Design, synthesis and biological evaluation of 4-fluoropyrrolidine-2- carbonitrile and octahydrocyclopenta[b]pyrrole-2-carbonitrile derivatives as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.08.059

Based on the previous work in our group and the principle of computer-aided drug design, a series of novel β-amino pyrrole-2-carbonitrile derivatives was designed and synthesized. Compounds 8l and 9l were efficacious and selective DPP4 inhibitors resulting in decreased blood glucose in vivo. Compound 8l had moderate DPP4 inhibitory activity (IC₅₀ = 0.05 μM) and good oral bioavailability (F = 53.2%). Compound 9l showed excellent DPP4 inhibitory activity (IC₅₀ = 0.01 μM), good selectivity (selective ratio: DPP8/DPP4 = 898.00; DPP9/DPP4 = 566.00) against related peptidases, and good efficacy in an oral glucose tolerance tests in ICR mice and moderate PK profiles (F = 22.8%, t_1/2 = 2.74 h). Moreover, compound 9l did not block hERG channel and exhibited no inhibition of liver metabolic enzymes such as CYP2C9.

Full text of DOI:10.1016/j.ejmech.2014.08.059

European Journal of Medicinal Chemistry 86 (2014) 242e256
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of 4-fluoropyrrolidine-2-
carbonitrile and octahydrocyclopenta[b]pyrrole-2-carbonitrile
derivatives as dipeptidyl peptidase IV inhibitors
,
b
,
,
,
Xun Ji a 1, Chunmei Xia a 1, Jiang Wang a, Mingbo Su a c, Lei Zhang a, Tiancheng Dong a,
Zeng Li a, Xia Wan a, Jingya Li a, Jia Li a *, Linxiang Zhao b, Zhaobing Gao a,
,
Hualiang Jiang a **, Hong Liu a
,
b
,
, *
a CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
People's Republic of China
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, People's Republic of China
c East China of Normal University, 3663 Zhongshan Road, Shanghai 200062, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the previous work in our group and the principle of computer-aided drug design, a series of
Received 1 April 2014
Received in revised form
15 August 2014
Accepted 16 August 2014
Available online 19 August 2014
novel
were efficacious and selective DPP4 inhibitors resulting in decreased blood glucose in vivo. Compound 8l
had moderate DPP4 inhibitory activity (IC50 ¼ 0.05 M) and good oral bioavailability (F ¼ 53.2%).
M), good selectivity (selective
ratio: DPP8/DPP4 ¼ 898.00; DPP9/DPP4 ¼ 566.00) against related peptidases, and good efficacy in an oral
glucose tolerance tests in ICR mice and moderate PK profiles (F ¼ 22.8%, t1/2 ¼ 2.74 h). Moreover,
compound 9l did not block hERG channel and exhibited no inhibition of liver metabolic enzymes such as
CYP2C9.
b-amino pyrrole-2-carbonitrile derivatives was designed and synthesized. Compounds 8l and 9l
m
Compound 9l showed excellent DPP4 inhibitory activity (IC50 ¼ 0.01
m
Keywords:
DPP4 inhibitors
GLP-1
© 2014 Elsevier Masson SAS. All rights reserved.
Selectivity
Oral bioavailability
Oral glucose tolerance tests
hERG
1. Introduction
sulfonylureas [3], and reducing hepatic glucose output with
biguanides [4], are associated with undesirable side effects
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease
that is usually accompanied by the complications, such as cardio-
vascular disease, blindness, kidney disorders and amputation [1].
Current therapeutic strategies, including reducing insulin resis-
tance using glitazones [2], increasing insulin secretion with
including hypoglycemia, b-cell apoptosis, and other gastrointes-
tinal tract effects. Therefore, discovery of safe and effective agents
to treat T2DM becomes more important. Evidence from numerous
of studies shows that dipeptidyl peptidase IV (DPPIV, DPP4, also
known as CD26) inhibitors represent a novel approach for treat-
ment T2DM [5].
DPP4 is an ubiquitous yet specific serine protease that cleaves N-
terminal dipeptides from polypeptides with L-proline or L-alanine
Abbreviations: DPP4, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1;
FAP, fibroblast activation protein; T2DM, type 2 diabetes mellitus; SARs, structur-
eeactivity relationships; OGTTs, oral glucose tolerance tests; PK, pharmacokinetics;
AUC, area under curve; ICR, institute of cancer research; KKAy mice, the yellow
obese gene (Ay) into mice.
at the penultimate position [6]. It exists as both a membrane-bound
protein and a soluble protein in plasma [7]. The most important
function of DPP4 is inactivation of glucagon-like peptide-1 (GLP-1)
in vivo. GLP-1 (7e36) is an important incretin that regulates blood
glucose levels [8]. After ingestion of nutrients, it is released from L-
cells to stimulate insulin secretion and biosynthesis [9], inhibits
glucose release [10], and delays gastric emptying, resulting in a
reduced appetite [11]. However, active GLP-1 (7e36) can be rapidly
degraded by DPP4, which cleaves the N-terminal two amino acids
* Corresponding authors.
** Corresponding author. CAS Key Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, People's Republic of China.
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), hljiang@mail.shcnc.ac.cn (H. Jiang),
hliu@mail.shcnc.ac.cn (H. Liu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.08.059
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
243
to generate the inactive GLP-1 (9e36). Hence, restoration of GLP-1
activity by inhibition of DPP4 represents a new approach for the
treatment of T2DM.
Recently, several DPP4 inhibitors [12e20] such as 1 (sitagliptin,
MK-0431) [21], 2 (vildagliptin, LAF-237) [22], 3 (saxagliptin, BMS-
477118) [23], 4 (alogliptin, SYR-322) [24], and 5 (linagliptin, BI-
1356) [25] (Fig. 1) have been approved. These agents have been
demonstrated to be capable to lower glucose and HBA1c levels and
to improve glucose tolerance in type 2 diabetic patients [26]. In our
From the binding mode of compound 8l, the 4-
fluoropyrrolidine-2-carbonitrile moiety occupied the S2 pocket
and the nitrile group could form a hydrogen bond with residue
Tyr547 via a water molecular bridge, and the nitrogen atom of 4-
fluoropyrrolidine-2-carbonitrile moiety could form hydrogen
bonds with residues Glu205 and Glu206 via water molecular bridge
(Fig. 3a). However, the fluorine atom could not form any specific
interaction with the enzyme. Furthermore, the S2 pocket still had
more space to fill. Therefore, a further design and modification to
enhance DPP4 inhibitory activities and selectivity was focused on
the pyrrolidine-2-carbonitrile moiety. In addition, the 2-azabicyclo
[3.1.0]hexane-3-carbonitrile fragment of saxagliptin [29] enhanced
the inhibitory activity and chemical stability. Moreover, octahy-
drocyclopenta[b]pyrrole as the predominant fragment was found in
launched drugs and active compounds, like ramipril [30] and
retagliptin [18]. Based on these strategies, a five-cycle substituted
pyrrolidine was introduced in subsequent design and modification
(Fig. 2).
group, we had presented aromatic substituted a-amino pyrrole-2-
carbonitrile derivatives as DPP4 inhibitors [27]. And we found
compound 6 showed excellent DPP4 inhibitory activity and good
oral glucose tolerance tests (OGTTs) efficacy in institute of cancer
research (ICR) mice and KKAy mice (the yellow obese gene (Ay) into
mice). However, compound 6 showed moderate oral bioavailability.
Therefore, compound 7 was designed and synthesized, and showed
excellent DPP4 inhibitory activity and selectivity and good OGTTs
efficacy in ICR mice. Moreover, the oral bioavailability was
increased [28]. Herein, we report the other modification results
based on compound 6, which was design, synthesis, structur-
eeactivity relationships (SARs), OGTTs and pharmacokinetics (PK)
evaluation of compound 9l as a novel, potent, and selective DPP4
inhibitor for treatment of T2DM.
2.1.2. Synthesis of target compounds
Compounds 8aem were synthesized according to Scheme 1.
Commercially available compound 10 was protected and then
fluorinated with diethylaminosulfur trifluoride (DAST) to generate
compound 12. Compound 16 [31] was obtained from compound 12
via demethylation, amidation, dehydration, and deprotection, fol-
lowed by a coupling reaction with N-Boc-b-amino acids (17aem) to
2. Results and discussion
yield the compounds 18aem. Deprotection of 18aem with tri-
fluoroacetic acid (TFA) produced the target compounds 8aem.
The synthetic route of compounds 9ael was shown in Scheme 2.
The important intermediate 24 was generated from starting ma-
terial 19 via protection, reduction, amidation, dehydration, and
deprotection as previously described. Coupling compound 24 with
various N-Boc-b-amino acids (17ael) yielded compounds 25ael,
which were deprotected with Et2OeHCl to afford final compounds
9ael.
2.1. Chemistry
2.1.1. Design of pyrrolidine-2-carbonitrile compounds
We had presented aromatic substituted
a-amino pyrrole-2-
carbonitrile derivatives as DPP4 inhibitors [27]. And we found
compound 6 showed excellent DPP4 inhibitory activity and good
OGTTs efficacy in ICR mice and KKAy mice. However, compound 6
showed moderate oral bioavailability. Therefore, the further
modification was carried out, and compound 7 showed excellent
DPP4 inhibitory activity and selectivity and good efficacy in OGTTs
in ICR mice. Moreover, the oral bioavailability was increased [28].
2.2. Biological evaluation
Besides, the other strategy was carried out by introduction of
b
-
2.2.1. In vitro enzyme inhibition studies
amino group based on compound 6. Moreover, 3-amino-4-
phenylbutanoic acid moiety as predominant fragment was found
in launched drugs, such as sitagliptin [21]. Therefore, a series of
All the synthesized compounds (8aem) were evaluated in vitro
for the capacity to inhibit DPP4 (Table 1). Due to the diversity of
serine proteases, the inhibitory activities of the other members of
the serine protease family (DPP7, DPP8, DPP9, and FAP) were also
evaluated. Selectivity against DPP8/9 was of particular importance
because inhibition of DPP8/9 had been associated with toxicity in
animal studies [32]. As shown in Table 1, all the compounds (8aem)
novel
b-amino pyrrolidine-2-carbonitrile derivatives
8 was
designed (Fig. 2). From the SARs of
b
-amino 4-fluoropyrrolidine-2-
carbonitrile derivatives, compound 8l showed moderate DPP4
inhibitory activity, however, the selectivity against DPP8/9 was very
low. Therefore, a further design and modification was carried out.
demonstrated
good
DPP4
inhibitory
activities
Fig. 1. Representative DPP4 inhibitors.

244
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
Fig. 2. Design and modification strategies of novel DPP4 inhibitors.
Fig. 3. Three-dimensional structural modes of inhibitors 8l (a) and 9l (b) to DPP4 (PDB ID: 2AJL) derived from the docking simulations. These two images were generated using the
Pymol program.
Scheme 1. Reagents and conditions: (a) (Boc)2O, NaHCO3, dioxane, 24 h; (b) DAST, CH2Cl2, ꢀ78 ꢁC to rt, 24 h; (c) LiOH, dioxane, H2O, overnight; (d) (Boc)2O, NH4HCO3, pyridine,
dioxane, 6 h; (e) cyanuric chloride, DMF, 1 h; (f) TsOH, CH3CN, rt, 24 h; (g) different group substituted N-Boc-b
-amino acids, EDCI, HOBt, TEA, DMF, 20 h; (h) CH2Cl2, TFA, 0 ꢁC to rt,
1 h.

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
245
Scheme 2. Reagents and conditions: (a) (Boc)2O, NaHCO3, dioxane, 24 h; (b) Pd/C, H2, CH3OH, overnight; (c) (Boc)2O, NH4HCO3, pyridine, dioxane, 6 h; (d) cyanuric chloride, DMF,
1 h; (e) TsOH, CH3CN, rt, 24 h; (f) different group substituted N-Boc-b
-amino acids, EDCI, HOBt, Et3N, DMF, 20 h; (g) Et2OeHCl, 0 ꢁC to rt, 24 h.
Table 1
Inhibitory activities and selectivity of b-amino 4-fluoropyrrolidine-2-carbonitrile derivatives.
Compd.
R
IC50
(m
M)a
SRb
DPP4
DPP7
DPP8
DPP9
FAP
DPP8/DPP4
DPP9/DPP4
8a
8b
8c
8d
8e
8f
8g
8h
H
2-Cl
2-Me
3-F
4-F
0.45 0.01
0.22 0.01
0.16 0.01
0.22 0.02
0.32 0.01
0.43 0.03
0.64 0.03
0.77 0.05
0.04 0.00
0.53 0.05
1.58 0.05
0.05 0.01
1.94 1.11
0.02 0.00
0.07 0.00
NIc
0.60 0.08
0.49 0.03
0.71 0.17
1.05 0.09
0.68 0.05
0.64 0.10
ND
0.17 0.04
0.12 0.02
0.03 0.01
0.62 0.06
0.19 0.03
0.14 0.02
ND
5.90 0.53
3.12 0.30
1.09 0.09
6.85 2.14
5.77 1.05
2.73 0.37
ND
1.33
2.23
4.44
4.77
2.13
1.49
/
0.38
0.55
0.19
2.82
0.59
0.33
/
129.71 24.4
NI
59.10 3.97
192.79 0.27
9.55 0.85
NDd
4-I
4-CF3
4-OMe
2,4-di-Cl
3,4-di-Cl
3,5-di-F
2,4,5-tri-F
H
ND
ND
ND
ND
/
/
8i
8j
8k
31.80 2.20
9.34 0.75
ND
50.62 6.04
ND
0.67 0.22
0.19 0.04
ND
1.01 0.11
ND
0.04 0.01
0.23 0.03
ND
0.33 0.04
ND
1.69 0.20
4.83 1.46
ND
3.38 0.12
ND
16.75
0.36
/
20.20
/
1.00
0.43
/
6.60
/
8l
8me
MK-0431
LAF-237
/
/
207.35 6.04
>100
67.72 10.3
1.96 0.23
67.41 9.3
0.20 0.03
30.39 2.94
3.72 0.31
3386.00
28.00
3370.50
2.86
a
Mean values of at least two experiments.
b
c
Selectivity Ratio (SR) ¼ DPP8 IC50/DPP4 IC50 and DPP9 IC50/DPP4 IC50
.
No inhibition.
Not detected.
d
e
The amino is S configuration.
(IC50 ¼ 0.04e1.94
at the phenyl ring, showed moderate inhibitory activity against
M). In order to improve the DPP4 inhibitory
activities in vitro, electron-withdrawing and electron-donating
groups were introduced at the ortho-, meta-, and para-positions
of the benzene ring. Introduction of chloro group (8b,
m
M). Compound 8a, which was non-substituted
essential for the para-position substituted compounds. For the di-
M), which was
di-chloro substituted at the ortho- and para-positions showed 11.3-
fold improvement in DPP4 inhibitory activity, and the selectivity
against DPP8/9 was also improved compared with compound 8a.
substituted compounds, compound 8i (IC50 ¼ 0.04
m
DPP4 (IC50 ¼ 0.45
m
However, compound 8j (IC50 ¼ 0.53
substituted at the meta- and para-positions, and compound 8k
M), which was di-fluoro substituted at the meta-po-
mM), which was di-chloro
IC50 ¼ 0.22
m
M), methyl group (8c, IC50 ¼ 0.16
mM), and fluoro group
M and 8e, IC50 ¼ 0.32 M) at the ortho-, meta-, and
(8d, IC50 ¼ 0.22
m
m
(IC50 ¼ 1.58
m
para-positions of the benzene ring resulted in slightly improve-
ment in DPP4 inhibition (2.0-, 2.8-, 2.0-, and 1.4-fold, respectively).
However, introduction of large groups at the para-position, the
inhibitory activities of compounds 8feg were equipotent or
reduced compared to compound 8a. For example, compound 8f
sitions, resulted in decreased inhibitory potency (1.2- and 3.5-fold,
respectively). Therefore, the meta-position was shown to be
important for potency, and the meta-position should not be
substituted simultaneously. Interestingly, introduction of the 2,4,5-
trifluoro group, resulted in 9.0-fold improvement in DPP4 inhibi-
(IC50 ¼ 0.43
tion was equipotent with compound 8a, and compounds 8g
(IC50 0.64 M) and 8h (IC50 0.77 M) containing tri-
fluoromethyl group and methoxyl group at the para-position
resulted in reduced potency (1.4-fold and 1.7-fold, respectively).
This indicated that introduction of a small group appeared to be
m
M) which contained an iodo group at the para-posi-
tory activity of compound 8l (IC50 ¼ 0.05
mM) compared with that
of compound 8a. Furthermore, investigation of the influence of the
configuration revealed that the S-configuration of compound 8m
¼
m
¼
m
(IC50 ¼ 1.94
This demonstrated that the R-configuration was particularly
important for the -amino compounds.
mM) was 4.3-fold less potent than that of compound 8a.
b

246
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
Compared with positive control compounds MK-0431 and LAF-
were very low. It indicated that the electronic effect and steric effect
might have greater influence for the series of compounds 8ael than
that of compounds 9aem. Moreover, as a consequence of the
introduction of the fluorine atom at the ortho-, meta-, or para-po-
sitions of the benzene ring, the DPP4 inhibitory activity of com-
pounds 8l and 9l were showed 9.0-fold and 44.0-fold improvement
compared to compounds 8a and 9a, respectively. This could be
ascribed to the fluorine atom attached to the benzene ring was
capable to form a hydrogen bond with residue Ser630 (see binding
modes section). Moreover, compared to compound 8l, the DPP4
inhibitory activity of compound 9l was 5.0-fold higher, and the
corresponding selectivity was 44.5-fold (DPP8/DPP4) and 85.8-fold
(DPP9/DPP4) greater, respectively. The probable reason was that
the octahydrocyclopenta[b]pyrrole fragment of compound 9l might
occupy more space in the S2 pocket and form hydrophobic inter-
action with the side chains of Phe357, Ser209, and Arg125 (Fig. 3b)
in comparison of the 4-fluoropyrrolidine-2-carbonitrile fragment
of compound 8l.
237, the inhibitory activity and selectivity of compound 8l were the
similar as LAF-237. However, the inhibitory activity and selectivity
of compound 8l were very lower than that of MK-0431 (Table 1).
To enhance the inhibitory activities, octahydrocyclopenta[b]
pyrrole-2-carbonitrile derivatives (9ael) were designed, synthe-
sized and evaluated. As shown in Table 2, Compound 9a
(IC50 ¼ 0.44
and excellent selectivity against other serine proteases. Introduc-
M), methyl group (9c,
M), and fluoro group (9d, IC50 ¼ 0.13 M and 9e,
M) at the ortho-, meta-, or para-positions of the ben-
mM) showed moderate inhibitory activity against DPP4
tion of the chloro group (9b, IC50 ¼ 0.07
m
IC50 ¼ 0.36
IC50 ¼ 0.27
m
m
m
zene ring, resulted in moderate enhancement in inhibitory potency
(6.3-, 1.2-, 3.4-, and 1.6-fold, respectively). Introduction of a large
group such as iodo group (9f, IC50 ¼ 8.80
mM) and trifluoromethyl
group (9g, IC50 ¼ 1.98 M) at the para-position resulted in a
m
dramatically decreased in inhibitory potency. Replacement of the
trifluoromethyl group with the methoxyl group (9h) at the para-
position resulted in no inhibitory activity. Therefore, for the octa-
hydrocyclopenta[b]pyrrole-2-carbonitrile derivatives, the electron-
donating group should not be substituted at the para-position,
particularly in the case of large group. For the di-substituted com-
In summary, by introduction of the predominant fragment
octahydrocyclopenta[b]pyrrole into the lead compound 8l, the
DPP4 inhibitory activities were improvement, such as compound 9l
(IC50 ¼ 0.01
mM). The important was the selectivity of compounds
9ael was enhancement compared to compounds 8aem.
pounds 9iek (compound 9i, IC50 ¼ 0.07
m
M; compound 9j,
IC50 ¼ 47.44
m
M; and compound 9k, IC50 ¼ 9.65
mM), the ortho-, and
para-position were beneficial while the meta-position was detri-
mental. Moreover, introduction of di-substituted groups at the
meta-position simultaneously resulted in a sharply reduced po-
tency. Interestingly, introduction of the 2,4,5-trifluoro group (9l,
2.2.2. Binding modes of compounds 8l and 9l
To gain structural information for further optimization, the
proposed 3D binding modes of compounds 8l and 9l to DPP4 were
generated based on docking simulation (Fig. 3a and b).
IC50 ¼ 0.01
mM) resulted in a 44-fold enhancement in inhibitory
The binding modes showed that compounds 8l and 9l were
bound to the active site of DPP4 with the amide moiety, which was
similar to the binding mode of sitagliptin with DPP4 [21]. The 2,4,5-
trifluorophenyl moiety fully occupied the S1 hydrophobic pocket,
and the fluorine atom formed a hydrogen bond with residue
Ser630. The 4-fluoropyrrolidine-2-carbonitrile and octahy-
drocyclopenta[b]pyrrole-2-carbonitrile moieties occupied the S2
pocket. Water molecular bridged the cyano nitrogen and the hy-
droxyl of Tyr547, and also interacted with the nitrogen of the pyr-
role-2-carbonitrile moiety and with the carbonyl of Glu205 and
activity compared to compound 9a, and the selectivity was also
improvement (SR: DPP8/DPP4 ¼ 898.00; DPP9/DPP4 ¼ 566.00).
In comparison of these two series of compounds 8aem and
9ael, the DPP4 inhibitory activities of compounds 8bef and 9bef
showed only slightly improvement in DPP4 inhibitory activities
compared with compounds 8a and 9a; while the inhibitory activ-
ities of compounds 8i and 9i showed 11.3- and 6.3-fold improve-
ment, respectively. However, compared to compounds 8feh and
8jek, the DPP4 inhibitory activities of compounds 9feh and 9jek
Table 2
Inhibitory activities and selectivity of b-amino octahydrocyclopenta[b]pyrrole-2-carbonitrile derivatives.
Compd.
R
IC50
(m
M)a
SRb
DPP4
DPP7
DPP8
DPP9
FAP
DPP8/DPP4
DPP9/DPP4
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
MK-0431
LAF-237
H
2-Cl
2-Me
3-F
4-F
0.44 0.02
0.07 0.00
0.36 0.03
0.13 0.01
0.27 0.01
8.80 0.86
1.98 0.18
NI
0.07 0.00
47.44 6.2
9.65 0.94
0.01 0.00
0.02 0.00
0.07 0.00
NIc
NI
NI
NI
NI
107.66 8.4
22.27 1.27
70.46 5.74
21.62 3.03
21.96 2.77
ND
ND
ND
0.88 0.16
ND
ND
NI
e
244.68
318.14
195.72
166.30
81.33
/
5.69 1.06
76.72 8.98
36.53 7.82
51.60 3.65
ND
ND
ND
2.12 0.51
ND
ND
34.41 2.87
109.88 6.53
NI
153.70 12.2
ND
ND
ND
3.85 0.38
ND
ND
81.29
213.11
281.00
191.11
/
/
/
30.29
/
/
898.00
3386.00
28.00
77.03 2.99
4-I
NDd
4-CF3
4-OMe
2,4-di-Cl
3,4-di-Cl
3,5-di-F
2,4,5-tri-F
/
ND
ND
28.26 1.02
ND
ND
71.68 2.13
207.35 6.0
>100
/
/
12.57
/
/
566.00
3370.50
2.86
8.98 0.83
67.72 10.34
1.96 0.23
5.66 0.44
67.41 9.26
0.20 0.03
12.46 0.47
30.39 2.94
3.72 0.31
/
a
Mean values of at least two experiments.
b
c
Selectivity Ratio (SR) ¼ DPP8 IC50/DPP4 IC50 and DPP9 IC50/DPP4 IC50
.
No inhibition.
Not detected.
d

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
247
Glu206. Furthermore, the (R)-
b
-amino group of compounds 8l and
although the free fatty acid and total cholesterol content were
unchanged. As expected, compound 9l and LAF-237 increased the
basal plasma insulin content significantly by 2.2-fold and 1.6-fold,
respectively (Table 4).
9l could form three hydrogen bonds with the side chains of a
tyrosine (Tyr662) and two glutamate residues (Glu205 and Glu206)
(Fig. 3a and b). Compared with the 4-fluoropyrrolidine-2-
carbonitrile moiety of compound 8l, the octahydrocyclopenta[b]
pyrrole-2-carbonitrile moiety could occupy more space in the S2
pocket and form hydrophobic interaction with the side chains of
Phe357, Ser209, and Arg125, which accounted for the increased
inhibitory activity of compound 9l compared with that of 8l.
Moreover, because of introduction of the fluorine atom, the inhib-
2.2.4. Pharmacokinetic evaluation of compounds 8l and 9l
The pharmacokinetic (PK) profiles of the selected compounds 8l
and 9l were assessed in SD rats (Table 5). Compound 8l showed a
high clearance (i.v.), high AUC and a high maximal concentration
(Cmax) when dosed orally. The half-life and the oral bioavailability of
compound 8l were 2.03 h and 53.2%, respectively (Table 5). Com-
pound 9l showed a lower AUC than that of compound 8l due to the
different dose administered. Compared to compound 8l, compound
9l showed lower clearance (i.v.) and a longer half-life (t1/2 ¼ 2.74 h).
However, the oral bioavailability of compound 9l decreased to
itory activities against DPP4 of compounds 8l (IC50 ¼ 0.05
9l (IC50 ¼ 0.01 M) showed 9.0-fold and 44.0-fold improvement
compared to compounds 8a (IC50 0.45 M) and 9a
(IC50 ¼ 0.44 M), respectively. From the binding modes of com-
mM) and
m
¼
m
m
pounds 8l and 9l, the fluorine atom [33] could form hydrogen
bonds with Ser630 (Fig. 3a and b), and this was consistent with the
observed enhancement in potency with this series compared to
compounds 8a and 9a.
22.8%. Otherwise, compared to the ratios of Cmax/Dose, AUC0et
/
Dose, and AUC0e∞/Dose of these two compounds 8l and 9l, com-
pound 8l had better PK properties than that of compound 9l when
dosage as p.o. However, compound 9l had higher AUC value when
dosage as i.v.
2.2.3. In vivo studies
On the basis of overall properties of in vitro potency and selec-
tivity, compounds 8l and 9l were selected for evaluation in OGTTs in
ICR mice. LAF-237 was used as positive control.
2.2.5. hERG testing of compounds 8l and 9l
Blockade of the hERG channel was
a significant hurdle
Compounds 8l and 9l were assessed for the ability to improve
glucose tolerance in ICR mice. Single dose administration (5 and
15 mg/kg) of compounds 8l and 9l to ICR mice at 30 min before an
oral glucose challenge produced a significant decrease in glucose
encountered in the drug discovery [34]. Because of being effica-
cious in vivo, compounds 8l and 9l were chosen to hERG testing
(Table 6). The IC50 values of compounds 8l and 9l on hERG were
larger than 40.00
values of compounds 8l and9l on hERG were 97.70
55.30 M using Thallium assay, respectively.
m
M using Patch-clamp experiment, and the IC50
excursion, showed by the reduction of area under curve (AUC)0e120
.
mM and
Compounds 8l and 9l reduced the blood glucose level significantly
at time points of 15, 30, 60, and 90 min after glucose challenge, and
showed in dose-dependent manner at the 15 min time point.
Collectively, the efficacy of 15 mg/kg of 8l (AUC0e120, 795.0 37.9)
m
2.2.6. Liver metabolic enzymes P450 testing of compounds 8l and 9l
Both compounds 8l and 9l showed no inhibition with liver
metabolic enzymes such as CYP2C9, and the inhibition activity of
liver metabolic enzymes such as CYP3A4 of compounds 8l and 9l
and 9l (AUC0e120, 847.3
29.8) was comparable to LAF-237 of
15 mg/kg (AUC0e120, 904.1 33.9) (Table 3), compounds 8l and 9l
improved the glucose tolerance capacity at 15 min better than that
of positive group LAF-237.
were 56.49 mM and 2.25 mM, respectively (Table 6).
Considering to the inhibitory activities against DPP4 (compound
8l, IC50 ¼ 0.05
m
M; compound 9l, IC50 ¼ 0.01
m
M) and selectivity
3. Conclusions
(compound 8l, selective ratio: DPP8/DPP4
¼
20.20; DPP9/
DPP4 ¼ 6.60; compound 9l, selective ratio: DPP8/DPP4 ¼ 898.00;
DPP9/DPP4 ¼ 566.00) in vitro, compound 9l was selected to do the
chronic effects experiment, although compound 8l had better data
in oral glucose tolerance tests than that of compound 9l in vivo.
Chronic effects of compound 9l were investigated in BKS db/db
mice dosed with 15 mg/kg/day in 5-weeks (Fig. 4). LAF-237 at the
same dose was included as positive control. Fig. 4 showed that
compound 9l significantly improved the glucose excursion during
the 60 mine120 min after oral gavage, as demonstrated by the
reduction of the AUC (3246.3 165.0 vs 3649.5 91.5 of the vehicle
group). The fasting blood glucose level and plasma triglyceride
content decreased obviously following compound 9l treatment,
A series of novel b-amino pyrrolidine-2-carbonitrile derivatives
was designed, synthesized and evaluated as potent and selective
DPP4 inhibitors. Compound 8l demonstrated a moderate DPP4
inhibitory activity, a good PK properties, and good in vivo efficacy
but poor selectivity. Then a further design and modification was
undertaken, and compound 9l showed excellent DPP4 inhibitory
activity, high selectivity against other related enzymes, a moderate
PK properties, and excellent in vivo efficacy in an OGTTs in ICR mice
and BKS db/db mice. Moreover, compounds 8l and 9l did not block
hERG channel and displayed no inhibition of liver metabolic en-
zymes such as CYP2C9. Further investigation of octahy-
drocyclopenta[b]pyrrole-2-carbonitrile derivatives is in progress.
Table 3
Single dose effect of compounds 8l and 9l on oral glucose tolerance tests in ICR mice.
Compd.
Dose (mg/kg)
Time (min)
0
AUC0e120 mmol/L/min
15
30
60
90
120
Veh
8l
e
6.0 1.1
5.8 0.3
5.4 0.2
6.0 0.2
5.9 0.3
5.6 0.1
13.2 1.3
15.8 3.5
11.5 2.7
8.5 0.3**
7.6 0.5**
9.2 0.6*
7.9 0.3**
8.2 0.4**
8.3 1.8
6.0 1.3
5.4 0.3
5.1 0.3
6.0 0.4
5.2 0.4
5.8 0.3
1175.5 216.4
876.6 32.3**
795.0 37.9**,¶
961.7 41.8*
5
15
5
15
15
10.3 0.5**
8.5 0.4**,¶,
11.2 0.5**
9.4 0.6**,¶,
11.2 0.6*
10.9 0.6**
10.2 0.7**
12.2 0.7**
10.6 0.5**
11.0 0.5**
6.2 0.3**
5.9 0.5**
6.8 0.5*
6.5 0.4*
6.9 0.4*
d
9l
f
f
847.3 29.8**,
904.1 33.9**
LAF-237
*p < 0.05 or **p < 0.01 was compared to the vehicle control group, and regarded as statistically significant; ¶p < 0.05 was compared to the positive control LAF-237 group;
dp < 0.05 was compared to 5 mg/kg 8l group; fp < 0.05 was compared to 5 mg/kg 9l group.

248
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
Fig. 4. Chronic effect of compound 9l on OGTT in BKS db/db mice. C57BKS db/db mice with 15 mg/kg/day 9l and LAF-237 treatment, oral glucose tolerance tests (1.5 g/kg) was
carried out after 6-h starvation of 5th-week treatment, the blood glucose level at 0, 30, 60, 90, and 120 min were recorded for the glucose tolerance capacity evaluation. The results
are presented as the mean SE. *p < 0.05 compared to vehicle group (n ¼ 5e8/group).
4. Experimental section
layer extracted with CH2Cl2, dried, filtered and concentrated. The
residue was purified by flash chromatography on silica gel, eluted
with a mixture of EA/PE (1:4, v/v), to afford 12 (1.6 g, 78%) as a
4.1. Chemistry
colorless oil. 1H NMR (CDCl3, 300 MHz):
d 5.18e5.34 (m, 2H), 3.72 (s,
The chemical reagents were purchased and used without
further purification. Nuclear magnetic resonance (NMR) spectros-
copy was performed on a Bruker AMX-400 and AMX-300 NMR (IS
3H), 3.46e3.65 (m, 2H), 2.03e2.29 (m, 2H), 1.40 (s, 9H). MS (ESI) m/
z 248 [MþH]þ.
as TMS). Chemical shifts were reported in parts per million (ppm, d)
4.1.3. (2S,4S)-1-[(tert-Butoxy)carbonyl]-4-fluoropyrrolidine-2-
carboxylic acid (13)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Low- and high-resolution mass spectra (LRMS
and HRMS) were given with electric, electrospray, and matrix-
assisted laser desorption ionization (EI and ESI) produced by a
A solution of compound 12 (2.46 g, 9.96 mmol) in dioxane
(20 mL), was added 10 mL of H2O followed by lithium hydroxide
hydrate (2.09 g, 49.8 mmol) at room temperature, the reaction was
stirred for 3 h (monitored by TLC). Then the solution was filtered
removing the insoluble substance, and the filtrate was removed the
solvent in vacuo, then the residue was added 10 mL of H2O and
acidified with concentrated HCl to pH 3e4, the product began to
precipitate, filtered and dried to afford 13 (2.2 g, 95%) as a white
Finnigan MAT-95, LCQ-DECA spectrometer and IonSpec 4.7 T. Op-
25
tical rotations were reported as follows: [
a
]
D
(c ¼ g/100 mL, in
solvent). Compounds 8aem and 9ael were confirmed ꢂ95% purity
(Supporting Information, Table S1). The details for purity analyses
of compounds 8aem and 9ael are described in the Supplementary
material.
solid. 1H NMR (CD3OD, 300 MHz):
d 5.12e5.30 (m, 1H), 4.38e4.42
(m, 1H), 3.59e3.72 (m, 2H), 2.39e2.46 (m, 2H), 1.47 (s, 9H). MS (ESI)
m/z 232 [MꢀH]ꢀ.
4.1.1. 1-tert-Butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-
dicarboxylate (11)
4.1.4. tert-Butyl (2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-
carboxylate (14)
A
solution of (2S,4R)-methyl 4-hydroxypyrrolidine-2-
carboxylate hydrochloride (10) (0.5 g, 2.75 mmol) in dioxane
(15 mL) was added (Boc)2O (721 mg, 3.3 mmol) and saturated
NaHCO3 (10.5 mL). The reaction was stirred at room temperature
overnight. The solvent was then removed in vacuo and CH2Cl2 was
added. The organics were washed with H2O twice and saturated
NaCl once, then dried, filtered and concentrated. The residue was
purified by flash chromatography on silica gel, eluted with a
mixture of EA/PE (1:5, v/v), to afford 11 (611 mg, 91%) as a white
A mixture of compound 13 (0.5 g, 1.86 mmol), (Boc)2O (0.61 g,
2.79 mmol), NH4HCO3 (0.22 g, 2.79 mmol) and pyridine (2 mL) in
dioxane (20 mL) was stirred at room temperature for 6 h. The
product was extracted with CH2Cl2, washed with 1 M HCl and
saturated NaCl, dried, filtrated, and concentrated. n-Hexane
(100 mL) was added and the product 14 (0.37 g, 85%) began to
precipitate using the ultrasound as a white solid. 1H NMR (CDCl3,
300 MHz):
d 5.59 (br, s, 1H), 5.13e5.31 (m, 1H), 4.36 (br, s, 1H),
solid. 1H NMR (CDCl3, 300 MHz):
d 4.35e4.48 (m, 2H), 3.72 (s, 3H),
3.52e3.81 (m, 2H), 2.31e2.78 (m, 2H), 1.48 (s, 9H). MS (ESI) m/z 233
[MþH]þ.
3.46e3.65 (m, 2H), 2.03e2.29 (m, 2H),1.39 (s, 9H). MS (ESI) m/z 246
[MþH]þ.
4.1.5. tert-Butyl (2S,4S)-2-cyano-4-fluoropyrrolidine-1-carboxylate
(15)
4.1.2. 1-tert-Butyl 2-methyl (2S,4S)-4-fluoropyrrolidine-1,2-
dicarboxylate (12)
A mixture of compound 14 (13 g, 56.0 mmol) and cyanuric
chloride (6.19 g, 33.6 mmol) in DMF (10 mL) was stirred at room
temperature for 2 h (monitored by TLC). After the reaction
completed, the solution was extracted with EtOAc, washed, dried,
concentrated, and purified by flash chromatography on silica gel,
eluted with a mixture of PE/EA (1/1, v/v), to afford 15 (9.7 g, 76%) as
Under the nitrogen protected, a solution of compound 11 (2 g,
8.2 mmol) in dry CH2Cl2 cooled to ꢀ78 ꢁC, was added DAST (1.97 g,
12.2 mmol). After stirring 3 h, the reaction slowly warmed to room
temperature overnight. Then the reaction solution was poured into
200 mL ice and NaHCO3 mixture solution and stirred acutely until
no CO2 evolution. The organic layer was separated and the aqueous
a white solid. 1H NMR (CDCl3, 300 MHz):
d 5.21e5.41 (m, 1H),

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
249
Table 4
for 20 h and then the saturated NaHCO3 was added. The mixture
was extracted with EtOAc and washed with saturated NaCl, dried
over Na2SO4 and concentrated. The residue was purified with flash
chromatography on silica gel, eluted with a mixture of PE/EA (4/1,
v/v) to afford 18a (112 mg, 85%) as a white solid. 1H NMR (CDCl3,
Chronic effect of 9l on blood glucose and plasma indicators in BKS db/db mice.
Biochemical indicator
Veh
23.3 1.0
23.0 1.0
1.95 0.14
1.01 0.09
1.46 0.10
2.22 0.14
LAF-237
22.2 1.8
9l
Random blood glucose (mM)
Fasting blood glucose (mM)
Plasma insulin (ng/mL)
Plasma triglyceride (mM)
Plasma free fatty acid (mEq/L)
Plasma total cholesterol
21.3 0.9
18.5 0.7*
3.16 0.4*
0.75 0.06*
1.07 0.07*
2.47 0.11
19.7 1.5*
4.31 0.65*
0.85 0.05*
1.43 0.08
2.40 0.13
300 MHz):
d 7.28e7.32 (m, 2H), 7.20e7.24 (m, 3H), 4.90 (d,
J ¼ 9.1 Hz, 1H), 4.11e4.15 (m, 1H), 3.59 (br, s, 1H), 3.45e3.49 (m, 1H),
3.05e3.10 (m, 1H), 2.85e2.89 (m, 1H), 2.62e2.66 (m, 1H), 2.39 (d,
J ¼ 4.8 Hz, 2H), 2.16e2.33 (m, 2H), 1.41 (s, 9H). MS (ESI) m/z 398
[MþNa]þ.
AUC0e120 of oGTT after 5-wks- 3649.5 91.5 3229.1 139.7* 3246.3 165.0*
treatment
C57BKS db/db mice with 15 mg/kg/day compound 9l treatment, oral glucose
tolerance tests (1.5 g/kg) was carried out after 6-h starvation of 5th-week treatment,
the blood glucose level was recorded for the glucose tolerance capacity evaluation.
The results are presented as the mean SE. *p < 0.05 compared to vehicle group
(n ¼ 5e8/group).
4.1.8. tert-Butyl N-[(2R)-1-(2-chlorophenyl)-4-[(2S,4S)-2-cyano-4-
fluoropyrrolidin-1-yl]-4-oxobutan-2-yl]carbamate (18b)
In the same manner as described for 18a, 18b was prepared from
Table 5
Pharmacokinetic properties of compounds 8l and 9l in SD rats.
Compd.
Admini.
Dose mg/kg
Tmax
h
Cmax ng/mL
AUC0et ng/mL*h
AUC0e∞ ng/mL*h
MRT h
t1/2
h
CLz L/h/kg
F %
8l
p.o.
i.v.
p.o.
i.v.
50
20
20
10
0.3
0.3
0.5
0.5
2692
2711
509
8838
5843
2040
4467
8935
5866
2361
4974
3.03
2.93
2.81
2.71
2.03
2.33
2.74
2.39
/
53.2
/
22.8
/
3.42
/
2.01
9l
1121
p.o., oral administration; i.v., intravenous injection.
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2-chlorophenyl)buta-
noic acid (17b). 1H NMR (CDCl3, 300 MHz):
7.28e7.36 (m, 2H),
Table 6
hERG and live metabolic enzymes P450 testing of compounds 8l and 9l.
d
7.12e7.19 (m, 2H), 5.27e5.46 (m, 2H), 4.95 (d, J ¼ 9.0 Hz, 1H), 4.24
(br, s, 1H), 3.45e3.84 (m, 2H), 3.08e3.14 (m, 2H), 2.21e2.73 (m, 4H),
1.38 (s, 9H). MS (ESI) m/z 432 [MþNa]þ.
Compd.
hERG (IC
,
50 mM)
CYP450 (IC50
, mM)
CYP3A4
CYP2C9
8l
9l
>40.00a
>40.00a
97.70b
55.30b
56.49
2.25
NIc
NI
4.1.9. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(2-methylphenyl)-4-oxobutan-2-yl]carbamate (18c)
a
Tested by Patch Clamp.
Using Thallium assay.
No inhibition.
b
c
In the same manner as described for 18a, 18c was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2-methylphenyl)buta-
noic acid (17c). 1H NMR (CDCl3, 300 MHz):
d 7.05e7.12 (m, 4H),
5.24e5.58 (m, 2H), 4.94 (d, J ¼ 9.0 Hz,1H), 4.18 (br, s,1H), 3.30e3.77
(m, 2H), 2.90e2.96 (m, 2H), 2.18e2.66 (m, 7H), 1.37 (s, 9H). MS (ESI)
m/z 412 [MþNa]þ.
4.62e4.76 (m, 1H), 3.49e3.93 (m, 2H), 2.63 (dd, J1 ¼ 15.0 Hz,
J2 ¼ 15.3 Hz, 1H), 2.30e2.44 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 215
[MþH]þ.
4.1.10. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(3-fluorophenyl)-4-oxobutan-2-yl]carbamate (18d)
4.1.6. (2S,4S)-4-fluoropyrrolidine-2-carbonitrile: 4-methylbenzene-
1-sulfonic acid (16)
In the same manner as described for 18a, 18d was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3-fluorophenyl)butanoic
A solution of compound 15 (7.9 g, 36.9 mmol) in CH3CN (50 mL)
was added 4-methylbenzenesulfonic acid hydrate (10.5 g,
55.32 mmol) and stirred at room temperature for 24 h. After the
reaction completed, the solution was removed in vacuo. The re-
sidual brown oil was dissolved in EtOAc (100 mL) and put into
fridge overnight, the product 16 (7.4 g, 71%) was precipitated as a
acid 17d. 1H NMR (CDCl3, 300 MHz):
d 7.22e7.28 (m, 1H), 6.88e6.93
(m, 3H), 5.27e5.44 (m, 2H), 4.95 (d, J ¼ 9.0 Hz, 1H), 4.15 (br, s, 1H),
3.39e3.84 (m, 2H), 2.90e2.97 (m, 2H), 2.18e2.68 (m, 4H), 1.39 (s,
9H). MS (ESI) m/z 416 [MþNa]þ.
brown crystal. 1H NMR (CD3OD, 400 MHz):
d
7.51 (dd, J1 ¼ 16.0 Hz,
J2 ¼ 8.0 Hz, 2H), 7.09e7.15 (m, 2H), 5.45e5.62 (m, 1H), 4.99e5.04
4.1.11. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(4-fluorophenyl)-4-oxobutan-2-yl]carbamate (18e)
(m, 1H), 3.42e3.67 (m, 2H), 2.30e2.70 (m, 3H), 3.17 (s, 3H). 13C NMR
(CD3OD, 100 MHz): d 143.3, 142.0, 130.0, 127.0, 116.4, 93.9, 92.5, 53.8
In the same manner as described for 18a, 18e was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-fluorophenyl)butanoic
(d, J ¼ 19.0 Hz), 46.8, 38.4 (d, J ¼ 17.0 Hz), 21.4. MS (EI) m/z 114 [M]þ.
HRMS (EI) m/z calcd C5H7 F N2 114.0593 [M]þ, found 114.0668.
acid (17e). 1H NMR (CDCl3, 300 MHz):
d 7.11e7.19 (m, 2H),
6.92e6.99 (m, 2H), 5.28e5.46 (m, 2H), 4.93 (d, J ¼ 9.0 Hz, 1H),
4.10e4.15 (m, 1H), 3.81e3.53 (m, 3H), 2.91e2.96 (m, 2H), 2.60e2.71
(m, 1H), 2.48e2.54 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z 416 [MþNa]þ.
4.1.7. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-4-oxo-1-phenylbutan-2-yl]carbamate (18a)
A
solution
of
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-
phenylbutanoic acid (compound 17a, 108 mg, 0.385 mmol) in
DMF (5 mL) was added HOBt (166 mg, 1.225 mmol) and EDCI
(154 mg, 0.805 mmol). After stirring for 30 min compound 16
(100 mg, 0.35 mmol) and additional TEA (0.17 mL, 1.225 mmol)
were added. This solution was allowed to stir at room temperature
4.1.12. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(4-iodophenyl)-4-oxobutan-2-yl]carbamate (18f)
In the same manner as described for 18a, 18f was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-iodophenyl)butanoic
acid (17f). 1H NMR (CDCl3, 300 MHz):
d 7.56e7.63 (m, 2H),

250
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
6.89e6.96 (m, 2H), 5.28e5.46 (m, 2H), 4.92 (d, J ¼ 9.0 Hz, 1H), 4.10
(br, s, 1H), 3.42e3.86 (m, 2H), 2.18e2.97 (m, 6H), 1.39 (s, 9H). MS
(ESI) m/z 524 [MþNa]þ.
4.1.19. tert-Butyl N-[(2S)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-4-oxo-1-phenylbutan-2-yl)carbamate (18m)
In the same manner as described for 18a, 18m was prepared
from (3S)-3-[[(tert-butoxy)carbonyl]amino]-4-phenylbutanoic acid
(17m). 1H NMR (CDCl3, 300 MHz):
d 7.20e7.31 (m, 5H), 5.22e5.60
4.1.13. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-4-oxo-1-[4-(trifluoromethyl)phenyl]butan-2-yl]carbamate (18g)
In the same manner as described for 18a, 18g was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-[4-(trifluoromethyl)
(m, 2H), 4.91 (d, J ¼ 9.0 Hz, 1H), 4.13 (br, s, 1H), 3.49e3.78 (m, 2H),
2.61e3.14 (m, 3H), 2.18e2.61 (m, 3H), 1.42 (s, 9H). MS (ESI) m/z 398
[MþNa]þ.
phenyl]butanoic acid (17g). 1H NMR (CDCl3, 300 MHz):
d 7.50e7.58
4.1.20. 2-Benzyl 1-tert-butyl (2S,3aS,6aS)-hexahydrocyclopenta[b]
pyrrole-1,2-dicarboxylate (20)
(m, 2H), 7.31e7.37 (m, 2H), 5.27e5.46 (m, 2H), 4.93 (d, J ¼ 9.0 Hz,
1H), 4.19 (br, s, 1H), 3.47e3.88 (m, 2H), 2.98e3.04 (m, 2H),
2.18e2.73 (m, 4H), 1.38 (s, 9H). MS (ESI) m/z 466 [MþNa]þ.
A solution of benzyl (2S,3aS,6aS)-octahydrocyclopenta[b]pyr-
role-2-carboxylate hydrochloride (19) (5 g, 17.74 mmol) in dioxane
(20 mL) was added (Boc)2O (4.65 g, 21.29 mmol) and saturated
NaHCO3 (15 mL). The reaction was stirred at room temperature
overnight. The solvent was then removed in vacuo and CH2Cl2 was
added. The organics were washed with H2O twice and saturated
NaCl once, and then dried, filtered and concentrated. The com-
pound 20 (6 g, 97%) was obtained as oil using in the next step
without further purification. MS (ESI) m/z 268 [MþNa]þ.
4.1.14. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(4-methoxyphenyl)-4-oxobutan-2-yl]carbamate (18h)
In the same manner as described for 18a, 18h was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-methoxyphenyl)buta-
noic acid (17h). 1H NMR (CDCl3, 300 MHz):
d 7.05e7.14 (m, 2H),
6.78e6.83 (m, 2H), 5.27e5.40 (m, 2H), 4.93 (d, J ¼ 9.0 Hz, 1H), 4.11
(br, s, 1H), 3.39e4.11 (m, 5H), 2.85e2.96 (m, 3H), 2.67 (t, J ¼ 9.0 Hz,
1H), 2.18e2.45 (m, 2H), 1.40 (s, 9H). MS (ESI) m/z 428 [MþNa]þ.
4.1.21. (2S,3aS,6aS)-1-[(tert-Butoxy)carbonyl]octahydrocyclopenta
[b]pyrrole-2-carboxylic acid (21)
A solution of compound 20 (6 g, 17.37 mmol) in CH3OH (30 mL)
was added 10% Pd/C. The mixture was stirred under H2 at room
temperature for 20 h, filtered, washed with CH3OH twice, and
concentrated to afford compound 21 as oil without further purifi-
cation. MS (ESI) m/z 254 [MꢀH]ꢀ.
4.1.15. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(2,4-dichlorophenyl)-4-oxobutan-2-yl)carbamate (18i)
In the same manner as described for 18a, 18i was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2,4-dichlorophenyl)
butanoic acid (17i). 1H NMR (CDCl3, 300 MHz):
d 7.31e7.39 (m, 1H),
7.18e7.22 (m, 2H), 5.31e5.63 (m, 2H), 4.93 (d, J ¼ 9.0 Hz, 1H), 4.20
(br, s, 1H), 3.51e3.88 (m, 2H), 2.70e2.78 (m, 2H), 2.19e2.73 (m, 4H),
1.35 (s, 9H). MS (ESI) m/z 466 [MþNa]þ.
4.1.22. tert-Butyl (2S,3aS,6aS)-2-carbamoyl-octahydrocyclopenta
[b]pyrrole-1-carboxylate (22)
In the same manner as described for compound 14, compound
22 was prepared from 21. The compound 22 was used in the next
step without further purification. MS (ESI) m/z 277 [MþNa]þ.
4.1.16. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(3,4-dichlorophenyl)-4-oxobutan-2-yl)carbamate (18j)
In the same manner as described for 18a, 18j was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3,4-dichlorophenyl)
4.1.23. tert-Butyl (2S,3aS,6aS)-2-cyano-octahydrocyclopenta[b]
pyrrole-1-carboxylate (23)
In the same manner as described for compound 15, compound
23 was prepared from 22. The compound 23 was used in the next
step without further purification. MS (ESI) m/z 259 [MþNa]þ.
butanoic acid (17j). 1H NMR (CDCl3, 300 MHz):
d
7.31e7.39 (m, 1H),
7.20e7.29 (m, 1H), 7.00e7.09 (m, 1H), 5.30e5.46 (m, 2H), 4.95 (d,
J ¼ 9.0 Hz, 1H), 4.10 (br, s, 1H), 3.46e3.88 (m, 2H), 2.90e2.98 (m,
2H), 2.18e2.73 (m, 4H), 1.38 (s, 9H). MS (ESI) m/z 466 [MþNa]þ.
4.1.24. (2S,3aS,6aS)-octahydrocyclopenta[b]pyrrole-2-carbonitrile:
4-methylbenzene-1-sulfonic acid (24)
4.1.17. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-(3,5-difluorophenyl)-4-oxobutan-2-yl)carbamate (18k)
In the same manner as described for 18a, 18k was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3,5-difluorophenyl)
In the same manner as described for compound 16, compound
24 was prepared from 23. 1H NMR (CD3OD, 400 MHz):
d 7.67e7.79
(m, 2H), 7.21e7.28 (m, 2H), 4.89 (s, 3H), 4.54e4.67 (m, 1H),
4.12e4.24 (m, 1H), 2.93e3.03 (m, 1H), 2.70e2.76 (m, 1H), 1.66e1.96
butanoic acid (17k). 1H NMR (CDCl3, 300 MHz):
d 6.66e6.76 (m,
(m, 9H). 13C NMR (CD3OD, 100 MHz):
d 143.4, 141.9, 129.9, 126.9,
3H), 5.30e5.45 (m, 2H), 4.94 (d, J ¼ 6.0 Hz, 1H), 4.11 (br, s, 1H),
3.47e3.86 (m, 2H), 2.90e2.96 (m, 2H), 2.17e2.73 (m, 1H), 1.40 (s,
9H). MS (ESI) m/z 434 [MþNa]þ.
115.6, 66.9, 48.2, 43.5, 36.9, 32.4, 31.5, 25.1, 21.4. MS (EI) m/z 136
[M]þ. HRMS (EI) m/z calcd C8H12N2 136.1000 [M]þ, found 136.1003.
25
[a
]
¼ ꢀ20.3 (c ¼ 0.385 g/100 mL, CH3OH).
D
4.1.18. tert-Butyl N-[(2R)-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (18l)
In the same manner as described for 18a, 18l was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2,4,5-trifluorophenyl)
4.1.25. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-4-oxo-1-phenylbutan-2-yl]
carbamate (25a)
A
solution
of
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-
butanoic acid (17l). 1H NMR (CDCl3, 400 MHz):
d
7.05e7.14 (m, 1H),
phenylbutanoic acid (compound 17a, 99.6 mg, 0.357 mmol) in
DMF (5 mL) was added HOBt (153 mg, 1.134 mmol) and EDCI
(160 mg, 0.81 mmol). After stirring for 30 min compound 24
(100 mg, 0.324 mmol) and TEA (0.16 mL, 1.134 mmol) were added.
This solution was allowed to stir at room temperature for 20 h and
then the saturated NaHCO3 was added. The mixture was extracted
with EtOAc and washed with saturated NaCl, dried over Na2SO4 and
concentrated. The residue was purified with flash chromatography
on silica gel, eluted with a mixture of PE/EA (4/1, v/v) to afford 25a
6.85e6.94 (m, 1H), 5.33e5.50 (m, 2H), 4.95 (d, J ¼ 16.0 Hz, 1H), 4.12
(br, s, 1H), 3.55e3.92 (m, 2H), 2.90e2.98 (m, 2H), 2.58e2.75 (m,
3H), 2.27e2.39 (m, 1H), 1.37 (s, 9H). 13C NMR (CD3OD, 100 MHz):
d
169.9, 162.2, 155.3, 121.7 (d, J ¼ 18.6 Hz), 119.2 (dd, J1 ¼ 6.1 Hz,
J2 ¼ 5.9 Hz), 117.4, 105.3 (dd, J1 ¼ 28.5 Hz, J2 ¼ 28.1 Hz), 92.8, 90.9,
79.6, 53.1 (d, J ¼ 24.1 Hz), 48.0, 45.3, 44.5, 37.2, 36.1 (d, J ¼ 21.2 Hz),
32.7, 28.2. MS (ESI) m/z 452 [MþNa]þ. HRMS (ESI) m/z calcd
C
20H23N3O3F4Na 452.1556 [MþNa]þ, found 452.1573.

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
251
(108 mg, 85%) as a white solid. 1H NMR (CDCl3, 300 MHz):
7.13e7.31 (m, 5H), 5.60e5.75 (m, 1H), 4.82e4.90 (m, 1H),
3.85e4.13 (m, 2H), 2.76e3.07 (m, 3H), 2.37e2.46 (m, 3H), 1.99e2.10
(m, 7H), 1.40 (s, 9H). MS (ESI) m/z 420 [MþNa]þ.
4.10e4.16 (m, 2H), 2.81e3.18 (m, 3H), 2.29e2.57 (m, 3H), 1.46e2.11
(m, 7H), 1.34 (s, 9H). MS (ESI) m/z 366 [MþH-Boc]þ, 488 [MþNa]þ.
d
4.1.32. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(4-methoxyphenyl)-4-
oxobutan-2-yl)carbamate (25h)
4.1.26. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(2-chlorophenyl)-4-
oxobutan-2-yl]carbamate (25b)
In the same manner as described for 25a, 25h was prepared
from (3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-methoxyphenyl)
butanoic acid (17h). 1H NMR (CDCl3, 300 MHz):
d 7.03e7.14 (m, 2H),
In the same manner as described for 25a, 25b was prepared
6.78e6.84 (m, 2H), 5.57e5.72 (dd, J1 ¼ 36.0 Hz, J2 ¼ 33.0 Hz, 1H),
4.81e4.90 (m, 1H), 4.05e4.13 (m, 2H), 3.77 (s, 3H), 2.78e2.91 (m,
3H), 2.29e2.52 (m, 3H), 1.49e2.11 (m, 7H), 1.40 (s, 9H). MS (ESI) m/z
328 [MþH-Boc]þ, 450 [MþNa]þ.
from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2-chlorophenyl)
7.29e7.32 (m, 1H),
butanoic acid (17b). 1H NMR (CDCl3, 300 MHz):
d
7.20e7.25 (m, 1H), 7.11e7.18 (m, 2H), 5.65e5.92 (m, 1H), 4.80e4.91
(m, 1H), 4.01e4.54 (m, 2H), 3.07e3.17 (m, 2H), 2.79e2.86 (m, 1H),
2.29e2.62 (m, 3H), 1.52e2.11 (m, 7H), 1.34 (s, 9H). MS (ESI) m/z 332
[MþH-Boc]þ, 454 [MþNa]þ.
4.1.33. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(2,4-dichlorophenyl)-4-
oxobutan-2-yl)carbamate (25i)
4.1.27. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-4-oxo-1-(2-methylphenyl)
butan-2-yl]carbamate (25c)
In the same manner as described for 25a, 25i was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2,4-dichlorophenyl)
butanoic acid (17i). 1H NMR (CDCl3, 300 MHz):
d 7.32e7.38 (m, 1H),
In the same manner as described for 25a, 25c was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2-methylphenyl)buta-
7.16e7.21 (m, 2H), 5.64e5.85 (m, 1H), 4.88e4.96 (m, 1H), 4.12e4.44
(m, 2H), 2.85e3.13 (m, 3H), 2.31e2.61 (m, 3H), 1.56e2.17 (m, 7H),
1.37 (s, 9H). MS (ESI) m/z 366 [MþH-Boc]þ, 488 [MþNa]þ.
noic acid (17c). 1H NMR (CDCl3, 300 MHz):
d 7.00e7.15 (m, 4H),
5.71e5.88 (m, 1H), 4.86e4.92 (m, 1H), 3.87e4.19 (m, 2H), 2.80e3.06
(m, 3H), 2.29e2.52 (m, 6H), 1.47e2.12 (m, 7H), 1.37 (s, 9H). MS (ESI)
m/z 312 [MþH-Boc]þ, 434 [MþNa]þ.
4.1.34. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(3,4-dichlorophenyl)-4-
oxobutan-2-yl)carbamate (25j)
4.1.28. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(3-fluorophenyl)-4-
oxobutan-2-yl]carbamate (25d)
In the same manner as described for 25a, 25j was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3,4-dichlorophenyl)
butanoic acid (17j). 1H NMR (CDCl3, 300 MHz):
d 7.31e7.39 (m, 2H),
In the same manner as described for 25a, 25d was prepared
7.07 (s, 1H), 5.57e5.71 (m, 1H), 4.88e4.92 (m, 1H), 4.10 (br, s, 2H),
2.81e3.05 (m, 3H), 2.32e2.59 (m, 3H), 1.61e2.14 (m, 7H), 1.39 (s,
9H). MS (ESI) m/z 366 [MþH-Boc]þ, 488 [MþNa]þ.
from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3-fluorophenyl)
7.19e7.24 (m, 1H),
butanoic acid (17d). 1H NMR (CDCl3, 300 MHz):
d
6.88e6.97 (m, 3H), 5.59e5.71 (dd, J1 ¼ 30.0 Hz, J2 ¼ 30.0 Hz, 1H),
4.84e4.92 (m, 1H), 3.92e4.15 (m, 2H), 2.80e3.05 (m, 3H),
2.27e2.56 (m, 3H), 1.52e2.15 (m, 7H), 1.38 (s, 9H). MS (ESI) m/z 316
[MþH-Boc]þ, 438 [MþNa]þ.
4.1.35. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(3,5-difluorophenyl)-4-
oxobutan-2-yl)carbamate (25k)
In the same manner as described for 25a, 25k was prepared
4.1.29. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(4-fluorophenyl)-4-
oxobutan-2-yl]carbamate (25e)
from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(3,5-
difluorophenyl)butanoic acid (17k). 1H NMR (CDCl3, 300 MHz):
d
6.62e6.75 (m, 3H), 5.59e5.71 (dd, J1 ¼ 30.0 Hz, J2 ¼ 27.0 Hz, 1H),
In the same manner as described for 25a, 25e was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-fluorophenyl)butanoic
4.84e4.91 (m,1H), 4.10 (br, s, 2H), 2.74e3.09 (m, 3H), 2.29e2.62 (m,
3H), 1.46e2.17 (m, 7H), 1.38 (s, 9H). MS (ESI) m/z 334 [MþH-Boc]þ,
456 [MþNa]þ.
acid (17e). 1H NMR (CDCl3, 300 MHz):
d 7.10e7.19 (m, 2H),
6.98e7.07 (m, 2H), 5.52e5.63 (m,1H), 4.81e4.88 (m,1H), 3.98e4.07
(m, 2H), 2.81e3.00 (m, 3H), 2.29e2.52 (m, 3H), 1.52e1.87 (m, 7H),
1.35 (s, 9H). MS (ESI) m/z 438 [MþNa]þ.
4.1.36. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-4-oxo-1-(2,4,5-trifluorophenyl)
butan-2-yl)carbamate (25l)
4.1.30. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-1-(4-iodophenyl)-4-oxobutan-
2-yl]carbamate (25f)
In the same manner as described for 25a, 25l was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(2,4,5-trifluorophenyl)
butanoic acid (17l). 1H NMR (CD3OD, 400 MHz):
d 7.00e7.10 (m,1H),
In the same manner as described for 25a, 25f was prepared from
(3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-iodophenyl)butanoic
6.81e6.90 (m, 1H), 5.59e5.74 (dd, J1 ¼ 48.0 Hz, J2 ¼ 52.0 Hz, 1H),
4.86e4.92 (m, 1H), 4.12 (br, s, 2H), 2.83e2.98 (m, 3H), 2.48e2.67
(m, 3H), 1.56e2.12 (m, 7H), 1.35 (s, 9H). 13C NMR (CD3OD,100 MHz):
acid (17f). 1H NMR (CDCl3, 300 MHz):
d 7.56e7.65 (m, 2H),
6.90e6.99 (m, 2H), 5.58e5.64 (m,1H), 4.81e4.90 (m,1H), 4.05e4.14
(m, 2H), 2.77e3.01 (m, 3H), 2.11e2.31 (m, 3H), 1.52e2.11 (m, 7H),
1.39 (s, 9H). MS (ESI) m/z 424 [MþH-Boc]þ, 546 [MþNa]þ.
d
170.1, 157.1, 155.3, 147.5 (d, J ¼ 12.6 Hz), 145.6 (d, J ¼ 9.8 Hz), 121.9
(d, J ¼ 14.4 Hz), 119.2, 119.0 (dd, J1 ¼ 9.7 Hz, J2 ¼ 9.6 Hz), 105.1 (dd,
J1 ¼ 22.9 Hz, J2 ¼ 22.8 Hz), 79.3, 63.9, 48.1, 46.3, 44.2, 36.7, 35.1, 34.1,
32.2, 29.6, 28.2, 25.8. MS (ESI) m/z 352 [MþH-Boc]þ, 474 [MþNa]þ.
HRMS (ESI) m/z calcd C23H29N3O3F3 452.2136 [MþH]þ, found
452.2161.
4.1.31. tert-Butyl N-[(2R)-4-[(2S,3aS,6aS)-2-cyano-
octahydrocyclopenta[b]pyrrol-1-yl]-4-oxo-1-(4-(trifluoromethyl)
phenyl)butan-2-yl)carbamate (25g)
In the same manner as described for 25a, 25g was prepared
from (3R)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-(trifluoromethyl)
4.1.37. (2S,4S)-1-[(3R)-3-amino-4-phenylbutanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8a)
A solution of 18a (120 mg) in dry CH2Cl2 (2 mL) was added
CF3COOH (1.0 mL) at ice-bathe and warmed to room temperature.
phenyl)butanoic acid (17g). 1H NMR (CDCl3, 300 MHz):
d
7.50e7.57
(m, 2H), 7.28e7.34 (m, 2H), 5.61e5.70 (m, 1H), 4.84e4.90 (m, 1H),

252
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
After 1 h, the mixture was concentrated, and the residue was added
10 mL Et2O. The white solid was precipitated, filtered and to afford
8a as TFA salt (105 mg). Yield: 88.2%. HPLC: 97.33%, tR ¼ 1.84 min. 1H
4.1.42. (2S,4S)-1-[(3R)-3-amino-4-(4-iodophenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8f)
In the same manner as described for 8a, 8f was prepared from
NMR (CD3OD, 400 MHz):
d 7.28e7.40 (m, 5H), 5.28e5.47 (m, 1H),
18f. Yield: 78.2%. HPLC: 95.88%, tR ¼ 1.83 min. 1H NMR (CD3OD,
4.98e5.03 (m, 1H), 3.78e3.90 (m, 2H), 3.58e3.70 (m, 1H),
400 MHz):
d
7.73 (d, J ¼ 8.0 Hz, 2H), 7.00e7.13 (m, 1H), 5.29e5.49
2.91e3.04 (m, 2H), 2.77e2.82 (m, 1H), 2.35e2.63 (m, 3H). 13C NMR
(m, 1H), 4.97e5.05 (m, 1H), 3.82e3.91 (m, 2H), 3.60e3.73 (m, 1H),
(CD3OD, 100 MHz):
d
169.5, 169.1, 135.3, 129.0, 128.8, 127.3, 117.6,
2.98 (d, J ¼ 8.0 Hz, 2H), 2.72e2.94 (m, 1H), 2.35e2.64 (m, 3H). 13
C
92.5 (d, J ¼ 141.3 Hz), 52.8 (d, J ¼ 18.9 Hz), 49.5, 44.6, 37.9, 35.8 (d,
NMR (CD3OD, 100 MHz): d 169.4, 169.0, 138.0, 135.1, 131.2, 117.6,
J ¼ 16.7 Hz), 34.6. MS (ESI) m/z 276 [MþH]þ. HRMS (ESI) calcd
92.5 (d, J ¼ 141.4 Hz), 92.4, 52.8 (d, J ¼ 18.8 Hz), 49.5, 44.6, 37.5, 35.8
25
C
15H19N3OF 276.1512 [MþH]þ, found 276.1502. [
a]
¼ ꢀ80.9
(d, J ¼ 16.7 Hz), 34.7. MS (ESI) m/z 402 [MþH]þ. HRMS (ESI) calcd
D
25
(c ¼ 0.11 g/100 mL, CH3OH).
C
15H18N3OFI 402.0479 [MþH]þ, found 402.0485. [
a
]
¼ ꢀ55.6
D
(c ¼ 0.18 g/100 mL, CH3OH).
4.1.38. (2S,4S)-1-[(3R)-3-amino-4-(2-chlorophenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8b)
4.1.43. (2S,4S)-1-[(3R)-3-amino-4-(4-(trifluoromethyl)phenyl)
butanoyl]-4-fluoropyrrolidine-2-carbonitrile (8g)
In the same manner as described for 8a, 8b was prepared from
18b. Yield: 80.5%. HPLC: 96.34%, tR ¼ 1.59 min. 1H NMR (CD3OD,
In the same manner as described for 8a, 8g was prepared from
400 MHz):
d 7.42e7.51 (m, 1H), 7.30e7.38 (m, 3H), 5.29e5.49 (m,
18g. Yield: 81.2%. HPLC: 100.00%, tR ¼ 1.89 min. 1H NMR (CD3OD,
1H), 4.98e5.03 (m, 1H), 3.91e3.98 (m, 1H), 3.57e3.89 (m, 2H),
400 MHz):
d
7.68e7.70 (d, J ¼ 8.0 Hz, 2H), 7.49e7.53 (m, 2H),
3.18e3.24 (m, 2H), 2.79e2.96 (m, 1H), 2.39e2.63 (m, 3H). 13C NMR
5.30e5.49 (m, 1H), 4.90e5.06 (m, 1H), 3.56e3.96 (m, 3H), 3.12 (d,
(CD3OD, 100 MHz):
d 169.3168.8, 133.6, 132.5, 131.5, 129.8, 129.2,
J ¼ 8.0 Hz, 2H), 2.76e2.96 (m, 1H), 2.37e2.64 (m, 3H). 13C NMR
127.4, 116.8, 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.9 Hz), 44.6, 35.8 (d,
(CD3OD, 100 MHz):
d
169.3, 168.9, 161.6 (d, J ¼ 27.3 Hz), 139.9, 129.8,
J ¼ 13.0 Hz), 35.5, 34.4. MS (ESI) m/z 310 [MþH]þ. HRMS (ESI) calcd
125.6, 117.6, 52.8 (d, J ¼ 18.8 Hz), 44.6, 37.7, 35.8 (d, J ¼ 16.7 Hz),
25
C15H18N3OFCl 310.1122 [MþH]þ, found 310.1114. [
(c ¼ 0.132 g/100 mL, CH3OH).
a
]
¼ ꢀ72.0
34.8. MS (ESI) m/z 344 [MþH]þ. HRMS (ESI) calcd C16H18N3OF4
D
25
344.1386 [MþH]þ, found 344.1380. [
a]
¼ ꢀ68.3 (c ¼ 0.12 g/
D
100 mL, CH3OH).
4.1.39. (2S,4S)-1-[(3R)-3-amino-4-(2-menthylphenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8c)
4.1.44. (2S,4S)-1-[(3R)-3-amino-4-(4-methoxyphenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8h)
In the same manner as described for 8a, 8c was prepared from
18c. Yield: 79.5%. HPLC: 95.32%, tR ¼ 1.70 min. 1H NMR (CD3OD,
In the same manner as described for 8a, 8h was prepared from
400 MHz):
d 7.18e7.24 (m, 4H), 5.28e5.47 (m, 1H), 4.99 (d,
18h. Yield: 79.7%. HPLC: 97.49%, tR ¼ 1.80 min. 1H NMR (CD3OD,
J ¼ 12.0 Hz, 1H), 3.78e3.89 (m, 2H), 3.56e3.70 (m, 1H), 3.01e3.09
400 MHz):
d
7.18e7.22 (m, 3H), 6.91e6.94 (d, J ¼ 12.0 Hz, 2H),
(m, 2H), 2.77e2.93 (m,1H), 2.41e2.63 (m, 3H), 2.37 (s, 3H). 13C NMR
5.29e5.48 (m, 1H), 4.97e5.04 (m, 1H), 3.77e3.85 (m, 5H),
3.58e3.73 (m, 1H), 2.90e2.96 (m, 2H), 2.69e2.82 (m, 1H),
(CD3OD, 100 MHz):
d 169.5, 169.1, 136.7, 133.4, 130.7, 129.8, 127.5,
126.2, 117.6, 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 44.6, 35.8 (d,
2.36e2.64 (m, 3H). 13C NMR (CD3OD, 100 MHz):
d 169.5, 169.2,
J ¼ 16.7 Hz), 35.5, 34.7, 18.1. MS (ESI) m/z 290 [MþH]þ. HRMS (ESI)
159.3, 130.1, 126.9, 117.6, 114.2, 92.5 (d, J ¼ 141.4 Hz), 54.3, 52.8 (d,
25
calcd C16H21N3OF 290.1669 [MþH]þ, found 290.1680. [
(c ¼ 0.104 g/100 mL, CH3OH).
a
]
¼ ꢀ65.4
J ¼ 18.8 Hz), 49.6, 44.6, 37.1, 35.8 (d, J ¼ 16.7 Hz), 34.7. MS (ESI) m/z
D
306 [MþH]þ. HRMS (ESI) calcd C16H21N3O2F 306.1618 [MþH]þ,
25
found 306.1596. [
a
]
¼ ꢀ75.0 (c ¼ 0.10 g/100 mL, CH3OH).
D
4.1.40. (2S,4S)-1-[(3R)-3-amino-4-(3-fluorophenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8d)
4.1.45. (2S,4S)-1-[(3R)-3-amino-4-(2,4-dichlorophenyl)butanoyl]-
4-fluoropyrrolidine-2-carbonitrile (8i)
In the same manner as described for 8a, 8d was prepared from
18d. Yield: 75.2%. HPLC: 96.25%, tR ¼ 1.72 min. 1H NMR (CD3OD,
In the same manner as described for 8a, 8i was prepared from
400 MHz):
d 7.35e7.45 (m, 1H), 7.00e7.08 (m, 3H), 5.30e5.49 (m,
18i. Yield: 69.3%. HPLC: 95.43%, tR ¼ 1.92 min. 1H NMR (CD3OD,
1H), 4.98e5.07 (m, 1H), 3.82e3.91 (m, 2H), 3.60e3.74 (m, 1H), 3.04
400 MHz): d 7.55 (s, 1H), 7.30e7.39 (m, 2H), 5.31e5.49 (m, 1H),
(d, J ¼ 4.0 Hz, 2H), 2.73e2.98 (m, 1H), 2.35e2.64 (m, 3H). 13C NMR
4.97e5.05 (m,1H), 3.84e3.98 (m, 2H), 3.55e3.74 (m,1H), 3.12e3.21
(CD3OD, 100 MHz):
d
169.4, 169.0, 163.1 (d, J ¼ 195.2 Hz), 138.0 (d,
(m, 2H), 2.74e2.95 (m, 1H), 2.35e2.64 (m, 3H). 13C NMR (CD3OD,
J ¼ 5.9 Hz), 130.6 (d, J ¼ 6.6 Hz), 124.9, 117.6, 115.8 (d, J ¼ 17.3 Hz),
114.1 (d, J ¼ 16.9 Hz), 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 49.3,
44.6, 37.6, 35.8 (d, J ¼ 16.7 Hz), 34.8. MS (ESI) m/z 294 [MþH]þ.
100 MHz): d 169.2, 168.9, 161.8, 134.9, 134.1, 132.5, 132.0, 129.5,
127.6, 117.6, 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 44.6, 35.8 (d,
J ¼ 16.7 Hz), 35.5, 34.5. MS (ESI) m/z 344 [MþH]þ, HRMS (ESI) calcd
25
HRMS (ESI) calcd C15H18N3OF2 294.1418[MþH]þ, found 294.1427.
C
15H17N3OFCl2 344.0733 [MþH]þ, found 344.0721. [
a
]
¼ ꢀ62.1
D
25
[
a]
¼ ꢀ66.0 (c ¼ 0.156 g/100 mL, CH3OH).
(c ¼ 0.132 g/100 mL, CH3OH).
D
4.1.41. (2S,4S)-1-[(3R)-3-amino-4-(4-fluorophenyl)butanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8e)
4.1.46. (2S,4S)-1-[(3R)-3-amino-4-(3,4-dichlorophenyl)butanoyl]-
4-fluoropyrrolidine-2-carbonitrile (8j)
In the same manner as described for 8a, 8e was prepared from
In the same manner as described for 8a, 8j was prepared from
18e. Yield: 84.4%. HPLC: 96.40%, tR ¼ 1.66 min. 1H NMR (CD3OD,
18j. Yield: 80.1%. HPLC: 95.42%, tR ¼ 1.84 min. 1H NMR (CD3OD,
400 MHz):
d
7.28e7.32 (m, 2H), 7.08e7.13 (m, 2H), 5.30e5.47 (m,
400 MHz): d 7.49e7.55 (m, 2H), 7.22e7.27 (m, 1H), 5.30e5.49 (m,
1H), 4.90e5.06 (m, 1H), 3.82e3.91 (m, 2H), 3.58e3.73 (m, 1H),
1H), 4.98e5.08 (m, 1H), 3.85e3.94 (m, 2H), 3.62e3.74 (m, 1H),
2.72e3.01 (m, 3H), 2.35e2.64 (m, 3H). 13C NMR (CD3OD, 100 MHz):
2.97e3.06 (m, 2H), 2.74e2.94 (m, 1H), 2.35e2.62 (m, 3H). 13C NMR
d
169.4, 169.0, 162.3 (d, J ¼ 194.5 Hz), 131.2, 130.8 (d, J ¼ 6.5 Hz),
(CD3OD, 100 MHz): d 169.3, 168.9, 161.5, 136.1, 132.5, 131.3, 131.2,
117.6, 115.5 (d, J ¼ 17.2 Hz), 92.5 (d, J ¼ 141.4 Hz), 52.8 (d,
J ¼ 18.8 Hz), 49.5, 44.6, 37.2, 35.8 (d, J ¼ 16.7 Hz), 34.7. MS (ESI) m/z
130.7, 128.9, 117.6, 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 49.2,
44.7, 37.0, 35.8 (d, J ¼ 16.7 Hz), 34.7. MS (ESI) m/z 344 [MþH]þ,
294 [MþH]þ. HRMS (ESI) calcd C15H18N3OF2 294.1418 [MþH]þ,
HRMS (ESI) calcd
C
15H17N3OFCl2 344.0733 [MþH]þ, found
25
25
found 294.1417. [
a
]
¼ ꢀ70.0 (c ¼ 0.13 g/100 mL, CH3OH).
344.0715. [
a
]
¼ ꢀ57.4 (c ¼ 0.108 g/100 mL, CH3OH).
D
D

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
253
4.1.47. (2S,4S)-1-[(3R)-3-amino-4-(3,5-difluorophenyl)butanoyl]-
4-fluoropyrrolidine-2-carbonitrile (8k)
31.7, 25.2. MS (ESI) m/z 332 [MþH]þ. HRMS (ESI) m/z calcd
C
18H23N3OCl 332.1530 [MþH]þ, found 332.1535.
In the same manner as described for 8a, 8k was prepared from
18k. Yield: 68.2%. HPLC: 100.00%, tR ¼ 1.76 min. 1H NMR (CD3OD,
4.1.52. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(2-methylphenyl)butanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9c)
400 MHz):
d 6.91e6.97 (m, 3H), 5.30e5.50 (m, 1H), 4.98e5.09 (m,
1H), 3.58e3.95 (m, 3H), 2.84e3.04 (m, 3H), 2.35e2.64 (m, 3H).13
NMR (CD3OD, 100 MHz):
C
In the same manner as described for 9a, 9c was prepared from
d
169.3, 168.9, 164.4 (d, J ¼ 10.3 Hz), 162.4
25c. Yield: 68.9%. HPLC: 95.09%, tR ¼ 2.01 min. 1H NMR (CD3OD,
(d, J ¼ 10.4 Hz), 161.8, 129.8, 125.6, 117.6, 112.1 (d, J ¼ 20.5 Hz), 92.5
400 MHz):
d 7.18e7.23 (m, 4H), 4.79e4.96 (m, 1H), 4.11e4.18 (m,
(d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 49.1, 44.6, 37.6, 35.8 (d,
1H), 3.80e3.89 (m, 1H), 2.76e3.11 (m, 4H), 2.32e2.37 (m, 5H),
J ¼ 16.7 Hz), 35.2. MS (ESI) m/z 312 [MþH]þ, HRMS (ESI) calcd
1.81e2.04 (m, 4H), 1.66e1.73 (m, 3H), 1.30 (m, 1H). 13C NMR
25
C
15H17N3OF3 312.1324 [MþH]þ, found 312.1329. [
a
]
¼ ꢀ72.0
D
(CD3OD, 100 MHz):
d 169.4, 136.8, 133.5, 130.7, 129.9, 127.3, 126.1,
(c ¼ 0.10 g/100 mL, CH3OH).
119.1, 63.9, 48.5, 46.4, 44.0, 35.4, 34.4, 34.1, 33.7, 31.7, 25.3, 18.1. MS
(ESI) m/z 312 [MþH]þ. HRMS (ESI) m/z calcd C19H26N3O 312.2076
[MþH]þ, found 312.2076.
4.1.48. (2S,4S)-1-[(3R)-3-amino-4-(2,4,5-trifluorophenyl)
butanoyl]-4-fluoropyrrolidine-2-carbonitrile (8l)
In the same manner as described for 8a, 8l was prepared from
4.1.53. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(3-fluorophenyl)butanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9d)
18l. Yield: 85.6%. HPLC: 99.18%, tR ¼ 1.73 min. 1H NMR (CD3OD,
400 MHz):
d 7.29e7.36 (m, 1H), 7.18e7.23 (m, 1H), 5.31e5.50 (m,
In the same manner as described for 9a, 9d was prepared from
1H), 4.97e5.09 (m, 1H), 3.87e3.96 (m, 2H), 3.57e3.85 (m, 1H), 3.06
25d. Yield: 76.5%. HPLC: 99.26%, tR ¼ 1.98 min. 1H NMR (CD3OD,
(d, J ¼ 8.0 Hz, 2H), 2.75e3.00 (m, 1H), 2.39e2.66 (m, 3H). 13C NMR
400 MHz):
d 7.36e7.41 (m, 1H), 7.03e7.11 (m, 3H), 4.86e4.96 (m,
(CD3OD, 100 MHz):
d
169.1, 168.8, 161.6 (d, J ¼ 27.2 Hz), 156.6 (d,
1H), 4.17e4.23 (m, 1H), 3.82e3.90 (m, 1H), 2.88e3.11 (m, 3H),
2.32e2.85 (m, 3H), 1.83e2.09 (m, 4H), 1.50e1.73 (m, 3H), 1.29e1.32
J ¼ 153.6 Hz), 149.8 (d, J ¼ 198.8 Hz), 146.9 (d, J ¼ 194.0 Hz), 119.1 (d,
J ¼ 16.0 Hz), 117.6, 105.7 (d, J ¼ 23.0 Hz), 92.5 (d, J ¼ 141.4 Hz), 52.8
(d, J ¼ 18.8 Hz), 48.2, 44.6, 35.8 (d, J ¼ 16.7 Hz), 34.8, 30.9. MS (ESI)
(m, 1H). 13C NMR (CD3OD, 100 MHz):
d
169.4, 163.1 (d, J ¼ 195.2 Hz),
138.2, 130.6, 125.0, 119.1, 115.8 (d, J ¼ 17.3 Hz), 114.0 (d, J ¼ 9.1 Hz),
63.9, 48.1, 46.4, 44.0, 37.6, 34.5, 33.7, 31.7, 25.2. MS (ESI) m/z 316
[MþH]þ. HRMS (ESI) m/z calcd C18H23N3OF 316.1825 [MþH]þ,
found 316.1816.
m/z 330 [MþH]þ. HRMS (ESI) calcd C15H16N3OF4330.1230 [MþH]þ,
25
found 330.1221. [
a
]
¼ ꢀ61.7 (c ¼ 0.12 g/100 mL, CH3OH).
D
4.1.49. (2S,4S)-1-[(3S)-3-amino-4-phenylbutanoyl]-4-
fluoropyrrolidine-2-carbonitrile (8m)
4.1.54. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(4-fluorophenyl)butanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9e)
In the same manner as described for 8a, 8m was prepared from
18m. Yield: 62.2%. HPLC: 95.81%, tR ¼ 1.78 min. 1H NMR (CD3OD,
In the same manner as described for 9a, 9e was prepared from
400 MHz),
d 7.26e7.37 (m, 5H), 5.32e5.45 (m, 1H), 4.89e4.93 (m,
25e. Yield: 78.6%. HPLC: 97.36%, tR ¼ 2.17 min. 1H NMR (CD3OD,
1H), 3.56e3.80 (m, 3H), 2.87e3.04 (m, 2H), 2.30e2.65 (m, 4H). 13
NMR (CD3OD, 100 MHz): 169.8, 169.1, 135.9, 129.0, 128.7, 127.1,
C
400 MHz):
d 7.27e7.32 (m, 2H), 7.07e7.13 (m, 2H), 4.86e4.96 (m,
d
1H), 4.18e4.24 (m, 1H), 3.80e3.89 (m, 1H), 2.89e3.08 (m, 3H),
2.32e2.83 (m, 3H), 1.84e2.07 (m, 4H), 1.51e1.73 (m, 3H), 1.28e1.34
117.7, 92.5 (d, J ¼ 141.4 Hz), 52.8 (d, J ¼ 18.8 Hz), 49.5, 44.6, 38.7, 35.8
(d, J ¼ 16.7 Hz), 35.3. MS (ESI) m/z 276 [MþH]þ. HRMS (ESI) calcd
(m, 1H). 13C NMR (CD3OD, 100 MHz):
d
169.4, 162.4 (d, J ¼ 218.8 Hz),
25
C
15H19N3OF 276.1512 [MþH]þ, found 276.1523. [
a]
¼ ꢀ12.8
D
131.6, 130.9, 119.1, 115.4 (d, J ¼ 16.3 Hz), 63.9, 49.6, 46.4, 44.0, 37.1,
34.5, 34.3, 33.7, 31.7, 25.2. MS (ESI) m/z 316 [MþH]þ. HRMS (ESI) m/z
calcd C18H23N3OF 316.1825 [MþH]þ, found 316.1843.
(c ¼ 0.125 g/100 mL, CH3OH).
4.1.50. (2S,3aS,6aS)-1-[(3R)-3-amino-4-phenylbutanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9a)
4.1.55. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(4-iodophenyl)butanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9f)
Compound 25a (108 mg) was suspended in Et2OeHCl at ice-
bathe, and warmed to room temperature. After stirring for 20 h,
the solvent was removed. The residue was added 30 mL Et2O, and
the white solid was precipitated, filtered, and dried to afford
compound 9a (64 mg) as HCl salt. Yield: 70.5%. HPLC: 98.28%,
In the same manner as described for 9a, 9f was prepared from
25f. Yield: 80.2%. HPLC: 95.87%, tR ¼ 2.23 min. 1H NMR (CD3OD,
400 MHz):
d 7.70e7.76 (m, 2H), 7.00e7.10 (m, 2H), 4.90e4.98 (m,
tR ¼ 2.01 min. 1H NMR (CD3OD, 400 MHz):
d 7.27e7.48 (m, 5H),
1H), 4.13e4.19 (m, 1H), 3.82e3.89 (m, 1H), 2.89e3.02 (m, 3H),
2.33e2.77 (m, 3H), 1.85e2.07 (m, 4H), 1.47e1.73 (m, 3H), 1.26e1.32
4.84e4.96 (m, 1H), 4.14e4.19 (m, 1H), 3.82e3.90 (m, 1H), 3.03e3.14
(m, 1H), 2.77e3.00 (m, 3H), 2.51e2.73 (m, 1H), 2.32e2.44 (m, 1H),
1.81e2.07 (m, 4H), 1.47e1.73 (m, 3H), 1.28e1.32 (m, 1H). 13C NMR
(m, 1H). 13C NMR (CD3OD, 100 MHz):
d 169.4, 160.9, 137.9, 135.2,
131.2, 119.1, 63.9, 49.4, 46.4, 44.0, 37.5, 34.5, 34.3, 33.6, 31.7, 25.3. MS
(ESI) m/z 424 [MþH]þ. HRMS (ESI) m/z calcd C18H23N3OI 424.0886
[MþH]þ, found 424.0897.
(CD3OD, 100 MHz):
d 169.5, 135.4, 129.0, 128.8, 127.3, 117.1, 63.9,
49.6, 46.4, 44.0, 37.9, 34.4, 34.1, 33.6, 31.7, 25.3. MS (ESI) m/z 298
[MþH]þ. HRMS (ESI) m/z calcd C18H24N3O 298.1919 [MþH]þ, found
298.1914.
4.1.56. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(4-(trifluoromethyl)
phenyl)butanoyl]octahydrocyclopenta[b]pyrrole-2-carbonitrile (9g)
In the same manner as described for 9a, 9g was prepared from
25g. Yield: 78.2%. HPLC: 97.95%, tR ¼ 2.12 min. 1H NMR(CD3OD,
4.1.51. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(2-chlorophenyl)butanoyl]
octahydrocyclopenta[b]pyrrole-2-carbonitrile (9b)
In the same manner as described for 9a, 9b was prepared from
400 MHz): d 7.65e7.74 (m, 2H), 7.42e7.49 (m, 2H), 4.86e4.96 (m,
25b. Yield: 75.2%. HPLC: 95.76%, tR ¼ 2.15 min. 1H NMR (CD3OD,
1H), 4.18e4.25 (m, 1H), 3.89e3.95 (m, 1H), 3.06e3.15 (m, 2H),
2.54e2.90 (m, 3H), 2.35e2.60 (m, 1H), 1.83e2.08 (m, 4H), 1.48e1.72
400 MHz):
d 7.43e7.52 (m, 1H), 7.29e7.35 (m, 3H), 4.83e4.96 (m,
1H), 4.12e4.21 (m, 1H), 3.91e4.01 (m, 1H), 3.12e3.24 (m, 2H),
2.34e2.92 (m, 4H), 1.83e2.08 (m, 4H), 1.47e1.72 (m, 3H), 1.29e1.34
(m, 3H), 1.29e1.34 (m, 1H).13C NMR (CD3OD, 100 MHz):
d 169.3,
160.9,140.1,129.8,125.6,123.7,119.1, 63.9, 48.1, 46.4, 44.0, 37.7, 34.4,
34.2, 33.7, 31.7, 25.2. MS (ESI) m/z 366 [MþH]þ. HRMS (ESI) m/z
calcd C19H23N3OF3 366.1793 [MþH]þ, found 366.1785.
(m, 1H). 13C NMR (CD3OD, 100 MHz):
d
169.4, 134.2, 133.2, 131.7,
129.7, 129.2, 127.4, 119.1, 63.9, 48.3, 46.4, 44.0, 35.6, 34.4, 34.1, 33.7,

254
X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
4.1.57. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(4-methoxyphenyl)
butanoyl]octahydrocyclopenta[b]pyrrole-2-carbonitrile (9h)
In the same manner as described for 9a, 9h was prepared from
25h. Yield: 68.7%. HPLC: 96.06%, tR ¼ 1.99 min. 1H NMR (CD3OD,
4.2. In vitro DPP4, DPP7, DPP8, DPP9 and FAP enzyme assay
4.2.1. Preparation of the DPPs enzyme
The DPP4, DPP7, DPP8, DPP9 and FAP enzymes were expressed
in high five cells using a baculoviral system (Bac-To-Bac; Life
Technologies) according to the literature [35], and his6-tagged re-
combinant proteins were purified by Ni-NTA resin individually.
400 MHz):
d 7.12e7.21 (m, 2H), 6.89e6.95 (m, 2H), 4.84e4.96 (m,
1H), 4.12e4.23 (m, 1H), 3.76e3.81 (m, 4H), 2.94e3.02 (m, 1H),
2.68e2.92 (m, 3H), 2.34e2.63 (m, 2H), 1.83e2.08 (m, 4H), 1.53e1.72
(m, 3H), 1.29e1.34 (m, 1H). 13C NMR (CD3OD, 100 MHz):
d 169.5,
159.3, 130.1, 127.0, 119.1, 114.1, 63.9, 54.3, 49.6, 46.4, 44.0, 37.3, 34.5,
34.1, 33.7, 31.7, 25.2. MS (ESI) m/z 328 [MþH]þ. HRMS (ESI) m/z
calcd C19H26N3O2 328.2025 [MþH]þ, found 328.2024.
4.2.2. Enzyme-based assay of DPP4
To measure the activity of DPP4, a continuous fluorometric assay
was employed using Ala-Pro-AMC, which is cleaved by the enzyme
to release the fluorescent aminomethylcoumarin (AMC). Liberation
of AMC was monitored using an excitation wavelength of 355 nm
and an emission wavelength of 460 nm using Envision microplate
reader (PerkinElmer). A typical reaction contained 50 pmol/L
4.1.58. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(2,4-dichlorophenyl)
butanoyl)octahydrocyclopenta[b]pyrrole-2-carbonitrile (9i)
In the same manner as described for 9a, 9i was prepared from
25i. Yield: 74.8%. HPLC: 95.61%, tR ¼ 2.24 min. 1H NMR (CD3OD,
enzyme, 10
compounds synthesized in this work, and assay buffer (100 mmol/L
HEPES, pH 7.5, 0.1 mg/mL BSA) in a total reaction volume of 50 L.
mmol/L Ala-Pro-AMC, different concentrations of the
400 MHz):
d 7.50e7.61 (m, 1H), 7.31e7.39 (m, 2H), 4.84e4.96 (m,
m
1H), 4.18e4.24 (m, 1H), 3.90e3.99 (m, 1H), 3.12e3.24 (m, 2H),
2.71e2.92 (m, 2H), 2.35e2.61 (m, 2H), 1.86e2.09 (m, 4H), 1.55e1.73
The DPP4 enzyme used in these studies was soluble human re-
combinant protein produced in a baculovirus expression system
(Bac-To-Bac; Life Technologies). The dose response of inhibition test
was carried out in duplicate. And the IC50 data was calculated using
the software GraphPad Prism 5, and chosen the equation
“sigmoidal doseeresponse (variable slope)” for curve fitting.
(m, 3H), 1.28e1.35 (m, 1H). 13C NMR (CD3OD, 100 MHz):
d 169.3,
135.0, 134.1, 132.7, 132.2, 129.3, 127.6, 119.1, 63.9, 46.4, 44.0, 35.3,
34.4, 34.1, 33.6, 31.7, 25.3. MS (ESI) m/z 366 [MþH]þ. HRMS (ESI) m/
z calcd C18H22N3OCl2 366.1140 [MþH]þ, found 366.1127.
4.2.3. Enzyme-based assay of DPPs inhibition selectivity
All DPPs proteins were expressed in high five cells using a
baculoviral system, and the activities of DPPs were assayed by
continuous fluorometric method. We used Nle-Pro-AMC as sub-
strate to measure the activities of DPP7 and FAP, and Ala-Pro-AMC
for DPP8 and DPP9 in the optimized pH (5.5 for DPP7 and 8.0 for
other members) assay system. The selective dose response of in-
hibition test on DPPs and data analysis is the same as DPP4 assay
system.
4.1.59. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(3,4-dichlorophenyl)
butanoyl]octahydrocyclopenta[b]pyrrole-2-carbonitrile (9j)
In the same manner as described for 9a, 9j was prepared from
25j. Yield: 68.3%. HPLC: 98.32%, tR ¼ 1.96 min. 1H NMR (CD3OD,
400 MHz):
d 7.49e7.54 (m, 2H), 7.18e7.23 (m, 1H), 4.86e4.94 (m,
1H), 4.20e4.28 (m, 1H), 3.82e3.90 (m, 1H), 2.77e3.04 (m, 5H),
2.36e2.62 (m, 2H), 1.87e2.06 (m, 4H), 1.56e1.73 (m, 3H), 1.29e1.34
(m, 1H). 13C NMR (CD3OD, 100 MHz):
d 169.2, 161.0, 136.3, 132.4,
131.2, 130.7, 129.0, 119.0, 63.9, 49.4, 46.5, 44.0, 37.1, 34.5, 34.4, 33.7,
31.7, 25.3. MS (ESI) m/z 366 [MþH]þ. HRMS (ESI) m/z calcd
4.2.4. Pharmacokinetic profiles in SD rats
C
18H22N3OCl2 366.1140 [MþH]þ, found 366.1125.
Compounds 8l and 9l were administered to SD rats. After oral
and intravenous administration, blood samples were collected. The
blood samples were centrifuged to obtain the plasma fraction. The
plasma samples were deproteinized with methanol containing an
internal standard. After centrifugation, the supernatant was diluted
with methanol and centrifuged again. The compound concentra-
tions in the supernatant were measured by LC/MS/MS.
4.1.60. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(3,5-difluorophenyl)
butanoyl]octahydrocyclopenta[b]pyrrole-2-carbonitrile (9k)
In the same manner as described for 9a, 9k was prepared from
25k. Yield: 71.2%. HPLC: 96.08%, tR ¼ 2.16 min. 1H NMR (CD3OD,
400 MHz):
d 6.90e6.99 (m, 3H), 4.91e4.97 (m, 1H), 4.22e4.29 (m,
1H), 3.89 (br, 1H), 3.00e3.08 (m, 1H), 2.57e2.94 (m, 3H), 2.35e2.45
(m,1H),1.85e2.09 (m, 4H),1.57e1.76 (m, 3H),1.28e1.32 (m,1H). 13
NMR (CD3OD, 100 MHz):
J ¼ 10.3 Hz), 139.8, 119.1, 112.1, 102.5, 63.9, 49.2, 46.4, 44.1, 37.5, 34.4,
34.1, 33.8, 31.7, 25.2. MS (ESI) m/z 334 [MþH]þ. HRMS (ESI) m/z
calcd C18H22N3OF2 334.1731 [MþH]þ, found 334.1726.
4.2.5. Oral glucose tolerance tests in ICR mice and chronic study in
C57BKS db/db mice
C
d
169.3, 164.3 (d, J ¼ 10.4 Hz), 132.4 (d,
All animal experiments were approved by the Animal Care and
Use Committee of Shanghai Institute of Materia Medica. For the
acute single dose study, vehicle (0.5% methylcellulose, 10 mL/kg),
compounds 8l (5 and 15 mg/kg), 9l (5 and 15 mg/kg) and LAF-237
(15 mg/kg) were administered to ICR mice after 16-h starvation,
then the oral glucose tolerance test (2.5 g/kg) was conducted after
30 min of the single dose, the blood glucose level at 0, 15, 30, 60, 90
and 120 min were recorded for area under curve calculation. For the
chronic study in C57BKS db/db mice with 15 mg/kg/day 9l treat-
ment, oral glucose tolerance test (1.5 g/kg) was carried out after 6-
h starvation of 5th-week treatment, the blood glucose level was
recorded for the glucose excursion capacity evaluation. Vehicle
(0.5% methylcellulose, 10 mL/kg/day) and LAF-237 (15 mg/kg/day)
were included as negative and positive control, respectively. At the
end of study, blood samples were collected and plasma insulin was
measured using ELISA kits (Linco Research), plasma triglyceride
and total cholesterol were assayed using kits from Shanghai Fudan-
zhangjiang, free fatty acid was determined by Wako Diagnostics
(for NEFA).
4.1.61. (2S,3aS,6aS)-1-[(3R)-3-amino-4-(2,4,5-trifluorophenyl)
butanoyl]octahydrocyclopenta[b]pyrrole-2-carbonitrile (9l)
In the same manner as described for 9a, 9l was prepared from
25l. Yield: 84.1%. HPLC: 96.67%, tR ¼ 1.96 min. 1H NMR (CD3OD,
400 MHz):
d 7.22e7.34 (m, 2H), 4.90e4.96 (m, 1H), 4.23e4.29 (m,
1H), 3.82e3.90 (m, 1H), 3.12e3.21 (m, 1H), 3.01e3.08 (m, 2H),
2.57e2.76 (m, 3H), 2.37e2.42 (m, 1H), 1.88e2.09 (m, 4H), 1.62e1.73
(m, 3H), 1.28e1.31 (m, 1H). 13C NMR (CD3OD, 100 MHz):
d 169.1,
161.6 (d, J ¼ 27.0 Hz), 156.6 (d, J ¼ 187.0 Hz), 149.6 (d, J ¼ 198.7 Hz),
146.7 (d, J ¼ 194.2 Hz), 119.2 (d, J ¼ 15.5 Hz), 119.1, 105.7 (d,
J ¼ 22.8 Hz), 63.9, 48.3, 46.4, 44.0, 35.7, 34.6, 34.4, 33.7, 31.7, 30.8.
MS (ESI) m/z 352 [MþH]þ. HRMS (ESI) m/z calcd C18H21N3OF3
25
352.1673 [MþH]þ, found 352.1619. [
a
]
¼ þ25.2 (c ¼ 0.23 g/
D
100 mL, CH3OH).

X. Ji et al. / European Journal of Medicinal Chemistry 86 (2014) 242e256
255
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The results are presented as the mean SE. Differences between
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p < 0.01 was regarded as statistically significant.
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Step1: Growing cells. CHO-hERG-ZG cells are grown in 75 cm2
flask with complete medium with 100 g/mL G418 and 100 g/mL
™ thallium assay
m
m
Hygromycin B until 80e90% confluency. Wash cells with PBS once.
Incubate cells with 1 mL 0.25% Trypsin until all cells are rounded
and can be easily dislodged from the surface. Add 10 mL complete
medium to stop Trypsin activity. Disassociate cells by thoroughly,
repetitively pipetting. Transfer them to 50 mL Falcon tube and spin
down at 1000 rpm for 5 min. Aspirate medium and resuspend cells
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density. Step 2: Cell seeding. CHO-hERG-ZG cells are plated into 96-
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37 ꢁC for 16e18 h. Cells will reach 80% confluency. After overnight
incubation the cells are media changed in loading buffer (old media
is tapped out) and incubated in the dark at RT for 90 min. Remove
the loading buffer and replace with assay buffer. Compounds 8l and
9l were added to the cell plate. The cell plate is incubated with
compounds for 20 min in the dark at RT. Load the cell plates on
FDSS. Fluorescent signals will be recorded every 2 s till 10 s. At 10 s,
stimulus buffer will be added to cells. Then fluorescent signals will
be recorded every second till 180 s, data QC on FDSS.
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The DPP4 protein was extracted from RCSB Protein Data Bank
(PDB ID: 2AJL). Compounds were generated using Sybyl program.
Gasteiger-Hückel charge was used and the conformation of each
compound was minimized using default parameters. Docking
studies were performed using Glide program. The DPP4 protein was
processed by minimal minimization with OPLS2005 force field. The
grid was sized to 15 Å in each direction at the center of the binding
pocket. Compounds were prepared for docking using Ligprep.
Ligand docking was performed in XP mode and flexible option,
with up to 100 poses saved per molecule. Glide score was consulted
for results analyzing.
Acknowledgments
[13] D.J. Madar, H. Kopecka, D. Pireh, H. Yong, Z. Pei, X. Li, P.E. Wiedeman,
S.W. Djuric, T.W. Von Geldern, M.G. Fickes, L. Bhagavatula, T. McDermott,
S. Wittenberger, S.J. Richards, K.L. Longenecker, K.D. Stewart, T.H. Lubben,
S.J. Ballaron, M.A. Stashko, M.A. Long, H. Wells, B.A. Zinker, A.K. Mika,
D.W. Beno, A.J. Kempf-Grote, J. Polakowski, J. Segreti, G.A. Reinhart, R.M. Fryer,
H.L. Sham, J.M. Trevillyan, Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (Grants 21021063, 91229204,
81125023 and 81025017), National S&T Major Projects
(2012ZX09103101-072, 2012ZX09301001-005, and 2013ZX09507-
001), sponsored by Program of Shanghai Subject Chief Scientist
(Grant 12XD1407100).
pyrrolidinyl]-2-oxoethyl]amino]-
4-methyl-1-piperidinyl]-4-
pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and
well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of
diabetes, J. Med. Chem. 49 (2006) 6416e6420.
[14] H. Sakashita, H. Kitajima, M. Nakamura, F. Akahoshi, Y. Hayashi, 1-((S)-g-
substituted prolyl)-(S)-2-cyanopyrrolidine as a novel series of highly potent
DPP-IV inhibitors, Bioorg. Med. Chem. Lett. 15 (2005) 2441e2445.
[15] H. Fukushima, A. Hiratate, M. Takahashi, A. Mikami, M. Saito-Hori,
E. Munetomo, K. Kitano, S. Chonan, H. Saito, A. Suzuki, Y. Takaoka,
K. Yamamoto, Synthesis and structure-activity relationships of potent 4-
fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors, Bioorg. Med.
Chem. 16 (2008) 4093e4106.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.059.
[16] P. Mattei, M. Boehringer, P. Di Giorgio, H. Fischer, M. Hennig, J. Huwyler,
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