
2 h
10 h
Trace
67%
13%
2
0%
12%
86%
3
ꢀ97%
20%
0%
Estimated structure
1a
a) Compound 1a was dissolved in pH 6.8 Britton–Robinson buffer solution and in-
cubated at 60 °C.
Reagents: (a) 4 M-HCl/AcOEt; (b) cyanuric chloride/DMF; (c) 2 M–HCl/H2O–
MeOH.
Chart 2
Reagents: (a) BocNR2CH(R1)CO2H, EDC, HOBt, DMF; (b) cyanuric chloride, DMF; or (CF3CO)2, N,N-diisopropylethylamine, THF; (c) 2 M-HCl/H2O–MeOH; (d)
FmocNR2CH(R1)CO2H, EDC, HOBt, DMF; (e) Et2NH, 1,2-dichloroethane; then HCl.
Chart 3

1112
Vol. 56, No. 8
Reagents: (a) R3-X, K2CO3, DMF; (b) 2 M-HCl/H2O–MeOH.
Chart 4
Table 3. DPP-IV Inhibitory Activity and Chemical Stability of 1a—k
Table 4. DPP-IV Inhibitory Activity of 1l—q
Remaining
Compd.
R1
R2
IC50 (nM)a)
amount (%)b)
Compd.
IC50 (nM)a)
1a
1b
1c
H
H
H
0.6
0.7
1.2
70
74
65
1l
0.8
2.2
1m
1n
1o
1p
1.2
2.5
1.7
1d
1e
H
H
0.6
1.4
93
52
1f
H
H
H
4.7
2.1
4.9
67
73
92
1g
1h
1q
3.3
a) DPP-IV inhibitory activity.
1i
H
0.7
1.1
41
68
—
1j
1k
H
Results and Discussion
The synthesized compounds were evaluated for DPP-IV-
inhibitory activity in human plasma using a fluorescence
assay with Gly-Pro-4-methylcoumaryl-7-amide.
Various sorts of a-amino acids were introduced at the P2
site. Valine derivative 1b and tert-butylglycine derivative 1d
exerted potent DPP-IV inhibitory activities (IC50ꢁ0.7,
Me
ꢀ100
a) DPP-IV inhibitory activity. b) Residual amount was measured after incubation
at 37 °C for 6 h in pH 6.8 aqueous buffer solution.
sine, O-methylthreonine, O-benzylthreonine and O-tert- 0.6 nM, respectively), similar to that of isoleucine derivative
butylthreonine) were used at the P2 site. 1a (IC50ꢁ0.6 nM). Cyclohexylglycine derivative 1j also
The cyano compounds 12a—f, 14i and 14j were obtained maintained an inhibitory activity (IC50ꢁ1.1 nM) (Table 3). On
from carbamoyl compounds 11a—f, 13i and 13j using cya- the other hand, threonine derivatives 1f, 1g and 1h exhibited
nuric chloride in DMF or trifluoroacetic anhydride and N,N- less potent activities (IC50ꢁ4.7, 2.1, 4.9 nM, respectively).
diisopropyl ethylamine.
X-ray crystallographic analysis of DPP-IV21,22) has sug-
Boc-protected compounds 12a—g and 12k—o were de- gested that the pocket to which the P2 site binds is lipophilic.
protected using hydrochloric acid to produce 1a—g and The oxygen atom on the threonine side chain seemed to have
1k—o. On the other hand, Fmoc-protected compounds difficulty binding tightly to the pocket. However, a benzyl
14h—j were converted to 1h—j using diethylamine.
ether side chain (1g) resulted in notable activity, possibly be-
7-Methoxytetrahydroquinoline derivative 12p and 7-car- cause of the affinity of the phenyl group. Compounds 1e and
bamoylmethoxy derivative 12q were obtained by alkylating 1i exhibited potent activities (IC50ꢁ1.4, 0.7 nM, respectively),
the 7-hydroxy derivative 12h; 12p and 12q were then treated and the long side chains did not reduce the compounds’
with hydrochloric acid to produce 1p and 1q.
As the activities of stereoisomers appeared interesting,
affinities.
Among the secondary amine derivatives, proline derivative
stereoisomers of 1d were synthesized. The synthetic route 1l had a potent inhibitory activity (IC50ꢁ0.8 nM) and tetrahy-
was similar to that for 1d. Four different hydroxy prolines (L- droisoquinoline derivatives 1m—q also had good potencies
trans, L-cis, D-trans and D-cis forms) and L- and D-N-Boc- (IC50ꢁ1.2—3.3 nM) (Table 4). However, the N-methyl iso-
tert-butylglycine were used to produce seven stereoisomers leucine derivative 1k had a very weak activity (IC50ꢀ
15—21.
100 nM).

August 2008
1113
Table 5. DPP-IV Inhibitory Activities of Stereoisomers of 1d
Chemical stability is an important characteristics in the
development of 2-cyanopyrrolidine DPP-IV inhibitors. Our
lead compound 1a was considerably stable in acidic solution,
but the residual amount of compound 1a decreased in neutral
solution. The stabilities of the compounds listed in Table 3
varied widely in a manner that was correlated with their
structures.
Streochemistry
Stereochemistry of
the pyrrolidine
Compd.
IC50 (nM)a)
of t-Bu Gly
The degradation of compound 1a under neutral conditions
might result from the attachment of a basic nitrogen to the
carbon atom of the cyano group, yielding a cyclic amidine.
By constructing a sterically hindered environment near the
nitrogen, this kind of degradation should be prevented.
tert-Butylglycine derivative 1d and O-tert-butylthreonine
1h were comparatively stable, as shown by their residual
amounts (93%, 92%, respectively) after 6 h of incubation
at 37 °C in a pH 6.8 buffer solution. Magnin reported that
a tert-butylglycine derivative exhibited the same chemical
stability as an isoleucine derivative in a cis-3,4-methano-
prolinenitrile series and better chemical stability than an
isoleucine derivative in a cis-4,5-methanoprolinenitrile se-
ries. Our result for fluoropyrrolidine was similar to that for
the cis-4,5-methanoprolinenitrile series.
1d (HCl)
L
L
(2S,4S)
(2S,4R)
(2R,4S)
(2R,4R)
(2S,4S)
(2S,4R)
(2R,4S)
(2R,4R)
0.6
246
ꢀ300
ꢀ300
42
ꢀ300
ꢀ300
ꢀ300
15
16
17
18
19
20
21
L
L
D
D
D
D
a) DPP-IV inhibitory activity.
In contrast, compounds 1e or 1i, which are not branched
at the carbon next to the a-position, yielded low residual
amounts (52%, 41%, respectively). Proline derivative 1l and
tetrahydroisoquinoline derivatives 1m—o also showed low
residual amounts, with the compounds listed in Table 4
showing the residual amounts of less than 20% under the Fig. 2. X-Ray Crystal Structure of Compound 22 (Free Base of 1d)
same condition. These compounds with cyclic side chains
easily undergo cyclization, since their nitrogen atoms can
come into proximity with the cyano group.
The plasma glucose level after glucose loading was signifi-
cantly lower in the 1d-treated group than in the vehicle-
To investigate the correlation between stereochemistry and treated control group (Figs. 3A, B). DPP-IV activity was al-
inhibitory activity, all the stereoisomers of 1d were synthe- most completely inhibited at 15 min after glucose loading,
sized. Compound 1d was selected for this investigation be- and this inhibitory effect was retained for 2 h (Fig. 3D).
cause it exhibited the best balance between stability and in- These results indicate that 1d may be useful for the treatment
hibitory activity.
of hyperglycemia, based on its inhibitory effect on plasma
We previously reported the structure–activity relationship DPP-IV activity. Insulin secretion was significantly enhanced
(SAR) for the stereochemistry of 4-fluoride16); here, we re- at 15 min after glucose loading (Fig. 3C). This finding sup-
port the SAR for that of 2-cyanide and a-amino acids at the ports the proposed mechanism that 1d might prevent the in-
P2 site as well as 4-fluoride. (4R)-Fluoride analogue 15 had a activation of active GLP-1 via DPP-IV inhibition and that an
400-fold lower potency (IC50ꢁ246 nM) than 1d, and (2R)- increase in GLP-1 activity might stimulate insulin secretion
cyanide analogue 16 had a more than 500-fold lower potency by acting upon b-cells in the pancreas, resulting in the sup-
(IC50ꢀ300 nM) (Table 5). D-tert-Butylglycine analogue 18 pression of hyperglycemia after glucose loading.
had a 70-fold lower potency (IC50ꢁ42 nM) than 1d, but the
genuine activity level of 18 was uncertain because the Conclusion
amount of commingled L-form 1d could not be determined.
We modified the P2 site of 2-cyano-4-fluoropyrrolidine de-
The other isomers did not exhibit inhibitory activity. We con- rivative 1a, which we previously reported, to obtain a more
cluded that all three chiral centers were important for activity useful DPP-IV inhibitor. As a result, we obtained a stable
because of the dramatic reductions in the activities of the and potent DPP-IV inhibitor, 1d, which is expected to be
stereoisomers of 1d.
useful as a therapeutic agent for lowering postprandial hyper-
We obtained a crystal of 22, the free base of 1d, for X-ray glycemia and treating type 2 diabetes mellitus. Subsequent
crystallographic analysis and observed that the configuration reports will describe the results of further investigations of 4-
of the cyano group and the fluorine atom was cis, while the fluoro-2-cyanopyrrolidines.
conformation of the amide bond was trans (Fig. 2).
Experimental
As 1d exhibited the most favorable inhibitory activity and
chemical stability profiles, the in vivo effect of 1d was exam-
ined in Zucker fatty rats, a model of obesity and impaired
glucose tolerance. Compound 1d was orally administered at
a dose of 1 mg/kg body weight at 30 min before glucose
loading; the plasma glucose, insulin and DPP-IV activity lev-
els were then monitored over time (Fig. 3).
1H-NMR spectroscopy was performed using a Varian VXR-300 or a
JEOL GX500 spectrometer. Chemical shifts were reported in parts per
million relative to tetramethylsilane as an internal standard (in NMR de-
scriptions: sꢁsinglet, dꢁdoublet, tꢁtriplet, qꢁquartet, mꢁmultiplet, and
brꢁbroad peak). 13C-NMR spectroscopy was performed using a JEOL
GX500 spectrometer. 19F-NMR spectroscopy was performed using a Varian
VXR-300 spectrometer. ESI mass spectra were recorded using a Shi-
madzu/Kratos HV-300. High resolution spectra were recorded on a Micro-

1114
Vol. 56, No. 8
Fig. 3. Effects of Oral Administration of 1d (1 mg/kg) on Plasma Glucose, Plasma Insulin and Plasma DPP-IV Activity during OGTT in Zucker Fatty Rats
##
Each point represents the meanꢅS.E. (nꢁ6). ∗ pꢆ0.05 vs. vehicle, Dunnett’s test. pꢆ0.01 vs. vehicle, Student’s t-test.
mass Q-TOF2 instrument. Melting points were measured using a Buchi 535
melting point apparatus without correction. Infrared spectra were recorded
using a Perkin-Elmer 1760 spectrometer. Elemental analyses were per-
formed using a Perkin-Elmer 240C analyzer (for carbon, hydrogen, and ni-
trogen) or a Yokokawa-Denki IC7000P analyzer (for halogens and sulfur).
Analytical thin-layer chromatography was conducted on precoated silica
gel 60 F254 plates (Merck). Chromatography was performed on 100- to
200-mesh silica gel C-200 (Wako Pure Chemical) using the solvent systems
(volume ratios) indicated below.
Degradation of 1a and HPLC Analysis Compound 1a (5 mg) was dis-
solved in pH 6.8 Britton–Robinson buffer solution (5 ml) and incubated at
60 °C. The solution was then analyzed using reverse phase HPLC using a
CAPCELL PAK UG120 (5 mm particle size, f 4.6ꢂ150 mm; SHISEIDO)
and eluted at 1.0 ml/min with acetonitrile–H2O (15 : 85 v/v, 10 mM ammo-
nium acetate solution); its UV absorbance was monitored at 210 nm. To ex-
amine chemical stability, the residual amount was also analyzed using HPLC
in a manner similar to the method described above.
(3S,7S)-7-Fluoro-1-imino-3-[(1S)-1-methylpropyl]hexahydropyr-
rolo[1,2-a]pyrazin-4(1H)-one Acetic Acid Salt (5) Compound 4 (free
base of 1a, 1.30 g, 5.72 mmol) was dissolved in EtOH (4.5 ml). AcOH
(0.36 ml, 6.29 mmol) and n-hexane (12 ml) were then added and the solution
was stirred at room temperature overnight. The resulting powder was col-
lected using filtration and dried in vacuo to yield the desired product (0.73 g,
44%), which was a colorless powder. mp 143—146 °C. 1H-NMR (500 MHz,
DMSO-d6) d 5.34 (1H, dt like, Jꢁ53.1, 3.8 Hz, H-7), 4.35 (1H, dd, Jꢁ11.8,
5.7 Hz, H-8a), 3.87 (1H, ddd, Jꢁ38.4, 14.4, 4.3 Hz, H-6), 3.66 (1H, d,
Jꢁ5.3 Hz, H-3), 3.41 (1H, dd, Jꢁ28.3, 14.4 Hz, H-6), 2.68 (1H, td like,
Jꢁ15.1, 5.6 Hz, H-8), 2.04—1.88 (1H, m, H-8), 1.84 (3H, s, AcOH), 1.77—
1.65 (1H, m), 1.52—1.42 (1H, m), 1.20—1.08 (1H, m), 0.87 (3H, d,
Jꢁ6.7 Hz), 0.85 (3H, t, Jꢁ7.3 Hz); 13C-NMR (125.4 MHz, DMSO-d6)
Jꢁ38.4, 14.3, 4.0 Hz, H-6), 3.55—3.44 (2H, m, H-3, H-6), 2.38 (1H, td like,
Jꢁ15.4, 5.9 Hz, H-8), 2.08 (1H, dddd, Jꢁ43.2, 15.3, 11.6, 3.7 Hz, H-8),
1.82—1.73 (1H, m), 1.57—1.47 (1H, m), 1.18—1.08 (1H, m), 0.89 (3H, d,
Jꢁ7.0 Hz), 0.87 (3H, t, Jꢁ7.3 Hz); 13C-NMR (125.4 MHz, DMSO-d6) d
168.0, 165.0, 90.4 (d, JC–Fꢁ171.5 Hz), 61.4, 55.8, 52.4 (d, JC–Fꢁ23.8 Hz),
38.7, 35.9 (d, JC–Fꢁ20.6 Hz), 24.6, 15.1, 10.9; 19F-NMR (282.2 MHz,
DMSO-d6) d ꢃ172.7. MS (ESI pos.) m/z 251 (MꢄNa)ꢄ; (ESI neg.) m/z 227
(MꢃH)ꢃ. Anal. Calcd for C11H17FN2O2: C, 57.88; H, 7.51; N, 12.27; F, 8.32.
Found: C, 57.86; H, 7.44; N, 12.26; F, 8.35. [a]D23 ꢄ76.0° (cꢁ0.3, MeOH).
tert-Butyl (2S,4S)-2-Cyano-4-fluoropyrrolidine-1-carboxylate (9)
tert-Butyl (2S,4S)-2-(aminocarbonyl)-4-fluoropyrrolidine-1-carboxylate
7
(697 mg, 3.00 mmol) was dissolved in DMF (3 ml), and cyanuric chloride
(332 mg, 1.80 mmol) was added, followed by stirring at room temperature
for 20 min. The reaction solution was taken up in water and extracted with
EtOAc. The organic phase was washed with a saturated aqueous NaCl solu-
tion and dried over MgSO4. The drying agent was removed by filtration, and
the filtrate was concentrated under reduced pressure to yield the desired
product (680 mg, quant.) as a light brown powder. 1H-NMR (300 MHz,
DMSO-d6) d 5.37 (1H, br d, Jꢁ53.3 Hz, H-4), 4.85—4.80 (1H, m, H-2),
3.66—3.38 (2H, m, H-5), 2.58—2.36 (2H, m, H-3), 2.64—2.36 (2H, m),
1.44 (9H, s). MS (ESI pos.) m/z 237 (MꢄNa)ꢄ; (ESI neg.) m/z 249
(MꢄCl)ꢃ.
(2S,4S)-4-Fluoropyrrolidine-2-carbonitrile Hydrochloride (10) Com-
pound 9 (450 mg, 2.10 mmol) was dissolved in MeOH (3 ml), and 4 M HCl
(3 ml) was added, followed by stirring at room temperature for 20 h. The re-
action solution was concentrated in vacuo to yield the desired product
(320 mg, quant.) as a light brown powder. This intermediate was used in the
next reaction without purification. 1H-NMR (300 MHz, DMSO-d6) d 5.53
(1H, br d, Jꢁ52.4 Hz, H-4), 4.97 (1H, dd, Jꢁ8.2, 4.3 Hz, H-2), 3.59 (1H, dd,
Jꢁ22.1, 13.7 Hz, H-5), 3.47 (1H, ddd, Jꢁ35.9, 13.7, 3.7 Hz, H-5), 2.64—
2.43 (2H, m, H-3). MS (ESI pos.) m/z 115 (MꢄH)ꢄ; (ESI neg.) m/z 149
(MꢄCl)ꢃ.
d
173.4, 167.2, 158.2, 90.5 (d, JC–Fꢁ171.5 Hz), 64.1, 54.4, 51.7 (d,
J
C–Fꢁ23.8 Hz), 39.4, 36.5 (d, JC–Fꢁ20.7 Hz), 24.7, 22.0, 15.5, 11.2; 19F-
NMR (282.2 MHz, DMSO-d6) d ꢃ173.4. MS (ESI pos.) m/z 228 (MꢄH)ꢄ,
250 (MꢄNa)ꢄ; (ESI neg.) m/z 226 (MꢃH)ꢃ. Anal. Calcd for C11H18FN3O
C2H4O2 0.5H2O: C, 52.69; H, 7.82; N, 14.18; F, 6.41. Found: C, 52.80; H,
7.93; N, 14.30; F, 6.45. [a]D23 ꢄ99.4° (cꢁ0.3, MeOH).
N-(tert-Butoxycarbonyl)-L-valyl-(4S)-4-fluoro-L-prolinamide
(11b)
(4S)-4-Fluoro-L-prolinamide hydrochloride 8 (500 mg, 2.97 mmol) and N-
(tert-butoxycarbonyl)-L-valine (645 mg, 2.97 mmol) were dissolved in DMF
(10 ml), and 1-hydroxybenzotriazole monohydrate (455 mg, 2.97 mmol),
1-(3,3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (598 mg,
3.12 mmol) and N,N-diisopropylethylamine (0.54 ml) were added while
cooling on ice. The temperature was then gradually increased, and the solu-
tion was stirred at room temperature overnight. The reaction solution was
poured into a saturated aqueous NaCl solution and extracted with EtOAc.
The organic phase was washed with a 10% aqueous KHSO4 solution, a 5%
aqueous NaHCO3 solution and a saturated aqueous NaCl solution, succes-
sively, and then dried over MgSO4. The drying agent was removed by filtra-
tion, and the filtrate was concentrated under reduced pressure to yield the
(3S,7S)-7-Fluoro-3-[(1R)-1-methylpropyl]hexahydropyrrolo[1,2-
a]pyrazine-1,4-dione (3) Compound 5 (240 mg, 1.28 mmol) was dis-
solved in pH 6.8 Britton–Robinson buffer solution (24 ml). The solution was
then stirred at 80 °C for 5 h. The reaction solution was extracted with
EtOAc, and the organic phase was successively washed with a saturated
aqueous NaCl solution and dried over Na2SO4. The drying agent was re-
moved by filtration, and the filtrate was concentrated under reduced pres-
sure. EtOAc and n-hexane were added to the residue, and the resulting pow-
der was collected using filtration and dried in vacuo to yield the desired
product (128 mg, 44%), which was a colorless powder. mp 129—131 °C. 1H-
NMR (500 MHz, DMSO-d6) d 8.48 (1H, d, Jꢁ4.0 Hz), 5.31 (1H, dt like,
Jꢁ53.0, 3.8 Hz, H-7), 4.36 (1H, dd, Jꢁ11.6, 6.1 Hz, H-8a), 3.83 (1H, ddd,
1
desired product (840 mg, 85%), as a colorless amorphous powder. H-NMR
(300 MHz, DMSO-d6) d 7.02 (1H, br s), 6.95—6.85 (2H, m), 5.33 (1H, br d,
Jꢁ53.6 Hz, H-4), 4.43 (1H, dd, Jꢁ9.8, 1.9 Hz, H-2), 4.09—3.73 (3H, m),

August 2008
1115
2.50—2.10 (2H, m, H-3), 2.02—1.84 (1H, m), 1.37 (9H, s), 0.94 (3H, d, H-2), 4.02—3.80 (3H, m), 2.60—2.25 (2H, m, H-3), 1.92—1.80 (1H, m),
Jꢁ6.7 Hz), 0.88 (3H, d, Jꢁ6.7 Hz). MS (ESI pos.) m/z 354 (MꢄNa)ꢄ; (ESI
neg.) m/z 330 (MꢃH)ꢃ.
(4S)-1-((2S)-2-Cyclohexyl-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]-
1.56—1.40 (1H, m), 1.34—1.15 (1H, m), 0.94 (3H, d, Jꢁ6.8 Hz), 0.91 (3H,
t, Jꢁ7.3 Hz). MS (ESI pos.) m/z 228 (MꢄH)ꢄ, 250 (MꢄNa)ꢄ; (ESI neg.)
m/z 262 (MꢄCl)ꢃ. HR-MS Calcd for C11H18FN3O (M)ꢄ 227.1434, Found
(m/z) 227.1438.
amino}acetyl)-4-fluoro-L-prolinamide
(13j) The
title
compound
(880 mg, quant.) was obtained as a colorless amorphous powder from 8
(2S,4S)-4-Fluoro-1-(3-methyl-L-valyl)pyrrolidine-2-carbonitrile Hy-
(300 mg, 1.78 mmol) and (2S)-cyclohexyl{[(9H-fluoren-9-ylmethoxy)car- drochloride (1d) The title compound was obtained as a colorless powder
bonyl]amino}acetic acid (709 mg, 1.87 mmol) in a manner similar to method in a manner similar to method used to prepare 1b. mp 265—269 °C (de-
used to prepare 11b. 1H-NMR (300 MHz, DMSO-d6) d 7.88 (2H, d, comp.). 1H-NMR (500 MHz, DMSO-d6) d 8.54 (3H, br s), 5.55 (1H, br d,
Jꢁ7.5 Hz), 7.73 (2H, d, Jꢁ7.4 Hz), 7.66 (1H, d, Jꢁ8.4 Hz), 7.41 (2H, t,
Jꢁ7.4 Hz), 7.32 (2H, t, Jꢁ7.4 Hz), 7.06 (1H, br s), 6.89 (1H, br s), 5.32 (1H,
Jꢁ51.5 Hz, H-4), 5.07 (1H, d, Jꢁ9.8 Hz, H-2), 4.15—3.93 (2H, m, H-5),
3.79 (1H, s), 2.55—2.32 (2H, m, H-3), 1.05 (9H, s); 13C-NMR (125.4 MHz,
br d, Jꢁ53.8 Hz, H-4), 4.44 (1H, dd, Jꢁ9.7, 2.1 Hz, H-2), 4.33—3.74 (6H, DMSO-d6) d 167.8, 118.3, 92.9 (d, JC–Fꢁ174.7 Hz), 57.5, 54.2 (d, JC–Fꢁ
m), 2.50—2.10 (2H, m, H-3), 1.90—0.90 (11H, m). MS (ESI pos.) m/z 516
22.8 Hz), 44.5, 35.4 (d, JC–Fꢁ20.7 Hz), 34.0, 25.7; 19F-NMR (282.2 MHz,
DMSO-d6) d ꢃ175.4. MS (ESI pos.) m/z 228 ([MꢄH]ꢄ), 250 ([MꢄNa]ꢄ);
(MꢄNa)ꢄ.
tert-Butyl ((1S)-1-{[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]carbon- (ESI neg.) m/z 262 (MꢄCl)ꢃ. HR-MS Calcd for C10H16FN3O(MꢄH)ꢄ
yl}-2-methylpropyl)carbamate (12b) The title compound (656 mg, 91%)
was obtained as colorless amorphous powder from 11b (760 mg,
228.1512, found (m/z) 228.1513. Anal. Calcd for C11H18FN3O HCl H2O:
a
C, 46.89; H, 7.51; N, 14.91. Found: C, 46.74; H, 7.56; N, 14.78. [a]D25
2.29 mmol) in a manner similar to method used to prepare 9. 1H-NMR ꢃ44.4 (cꢁ0.3, MeOH).
(300 MHz, DMSO-d6)
Jꢁ51.5 Hz, H-4), 5.01 (1H, dd, Jꢁ7.3, 2.0 Hz, H-2), 4.11—3.76 (3H, m),
d
7.17 (1H, br d, Jꢁ7.9 Hz), 5.49 (1H, br d,
Benzyl {(5S)-5-Amino-6-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-6-
oxohexyl}carbamate Hydrochloride (1e) The title compound was ob-
2.60—2.25 (2H, m, H-3), 2.01—1.83 (1H, m), 1.36 (9H, s), 0.94 (3H, d, tained as a colorless powder in a manner similar to method used to pre-
Jꢁ6.5 Hz), 0.88 (3H, d, Jꢁ6.8 Hz). MS (ESI pos.) m/z 336 (MꢄNa)ꢄ; (ESI pare 1b. 1H-NMR (300 MHz, DMSO-d6) d 8.42 (3H, br s), 7.42—7.21
neg.) m/z 312 (MꢃH)ꢃ.
(6H, m), 5.54 (1H, br d, Jꢁ51.0 Hz, H-4), 5.08 (1H, br d, Jꢁ8.2 Hz, H-2),
9H-Fluoren-9-ylmethyl ((1S,2R)-2-tert-Butoxy-1-{[(2S,4S)-2-cyano-4- 5.00 (2H, s), 4.06—3.66 (3H, m), 3.09—2.90 (2H, m), 2.62—2.26 (2H,
fluoropyrrolidin-1-yl]carbonyl}propyl)carbamate (14h) The title com- m, H-3), 1.85—1.65 (2H, m), 1.50—1.25 (4H, m). MS (ESI pos.) m/z 399
pound (870 mg, 88%) was obtained as a colorless amorphous powder from
(MꢄNa)ꢄ; (ESI neg.) m/z 411 (MꢄCl)ꢃ, 375 (MꢃH)ꢃ. HR-MS Calcd for
10 (301 mg, 2.0 mmol) and O-(tert-butyl)-N-[(9H-fluoren-9-ylmethoxy)car- C19H25FN4O3 (M)ꢄ 376.1911, Found (m/z) 376.1930.
bonyl]-L-threonine (795 mg, 2.0 mmol) in a manner similar to method used
to prepare 11b. H-NMR (300 MHz, DMSO-d6) d 7.89 (2H, d, Jꢁ7.3 Hz),
7.74 (2H, dd, Jꢁ7.3, 2.2 Hz), 7.41 (2H, t, Jꢁ7.3 Hz), 7.37—7.28 (2H, m),
(2S,4S)-4-Fluoro-1-(O-methyl-L-threonyl)pyrrolidine-2-carbonitrile
1
Hydrochloride (1f) The title compound was obtained as a colorless pow-
der in a manner similar to method used to prepare 1b. mp 149—152 °C. 1H-
5.48 (1H, br d, Jꢁ51.6 Hz, H-4), 5.04—4.97 (1H, m, H-2), 4.36—4.16 (4H, NMR (500 MHz, DMSO-d6) d 8.62 (3H, br s), 5.54 (1H, br d, Jꢁ51.7 Hz, H-
m), 3.94—3.70 (2H, m, H-5), 2.60—2.30 (2H, m, H-3), 1.16 (9H, s), 1.09 4), 5.07 (1H, d, Jꢁ8.8 Hz, H-2), 4.17—3.93 (3H, m), 3.70 (1H, quintet,
(3H, d, Jꢁ6.2 Hz). MS (ESI pos.) m/z 516 (MꢄNa)ꢄ.
Jꢁ6.4 Hz), 3.33 (3H, s), 2.55—2.33 (2H, m, H-3), 1.17 (3H, d, Jꢁ6.4 Hz);
9H-Fluoren-9-ylmethyl {(1S)-2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin- 13C-NMR (125.4 MHz, DMSO-d6) d 166.5, 118.2, 92.9 (d, JC–Fꢁ174.7 Hz),
1-yl]-1-cyclohexyl-2-oxoethyl}carbamate (14j) Compound 13j (860 mg,
75.5, 56.5, 54.3, 53.4 (d, JC–Fꢁ22.7 Hz), 44.9, 35.4 (d, JC–Fꢁ19.6 Hz), 14.6;
1.74 mmol) was dissolved in THF (20 ml), and trifluoroacetic anhydride 19F-NMR (282.2 MHz, DMSO-d6) d ꢃ174.8. MS (ESI pos.) m/z 252
(0.49 ml, 3.48 mmol) was added while cooling on ice. The solution was then (MꢄNa)ꢄ; (ESI neg.) m/z 264 (MꢄCl)ꢃ. HR-MS Calcd for C10H17FN3O2
stirred on ice for 1 h. The reaction solution was concentrated in vacuo, and (MꢄH)ꢄ 230.1305, Found (m/z) 230.1308. Anal. Calcd for C10H16FN3O2
the residue was purified by silica gel column chromatography (developing HCl 1/3H2O: C, 44.20; H, 6.55; N, 15.46; F, 6.99; Cl, 13.05. Found: C,
solvent: hexane–EtOAcꢁ3 : 1—1 : 2) to yield the desired product (760 mg,
44.58; H, 6.77; N, 15.03; F, 6.87; Cl,12.71. [a]D25 ꢃ80.8° (cꢁ0.3, MeOH).
(2S,4S)-1-(O-Benzyl-L-threonyl)-4-fluoropyrrolidine-2-carbonitrile
1
92%) as a colorless amorphous powder. H-NMR (300 MHz, DMSO-d6) d
7.90—7.82 (3H, m), 7.72 (2H, d, Jꢁ7.3 Hz), 7.45—7.28 (5H, m), 5.49 (1H, Hydrochloride (1g) The title compound was obtained as a brown powder
br d, Jꢁ51.6 Hz, H-4), 5.04—4.97 (1H, m, H-2), 4.32—3.78 (6H, m), in a manner similar to method used to prepare 1b. 1H-NMR (300 MHz,
2.60—2.25 (2H, m, H-3), 1.92—0.92 (11H, m). MS (ESI pos.) m/z 498 DMSO-d6)
d 8.57 (3H, br s), 7.42—7.23 (5H, m), 5.53 (1H, br d,
(MꢄNa)ꢄ.
Jꢁ52.1 Hz, H-4), 5.12 (1H, br d, Jꢁ8.4 Hz, H-2), 4.62 and 4.56 (2H, ABq,
(2S,4S)-4-Fluoro-1-L-valylpyrrolidine-2-carbonitrile
(1b) MeOH (10.9 ml) and 4 M aqueous HCl solution (10.9 ml) were added
to compound 12b (1.70 g, 5.43 mmol) while cooling on ice, then stirred at
Hydrochloride Jꢁ11.8 Hz), 4.20—3.85 (4H, m), 2.60—2.30 (2H, m, H-3), 1.23 (3H, d,
Jꢁ6.4 Hz). MS (ESI pos.) m/z 306 (MꢄH)ꢄ, 328 (MꢄNa)ꢄ; (ESI neg.) m/z
340 (MꢄCl)ꢃ. HR-MS Calcd for C16H21FN3O2 (MꢄH)ꢄ 306.1618, Found
room temperature overnight. Four molar aqueous NaOH solution (12 ml) (m/z) 306.1615.
and an excess amount of NaCl were added to the solution, and the resulting
(2S,4S)-4-Fluoro-1-(N-methyl-L-isoleucyl)pyrrolidine-2-carbonitrile
solution was extracted with EtOAc. The organic phase was washed with a Hydrochloride (1k) The title compound was obtained as a colorless amor-
1
saturated aqueous NaCl solution and dried over Na2SO4. After removing the phous powder in a manner similar to method used to prepare 1b. H-NMR
drying agent using filtration, the filtrate was concentrated under reduc- (300 MHz, CD3OD) d 5.48 (1H, br d, Jꢁ51.9 Hz, H-4), 5.12 (1H, br d,
ed pressure to obtain (2S,4S)-4-fluoro-1-L-valylpyrrolidine-2-carbonitrile Jꢁ8.2 Hz, H-2), 4.19—3.80 (3H, m), 2.71—2.33 (2H, m, H-3), 2.68 (3H, s),
(800 mg, 70%), 600 mg of which was then dissolved in MeOH (1.2 ml) and
4 M HCl/EtOAc (0.77 ml) while cooling on ice, stirred for 1 h, and poured
2.10—1.93 (1H, m), 1.79—1.63 (1H, m), 1.37—1.17 (1H, m), 1.13 (3H, d,
Jꢁ7.0 Hz), 1.02 (3H, t, Jꢁ7.3 Hz). MS (ESI pos.) m/z 242 (MꢄH)ꢄ, 264
into diisopropyl ether (20 ml). The precipitated insoluble substance was col- (MꢄNa)ꢄ; (ESI neg.) m/z 276 (MꢄCl)ꢃ.
lected using filtration to yield the desired product (750 mg) as a colorless
powder. mp 256—259 °C (decomp.). 1H-NMR (500 MHz, DMSO-d6) d 8.57
(3H, br s), 5.55 (1H, br d, Jꢁ51.8 Hz, H-4), 5.06 (1H, d, Jꢁ9.2 Hz, H-2),
(2S,4S)-4-Fluoro-1-L-prolylpyrrolidine-2-carbonitrile Hydrochloride
(1l) The title compound was obtained as a pale orange powder in a manner
similar to method used to prepare 1b. 1H-NMR (300 MHz, DMSO-d6) d
4.08—3.90 (2H, m, H-5), 3.83 (1H, d, Jꢁ7.3 Hz), 2.55—2.34 (2H, m, H-3), 5.56 (1H, br d, Jꢁ51.6 Hz, H-4), 5.12—5.05 (1H, m, H-2), 4.37 (1H, t,
2.17—2.07 (1H, m), 1.01 (3H, d, Jꢁ6.7 Hz), 0.98 (3H, d, Jꢁ6.7 Hz); 13C-
NMR (125.4 MHz, DMSO-d6) d 168.2, 118.3, 92.9 (d, JC–Fꢁ174.7 Hz),
Jꢁ7.3 Hz), 3.96—3.74 (2H, m, H-5), 2.62—2.32 (2H, m), 2.00—1.87 (2H,
m), 1.85—1.71 (1H, m). MS (ESI pos.) m/z 212 (MꢄH)ꢄ, 234 (MꢄNa)ꢄ;
55.2, 53.5 (d, JC–Fꢁ21.7 Hz), 44.7, 35.4 (d, JC–Fꢁ20.7 Hz), 29.9, 17.8, 17.7; (ESI neg.) m/z 246 (MꢄCl)ꢃ. HR-MS Calcd for C10H14FN3O (M)ꢄ
19F-NMR (282.2 MHz, DMSO-d6) d ꢃ175.2. MS (ESI pos.) m/z 214
(MꢄH)ꢄ, 236 (MꢄNa)ꢄ; (ESI neg.) m/z 248 (MꢄCl)ꢃ. HR-MS Calcd for
211.1121, Found (m/z) 211.1129.
(2S,4S)-4-Fluoro-1-[(3S)-1,2,3,4-tetrahydroisoquinolin-3-ylca-
C10H17FN3O (MꢄH)ꢄ 214.1356, Found (m/z) 214.1355. Anal. Calcd for rbonyl]pyrrolidine-2-carbonitrile Hydrochloride (1m) The title com-
C10H16FN3O HCl H2O: C, 44.86; H, 7.15; N, 15.69; F, 7.10; Cl, 13.24. pound was obtained as a colorless amorphous powder in a manner similar to
Found: C, 44.67; H, 7.09; N, 15.75; F, 6.97; Cl,12.90. [a]D25 ꢃ68.2° (cꢁ0.3, method used to prepare 1b. 1H-NMR (300 MHz, DMSO-d6) d 9.94 (2H,
MeOH).
(2S,4S)-1-L-Alloisoleucyl-4-fluoropyrrolidine-2-carbonitrile Hydro-
br s), 7.34—7.24 (4H, m), 5.59 (1H, br d, Jꢁ52.7 Hz, H-4), 5.19—5.13 (1H,
m, H-2), 4.50 (1H, dd, Jꢁ12.1, 4.4 Hz), 4.32 (2H, s), 4.18—3.87 (2H, m, H-
chloride (1c) The title compound was obtained as a colorless powder in a 5), 3.40 (1H, dd, Jꢁ16.6, 4.4 Hz), 3.01 (1H, dd, Jꢁ16.6, 12.1 Hz), 2.63—
1
manner similar to method used to prepare 1b. H-NMR (300 MHz, DMSO-
2.34 (2H, m, H-3). MS (ESI pos.) m/z 274 (MꢄH)ꢄ, 296 (MꢄNa)ꢄ; (ESI
d6) d 8.35 (3H, br s), 5.55 (1H, br d, Jꢁ51.0 Hz, H-4), 5.12—5.06 (1H, m, neg.) m/z 272 (MꢃH)ꢃ, 308 (MꢄCl)ꢃ. HR-MS Calcd for C15H17FN3O

1116
Vol. 56, No. 8
(MꢄH)ꢄ 274.1356, Found (m/z) 274.1339.
(300 MHz, DMSO-d6) d 7.47 (1H, br s), 7.36 (1H, br s), 7.23—7.11 (1H, m),
6.88—6.75 (2H, m), 5.55 (1H, br d, Jꢁ53.9 Hz, H-4), 5.07—4.46 (3H, m),
4.39 (2H, s), 4.35—3.60 (3H, m), 3.18—3.02 (1H, m), 2.94—2.70 (1H, m),
2.58—2.20 (2H, m), 1.43, 1.38 and 1.33 (total 9H, each s). MS (ESI pos.)
m/z 469 (MꢄNa)ꢄ; (ESI neg.) m/z 445 (MꢃH)ꢃ.
(2S,4S)-4-Fluoro-1-{[(3S)-7-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-
yl]carbonyl}pyrrolidine-2-carbonitrile Hydrochloride (1n) The title
compound was obtained as a colorless amorphous powder in a manner simi-
lar to method used to prepare 1b. 1H-NMR (300 MHz, DMSO-d6) d 9.81
(2H, br s), 9.57 (1H, s), 7.09 (1H, d, Jꢁ8.4 Hz), 6.72 (1H, dd, Jꢁ8.4,
2.4 Hz), 6.65 (1H, d, Jꢁ2.4 Hz), 5.57 (1H, br d, Jꢁ52.1 Hz, H-4), 5.18—
5.12 (1H, m, H-2), 4.43 (1H, dd, Jꢁ12.1, 4.2 Hz), 4.22 (2H, s), 4.14—3.87
(2H, m, H-5), 3.27 (1H, dd, Jꢁ16.2, 4.2 Hz), 2.88 (1H, dd, Jꢁ16.2,
12.1 Hz), 2.62—2.32 (2H, m, H-3). MS (ESI pos.) m/z 290 (MꢄH)ꢄ, 312
(MꢄNa)ꢄ; (ESI neg.) m/z 288 (MꢃH)ꢃ, 324 (MꢄCl)ꢃ. HR-MS Calcd for
C15H17FN3O2 (MꢄH)ꢄ 290.1305, Found (m/z) 290.1304.
(2S,4S)-1-{[(3S)-6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-3-yl]-
carbonyl}-4-fluoropyrrolidine-2-carbonitrile Hydrochloride (1o) The
title compound was obtained as a colorless amorphous powder in a man-
ner similar to method used to prepare 1b. 1H-NMR (300 MHz, DMSO-d6) d
9.86 (2H, br s), 6.91 (1H, s), 6.87 (1H, s), 5.56 (1H, br d, Jꢁ52.4 Hz, H-4),
5.19—5.13 (1H, m, H-2), 4.44 (1H, dd, Jꢁ12.4, 4.4 Hz), 4.21 (2H, s),
4.15—3.85 (2H, m, H-5), 3.733 (3H, s), 3.730 (3H, s), 3.35—3.25 (1H, m),
2.91 (1H, dd, Jꢁ16.1, 12.4 Hz), 2.63—2.34 (2H, m, H-3). MS (ESI pos.)
m/z 334 (MꢄH)ꢄ, 356 (MꢄNa)ꢄ; (ESI neg.) m/z 368 (MꢄCl)ꢃ. HR-MS
Calcd for C17H21FN3O3 (MꢄH)ꢄ 334.1567, Found (m/z) 334.1566.
(2S,4S)-1-[O-(tert-Butyl)-L-threonyl]-4-fluoropyrrolidine-2-carboni-
trile Hydrochloride (1h) Compound 14h (710 mg, 1.44 mmol) was dis-
solved in THF (7 ml), diethylamine (0.7 ml) was added, and the solution was
continuously stirred at room temperature for 2 h. The solution was concen-
trated in vacuo, and the residue was purified by silica gel column chro-
matography (developing solvent: CHCl3–MeOHꢁ100 : 3—100 : 5). The re-
sulting residue was dissolved in diethyl ether (20 ml) and, after the addition
of 4-M HCl/EtOAc (0.36 ml), the resulting salt was collected using filtration
and dried in vacuo to yield the desired product (278 mg, 59%) as a colorless
powder. 1H-NMR (300 MHz, DMSO-d6) d 8.38 (3H, br s), 5.53 (1H, br d,
Jꢁ52.4 Hz, H-4), 5.09 (1H, br d, Jꢁ8.2 Hz, H-2), 4.33—3.83 (4H, m),
2.58—2.25 (2H, m, H-3), 1.18 (9H, s) , 1.15 (3H, d, Jꢁ6.2 Hz). MS (ESI
pos.) m/z 294 (MꢄNa)ꢄ; (ESI neg.) m/z 306 (MꢄCl)ꢃ. HR-MS Calcd for
C13H23FN3O2 (MꢄH)ꢄ 272.1774, Found (m/z) 272.1793.
Benzyl 6-({5-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-(4S)-4-amino-
5-oxopentanoyl}amino)hexanoate (1i) The title compound was obtained
as a colorless powder in a manner similar to method used to prepare 1h. 1H-
NMR (300 MHz, DMSO-d6) d 8.49 (3H, br s), 8.06 (1H, t, Jꢁ5.6 Hz),
7.42—7.29 (5H, m), 5.54 (1H, br d, Jꢁ51.9 Hz, H-4), 5.08 (2H, s), 5.08—
5.04 (1H, m, H-2), 4.13—3.66 (3H, m), 3.10—2.96 (2H, m), 2.55—2.18
(6H, m), 2.00—1.83 (2H, m), 1.60—1.48 (2H, m), 1.45—1.34 (2H, m),
1.32—1.20 (2H, m). MS (ESI pos.) m/z 469 (MꢄNa)ꢄ; (ESI neg.) m/z 481
(MꢄCl)ꢃ. HR-MS Calcd for C23H31FN4O4 (M)ꢄ 446.2329, Found (m/z)
446.2328.
(2S,4S)-1-[(2S)-2-Amino-2-cyclohexylacetyl]-4-fluoropyrrolidine-2-
carbonitrile Hydrochloride (1j) The title compound was obtained as a
colorless powder in a manner similar to method used to prepare 1h. 1H-
NMR (300 MHz, DMSO-d6) d 8.48 (3H, br s), 5.54 (1H, br d, Jꢁ51.6 Hz, H-
4), 5.06 (1H, d, Jꢁ8.7 Hz, H-2), 4.10—3.78 (3H, m), 2.60—2.26 (2H, m, H-
3), 1.86—1.51 (6H, m), 1.30—1.00 (5H, m). MS (ESI pos.) m/z 276
(MꢄNa)ꢄ; (ESI neg.) m/z 288 (MꢄCl)ꢃ. HR-MS Calcd for C13H20FN3O
(M)ꢄ 253.1590, Found (m/z) 253.1605.
(2S,4S)-4-Fluoro-1-{[(3S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-
yl]carbonyl}pyrrolidine-2-carbonitrile Hydrochloride (1p) The title
compound was obtained as a colorless amorphous powder in a manner simi-
lar to method used to prepare 1b. 1H-NMR (300 MHz, DMSO-d6) d 9.96
(2H, br s), 7.21 (1H, d, Jꢁ9.3 Hz), 6.90—6.85 (2H, m), 5.58 (1H, br d,
Jꢁ52.3 Hz, H-4), 5.19—5.13 (1H, m, H-2), 4.46 (1H, dd, Jꢁ12.2, 4.3 Hz),
4.28 (2H, s), 4.16—3.87 (2H, m, H-5), 3.74 (3H, s), 3.34 (1H, dd, Jꢁ16.4,
4.3 Hz), 2.92 (1H, dd, Jꢁ16.4, 12.2 Hz), 2.63—2.33 (2H, m, H-3). MS (ESI
pos.) m/z 304 (MꢄH)ꢄ, 326 (MꢄNa)ꢄ; (ESI neg.) m/z 338 (MꢄCl)ꢃ. HR-
MS Calcd for C16H19FN3O2 (MꢄH)ꢄ 304.1461, Found (m/z) 304.1456.
2-[((3S)-3-{[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]carbonyl}-
1,2,3,4-tetrahydroisoquinolin-7-yl)oxy]acetamide Hydrochloride (1q)
The title compound was obtained as a colorless amorphous powder in a
1
manner similar to method used to prepare 1b. H-NMR (300 MHz, DMSO-
d6) d 9.78 (2H, br s), 7.52 (1H, br s), 7.39 (1H, br s), 7.23 (1H, d, Jꢁ8.4 Hz),
6.94—6.85 (2H, m), 5.58 (1H, br d, Jꢁ52.1 Hz, H-4), 5.19—5.13 (1H, m,
H-2), 4.47 (1H, dd, Jꢁ11.8, 4.0 Hz), 4.42 (2H, s), 4.27 (2H, s), 4.16—3.84
(2H, m, H-5), 3.41—3.25 (1H, m), 2.92 (1H, dd, Jꢁ16.6, 12.4 Hz), 2.60—
2.34 (2H, m, H-3). MS (ESI pos.) m/z 347 (MꢄH)ꢄ, 369 (MꢄNa)ꢄ; (ESI
neg.) m/z 345 (MꢃH)ꢃ, 381 (MꢄCl)ꢃ. HR-MS Calcd for C17H20FN4O3
(MꢄH)ꢄ 347.1519, Found (m/z) 347.1527.
(2S,4R)-4-Fluoro-1-(3-methyl-L-valyl)pyrrolidine-2-carbonitrile (15)
The title compound was obtained as a colorless amorphous powder in a
1
manner similar to method used to prepare 1d. H-NMR (500 MHz, DMSO-
d6) d 5.37 (1H, br d, Jꢁ52.1 Hz, H-4), 4.75 (1H, dd, Jꢁ9.4, 7.9 Hz, H-2),
4.13 (1H, ddd, Jꢁ21.1, 12.7, 2.0 Hz, H-5), 3.72 (1H, ddd, Jꢁ38.5, 12.6,
2.8 Hz, H-5), 3.22 (1H, s), 2.73—2.63 (1H, m, H-3), 2.55—2.40 (1H, m, H-
3), 1.63 (2H, br s), 0.91 (9H, s); 13C-NMR (125.4 MHz, DMSO-d6) d 174.2,
118.5, 91.8 (d, JC–Fꢁ176.7 Hz), 59.2, 53.3 (d, JC–Fꢁ21.7 Hz), 44.3, 35.4 (d,
J
C–Fꢁ21.7 Hz), 35.2, 25.9; 19F-NMR (282.2 MHz, DMSO-d6) d ꢃ176.9. MS
(ESI pos.) m/z 250 (MꢄNa)ꢄ; (ESI neg.) m/z 226 (MꢃH)ꢃ. HR-MS Calcd
for C11H19FN3O (MꢄH)ꢄ 228.1512, Found (m/z) 228.1517. Anal. Calcd for
C11H18FN3O: C, 58.13; H, 7.98; N, 18.49; F, 8.36. Found: C, 57.93; H, 8.00;
N, 18.34; F, 8.60. [a]D25 ꢃ60.1° (cꢁ0.3, MeOH).
(2R,4R)-4-Fluoro-1-(3-methyl-L-valyl)pyrrolidine-2-carbonitrile (17)
The title compound was obtained as a colorless amorphous powder in a
manner similar to method used to prepare 1d. H-NMR (500 MHz, DMSO-
1
d6) d 5.45 (1H, br d, Jꢁ48.8 Hz, H-4), 4.96—4.88 (1H, m, H-2), 4.08 (1H,
dd, Jꢁ25.0, 12.2 Hz, H-5), 3.73 (1H, ddd, Jꢁ38.8, 12.2, 3.4 Hz, H-5), 3.12
(1H, s), 2.58—2.35 (2H, m, H-3), 1.70 (2H, br s), 0.89 (9H, s); 13C-NMR
(125.4 MHz, DMSO-d6) d 174.2, 118.8, 93.1 (d, JC–Fꢁ174.3 Hz), 59.3, 53.2
(d, JC–Fꢁ22.7 Hz), 44.4, 35.6 (d, JC–Fꢁ20.6 Hz), 34.7, 26.0; 19F-NMR
(282.2 MHz, DMSO-d6) d ꢃ174.3. MS (ESI pos.) m/z 250 (MꢄNa)ꢄ; (ESI
neg.) m/z 226 (MꢃH)ꢃ. HR-MS Calcd for C11H19FN3O (MꢄH)ꢄ 228.1512,
Found (m/z) 228.1515. Anal. Calcd for C11H18FN3O: C, 58.13; H, 7.98; N,
18.49; F, 8.36. Found: C, 58.01; H, 8.04; N, 18.45; F, 8.35. [a]D25 ꢄ163.4°
(cꢁ0.3, MeOH).
(2S,4R)-4-Fluoro-1-(3-methyl-D-valyl)pyrrolidine-2-carbonitrile (19)
The title compound was obtained as a colorless amorphous powder in a
1
tert-Butyl (3S)-3-{[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]carbonyl}-
7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (12p) Com-
pound 12n (630 mg, 1.62 mmol) was dissolved in DMF (6 ml) and K2CO3
(250 mg, 1.81 mmol) and methyl iodide (0.15 ml, 2.41 mmol) were added;
the solution was then stirred at room temperature overnight. The reaction so-
lution was diluted with EtOAc and washed with 0.5 M HCl aqueous solution,
NaHCO3 solution and a saturated aqueous NaCl solution, successively. The
organic phase was then dried over Na2SO4. After the removal of the drying
agent using filtration, the solvent was evaporated in vacuo and diisopropyl
ether was added to the residue. The resulting precipitate was collected using
filtration and dried in vacuo to yield the desired product (420 mg, 64%) as a
pale yellow powder. 1H-NMR (300 MHz, DMSO-d6) d 7.22—7.10 (1H, m),
6.88—6.73 (2H, m), 5.56 (1H, br d, Jꢁ52.5 Hz, H-4), 5.06—3.78 (6H, m),
3.73 (3H, s), 3.20—2.25 (4H, m), 1.43, 1.37 and 1.33 (total 9H, each s). MS
(ESI pos.) m/z 426 (MꢄNa)ꢄ.
manner similar to method used to prepare 1d. H-NMR (500 MHz, DMSO-
d6) d 5.39 (1H, br d, Jꢁ52.1 Hz, H-4), 4.79 (1H, t, Jꢁ8.6 Hz, H-2), 4.08
(1H, ddd, Jꢁ20.3, 13.0, 1.8 Hz, H-5), 3.79 (1H, ddd, Jꢁ37.8, 12.8, 3.1 Hz,
H-5), 3.19 (1H, s), 2.73—2.63 (1H, m, H-3), 2.53—2.38 (1H, m, H-3), 1.68
(2H, br s), 0.90 (9H, s); 13C-NMR (125.4 MHz, DMSO-d6) d 174.2, 118.7,
91.9 (d, JC–Fꢁ175.7 Hz), 59.0, 52.9 (d, JC–Fꢁ21.7 Hz), 44.3, 35.6 (d,
J
C–Fꢁ21.7 Hz), 34.6, 25.9; 19F-NMR (282.2 MHz, DMSO-d6) d ꢃ178.0. MS
(ESI pos.) m/z 250 (MꢄNa)ꢄ; (ESI neg.) m/z 226 (MꢃH)ꢃ. HR-MS Calcd
for C11H19FN3O (MꢄH)ꢄ 228.1512, Found (m/z) 228.1514. Anal. Calcd for
C11H18FN3O: C, 58.13; H, 7.98; N, 18.49; F, 8.36. Found: C, 57.99; H, 8.05;
N, 18.42; F, 8.39. [a]D25 ꢃ190.0° (cꢁ0.3, MeOH).
(2R,4R)-4-Fluoro-1-(3-methyl-D-valyl)pyrrolidine-2-carbonitrile (21)
The title compound was obtained as a colorless amorphous powder in a
manner similar to method used to prepare 1d. H-NMR (500 MHz, DMSO-
1
d6) d 5.47 (1H, br d, Jꢁ52.5 Hz, H-4), 4.98 (1H, br d, Jꢁ5.5 Hz, H-2), 3.98
(1H, dd like, Jꢁ25.7, 12.5 Hz, H-5), 3.82 (1H, ddd, Jꢁ39.6, 12.5, 3.3 Hz, H-
5), 3.14 (1H, s), 2.48—2.33 (2H, m, H-3), 1.60 (2H, br s), 0.93 (9H, s); 13C-
NMR (125.4 MHz, DMSO-d6) d 174.1, 118.8, 93.2 (d, JC–Fꢁ174.3 Hz),
58.9, 53.4 (d, JC–Fꢁ21.7 Hz), 44.1, 35.3 (d, JC–Fꢁ20.6 Hz), 35.0, 25.8; 19F-
NMR (282.2 MHz, DMSO-d6) d ꢃ174.5. MS (ESI pos.) m/z 250 (MꢄNa)ꢄ;
tert-Butyl (3S)-7-(2-Amino-2-oxoethoxy)-3-{[(2S,4S)-2-cyano-4-fluoro-
pyrrolidin-1-yl]carbonyl}-3,4-dihydroisoquinoline-2(1H)-carboxylate
(12q) The title compound (550 mg, 80%) was obtained as a colorless
powder from 12n (600 mg, 1.54 mmol) and 2-bromoacetamide (320 mg,
2.27 mmol) in a manner similar to method used to prepare 12p. 1H-NMR

August 2008
1117
(ESI neg.) m/z 226 (MꢃH)ꢃ. HR-MS Calcd for C11H19FN3O (MꢄH)ꢄ
228.1512, Found (m/z) 228.1517. Anal. Calcd for C11H18FN3O: C, 58.13; H,
7.98; N, 18.49; F, 8.36. Found: C, 57.78; H, 8.06; N, 18.42; F, 8.38. [a]D25
ꢄ33.7° (cꢁ0.3, MeOH).
(1993).
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(2000).
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(1999).
9) Holst J. J., Deacon C. F., Diabetes, 47, 1663—1670 (1998).
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gold B. L., Mone M. D., Russell M. E., Weldon S. C., Hughes T. E., J.
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S. P., Abboa-Offei B., Cap M., Xin L., Tao L., Tozzo E., Welzel G. E.,
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X-Ray Crystal Analysis Single crystals of 22 (free base of 1d) that
were suitable for X-ray crystallography were grown by crystallization
from chloroform/ethyl acetate. Data were collected at 288 K on a Mac Sci-
ence/Bruker axs MXC18 four-circle automated diffractometer using CuKa
radiation (lꢁ1.54178). The compound with the chemical formula of
C11H18F1N3O1 crystallized in the monoclinic space group P21, aꢁ9.642(2),
bꢁ10.662(2), cꢁ6.1103(11) Å, bꢁ106.793(13)°, Vꢁ601.4(2) Å3, Zꢁ2. The
structure was solved by a direct method using the program maXus and was
refined using the full-matrix least square method. All H-atom positions were
located from a difference Fourier synthesis. All non-hydrogen atoms were
refined anisotropically, and all H-atoms were refined isotropically. The final
R and Rw values were 0.039 and 0.068, respectively.
DPP-IV Inhibitory Activity The inhibition of DPP-IV activity was
tested using a method described by Deacon et al.23) Plasma containing DPP-
IV was prepared by centrifuging blood collected from healthy human volun-
teers. Enzyme reactions were carried out using 96-flat-bottom-well plates in
a buffer solution of pH 7.8 containing 25 mM HEPES, 140 mM NaCl, and 1%
BSA. To a mixture of 25 ml of 100 mM Gly-Pro-4-methylcoumaryl-7-amide
solution (manufactured by Peptide Institute, Inc.), 7.5 ml of 133 mM MgCl2
solution, 5 ml of the test compound, and 12.5 ml of plasma diluted to 1/100
with the above buffer solution were added. The solution was allowed to react
at room temperature for 2 h, and 50 ml of 25% aqueous acetic acid solution
was added to stop the reaction. The fluorescence intensity of the liberated 7-
amino-4-methylcoumarin was determined using a fluorescence plate reader
(1420 ARVOTM Multilabel Counter manufactured by Wallac Oy; Excitation:
390 nm; Emission: 460 nm).
Oral Glucose Tolerance Test (OGTT) in Zucker Fatty Rats An
OGTT in Zucker fatty rats was carried out based on the method described by
Balkan et al.24) Food was withheld overnight from male Zucker fatty and
lean rats (10 weeks of age; nꢁ6). Compound 1d was then dissolved in dis-
tilled water and administered orally. After 30 min, a glucose solution (2 g/kg
body weight) was orally administered. Blood samples were collected from
the orbital venous sinus under ether anesthesia at the indicated times, and
plasma samples were prepared. The plasma glucose concentration, plasma
insulin concentration, and plasma DPP-IV activity were measured.
18) Magnin D. R., Robl J. A., Sulsky R. B., Augeri D. J., Huang Y., Simp-
kins L. M., Taunk P. C., Betebenner D. A., Robertson J. G., Abboa-
Offei B. E., Wang A., Cap M., Xin L., Tao L., Sitkoff D. F., Malley M.
F., Gougoutas J. Z., Khanna A., Huang Q., Han S., Parker R. A.,
Hamann L. G., J. Med. Chem., 47, 2587—2598 (2004).
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20) Maetz P., Rodriguez M., Tetrahedron Lett., 38, 4221—4222 (1997).
21) Rasmussen H. B., Branner S., Wiberg F. C., Wagtmann N., Nat. Struct.
Biol., 10, 19—25 (2003).
Acknowledgment The authors wish to thank the following individuals
for their assistance with this work: Keita Matsumoto, Atsushi Okada, Noriko
Takahashi, Ayumi Koda, and Miho Kawanishi.
22) Hiramatsu H., Kyono K., Higashiyama Y., Fukushima C., Shima H.,
Sugiyama S., Inaka K., Yamamoto A., Shimizu R., Biochem. Biophys.
Res. Commun., 302, 849—854 (2003).
23) Deacon C. F., Hughes T. E., Holst J. J., Diabetes, 47, 764—769 (1998).
24) Balkan B., Kwasnik L., Miserendino R., Holst J. J., Li X., Diabetolo-
gia, 42, 1324—1331 (1999).
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