Design and synthesis of 4-(2,4,5-Trifluorophenyl)butane-1,3-diamines as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1002/cmdc.201300104

The worldwide prevalence of diabetes has spurred numerous studies on the development of new antidiabetic medicines. As a result, dipeptidyl peptidaseIV (DPP4) has been recognized as a validated target. In our efforts to discover new DPP4 inhibitors, we analyzed the complexed structures of DPP4 available in Protein Data Bank and designed a series of triazole compounds. After enzyme activity assays and crystallographic verification of the binding interaction patterns, we found that the triazole compounds can inhibit DPP4 with micromolar IC₅₀ values. Liver microsome stability and cytochrome P450 metabolic tests were performed on this series, revealing undesirable pharmacokinetic profiles for the triazole compounds. To overcome this liability, we substituted the triazole ring with an amide or urea group to produce a new series of DPP4 inhibitors. Based on its enzyme activity, metabolic stability, and selectivity over DPP8 and DPP9, we selected compound 21r for further study of its invivo effects in mice using an oral glucose tolerance test (OGTT). The results show that 21r has efficacy similar to that of sitagliptin at a dose of 3mgkg^-1. The crystal structure of 21r bound to DPP4 also reveals that the trifluoromethyl group is directed toward a subpocket different from the subsite bound by sitagliptin, providing clues for the design of new DPP4 inhibitors.

Full text of DOI:10.1002/cmdc.201300104

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Blocking IV for type II: A series of tria-
zole compounds were designed and
synthesized based on analysis of DPP4
complex structures in the PDB. Enzyme
assays and in vitro DMPK tests showed
that these compounds are moderately
active against DPP4, yet they also have
undesirable metabolic properties related
to CYP2D6. We therefore substituted
the triazole group with an amide
L. Zhu, Y. Li, Q. Ling, M. Su, X. Wang,
C. Xia, Q. Yi, J. Li, J. Li,* B. Xiong,*
J. Shen*
&& – &&
Design and Synthesis of 4-(2,4,5-
Trifluorophenyl)butane-1,3-diamines
as Dipeptidyl Peptidase IV Inhibitors
moiety to obtain a new series of DPP4
inhibitors with excellent selectivity and
in vivo PK and PD efficacies similar to
those of sitagliptin.
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DOI: 10.1002/cmdc.201300104
Design and Synthesis of 4-(2,4,5-Trifluorophenyl)butane-
1,3-diamines as Dipeptidyl Peptidase IV Inhibitors
Linrong Zhu,[a] Yuanyuan Li,[b] Qiu Ling,[c] Mingbo Su,[b] Xin Wang,[a] Chunmei Xia,[b] Qu Yi,[c]
Jingya Li,[b] Jia Li,*[b] Bing Xiong,*[a] and Jingkang Shen*[a]
The worldwide prevalence of diabetes has spurred numerous
studies on the development of new antidiabetic medicines. As
a result, dipeptidyl peptidase IV (DPP4) has been recognized as
a validated target. In our efforts to discover new DPP4 inhibi-
tors, we analyzed the complexed structures of DPP4 available
in Protein Data Bank and designed a series of triazole com-
pounds. After enzyme activity assays and crystallographic veri-
fication of the binding interaction patterns, we found that the
triazole compounds can inhibit DPP4 with micromolar IC50
values. Liver microsome stability and cytochrome P450 meta-
bolic tests were performed on this series, revealing undesirable
pharmacokinetic profiles for the triazole compounds. To over-
come this liability, we substituted the triazole ring with an
amide or urea group to produce a new series of DPP4 inhibi-
tors. Based on its enzyme activity, metabolic stability, and se-
lectivity over DPP8 and DPP9, we selected compound 21r for
further study of its in vivo effects in mice using an oral glucose
tolerance test (OGTT). The results show that 21r has efficacy
similar to that of sitagliptin at a dose of 3 mgkgÀ1. The crystal
structure of 21r bound to DPP4 also reveals that the trifluoro-
methyl group is directed toward a subpocket different from
the subsite bound by sitagliptin, providing clues for the design
of new DPP4 inhibitors.
Introduction
According to estimates from the World Health Organization
(WHO), more than 346 million people worldwide currently
suffer from diabetes,[1] a progressive disease characterized by
high blood glucose levels resulting from insulin resistance or
impairment of insulin secretion. The search for novel mechanis-
tic approaches to control this chronic metabolic disease has
found that the infusion of an endogenous hormone known as
glucagon-like peptide-1 (GLP-1) can delay the development of
diabetes or can reverse recent-onset diabetes in non-obese di-
abetic (NOD) mice.[2] Secreted from the L cells of the small in-
testine, GLP-1 stimulates a range of activities, including glu-
cose-dependent stimulation of insulin secretion,[3] suppression
of postprandial glucagon secretion, retardation of gastric emp-
tying,[4] induction of satiety,[5] and stimulation of the formation
of new b cells in the long term.[6] However, the active form of
GLP-1, the GLP-1(7–37) peptide, is rapidly inactivated by dipep-
tidyl peptidase IV (DPP4), a serine protease, via cleavage of a di-
peptide from the N terminus[7] of GLP-1. Therefore, inhibition
of DPP4 enhances endogenous GLP-1 activity by decreasing
the rate of GLP-1 degradation, which represents a promising
approach to the treatment of type II diabetes.[8] Indeed, several
drugs such as sitagliptin,[9] vildagliptin,[10] saxagliptin,[11] aloglip-
tin,[12] and linagliptin[13] directed to this approach have been
approved in certain countries for the treatment of type II dia-
betes (Figure 1).
More than 80 crystal structures of DPP4 in complex with
bound inhibitors have been solved. These complex structures
have shown that a subpocket formed by the catalytic residue
Ser630 and nearby residues is usually occupied by a hydropho-
[a] L. Zhu,+ Prof. X. Wang, Prof. B. Xiong, Prof. J. Shen
State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203 (China)
E-mail: bxiong@simm.ac.cn
jkshen@simm.ac.cn
[b] Dr. Y. Li,+ Dr. M. Su, C. Xia, Prof. J. Li, Prof. J. Li
National Center for Drug Screening, State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
189 Guo Shou Jing Road, Shanghai 201203 (China)
E-mail: jli@mail.shcnc.ac.cn
[c] Dr. Q. Ling, Dr. Q. Yi
Department of Geriatrics, Central Hospital of Xuhui District
Shanghai, 966 Huaihai Middle Road, Shanghai 200031 (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300104.
Figure 1. Representative DPP4 inhibitors.
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bic ring group such as the tri-
fluorobenzyl group of sitagliptin
or the benzonitrile of alogliptin.
Invariably, there are two acidic
residues—Glu205 and Glu206—
in the binding site of DPP4 that
must be stabilized by an amine
group of an inhibitor. In addition
to these vital interactions, inhibi-
tors can use different functional
groups to further strengthen the
binding to DPP4.[14] With this
structural information we hy-
pothesized that the linker and
right terminal portions of sita-
gliptin could be modified to
generate a new series of DPP4
inhibitors.
Using the well-established
click chemistry method, we ini-
tially performed an isosteric re-
placement of the amide group
with a triazole ring to quickly ex-
plore the feasibility of modifying
Scheme 1. Synthesis of triazoles. Reagents and conditions: a) TMG, CH2Cl2, RT, overnight; b) [(R,R)-Et-DuPHOS-
(COD)Rh]+TfOÀ, H2 (5 bar), MeOH, 18 h; c) LiBH4, THF, RT, 2 h; d) MsCl, Et3N, CH2Cl2, RT, 30 min; e) NaN3, DMF,
678C, 18 h; f) for 15a–c and 16a–g: alkynes, CuI, sodium ascorbate, tBuOH, H2O, 808C, 3 h; g) for 15d–f and 16h:
alkynes, Cp*RuCl(PPh3)2, THF, reflux, 5 h; h) 4n HCl in dioxane, RT, 2–14 h.
the linker and terminal parts of sitagliptin.[15] The structure–ac-
tivity relationships (SARs) of this triazole series clearly indicate
a high tolerance for various functional groups at that range of
the binding site of DPP4. Unfortunately, in vitro pharmacoki-
netics tests revealed undesirable properties of triazole com-
pounds. To address this issue, based on the crystal structure
obtained for one of the triazole compounds with DPP4, we
substituted the triazole group with amide or urea groups to
generate a new series of DPP4 inhibitors bearing a 1,3-diamine
linker. After optimization of this series, compound 21r
emerged as showing good enzyme inhibitory activity and ex-
cellent selectivity for DPP4 over two dipeptidyl peptidase
family homologues: DPP8 and DPP9.[16] Inhibitor 21r was then
advanced to in vivo studies to assess the utility of the 1,3-dia-
mine series; it shows efficacy similar to that of sitagliptin in the
oral glucose tolerance test.
chemistry step, the use of copper(I) iodide as a catalyst result-
ed in the formation of 1,4-regioisomers 15a–c and 16a–g,
whereas the ruthenium catalyst Cp*RuCl(PPh3)2 resulted in 1,5-
regioisomers 15d–f and 16h.[18] Deprotection of 15a–f and
16a–h provided the desired triazoles 17a–f and 18a–h, re-
spectively.
As shown in Scheme 2, hydrogenation of azide 14 afforded
amine 19. Acylation of 19 with acid chlorides, acids, isocya-
nate, and 4-nitrophenyl carbamates gave the corresponding N-
substituted derivatives 20a–s. Treatment of 20s with 3-chloro-
perbenzoic acid (mCPBA) yielded 20t. Boc deprotection of
20a–t with hydrochloric acid provided the desired 1,3-diamine
derivatives 21a–t.
Results and Discussion
Sitagliptin was approved by the US Food and Drug Administra-
tion (FDA) in 2006 as the first drug to target DPP4; it has
a unique interaction pattern with its target. As illustrated in
Figure 2, the trifluorobenzyl group of sitagliptin is situated in
the subpocket of the binding site of DPP4, which is formed by
catalytic residues Ser630, His740, and four aromatic residues:
Trp659, Tyr631, Tyr662, and Tyr666. The vital amine group of
sitagliptin forms strong salt-bridge interactions with two acidic
residues, Glu205 and Glu206. The amide group undergoes
a water-bridged hydrogen bonding interaction with the side
chain of Tyr547, and the terminal fused heterocyclic ring not
only interacts with aromatic residue Phe357 through p–p
stacking, but also uses the more negatively charged trifluoro-
methyl group to interact with the positively charged Arg358
residue.[9]
Chemistry
The synthesis of triazoles is illustrated in Scheme 1. The Wittig–
Horner reaction between commercial glycine ester 6 and 2,4,5-
trifluorobenzaldehyde 7 in the presence of 1,1,3,3-tetramethyl-
guanidine (TMG) furnished the desired (Z)-dehydrophenylala-
nine 8 exclusively. Asymmetric hydrogenation of 8 using [(R,R)-
Et-DuPHOS(COD)Rh]+TfOÀ as catalyst provided the chiral a-(R)-
amino ester 9.[17] The b-(R)-amino ester 10 was synthesized
from 9 according to a procedure published by Merck research-
ers.[9] Reduction of amino esters 9 and 10 with lithium borohy-
dride afforded alcohols 11 and 12, respectively. These two al-
cohols were converted into the corresponding azides 13 and
14 by successive mesylation and azidation. Azides 13 and 14
were allowed to react with alkynes to form the corresponding
1,2,3-triazoles 15a–f and 16a–h, respectively. In this click
As revealed by these interactions between sitagliptin and
DPP4, and given the large unoccupied space around the right
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ture of DPP4 in complex with in-
hibitor 18d. As shown in
Figure 3, the trifluorobenzyl and
amine groups occupy the same
positions as sitagliptin in the
DPP4 binding site. The triazole
group is stacked with the side
chain
of
Phe357,
further
strengthening the binding inter-
actions. The notable difference is
that the attached 3-methylphen-
yl group is oriented in a different
direction, making the ligands
occupy a much larger subpocket
than the subsite that accommo-
dates the trifluoromethyl group
of sitagliptin. This is consistent
with the SAR of these triazole
Scheme 2. Synthesis of 25a–t. Reagents and conditions: a) Pd/C, H2, MeOH, RT, 5 h; b) acid chlorides, Et3N, CH2Cl2,
RT, 1 h; c) acids, EDCI, HOBt, DIPEA, CH2Cl2, RT, 14 h; d) 4-isocyanatobenzonitrile, CH2Cl2, RT, 18 h; e) 4-nitrophenyl
carbamates, K2CO3, DMF, 1008C, 14 h; f) mCPBA, CH2Cl2, RT, 16 h; g) 4n HCl in dioxane, RT, 2–14 h.
Table 1. DPP4 inhibitory activities of compounds 17a–18h.
Compd
n
R1
R2
IC50 [mm][a]
17a
17b
17c
17d
17e
17 f
18h
18a
18b
18c
18d
18e
18 f
18g
1
1
1
1
1
1
2
2
2
2
2
2
2
2
Ph
CH2OH
4-MeOPh
H
H
H
H
4-MeOPh
Ph
CH2OH
3-MePh
1-methyl-1H-imidazol-2-yl
cHex
H
H
H
Ph
CH2OH
4-MeOPh
4-MeOPh
H
H
H
H
H
H
H
20.1Æ1.4
17.9Æ0.8
12.0Æ1.8
32.3Æ1.5
16.6Æ1.6
56.8Æ4.8
0.34Æ0.07
0.19Æ0.02
0.30Æ0.02
0.57Æ0.04
0.3Æ0.04
0.16Æ0.01
0.48Æ0.03
0.15Æ0.02
Figure 2. The crystal structure of sitagliptin bound to DPP4 (PDB ID: 1X70).
The protein is depicted in surface and ribbon model, while sitagliptin is
shown in stick model. Water molecules are shown as red spheres.
terminal portion (the fused heterocyclic ring), we expected
that these parts could be modified to identify new patentable
DPP4 inhibitors. With this rationale, we used click chemistry to
rapidly explore the SAR of the linker and the terminal portion
of sitagliptin. The synthesis of triazole compounds is outlined
in Scheme 1, and the enzymatic activities of these target com-
pounds are listed in Table 1.
COOH
[a] The IC50 value of sitagliptin is 20 nm under these assay conditions.
In general, the triazole compounds demonstrated micromo-
lar or sub-micromolar activities toward DPP4. The longer mole-
cules 18a–h are more potent than compounds one carbon
unit shorter. As exemplified by analogues with the same 5-
phenyl triazole functional group, 18b is ~60-fold more active
than 17a. From the SAR of the triazole analogues with n=2,
this series was found to have a high degree of tolerance at the
right terminal, as various substituted triazoles showed similar
activities in the range of 0.15–0.6 mm. The negatively charged
inhibitor 18g and 4-methoxyphenyl-substituted compound
18a have nearly the same activities toward DPP4, further indi-
cating that the charge properties at that portion are not deter-
mining factors for potency.
Figure 3. Superposition of the crystal structure of 18d bound to DPP4 (PDB
ID: 4DSA) with the crystal structure of sitagliptin bound to DPP4 (PDB ID:
1X70). The protein is depicted in surface and ribbon model, while 18d and
sitagliptin are shown in stick model, colored grey and green, respectively.
To explore the interaction patterns of these triazole com-
pounds, X-ray crystallography was applied to solve the struc-
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compounds, that various substituents can be attached to the
triazole moiety.
To further assess the lead-like properties of these inhibitors,
in vitro pharmacokinetic profiles on 18a and 18d were per-
formed. Results of human and rat liver microsome stability
tests revealed that the compounds are relatively stable at
a concentration of 10 mm, particularly compound 18a (HLM
and RLM clearance rates are all <10 mLminÀ1 mgÀ1). Five repre-
sentative cytochrome P450 (CYP) enzymes were used to fur-
ther determine the metabolic stability of these compounds. As
listed in Table 3 below, although compounds 18a and 18d
have statistically negligible inhibition toward four CYPs at
10 mm, they show ~50% inhibition on an important subtype,
CYP2D6.
Figure 4. Superposition of the crystal structure of 21r bound to DPP4 (PDB
ID: 4J3J) with the crystal structure of sitagliptin bound to DPP4 (PDB ID:
1X70). The protein is depicted in surface and ribbon model, while 21r and
sitagliptin are shown in stick model, colored grey and green, respectively.
To discover inhibitors with better metabolic stability proper-
ties, we recovered the amide group to substitute the triazole
group, and synthesized 20 compounds as listed in Table 2.
From the DPP4 inhibitory activities of these compounds, it was
found that the amide inhibitors can maintain activities similar
to those of the triazole compounds, such as 21a (IC50 =
0.26 mm) versus 18b (IC50 =0.30 mm). Substitutions on the
phenyl group of 21a have minimal effect on potency (21b–f).
upon interacting with DPP4. In sitagliptin, the trifluoromethyl
group points toward the positively charged side chain of
Arg358, while in the 21r complex structure, the trifluorometh-
yl heterocycle rotates ~1808, forcing the trifluoro-
methyl group to interact with Tyr585 and Arg356.
This comes as a surprise, given that 21r and sitaglip-
tin differ only at the linker portion; however, this
binding mode is reasonable in that the trifluorometh-
yl group of sitagliptin is very close to the residues
lining the binding site, and would not have much
Table 2. DPP4 inhibitory activities of compounds 21a–t.
Compd
R
IC50 [mm]
Compd
R
IC50 [mm]
room for the extended ligand of 21r. This new bind-
ing mode may open a new direction for the design
of new compounds that interact with this under-de-
scribed subpocket.
21a
21b
21c
21d
21e
21 f
21g
Ph
2,4-di-FPh
4-MeOPh
4-MeSO2Ph
4-NH2SO2Ph
4-CF3Ph
0.26Æ0.03
0.35Æ0.04
0.11Æ0.02
0.11Æ0.01
0.18Æ0.01
0.21Æ0.02
21k
21l
thiophen-3-yl
quinolin-6-yl
CH2Ph
0.19Æ0.01
0.11Æ0.02
0.75Æ0.1
0.4Æ0.03
0.59Æ0.04
0.39Æ0.03
0.3Æ0.04
21m
21n
21o
21p
21q
CH2-4-FPh
CH2-4-MeOPh
CH2-4-CF3Ph
NH-4-CNPh
Regarding the triazoles, we also assessed the in vi-
tro metabolic stability of these amide compounds. As
listed in Table 3, it was found that 21g, 21i, and 21r
are stable in human and rat liver microsomes, with
clearance ratios <100 mLminÀ1 mgÀ1. Similar to the
triazole compounds, 21g and 21i show an inhibition
ratio of >80% for CYP2D6 at a concentration of
10 mm. Interestingly, compound 21r has very clean
profiles in these in vitro DMPK tests, and is a promis-
ing compound for further investigation.
4-(1H-1,2,4-triazol-1- 0.08Æ0.01
yl)Ph
1H-benzo[d]imidazol-
0.11Æ0.01
21h
21r
0.031Æ0.002
6-yl
1H-benzo[d]-
[1,2,3]triazol-6-yl
21i
21j
0.051Æ0.002 21s
0.22Æ0.01
0.11Æ0.01
Based on potency and structural diversity, we se-
lected six compounds from this series to test the se-
lectivity across enzymes DPP4, DPP8, and DPP9. Al-
though DPP8 and DPP9 are less studied than DPP4,
they are associated with several diseases such as cancer[19] and
asthma.[20] As reported in previous studies, selective inhibition
of DPP8/DPP9 in animals resulted in severe toxic reactions, in-
cluding alopecia, thrombocytopenia, anemia, enlarged spleen,
multiple histological pathologies, and increased mortality.[16]
Therefore it is important for DPP4 inhibitors to be selective
over these two homologous enzymes. From the selectivity pro-
files, most of the compounds have >100-fold selectivity for
DPP4 over DPP8 and DPP9. Compound 21i is >800-fold more
active toward DPP4 than DPP8, and shows ~320-fold selectivi-
pyridin-3-yl
0.15Æ0.02
21t
A benzyl group (in 21m) effects about twofold lower activity
than 21a, and various attached groups on the benzyl moiety
(21n–q) are also less potent than the phenyl group analogues
(21b–f). The compounds containing a fused heterocycle at the
right terminus have higher activities for DPP4, especially com-
pounds 21i and 21r. As depicted by the co-crystal structure of
21r bound to DPP4 (Figure 4), the trifluorobenzyl and amine
groups of 21r situate at similar positions to those occupied by
sitagliptin, whereas the trifluoromethyl-containing heterocycle
adopts different conformations between the two compounds
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a positive control. Single-dose administration (1, 3, 10 mgkgÀ1
)
Table 3. In vitro pharmacokinetic profiles of selected compounds.
of compound 21r to mice 30 min before an oral glucose chal-
lenge produced a significant decrease in glucose excursion, as
shown by the decrease in area under the curve (AUC)0–120 (Fig-
ure 5b). Compound 21r (3 and 10 mgkgÀ1) decreased blood
glucose levels significantly 15, 30, 60, and 90 min after glucose
challenge (Figure 5a). Collectively, although the lowest dose
group (AUC0–120, 1454.00Æ59.47 versus control) were un-
Compd
CLint [mLminÀ1 (mgprotein)À1
]
Direct CYP inhibition [%][c]
3A4 2D6 2C9 1A2 2C19
HLM[a]
RLM[b]
18a
18d
21g
21i
3
47
95
9
1
71
4
59
4
19
24
11
NI
7
49
85
95
82
18
22
10
14
17
4
17
13
36
10
7
36
26
18
24
15
21r
15
changed, the efficacy of 3 or 10 mgkgÀ1 of 21r (AUC0–120
:
[a] Human liver microsomes. [b] Rat liver microsomes. [c] Determined at
a compound concentration of 10 mm; NI: no inhibition.
1094.63Æ44.38, p<0.01; 1019.63Æ99.09, p<0.01) is similar to
that of sitagliptin at 3 mgkgÀ1 (AUC0–120: 1063.25Æ76.21, p<
0.01).
ty over DPP9. Compound 21r demonstrated excellent selectivi-
ty of >1000-fold toward DPP4, which is similar to the selectivi-
ty profile of sitagliptin, making 21r suitable for further assess-
ment in animal models (Table 4).
Conclusions
In summary, based on the analysis of a large volume of crystal
structure data available in the PDB, we designed and elaborat-
ed a series of 4-(2,4,5-trifluorophenyl)butane-1,3-diamines as
DPP4 inhibitors. Click chemistry was used to quickly explore
the SAR of the linker and the right terminal part of sitagliptin,
Table 4. Inhibitory activities of selected compounds against DPP4 and
other homologous enzymes DPP8 and DPP9.
Compd
IC50 [mm]
DPP4
DPP8
DPP9
21c
21h
21i
21j
21o
21r
0.11Æ0.02
0.11Æ0.01
0.051Æ0.002
0.15Æ0.02
0.59Æ0.04
0.031Æ0.002
12.47Æ2.01
6.52Æ1.10
45.87Æ5.93
48.07Æ4.51
36.96Æ5.85
78.57Æ9.38
18.95Æ2.64
11.33Æ1.45
16.34Æ1.77
38.45Æ5.9
11.57Æ2.22
41.6Æ3.34
On the basis of the overall properties of in vitro potency,
pharmacokinetics, and selectivity, compound 21r was selected
for further evaluation of in vivo pharmacokinetics. As shown in
Table 5, compound 21r and sitagliptin have overall similar
Table 5. In vivo pharmacokinetics study of 21r and the control com-
pound sitagliptin.
Compd
tmax [h]
Cmax [ngmLÀ1
]
MRT [h]
t1/2 [h]
sitagliptin
0.500
2.00
1314
719
2.16
2.72
1.20
1.03
21r
pharmacokinetics profiles in the ICR mice model. Compound
21r has a slightly shorter t1/2 than sitagliptin, yet a longer
mean residence time (MRT) than sitagliptin. The most notable
difference is the parameter tmax, which shows that compound
21r is about fourfold slower than sitagliptin in reaching maxi-
mum concentration in serum. Notably, the dose of sitagliptin is
once per day, which may indicate that sitagliptin has very dif-
ferent profiles in various species. Therefore, in future develop-
ment efforts, other in vivo models should be used to further
assess the pharmacokinetics of 21r.
Figure 5. Effect of acute administration of 21r on glucose levels in normal
mice: a) 21r, sitagliptin, or vehicle were administered orally to 5-h-fasted
normal ICR mice 30 min prior to oral glucose load (3 gkgÀ1); n=6 in each
case. b) AUC: data are the mean ÆSEM; *p<0.05, **p<0.01 vs. control.
We also evaluated the pharmacodynamics of 21r by OGTT
experiments in lean (ICR) mice, and sitagliptin was used as
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3.55 (m, 1H), 2.90–2.75 (m, 2H), 2.20 (brs, 1H), 1.39 ppm (s, 9H);
and two rounds of medicinal chemistry optimization were car-
ried out by integrating the DPP4–inhibitor interaction informa-
tion obtained from co-crystal structures of 18d and 21r. Final-
ly, several compounds were discovered to have inhibitory ac-
tivity similar to that of sitagliptin. In vitro metabolic stability
profiling and selectivity tests were performed on some potent
inhibitors, and 21r was identified as a promising lead for
in vivo study. From the OGTT experiments, it was found that
21r has similar efficacy to that of sitagliptin at a dose of
3 mgkgÀ1, making it suitable for further drug development.
+
MS: m/z 306 [M+H]
.
(R)-tert-Butyl (4-hydroxy-1-(2,4,5-trifluorophenyl)butan-2-yl)car-
bamate (12): Compound 12 was prepared from (R)-methyl 3-((tert-
butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoate 10 fol-
lowing the procedure of 11 in 87% yield (white solid, mp: 89–
918C). 1H NMR (300 MHz, CDCl3): d=7.10–7.00 (m, 1H), 6.96–6.86
(m, 1H), 4.57 (d, J=8.8 Hz, 1H), 4.10–3.95 (m, 1H), 2.71–2.65 (m,
2H), 2.85–2.70 (m, 2H), 1.93–1.80 (m, 1H), 1.50–1.41 (m, 1H),
1.40 ppm (s, 9H); MS: m/z 320 [M+H]+.
(R)-tert-Butyl (1-azido-3-(2,4,5-trifluorophenyl)propan-2-yl)carba-
mate (13): Methanesulfonyl chloride (376 mg, 3.3 mmol) was
added dropwise to a solution of 11 (915 mg, 3 mmol) and Et3N
(365 mg, 3.6 mmol) in dry CH2Cl2 (20 mL) at 08C. The reaction mix-
ture was allowed to reach room temperature and stirred for
30 min before it was quenched with 1n HCl (5 mL) and extracted
with CH2Cl2 (2ꢁ20 mL). The combined organic layers were washed
with sat. NaHCO3 (10 mL) and brine (10 mL), dried over Na2SO4,
and concentrated to give 1.07 g of mesylate as a light-brown solid,
which was used without further purification. A mixture of the me-
sylate (1 g, 2.61 mmol) and NaN3 (650 mg, 10 mmol) in dry DMF
(10 mL) was heated at 678C for 18 h. The reaction mixture was
poured into Et2O (60 mL), washed with H2O (2ꢁ20 mL) and brine
(20 mL), dried over Na2SO4, and concentrated. The residue was pu-
rified by column chromatography (PE/EtOAc 6:1) to yield the title
compound 13 (764 mg, 82% over two steps) as a white solid (mp:
95–978C). 1H NMR (300 MHz, CDCl3): d=7.08–7.99 (m, 1H), 6.96–
6.87 (m, 1H), 4.66 (d, J=6.7 Hz, 1H), 3.98–3.90 (m, 1H), 3.51–3.37
Experimental Section
General synthesis procedures
THF was dried over Na0. DMF and CH2Cl2 were dried over CaH2.
Other commercial reagents and solvents were used without further
purification. 1H NMR spectra were recorded on a Varian Mercury
300 MHz spectrometer or a Bruker DPX 400 MHz spectrometer.
Chemical shifts (d) are reported in ppm relative to the solvent
peak of TMS. Low-resolution MS data were determined on
a Thermo Finnigan LCQDECAxP LC–MS instrument. HRMS-ESI data
were recorded on a Waters Q-TOF Ultima Global instrument. Melt-
ing points (SGW X-4, Shanghai Precision & Scientific Instrument
Co., Ltd.) are uncorrected. Column chromatography was performed
with silica gel (200–300 mesh, Qingdao Haiyang Chem, China).
(Z)-Methyl 2-((tert-butoxycarbonyl)amino)-3-(2,4,5-trifluorophe-
nyl)acrylate (8): Methyl 2-((tert-butoxycarbonyl)amino)-2-(dime-
thoxyphosphoryl)acetate 6 (3.27 g, 11 mmol) was added portion-
wise to a solution of 2,4,5-trifluorobenzaldehyde 7 (1.6 g, 10 mmol)
and 1,1,3,3-tetramethylguanidine (1.38 g, 12 mmol) in CH2Cl2
(30 mL) at room temperature. The resulting mixture was stirred at
room temperature overnight. Concentration followed by column
chromatography (petroleum ether (PE)/EtOAc 1:6) afforded com-
pound 8 (3.04 g, 92%) as a white solid (mp: 87–898C). 1H NMR
(300 MHz, CDCl3): d=7.48–7.37 (m, 1H), 6.98–6.87 (m, 1H), 6.52 (s,
1H), 3.87 (s, 3H), 1.41 ppm (s, 9H); MS: m/z 332 [M+H]+.
(m, 2H), 2.89–3.73 (m, 2H), 1.39 ppm (s, 9H); MS: m/z 230 [M+H
+
-Boc]
.
(R)-tert-Butyl (4-azido-1-(2,4,5-trifluorophenyl)butan-2-yl)carba-
mate (14): Compound 14 was prepared from 12, following the
procedure for 13, in 64% yield as a white solid (mp: 96–988C).
1H NMR (300 MHz, CDCl3): d=7.10–6.69 (m, 1H), 6.97–6.86 (m, 1H),
4.43 (d, J=8.7 Hz, 1H), 3.96–3.82 (m, 1H), 3.51–3.34 (m, 2H), 2.88–
2.68 (m, 2H), 1.88–1.75 (m, 1H), 1.73–1.59 (m, 1H), 1.39 ppm (s,
9H); MS: m/z 345 [M+H]+.
(R)-Methyl 2-((tert-butoxycarbonyl)amino)-3-(2,4,5-trifluorophe-
nyl)propanoate (9): Compound 8 (3 g, 9.06 mmol) was dissolved
in dry MeOH (30 mL, 0.3m), and catalyst [(R,R)-Et-DuPHOS-
(COD)Rh]+TfOÀ (65 mg, 0.009 mmol, 0.1 mol%) was added. The
mixture was deoxygenated with argon, and hydrogenation was
performed at 5 bar for 18 h. The solvent was evaporated, and the
residue was purified through a short pad of silica gel (PE/EtOAc
4:1) to give 2.87 g crude product (94.5% ee), which was further pu-
rified by recrystallization in PE to give the title compound 9
(2.56 g, 98.5% ee, 85%) as a white solid (mp: 58–608C). 1H NMR
(300 MHz, CDCl3): d=6.84–7.03 (m, 2H), 5.06 (d, J=7.8 Hz, 1H),
4.49–4.59 (m, 1H), 3.74 (s, 3H), 3.17 (dd, J=5.7, 13.7 Hz, 1H), 2.97
(dd, J=6.4, 13.7 Hz, 1H), 1.41 ppm (s, 9H); MS: m/z 334 [M+H]+.
General procedure for 15a–c, 16a–g
A mixture of azide (0.2 mmol), alkyne (0.24 mmol), sodium ascor-
bate (0.12 mmol), and CuI (0.05 mmol) in tBuOH/H2O (1:1, 2 mL)
was heated at 808C under N2 for 3 h. The reaction mixture was di-
luted with H2O (5 mL) and extracted with EtOAc (3ꢁ10 mL). The
combined organic layers were dried over Na2SO4 and concentrated.
The residue was purified by column chromatography to yield the
desired 1,4-disubstitued triazole.
(R)-tert-Butyl (1-(4-phenyl-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluoro-
phenyl)propan-2-yl)carbamate (15a): Compound 15a was pre-
pared from 13 and ethynylbenzene (75 mg, 87%, white solid, mp:
(R)-tert-Butyl (1-hydroxy-3-(2,4,5-trifluorophenyl)propan-2-yl)car-
bamate (11): LiBH4 (158 mg, 7.2 mmol) was added portionwise to
a solution of 9 (1.2 g, 3.6 mmol) in dry THF (25 mL) at 08C. The re-
action mixture was allowed to reach room temperature and stirred
for 2 h before it was quenched with 1n HCl and extracted with
EtOAc (2ꢁ25 mL). The combined organic layers were washed with
brine (20 mL), dried over Na2SO4, and concentrated. The residue
was purified by column chromatography (PE/EtOAc 2:1) to yield
the title compound 11 (990 mg, 90%) as a white solid (mp: 121–
1
206–2098C, dec.). H NMR (400 MHz, CDCl3): d=7.84–7.81 (m, 2H),
7.79 (s, 1H), 7.46–7.42 (m, 2H), 7.37–7.33 (m, 1H), 7.15–7.08 (m,
1H), 6.97–6.90 (m, 1H), 4.86 (brd, 1H), 4.62–4.54 (m, 2H), 4.26–4.18
(m, 1H), 2.94–2.78 (m, 2H), 1.37 ppm (s, 9H); MS: m/z 433 [M+H]+
.
(R)-tert-Butyl
(1-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-3-
(2,4,5-trifluorophenyl)propan-2-yl)carbamate (15b): Compound
15b was prepared from 13 and propargyl alcohol (61 mg, 79%,
white solid). 1H NMR (400 MHz, CDCl3): d=7.54 (s, 1H), 7.16–7.08
(m, 1H), 6.96–6.89 (m, 1H), 5.09 (d, J=6.4 Hz, 1H), 4.78 (s, 2H),
1
1238C). H NMR (300 MHz, CDCl3): d=7.11–7.03 (m, 1H), 6.95–6.86
(m, 1H), 4.80 (br, 1H), 3.89–3.78 (m, 1H), 3.73–3.66 (m, 1H), 3.62–
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4.57–4.47 (m, 2H), 4.22–4.14 (m, 1H), 2.90–2.80 (m, 2H), 1.38 ppm
(s, 9H); MS: m/z 387 [M+H]+.
was prepared from 14 and propiolic acid (63 mg, 76%, white solid,
mp: 167–1698C, dec.). H NMR (300 MHz, [D6]acetone): d=8.48 (s,
1
1H), 7.33–7.11 (m, 2H), 6.18 (d, J=9.3 Hz, 1H), 4.66–4.48 (m, 2H),
3.95–3.80 (m, 1H), 2.94 (dd, J=5.0, 13.7 Hz, 1H), 2.77 (dd, J=9.3,
13.1 Hz, 1H), 2.35–2.10 (m, 2H), 1.31 ppm (s, 9H); MS: m/z 415
[M+H]+.
(R)-tert-Butyl
(1-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-3-
(2,4,5-trifluorophenyl)propan-2-yl)carbamate (15c): Compound
15c was prepared from 13 and 1-ethynyl-4-methoxybenzene
(77 mg, 83%, white solid, mp: 200–2048C, dec.). H NMR (400 MHz,
1
CDCl3): d=7.77–7.72 (m, 2H), 7.69 (s, 1H), 7.14–7.07 (m, 1H), 6.98–
6.89 (m, 3H), 4.85 (brd, 1H), 4.60–4.53 (m, 2H), 4.25–4.16 (m, 1H),
General procedure for 15d–f, 16h
3.84 (s, 3H), 2.92–2.77 (m, 2H), 1.36 ppm (s, 9H); MS: m/z 463 [M+
+
A mixture of azide (0.1 mmol), alkyne (0.11 mmol), and Cp*RuCl-
(PPh3)2 (0.01 mmol) in THF (5 mL) was heated at reflux under N2 for
5 h. The solvent was evaporated, and the residue was purified by
column chromatography to obtain the desired 1,5-disubstituted tri-
azole.
H]
.
(R)-tert-Butyl
(4-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1-
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (16a): Compound
16a was prepared from 14 and 1-ethynyl-4-methoxybenzene
1
(66 mg, 69%, white solid, mp: 185–1898C, dec.). H NMR (300 MHz,
(R)-tert-Butyl (1-(5-phenyl-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluoro-
phenyl)propan-2-yl)carbamate (15d): Compound 15d was pre-
pared from 13 and ethynylbenzene (36 mg, 84%, white solid, mp:
CDCl3): d=7.70–7.78 (m, 2H), 6.83–7.04 (m, 4H), 3.34–3.60 (m, 3H),
3.84–3.92 (m, 1H), 3.83 (s, 3H), 2.67–2.85 (m, 2H), 1.98–2.28 (m,
2H), 1.36 ppm (s, 9H); MS: m/z 477 [M+H]+.
1
98–998C). H NMR (300 MHz, CDCl3): d=7.70 (s, 1H), 7.49–7.43 (m,
(R)-tert-Butyl (4-(4-phenyl-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluoro-
phenyl)butan-2-yl)carbamate (16b): Compound 16b was pre-
pared from 14 and ethynylbenzene (67 mg, 75%, white solid, mp:
3H), 7.36–7.30 (m, 2H), 6.95–6.78 (m, 2H), 5.13 (d, J=8.2 Hz, 1H),
4.50–4.34 (m, 2H), 4.25–4.10 (m, 1H), 2.72 (d, J=7.2 Hz, 2H),
1.32 ppm (s, 9H); MS: m/z 433 [M+H]+.
1
183–1878C, dec.). H NMR (300 MHz, CDCl3): d=7.78–7.88 (m, 3H),
(R)-tert-Butyl
(1-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-3-
7.29–7.46 (m, 3H), 6.94–7.04 (m, 1H), 6.83–6.93 (m, 1H), 4.36–4.60
(m, 3H), 2.79–2.93 (m, 1H), 2.67–2.86 (m, 2H), 2.15–2.29 (m, 1H),
1.95–2.14 (m, 1H), 1.36 ppm (s, 9H); MS: m/z 447 [M+H]+.
(2,4,5-trifluorophenyl)propan-2-yl)carbamate (15e): Compound
15e was prepared from 13 and propargyl alcohol (28 mg, 73%,
1
white solid, mp: 127–1298C). H NMR (300 MHz, CDCl3): d=7.59 (s,
1H), 7.15–7.05 (m, 1H), 6.98–6.88 (m, 1H), 5.08 (d, J=8.2 Hz, 1H),
4.83–4.68 (m, 2H), 4.58–4.42 (m, 2H), 4.40–4.29 (m, 1H), 4.08 (brs,
1H), 2.97 (dd, J=4.7, 14.2 Hz, 1H), 2.82 (dd, J=8.5, 14.2 Hz, 1H),
1.31 ppm (s, 9H); MS: m/z 387 [M+H]+.
(R)-tert-Butyl
(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-1-
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (16c): Compound
16c was prepared from 14 and propargyl alcohol (50 mg, 63%,
white solid, mp: 98–1008C). H NMR (300 MHz, CDCl3): d=7.64 (s,
1
1H), 7.05–6.95 (m, 1H), 6.95–6.85 (m, 1H), 4.79 (s, 2H), 4.57–4.45
(m, 1H), 4.44–4.28 (m, 1H), 3.88–3.71 (m, 1H), 2.85–2.66 (m, 2H),
2.24–2.11 (m, 1H), 2.08–1.94 (m, 1H), 1.39 ppm (s, 9H); MS: m/z
401 [M+H]+.
(R)-tert-Butyl
(1-(5-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-3-
(2,4,5-trifluorophenyl)propan-2-yl)carbamate (15 f): Compound
15 f was prepared from 13 and 1-ethynyl-4-methoxybenzene
(40 mg, 87%, white solid, mp: 71–738C). H NMR (300 MHz, CDCl3):
1
d=7.65 (s, 1H), 7.28–7.21 (m, 2H), 7.00–6.78 (m, 4H), 5.14 (d, J=
8.0 Hz,1H), 4.43–4.34 (m, 2H), 4.24–4.11 (m, 1H), 3.86 (s, 3H), 2.72
(d, J=7.1 Hz, 2H), 1.33 ppm (s, 9H); MS: m/z 463 [M+H]+.
(R)-tert-Butyl (4-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluor-
ophenyl)butan-2-yl)carbamate (16d): Compound 16d was pre-
pared from 14 and 1-ethynyl-3-methylbenzene (58 mg, 63%, white
1
solid, mp: 165–1688C, dec.). H NMR (300 MHz, CDCl3): d=7.82 (s,
(R)-tert-Butyl
(4-(5-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1-
1H), 7.67 (s, 1H), 7.58 (d, J=7.8 Hz, 1H), 7.31 (t, J=7.5 Hz, 1H),
7.15 (d, J=7.1 Hz, 1H), 7.03–6.93 (m, 1H), 6.93–6.83 (m, 1H), 4.60–
4.37 (m, 3H), 3.92–3.77 (m, 1H), 2.86–2.68 (m, 2H), 2.39 (s, 3H),
2.26–2.14 (m, 1H), 2.14–2.00 (m, 1H), 1.36 ppm (s, 9H); MS: m/z
461 [M+H]+.
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (16h): Compound
16h was prepared from 14 and 1-ethynyl-4-methoxybenzene
(28 mg, 59%, white solid). 1H NMR (300 MHz, CDCl3): d=7.63 (s,
1H), 7.25 (d, J=8.5 Hz, 2H), 7.01–6.81 (m, 4H), 4.48–4.30 (m,3H),
3.86 (s, 3H), 3.75–3.63 (m, 1H), 2.79–2.64 (m, 2H), 2.15–1.90 (m,
2H), 1.34 ppm (m, 9H); MS: m/z 477 [M+H]+.
(R)-tert-Butyl (4-(4-(1-methyl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-
yl)-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (16e): Com-
pound 16e was prepared from 14 and 2-ethynyl-1-methyl-1H-imi-
dazole (58 mg, 64%, white solid). 1H NMR (400 MHz, [D6]DMSO):
d=8.38 (s, 1H), 7.85 (s, 1H), 7.46–7.39 (m, 1H), 7.31–7.25 (m, 1H),
7.22 (s, 1H), 6.87 (d, J=9.1 Hz, 1H), 4.45–4.38 (m, 2H), 3.81 (s, 3H),
3.67–3.58 (m, 1H), 2.82 (dd, J=4.6, 13.8 Hz, 1H), 2.57 (dd, J=9.4,
13.8 Hz, 1H), 2.14–2.06 (m, 1H), 2.01–1.91 (m, 1H), 1.26 ppm (s,
9H); MS: m/z 451 [M+H]+.
General procedure for 17a–f, 18a–h
HCl in dioxane (4n, 20 equiv) was added to a solution of 15/16 in
dioxane (0.2m). After being stirred at room temperature for 2–
14 h, the solution was concentrated. The residue was treated with
Et2O, filtered, and dried in vacuo to give 17/18.
(R)-1-(4-Phenyl-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluorophenyl)pro-
pan-2-amine·HCl (17a): 1H NMR (400 MHz, CD3OD): d=8.38 (s,
1H), 7.86–7.83 (m, 2H), 7.48–7.45 (m, 2H), 7.41–7.35 (m, 2H), 7.24–
7.18 (m, 1H), 4.72–4.57 (m, 2H), 3.97–3.91 (m, 1H), 3.06–2.91 ppm
(m, 2H); MS: m/z 333 [M+H]+; HRMS: m/z [M+H]+ calcd for
C17H15F3N4: 333.1327, found: 333.1324.
(R)-tert-Butyl
(4-(4-cyclohexyl-1H-1,2,3-triazol-1-yl)-1-(2,4,5-tri-
fluorophenyl)butan-2-yl)carbamate (16 f): Compound 16 f was
prepared from 14 and ethynylcyclohexane (69 mg, 76%, white
solid, mp: 159–1618C, dec.). H NMR (300 MHz, CDCl3): d=7.30 (s,
1H), 7.04–6.85 (m, 2H), 4.59–4.28 (m, 3H), 3.88–3.74 (m, 1H), 2.85–
2.66 (m, 3H), 2.23–1.95 (m, 4H), 1.86–1.68 (m, 4H), 1.49–1.15 ppm
(m, 13H); MS: m/z 453 [M+H]+.
1
(R)-(1-(2-Amino-3-(2,4,5-trifluorophenyl)propyl)-1H-1,2,3-triazol-
4-yl)methanol·HCl (17b): 1H NMR (400 MHz, CD3OD): d=8.15 (s,
1H), 7.44–7.38 (m, 1H), 7.28–7.21 (m, 1H), 4.83–4.74 (m, 4H), 4.19–
4.12 (m, 1H), 3.13–3.03 ppm (m, 2H); MS: m/z 287 [M+H]+.
(R)-1-(3-((tert-Butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)bu-
tyl)-1H-1,2,3-triazole-4-carboxylic acid (16g): Compound 16g
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(R)-1-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluor-
ophenyl)propan-2-amine·HCl (17c): 1H NMR (400 MHz, CD3OD):
d=8.55 (s, 1H), 7.75 (d, J=8.6 Hz, 2H), 7.46–7.40 (m, 1H), 7.25–
7.18 (m, 1H), 7.03 (d, J=8.6 Hz, 2H), 4.88–4.78 (m, 2H), 4.26–4.19
(m, 1H), 3.83 (s, 3H), 3.18–3.08 ppm (m, 2H); MS: m/z 363 [M+H]+
; HRMS: m/z [M+H]+ calcd for C18H17F3N4O: 363.1433, found:
363.1427.
14.5 Hz, 1H), 2.96–2.88 (m, 1H), 2.46–2.32 (m, 2H), 2.11–2.02 (m,
2H), 1.89–1.83 (m, 2H), 1.79–1.75 (m, 1H), 1.56–1.41 (m, 4H), 1.38–
1.28 ppm (m, 1H); MS: m/z 353 [M+H]+; HRMS: m/z [M+H]+
calcd for C18H23F3N4: 353.1953, found: 353.1959.
(R)-1-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-1H-1,2,3-triazole-4-
1
carboxylic acid·HCl (18g): H NMR (400 MHz, CD3OD): d=8.47 (s,
1H), 7.35–7.29 (m, 1H), 7.23–7.17 (m, 1H), 4.70–4.64 (m, 2H), 3.57–
3.49 (m, 1H), 3.10 (dd, J=5.3, 13.8 Hz, 1H), 3.01 (dd, J=6.9,
13.8 Hz, 1H), 2.34–2.27 ppm (m, 2H); MS: m/z 315 [M+H]+; HRMS:
m/z [M+H]+ calcd for C13H13F3N4O2: 315.1069, found: 315.1068.
(R)-1-(5-Phenyl-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluorophenyl)pro-
pan-2-amine·HCl (17d): 1H NMR (400 MHz, CD3OD): d=7.94 (s,
1H), 7.54–7.45 (m, 3H), 7.40–7.38 (m, 2H), 7.18–7.11 (m, 2H), 4.72–
4.60 (m, 2H), 3.96–3.91 (m, 1H), 3.02 (dd, J=5.6, 14.2 Hz, 1H),
2.89 ppm (dd, J=9.2, 14.2 Hz, 1H); MS: m/z 333 [M+H]+.
(R)-4-(5-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluor-
1
ophenyl)butan-2-amine·HCl (18h): H NMR (400 MHz, CD3OD): d=
7.97 (s, 1H), 7.40 (d, J=8.7 Hz, 2H), 7.26–7.14 (m, 2H), 7.07 (d, J=
8.7 Hz, 2H), 4.69–4.62 (m, 2H), 3.87 (s, 3H), 3.50–3.47 (m, 1H), 2.95
(d, J=7.0 Hz, 2H), 2.22–2.10 ppm (m, 2H); MS: m/z 377 [M+H]+.
(R)-(1-(2-Amino-3-(2,4,5-trifluorophenyl)propyl)-1H-1,2,3-triazol-
5-yl)methanol·HCl (17e): 1H NMR (400 MHz, CD3OD): d=7.98 (s,
1H), 7.47–7.44 (m, 1H), 7.30–7.23 (m, 1H), 4.82–4.72 (m, 4H), 4.28–
4.21 (m, 1H), 3.17 ppm (d, J=7.2 Hz, 2H); MS: m/z 287 [M+H]+.
(R)-tert-Butyl (4-amino-1-(2,4,5-trifluorophenyl)butan-2-yl)carba-
mate (19): To a solution of 14 (3.5 g, 10.2 mmol) in MeOH (50 mL)
was added 10% Pd/C (150 mg, 0.14 mmol) under N2. The suspen-
sion was then stirred under H2 atmosphere (1 atm) at room tem-
perature for 5 h, and the reaction mixture was filtered through
a pad of Celite and concentrated to furnish the title compound 19
(R)-1-(5-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-3-(2,4,5-trifluor-
ophenyl)propan-2-amine·HCl (17 f): 1H NMR (400 MHz, CD3OD):
d=7.96 (s, 1H), 7.34–7.30 (m, 2H), 7.20–7.12 (m, 2H), 7.03–6.99 (m,
2H), 4.71 (dd, J=8.1, 15.2 Hz, 1H), 4.62 (dd, J=3.6, 15.2 Hz, 1H),
3.93–3.87 (m, 1H+s, 3H), 3.04 (dd, J=5.5, 14.4 Hz, 1H), 2.90 ppm
(dd, J=9.6, 14.4 Hz, 1H); MS: m/z 363 [M+H]+; HRMS: m/z [M+
H]+ calcd for C18H17F3N4O: 363.1433, found: 363.1430.
1
(3.04 g, 94%) as a grey solid. H NMR (300 MHz, CDCl3): d=7.01–
6.97 (m, 1H), 6.93–6.83 (m, 1H), 4.79 (d, J=8.6 Hz, 1H), 2.97–2.83
(m, 1H), 2.85–2.64 (m, 4H), 1.71–1.58 (m, 1H), 1.52–1.39 (m, 1H),
1.37 ppm (s, 9H); MS: m/z 319 [M+H]+.
(R)-4-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluor-
1
ophenyl)butan-2-amine·HCl (18a): H NMR (400 MHz, CD3OD): d=
8.39 (s, 1H), 7.72 (d, J=8.6 Hz, 2H), 7.36–7.31 (m, 1H), 7.21–7.14
(m, 1H), 7.02 (d, J=8.6 Hz, 2H), 4.70 (t, J=6.5 Hz, 2H), 3.84 (s, 3H),
3.59–3.54 (m, 1H), 3.13–3.00 (m, 2H), 2.35–2.33 ppm (m, 2H); MS:
m/z 377 [M+H]+; HRMS: m/z [M+H]+ calcd for C19H19F3N4O:
377.1589, found: 377.1584.
General procedure of 20a,b, 20m,n
Et3N (1.2 equiv) and acid chloride (1 equiv) were added successive-
ly to a solution of 19 (32 mg, 0.1 mmol, 1 equiv) in dry CH2Cl2
(0.1m) at 08C. The resulting mixture was allowed to reach room
temperature and stirred for 1 h before it was quenched with 1n
HCl and extracted with CH2Cl2. The organic layer was washed with
sat. NaHCO3 and brine, dried over Na2SO4, and concentrated. The
residue was purified by column chromatography to give the de-
sired compound.
(R)-4-(4-Phenyl-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluorophenyl)bu-
1
tan-2-amine·HCl (18b): H NMR (400 MHz, CD3OD): d=8.36 (s, 1H),
7.85–7.82 (m, 2H), 7.49–7.45 (m, 2H), 7.41–7.32 (m, 2H), 7.23–7.17
(m, 1H), 4.72 (t, J=6.9 Hz, 2H), 3.59–3.52 (m, 1H), 3.14 (dd, J=6.6,
14.4 Hz, 1H), 3.04 (dd, J=7.2, 14.4 Hz, 1H), 2.42–2.29 ppm (m, 2H);
MS: m/z 347 [M+H]+.
(R)-(1-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-1H-1,2,3-triazol-4-
yl)methanol·HCl (18c): H NMR (400 MHz, CD3OD): d=8.47 (s, 1H),
7.39–7.33 (m, 1H), 7.25–7.19 (m, 1H), 4.87 (s, 2H), 4.75 (t, J=6.9 Hz,
2H), 3.59–3.54 (m, 1H), 3.13–3.00 (m, 2H), 2.39–2.30 ppm (m, 2H);
MS: m/z 301 [M+H]+.
(R)-tert-Butyl (4-benzamido-1-(2,4,5-trifluorophenyl)butan-2-yl)-
carbamate (20a): Benzoyl chloride as acid chloride, compound
20a was obtained as a white solid (38 mg, 89%, mp: 157–1608C,
dec.). 1H NMR (300 MHz, CDCl3): d=7.85 (d, J=6.7 Hz, 2H), 7.56–
7.42 (m, 3H), 7.08–6.86 (m, 2H), 4.55 (d, J=9.2 Hz, 1H), 4.05–3.89
(m, 2H), 3.16–3.04 (m, 1H), 2.85–2.68 (m, 2H), 2.00–1.83 (m, 1H),
1.62–1.50 (m, 1H), 1.41 ppm (s, 9H); MS: m/z 423 [M+H]+.
1
(R)-4-(4-(m-Tolyl)-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluorophenyl)-
butan-2-amine·HCl (18d): 1H NMR (400 MHz, CD3OD): d=8.55 (s,
1H), 7.64 (s, 1H), 7.59 (d, J=7.6 Hz, 1H), 7.38–7.14 (m, 4H), 4.74 (t,
J=7.0 Hz, 2H), 3.60–3.55 (m, 1H), 3.15–3.00 (m, 2H), 2.41 (s, 3H),
2.38–2.34 ppm (m, 2H); MS: m/z 361 [M+H]+; HRMS: m/z [M+H]+
calcd for C19H19F3N4: 361.1640, found: 361.1644.
(R)-tert-Butyl
(4-(2,3-difluorobenzamido)-1-(2,4,5-trifluorophe-
nyl)butan-2-yl)carbamate (20b): 2,3-Difluorobenzoyl chloride as
acid chloride, compound 20b was obtained as a white solid
1
(40 mg, 88%, mp: 140–1438C). H NMR (300 MHz, CDCl3): d=7.80–
(R)-4-(4-(1-Methyl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-yl)-1-
7.70 (m, 1H), 7.35–7.22 (m, 2H), 7.22–7.12 (m, 1H), 7.06–6.95 (m,
1H), 6.94–6.84 (m, 1H), 4.50 (d, J=9.0 Hz, 1H), 3.98–3.80 (m, 2H),
3.28–3.15 (m, 1H), 2.82–2.64 (m, 2H), 1.97–1.85 (m, 1H), 1.65–1.48
(m, 1H), 1.38 ppm (s, 9H); MS: m/z 459 [M+H]+.
(2,4,5-trifluorophenyl)butan-2-amine·HCl
(18e):
1H NMR
(400 MHz, CD3OD): d=9.06 (s, 1H), 8.62 (s, 1H), 7.91 (d, J=1.3 Hz,
1H), 7.40–7.33 (m, 1H), 7.28–7.21 (m, 1H), 4.76 (t, J=7.1 Hz, 2H),
4.14 (s, 3H), 3.65–3.58 (m, 1H), 3.14 (dd, J=6.6, 14.6 Hz, 1H), 3.04
(dd, J=7.1, 14.6 Hz, 1H), 2.41–2.34 ppm (m, 2H); MS: m/z 351 [M+
H]+; HRMS: m/z [M+H]+ calcd for C16H17F3N6: 351.1545, found:
351.1549.
(R)-tert-Butyl (4-(2-phenylacetamido)-1-(2,4,5-trifluorophenyl)bu-
tan-2-yl)carbamate (20m): Phenylacetyl chloride as acid chloride,
compound 20m was obtained as a white solid (38 mg, 87%, mp:
1
159–1618C, dec.). H NMR (300 MHz, CDCl3): d=7.38–7.24 (m, 5H),
(R)-4-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-1-(2,4,5-trifluorophenyl)-
butan-2-amine·HCl (18 f): 1H NMR (400 MHz, CD3OD): d=8.48 (s,
1H), 7.42–7.36 (m, 1H), 7.27–7.20 (m, 1H), 4.80 (t, J=7.3 Hz, 2H),
3.64–3.58 (m, 1H), 3.12 (dd, J=6.8, 14.5 Hz, 1H), 3.05 (dd, J=7.3,
6.20 (br, 1H), 6.99–6.83 (m, 2H), 4.42 (d, J=9.4 Hz, 1H), 3.78–3.55
(m, 2H), 3.54 (s, 2H), 2.96–2.83 (m, 1H), 2.74–2.59 (m, 2H), 1.77–
1.66 (m, 1H), 1.49–1.38 (m, 1H), 1.35 ppm (s, 9H); MS: m/z 437
[M+H]+.
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(R)-tert-Butyl (4-(2-(4-fluorophenyl)acetamido)-1-(2,4,5-trifluoro-
phenyl)butan-2-yl)carbamate (20n): 4-Fluorophenylacetyl chloride
as acid chloride, compound 20n was obtained as a white solid
(R)-tert-Butyl (4-(1H-benzo[d]imidazole-5-carboxamido)-1-(2,4,5-
trifluorophenyl)butan-2-yl)carbamate (20h): 1H-Benzo[d]imida-
zole-5-carboxylic acid as acid, compound 20h was obtained as
a
white solid (35 mg, 75%, mp: 164–1678C, dec.). 1H NMR
1
(36 mg, 79%, mp: 155–1588C, dec.). H NMR (400 MHz, CDCl3): d=
7.26–7.23 (m, 2H), 7.04–6.86 (m, 4H), 6.31 (br, 1H), 4.42–4.40 (d,
J=9.1 Hz, 1H), 3.73–3.65 (m, 2H), 3.51 (s, 2H), 2.92–2.82 (m, 1H),
2.76–2.62 (m, 2H), 1.77–1.66 (m, 1H), 1.42–1.36 ppm (m, 10H); MS:
m/z 455 [M+H]+.
(300 MHz, CDCl3): d=8.19 (s, 1H), 8.11 (s, 1H), 7.75–7.52 (m, 3H),
7.05–6.94 (m, 1H), 6.89–6.78 (m, 1H), 4.89 (d, J=9.7 Hz, 1H), 4.04–
3.85(m, 2H), 3.30–3.15 (m, 1H), 2.85–2.65 (m, 2H), 2.00–1.85(m,
1H), 1.69–1.53 (m, 1H), 1.37 ppm (s, 9H); MS: m/z 463 [M+H]+.
(R)-tert-Butyl
(4-(1H-benzo[d][1,2,3]triazole-5-carboxamido)-1-
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (20i): 1H-Benzo[d]-
[1,2,3]triazole-5-carboxylic acid as acid, compound 20i was ob-
tained as a light-brown solid (27 mg, 59%). 1H NMR (300 MHz,
[D6]acetone): d=8.54–7.86 (m, 3H), 7.37–7.26 (m, 1H), 7.22–7.11
(m, 1H), 6.20 (d, J=9.2 Hz, 1H), 4.03–3.91 (m, 1H), 3.79–3.67 (m,
1H), 3.41–3.28 (m, 1H), 2.94 (dd, J=4.7, 14.0 Hz, 1H), 2.77 (dd, J=
9.5, 13.9 Hz, 1H), 2.02–1.88 (m, 1H), 1.87–1.74 (m, 1H), 1.32 ppm (s,
9H); MS: m/z 464 [M+H]+.
General procedure for 20c–l, 20o,p
DIPEA (2 equiv), EDCI (1.2 equiv), and HOBt (1 equiv) were added
successively to a solution of acid (1.1 equiv) in dry CH2Cl2 (0.1m) at
08C. After stirring at the same temperature for 0.5 h, 19 (32 mg,
0.1 mmol, 1 equiv) was added. The resulting mixture was allowed
to reach room temperature and stirred for 14 h before it was
quenched with 1n HCl and extracted with CH2Cl2. The organic
layer was washed with sat. NaHCO3 and brine, dried over Na2SO4,
and concentrated. The residue was purified by column chromatog-
raphy to give the desired compound.
(R)-tert-Butyl (4-(nicotinamido)-1-(2,4,5-trifluorophenyl)butan-2-
yl)carbamate (20j): Nicotinic acid as acid, compound 20j was ob-
tained as a white solid (29 mg, 69%, mp: 142–1458C). 1H NMR
(300 MHz, CDCl3): d=9.09 (s, 1H), 8.72 (br, 1H), 8.16 (d, J=7.9 Hz,
1H), 7.64 (m, 1H), 7.38 (dd, J=4.8, 7.4 Hz, 1H), 7.06–6.95 (m, 1H),
6.95–6.85 (m, 1H), 4.59 (d, J=9.3 Hz, 1H), 4.07–3.85 (m, 2H), 3.11–
3.00 (m, 1H), 2.83–2.66 (m, 2H), 1.99–1.82 (m, 2H), 1.39 ppm (s,
9H); MS: m/z 424 [M+H]+.
(R)-tert-Butyl (4-(4-methoxybenzamido)-1-(2,4,5-trifluorophenyl)-
butan-2-yl)carbamate (20c): 4-Methoxybenzoic acid as acid, com-
pound 20c was obtained as a white solid (34 mg, 75%, mp: 147–
1
1508C). H NMR (300 MHz, CDCl3): d=7.80 (d, J=8.6 Hz, 2H), 7.21
(br, 1H), 7.05–6.95 (m, 1H), 6.95–6.83 (m, 3H), 4.57 (d, J=9.8 Hz,
1H), 4.01–3.88 (m, 2H), 3.84 (s, 3H), 3.10–2.97 (m, 1H), 2.82–2.65
(m, 2H), 1.95–1.83 (m, 1H), 1.58–1.42 (m, 1H), 1.39 ppm (s, 9H);
MS: m/z 453 [M+H]+.
(R)-tert-Butyl (4-(thiophene-3-carboxamido)-1-(2,4,5-trifluorophe-
nyl)butan-2-yl)carbamate (20k): Thiophene-3-carboxylic acid as
acid, compound 20k was obtained as a white solid (33 mg, 77%).
1H NMR (300 MHz, [D6]DMSO): d=8.20 (br, 1H), 8.07–8.04 (m, 1H),
7.57–7.54 (m, 1H), 7.47–7.25 (m, 3H), 6.78 (d, J=9.2 Hz, 1H), 3.72–
3.62 (m, 1H), 3.36–3.08 (m, 2H), 2.84–2.76 (m, 1H), 2.59–2.50 (m,
1H), 1.73–1.55 (m, 2H), 1.26 ppm (s, 9H); MS: m/z 429 [M+H]+.
(R)-tert-Butyl (4-(4-(methylsulfonyl)benzamido)-1-(2,4,5-trifluoro-
phenyl)butan-2-yl)carbamate (20d): 4-(Methylsulfonyl)benzoic
acid as acid, compound 20d was obtained as a white solid (39 mg,
77%, mp: 200–2028C, dec.). 1H NMR (300 MHz, CDCl3): d=8.10–
7.99 (m, 4H), 7.78–7.69 (m, 1H), 7.07–6.86 (m, 2H), 4.61 (d, J=
9.6 Hz, 1H), 4.09–3.86 (m, 2H), 3.15–3.00 (m, 4H), 2.84–2.69 (m,
2H), 2.01–1.85 (m, 1H), 1.60–1.44 (m, 1H), 1.41 ppm (s, 9H); MS:
m/z 501 [M+H]+.
(R)-tert-Butyl (4-(quinoline-6-carboxamido)-1-(2,4,5-trifluorophe-
nyl)butan-2-yl)carbamate (20l): Quinoline-6-carboxylic acid as
acid, compound 20l was obtained as a white solid (36 mg, 75%,
1
mp: 161–1628C, dec.). H NMR (300 MHz, CDCl3): d=8.99 (dd, J=
(R)-tert-Butyl
(4-(4-sulfamoylbenzamido)-1-(2,4,5-trifluorophe-
1.4, 4.2 Hz, 1H), 8.39 (s, 1H), 8.25 (dd, J=1.4, 8.3 Hz, 1H), 8.19–8.12
(m, 2H), 7.62 (br, 1H), 7.46 (dd, J=4.2, 8.3 Hz, 1H), 7.04–6.96 (m,
1H), 6.95–6.83 (m, 1H), 4.58 (d, J=9.9 Hz, 1H), 4.11–3.91 (m, 2H),
3.19–3.07 (m, 1H), 2.83–2.71 (m, 2H), 2.02–1.90 (m, 1H), 1.62–1.47
(m, 1H), 1.41 ppm (s, 9H); MS: m/z 474 [M+H]+.
nyl)butan-2-yl)carbamate (20e): 4-Sulfamoylbenzoic acid as acid,
compound 20e was obtained as a white solid (37 mg, 73%, mp:
196–1998C, dec.). 1HNMR (300 MHz, [D6]acetone): d=8.17 (t, 1H),
8.05–7.99 (m, 2H), 7.98–7.93 (m, 2H), 7.36–7.25 (m, 1H), 7.22–7.12
(m, 1H), 6.18 (d, J=9.4 Hz, 1H), 4.03–3.88 (m, 1H), 3.75–3.62 (m,
1H), 3.37–3.24 (m, 1H), 2.92 (dd, J=4.8, 14.0 Hz, 1H), 2.76 (dd, J=
9.6, 13.9 Hz, 1H), 1.99–1.74 (m, 2H), 1.32 ppm (s, 9H); MS: m/z 502
[M+H]+.
(R)-tert-Butyl
(4-(2-(4-methoxyphenyl)acetamido)-1-(2,4,5-tri-
fluorophenyl)butan-2-yl)carbamate (20o): 4-Methoxyphenylacetic
acid as acid, compound 20o was obtained as a white solid (37 mg,
1
79%, mp: 155–1578C, dec.). H NMR (400 MHz, CDCl3): d=7.18 (d,
(R)-tert-Butyl (4-(4-(trifluoromethyl)benzamido)-1-(2,4,5-trifluoro-
phenyl)butan-2-yl)carbamate (20 f): 4-(Trifluoromethyl)benzoic
acid as acid, compound 20 f was obtained as a white solid (40 mg,
81%).1HNMR (300 MHz, CD3OD): d=7.97 (d, J=8.0 Hz, 2H), 7.76 (d,
J=8.0 Hz, 2H), 7.23–7.05 (m, 2H), 3.88–3.78 (m, 1H), 3.62–3.53 (m,
1H), 3.35–3.30 (m, 1H), 2.93–2.85 (m, 1H), 2.66–2.57 (m, 1H), 1.94–
1.82 (m, 1H), 1.76–1.64 (m, 1H), 1.33 ppm (s, 9H); MS: m/z 491
[M+H]+, 391 [M+HÀBoc]+.
J=8.4 Hz, 2H), 6.99–6.85 (m, 4H), 6.13 (br, 1H), 4.43 (d, J=9.2 Hz,
1H), 3.79 (s, 3H), 3.76–3.68 (m, 1H), 3.64–3.54 (m, 1H), 3.48 (s, 2H),
2.97–2.87 (m, 1H), 2.76–2.62 (m, 2H), 1.76–1.68 (m, 1H), 1.47–1.40
(m, 1H), 1.35 ppm (s, 9H); MS: m/z 467 [M+H]+.
(R)-tert-Butyl
(4-(2-(4-(trifluoromethyl)phenyl)acetamido)-1-
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (20p): 4-(Trifluorome-
thyl)phenylacetic acid as acid, compound 20o was obtained as
1
a white solid (42 mg, 83%). H NMR (400 MHz, CDCl3): d=7.59 (d,
(R)-tert-Butyl (4-(4-(1H-1,2,4-triazol-1-yl)benzamido)-1-(2,4,5-tri-
fluorophenyl)butan-2-yl)carbamate (20g): 4-(1H-1,2,4-Triazol-1-yl)-
benzoic acid as acid, compound 20g was obtained as a white solid
J=8.1 Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 6.98–6.86 (m, 2H), 6.46 (br,
1H), 4.39 (d, J=9.0 Hz, 1H), 3.79–3.66 (m, 1H), 3.59 (s, 2H), 2.89–
2.79 (m, 1H), 2.75–2.62 (m, 2H), 1.79–1.70 (m, 1H), 1.47–1.31 ppm
(m, 10H); MS: m/z 505 [M+H]+.
1
(39 mg, 80%, mp: 210–2148C, dec.). H NMR (300 MHz, CDCl3): d=
8.63 (s, 1H), 8.12 (s, 1H), 8.02 (d, J=8.3 Hz, 2H), 7.78 (d, J=8.3 Hz,
2H), 7.59 (brs, 1H), 7.06–6.84 (m, 2H), 4.59 (d, J=9.5 Hz, 1H), 4.09–
3.87 (m, 2H), 3.12–2.99 (m, 1H), 2.83–2.67 (m, 2H), 2.01–1.87 (m,
1H), 1.58–1.45 (m, 1H), 1.40 ppm (s, 9H); MS: m/z 490 [M+H]+.
(R)-tert-Butyl (4-(3-(4-cyanophenyl)ureido)-1-(2,4,5-trifluorophe-
nyl)butan-2-yl)carbamate (20q): 4-Isocyanatobenzonitrile (32 mg,
0.22 mmol) was added to a solution of 19 (64 mg, 0.2 mmol) in dry
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CH2Cl2 (5 mL). After stirring at room temperature for 18 h, the solu-
tion was concentrated, and the residue was purified by column
chromatography (PE/EtOAc 3:1) to furnish the title compound 20q
(65 mg, 70%). 1H NMR (300 MHz, CDCl3): d=7.57–7.43 (m, 4H),
7.15 (brs, 1H), 7.05–6.83 (m, 2H), 5.85 (brt, 1H), 4.49 (d, J=9.1 Hz,
1H), 3.97–3.82 (m, 1H), 3.75–3.63 (m, 1H), 3.00–2.87 (m, 1H), 2.82–
2.63 (m, 2H), 1.91–1.77 (m, 1H), 1.52–1.40 (m, 1H), 1.36 ppm (s,
9H); MS: m/z 463 [M+H]+.
Synthesis of 21a–t
Compounds 21a–t were prepared from the corresponding inter-
mediates 20a–t by following the same deprotection procedure for
17 and 18.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)benzamide·HCl
1
(21a): H NMR (400 MHz, CD3OD): d=7.82–7.80 (m, 2H), 7.57–7.53
(m, 1H), 7.48–7.44 (m, 2H), 7.35–7.28 (m, 1H), 7.21–7.14 (m, 1H),
3.65–3.56 (m, 1H), 3.50–3.40 (m, 2H), 3.04–2.92 (m, 2H), 1.96–
1.78 ppm (m, 2H) LC-MS: m/z 323 [M+H]+; HRMS: m/z [M+H]+
calcd for C17H17F3N2O: 323.1371, found: 323.1370.
(R)-tert-Butyl
(4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-
[1,2,4]triazolo[4,3-a]pyrazine-7-carboxamido)-1-(2,4,5-trifluoro-
phenyl)butan-2-yl)carbamate (20r): Et3N (120 mg, 1.2 mmol) and
4-nitrophenyl chloroformate (200 mg, 1 mmol) were added succes-
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-2,3-difluorobenza-
mide·HCl (21b): H NMR (400 MHz, CD3OD): d=7.49–7.17 (m, 5H),
3.63–3.47 (m, 3H), 3.09–2.99 (m, 2H), 1.99–1.86 ppm (m, 2H); MS:
m/z 359 [M+H]+; HRMS: m/z [M+H]+ calcd for C17H15F5N2O:
359.1183, found: 359.1189.
1
sively to
a
solution of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-
[1,2,4]triazolo[4,3-a]pyrazine (192 mg 1 mmol) in dry CH2Cl2 (10 mL)
at 08C. After stirring at room temperature for 2 h, the solution was
diluted with CH2Cl2 (30 mL), washed with H2O (2ꢁ20 mL) and brine
(10 mL), dried over Na2SO4, and concentrated to give the 4-nitro-
phenyl carbamate as a yellow solid, which was used without fur-
ther purification. A mixture of 19 (64 mg, 0.2 mmol), 4-nitrophenyl
carbamate obtained from last step (79 mg, 0.22 mmol), and K2CO3
(33 mg, 0.24 mmol) in dry DMF (2 mL) was stirred at 1008C for
14 h. The reaction mixture was concentrated. Et2O (25 mL) and H2O
(10 mL) were added to the residue, and the phases were separat-
ed. The aqueous phase was extracted with Et2O (2ꢁ10 mL), and
the combined organic layers were washed with 1n NaOH(aq) (2ꢁ
10 mL) and brine (10 mL), and dried over Na2SO4. After removal of
the solvent, the residue was purified by column chromatography
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-4-methoxybenza-
1
mide·HCl (21c): H NMR (400 MHz, CD3OD): d=7.82–7.79 (m, 2H),
7.37–7.30 (m, 1H), 7.23–7.17 (m, 1H), 7.01–6.98 (m, 2H), 3.85 (s,
3H), 3.64–3.57 (m, 1H), 3.47–3.41 (m, 2H), 3.02 (d, J=6.9 Hz, 2H),
1.98–1.81 ppm (m, 2H); MS: m/z 353 [M+H]+; HRMS: m/z [M+H]+
calcd for C18H19F3N2O2: 353.1477, found: 353.1473.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-4-(methylsulfonyl)-
1
benzamide·HCl (21d): H NMR (400 MHz, CD3OD): d=8.05 (s, 4H),
7.39–7.32 (m, 1H), 7.22–7.16 (m, 1H), 3.65–3.48 (m, 3H), 3.16 (s,
3H), 3.08–2.98 (m, 2H), 2.01–1.87 ppm (m, 2H); MS: m/z 401 [M+
H]+; HRMS: m/z [M+H]+ calcd for C18H19F3N2O3S: 401.1147, found:
401.1146.
1
(PE/EtOAc 1:2) to give 20r (66 mg, 62%) as a white solid. H NMR
(300 MHz, CDCl3): d=7.05–6.85 (m, 2H), 6.43–6.34 (m, 1H), 4.92 (d,
J=17.0 Hz, 1H), 4.85 (d, J=17.0 Hz, 1H), 4.57 (d, J=9.5 Hz, 1H),
4.17 (t, J=5.4 Hz, 2H), 3.93 (t, J=5.4 Hz, 2H), 3.90–3.80 (m, 1H),
3.77–3.65 (m, 1H), 3.02–2.87 (m, 1H), 2.81–2.65 (m, 2H), 1.90–1.78
(m, 1H), 1.50–1.39 (m, 1H), 1.37 ppm (s, 9H); MS: m/z 537 [M+H]+
.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-4-sulfamoylbenza-
mide·HCl (21e): H NMR (400 MHz, CD3OD): d=7.97 (s, 4H), 7.38–
7.32 (m, 1H), 7.22–7.16 (m, 1H), 3.64–3.57 (m, 1H), 3.54–3.47 (m,
1
2H), 3.08–2.98 (m, 2H), 2.00–1.86 ppm (m, 2H); MS: m/z 402 [M+
+
H]+; HRMS: m/z [M+H]
calcd for C17H18F3N3O3S: 402.1099,
found: 402.1097.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-4-(trifluorome-
1
thyl)benzamide·HCl (21 f): H NMR (400 MHz, CD3OD): d=7.99 (d,
(R)-tert-Butyl
(4-(thiomorpholine-4-carboxamido)-1-(2,4,5-tri-
J=8.1 Hz, 2H), 7.79 (d, J=8.1 Hz, 2H), 7.37–7.31 (m, 1H), 7.23–7.16
(m, 1H), 3.65–3.57 (m, 1H), 3.53–3.46 (m, 2H), 3.07–2.97 (m, 2H),
1.98–1.89 ppm (m, 2H); MS: m/z 391 [M+H]+.
fluorophenyl)butan-2-yl)carbamate (20s): According to the
method described for 20r, replacing 3-(trifluoromethyl)-5,6,7,8-
tetrahydro[1,2,4]triazolo[4,3-a]pyrazine with thiomorpholine, com-
pound 20s was obtained from 95 mg (0.3 mmol) 19 as a white
solid (79 mg, 59%). 1H NMR (400 MHz, CDCl3): d=7.04–6.98 (m,
1H), 6.94–6.87 (m, 1H), 5.61 (br, 1H), 4.47 (d, J=9.6 Hz, 1H), 3.92–
3,82 (m, 1H), 3.76–3.64 (m, 5H), 2.95–2.86 (m, 1H), 2.79–2.66 (m,
2H), 2.61–2.58 (m, 4H), 1.85–1.77 (m, 1H), 1.44–1.35 ppm (m, 10H);
MS: m/z 448 [M+H]+.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-4-(1H-1,2,4-triazol-
1
1-yl)benzamide·HCl (21g): H NMR (400 MHz, CD3OD): d=10.02 (s,
1H), 8.74 (s, 1H), 8.11–8.08 (m, 2H), 8.05–8.02 (m, 2H), 7.40–7.34
(m, 1H), 7.22–7.16 (m, 1H), 3.65–3.45 (m, 3H), 3.09–2.99 (m, 2H),
2.02–1.88 ppm (m, 2H); MS: m/z 390 [M+H]+.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-1H-benzo[d]imida-
zole-6-carboxamide·HCl (21h): 1H NMR (400 MHz, CD3OD): d=
9.51 (s, 1H), 8.41 (d, J=0.8 Hz, 1H), 8.12 (dd, J=1.5, 8.7 Hz, 1H),
7.94 (d, J=8.7 Hz, 1H), 7.42–7.35 (m, 1H), 7.20–7.14 (m, 1H), 3.67–
3.51 (m, 3H), 3.11–3.00 (m, 2H), 2.05–1.92 ppm (m, 2H); MS: m/z
363 [M+H]+.
(R)-tert-Butyl (4-(1,1-dioxidothiomorpholine-4-carboxamido)-1-
(2,4,5-trifluorophenyl)butan-2-yl)carbamate (20t): To a solution
of 20s (45 mg, 0.1 mmol) in dry CH2Cl2 (5 mL) was added 3-chloro-
perbenzoic acid (mCPBA; 77%, 56 mg, 0.25 mmol) at 08C. After
stirring at room temperature for 16 h, the solution was diluted
with CH2Cl2 (20 mL), washed with sat. NaHCO3 (2ꢁ10 mL) and
brine (10 mL), dried over Na2SO4, and concentrated. The residue
was purified by column chromatography (CH2Cl2/MeOH 20:1) to
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-1H-benzo[d]-
[1,2,3]triazole-6-carboxamide·HCl (21i): 1H NMR (400 MHz,
CD3OD): d=8.45 (s, 1H), 7.98 (dd, J=1.4, 8.7 Hz, 1H), 7.91 (d,
J=8.7 Hz, 1H), 7.40–7.35 (m, 1H), 7.23–7.16 (m, 1H), 3.68–3.62
(m, 1H), 3.57–3.52 (m, 2H), 3.10–2.98 (m, 2H), 2.00–1.92 ppm
(m, 2H); MS: m/z 364 [M+H]+.
1
give 20t (27 mg, 56%) as a white solid. H NMR (400 MHz, CDCl3):
d=7.03–6.90 (m, 2H), 6.22 (br, 1H), 4.45 (d, J=9.6 Hz, 1H), 3.99–
3.82 (m, 5H), 3.77–3.70 (m, 1H), 3.02 (t, J=5.1 Hz, 4H), 2.92–2.84
(m, 1H), 2.79–2.67 (m, 2H), 1.88–1.81 (m, 1H), 1.43–1.35 ppm (m,
10H); MS: m/z 480 [M+H]+.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)nicotinamide·HCl
(21j): 1H NMR (400 MHz, CD3OD): d=9.32 (s, 1H), 9.03–9.01 (m,
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2H), 8.19 (dd, J=5.9, 8.0 Hz, 1H), 7.44–7.37 (m, 1H), 7.23–7.16 (m,
1H), 3.62–3.57 (m, 3H), 3.11–3.00 (m, 2H), 2.02–1.96 ppm (m, 2H);
MS: m/z 324 [M+H]+; HRMS: m/z [M+H]+ calcd for C16H16F3N3O:
324.1324, found: 324.1327.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)thiomorpholine-4-
carboxamide 1,1-dioxide·HCl (21t): 1H NMR (400 MHz, CD3OD):
d=7.39–7.33 (m, 1H), 7.29–7.23 (m, 1H), 3.95–3.87 (m, 4H), 3.50–
3.39 (m, 2H), 3.31–3.25 (m, 1H), 3.10 (t, J=5.1 Hz, 4H), 3.01 (d, J=
6.8 Hz, 2H), 1.87–1.73 ppm (m, 2H); MS: m/z 380 [M+H]+.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)thiophene-3-car-
boxamide·HCl (21k): 1H NMR (400 MHz, D2O): d=7.88 (m, 1H),
7.41–7.39 (m, 1H), 7.27 (m, 1H), 7.15–7.09 (m, 1H), 6.99–6.92 (m,
1H), 3.49–3.35 (m, 3H), 2.96–2.85 (m, 2H), 1.89–1.80 ppm (m, 2H);
MS: m/z 329 [M+H]+.
Cloning, expression, and purification
The extracellular domain of DPP4 (residues 39–766) was cloned
into a mammalian expression vector pTT28 with an N-terminal
fusion to a secretory signal peptide and a C-terminal fusion to
a His6 affinity tag. The protein was expressed in FreeStyle 293-F
cells (Invitrogen) and secreted at a low level of 2 mgLÀ1. Freestyle
293 expression medium (50 mL; Invitrogen) containing 1 mg plas-
mid and 50 mL medium containing 3 mg polyethylenimine (Poly-
sciences) were mixed and incubated at room temperature for
15 min. Cell culture at 1.2ꢁ106 cell density was transfected by
adding 100 mL mixture to a final volume of 1 L. The cells were
grown at 378C for another 96 h, after which the supernatant was
harvested by centrifugation at 4000 g for 25 min at 48C. The super-
natant was loaded onto a Ni-NTA column (Qiagen) that was equili-
brated with a buffer containing 50 mm Tris·HCl pH 7.5, 150 mm
NaCl, 0.5% (v/v) CHAPS, 5 mm 2-mercaptoethanol. The column was
washed with five column volumes of a buffer containing 50 mm
Tris·HCl pH 7.5, 150 mm NaCl, 0.5% (v/v) CHAPS, 5 mm 2-mercap-
toethanol, 20 mm imidazole, then eluted with a buffer containing
50 mm Tris·HCl pH 7.5, 150 mm NaCl, 0.5% (v/v) CHAPS, 5 mm 2-
mercaptoethanol, 250 mm imidazole. The eluted protein was con-
centrated to ~1 mL using Centricon YM-10 (Millipore) and further
purified by elution with SEC buffer (50 mm Tris·HCl pH 7.5, 150 mm
NaCl, 0.5% (v/v) CHAPS, 5 mm 2-mercaptoethanol) over a Hiload
10/300 Superdex 200 GL column to 95% purity, as assessed by
SDS-PAGE (Coomassie Brilliant Blue R-250 staining), and concentrat-
ed to 10–15 mgmLÀ1 in the SEC buffer. The DPP4 was homoge-
nous on SDS-PAGE with a molecular weight (Mr) of 110 kDa. N-ter-
minal amino acid sequencing verified the sequence starting at po-
sition 39. The Mr of the purified recombinant DPP4(39–766) was deter-
mined to be 104400 Da by MS. The theoretical Mr for non-glycosy-
lated DPP4(39–766) is 84400 Da. Therefore, glycosylation can be
estimated to account for 24% Mr of the whole glycosylated pro-
tein.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)quinoline-6-carbox-
1
amide·HCl (21l): H NMR (400 MHz, CD3OD): d=9.34–9.33 (m, 2H),
8.87 (s, 1H), 8.58 (d, J=8.6 Hz, 1H), 8.36 (d, J=8.6 Hz, 1H), 8.33–
8.15 (m, 1H), 7.43–7.36 (m, 1H), 7.22–7.15 (m, 1H), 3.68–3.56 (m,
3H),3.13–3.00 (m, 2H), 2.05–1.96 ppm (m, 2H); MS: m/z 374 [M+
H]+; HRMS: m/z [M+H]+ calcd for C20H18F3N3O: 384.1480, found:
384.1480.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-2-phenylacetami-
de·HCl (21m): 1H NMR (400 MHz, CD3OD): d=7.32–7.17 (m, 7H),
3.53 (d, J=14.2 Hz, 1H), 3.49 (d, J=14.2 Hz, 1H), 3.45–3.37 (m, 1H),
3.27–3.22 (m, 2H), 2.92 (d, J=7.0 Hz, 2H), 1.79–1.74 ppm (m, 2H);
+
MS: m/z 337 [M+H]+; HRMS: m/z [M+H] calcd for C18H19F3N4O:
337.1528, found: 337.1529.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-2-(4-fluoropheny-
l)acetamide·HCl (21n): 1H NMR (400 MHz, CD3OD): d=7.35–7.19
(m, 4H), 7.09–7.03 (m, 2H), 3.53 (s, 2H), 3.46–3.39 (m, 1H), 3.32–
3.27 (m, 2H), 2.98 (d, J=7.0 Hz, 2H), 1.84–1.79 ppm (m, 2H); MS:
m/z 355 [M+H]+; HRMS: m/z [M+H]+ calcd for C18H18F4N2O:
355.1434, found: 355.1428.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-2-(4-methoxyphe-
1
nyl)acetamide·HCl (21o): H NMR (400 MHz, CD3OD): d=7.26–7.15
(m, 4H), 6.87–6.83 (m, 2H), 3.76 (s, 3H), 3.44 (s, 2H), 3.41–3.36 (m,
1H), 3.29–3.22 (m, 2H), 2.93 (d, J=7.0 Hz, 2H), 1.80–1.75 ppm (m,
2H); MS: m/z 367 [M+H]+. HRMS: m/z [M+H]+ calcd for
C19H21F3N2O2: 367.1633, found: 367.1638.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-2-(4-(trifluorome-
1
thyl)phenyl)acetamide·HCl (21p): H NMR (400 MHz, CD3OD): d=
7.61 (d, J=8.1 Hz, 2H), 7.48 (d, J=8.1 Hz, 2H), 7.29–7.15 (m, 2H),
3.62 (s, 2H), 3.44–3.37 (m, 1H), 3.31–3.25 (m, 2H), 2.94 (d, J=
7.0 Hz, 2H), 1.81–1.76 ppm (m, 2H); MS: m/z 405 [M+H]+.
(R)-1-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-3-(4-cyanopheny-
l)urea·HCl (21q): H NMR (400 MHz, CD3OD): d=7.58 (s, 4H), 7.39–
Crystallization and structure determination
1
Crystals of the DPP4–compound 18d complex were obtained by
sitting drop vapor diffusion at 188C with a drop composed of
0.5 mL of DPP4–compound C2 mixture and 0.8 mL of crystallization
solution containing 0.03m NH4OAc, 28% (w/v) PEG 1500, and 0.1m
MES pH 5.5. Crystals were soaked in a cryoprotectant solution con-
taining crystallization solution plus 20% (v/v) glycerol, and were
flash-frozen in liquid nitrogen. The diffraction data were collected
at APS beam line 21-ID-D and processed to 3.2 ꢂ in space group
P32 using HKL2000.[21] The structure was solved by molecular re-
placement using Phaser[22] with the structure of human DPP4 (PDB
ID: 3HAC) as the search model. There is a DPP4 dimer in an asym-
metric unit and two copies of compound 18d in the refined struc-
ture. The structure has been deposited at the PDB (PDB ID: 4DSZ).
Detailed data-processing and structure-refinement statistics are
listed in the Supporting Information.
7.32 (m, 1H), 7.23–7.16 (m, 1H), 3.54–3.47 (m, 1H), 3.45–3.32 (m,
2H), 3.05–2.96 (m, 2H), 1.90–1.76 ppm (m, 2H); MS: m/z 363 [M+
H]+; HRMS: m/z [M+H]+ calcd for C18H17F3N4O: 363.1433, found:
363.1432.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)-3-(trifluorometh-
yl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxami-
de·HCl (21r): 1H NMR (400 MHz, CD3OD): d=7.38–7.31 (m, 1H),
7.24–7.17 (m, 1H), 4.87 (s, 2H), 4.24 (t, J=5.4 Hz, 2H), 4.01–3.88 (m,
2H), 3.51–3.38 (m, 1H), 3.34–3.27 (m, 1H), 2.99 (d, J=7.0 Hz, 2H),
1.89–1.72 ppm (m, 2H); MS: m/z 437 [M+H]+; HRMS: m/z [M+H]
+
calcd for C17H18F6N6O: 437.1525, found: 437.1528.
(R)-N-(3-Amino-4-(2,4,5-trifluorophenyl)butyl)thiomorpholine-4-
carboxamide·HCl (21s): 1H NMR (400 MHz, CD3OD): d=7.36–7.29
(m, 1H), 7.25–7.19 (m, 1H), 3.70–3.65 (m, 4H), 3.45–3.36 (m, 2H),
3.27–3.21 (m, 1H), 3.03–2.93 (m, 2H), 2.56–2.53 (m, 4H), 1.85–
Crystals of the DPP4–compound 21r complex were obtained by
sitting drop vapor diffusion at 188C with a drop composed of
01 mL of DPP4–compound C5 mixture and 1 mL of crystallization
+
1.68 ppm (m, 2H); MS: m/z 348 [M+H]+; HRMS: m/z [M+H]
calcd for C15H20F3N3OS: 348.1357, found: 348.1362.
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solution containing 28% (w/v) PEG 1500. The diffraction data were
collected at APS beam line 21-ID-D and processed using HKL2000
to 3.2 ꢂ in space group P32. The structure determination procedure
was similar to that described for the DPP4–compound C1 struc-
ture. There is a DPP4 dimer in an asymmetric unit and two copies
of compound 21r in the refined structure. The structure has been
deposited at the PDB (PDB ID: 4J3J). Detailed data-processing and
structure-refinement statistics are listed in the Supporting Informa-
tion.
enytoin 100 mm) and 10 mm test compound or positive control
cocktail (ketoconazole 10 mm, quinidine 10 mm, sulfaphenazole
100 mm, a-naphthoflavone 10 mm, tranylcypromine 1000 mm), or
negative control (PBS). The final concentration of organic reagent
in incubation mixtures was <1% v/v. There was a 5 min pre-incu-
bation period at 378C before the reaction was initiated by adding
a NADPH-generating system. Reactions were conducted for 20 min
for CYP3A4, CYP2D6, and CYP1A2. For each probe drug, the per-
centage of metabolite conversion was <20% of substrate added.
Percent inhibition was calculated as: (formation of the metabolite
of probe substrates with 10 mm test compound)/(formation of the
metabolite of probe substrates with PBS)ꢁ100.
Enzyme activity assays
To measure the activity of DPPs, a continuous fluorimetric assay
was employed using Ala-Pro-AMC, which is cleaved by the enzyme
to release the fluorescent aminomethylcoumarin (AMC). Liberation
of AMC was monitored at an excitation wavelength of 355 nm and
an emission wavelength of 460 nm using an Envision microplate
reader (PerkinElmer). A typical reaction contained 50 pmolLÀ1
enzyme, 10 mmolLÀ1 Ala-Pro-AMC, various test compound concen-
trations, and assay buffer (100 mmolLÀ1 HEPES pH 7.5, 0.1 mgmLÀ1
BSA) in a total reaction volume of 50 mL. DPP4, DPP8, and DPP9 en-
zymes were expressed in high five cells using a baculoviral system
(Bac-To-Bac; Life Technologies) according to an earlier publica-
tion,[25] and His6-tagged recombinant proteins were purified by Ni-
NTA resin individually.
Pharmacokinetics and oral glucose tolerance tests in normal
ICR mice
All animal experiments were approved by the Animal Care and Use
Committee of the Shanghai Institute of Materia Medica. For the
pharmacokinetics studies, compound 21r (hydrochloride salt;
21.6 mgkgÀ1) and sitagliptin (phosphate salt; 25.7 mgkgÀ1) were
administered orally to ICR mice after starvation for 16 h. Blood
samples were then taken at 0.25, 0.5, 1, 2, 4, 8, and 24 h for analy-
sis by LC–MS–MS (Shimadzu UFLC 20-AD/API-4000). The pharmaco-
kinetic parameters were obtained from raw data with WinNonlin
software.
For the acute single-dose study, compound 21r was administered
to ICR mice after starvation for 5 h. The oral glucose tolerance test
(3 gkgÀ1) was then conducted after 30 min of the single dose, and
blood glucose levels at 0, 15, 30, 60, 90, and 120 min were record-
ed for area under the curve (AUC) calculations. Sitagliptin was used
as a positive control. The results are presented as the mean ÆSEM.
Differences between the groups were analyzed by Student’s t test;
*p<0.05 or **p<0.01 were regarded as statistically significant.
In vitro pharmacokinetics assays
Reactions were initiated by the addition of enzyme preparations as
microsome fractions from HEK293 cells at a final concentration of
80 mgmLÀ1 for 11b-HSD1. Following 60 min incubation at 378C,
the reaction was stopped by adding 35 mL of 10 mgmLÀ1 protei-
n A-coated SPA beads (GE, Piscataway, NJ, USA) suspended in Su-
perblock Blocking Buffer (Pierce, Rockford, IL, USA) with 3 mgmLÀ1
of murine monoclonal cortisol antibody (East Coast Biologics,
North Berwick, ME, USA) and 314 mm glycyrrhetinic acid (Sigma–Al-
drich, St. Louis, MO, USA). The plates were incubated under plastic
film on an orbital shaker for 120 min at room temperature before
counting. The amount of [3H]cortisol generated by 11b-HSD1 was
captured on the beads and measured in a microplate liquid scintil-
lation counter. Percent inhibition was calculated relative to unin-
hibited control. Data were obtained from at least three independ-
ent experiments. IC50 values were calculated by using Prism Ver-
sion 4 (GraphPad Software, San Diego, CA, USA).
Acknowledgements
The authors thank Dr. Haiyan Liu and Dr. Xuejun Liu for assis-
tance with pharmacokinetics assays and Dr. Fan Jiang and Dr.
Jianhua Cai for their help in crystallization and X-ray crystallog-
raphy. We are grateful for financial support from the Program of
Excellent Young Scientist of the Chinese Academy of Sciences to
B.X. (grant KSCX2-EW-Q-3-01) and the National Natural Science
Foundation of China (grant 30600784).
Microsomes (human: Xenotech, Lot No. H0610; rat: Xenotech, Lot
No. R1000) (0.5 mgmLÀ1) were pre-incubated with 1 mm test com-
pound for 5 min at 378C in 0.1m phosphate buffer (pH 7.4) with
1 mm EDTA and 5 mm MgCl2. The reactions were initiated by
adding pre-warmed cofactors (1 mm NADPH). After 0, 5, 10, and
30 min incubations at 378C, the reactions were stopped by adding
an equal volume of cold CH3CN. The samples were vortexed for
10 min and then centrifuged at 10000 g for 10 min. Supernatants
were analyzed by LC–MS–MS for the amount of parent compound
remaining, and the corresponding loss of parent compound was
also determined by LC–MS–MS.
Keywords: 1,3-diamines
peptidase IV · inhibitors
· crystal structures · dipeptidyl
[1] Diabetes Fact Sheet No. 312, World Health Organization, http://
www.WHO.int/mediacentre/factsheets/fs312/en/ (accessed April 24,
2013).
[2] J. Zhang, Y. Tokui, K. Yamagata, J. Kozawa, K. Sayama, H. Iwahashi, K.
Okita, M. Miuchi, H. Konya, T. Hamaguchi, M. Namba, I. Shimomura, J. I.
Miyagawa, Diabetologia 2007, 50, 1900–1909.
[3] S. Mojsov, G. C. Weir, J. F. Habener, J. Clin. Invest. 1987, 79, 616–619.
[4] J. Schirra, M. Katschinski, C. Weidmann, T. Schafer, U. Wank, R. Arnold, B.
Goke, J. Clin. Invest. 1996, 97, 92–103.
[5] P. J. Shughrue, M. V. Lane, I. Merchenthaler, Endocrinology 1996, 137,
5159–5162.
Activities of the five CYP isoforms (3A4, 2D6, 2C9, 1A2, and 1C19)
were characterized based on their probe reactions. Incubation mix-
tures were prepared in a total volume of 100 mL as follows:
0.2 mgmLÀ1 microsome (human: Xenotech, Lot No. H0610),
NADPH (1 mm), 100 mm phosphate buffer (pH 7.4), probe sub-
strates cocktail (midazolam 10 mm, testosterone 100 mm, dextrome-
thorphan 10 mm, diclofenac 20 mm, phenacetin 100 mm, and meph-
[6] D. J. Drucker, Mol. Endocrinol. 2003, 17, 161–171.
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CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[7] a) R. Mentlein, B. Gallwitz, W. E. Schmidt, Eur. J. Biochem. 1993, 214,
829–835; b) T. J. Kieffer, C. H. McIntosh, R. A. Pederson, Endocrinology
1995, 136, 3585–3596.
K. A. Lyons, H. O. Ok, R. A. Patel, A. N. Petrov, K. A. Pryor, X. Qian, L.
Reigle, A. Woods, J. K. Wu, D. Zaller, X. Zhang, L. Zhu, A. E. Weber, N. A.
Thornberry, Diabetes 2005, 54, 2988–2994.
[8] L. B. Knudsen, J. Med. Chem. 2004, 47, 4128–4134.
[17] M. J. Burk, J. E. Feaster, W. A. Nugent, R. L. Harlow, J. Am. Chem. Soc.
1993, 115, 10125–10138.
[18] B. C. Boren, S. Narayan, L. K. Rasmussen, L. Zhang, H. Zhao, Z. Lin, G. Jia,
V. V. Fokin, J. Am. Chem. Soc. 2008, 130, 8923–8930.
[19] C. H. Wilson, C. A. Abbott, Int. J. Oncol. 2012, 41, 919–932.
[20] J. Schade, M. Stephan, A. Schmiedl, L. Wagner, A. J. Niestroj, H. U.
Demuth, N. Frerker, C. Klemann, K. A. Raber, R. Pabst, S. von Horsten, J.
Histochem. Cytochem. 2008, 56, 147–155.
[21] Z. Otwinowski, D. Borek, W. Majewski, W. Minor, Acta Crystallogr. Sect. A
2003, 59, 228–234.
[22] A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storo-
ni, R. J. Read, J. Appl. Crystallogr. 2007, 40, 658–674.
[23] G. N. Murshudov, P. Skubak, A. A. Lebedev, N. S. Pannu, R. A. Steiner,
R. A. Nicholls, M. D. Winn, F. Long, A. A. Vagin, Acta Crystallogr. Sect. D
2011, 67, 355–367.
[9] D. Kim, L. Wang, M. Beconi, G. J. Eiermann, M. H. Fisher, H. He, G. J.
Hickey, J. E. Kowalchick, B. Leiting, K. Lyons, F. Marsilio, M. E. McCann,
R. A. Patel, A. Petrov, G. Scapin, S. B. Patel, R. S. Roy, J. K. Wu, M. J. Wyv-
ratt, B. B. Zhang, L. Zhu, N. A. Thornberry, A. E. Weber, J. Med. Chem.
2005, 48, 141–151.
[10] E. B. Villhauer, J. A. Brinkman, G. B. Naderi, B. E. Dunning, B. L. Mangold,
M. D. Mone, M. E. Russell, S. C. Weldon, T. E. Hughes, J. Med. Chem.
2002, 45, 2362–2365.
[11] D. J. Augeri, J. A. Robl, D. A. Betebenner, D. R. Magnin, A. Khanna, J. G.
Robertson, A. Wang, L. M. Simpkins, P. Taunk, Q. Huang, S. P. Han, B.
Abboa-Offei, M. Cap, L. Xin, L. Tao, E. Tozzo, G. E. Welzel, D. M. Egan, J.
Marcinkeviciene, S. Y. Chang, S. A. Biller, M. S. Kirby, R. A. Parker, L. G.
Hamann, J. Med. Chem. 2005, 48, 5025–5037.
[12] J. Feng, Z. Zhang, M. B. Wallace, J. A. Stafford, S. W. Kaldor, D. B. Kassel,
M. Navre, L. Shi, R. J. Skene, T. Asakawa, K. Takeuchi, R. Xu, D. R. Webb,
S. L. Gwaltney II, J. Med. Chem. 2007, 50, 2297–2300.
[24] P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr. Sect. D
2010, 66, 486–501.
[13] M. Eckhardt, E. Langkopf, M. Mark, M. Tadayyon, L. Thomas, H. Nar, W.
Pfrengle, B. Guth, R. Lotz, P. Sieger, H. Fuchs, F. Himmelsbach, J. Med.
Chem. 2007, 50, 6450–6453.
[25] X. Zhang, J. Wang, M. Su, Z. Li, J. Li, H. Liu, Chem. Biol. Drug Des. 2012,
80, 843–852.
[14] B. Kuhn, M. Hennig, P. Mattei, Curr. Top. Med. Chem. 2007, 7, 609–620.
[15] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056–
2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
Received: March 4, 2013
Revised: April 19, 2013
[16] G. R. Lankas, B. Leiting, R. S. Roy, G. J. Eiermann, M. G. Beconi, T. Biftu,
C. C. Chan, S. Edmondson, W. P. Feeney, H. He, D. E. Ippolito, D. Kim,
Published online on && &&, 0000
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