Synthesis and biological evaluation of triazole based uracil derivatives as novel DPP-4 inhibitors

Article abstract of DOI:10.1039/c6ob01818a

A series of triazole based uracil derivatives were designed and synthesized as novel DPP-4 inhibitors. Compound A01 was identified as a lead compound for SAR studies focused on the structural modification at the S_2′ subsite of DPP-4. The novel analogues A02-A25 were obtained by modifying the substituents at the phenyl group, and B01-B09, by introducing the carbonyl group. On screening in DPP-4, compounds B03, B04 and B08 showed a significant improvement in DPP-4 inhibitory activities compared to compound A01 and showed comparable activities to the marketed DPP-4 inhibitor, alogliptin. Docking studies revealed new favorable binding modes of designed compounds in the S_2′ subsite and proved that structural modifications in the S_2′ subsite were an effective option to increase the inhibition of DPP-4. In vitro DPP-8 and DPP-9 tests indicated that all compounds showed excellent selectivity against DPP-8 and DPP-9. Further in vivo evaluation showed that compound B04 could significantly improve oral glucose tolerance in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice. These data suggest that compound B04 could be a promising DPP-4 inhibitor for future treatment of T2DM.

Full text of DOI:10.1039/c6ob01818a

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Page 1 of 14
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DOI: 10.1039/C6OB01818A
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ARTICLE
Synthesis and Biological Evaluation of Triazole Based Uracil
Derivatives as Novel DPP-4 inhibitors
#
a,b
#a
c
c
a
Qing Li,
Li Han, Bin Zhang, Jinpei Zhou and Huibin Zhang *
Received 00th January 20xx,
Accepted 00th January 20xx
A series of triazole based uracil derivatives were designed and synthesized as novel DPP-4 inhibitors. Compound A01 was
firstly identified as a lead compound for SAR studies, which focused on structural modification in S2’ subsite of DPP-4. Then
novel analogues A02-A25 by modifying substituents at phenyl group and B01-B09 by introducing carbonyl group were
designed. By screening in DPP-4, compounds B03, B04 and B08 showed a significant improvement in DPP-4 inhibitory
activities compared with compound A01 and a comparable activities with marketed DPP-4 inhibitor alogliptin. Docking study
DOI: 10.1039/x0xx00000x
www.rsc.org/
revealed a new favorable binding modes of designed compounds in S ’ subsite, and proved structural modifications in S2’
2
subsite were an effective option to increase the inhibition of DPP-4. In vitro DPP-8 and DPP-9 tests displayed that all
compounds showed excellent selectivity against DPP-8 and DPP-9. Further in vivo evaluation showed compound B04 could
significantly improve oral glucose tolerance in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic
C57BL/6 mice. These data suggest that compound B04 could be a promising DPP-4 inhibitor for future treatment of T2DM. .
Introduction
Type 2 diabetes mellitus (T2DM) is growing metabolic disorder,
which is expected to affect 366 million people by 2030 worldwide.1
Currently, Dipeptidyl peptidase-IV (DPP-4) has been validated as one
of the most effective targets for T2DM treatment. DPP-4 is a serine
peptidase, which rapidly inactivates endogenous glucagon-like
peptide-1 (GLP-1) with a very short half-life 1-2 min.2, 3 GLP-1
secreted by intestinal endocrine cells in response to the presence of
nutrients can increase the production and release of insulin from
4
, 5
pancreatic β cells. Inhibition of DPP-4 can prevent the degradation
of GLP-1, and thus enhance insulin secretion and improve the glucose
6
tolerance. Various DPP-4 inhibitors, such as aloglitptin and
linagliptin (Fig. 1), have already been released as therapeutic drugs Fig. 1 The structures of alogliptin, linagliptin and yogliptin, and the
7
for T2DM. However, some of DPP-4 inhibitors has been reported to design of triazole based uracil derivatives.
8
, 9
increase risk of hospitalisation for heart failure,
and also may be
pocket consists of catalytic triad (Ser630, Asn710 and His740) and the
S2 subsite involves key interactions with Glu205 and Glu206 and
10
associated with severe joint pain side effect. Thus it is necessity to
develop novel DPP-4 inhibitors with more safety and less side effects
for the treatment of T2DM.
Arg125. The S
the S ’, and S
crystallographic structure (PDB ID: 2RGU) indicates that the S
subsite is highly hydrophobic subsite is made up of Tyr547 and Ser630. The S ’ subsite is a larger
cavity surrounded by residues of Trp629 Trp627, Tyr752, His740,
Ile742 and His748 (Fig. 2B). Only few inhibitors can bind into this S
subsite to increase their inhibitory activities, and thus leaving more
1
and S2 subsites have been extensively studied, but
12
1
2
’ subsites have not been clearly defined. The
The binding site of DPP-4 enzyme has four subsites, including S
1
,
1
’
1
1
2
S , S
1
’, and S
2
’ subsites (Fig. 2). The S
1
2
2
’
1
1
to be explored.
a.Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic
Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu
Among all the DPP-4 inhibitors, alogliptin with IC50 value of 8 nM
2
10009, China. E-mail: zhanghb80@cpu.edu.cn
b.
c.
Pharmaceutical College, Guangxi Medical University, Nanning, Guangxi, PR China.
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia
Xiang, Nanjing 210009, PR China.
and linagliptin with IC50 value of 1 nM are approved by US FDA (Fig.
13, 14
1).
The aligned crystallographic structures (PDB ID: 3G0B and
Fax: +86 25 83271480; Tel: +86 25 83271302
Electronic Supplementary Information
DOI: 10.1039/x0xx00000x
These authors contributed equally to this work
2RGU) revealed that alogliptin and linagliptin have the similar
binding modes in S , S , and S ’ subsites. Both incorporate (R)-3-
†
(ESI)
available
here.
See
1
2
1
aminopiperidine to form salt bridges with Glu205 and Glu206 in S
subsite, hydrophobic cyanobenzyl group and butynyl group bind to
2
#
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ARTICLE
than linagliptin may be because of the different binding interactions
in the S ’ subsite, which suggested that modification of substituent
in S ’ subsite can be a good strategy for design of new DPP-4
inhibitors.
A
2
2
With those information in mind, we chose the uracil ring as
scaffold to interact with S ’ subsite, and 2-butynyl group of linagliptin
was introduced at N3-position of uracil to bind with S subsite, while
-(R)-aminopiperidine group at C6-position of uracil was remained to
1
1
3
2
form salt bridges with Glu205 and Glu206 in S subsite. 1,2,3-triazole
ring was introduced to N3-position of uracil to bind with the S ’
2
subsite (Fig. 1) because of two reasons: one is the 1,2,3-triazole itself
may provide π-π interaction with Trp629 as quinazoline ring of
linagliptin does, and the substituent in 1-position of 1,2,3-triazole can
be easily modified for structure-activity studies. Compound A01 with
B
1-phenyl-1,2,3-triazole group was firstly synthesized and showed a
DPP-4 potency of 185.24 nM and excellent selectivity against DPP-8
and DPP-9 (both >100 μM, Fig. 1). Docking study reavealed that
compound A01 displayed expected binding modes with DPP-4. As
shown in Fig. 2, the uracil ring, 2-butynyl group and 3-(R)-
aminopiperidine group interacted with DPP-4 in a similar way with
linagliptin, 1-phenyl-1,2,3-triazole provided π-π interaction with
Trp629 in the S
potent than that of marketed alogliptin and linagliptin. However,
modification of 1-phenyl-1,2,3-triazole group of compound A01 in S
subsite might be good way to increase their inhibitory
2
’ subsite. DPP-4 potency of compound A01 was less
2
’
a
activities.Thus we continued to design and synthesize novel
analogues A02-A25 and B01-B09 with diverse groups in N-1 position
of triazole rings. Herein, we reported the design, synthesis and
biological evaluation those 1,2,3-triazole based uracil derivatives as
novel DPP-4 inhibitors.
C
Results and discussion
Chemistry
The synthetic route adopted to obtain compounds A01-A25 is
depicted in Scheme 1. The selective alkylation of commercially
available material 6-chlorouracil with 1-bromo-2-butyne and
ethyldiisopropylamine at room temperature produced compound C.
Compound C was further alkylated by 3-bromopropyne to afford
compound D. Amination of compound D with 3-(R)-Boc-amino-
Fig. 2 A, the aligned crystallographic structures of alogliptin (blue)
and linagliptin (green); B, the residues (purple) of the S
the docking binding modes of compound A01 (cyan).
2
’ subsite; C,
o
piperidine at 60 C furnished compound E. Compounds H01-22 were
S
1
subsite (Fig. 2). This salt bridge interaction in S
hydrophobic binding with S subsite are two key features of DPP-4 NaN
inhibitors. In addition, the uracil and purine rings form same π-π Subsequently, compound E was treated with compounds H01-22 at
2
subsite and obtained by treating compounds G01-22 with NaNO
2
followed by
17
1
3
in 6 M hydrochloric acid according to literature procedures.
11
interactions with Tyr547 and hydrogen bond with Ser630 in the S
subsite. However, they have different binding interactions in the S
1
’
’
room temperature in the presence of catalytic amount of copper
sulfate and sodium ascorbate in methanol and water (v/v, 4/1) for 12
2
subsite. Alogliptin does not have binding interaction with S
while the quinazoline substituent of linagliptin forms π-π interaction compounds F01-22 with HCl gas at 0 C furnished the target
2
’ subsite, h to provide compounds F01-22. Deprotection of Boc group of
o
with Trp629 in the S
2
’ subsite. Yogliptin is discovered as a more compounds A01-A22. The compounds A23-25 were prepared from
potent (IC50 = 0.05 nM), long-acting DPP-4 inhibitor,1
5, 16
which may F20-22 by cleavage of Boc and ester groups.The structures of target
take more efficient binding with S ’ subsite via benzo[d]thiazole ring compounds A01-A25 obtained were listed in Table 1 and Table 2.
2
than quinazoline of linagliptin. The reason why linagliptin has 8-fold
higher activity than alogliptin and yogliptin are 20-fold more potent
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DOI: 10.1039/C6OB01818A
Table 1 In vitro DPP-4 inhibitory activities of compounds A01-19
Compounds
A01
R
1
%Inhibition at 100nM IC50 (nM) a,b
H
37.26±4.31
75.56±2.76
25.03±2.48
46.44±1.71
35.00±2.91
26.88±0.78
8.18±2.44
185.24
64.05
NT
A02
2’-F
A03
3’-F
Scheme
1 Synthesis of compounds A01-A25. Reagents and
A04
4’-F
135.45
243.67
NT
conditions: (a) 1-bromobut-2-yne, DIPEA, DMF, r.t., 12 h; (b) 3-
A05
2’,4’-diF
2’-Me, 4’-F
2’-Cl
bromoprop-1-yne, K
-ylcarbamate, K CO
2
CO
3
, DMF, r.t. 12 h; (c) tert-butyl (R)-piperidin-
A06
o
3
2
3
, DMF, 60 C, 6h; (d) compounds H, CuSO
4
·5H
2
O,
A07
NT
Sodium ascorbate, MeOH/H
2
O (4:1), r.t., 12 h; (e) HCl gas, EA/ether,
A08
3’-Cl
46.90±1.48
3.72±0.71
168.63
NT
o
o
o
0
H
C; (f) (i) NaNO
O/MeOH, 6h.
2 2 3 2
, HCl, H O, 0 C; (ii) NaN , H O , 0 C, 2h; (g) NaOH,
A09
4’-Cl
2
A10
2’,5’-diCl
2’-Me
4’- Me
2’,5’-diMe
2’-Me, 5-Cl
2’-OH, 4’-Me
4’-t-Bu
2’-MeO
3’-MeO
4’-MeO
-
23.51±5.22
13.71±13.86
32.12±9.64
30.94±16.65
28.88±7.12
23.29±6.41
23.72±3.10
40.61±0.70
52.94±0.00
23.79±6.87
86.46±5.22
98.46±5.22
NT
The synthetic route to compounds B01-09 is outlined in Scheme 2.
The starting materials compounds I01-05 were treated with NaN in
acetone/H O to give compounds J01-05. Compounds J01-05 reacted
A11
NT
A12
NT
3
A13
NT
2
with compound E in similar condition depicted in Scheme 1 to give
compounds K01-05. Compounds K01-05 were hydrolyzed with NaOH
and removed protection group with HCl gas to afford the target
compounds B01, 03, 05, 07 and 09. Deprotection of compounds K01-
A14
NT
A15
NT
A16
NT
A17
219.42
88.53
NT
05 furnished the target compounds B02, 04, 06 and 08.The structures
A18
of target compounds B01-B09 were listed in Table 2.
A19
alogliptin
6.85
1.25
In vitro DPP-4 inhibition studies and SAR analysis of target
compounds
linagliptin
-
a
Measured in three independent experiments.
NT: not tested
b
The synthesized compounds were evaluated in vitro for their
capacity to inhibit human recombinant DPP-4 using linagliptin and
alogliptin as the positive controls. Inhibitory potency were
measuredby following the increase of fluorimetric intensity at 460
nm upon hydrolysis of H-Gly-Pro-aminomethylcoumarin (H-Gly-Pro-
AMC). The compounds with good inhibition rates at 100 nM were
further selected to determine their IC50 values. The inhibitory
activities were depicted in Table 1.
Compound A01 with the IC50 of 185.24 nM was selected as
starting point for our initial SAR studies, which focused on phenyl
group in N-1 position of triazole ring (compounds A01-19). A wide
variety of substituents were introduced to benzene ring. As shown in
Table 1, most of the synthesized compounds demonstrated
significant in vitro DPP-4 inhibitory activities. The mono-fluoro
substituted at ortho or para position of benzene ring (compounds
A02 and A04) led to increase of activities, compound A02 showed 3-
fold more potent inhibitory activity (with the IC50 of 64.05 nM)
compared to compound A01. Compound A04 exhibited DPP-4
potency of 135.45 nM, which was slightly more potent than
compound A01. However, addition of mono-fluorine atom at meta
position or introduction of two fluorine atoms at ortho- or para-
position of benzene ring reduced the DPP-4 inhibitory potency (A03
and A05). Chloro group at meta position of the benzene ring
tolerated DPP-4 inhibition (compound A08, IC50 = 168.63 nM),
introduction of chloro group at other positions led to decrease of
activities (compound A07, A09 and A10). In addition, presence of
methyl group at benzene ring slightly reduced inhibitory potency
Scheme 2 Synthesis of compounds B01-B09. Reagents and
conditions: (a) NaN , Acetone/H O, 60 °C, 8h; (b) compound E,
3 2
CuSO
4
·5H
2
O, Sodium ascorbate, MeOH/H
2
O (4:1), r.t., 12h; (c) NaOH,
o
H
2
O/MeOH, 6h; (d) HCl gas, EA/ether, 0 C.
(
compounds A11-15). The methoxy at meta-position ( compound
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Table 2 In vitro DPP-4 inhibitory activities of compounds A20-25 and showed that compound A02 and A18 exhibited desirable inhibitory,
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DOI: 10.1039/C6OB01818A
B01-09
but their acitivities were still less potent than reference drugs
linagliptin (IC50 = 1.25 nM) and alogliptin (IC50 = 6.85 nM). Thus
further optimization was performed to identify a compound with
enhanced inhibitory activity.
Published X-ray co-crystal structure suggested that some DPP-4
inhibitors, such as aloglitpin18 and nicotinic acid derivative , were
19
2
interacted with Arg125 residue of DPP-4 in S subsite, and docking
study revealed Arg125 is located near the triazole ring of compound
A01 (Fig. 1). Thus Arg125 is a potential target residue for achieving
potent DPP-4 inhibitory activity in our design. Some known DPP-4
inhibitors generate a hydrogen-bond between the carbonyl oxygen
and Arg125 . Therefore, we further designed a series of triazole-
based uracil derivatives by introducing an additional carbonyl oxygen
that could make this desired hydrogen-bond interaction (compound
A20-25 and B01-09). The results were shown in Table 2.
IC50(nM)a,b
Compounds
A20
R
2
%Inhibition at 100nM
2
2
8.94±1.53
4.07±6.16
345.32
2
0
NT
A21
A22
A23
3
6.84±13.30
172.53
65.63
Carbonyl groups were firstly introduced to the benzene ring of
compound A01 to afford compounds A20-25, in which compounds
A23-25 were added with carboxylic acid group and compound A20-
7
2.73±1.32
7.88±0.00
22 with corresponding carboxylic ester. Compound A23 with
carboxylic acid at ortho-position showed IC50 value of 65.63 nM,
manifested 3-fold more potent inhibitory activity compared with
compound A01. The carboxylic acid at meta- and para-positions and
corresponding ester led to decrease of potency.
1
NT
A24
2
5
0.96±2.60
8.73±2.86
NT
A25
B01
Some aliphatic carboxylic acids with different carbon chain
lengths (2-4) and corresponding esters were also introduced at 1-
position of triazole ring (B01-09). As shown in Table 2, introduction
of aliphatic carboxylic acids led to a significant increase of DPP-4
potency. Compounds bearing acetic acid (B01, IC50 = 84.72 nM) or 2-
propanoic acid (B06, IC50 = 71.62 nM) exhibited an increase in
potency by more than 2- and 3-fold compared to compound A01.
Remarkably, compound B03 with 3-propanoic acid group exhibited
low nanomolar inhibitory activity with the IC50 of 12.45 nM, which
was 15-fold more potent than that of compound A01. Compound
B08 bearing (E)-but-2-enoic acid exhibited the most potent activity
with the IC50 of 9.56 nM, its activity was comparable with alogliptin.
These results clearly indicated the importance of chain lengths of the
carboxylic acid for the activity. Replacement of carboxylic acids with
their corresponding ester resulted in substantial potency loss.
Compound B04 (IC50 = 64.31 nM), which has methyl propionate,
showed better potency relative to the rest of the ester.
84.72
22.84±8.30
79.55±2.24
58.39±0.50
77.28±1.14
NT
B02
B03
B04
B05
12.45
64.31
26.81
5
9.24±2.08
71.62
NT
B06
2
8.82±0.09
B07
B08
Docking Study
8
3.82±0.93
9.56
To understand the binding modes of the triazole-based derivatives in
2
S ’ subsite, compounds A02, B03 and B08 were selected for docking
4
7.24±2.5
105.56
B09
study (Fig.34). We used the molecular docking program GLIDE 5.9 to
dock compounds into a DPP-4 crystal structure (PDB ID: 2RGU). The
docking results were shown in Fig. 3, all compunds display favorable
binding modes. As shown in Fig. 3-A, the overlay of A02 against
linagliptin shows the uracil ring, 2-butynyl group and (3R)-
aminopiperidine group interact with DPP-4 in a similar way with
linagliptin. However, compared with conjugated phenyl moiety of
the quinazoline ring in linagliptin, nonconjugated 1-phenyl-triazole
moiety of compound A02 take poor π-π stacking interaction with
Trp629. The difference may explain the observed potency decrease
-
-
86.46±5.22
98.46±5.22
8.85
1.47
alogliptin
linagliptin
a
Measured in three independent experiments.
NT: not tested
b
A18) exhibited 2-fold more potent inhibitory activity (IC50 = 88.53 nM)
compared to compound A01. The methoxy at ortho- or para-position
reduced inhibitory potency. Finally, an investigation of the inhibitory
activities of these N-1 phenyl triazole-based uracil derivatives
4
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of compounds A01-19 compared to linagliptin. Docking results of Table 3 Selectivity for DPP-4 over DPP-8 and DPP-9.
ARTICLE
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DOI: 10.1039/C6OB01818A
compounds B03 and B08 (Fig. 3-B) showed that the uracil ring, 2-
butynyl and (3R)-aminopiperidine groups also have similar binding
modes to those shown by linaglitpin, but the carboxy groups with
DPP-4
nM)
DPP-8
DPP-9
DPP-
DPP-
Compounds
(
(μM)
(μM)
8/DPP-4
9/DPP-4
different carbon chain lengths form different binding modes in S
subsite. The carboxy group of compound B03 with two carbon
lengths can interact with Arg125 in S subsite by hydrogen bond as
we expected. However, compound B08 with three carbon atoms
lengths displays new conformations in S ’ subsite that the carboxy
groups can interact with Tyr752 with hydrogen bond, and ethenyl
chain has hydrophobic interaction with Trp629. The most potent
activity of compound B08 can be explained by the hydrogen-bonding
2
’
A01
185.24
64.05
65.63
84.72
12.45
64.31
9.56
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.2
>540
>540
A02
>1560
>1540
>1190
>8000
>1560
>10,000
>950
>1560
>1540
>1190
>8000
>1560
>10,000
>950
2
A23
2
B01
B03
B04
2
interaction and the increase in hydrophobic interaction in S ’ subsite.
Therefore, analysis of compounds A01-25 and B01-09 yielded the
novel and important finding that modifications of substituent in S2’
subsite were an effective and useful option to increase the inhibition
of DPP-4.
B08
B09
105.56
6.85
Alogliptin
Saxagliptin
>14,000
66
>14,000
33
Selectivity over DPP-8 and DPP-9
6.00
Selected compounds were evaluated for its inhibition of DPP-4
homologues, including DPP-8 and DPP-9, and the results were
compared to those of marketed DPP-4 inhibitors. As shown in Table
selected compounds showed no inhibition of DPP-8 or DPP-9 with
the IC50 > 100 μM, which showed excellent selectivity against DPP-8
and DPP-9.
3, Saxagliptin inhibited both DPP-8 and DPP-9 with a selectivity ratio
for DPP-4 over DPP-9 of only about 30-fold. On the other hand, all
Hypoglycemic effect of B03, B04, B08 and B09 in ICR mice
Based on in vitro potency and selectivity analysis, compounds B03,
B04, B08 and B09 were selected for acute efficacy evaluation by the
oral glucose tolerance test (OGTT) in ICR mice. A single dose of
compounds B03, B04, B08 and B09 (3 mg/kg) were administered to
ICR mice. As shown in Fig. 4, the OGTT produced a significant
decrease in glucose level after half hour compared with the vehicle
group. Alogliptin, which was used as a positive control, reduced the
area under curve from 0 to 120 min (AUC) 0-120 min to 38.2%
A
(
alogliptin, 842±103, vehicle control, 1,363±157). Compared
compound B03 with B04, compound B03 showed more potent in
vitro activity than compound B04, but compound B04 was more
potent in vivo. Compound B04 reduced the value to 31.4% (935±125),
and compound B03 reduced only 21.2% (1150±130). The reason may
be low membrane permeability of compound B03 caused by the
formation of a zwitterion between the carboxyl group and the amino
group. Compound B04 was the methyl ester of compound B03, the
more lipophilic compound B04 have higher membrane permeability
to improve the bioavailability, and compound B04 can probably act
as a prodrug that metabolized into compound B03, as reported in
B
21-23
literature,
both improved in vivo activity of compound B04. In
addtion, compounds B08 and B09 showed similar potency in vivo,
B08 and B09 reduced the value to 28.1% (B08, 981±98) and 25.2%
(
1091±120), respectively.
In order to verify the hypoglycemic effect, compounds B04 and
B09 were selected for acute efficacy evaluation in ICR mice at a
higher dose of 10 mg/kg (Fig. 5). Compound B04 reduced area under
the curve from 0 to 120 min (AUC)0-120 min to 40.4% (B04, 947±85;
Fig. 3. A docking study of DPP-4 protein and compounds A02 (orange
carbons), B03 (yellow carbons) and B08 (salmon carbons).
Linaglipitin (blue carbons) and the amino acids (green carbons, white
text) are indicated. The hydrogen bonds are shown as black dotted vehicle control, 1,589±139), which was slightly less potent than the
lines.
hypoglycemic effect of alogliptin (42.5%, 913±73). But the activity of
compound B09 (29.2%, 1105±124) was still much lower than that of
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J. Name., 2013, 00, 1-3 | 5
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Org aP nl ei ca s &e dB oi o nmo to al ed cj uu sl at rm Ca hr ge imn si stry
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ARTICLE
Journal Name
alogliptin at higher dose. Thus compound B04 with best potency in
View Article Online
vivo was then considered for further evaluation.
DOI: 10.1039/C6OB01818A
Antihyperglycemic effect of compound B04 in type 2 diabetic
C57BL/6 mice
The antihyperglycemic effect of B04 was evaluated in type 2 diabetic
C57BL/6 mice. As shown in Fig. 6, Compund B04 at dose of 1-10
mg/kg was orally administrated in diabetic C57BL/6 mice prior to
glucose challenge. The results indicated that B04 significantly
reduced blood glucose at a dose of 3 mg/kg and oral administration
of B04 dose-dependently reduced blood glucose in type 2 diabetic
C57BL/6 mice.
The toxicity evaluation of compound B04
To evaluation of the potential toxicity of compound B04, the ICR mice
and diabetic C57BL/6 mice after OGTT at the dose of 10 mg/kg were
kept under conventional conditions of controlled temperature,
humidity, and lighting for additional 1 week. All animals survived
until the end of the study. The results suggested that the compound
was non-toxic at the tested dose.
Fig. 5 Effect of Compounds B04 and B09 (10 mg/kg) during an OGTT
in male ICR mice and AUC0–120 min of blood glucose levels. Values are
mean ± SEM (n = 8). **P≤0.01 and ***P ≤0.001 compared to vehicle-
treated ICR mice by Student’s t test.
Conclusions
In summary, we designed and optimized 1,2,3-triazole based uracil
derivatives to obtain novel DPP-4 inhibitors. Firstly, Compound A01
with phenyl group was firstly identified as a lead compound, then
substituents were introduced to phenyl group at N-1 position of
1
,2,3-triazole, and the resulting compounds manifested weak to
moderate DPP-4 inhibitory activities. Then we designed the
introduction of a carboxy group that could increase the inhibitory
activities by interacting with Arg125. Compounds B03, B04 and B08
possessing a carboxy group actually showed improved DPP-4
inhibitory activities. Docking study revealed that the carboxy group
2
at 1,2,3-triazole ring show new favorable conformations in S ’ subsite,
and suggested modifications of substituent in S2’ subsite were an
effective way to increase the inhibition of DPP-4. Furthermore,
Compounds B03, B04 and B08 also showed good selectivity over
DPP-8 and DPP-9. In addtion, compound B04 could significantly
improve oral glucose tolerance in ICR mice and dose-dependently
reduced glucose levels in type 2 diabetic C57BL/6 mice. These data
suggest that compound B04 could be a promising DPP-4 inhibitor for
future treatment of T2DM.
Experimental
General chemistry
Fig. 4 Effect of Compounds B03, B04, B08 and B09 (3 mg/kg) during
an OGTT in male ICR mice and AUC0–120 min of blood glucose levels. All reagents were purchased from commercial sources and used
Values are mean ± SEM (n = 8). * P≤0.05, **P≤0.01, ***P ≤0.001 without further purification. Reactions were monitored by TLC on
Compared to vehicle-treated ICR mice by Student’s t test.
silica gel 60 F254 plates (Qingdao Ocean Chemical Company, China).
Column chromatography was performed on silica gel (200-300 mesh,
Qingdao Ocean Chemical Company, China). Melting points were
6
| J. Name., 2012, 00, 1-3
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Page 7 of 14
Org aP nl ei ca s &e dB oi o nmo ot la ed cj uu sl at rm Ca hr ge imn si stry
Journal Name
ARTICLE
s, 1H), 4.74 (s, 2H), 4.51 (s, 2H), 3.17 (s, 1H), 1.80 (s, 3H). HR-MS (ESI)
(
View Article Online
DOI: 10.1039/C6OB01818A
+
10 2 2
m/z: calculated C11H N O Cl [M+H] 237.1324, found 237.1318.
Tert-butyl
(R)-(1-(3-(but-2-yn-1-yl)-2,6-dioxo-1-(prop-2-yn-1-yl)-
1
,2,3,6-tetra- hydropyrimidin-4-yl)piperidin-3-yl)carbamate (E)
A mixture of C (8.0 g, 33.9 mmol), (R)-3-(N-Boc-amino)piperidine
8.13 g, 40.68 mmol) and K CO (10.76 g, 77.90 mmol) in DMF (40 mL)
(
2
3
was stirred at 65 °C for 5 h. After cooling to r.t., the mixture was
poured into water (1000 mL). The precipitate was collected by
filtration, washed with water, and dried to give compound E as a
1
brown solid (13.5 g, 99 % ). mp: 87-89 °C . H NMR (300 MHz, CDCl
3
):
δ 5.27 (s, 1H), 4.92-4.76 (m, 1H), 4.70-4.66 (m, 3H), 4.49 (d, J = 17.1
Hz, 1H), 3.88-3.82 (m, 1H), 3.35-3.25 (m, 1H), 3.17-3.02 (m, 1H), 2.94-
2
1
.78 (m, 1H), 2.75-2.54 (m, 1H), 2.18 (t, J = 2.4 Hz, 1H), 1.97-1.85 (m,
H), 1.82 (s, 3H), 1.74-1.67 (m, 2H), 1.45 (s, 9H). HR-MS (ESI) m/z:
+
calculated C21
H
29
N
4
O
4
[M+H] 401.4327, found 401.4319.
azidobenzene (H01)
Aniline (5.62 g, 18 mmol) was dissolved in 6 mol/L hydrochloric
acid (20 mL). To this solution, sodium nitrite (0.89 g, 12.9 mmol) was
slowly added at 0 to 5 °C within 30 min. The solution was vigorously
stirred at 0-5 °C for 30 min. Sodium azide (0.91 g, 13.9 mmol,
dissolved in 2 mL of water) was slowly added into the reaction
mixture at 0 °C. The resulting solution was stirred at 0 °C for 2 h
followed by diluting with ice water (50 mL) and extracting with EtOAc
Fig. 6 Effect of B04 during an OGTT in diabetic C57BL/6 mice. (A)
show time-dependent changes of blood glucose after oral
administration of B04, followed by 1 g/kg oral glucose challenge.
Date in (B) represent AUC0–120 min of blood glucose levels. Values are
mean ± SEM (n = 6). *P≤0.05, **P ≤0.01 and ***P ≤0.001 compared
to vehicle-treated C57BL/6 mice by Student’s t test.
(
5
3 × 50 mL). The combined organic layer was washed with water (3 ×
0 mL), saturated aqueous NaHCO (50 mL × 3) and brine (100 mL),
, filtered and concentrated to afford
3
dried over anhydrous Na
2
SO
4
1
yellow oil (0.84 g, 65%). H NMR (300 MHz, CDCl
Hz, 2H), 7.08-7.05 (m, 1 H), 6.96 (m, 2 H).
3
): δ 7.27 (t, J = 7.2
measured on capillary tube and were uncorrected. IR spectra (in KBr
pellets) were taken using Shimadzu FT-IR-8400S spectrophotometer.
tert-butyl (R)-(1-(3-(but-2-yn-1-yl)-2,6-dioxo-1-((1-phenyl-1H-1,2,3-
triazol-4-yl) methyl)-1,2,3,6-tetrahydropyrimidin-4-yl)piperidin-3-
yl)carbamate (F01)
1
13
H NMR and C NMR spectra (DMSO-d
6 3
, CDCl ) were recorded with
a Bruker AV-300 spectrometer in the indicated solvents (TMS as
internal standard): the values of the chemical shifts are expressed in To the solution of compund E (212 mg, 0.53 mmol) and H01 (76 mg,
δ values (ppm) and the coupling constants (J) in Hz. High-resolution 0.64 mmol) in 80% methanol (10 mL), CuSO ·5H O (6.6 mg, 0.027
4
2
mass spectra were recorded using an Agilent QTOF 6520.
mmol) and ascorbate sodium (30 mg) were added, respectively. The
reaction solution was stirred at room temperature for 12 h. The
mixture was conentrated under reduced presure, water was added
1-(but-2-ynyl)-6-chloropyrimidine-2,4(1H,3H)-dione (C)
To a mixture of 6-chlorouracil (10 g, 69 mmol) and DIPEA (9.7 g, 75 and (20 mL) and extracted with DCM (3 × 20 mL). The combined
mmol) in DMF (30 mL) was added 1-bromo-2-butyne (9.9 g, 75 mmol). organic layer was washed with saturated brine, dried over anhydrous
The reaction mixture was stirred at r.t for 12 h. Water (150 mL) was Na
added. The precipitate was collected by filtration, washed
2 4
SO , and concentrated. The crude product was purified by flash
chromatography (petroleum ether/ethyl acetate, 10:1~ 3:1) to give
1
compound F01 as a light yellow solid (234 mg, 85%). H NMR (300
MHz, CDCl ): δ 7.71 (s, 1H), 7.28-7.25 (m, 2H), 7.08-7.00 (m, 3H), 5.32
3
with water and EtOH, and dried to give compound C as a light yellow
1
solid (10.8 g, 80% ). mp: 216-217 °C . H NMR (300 MHz, DMSO-d
6
): δ
(
(
(
s, 2H), 5.28 (s, 1H), 4.83-4.74 (m, 1H), 4.67 (d, J = 15.9 Hz, 1H), 4.49
d, J = 15.9 Hz, 1H), 3.87-3.78 (m, 1H), 3.33-3.25 (m, 1H), 3.14-3.03
m, 1H), 2.88-2.81 (m, 1H), 2.71-2.58 (m, 1H), 1.97-1.85 (m, 1H), 1.82
1
1.71 (s, 1H), 5.99 (s, 1H), 4.65 (s, 2H), 1.80 (s, 3H). HR-MS (ESI) m/z:
+
8 8 2 2
calculated C H N O Cl [M+H] 199.0274, found 199.0276.
1-(but-2-ynyl)-6-chloro-3-(prop-2-ynyl)pyrimidine-2,4(1H,3H)-
(s, 3H), 1.74-1.67 (m, 2H), 1.45 (s, 9H).
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-phenyl-1H-
2 3
To a suspension of B (9 g, 45.5 mmol) and K CO
(14.4 g, 104.53 mmol) 1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
dione (D)
(
in DMF (36 mL) was added 3-bromopropyne (6.5 g, 54.5 mmol). The hydrochloride (A01)
reaction mixture was stirred at r.t for 12 h. The reaction mixture was
poured into water. The precipitate was collected by filtration,
Compund F01 (200 mg, 0.38 mmol) was dissolved in EtOAc (5 mL)
and ether (30 mL). Freshly prepared HCl gas was bubbled into the
solution at °C. After TLC analysis indicated the completed
consumption of starting materials, the precipitate was collected by
washed with water, and dried to give compound D as a yellow solid
1
(
8.48 g, 79%). mp: 147-149 °C . H NMR (300 MHz, DMSO-d
6
): δ 6.21
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Page 8 of 14
ARTICLE
Journal Name
filtration, dried in vacuo to afford compound A01 as a white solid (R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2,4-difluoro
View Article Online
-
1
DOI: 10.1039/C6OB01818A
(
135 mg, 77%). mp: 83-86 °C; IR (vmax cm ): 3438, 2949, 1704, 1654, phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
1
1
8
5
3
1
1
8
3
503, 1441, 805, 762; H NMR (300MHz, DMSO-d
6
): δ 8.64 (s, 1H), hydrochloride(A05)
.34 (brs, 3H), 7.89-7.86 (m, 2H), 7.61-7.55 (m, 2H), 7.50-7.45 (m, 1H),
.28 (s, 1H), 5.09 (s, 2H), 4.70-4.64 (m, 1H), 4.50-4.44 (m, 1H), 3.38-
.30 (m, 2H), 3.12-3.01 (m, 2H), 2.92-2.85 (m, 1H), 1.97-1.88 (m, 2H),
.78 (s, 3H), 1.68-1.62 (s, 2H); C NMR (75MHz, DMSO-d ): δ 161.40,
6
59.04, 151.39, 144.03, 136.51, 129.82, 128.58, 121.45, 119.96,
Following a similar procedure for the preparation of A01, A05
was prepared starting from 2,4-difluoroaniline. White solid (112 mg);
mp: 84-86 °C; IR (vmax cm ): 3442, 2922, 1706, 1654, 1609, 1440, 808;
6
H NMR (300MHz, DMSO-d ): δ 8.41 (s, 1H), 8.34 (brs, 3H), 7.91-7.83
m, 1H), 7.71-7.63 (m, 1H), 7.38-7.30 (m, 1H), 5.28 (s, 1H), 5.11 (s,
H), 4.67 (d, J = 17.4 Hz, 1H), 4.47 (d, J = 17.4 Hz, 1H), 3.39-3.28 (m,
H), 3.15-2.98 (m, 2H), 2.93-2.84 (m, 1H), 1.97-1.88 (m, 2H), 1.78 (s,
H), 1.70-1.59 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.38, 159.05,
6
-1
13
1
(
8.33, 79.72, 74.57, 52.04, 51.41, 45.94, 35.79, 35.55, 26.91, 21.21,
2
2
3
+
.13; HR-MS (ESI) m/z: calculated for C22
H
25
N
7
O
2
[M+H] : 420.2142,
found: 420.2140.
13
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2-fluoro
151.38, 143.39, 127.54, 124.95, 112.81, 112.51, 105.96, 105.60,
105.28, 88.34, 79.72, 74.54, 52.07, 51.38, 45.93, 35.63, 35.54, 26.89,
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A02)
21.18, 3.12; HR-MS (ESI) m/z: calculated for
22 23 2 7 2
C H F N O
+
[
M+H] :456.1954, found:456.1953.
Following a similar procedure for the preparation of A01, A02 was
prepared starting from 2-fluoroaniline. White solid (140 mg). mp: 71- (R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(4-fluoro-2-
-
1
7
5 °C; IR (vmax cm ): 3438, 1704, 1654, 1600, 1510, 1474, 1441, 764; methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
1
6
H NMR (300MHz, DMSO-d ): δ 8.41 (s, 1H), 8.28 (brs, 3H), 7.83-7.78 dione hydrochloride (A06)
(
m, 1H), 7.63-7.51 (m, 2H), 7.45-7.40 m, 1H), 5.29 (s, 1H), 5.12 (s, 2H),
.66 (d, J = 18.0 Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 3.35-3.27 (m, 2H),
Following a similar procedure for the preparation of A01, A06 was
4
3
1
1
1
2
prepared starting from 4-fluoro-2-methylaniline. White solid (125
.12-2.98 (m, 2H), 2.92-2.84 (m, 1H), 1.98-1.88 (m, 2H), 1.78 (s, 3H),
-1
mg); mp: 92-95°C; IR (vmax cm ): 3442, 1705, 1651, 1508, 1441, 1231,
13
6
.69-1.61 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.40, 159.05,
1
8
1
4
3
1
09; H NMR (300MHz, DMSO-d
6
) δ: 8.33-8.29 (m, 4H), 7.50-7.45 (m,
51.40, 143.42, 131.12, 125.84, 125.51, 125.46, 124.75, 117.19,
16.93, 88.45, 79.72, 74.52, 52.12, 51.37, 46.00, 35.64, 35.52, 26.94,
H), 7.39-7.35 (m, 1H), 7.27-7.21 (m, 1H), 5.29 (s, 1H), 5.11 (s, 2H),
.67 (d, J = 15.0 Hz, 1H), 4.47 (d, J = 15.0 Hz, 1H), 3.38-3.27 (m, 2H),
.13-3.00 (m, 2H), 2.93-2.86 (m, 1H), 2.11 (s, 3H), 1.97-1.86 (m, 2H),
1.28, 3.09; HR-MS (ESI) m/z: calculated for
M+H] :438.2048, found: 438.2056.
22 7 2
C H24FN O
+
[
13
6
.78 (s, 3H), 1.70-1.61 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.40,
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(3-
159.01, 151.39, 142.89, 132.66, 128.24, 125.14, 117.85, 117.54,
113.82, 113.52, 88.43, 79.69, 74.54, 52.10, 51.38, 45.99, 35.71, 35.50,
fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
dione hydrochloride (A03)
26.93, 21.27, 17.34, 3.10; HR-MS (ESI) m/z: calculated for
+
C
23
H
7
26FN O
2
[M+H] : 452.2205, found: 452.2200
Following a similar procedure for the preparation of A01, A03 was
prepared starting from 3-fluoroaniline. White solid (135 mg); mp: 73- (R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2-chloro
7
-1
1
7 °C; IR (vmax cm ): 3435, 2950, 1705, 1651, 1604, 871, 784; H NMR phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(
300MHz, DMSO-d ) δ 8.71 (brs, 1H), 8.30 (s, 3H), 7.87-7.75 (m, 2H), hydrochloride (A07)
6
7
.67- 7.59(m, 1H), 7.37-7.30 (m, 1H), 5.29 (s, 1H), 5.10 (s, 2H), 4.67
Following a similar procedure for the preparation of A01, A07 was
prepared starting from 2-chloroaniline. White solid (112 mg); mp:80-
(
d, J = 18.0 Hz, 1H), 4.48 (d, J = 18.0 Hz, 1H), 3.40-3.29 m, 2H), 3.15-
2
1
1
5
.98 (m, 2H), 2.92-2.85 (m, 1H), 1.97-1.88 (m, 2H), 1.78 (s, 3H), 1.69-
-
1
1
8
(
7
3 °C; IR (vmax cm ):3442, 2949, 1704, 1654, 1440, 766; H NMR
300MHz, DMSO-d ) δ 8.37 (s, 1H), 8.27 (brs, 3H), 7.77-7.74 (m, 1H),
.67-7.62 (m, 2H), 7.60-7.55 (m, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 4.66
13
6
.62 (m, 2H). C NMR (75MHz, DMSO-d ): δ 161.40, 159.04, 151.39,
6
44.03, 136.51, 129.82, 128.58, 121.45, 119.96, 88.33, 79.72, 74.57,
2.04, 51.41, 45.94, 35.79, 35.54, 26.91, 21.21, 3.13; HR-MS (ESI) m/z:
(
2
d, J =15.0 Hz, 1H), 4.47 (d, J = 15.0 Hz, 1H), 3.33-3.27 (m, 2H), 3.13-
.97 (m, 2H), 2.91-2.84 (m, 1H), 1.98-1.88 (m, 2H), 1.78 (s, 3H), 1.69-
+
calculated for C22
H
24FN
7
O
2
[M+H] : 438.2048, found: 438.2048.
13
(R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(4-fluoro
6
1.60 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.37, 159.03, 151.40,
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A04)
142.87, 134.44, 131.52, 130.50, 128.38, 128.28, 125.46, 88.46, 79.70,
74.54, 52.13, 51.37, 46.00, 35.66, 35.50, 26.95, 21.29, 3.10; HR-MS
+
(
ESI) m/z: calculated for
C
22
H
7
24ClN O
2
[M+H] :454.1753,
Following a similar procedure for the preparation of A01, A04
was prepared starting from 4-fluoroaniline. White solid (127 mg); mp:
found:454.1756.
-
1
1
8
3-87 °C; IR (vmax cm ): 3442, 1705, 1651, 1517, 1441, 1047, 807; H (R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(3-chlro
NMR (300MHz, DMSO-d ) δ 8.62 (s, 1H), 8.28 (brs, 3H), 7.94-7.90 (m, phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
H), 7.45-7.41 (m, 2H), 5.29 (s, 1H), 5.09 (s, 2H), 4.66 (d, J = 15.0 Hz, hydrochloride (A08)
6
2
1
2
H), 4.48 (d, J = 15.0 Hz, 1H), 3.33-3.28 (m, 2H), 3.14-2.99 (m, 2H),
.92-2.84 (m, 1H), 1.97-1.87 (m, 2H), 1.78 (s, 3H), 1.69-1.60 (m, 2H);
C NMR (75MHz, DMSO-d ): δ 161.39, 159.04, 151.39, 144.03,
6
Following a similar procedure for the preparation of A01, A08
was prepared starting from 3-chloroaniline. White solid (108 mg,
13
-
1
4
7
8
9%), mp:83-87 °C; IR (vmax cm ):3439, 2921, 1704, 1652, 1595, 1441,
1
5
33.09, 122.43, 121.74, 116.77, 116.46, 88.44, 79.73, 74.53, 52.10,
1
90; H NMR (300MHz, DMSO-d
.02 (m, 1H), 7.92-7.90 (m, 1H), 7.64-7.54 (m, 2H), 5.29 (s, 1H), 5.10
6
) δ 8.74 (s, 1H), 8.32 (brs, 3H), 8.04-
1.40, 46.01, 35.76, 35.52, 26.96, 21.29, 3.10; HR-MS (ESI) m/z:
+
calculated for C22
H
24FN
7
O
2
[M+H] :438.2048, found:438.2050.
8
| J. Name., 2012, 00, 1-3
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Page 9 of 14
Org aP nl ei ca s &e dB oi o nmo ot la ed cj uu sl at rm Ca hr ge imn si stry
Journal Name
ARTICLE
(
(
(
1
1
2
s, 2H), 4.67 (d, J = 17.1 Hz, 1H), 4.48 (d, J = 17.1 Hz, 1H), 3.40-3.29 Following a similar procedure for the preparation of A01, A12 was
m, 2H), 3.14-2.99 (m, 2H), 2.93-2.85 (m, 1H), 1.98-1.89 (m, 2H), 1.79 prepared starting from 4-methylaniline. White solid (116 mg); mp:
View Article Online
DOI: 10.1039/C6OB01818A
s, 3H), 1.70-1.61 (m, 2H); 13C NMR (75MHz, DMSO-d
): δ 161.37, 81-86 °C; IR (vmax cm ):3447, 2920, 1705, 1655, 1440, 818, 786; H
59.05, 151.39, 144.26, 137.58, 134.13, 131.53, 128.36, 121.63, NMR (300MHz, DMSO-d ) δ 8.57 (s, 1H), 8.30 (brs, 3H), 7.75 (d, J =
19.72, 118.55, 88.42, 79.73, 74.53, 52.10, 51.41, 46.00, 35.79, 35.55, 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 5.28 (s, 1H), 5.09 (s, 2H), 4.66 (d,
-1
1
6
6
6.96, 21.29, 3.11; HR-MS (ESI) m/z: calculated for C22
M+H] : 454.1753, found:454.1751.
H
24ClN
7
O
2
J = 18.0 Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 3.40-3.28 (m, 2H), 3.14-2.98
(m, 2H), 2.92-2.85 (m, 1H), 2.37 (s, 3H), 1.98-1.86 (m, 2H), 1.78 (s,
+
[
1
3
3
1
7
6
H), 1.68-1.59 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.40, 159.03,
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(4-chloro
51.39, 143.89, 138.15, 134.31, 130.14, 121.30, 119.84, 88.40, 79.71,
4.56, 52.09, 51.40, 45.98, 35.80, 35.53, 26.95, 21.28, 20.51, 3.12;
HR-MS (ESI) m/z: calculated for C23
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride(A09)
+
H
27
7
N O
2
[M+H] : 434.2299,
Following a similar procedure for the preparation of A01, A09 found:434.2297.
was prepared starting from 4-chloroaniline. White solid (150 mg); mp:
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2,6-dimethyl
-1
1
8
8-91 °C; IR (vmax cm ): 3442, 1705, 1651, 1502, 1442, 1232; H NMR
300MHz, DMSO-d ) δ 8.68 (s, 1H), 8.33 (brs, 3H), 7.93 (d, J = 9.0 Hz,
H), 7.66 (d, J = 9.0 Hz, 2H), 5.29 (s, 1H), 5.10 (s, 2H), 4.67 (d, J = 18.0
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A13)
(
6
2
Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 3.32-3.27 (m, 2H), 3.14-2.98 (m, 2H), Following a similar procedure for the preparation of A01, A13 was
2
.92-2.85 (m, 1H), 1.98-1.88 (m, 2H), 1.78 (s, 3H), 1.70-1.60 (m, 2H); prepared starting from 2,6-dimethylaniline. White solid (122 mg); mp:
13
-1
1
C NMR (75MHz, DMSO-d
35.31, 132.81, 129.76, 121.68, 121.56, 88.43, 79.73, 74.54, 52.10, NMR (300MHz, DMSO-d
6
): δ 161.38, 159.05, 151.39, 144.21, 95-98°C; IR (vmax cm ): 3438, 2953, 1705, 1654, 1440, 1230, 786; H
) δ: 8.26 (brs, 3H), 8.20 (s, 1H), 7.41-7.35 (m,
1
5
6
1.40, 46.00, 35.77, 35.54, 26.96, 21.31, 3.11; HR-MS (ESI) m/z: 1H), 7.28-7.26 (m, 2H), 5.29 (s, 1H), 5.11 (s, 2H), 4.66 (d, J = 15.0 Hz,
+
calculated for C22
H
24ClN
7
O
2
[M+H] : 454.1753, found:454.1750.
1H), 4.46 (d, J = 15.0 Hz, 1H), 3.34-3.25 (m, 2H), 3.13-2.98 (m, 2H),
2
1
1
5
.91-2.84 (m, 1H), 1.97-1.91 (m, 2H), 1.89 (s, 6H), 1.77 (s, 3H), 1.69-
.61 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.38, 158.97, 151.34,
6
42.81, 135.75, 134.75, 129.83, 128.28, 125.16, 88.37, 79.61, 74.55,
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2,6-dichloro
13
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A10)
2.07, 51.37, 45.94, 35.78, 35.47, 26.91, 21.19, 16.84, 3.10; HR-MS
+
Following a similar procedure for the preparation of A01, A10 (ESI) m/z: calculated for
C
24
H
29
N
7
O
2
[M+H] :448.2455,
was prepared starting from 2,6-dichloroaniline. White solid (142 mg); found:448.2448.
-1
mp: 100-104°C; IR (vmax cm ): 3442, 1705, 1655, 1601, 1483, 1441,
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(5-chloro-2-
1
1
7
231, 795; H NMR (300MHz, DMSO-d
.79-7.76 (m, 2H), 7.70-7.64 (m, 1H), 5.30 (s, 1H), 5.12 (s, 2H), 4.66
6
) δ: 8.38 (s, 1H), 8.26 (s, 3H),
methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
dione hydrochloride (A14)
(
(
(
d, J = 18 Hz, 1H), 4.47 (d, J = 18 Hz, 1H), 3.32-3.27 (m, 2H), 3.13-3.00
m, 2H), 2.92-2.85 (m, 1H), 1.96-1.86 (m, 2H), 1.78 (s, 3H), 1.69-1.59 Following a similar procedure for the preparation of A01, A14 was
13
6
m, 2H); C NMR (75MHz, DMSO-d ): δ 161.33, 159.03, 151.36, prepared starting from 5-chloro-4-methylaniline. White solid (137
-1
1
5
42.93, 132.81, 132.60, 132.54, 129.16, 125.88, 88.41, 79.70, 74.51, mg); mp: 94-96°C; IR (vmax cm ):3448, 2922, 1705, 1654, 1604, 1498,
1
2.12, 51.37, 45.98, 35.68, 35.51, 26.93, 21.27, 3.11; HR-MS (ESI) m/z: 1440, 828, 809, 786; H NMR (300MHz, DMSO-d
6
) δ: 8.36 (s, 1H), 8.29
+
calculated for C22
H
23Cl
2
7
N O
2
[M+H] :488.1363, found:488.1363.
(brs, 3H), 7.57-7.48 (m, 3H), 5.29 (s, 1H), 5.11 (s, 2H), 4.66 (d, J = 18
Hz, 1H), 4.47 (d, J = 18 Hz, 1H), 3.34-3.28 (m, 2H), 3.12-2.97 (m, 2H),
2
1
1
(R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(o-tolyl)-1H-
.92-2.85 (m, 1H), 2.12 (s, 3H), 1.97-1.87 (m, 2H), 1.78 (s, 3H), 1.69-
1
,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
13
.61 (m, 2H); C NMR (75MHz, DMSO-d
6
): δ 161.38, 159.01, 151.38,
hydrochloride (A11)
42.98, 137.03, 132.91, 132.07, 130.76, 129.49, 125.60, 125.00,
Following a similar procedure for the preparation of A01, A11 88.42, 79.69, 74.55, 52.10, 51.39, 45.98, 35.70, 35.51, 26.93, 21.26,
+
was prepared starting from 2-methylaniline. White solid (120 mg); 17.06, 3.11; HR-MS (ESI) m/z: calculated for C23
mp: 81-85 °C; IR (vmax cm ): 3439, 1705, 1651, 1441, 1230; H NMR 468.1909, found: 468.1912.
H
7
26ClN O
2
[M+H] :
-1
1
(
(
1
2
300MHz, DMSO-d
m, 2H), 5.28 (s, 1H), 5.11 (s, 2H), 4.66 (d, J = 18.0 Hz, 1H), 4.47 (d, J =
8.0 Hz, 1H), 3.39-3.28 (m, 2H), 3.13-2.97 (m, 2H), 2.92-2.85 (m, 1H),
.12 (s, 3H), 1.97-1.85 (m, 2H), 1.78 (s, 3H), 1.69-1.60 (m, 2H);
6
) δ 8.35-8.29 (m, 4H), 7.48-7.44 (m, 2H), 7.40-7.39
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2-hydroxy-4-
methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-
dione hydrochloride (A15)
1
3
C
NMR (75MHz, DMSO-d
6
): δ 161.40, 159.00, 151.40, 142.84, 136.18,
Following a similar procedure for the preparation of A01, A15
1
5
32.92, 131.29, 129.67, 126.89, 125.87, 124.88, 88.43, 79.67, 74.56, was prepared starting from 2-hydroxy-4-methylaniline. White solid
-1
2.11, 51.38, 45.99, 35.74, 35.50, 26.94, 21.39, 17.37, 3.11; HR-MS (117 mg); mp: 107-110°C; IR (vmax cm ):3442, 2950, 1703, 1649, 1441,
+
1
(
ESI) m/z: calculated for
C
23
H
27
7
N O
2
[M+H] : 434.2299, 806; H NMR (300MHz, DMSO-d
6
) δ: 10.51 (s, 1H), 8.28 (brs, 3H), 8.24
found:434.2298.
(s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 6.94 (s, 1H), 6.77 (d, J = 8.1 Hz, 1H),
5
1
3
.28 (s, 1H), 5.09 (s, 2H), 4.65 (d, J = 18.0 Hz, 1H), 4.47 (d, J = 18.0 Hz,
(
1
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(p-tolyl)-1H-
,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
H), 3.40-3.27 (m, 2H), 3.11-2.98 (m, 2H), 2.91-2.85 (m, 1H), 2.29 (s,
13
H), 1.96-1.86 (m, 2H), 1.78 (s, 3H), 1.68-1.62 (m, 2H); C NMR
hydrochloride (A12)
(
75MHz, DMSO-d
6
): δ 161.42, 158.99, 151.36, 149.17, 142.41, 139.74,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Org aP nl ei ca s &e dB oi o nmo to al ed cj uu sl at rm Ca hr ge imn si stry
Page 10 of 14
ARTICLE
Journal Name
-
1
1
5
24.74, 124.62, 122.09, 120.11, 117.26, 88.37, 79.71, 74.55, 52.06, mp: 94-97 °C; IR (vmax cm ): 3441, 2949, 1705, 1655, 1605, 1441,
View Article Online
DOI: 10.1039/C6OB01818A
1
1.35, 45.93, 35.71, 35.50, 26.91, 21.23, 20.80, 3.12; HR-MS (ESI) m/z: 1230, 836; H NMR (300MHz, DMSO-d
6
) δ: 8.51 (s, 1H), 8.29 (brs, 3H),
+
calculated for C23
H
27 7
N O
3
[M+H] : 450.2248, found: 450.2241.
7.77 (d, J = 9.0 Hz, 2H), 7.11 (d, J = 9.0 Hz, 2H), 5.28 (s, 1H), 5.08 (s,
2
3
2
1
H), 4.67 (d, J = 18.0 Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 3.82 (s, 3H),
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(4-(tert-butyl)
.39-3.27 (m, 2H), 3.14-2.96 (m, 2H), 2.92-2.85 (m, 1H), 1.99-1.87 (m,
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A16)
13
H), 1.78 (s, 3H), 1.69-1.58 (m, 2H); C NMR (75MHz, DMSO-d
6
): δ
61.40, 159.16, 159.02, 151.39, 143.75, 129.98, 121.65, 121.42,
Following a similar procedure for the preparation of A01, A16 114.79, 88.41, 79.71, 74.56, 55.52, 52.08, 51.40, 45.98, 35.80, 35.52,
27 7 3
was prepared starting from 4-(tert-buthyl)aniline. White solid (132 26.95, 21.28, 3.12; HR-MS (ESI) m/z: calculated for C23H N O
-
1
+
mg); mp: 101-103 °C; IR (vmax cm ):3435, 2959, 2867, 1705, 1655, [M+H] : 450.2248, found: 450.2245
1
1
7
2
2
3
1
1
3
440, 839; H NMR (300MHz, DMSO-d
6
) δ: 8.58 (s, 1H), 8.31 (brs, 3H),
ethyl
(R)-2-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
.77 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 5.28 (s, 1H), 5.09 (s,
H), 4.66 (d, J = 16.8 Hz, 1H), 4.47 (d, J = 16.8 Hz, 1H), 3.39-3.28 (m,
H), 3.12-2.93 (m, 2H), 2.92-2.85 (m, 1H), 1.99-1.83 (m, 2H), 1.78 (s,
H), 1.67-1.63 (m, 2H), 1.32 (s, 9H); C NMR (75MHz, DMSO-d
61.41, 159.03, 151.39, 151.18, 143.83, 134.22, 126.51, 121.42, was prepared starting from ethyl 2-aminobenzoate. White solid (97
19.73, 88.34, 79.71, 74.57, 52.04, 51.40, 45.94, 35.79, 35.54, 34.44, mg); mp: 171-174 °C; IR (vmax cm ): 3434, 2949, 1707, 1654, 1604,
0.95, 26.89, 21.20, 3.14; HR-MS (ESI) m/z: calculated for C26
M+H] : 476.2768, found: 476.2768.
dioxo-3,6-dihydropyrim idin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)benzoate hydrochloride (A20)
1
3
6
): δ
Following a similar procedure for the preparation of A01, A20
-1
1
H
N
33 7
O
2
1440, 1295, 1131, 766; H NMR (300MHz, DMSO-d
4H), 7.91-7.88 (m, 1H), 7.81-7.76 (m, 1H), 7.71-7.63 (m, 2H), 5.29 (s,
6
) δ: 8.35-8.32 (m,
+
[
1
4
2
H), 5.10 (s, 2H), 4.66 (d, J = 16.5 Hz, 1H), 4.46 (d, J = 16.5 Hz, 1H),
.00 (q, J = 7.2 Hz, 2H), 3.39-3.27 (m, 2H), 3.13-2.96 (m, 2H), 2.91-
.84 (m, 1H), 1.98-1.87 (m, 2H), 1.77 (s, 3H), 1.63-1.59 (m, 2H), 0.91
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(2-methoxy
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A17)
13
(
6
t, J = 7.2 Hz, 3H); C NMR (75MHz, DMSO-d ): δ 165.34, 161.33,
Following a similar procedure for the preparation of A01, A17 159.00, 151.33, 143.02, 135.09, 132.80, 130.40, 129.96, 127.66,
was prepared starting from 2-methoxyaniline. White solid (123 mg); 126.21, 124.82, 88.40, 79.68, 74.54, 61.13, 52.08, 51.38, 45.93, 35.46,
-1
29 7 4
mp: 80-84 °C; IR (vmax cm ):3435, 2949, 1705, 1654, 1602, 1507, 1475, 26.91, 21.21, 13.39, 3.10; HR-MS (ESI) m/z: calculated for C25H N O
1
+
1
3
(
3
2
441, 1232, 806; H NMR (300MHz, DMSO-d
6
) δ 8.58 (s, 1H), 8.31 (brs, [M+H] :492.2354, found:492.2353.
H), 7.77 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 5.28 (s, 1H), 5.09
s, 2H), 4.66 (d, J = 15.0 Hz, 1H), 4.47 (d, J = 15.0 Hz, 1H), 3.58 (s, 3H),
.38-3.28 (m, 2H), 3.13-2.99 (m, 2H), 2.92-2.85 (m, 1H), 1.97-1.88 (m,
H), 1.78 (s, 3H), 1.69-1.61 (m, 2H); C NMR (75MHz, DMSO-d
ethyl (R)-3-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
dioxo-3,6- dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)benzoate hydrochloride (A21)
13
6
): δ
1
61.42, 159.01, 151.39, 142.49, 130.67, 125.71, 125.57, 125.26,
Following a similar procedure for the preparation of A01, A21
1
4
20.82, 112.95, 100.78, 88.37, 79.69, 74.57, 56.07, 52.07, 51.36, was prepared starting from ethyl 3-aminobenzoate. Yellow solid (87
-1
5.94, 35.65, 35.50, 26.90, 21.17, 3.11; HR-MS (ESI) m/z: calculated mg); mp: 86-89 °C; IR (vmax cm ): 3434, 2952, 1708, 1654, 1441, 757;
+
1
for C23
H
27 7
N O
3
[M+H] :450.2248, found:450.2245.
H NMR (300MHz, DMSO-d
6
) δ: 8.79 (s, 1H), 8.38 (brs, 4H), 8.18 (d, J
=
1
4
2
9.0 Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.74 (t, J = 9.0 Hz, 1H), 5.28 (s,
(
R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(3-methoxy
H), 5.11 (s, 2H), 4.68 (d, J = 15.6 Hz, 1H), 4.48 (d, J = 15.6 Hz, 1H),
.37 (q, J = 6.0 Hz, 2H), 3.40-3.30 (m, 2H), 3.14- 2.99 (m, 2H), 2.93-
.86 (m, 1H), 1.98-1.89 (m, 2H), 1.78 (s, 3H), 1.69-1.61 (m, 2H), 1.35
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A18)
13
Following a similar procedure for the preparation of A01, A18 (t, J = 6.0 Hz, 3H); C NMR (75MHz, DMSO-d
6
): δ 164.74, 161.40,
was prepared starting from 3-methoxyaniline. White solid (114 mg); 159.06, 151.40, 144.33, 136.76, 131.44, 130.51, 128.93, 124.45,
-1
mp: 81-85 °C; IR (vmax cm ):3434, 2945, 1706, 1651, 1609, 1440, 782; 121.69, 120.09, 88.31, 79.72, 74.57, 61.29, 52.03, 51.42, 45.92, 35.81,
1
H NMR (300MHz, DMSO-d
6
) δ: 8.66 (s, 1H), 8.32 (brs, 3H), 7.51-7.44 35.57, 26.91, 21.16, 14.08, 3.14; HR-MS (ESI) m/z: calculated for
+
(
m, 3H), 7.05-7.02 (m, 1H), 5.28 (s, 1H), 5.09 (s, 2H), 4.67 (d, J = 18.0
C
25
H
29
7
N O
4
[M+H] : 492.2354, found:492.2360.
Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 3.84 (s, 3H), 3.39-3.29 (m, 2H), 3.13-
ethyl
(R)-4-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
2
1
1
8
2
4
.97 (m, 2H), 2.93-2.86 (m, 1H), 1.97-1.86 (m, 2H), 1.78 (s, 3H), 1.70-
.61 (m, 2H); C NMR (75MHz, DMSO-d ): δ 161.41, 160.12, 159.04,
6
51.39, 143.95, 137.58, 130.75, 121.57, 114.29, 111.99, 105.62,
dioxo-3,6- dihydropyrim idin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)benzoate (A22)
13
8.40, 79.73, 74.54, 55.59, 52.08, 51.40, 45.99, 35.79, 35.53, 26.94,
Following a similar procedure for the preparation of A01, A22
+
1.27, 3.11; HR-MS (ESI) m/z: calculated for C23
H
27
7
N O
3
[M+H] : was prepared starting from ethyl 4-aminobenzoate. Yellow solid (94
-1
50.2248, found: 450.2244.
mg); mp: 95-99 °C; IR (vmax cm ):3435, 1713, 1651, 1608, 1519, 1442,
1
1
8
2
232, 770; H NMR (300MHz, DMSO-d
.13 (d, J =9.0 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H), 5.29 (s, 1H), 5.11 (s,
H), 4.67 (d, J = 18.0 Hz, 1H), 4.47 (d, J = 18.0 Hz, 1H), 4.34 (q, J = 7.2
6
) δ: 8.79 (s, 1H), 8.35 (brs, 3H),
(R)-6-(3-aminopiperidin-1-yl)-1-(but-2-yn-1-yl)-3-((1-(4-methoxy
phenyl)-1H-1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (A19)
Hz, 2H), 3.41-3.29 (m, 2H), 3.18-2.99 (m, 2H), 2.92-2.85 (m, 1H), 1.97-
Following a similar procedure for the preparation of A01, A19 1.85 (m, 2H), 1.78 (s, 3H), 1.69-1.59 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H);
13
was prepared starting from 4-methoxyaniline. White solid (108 mg);
6
C NMR (75MHz, DMSO-d ): δ 164.79, 161.39, 159.06, 151.38,
1
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 11 of 14
Org aP nl ei ca s &e dB oi o nmo ot la ed cj uu sl at rm Ca hr ge imn si stry
Journal Name
ARTICLE
1
6
3
44.47, 139.63, 130.87, 129.46, 121.60, 119.75, 88.31, 79.73, 74.55, (m, 2H), 1.78 (s, 3H), 1.68- 1.59 (m, 2H); 13C NMR (75MHz, DMSO-d
):
View Article Online
6
DOI: 10.1039/C6OB01818A
1.06, 52.03, 51.42, 45.93, 44.15, 35.78, 35.58, 26.93, 21.20, 14.11, δ 166.35, 161.39, 159.05, 151.38, 144.41, 139.42, 131.02, 130.47,
+
.14; HR-MS (ESI) m/z: calculated for C25
H
29 7
N O
4
[M+H] : 492.2354, 121.58, 119.66, 88.33, 79.73, 74.56, 52.05, 51.41, 45.94, 35.77, 35.57,
found:492.2347.
R)-2-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid hydrochloride (A23)
To the solution of Boc precursor of A20 (160 mg, 0.27 mmol) in acetone (50 mL), was added dropwise the solution of NaN
26.92, 21.18, 3.14; HR-MS (ESI) m/z: calculated for C23
H
25
N
7
O
4
+
[
M+H] : 464.2041, found:464.2047.
(
3
ethyl 2-azidoacetate (J01)
To the solution of ethyl bromoacetate (10 g, 60 mmol) in
(9.73 g,
3
methanol (5 mL), was added NaOH (1M, 2 mL). the resulting mixture 150 mmol) in water (40 mL) at 0–5 °C. The mixture was stirred at
was stirred at r.t. for 2 h. After evaporation of solvent, the residue 60 °C for 4 h. After cooling to r.t., the solvent was evaporated. Water
was dissolved in water (3 mL) and acidified with HCl (1M, 2 mL) to was added, and extracted with with dichloromethane (4 × 40 mL),
adjust the pH value to 3. The precipitate was collected by filtration, washed with saturated NaHCO
purified by flash chromatography (dichloromethane/methanol, anhydrous Na SO and concentrated in vacuo to give J01 as
00:1~ 30:1) to give Boc precursor of A23, which was dissolved in colourless oil (7 g, 90%), MS (ESI) m/z: 130.1 [M+H] ; H NMR (300
3
(3 × 20 mL) and brine, dried over
2
4
+
1
1
EtOAc (5 mL) and ether (30 mL), and bubbled with freshly prepared MHz, CDCl
HCl gas at °C. After TLC indicated consumption of starting materials, 3H).
the precipitate was collected by filtration, dried in vacuo to to give
3
): δ 4.25 (q, J = 7.2 Hz, 2H), 3.83 (s, 2H), 1.27 (t, J = 7.2 Hz,
ethyl (R)-2-(4-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)amino)
compound A23 as a white solid (70 mg, yield 62%). mp:111-116 °C;
IR (vmax cm ):3448, 2921, 1705, 1651, 1442, 800,767; H NMR
piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-
-1
1
1H-1,2,3-triazol-1-yl)acetate (K01)
(
6
300MHz, DMSO-d ) δ: 13.21 (brs, 1H), 8.38 (brs, 3H), 8.32 (s, 1H),
7
7
1
2
.89 (d, J = 7.8 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), Following a similar procedure for the preparation of H01, J01 was
.57 (d, J = 7.8 Hz, 1H), 5.27 (s, 1H), 5.09 (s, 2H), 4.67 (d, J = 18.0 Hz, prepared starting from compound D (5 g, 12.5 mmol) and compoud
H), 4.47 (d, J = 18.0 Hz, 1H), 3.40- 3.28 (m, 2H), 3.13- 2.98 (m, 2H), I01 (1.93 mg, 15.0 mmol). The crude product was purified by flash
.93-2.86 (m, 1H), 1.97-1.85 (m, 2H), 1.78 (s, 3H), 1.69- 1.60 (m, 2H); chromatography (EtOAc/ether 100:1~30:1 ) to give K01 as a white
1
3
1
C NMR (75MHz, DMSO-d
6
): δ 166.57, 161.36, 159.00, 151.39, solid (4.3 g, 65%). mp: 78-80 °C; H NMR (300 MHz, CDCl
3
): δ 7.71 (s,
1
8
3
42.82, 135.06, 132.29, 130.28, 129.76, 128.54, 126.25, 124.64, 1H), 5.32 (s, 2H), 5.28 (s, 1H), 5.02 (s, 2H), 4.83-4.74 (m, 1H), 4.67 (d,
8.32, 79.73, 74.58, 52.04, 51.39, 45.92, 35.72, 35.56, 26.90, 21.23, J = 15.3 Hz, 1H), 4.48 (d, J = 15.3 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.87-
+
.14; HR-MS (ESI) m/z: calculated for C23
H
25 7
N O
4
[M+H] :464.2041, 3.78 (m, 1H), 3.34-3.21 (m, 1H), 3.15-3.02 (m, 1H), 2.86-2.80 (m, 1H),
2.72-2.59 (m, 1H), 1.96-1.87 (m, 1H), 1.82 (s, 3H), 1.74-1.67 (m, 2H),
found:464.2044.
1
C
.45 (s, 9H), 1.26 (t, J = 7.2 Hz, 3H). HR-MS (ESI) m/z: calculated for
(
3
R)-3-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
+
25
H
36
7
N O
6
[M+H] : 529.4512, found: 529.4516.
yl)benzoic acid hydrochloride (A24)
(R)-2-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-yl)acetic
acid hydrochloride (B01)
3
Following a similar procedure for the preparation of A23, A24 was
prepared starting from Boc precursor of A21. White solid (65 mg);
-1
1
mp: 115-119 °C; IR (vmax cm ): 3448, 1706, 1643, 1443, 1232, 760; H Following a similar procedure for the preparation of A23, B01 was
NMR (300MHz, DMSO-d ) δ: 8.78 (s, 1H), 8.36 (s, 1H), 8.32 (brs, 3H), prepared starting from K01 (3.5 g, 6.6 mmol). White solid (86 mg,
6
-1
8
5
3
2
1
1
4
.15 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 65%); mp: 114-117 °C; IR (vmax cm ): 3448, 2978, 1743, 1704, 1650,
.29 (s, 1H), 5.10 (s, 2H), 4.67 (d, J = 17 Hz, 1H), 4.47 (d, J = 17 Hz, 1H), 1442, 1233, 1053, 815; H NMR (300MHz, DMSO-d
.35- 3.28 (m, 2H), 3.14-3.01 (m, 2H), 2.93-2.85 (m, 1H), 1.95-1.85 (m, 1H), 8.18 (brs, 3H), 7.93 (s, 1H), 5.28 (s, 1H), 5.22 (s, 2H), 5.01 (s, 2H),
H), 1.78 (s, 3H), 1.70-1.59 (m, 2H); C NMR (75MHz, DMSO-d
66.29, 161.41, 159.07, 151.41, 144.33, 136.68, 132.43, 130.35, 3.10-3.00 (m, 2H), 2.90-2.83 (m, 1H), 1.95-1.87 (m, 2H), 1.79 (s, 3H),
6
29.10, 124.07, 121.57, 120.24, 88.31, 79.71, 74.58, 52.02, 51.42, 1.69-1.61 (m, 2H); C NMR (75MHz, DMSO-d ): δ 168.51, 161.34,
1
6
) δ: 13.37 (brs,
13
6
): δ 4.64 (d, J = 18.0 Hz, 1H), 4.45 (d, J = 18.0 Hz, 1H), 3.31-3.23 (m, 2H),
13
5.92, 35.86, 35.58, 26.90, 21.19, 3.14; HR-MS (ESI) m/z: calculated 158.98, 151.29, 142.55, 124.86, 88.36, 79.74, 74.50, 64.87, 59.71,
+
for C23
H
25 7
N O
4
[M+H] : 464.2041, found:464.2046.
52.07, 51.34, 50.34, 45.93, 35.73, 35.47, 34.08, 26.90, 21.25, 20.72,
1
5.12, 14.03, 3.13; HR-MS (ESI) m/z: calculated for C18
H
N
23 7
O
4
(
3
R)-4-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
+
[
M+H] : 402.1884, found: 402.1881.
yl)benzoic acid hydrochloride (A25)
ethyl
(R)-2-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
dioxo-3,6-dihydr- opyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)acetate hydrochloride (B02)
Following a similar procedure for the preparation of A23, A25 was
prepared starting from Boc precursor of A22. White solid (86 mg);
-
1
mp: 122-126 °C; IR (vmax cm ):3457, 2950, 1705, 1647, 1608, 1443, Following a similar procedure for the preparation of A23, B02 was
1
7
8
1
3
99, 773; H NMR (300MHz, DMSO-d
6
) δ: 13.22 (brs, 1H), 8.77 (s, 1H), prepared starting from K01. White solid (120 mg, yield: 78%); mp:
-1
1
.25 (brs, 3H), 8.12 (d, J = 8.1 Hz, 2H), 8.04 (d, J = 8.1 Hz, 2H), 5.30 (s, 71-73 °C; IR (vmax cm ): 3435, 2926, 1751, 1651, 1439, 750; H NMR
H), 5.11 (s, 2H), 4.66 (d, J = 15.0 Hz, 1H), 4.47 (d, J = 15.0 Hz, 1H), (300MHz, DMSO-d ): δ 8.36 (brs, 3H), 7.96 (s, 1H), 5.33 (s, 2H), 5.26
.28-3.19 (m, 2H), 3.12- 3.01 (m, 2H), 2.91-2.84 (m, 1H), 1.96- 1.85 (s, 1H), 5.02 (s, 2H), 4.65 (d, J = 18.0 Hz, 1H), 4.45 (d, J = 18.0 Hz, 1H),
6
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Org aP nl ei ca s &e dB oi o nmo to al ed cj uu sl at rm Ca hr ge imn si stry
Page 12 of 14
ARTICLE
Journal Name
4
2
.16 (q, J = 6.6 Hz, 2H), 3.40-3.27 (m, 2H), 3.12-2.97 (m, 2H), 2.91- Following a similar procedure for the preparation of B01. B05 was
View Article Online
DOI: 10.1039/C6OB01818A
.84 (m, 1H), 1.96-1.85 (m, 2H), 1.78 (s, 3H), 1.68-1.57 (m, 2H), 1.20 prepared starting from ethyl 2-bromopropionate. White solid (103 g);
13
-1
1
(
t, J = 6.6 Hz, 3H); C NMR (75MHz, DMSO-d
6
): δ 167.21, 161.35, mp: 178-181 °C; IR (vmax cm ): 3447, 2948, 1704, 1650, 1231, 805; H
1
5
59.00, 151.29, 142.68, 124.96, 88.36, 79.74, 74.50, 61.40, 52.07, NMR (300MHz, DMSO-d ): δ 8.41 (brs, 3H), 8.00 (s, 1H), 5.47-5.45 (m,
1.36, 50.21, 45.93, 35.72, 35.48, 26.88, 21.18, 13.93, 3.14; HR-MS 1H), 5.25 (s, 1H), 5.01 (s, 2H), 4.66 (d, J = 18.0 Hz, 1H), 4.46 (d, J = 18.0
6
+
(
ESI) m/z: calculated for
C
20
H
27
N
7
O
4
[M+H] :430.2197, Hz, 1H), 3.34-3.20 (m, 2H), 3.07-2.97 (m, 2H), 2.92-2.89 (m, 1H), 1.96-
13
found:430.2198.
1.92 (m, 2H), 1.79 (s, 3H), 1.79-1.68 (m, 5H); C NMR (75MHz,
DMSO-d ): δ 170.81, 161.36, 158.97, 151.32, 142.41, 123.14, 88.39,
9.73, 74.53, 57.41, 52.08, 51.38, 45.92, 35.82, 35.47, 26.91, 21.11,
6
(R)-3-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
7
1
3
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
7.07, 3.13; HR-MS (ESI) m/z: calculated for
19 25 7 4
C H N O
yl)propanoic acid hydrochloride(B03)
+
[
M+H] :416.2041, found:416.2032.
Following a similar procedure for the preparation of B01. B03 was
ethyl
2-(4-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
prepared starting from methyl 3-bromopropionate. White solid (78
dioxo-3,6-dihy- dropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)propanoate hydrochloride (B07)
-1
mg), mp: 74-76°C; IR (vmax cm ): 3447, 2956, 1704, 1647, 1443, 1231,
1
8
07; H NMR (300MHz, DMSO-d
6
) δ: 8.45 (brs, 3H), 7.92 (s, 1H), 5.24
(
(
(
1
5
s, 1H), 4.98 (s, 2H), 4.70-4.61 (m, 1H), 4.51-4.41 (m, 3H), 3.38-3.30 Following a similar procedure for the preparation of B02. B07 was
m, 2H), 3.11-2.96 (m, 2H), 2.88-2.83 (m, 3H), 1.93-1.85 (m, 2H), 1.79 prepared starting from ethyl 2-bromopropionate. White solid (126
s, 3H), 1.66-1.62 (m, 2H); 13C NMR (75MHz, DMSO-d
): δ 171.74, mg); mp: 146-149 °C; IR (vmax cm ):3434, 2947, 2360, 1746, 1705,
-1
6
1
61.34, 158.96, 151.28, 142.57, 123.57, 88.35, 79.72, 74.53, 52.07, 1633, 1441, 856; H NMR (300MHz, DMSO-d
6
): δ 8.24 (brs, 3H), 8.03
1.36, 45.92, 45.23, 35.76, 35.45, 33.93, 26.87, 21.26, 3.13. HR-MS (s, 1H), 5.58 (q, J = 7.2 Hz, 1H), 5.27 (s, 1H), 5.04 (s, 2H), 4.64 (d, J =
+
(
ESI) m/z: calculated for
C
19
H
25
N
7
O
4
[M+H] :416.2041, 16.2 Hz, 1H), 4.46 (d, J = 16.2 Hz, 1H), 4.14 (q, J = 7.2 Hz, 2H), 3.34-
found:416.2041.
3.22 (m, 2H), 3.12-2.96 (m, 2H), 2.91-2.82 (m, 1H), 1.99-1.85 (m, 2H),
1
.79 (s, 3H), 1.71 (d, J = 7.2 Hz, 3H), 1.65-1.55 (m, 2H), 1.17 (t, J = 7.2
methyl (R)-3-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
dioxo-3,6-dihy dropyri midin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)propanoate hydrochloride (B04)
13
Hz, 3H); C NMR (75MHz, DMSO-d
1
5
6
): δ: 183.31, 169.25, 161.35,
58.98, 151.31, 142.57, 123.25, 88.36, 79.72, 74.52, 61.55, 57.30,
2.07, 51.38, 45.93, 35.80, 35.49, 26.91, 21.21, 17.04, 13.83, 3.13;
+
Following a similar procedure for the preparation of B02. B04 was HR-MS (ESI) m/z: calculated for
C
21
H
29
N
7
O
4
[M+H] :444.2354,
prepared starting from methyl 3-bromopropionate. White solid (113 found:444.2344.
-1
mg); mp: 73-76°C; IR (vmax cm ): 3445, 2936, 1705, 1643, 1447, 1236,
(
3
R,E)-4-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-yl)but-2-
enoic acid hydrochloride (B08)
1
806; H NMR (300MHz, DMSO-d
6
): δ 8.45 (brs, 3H), 7.93 (s, 1H), 5.24
(
s, 1H), 4.98 (s, 2H), 4.66 (d, J = 18.0 Hz, 1H), 4.53 (t, J = 6.6 Hz, 2H),
4
2
(
.45 (d, J = 18.0 Hz, 1H), 3.59 (s, 3H), 3.38-3.30 (m, 2H), 3.12-3.03 (m,
H), 2.95 (t, J = 6.6 Hz, 2H), 2.89-2.83 (m, 1H), 1.98-1.87 (m, 2H), 1.79 Following a similar procedure for the preparation of B01. B08 was
): δ 170.78, prepared starting from ethyl 4-bromocrotonate. White solid (113
s, 3H), 1.69-1.62 (m, 2H); 13C NMR (75MHz, DMSO-d
6
-1
1
5
61.34, 158.97, 151.28, 142.60, 123.64, 88.35, 79.72, 74.53, 52.06, mg); mp: 210-213 °C; IR (vmax cm ): 3435, 2950, 1705, 1650, 1442,
1
1.60, 51.37, 45.93, 45.05, 35.74, 35.46, 33.62, 26.88, 21.18, 3.14; 806; H NMR (300MHz, DMSO-d
6
): δ 8.44 (s, 3H), 8.35 (s, 0.5 H), 7.98
+
HR-MS (ESI) m/z: calculated for
found:430.2196.
C
20
H
27
N
7
O
4
[M+H] :430.2197, (s, 0.5 H), 7.40 (d, J = 14.4 Hz, 0.5 H), 6.88 (dt, J = 15.3, 5.1 Hz, 0.5 H),
6.47-6.32 (m, 0.5 H), 5.66 (d, J = 14.4 Hz, 1H), 5.25 (s, 1H), 5.18 (d, J =
5
1
2
.1 Hz, 1H), 5.01 (s, 2H), 4.66 (d, J = 17.7 Hz, 1H), 4.45 (d, J = 17.7 Hz,
H), 3.38-3.29 (m, 2H), 3.23 (d, J = 7.4 Hz, 1H), 3.06-3.00 (m, 2H),
.95-2.80 (m, 1H), 1.94-1.89 (m, 2H), 1.78 (s, 3H), 1.71-1.55 (m, 2H);
(
3
R)-4-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)butanoic acid hydrochloride (B05)
13
6
C NMR (75MHz, DMSO-d ): δ 172.02, 171.81, 161.35, 159.02,
Following a similar procedure for the preparation of B01. B05 was 151.28, 143.46, 143.00, 142.57, 141.45, 126.15, 124.37, 123.95,
prepared starting from ethyl 4-bromobutyrate. White solid (68 mg); 123.85, 120.61, 119.21, 115.49, 88.35, 79.69, 75.89, 74.51, 56.84,
-1
1
mp: 160-163 °C; IR (vmax cm ): 3448, 2952, 1706, 1651, 1442, 805; H 52.06, 51.35, 49.60, 45.93, 36.72, 35.75, 35.52, 35.45, 34.13, 26.88,
NMR (300MHz, DMSO-d
6
) δ: 8.33 (s, 3H), 7.94 (s, 1H), 5.25 (s, 1H), 21.20, 14.04, 3.12. HR-MS (ESI) m/z: calculated for C20
H
25
N
7
O
4
+
4
7
2
2
1
3
.99 (s, 2H), 4.65 (d, J = 18 Hz, 1H), 4.45 (d, J = 18 Hz, 1H), 4.32 (t, J = [M+H] : 428.2041, found: 428.2038.
.0 Hz, 2H), 3.43-3.33 (m, 2H), 3.13-2.95 (m, 2H), 2.92-2.83 (m, 1H),
ethyl (R,E)-4-(4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-
dioxo-3,6-di hydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-
yl)but-2-enoate hydrochloride (B09)
.21 (t, J = 7.1 Hz, 2H), 2.01-1.90 (m, 4H), 1.79 (s, 3H), 1.70-1.58 (m,
13
H); C NMR (75MHz, DMSO-d
6
): δ 173.53, 161.36, 158.96, 151.30,
42.72, 123.33, 88.37, 79.69, 74.54, 52.07, 51.36, 48.50, 45.94, 35.80,
5.43, 30.34, 26.89, 25.20, 21.22, 3.12; HR-MS (ESI) m/z: calculated Following a similar procedure for the preparation of B02. B09 was
+
for C20
H
27 7
N O
4
[M+H] : 430.2197, found: 430.2193.
prepared starting from ethyl ethyl 4-bromocrotonate. White solid
-
1
(
134 mg, yield 76%); mp: 177-180 °C; IR (vmax cm ): 3435, 2954, 1708,
2
3
-(4-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-
,6-dihydropyrimidin-1(2H)-yl)methyl)-1H-1,2,3-triazol-1-yl)
1
1
1
1
651, 1442, 805; H NMR (300MHz, DMSO-d
6
): δ 8.29 (s, 3H), 8.00 (s,
H), 6.96 (dt, J = 15.0, 6.0 Hz, 1H), 5.73 (d, J = 15.0 Hz, 1H), 5.26 (s,
H), 5.20 (d, J = 6.0 Hz, 2H), 5.01 (s, 2H), 4.64 (d, J = 15.0 Hz, 1H), 4.45
propanoic acid hydrochloride (B06)
1
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 13 of 14
Org aP nl ei ca s &e dB oi o nmo ot la ed cj uu sl at rm Ca hr ge imn si stry
Journal Name
ARTICLE
(
d, J = 15.0 Hz, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.36-3.25 (m, 2H), 3.12- solution, 2.5 g/kg). The blood glucose measured for the grouping of
View Article Online
DOI: 10.1039/C6OB01818A
2
1
1
8
2
.97 (m, 2H), 2.91-2.82 (m, 1H), 1.96-1.85 (m, 2H), 1.78 (s, 3H), 1.69- the animals was used as the data before administration of vehicle,
13
6
.61 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H); C NMR (75MHz, DMSO-d ): δ alogliptin or compounds (time -30). Blood samples were collected
64.83, 161.36, 158.98, 151.30, 143.02, 142.16, 123.90, 122.86, time 0, 15, 30, 45, 60 and 120 min. The blood glucose was measured
8.35, 79.69, 74.52, 60.22, 52.06, 51.37, 49.61, 45.93, 35.77, 35.47, by blood glucose test strips (SanNuo ChangSha, ChangSha, China).
6.90, 21.21, 14.00, 3.13; HR-MS (ESI) m/z: calculated for C22
M+H] :456.2354, found:456.2354.
H
N
29 7
O
4
In vivo oral glucose tolerance test (OGTT) in C57BL/6 mice
+
[
Male C57BL/6 mice left to acclimatize for 1 week were fed with high-
fat diet (MD 12032, rodent diet with 45 kcal% fat, from Mediscience
In vitro assay for inhibition of DPP-4, DPP-8 and DPP-9
The DPP-4 Drug Discovery Kit (Enzo Life Sciences International, Inc.) Ltd., Yangzhou, China) for 12 weeks to induce insulin resistance. The
was used for the assay of inhibition of DPP-4 activity. The assay is mice with fasting blood glucose level 10 mmol/L or higher were
based on the cleavage of 7-amino-4-methylcoumarin (AMC) moiety considered as type 2 diabetic model and selected for acute oral
from the C-terminus of the peptide substrate (H-Gly-Pro-AMC), glucose tolerance test. Type 2 diabetic C57BL/6 mice were fasted for
which increases its fluorescence intensity at 460 nm. The DPP-4 12 h, weighted, randomized into groups (n = 6). Mice were dosed
inhibitor P32/98 was selected as a control. The substrate and DPP-4 orally with single doses of vehicle (ultrapure water), test compounds
enzyme were diluted 1/50 with assay buffer (50mM Tris, pH=7.5). 25 (suspended in vehicle; 10 mL/kg; 1-10 mg/kg), 30 min prior to oral
μL of assay buffer, 15μL enzyme solution and 10 μL of appropriately glucose load (10% aqueous glucose solution, 1 g/kg). The blood
diluted solutions of the test compounds were added sequently to 96- glucose levels were measured by blood glucose test strips (SanNuo
o
well microtiter plates. After incubation at 37 C for 10 min, 50 μL of GA-3 type, ChangSha, China) before administration of vehicle,
diluted substrate solution was added. Fluorescence was measured alogliptin or test compounds (–30 min). After glucose oral
using an excitation wavelength of 380 nm and an emission adminstration, blood glucose were measured at 0, 15, 30, 60 and 120
wavelength of 460 nm by a Synergy H1 MultMode Reader (BioTek, min. The blood glucose was measured by blood glucose test strips
USA). The inhibitory rate relative to the control without inhibitor was (SanNuo ChangSha, ChangSha, China).
calculated and IC50 value was determined by nonlinear regression
Acknowledgements
fitted by GraphPad Prism 5. The assays for inhibition of DPP-8 and
DPP-9 activity were performed in similar procedure by using DPP4-
Glo™ Assay kit (Promega, Cat.No.G8531), DPP-8 and DPP-9 enzymes
This study was supported by the China National Key H-Tech
Innovation Project for the R&D of Novel Drugs (No.
(
BPS, Cat. NO. 80080 and 80080).
2
013ZX09301303-002), and supported by Natural Science
Foundation of Jiangsu Province (No. BK20141349).
Molecular modeling
Docking studies were carried out using Glide 5.9 in Schrödinger 2013
suite. The DPP-4 protein was extracted from RCSB Protein Data Bank Notes and references
(
PDB ID: 2RGU). Protein structures were prepared using Maestro
protein preparation wizard applying the default parameters. Ligands
were built using Maestro build panel and prepared by LigPrep
application using default parameters. A docking grid was constructed
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