R-3-Amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives as potent inhibitors of dipeptidyl peptidase-4: Design, synthesis, biological evaluation, and molecular modeling

Article abstract of DOI:10.1016/j.bmc.2014.09.051

A series of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives was designed, synthesized, and evaluated as novel inhibitors of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes. Most of the synthesized compounds demonstrated good inhibition activities against DPP-4. Among these, compounds 3e, 4c, 4l, and 4n exhibited prominent inhibition activities against DPP-4, with IC₅₀s of 0.07, 0.07, 0.14, and 0.17 μM, respectively. The possible binding modes of compounds 3e and 4n with dipeptidyl peptidase-4 were also explored by molecular docking simulation. These potent DPP-4 inhibitors were optimized for the absorption, distribution, metabolism, and excretion (ADME) properties, and compound 4n displayed an attractive pharmacokinetic profile (F = 96.3%, t_1/2 = 10.5 h).

Full text of DOI:10.1016/j.bmc.2014.09.051

Bioorganic & Medicinal Chemistry 22 (2014) 6684–6693
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(R)-3-Amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-
trifluorophenyl)butan-1-one derivatives as potent inhibitors
of dipeptidyl peptidase-4: Design, synthesis, biological evaluation,
and molecular modeling
⇑
⇑
Sinan Wang , Mingbo Su , Jiang Wang , Zeng Li, Lei Zhang, Xun Ji, Jingya Li, Jia Li , Hong Liu
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi
Road, Shanghai 201203, 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:
Received 23 July 2014
Revised 24 September 2014
Accepted 24 September 2014
Available online 23 October 2014
A
series of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one
derivatives was designed, synthesized, and evaluated as novel inhibitors of dipeptidyl peptidase-4
(DPP-4) for the treatment of type 2 diabetes. Most of the synthesized compounds demonstrated good
inhibition activities against DPP-4. Among these, compounds 3e, 4c, 4l, and 4n exhibited prominent inhi-
bition activities against DPP-4, with IC50s of 0.07, 0.07, 0.14, and 0.17 lM, respectively. The possible bind-
ing modes of compounds 3e and 4n with dipeptidyl peptidase-4 were also explored by molecular docking
simulation. These potent DPP-4 inhibitors were optimized for the absorption, distribution, metabolism,
and excretion (ADME) properties, and compound 4n displayed an attractive pharmacokinetic profile
(F = 96.3%, t1/2 = 10.5 h).
Keywords:
DPP-4
Type 2 diabetes
Inhibitor
Binding mode
Oral bioavailability
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
potent and selective inhibitors of DPP-4. However, their low oral
bioavailability and short half-life raised concerns regarding their
Type 2 diabetes is a progressive metabolic disorder character-
ized by high blood glucose in the presence of high endogenous
insulin levels, causing serious vascular complications, heart dis-
ease, renal failure, blindness, significant morbidity, and mortal-
ity.1,2 This metabolic disease has become one of the fastest
growing health concerns in the world.3 Glucagon-like peptide-1
(GLP-1) is a potent antihyperglycemic hormone, inducing glu-
cose-dependent stimulation of insulin secretion, while inhibiting
glucagon secretion.4–7 However, active GLP-1 is rapidly degraded
by the serine protease dipeptidyl peptidase-4 (DPP-4) which selec-
tively cleaves a dipeptide from the N-terminus of GLP-1 to produce
the inactive GLP-1[9–36] amide.8,9 Consequently, inhibition of
DPP-4 has emerged as a new potential approach for the treatment
of type 2 diabetes.10–13 To date, a large number of DPP-4 inhibitors
have been reported.14–18
pharmacokinetic suitability for oral administration. As discussed
previously,19 we hypothesized that a larger hydration sphere car-
ried by the adjacent amides might account for this issue. In our
search for structural modifications to overcome this, while
maintaining favorable properties, we envisaged to replace the side
chain amide with
a bioisosteric (3aS,7aS)-octahydro-1H-indol
(3a–f and 4a–n, Fig. 1). Herein, we report our efforts towards the
design, synthesis, biological evaluation, and molecular modeling
of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-tri-
fluorophenyl)butan-1-one derivatives as a novel series of DPP-4
inhibitors.
2. Results and discussion
2.1. Design
In our previous work, we described a series of 4-fluoropyrroli-
dine-2-carbonitrile and octahydrocyclopenta[b]pyrrole-2-carboni-
trile derivatives (1 and 2).19 Early examples were found to be
The X-ray crystal study of various inhibitors in complex with
DPP-4 revealed that the binding site of DPP-4 mainly comprises
three parts: a S1 pocket, the N-terminal recognition region, and a
S2 pocket (Fig. 2).14,20 The hydrophobic S1 pocket is formed by res-
idues of Ser630, Tyr631, Tyr547, Val711, Asn710, and Arg125. The
N-terminal recognition region is formed by Glu205, Glu206, and
⇑
Corresponding authors. Tel.: +86 21 50801552; fax: +86 21 50800721 (J.L.); tel./
fax: +86 21 50807042 (H.L.).
E-mail addresses: jli@simm.ac.cn (J. Li), hliu@simm.ac.cn (H. Liu).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.09.051
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
6685
F
F
F
F
S1 pocket
F
S1 pocket
F
NH2
O
NH2
O
N
N
F
X
NC
R
O
S2 pocket
S2 pocket
1
X = O,NH
3a-f
F
F
NH2
O
S1 pocket
F
S1 pocket
F
N
NH2
O
F
NC
N
F
S2 pocket
X
R
S2 pocket
2
X = O,NH
4a-n
Figure 1. Design of novel (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives as DPP-4 inhibitors.
a variety of substituents X and R. Deprotection of compounds 10a–
f using TFA in dichloromethane afforded target compounds 3a–f.
Scheme 2 depicts the sequence of reactions that led to the prep-
aration of compounds 4a–f using (2S,3aS,7aS)-1-(tert-butoxycar-
bonyl)octahydro-1H-indole-2-carboxylic acid (6) as the starting
material. In general, reducing the carboxyl group in compound 6
to hydroxyl group using LiAlH4 in tetrahydrofuran (THF) afforded
derivative 11, which was then reacted with alkyl or aryl acyl chlo-
ride to give compounds 12a–f, respectively. The target compounds
13a–f were prepared from 12a–f and (R)-3-(tert-butoxycarbonyla-
mino)-4-(2,4,5-trifluorophenyl)butanoic acid
9 using standard
coupling conditions followed by deprotection of the amines 14a–
f, which afforded target compounds 4a–f.
Compounds 4g–n were synthesized according to Scheme 3.
Compound 11 was transformed to 16 using p-toluenesulfonyl chlo-
ride followed by NaN3. After deprotection, condensation of 17 with
(R)-3-(tert-butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)buta-
noic acid 9 provided compound 18, which was then reduced by H2,
Pd/C in MeOH to give compound 19. Condensation of compound 19
with various acyl chloride or sulfonyl chloride afforded compounds
20g–n. Deprotection of Boc derivatives 20g–n by treatment with
TFA in CH2Cl2 provided target compounds 4g–n.
Figure 2. The binding pocket of DPP-4 (PDB ID: 1X70). This image was generated
using the Pymol program.20
Tyr662. The hydrophobic S2 pocket, which is bigger compared
with S1, is surrounded by residues Phe357, Arg358, Ser209, and
Tyr666. Investigation of the structure–activity relationships (SARs)
of the DPP-4 inhibitors previously reported,14,21,22 indicated that
the S2 pocket can tolerate different segments, thus we selected
an (3aS,7aS)-octahydro-1H-indole moiety to fill in the S2 pocket
and introduced various functional groups on it. To improve
pharmacokinetic properties of DPP-4 inhibitors,19 a novel series
of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-tri-
fluorophenyl)butan-1-one derivatives (3a–f and 4a–n) was
designed (Fig. 1).
2.3. In vitro enzyme inhibition studies and structure–activity
relationships
All the synthesized compounds were evaluated in vitro for inhi-
bition of human recombinant DPP-4. Inhibitory potencies were
measured by monitoring the hydrolytic reaction of Ala-Pro-AMC
by human DPP-4 with Sitagliptin as the positive control. The
results were reported as concentrations for 50% inhibition (IC50).
DPP-7, DPP-8, DPP-9, and fibroblast activation protein (FAP) data
were also presented because the importance of selectivity over
DPP-8/9 has been frequently mentioned in animal toxicity stud-
ies.23–27 SARs for these compounds are summarized in Tables 1–3.
To obtain good inhibitory activity against DPP-4, various amides
and esters were prepared. As shown in Table 1, introduction of the
simple N-phenyl-acetamide 3a resulted in DPP-4 inhibitory
2.2. Chemistry
The synthesis of compounds 3a–f is outlined in Scheme 1. Pro-
tection of the commercially available compound 5 formed com-
pound 6. Condensation of compound 6 with various amines
(X = N) or alcohols (X = O) provided 7a–f, followed by deprotection
using trifluoroacetic acid (TFA) to form compounds 8a–f, which
were condensed with (R)-3-(tert-butoxycarbonylamino)-4-(2,4,5-
trifluorophenyl)butanoic acid 9 to provide compounds 10a–f with
activity with an IC50 value of 5.81
acetamide 3b increased the activity (IC50 = 0.34
l
M. Substitution of N-benzyl-
M) by approxi-
l
mately 17-fold compared to that of 3a. In case of three-membered
N-cyclopropyl-acetamide 3c and N-(cyclopropylmethyl)-acetam-
ide 3d increased potency by 15.3 and 7.6 fold compared with
N-phenyl-acetamide 3a. Generally, introduction of an aliphatic ring

6686
S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
O
O
X
b
c
a
COOH
+
COOH
N
N
N
H
X
R
R
N
H
Boc
Boc
X = O,NH
X = O,NH
7a-f
8a-f
6
d
5
O
F
F
O
O
F
F
F
F
O
F
F
F
HN
O
NH2
O
HN
O
e
N
N
OH
X
X
R
R
O
O
X = O,NH
10a-f
X = O,NH
3a-f
9
Scheme 1. Reagents and conditions: (a) (Boc)2O, TEA, CH2Cl2; (b) HOBT, EDCI, DMAP, TEA, CH2Cl2, overnight; (c) CF3COOH, CH2Cl2, overnight; (d) EDCI, DMAP, TEA, CH2Cl2,
overnight; and (e) CF3COOH, CH2Cl2, overnight.
OH
b
c
a
COOH
+
N
N
O
R
N
H
O
R
N
Boc
Boc
Boc
11
6
12a-f
13a-f
O
O
F
F
F
F
F
O
O
O
F
F
F
HN
HN
O
NH2
O
d
e
OH
N
N
F
O
O
R
R
9
14a-f
4a-f
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, overnight; (b) acyl chloride, TEA, CH2Cl2, overnight; (c) CF3COOH, CH2Cl2, overnight; (d) EDCI, DMAP, TEA, CH2Cl2,
overnight; (e) CF3COOH, CH2Cl2, overnight.
O
S
O
N3
N3
OH
c
b
O
a
+
N
H
N
N
N
Boc
Boc
Boc
11
15
16
F
17
O
O
F
F
F
O
O
O
O
O
F
F
F
HN
O
O
HN
F
d
e
HN
N
N
OH
F
H2N
19
N3
18
9
O
F
F
F
F
O
O
F
F
NH2
O
HN
g
f
N
N
RHN
4g-n
RHN
20g-n
Scheme 3. Reagents and conditions: (a) p-toluenesulfonyl chloride, pyridine, overnight; (b) NaN3, DMF, 65 °C, overnight; (c) CF3COOH, CH2Cl2, overnight; (d) EDCI, DMAP,
TEA, CH2Cl2, overnight; (e) H2, Pd/C, MeOH, 2 h; (f) acyl chloride or sulfonyl chloride, TEA, CH2Cl2, overnight; and (g) CF3COOH, CH2Cl2, overnight.
performed better than introduction of an aromatic group. Surpris-
ingly, the ethyl ester 3e significantly increased DPP-4 inhibitory
Among compounds 4a–f, introduction of an acetyl group (4a)
showed excellent DPP-4 inhibitory activity (IC50 = 0.07 M). The
l
activity (IC50 = 0.07
Compound 3f, which was a carboxylic acid lost its activity against
DPP-4 (IC50 = 3.66 M).
l
M) being 5-fold more active than 3d.
DPP-4 inhibitory activities of hydroxyl derivatives (4b, 4c, and
4d) were directly correlated with the number of the ring size
(the IC50 values of cyclopropyl, cyclobutyl, and cyclopentyl were
l

S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
6687
Table 1
Table 2
Enzymatic activities of compounds 3a–f against DPP-4
Enzymatic activities of compounds 4a–n against DPP-4
F
F
F
F
NH2
O
NH2
O
N
N
F
F
X
R
X
R
O
a
Compd
X
R
DPP-4 IC50
0.07
(lM)
a
Compd
X
R
DPP-4 IC50
5.81
(lM)
4a
O
O
O
O
3a
NH
4b
4c
0.08
0.07
3b
NH
0.34
3c
NH
NH
0.38
0.76
O
O
O
O
3d
3e
3f
O
O
—
0.07
3.66
0.02
4d
4e
0.18
0.32
H
—
Sitagliptin
a
In vitro activity.
O
O
4f
OH
NH
—
0.51
0.29
4g
0.08, 0.07, and 0.18
pounds 4e (benzoyl moiety) and 4f (carboxylic acid) were less
potent with IC50 values of 0.32 and 0.51 M. The activity
lM, respectively). In the SAR, we found com-
O
O
l
4h
4i
NH
NH
0.54
0.33
decreased with the expansion of the ring and compounds with
aromatic groups were relatively less potent. Replacement of oxy-
gen in the hydroxyl linker to nitrogen showed a similar activity
pattern, which was dependent on the bulkiness of direct substi-
tuent to nitrogen (4g, 4h, 4i, 4j, and 4k). Generally, the amide
series were less potent than the ester series. Finally, incorporat-
ing a sulfonyl group instead of the acyl resulted in highly potent
DPP-4 inhibitory activity (4l, 4m, and 4n). Investigation of the
inhibitory activities of compounds 3a–f and 4a–n showed that
compounds 3e, 4a, 4b, 4c, 4l, and 4n exhibited good activity.
However, the inhibitory activity was lower than that of Sitaglip-
O
O
4j
NH
NH
0.43
0.61
4k
O
S
tin (IC50 = 0.02 lM).
Inhibition of DPP-8 and DPP-9 might be connected with toxic-
ity, selected compounds were tested for their selectivity profiles
against the DPP-4 homologs DPP-7, DPP-8, DPP-9, and fibroblast
activation protein (FAP). Data are presented in Table 3. Compound
3e exhibited good selectivity (SR: DPP-8/DPP-4 = 53; DPP-9/DPP-
4 = 246). The other selected compounds exhibited moderate selec-
tivities against DPP-8 and DPP-9.
4l
NH
NH
0.14
0.43
O
O
O
S
O
4m
CF3
S
4n
NH
—
0.17
0.02
O
O
Sitagliptin
In vitro activity.
—
a
2.4. Binding mode of compounds 3e, 4n, and Sitagliptin
To gain structural information for further optimization, the 3D
binding modes of compounds 3e, 4n, and Sitagliptin to DPP-4
(PDB ID: 1X70) were generated based on docking simulations
(Fig. 3). The binding modes indicated that the 2,4,5-triflurophenyl
moiety of compounds 3e, 4n, and Sitagliptin effectively binds to
DPP-4 in the S1 hydrophobic pocket. The b-amino group forms
three hydrogen bonds with the side chains of Glu205, Glu206,
and Tyr662. The triazolopiperazine of Sitagliptin is stacked against
the side chain of Phe357 and the trifluoromethyl substituent inter-
acts with the side chains of Arg358 and Ser209 in the S2 pocket.
Meanwhile, the simulations indicated that the (3aS,7aS)-octahy-
dro-1H-indole moiety of compounds 3e and 4n could not be
stacked against the side chain of Phe357, respectively, compared
with Sitagliptin, which could be the main reason for the decrease
in inhibitory activity.
2.5. Pharmacokinetic evaluation of compounds 3e, 4l, and 4n
The pharmacokinetic (PK) profiles of the selected compounds
3e, 4l, and 4n were assessed in Sprague–Dawley (SD) rats (Table 4).
The Cmax of compound 3e at 1 h was 194 ng/mL, with an AUC0–t
621.6 ng/mL*h at a dose of 20 mg/kg. Moreover, compound 3e
demonstrated high clearance in SD rats. The absolute oral bioavail-
ability for compounds 3e and 4l were moderate (22.4% and 43.7%),
while compound 4l had a lower half-life than compound 3e (2.52 h
vs 2.80 h, respectively). Compound 4n exhibited an attractive
pharmacokinetic profile likely suitable for once daily dosing in
humans. High oral bioavailability (96.3%) and exposure
(5518.9 ng/mL*h) was accompanied by
a reasonable half-life
(10.5 h), which was much better than Sitagliptin.

6688
S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
Table 3
Potencies and selectivities of selected DPP-4 inhibitors
Compd
IC50
(
l
M)a
SRb
DPP-4
DPP-7
DPP-8
DPP-9
FAP
3.35
DPP-8/DPP-4
DPP-9/DPP-4
3e
4a
4b
4c
4d
4g
4l
4n
0.07
0.07
0.08
0.07
0.18
0.29
0.14
0.17
0.02
NIc
3.42
1.82
0.75
0.88
1.75
4.17
1.47
2.90
56.33
15.96
3.06
2.18
3.30
1.96
6.27
0.32
5.26
56.72
53
25
10
13
10
14
11
17
2817
246
42
28
49
11
22
2
32
2836
59.83
31.94
23.10
24.34
NI
6.85
14.00
207.35
5.83
3.29
3.92
1.71
6.36
2.06
4.03
34.18
Sitagliptin
a
b
c
In vitro activity.
Selectivity Ratio.
NI: no inhibition.
Figure 3. Three-dimensional structural modes of inhibitors 3e (a), 4n (b), and Sitagliptin (c) to DPP-4 (PDB ID: 1X70) derived from the docking simulations. These three
images were generated using the Pymol program.20
Table 4
Pharmacokinetic properties of compounds 3e, 4l, 4n, and Sitagliptin in SD rats
Compd
Admin
Dose
mg/kg
Tmax
h
Cmax
ng/mL
AUC0–t
ng/mL*h
MRT
h
t1/2
h
CLz
L/h/kg
F
%
3e
p.o.a
i.v.
p.o.
i.v.
p.o.
i.v.
20
10
20
10
20
10
2
1
0.25
2
0.25
2
0.25
/
/
194
618.7
360.3
620.4
851.7
629.1
/
621.6
1387.4
1791.5
2049.4
5518.9
2865.0
/
2.67
1.90
3.27
2.57
4.13
3.28
/
2.80
1.56
2.52
1.95
10.5
4.51
1.7
/
22.4
/
43.7
/
96.3
/
76
/
a
7.01
/
4.52
/
2.42
/
/
4l
4n
Sitagliptinb
p.o.
i.v.
1
/
/
/
/
a
p.o., oral administration; i.v., intravenous injection.
Ref. 14.
b
3. Conclusion
4. Experiments
4.1. Chemistry
In conclusion, we designed, synthesized, and evaluated a series
of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-tri-
fluorophenyl)butan-1-ones as novel DPP-4 inhibitors. In vitro
evaluation found that most of the compounds exhibited good
in vitro potency against DPP-4 over a low micromolar range.
The reagents (chemicals) were purchased from commercial
sources, and used without further purification. Analytical-thin
layer chromatography was HSGF254 (0.15–0.2 mm thickness).
Yields were not optimized. Nuclear magnetic resonance (NMR)
spectroscopy were given on a Brucker AMX-400 and AMX-300
instruments (using tetramethylsilane as internal standard). Chem-
ical shifts were reported in parts per million (ppm, delta) down-
field from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multi-
plet (m), and broad (br). Low-resolution mass spectroscopy were
carried out on an electric ionization (ESI) electrospray and a
LCQ-DECA spectrometer produced by Finnigan MAT-95.
Among these compounds, 3e (IC50 = 0.07
4l (IC50 = 0.14 M), and 4n (IC50 = 0.17
l
M), 4c (IC50 = 0.07
lM),
l
lM) showed good DPP-4
potency. The pharmacokinetic profiles of compounds 3e, 4l, and
4n are suitable for clinical use. In particular, compound 4n exhib-
ited an attractive PK profile (F = 96.3%, t1/2 = 10.5 h). These potent
compounds represent a novel scaffold for the development of
DPP-4 inhibitors. Current efforts are aimed at further modifying
these compounds to achieve more potent and selective DPP-4
inhibitors.

S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
6689
4.1.1. General procedures for the synthesis of compounds 3a–f
3.76–3.68 (m, 1H), 3.50–3.42 (m, 1H), 2.75–2.69 (m, 1H), 2.63–
2.56 (m, 1H), 2.45–2.39 (m, 2H), 2.34–2.25 (m, 1H), 1.99–1.90
(m, 4H), 1.75–1.61 (m, 4H), 1.52–1.36 (m, 2H); 13C NMR (CDCl3,
75 MHz): d 171.8, 170.3, 157.4, 157.3, 154.9, 154.8, 150.2, 150.1,
149.9, 147.9, 147.8, 147.7, 147.6, 147.4, 145.5, 145.4, 138.5,
128.5, 127.5, 127.2, 121.8, 121.6, 119.2, 119.1, 119.0, 119.0,
105.7, 105.5, 105.4, 105.2, 59.8, 58.9, 48.9, 43.4, 39.8, 37.1, 35.6,
29.5, 27.7, 25.6, 23.8, 19.8; ESI-MS m/z calcd for C26H31F3N3O2
[M+H]+ 474.2, found 474.1.
(a) To a solution of compound 5 in CH2Cl2 was added triethyl-
amine and (Boc)2O. The reaction was monitored by TLC.
After the starting materials were completely consumed,
the mixture was diluted with CH2Cl2, washed sequentially
with 1 N HCl and brine. The organic layer was dried over
Na2SO4, filtered, and evaporated to afford compound 6 as
oil (94% yield).
(b) To a solution of compound 6 in CH2Cl2 was added HOBt,
EDCI, DMAP, and triethylamine. After stirring for 30 min ani-
line was added. The reaction mixture was stirred overnight.
The reaction was monitored by TLC. After the starting mate-
rials were completely consumed, the mixture was diluted
with CH2Cl2, washed sequentially with 1 N HCl and brine.
The organic layer was dried over Na2SO4, filtered, and evap-
orated. The residue was purified by a flash column chroma-
tography (CH2Cl2 as an eluent) to afford compound 7a as a
white solid (68% yield).
(c) To a solution of 7a in dry CH2Cl2 was added CF3COOH. After
stirring the reaction overnight, the solvent was removed
under reduced pressure. The mixture was diluted with ethyl
acetate, washed sequentially with saturated Na2CO3 and
brine. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure to afford compound
8a as a yellow solid (85% yield).
(d) To a solution of (R)-3-(tert-butoxycarbonylamino)-4-(2,4,5-
trifluorophenyl)butanoic acid 9 in CH2Cl2 was added HOBt,
EDCI, DMAP, and triethylamine. After stirring for 30 min 8a
was added. The reaction mixture was stirred overnight.
The reaction was monitored by TLC. After the starting mate-
rials were completely consumed, the mixture was diluted
with CH2Cl2, washed sequentially with 1 N HCl and brine.
The organic layer was dried over Na2SO4, filtered, and evap-
orated. The residue was purified by a flash column chroma-
tography (petroleum ether/ethyl acetate = 4:1, v/v, as an
eluent) to afford compound 10a as a white solid (72% yield).
(e) To a solution of 10a in dry CH2Cl2 was added CF3COOH. After
stirring the reaction overnight, the solvent was removed
under reduced pressure. The mixture was diluted with ethyl
acetate, washed sequentially with saturated Na2CO3 and
brine. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure to afford compound
3a as oil (80% yield).
4.1.4. (2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)-N-cyclopropyloctahydro-1H-indole-
2-carboxamide (3c)
1H NMR (CDCl3, 300 MHz): d 7.26–7.14 (m, 1H), 6.96–6.88 (m,
1H), 4.38–4.35 (m, 1H), 3.71–3.59 (m, 2H), 3.15–3.11 (m, 2H),
2.79–2.72 (m, 2H), 2.35–2.33 (m, 1H), 2.09–2.06 (m, 3H), 1.72–
1.25 (m, 8H), 0.46–0.44 (m, 2H), 0.18–0.16 (m, 2H); 13C NMR
(MeOD-d4, 75 MHz): d 172.8, 169.5, 157.5, 157.4, 155.0, 154.9,
150.8, 150.7, 150.6, 148.4, 148.3, 148.2, 148.2, 148.0, 145.4,
145.3, 119.5, 119.4, 119.3, 119.2, 119.0, 118.9, 105.8, 105.7,
105.6, 105.5, 61.7, 59.9, 49.5, 36.8, 35.3, 35.0, 33.4, 30.6, 26.6,
25.8, 25.4, 23.6, 6.2, 6.2; ESI-MS m/z calcd for C22H29F3N3O2
[M+H]+ 424.2, found 424.1.
4.1.5. (2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)-N-(cyclopropylmethyl)octahydro-
1H-indole-2-carboxamide (3d)
1H NMR (CDCl3, 300 MHz): d 7.22–7.13 (m, 1H), 6.96–6.88 (m,
1H), 4.41–4.30 (m, 1H), 3.72–3.68 (m, 1H), 3.61–3.56 (m, 1H),
3.18–3.11 (m, 2H), 3.05–2.88 (m, 2H), 2.79–2.72 (m, 1H), 2.35–
2.31 (m, 1H), 2.11–2.04 (m, 3H), 1.72–1.48 (m, 6H), 1.31–1.09
(m, 2H), 0.94–0.88 (m, 1H), 0.46–0.44 (m, 2H), 0.18–0.16 (m,
2H); 13C NMR (CDCl3, 75 MHz): d 171.8, 169.0, 157.5, 157.4,
155.0, 154.9, 151.0, 150.8, 150.6, 148.4, 148.3, 148.2, 148.2,
148.1, 145.7, 145.6, 119.6, 119.4, 119.2, 119.1, 119.0, 118.9,
106.0, 105.7, 105.6, 105.5, 60.3, 59.0, 49.2, 44.2, 37.0, 33.6, 31.4,
31.0, 27.1, 25.4, 23.6, 19.8, 10.4, 3.2, 3.2; ESI-MS m/z calcd for C23-
H31F3N3O2 [M+H]+ 438.2, found 438.1.
4.1.6. (2S,3aS,7aS)-Ethyl-1-((R)-3-amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indole-2-carboxylate
(3e)
1H NMR (CDCl3, 300 MHz): d 7.09–7.04 (m, 1H), 6.91–6.82 (m,
1H), 4.36–4.30 (m, 1H), 4.15 (q, J = 7.5 Hz, 2H), 3.75–3.67 (m,
1H), 3.52–3.52 (m, 1H), 2.77–2.60 (m, 2H), 2.37–2.32 (m, 2H),
2.12–2.05 (m, 1H), 1.98–1.87 (m, 1H), 1.74–1.45 (m, 6H), 1.25–
1.09 (m, 6H); 13C NMR (CDCl3, 75 MHz): d 172.5, 169.2, 157.5,
157.3, 155.0, 154.9, 150.5, 150.2, 150.1, 148.0, 147.9, 147.8,
147.6, 147.4, 145.6, 145.4, 121.0, 120.8, 119.4, 119.3, 119.1,
118.9, 105.7, 105.5, 105.2, 104.9, 61.2, 59.0, 58.2, 48.9, 37.4, 34.7,
33.8, 30.5, 27.7, 25.6, 23.6, 19.9, 14.1; ESI-MS m/z calcd for C21H28-
F3N2O3 [M+H]+ 413.2, found 413.1.
4.1.2. (2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)-N-phenyloctahydro-1H-indole-2-
carboxamide (3a)
1H NMR (CDCl3, 300 MHz): d 7.67 (d, J = 8.1 Hz, 2H), 7.28–7.27
(m, 1H), 7.23–7.22 (m, 1H), 7.19–7.11 (m, 1H), 7.05–7.00 (m,
1H), 6.95–6.87 (m, 1H), 4.71–4.65 (m, 1H), 3.66–3.61 (m, 2H),
2.97–2.76 (m, 2H), 2.55–2.49 (m, 1H), 2.42–2.34 (m, 1H), 2.18–
2.15 (m, 2H), 1.76–1.46 (m, 8H), 1.14–1.09 (m, 1H); 13C NMR
(MeOD-d4, 75 MHz): d 173.1, 170.4, 159.4, 159.4, 157.5, 157.4,
152.5, 152.4, 152.3, 150.5, 150.4, 150.3, 149.8, 149.6, 147.8,
147.7, 140.4, 130.4, 125.8, 121.8, 121.7, 121.3, 121.2, 121.1,
121.1, 107.7, 107.5, 107.5, 107.3, 62.8, 60.3, 50.8, 39.2, 35.8, 32.7,
32.7, 29.3, 27.1, 25.2, 21.6; ESI-MS m/z calcd for C25H29F3N3O2
[M+H]+ 460.2, found 460.1.
4.1.7. (2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indole-2-carboxylic
acid (3f)
1H NMR (CDCl3, 300 MHz): d 7.14–7.06 (m, 1H), 6.96–6.87 (m,
1H), 4.41–4.35 (m, 1H), 3.80–3.72 (m, 1H), 3.60–3.57 (m, 1H),
2.78–2.65 (m, 2H), 2.45–2.42 (m, 2H), 2.15–2.10 (m, 1H), 2.03–
1.92 (m, 1H), 1.79–1.50 (m, 6H), 1.30–1.25 (m, 3H); 13C NMR
(CDCl3, 75 MHz): d 175.9, 170.2, 157.4, 157.2, 155.0, 154.9, 150.3,
150.2, 150.1, 148.1, 148.0, 147.9, 147.5, 147.4, 145.5, 145.4,
121.0, 120.8, 119.2, 119.1, 119.0, 118.9, 105.5, 105.4, 105.2,
105.0, 62.3, 57.9, 50.4, 37.4, 36.2, 35.5, 32.6, 30.2, 26.7, 24.8,
23.0; ESI-MS m/z calcd for C19H24F3N2O3 [M+H]+ 385.2, found
385.1.
4.1.3. (2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)-N-benzyloctahydro-1H-indole-2-
carboxamide (3b)
1H NMR (CDCl3, 300 MHz): d 7.32–7.26 (m, 4H), 7.23–7.20 (m,
1H), 7.06–6.98 (m, 1H), 6.94–6.86 (m, 1H), 4.51–4.41 (m, 3H),

6690
S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
4.1.8. General procedures for the synthesis of compounds 4a–f
1.34–1.25 (m, 2H), 1.00–0.96 (m, 2H), 0.87–0.85 (m, 2H); 13C
NMR (CDCl3, 75 MHz): d 174.2, 169.5, 156.7, 156.6, 154.7, 154.7,
149.5, 149.4, 149.3, 147.4, 147.3, 147.2, 147.1, 147.0, 145.2,
145.0, 121.0, 120.9, 118.8, 118.8, 118.7, 118.6, 105.1, 105.0,
104.9, 104.8, 63.8, 58.0, 55.8, 48.6, 38.6, 36.0, 34.3, 29.0, 28.0,
25.2, 23.4, 19.6, 12.3, 8.0, 7.9; ESI-MS m/z calcd for C23H30F3N2O3
[M+H]+ 439.2, found 439.1.
(a) Compound 6 in dry THF was added 1 N LiAlH4 slowly at 0 °C.
The reaction mixture was stirred overnight at room temper-
ature. After the starting materials were completely con-
sumed, the mixture was diluted with EA, washed
sequentially with 1 N HCl and brine. The organic layer was
dried over Na2SO4, filtered, and evaporated under reduced
pressure to afford compound 11 as oil (60% yield).
4.1.11. ((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
(b) To a solution of 11 and triethylamine in CH2Cl2 was added
acetyl chloride. The reaction mixture was stirred overnight.
The reaction was monitored by TLC. After the starting mate-
rials were completely consumed, the mixture was purified
by a flash column chromatography (petroleum ether/ethyl
acetate = 1:1, v/v, as an eluent) to afford compound 12a as
a white solid (79% yield).
(c) To a solution of 12a in dry CH2Cl2 was added CF3COOH. After
stirring the reaction overnight, the solvent was removed
under reduced pressure. The mixture was diluted with ethyl
acetate, washed sequentially with saturated Na2CO3 and
brine. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure to afford compound
13a as a yellow solid (88% yield).
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl
cyclobutanecarboxylate (4c)
1H NMR (CDCl3, 300 MHz): d 7.14–7.05 (m, 1H), 6.96–6.87 (m,
1H), 4.34–4.31 (m, 2H), 4.22–4.17 (m, 1H), 3.67–3.54 (m, 2H),
3.17–3.11 (m, 1H), 2.79–2.74 (m, 2H), 2.32–2.17 (m, 10H), 1.92–
1.85 (m, 3H), 1.73–1.62 (m, 4H), 1.31–1.25 (m, 2H); 13C NMR
(CDCl3, 75 MHz): d 174.7, 169.2, 156.7, 156.6, 154.7, 154.7, 149.7,
149.6, 149.5, 147.6, 147.5, 147.4, 147.3, 147.2, 145.3, 145.2,
120.2, 120.0, 119.0, 118.9, 118.8, 118.8, 105.2, 105.1, 105.0,
104.8, 63.3, 57.9, 55.9, 48.6, 48.5, 37.5, 35.9, 33.0, 29.2, 28.8,
27.8, 25.0, 24.9, 24.6, 23.3, 19.6; ESI-MS m/z calcd for C24H32F3N2-
O3 [M+H]+ 453.2, found 453.1.
4.1.12. ((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
(d) To a solution of (R)-3-(tert-butoxycarbonylamino)-4-(2,4,5-
trifluorophenyl)butanoic acid 9 in CH2Cl2 was added HOBt,
EDCI, DMAP, and triethylamine. After stirring for 30 min
13a was added. The reaction mixture was stirred overnight.
The reaction was monitored by TLC. After the starting mate-
rials were completely consumed, the mixture was diluted
with CH2Cl2, washed sequentially with 1 N HCl and brine.
The organic layer was dried over Na2SO4, filtered, and evap-
orated. The residue was purified by a flash column chroma-
tography to afford compound 14a as a white solid (74%
yield).
(e) To a solution of 14a in dry CH2Cl2 was added CF3COOH. After
stirring the reaction overnight, the solvent was removed
under reduced pressure. The mixture was diluted with ethyl
acetate, washed sequentially with saturated Na2CO3 and
brine. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure to afford compound
4a as oil (85% yield).
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl
cyclopentanecarboxylate (4d)
1H NMR (CDCl3, 300 MHz): d 7.15–7.06 (m, 1H), 6.95–6.87 (m,
1H), 4.37–4.20 (m, 3H), 3.69–3.61 (m, 1H), 3.57–3.54 (m, 1H),
2.80–2.70 (m, 3H), 2.44–2.41 (m, 2H), 2.35–2.28 (m, 3H), 1.90–
1.55 (m, 13H), 1.34–1.12 (m, 3H); 13C NMR (CDCl3, 75 MHz): d
176.0, 169.4, 156.7, 156.6, 154.7, 154.7, 149.5, 149.4, 149.3,
147.5, 147.4, 147.3, 147.2, 147.1, 145.3, 145.2, 120.7, 120.6,
118.8, 118.8, 118.7, 118.6, 105.1, 105.0, 104.9, 104.8, 63.6, 57.9,
55.8, 48.5, 43.3, 38.0, 35.9, 33.9, 29.7, 29.6, 29.4, 28.9, 27.9, 25.3,
23.3, 19.6; ESI-MS m/z calcd for C25H34F3N2O3 [M+H]+ 467.3, found
467.1.
4.1.13. ((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl
benzoate (4e)
1H NMR (CDCl3, 300 MHz): d 8.02 (d, J = 8.1 Hz, 2H), 7.59–7.54
(m, 1H), 7.46–7.41 (m, 2H), 7.13–7.05 (m, 1H), 6.94–6.86 (m,
1H), 4.67–4.62 (m, 1H), 4.55–4.50 (m, 1H), 4.38–4.32 (m, 1H),
3.74–3.66 (m, 1H), 3.57–3.51 (m, 1H), 2.82–2.65 (m, 2H), 2.42–
2.40 (m, 2H), 2.31–2.24 (m, 1H), 2.01–1.91 (m, 5H), 1.75–1.63
(m, 3H), 1.38–1.25 (m, 2H); 13C NMR (CDCl3, 75 MHz): d 169.8,
165.9, 156.7, 156.6, 154.7, 154.7, 149.4, 149.3, 149.2, 147.4,
147.3, 147.2, 147.1, 147.1, 145.2, 145.1, 132.5, 129.6, 129.0,
127.9, 121.4, 121.3, 118.7, 118.6, 118.5, 118.5, 105.1, 105.0,
104.9, 104.7, 64.7, 58.0, 55.7, 48.5, 39.8, 36.1, 35.1, 29.3, 28.4,
25.2, 23.4, 19.6; ESI-MS m/z calcd for C26H30F3N2O3 [M+H]+
475.2, found 475.1.
4.1.9. ((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl
acetate (4a)
1H NMR (CDCl3, 300 MHz): d 7.12–7.03 (m, 1H), 6.94–6.85 (m,
1H), 4.32–4.24 (m, 2H), 4.20–4.15 (m, 1H), 3.69–3.61 (m, 1H),
3.55–3.52 (m, 1H), 2.77–2.65 (m, 4H), 2.41–2.38 (m, 1H), 2.25–
2.19 (m, 1H), 2.04 (s, 3H), 1.92–1.86 (m, 2H), 1.72–1.66 (m, 4H),
1.52–1.48 (m, 1H), 1.28–1.23 (m, 2H); 13C NMR (CDCl3, 75 MHz):
d 171.1, 169.8, 157.4, 157.3, 155.0, 154.9, 150.6, 150.5, 150.4,
148.1, 148.0, 148.0, 147.8, 147.7, 145.6, 145.5, 120.5, 120.3,
119.6, 119.5, 119.3, 119.1, 105.6, 105.5, 105.3, 105.2, 64.2, 61.6,
59.2, 49.0, 36.2, 36.0, 33.4, 29.7, 28.5, 25.7, 25.6, 23.8, 20.1; ESI-
MS m/z calcd for C21H28F3N2O3 [M+H]+ 413.2, found 413.1.
4.1.14. (R)-3-Amino-1-((2S,3aS,7aS)-2-
(hydroxymethyl)octahydro-1H-indol-1-yl)-4-(2,4,5-
trifluorophenyl)butan-1-one (4f)
1H NMR (CDCl3, 300 MHz): d 7.14–7.05 (m, 1H), 6.93–6.87 (m,
1H), 4.19–4.09 (m, 1H), 3.75–3.67 (m, 2H), 3.57–3.50 (m, 2H),
2.80–2.74 (m, 2H), 2.46–2.42 (m, 2H), 2.28–2.22 (m, 1H), 1.94–
1.88 (m, 2H), 1.71–1.33 (m, 8H); 13C NMR (CDCl3, 75 MHz): d
173.4, 157.5, 157.4, 155.0, 154.9, 150.5, 150.4, 150.3, 148.1,
148.0, 147.9, 147.8, 147.7, 145.6, 145.5, 120.3, 120.2, 119.6,
119.5, 119.2, 119.1, 105.8, 105.7, 105.6, 105.5, 63.8, 63.7, 61.2,
49.5, 37.0, 35.9, 33.7, 31.6, 29.8, 27.4, 25.8, 21.8; ESI-MS m/z calcd
for C19H26F3N2O2 [M+H]+ 371.2, found 371.1.
4.1.10. ((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl
cyclopropanecarboxylate (4b)
1H NMR (CDCl3, 300 MHz): d 7.14–7.06 (m, 1H), 6.96–6.87 (m,
1H), 4.33–4.32 (m, 2H), 4.24–4.18 (m, 1H), 3.69–3.64 (m, 1H),
3.56–3.50 (m, 1H), 2.78–2.69 (m, 2H), 2.41–2.39 (m, 2H),
2.25–2.21 (m, 1H), 1.94–1.92 (m, 4H), 1.69–1.55 (m, 5H),

S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
6691
4.1.15. General procedures for the synthesis of compounds 4g–n
75 MHz): d 173.3, 172.4, 159.0, 159.0, 157.1, 157.0, 151.9, 151.8,
151.7, 149.9, 149.8, 149.7, 149.6, 149.5, 147.6, 147.5, 123.5,
123.4, 121.1, 121.0, 120.9, 120.9, 107.6, 107.5, 107.4, 107.2, 61.2,
59.4, 50.8, 48.5, 41.9, 38.2, 37.3, 33.2, 30.9, 27.4, 25.8, 25.1, 21.8;
ESI-MS m/z calcd for C21H29F3N3O2 [M+H]+ 412.2, found 412.1.
(a) Compound 11 was dissolved in pyridine, after a few minutes,
p-toluenesulfonyl chloride was added. Stirring the reaction
overnight, the mixture was diluted with EA, washed sequen-
tially with 1 N HCl and brine. The organic layer was dried
over Na2SO4, filtered, and evaporated under reduced pres-
sure to afford compound 15 as oil (80% yield).
4.1.17. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-
(b) The crude product 15 was dissolved in DMF, NaN3 was
added. The reaction mixture was stirred overnight at 65 °C.
After the starting materials were completely consumed,
the mixture was diluted with EA, washed sequentially with
1 N HCl and brine. The organic layer was dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was purified by a flash column chromatography (petroleum
ether/ethyl acetate = 8:1, v/v, as an eluent) to afford com-
pound 16 as a white solid (66% yield).
(c) To a solution of 16 in dry CH2Cl2 was added CF3COOH. The
reaction was monitored by TLC. After the starting materials
were completely consumed, the solvent was removed under
reduced pressure. The mixture was diluted with EA, washed
sequentially with saturated Na2CO3 and brine. The organic
layer was dried over Na2SO4, filtered, and evaporated under
reduced pressure to afford compound 17 as a yellow solid
(85% yield).
(d) To a solution of (R)-3-(tert-butoxycarbonylamino)-4-(2,4,5-
trifluorophenyl)butanoic acid 9 in CH2Cl2 was added HOBt,
EDCI, DMAP, and triethylamine. After stirring for 30 min 17
was added. The reaction mixture was stirred overnight.
The reaction was monitored by TLC. After the starting mate-
rials were completely consumed, the mixture was diluted
with CH2Cl2, washed sequentially with 1 N HCl and brine.
The organic layer was dried over Na2SO4, filtered, and evap-
orated. The residue was purified by a flash column chroma-
tography (petroleum ether/ethyl acetate = 4:1, v/v, as an
eluent) to afford compound 18 as a white solid (62% yield).
(e) Compound 18 was dissolved in MeOH and 10% Pd–C was
added. The resulting mixture was stirred under hydrogen
atmosphere at room temperature for 2 h. The reaction mix-
ture was filtered and evaporated in vacuo to give 19 as a
white solid (94% yield).
(f) To a solution of compound 19 and triethylamine in CH2Cl2
was added various acetyl chloride and sulfonyl chloride.
The reaction mixture was stirred overnight. The reaction
was monitored by TLC. After the starting materials were
completely consumed, the mixture was purified by a flash
column chromatography (petroleum ether/ethyl ace-
tate = 1:1, v/v, as an eluent) to afford compound 20.
yl)methyl)cyclopropanecarboxamide (4h)
1H NMR (CDCl3, 300 MHz): d 7.81 (br s, 1H), 7.11–7.06 (m, 1H),
6.93–6.88 (m, 1H), 4.11–4.06 (m, 1H), 3.69–3.43 (m, 4H), 3.20–3.13
(m, 1H), 2.83–2.76 (m, 2H), 2.47–2.42 (m, 2H), 2.23–2.16 (m, 1H),
2.04–1.96 (m, 1H), 1.77–1.60 (m, 4H), 1.53–1.42 (m, 2H), 1.29–1.10
(m, 3H), 0.89–0.89 (m, 2H), 0.69–0.68 (m, 2H); 13C NMR (CDCl3,
75 MHz): d 175.8, 173.1, 159.1, 159.0, 157.1, 157.1, 152.0, 151.9,
151.8, 150.0, 149.9, 149.8, 149.6, 149.5, 147.7, 147.6, 123.2,
123.1, 121.1, 121.1, 121.0, 120.9, 107.7, 107.5, 107.4, 107.3, 61.2,
59.7, 50.9, 48.1, 41.2, 38.2, 36.8, 33.1, 30.9, 27.4, 25.8, 21.8, 16.6,
8.9, 8.7; ESI-MS m/z calcd for C23H31F3N3O2 [M+H]+ 438.2, found
438.1.
4.1.18. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-
yl)methyl)cyclobutanecarboxamide (4i)
1H NMR (CDCl3, 300 MHz): d 7.64 (br s, 1H), 7.13–7.05 (m, 1H),
6.97–6.88 (m, 1H), 4.13–4.05 (m, 1H), 3.75–3.68 (m, 2H), 3.56–
3.52 (m, 1H), 3.12–2.97 (m, 2H), 2.82–2.76 (m, 1H), 2.71–2.63
(m, 1H), 2.43–2.41 (m, 2H), 2.26–2.11 (m, 4H), 2.04–1.94 (m,
5H), 1.78–1.54 (m, 5H), 1.29–1.10 (m, 3H); 13C NMR (CDCl3,
75 MHz): d 177.1, 173.4, 159.0, 159.0, 157.1, 157.0, 151.8, 151.7,
151.6, 149.8, 149.7, 149.6, 149.5, 147.6, 147.5, 123.8, 123.7,
121.0, 121.0, 120.9, 120.8, 107.6, 107.4, 107.4, 107.2, 61.2, 59.4,
50.9, 48.6, 42.7, 41.9, 38.2, 37.8, 33.2, 30.9, 27.4, 27.3, 27.2,
25.9, 21.8, 20.0; ESI-MS m/z calcd for C24H33F3N3O2 [M+H]+
452.3, found 452.1.
4.1.19. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-trifluoro-
phenyl)butanoyl)octahydro-1H-indol-2-
yl)methyl)cyclopentanecarboxamide (4j)
1H NMR (CDCl3, 300 MHz): d 7.58 (br s, 1H), 7.15–7.07 (m, 1H),
6.97–6.88 (m, 1H), 4.11–4.03 (m, 1H), 3.71–3.65 (m, 2H), 3.61–3.57
(m, 1H), 3.20–3.13 (m, 2H), 2.84–2.79 (m, 2H), 2.54–2.47 (m, 3H),
2.24–2.18 (m, 1H), 2.06–1.97 (m, 1H), 1.83–1.65 (m, 9H), 1.59–1.49
(m, 4H), 1.31–1.14 (m, 3H); 13C NMR (CDCl3, 75 MHz): d 178.5,
173.1, 159.1, 159.0, 157.1, 157.1, 151.9, 151.8, 151.7, 149.9,
149.8, 149.7, 149.6, 149.5, 147.7, 147.6, 123.3, 123.2, 121.1,
121.1, 121.0, 120.9, 107.7, 107.5, 107.4, 107.3, 61.2, 59.6, 50.9,
48.1, 47.9, 41.5, 38.2, 37.0, 33.1, 32.3, 32.3, 30.9, 27.7, 27.4, 25.8,
21.8; ESI-MS m/z calcd for C25H35F3N3O2 [M+H]+ 466.3, found
466.1.
(g) To a solution of compound 20g–n in dry CH2Cl2 was added
CF3COOH. The reaction was monitored by TLC. The reaction
mixture was stirred overnight. After the starting materials
were completely consumed, the solvent was removed under
reduced pressure. The mixture was diluted with EA, washed
sequentially with saturated Na2CO3 and brine. The organic
layer was dried over Na2SO4, filtered, and evaporated under
reduced pressure to afford compounds 4g–n.
4.1.20. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-trifluoro-
phenyl)butanoyl)octahydro-1H-indol-2-yl)methyl)benzamide
(4k)
1H NMR (CDCl3, 300 MHz): d 8.72 (br s, 1H), 7.86 (d, J = 7.8 Hz,
2H), 7.46–7.42 (m, 3H), 7.15–7.06 (m, 1H), 6.94–6.86 (m, 1H),
4.29–4.21 (m, 1H), 3.94–3.87 (m, 1H), 3.74–3.68 (m, 1H), 3.65–
3.61 (m, 1H), 3.33–3.26 (m, 1H), 3.12 (br s, 2H), 2.87–2.85 (m,
2H), 2.65–2.57 (m, 1H), 2.52–2.46 (m, 1H), 2.27–2.23 (m, 1H),
2.17–2.06 (m, 1H), 1.85–1.50 (m, 6H), 1.32–1.16 (m, 3H); 13C
NMR (CDCl3, 75 MHz): d 173.7, 169.0, 159.0, 159.0, 157.1, 157.1,
151.9, 151.8, 151.7, 149.9, 149.8, 149.7, 149.6, 149.5, 147.7,
147.5, 136.0, 133.2, 130.4, 129.0, 123.4, 123.3, 121.1, 121.1,
120.9, 120.9, 107.6, 107.5, 107.4, 107.2, 61.4, 59.2, 51.0, 49.9,
42.0, 38.2, 37.2, 33.3, 31.0, 27.4, 25.9, 21.8; ESI-MS m/z calcd for
4.1.16. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-trifluoro-
phenyl)butanoyl)octahydro-1H-indol-2-yl)methyl)acetamide
(4g)
1H NMR (CDCl3, 300 MHz): d 7.76 (br s, 1H), 7.14–7.05 (m, 1H),
6.97–6.88 (m, 1H), 4.13–4.05 (m, 1H), 3.74–3.67 (m, 2H), 3.60–3.54
(m, 1H), 3.12–3.04 (m, 1H), 2.85–2.78 (m, 1H), 2.74–2.67 (m, 1H),
2.46–2.44 (m, 2H), 2.22–2.16 (m, 2H), 2.08–2.01 (m, 2H), 1.97 (s,
3H), 1.77–1.55 (m, 4H), 1.30–1.16 (m, 3H); 13C NMR (CDCl3,
C
26H31F3N3O2 [M+H]+ 474.2, found 474.1.

6692
S. Wang et al. / Bioorg. Med. Chem. 22 (2014) 6684–6693
4.1.21. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl)-4-
methylbenzenesulfonamide (4l)
4.2.3. Enzyme-based assay of inhibition selectivity against DPPs
The inhibitory effect of each compounds on DPPs were assayed
by continuous fluorometric method. We used Nle-Pro-AMC as sub-
strate to measure the activities of DPP-7 and FAP, and Ala-Pro-AMC
for DPP-8 and DPP-9 in the optimized pH (5.5 for DPP-7 and 8.0 for
other members) assay system.
1H NMR (CDCl3, 300 MHz): d 7.72 (d, J = 8.4 Hz, 2H), 7.25 (d,
J = 6.6 Hz, 2H), 7.13–7.04 (m, 1H), 6.97–6.88 (m, 1H), 6.75 (br s,
1H), 4.00–3.92 (m, 1H), 3.63–3.57 (m, 1H), 3.54–3.48 (m, 1H),
3.31–3.27 (m, 1H), 2.96–2.90 (m, 1H), 2.81–2.75 (m, 1H), 2.72–
2.65 (m, 1H), 2.39 (s, 3H), 2.37–2.34 (m, 2H), 2.19–2.13 (m, 3H),
1.95–1.89 (m, 1H), 1.73–1.47 (m, 6H), 1.25–1.10 (m, 3H); 13C
NMR (CDCl3, 75 MHz): d 173.4, 159.0, 159.0, 157.1, 157.0, 151.7,
151.6, 151.5, 149.8, 149.7, 149.5, 149.4, 147.6, 147.5, 144.9,
139.2, 131.4, 128.9, 124.0, 123.8, 121.1, 121.0, 120.9, 120.9,
107.6, 107.4, 107.3, 107.2, 60.9, 59.4, 50.9, 50.7, 42.6, 38.1, 37.9,
33.0, 30.7, 27.4, 25.8, 23.3, 21.8; ESI-MS m/z calcd for C26H33F3N3-
O3S [M+H]+ 524.2, found 524.1.
4.3. Binding studies
The DPP-4 protein was extracted from RCSB Protein Data Bank
(PDB ID: 1X70). Compounds were generated using Sybyl program.
Gasteiger–Hückel charge was used and the conformation of each
compound was minimized using default parameters. Docking stud-
ies were performed using Glide program. The DPP-4 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 Lig-
prep. 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.
4.1.22. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-
yl)methyl)methanesulfonamide (4m)
1H NMR (CDCl3, 300 MHz): d 7.20–7.12 (m, 1H), 6.99–6.90 (m,
1H), 4.07–4.01 (m, 1H), 3.83–3.78 (m, 1H), 3.57–3.52 (m, 1H),
3.37–3.32 (m, 1H), 3.23–3.19 (m, 1H), 2.92 (s, 3H), 2.85–2.71 (m,
3H), 2.62–2.56 (m, 1H), 2.28–2.23 (m, 1H), 2.03–2.00 (m, 1H),
1.84–1.48 (m, 6H), 1.25–1.08 (m, 3H); 13C NMR (CDCl3, 75 MHz):
d 169.6, 156.7, 156.7, 154.8, 154.7, 150.1, 150.0, 149.9, 148.1,
148.0, 147.9, 147.4, 147.3, 145.4, 145.3, 119.0, 118.9, 118.5,
118.3, 105.4, 105.3, 105.2, 105.0, 58.8, 57.9, 48.8, 46.6, 39.2, 35.5,
33.4, 30.8, 30.3, 27.9, 24.8, 23.2, 19.3; ESI-MS m/z calcd for C20H29-
F3N3O3S [M+H]+ 448.2, found 448.1.
4.4. Pharmacokinetic profile in SD rats
Compounds 3e, 4l, and 4n 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
concentrations in the supernatant were measured by LC/MS/MS.
4.1.23. N-(((2S,3aS,7aS)-1-((R)-3-Amino-4-(2,4,5-
trifluorophenyl)butanoyl)octahydro-1H-indol-2-yl)methyl)-
1,1,1-trifluoromethanesulfonamide (4n)
Acknowledgments
1H NMR (CDCl3, 300 MHz): d 7.16–7.09 (m, 1H), 7.00–6.91 (m,
1H), 4.09–4.05 (m, 1H), 3.83–3.78 (m, 1H), 3.59–3.50 (m, 2H),
3.27–3.23 (m, 1H), 3.10–2.99 (m, 2H), 2.85–2.77 (m, 1H), 2.65–
2.61 (m, 1H), 2.30–2.26 (m, 1H), 2.09–1.99 (m, 1H), 1.80–1.51
(m, 6H), 1.31–1.13 (m, 3H); 13C NMR (CDCl3, 75 MHz): d 170.1,
161.6, 156.7, 156.7, 154.8, 154.7, 150.2, 150.1, 150.0, 148.2,
148.1, 148.0, 147.4, 147.3, 145.5, 145.4, 118.9, 118.7, 118.1,
118.0, 105.5, 105.4, 105.3, 105.2, 59.0, 58.1, 48.8, 48.3, 35.4, 33.4,
30.8, 30.2, 27.9, 24.7, 23.1, 19.3; ESI-MS m/z calcd for C20H26F6N3-
O3S [M+H]+ 502.2, found 502.1.
We gratefully acknowledge financial support from the
National Natural Science Foundation of China (Grants 21021063,
91229204, 81302633, and 81025017), National S&T Major Projects
(2012ZX09103101-072, 2012ZX09301001-005, 2013ZX09507-001,
and 2014ZX09507002-001).
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