Discovery of Highly Polar β-Homophenylalanine Derivatives as Nonsystemic Intestine-Targeted Dipeptidyl Peptidase IV Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01649

Although intensively expressed within intestine, the precise roles of intestinal dipeptidyl peptidase IV (DPPIV) in numerous pathologies remain incompletely understood. Here, we first reported a nonsystemic intestine-targeted (NSIT) DPPIV inhibitor with β-homophenylalanine scaffold, compound 7, which selectively inhibited the intestinal rather than plasmatic DPPIV at an oral dosage as high as 30 mg/kg. We expect that compound 7 could serve as a qualified tissue-selective tool to determine undetected physiological or pathological roles of intestinal DPPIV.

Full text of DOI:10.1021/acs.jmedchem.9b01649

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Brief Article
Discovery of Highly Polar #-homophenylalanine Derivatives as
Non-systemic Intestine-Targeted Dipeptidyl Peptidase IV Inhibitors
Fubao Huang, Mengmeng Ning, Kai Wang, Jia Liu, Wen bo Guan, Ying Leng, and Jianhua Shen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01649 • Publication Date (Web): 20 Nov 2019
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Journal of Medicinal Chemistry
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Discovery of Highly Polar β-homophenylalanine Derivatives as Non-systemic Intestine-Targeted
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Dipeptidyl Peptidase IV Inhibitors
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Fubao Huang§‡, Mengmeng Ning§‡, Kai Wang§, Jia Liu§, Wenbo Guan§, Ying Leng§*, and Jianhua Shen§*
State Key Laboratory of Drug Research, §Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555
Zuchongzhi Road, Shanghai 201203, China.
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China
KEYWORDS Non-systemic intestine-targeted, DPPIV, Inhibitors, β-homophenylalanine, Highly polar
ABSTRACT: Although intensively expressed within intestine, the precise roles of intestinal dipeptidyl peptidase IV (DPPIV) in numerous
pathologies remain incompletely understood. Here, we firstly reported a non-systemic intestine-targeted (NSIT) DPPIV inhibitor with β-
homophenylalanine scaffold, compound 7, which selectively inhibited the intestinal rather than plasmatic DPPIV at an oral dosage as high as
30 mg/kg. We expect that compound 7 could serve as a qualified tissues-selective tool to determine undetected physiological or pathological
roles of intestinal DPPIV.
inhibitors are currently approved to treat type 2 diabetes7, 8. Research
on DPPIV inhibitors’ glucoregulatory mechanisms found that
changes to the incretin hormones pathway, such as preventing the
inactivation of glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropic polypeptide (GIP), and several incretin-
independent pathways collectively contribute to the antidiabetic
INTRODUCTION
Dipeptidyl peptidase IV (DPPIV), also referred as T-cell antigen
CD26, is a 110-kDa glycoprotein that exists in two major isoforms, a
membrane-anchored form and a soluble form1. DPPIV is viewed as
a complex protein and plays important roles in many physiological
processes1. For example, as a serine peptidase, DPPIV can regulate
the catabolism of over 40 bioactive peptides, which are related to
metabolism, nociception, psychoneuroendocrine regulation,
cardiovascular adaptation, and so on2. In addition, it is also involved
in cell adhesion and the immune response through its non-
enzymatic functions via interactions with contiguous membrane or
extra-cellular matrix proteins2. Furthermore, the extensive
distribution of the DPPIV protein further amplifies its complexity.
Soluble DPPIV principally circulates in body fluids, such as plasma,
cerebrospinal fluid and seminal fluid, while the membrane-bound
form can be found in numerous tissues including the lung, intestine,
brain, pancreas, kidney, blood vessels, liver, lymph nodes, and
spleen, and is predominately situated on the surface of epithelial,
endothelial and immune cells3.
actions of DPPIV1, 9, 10
.
Usually, the biological roles of DPPIV will change with its
location and the molecular targets it interacts with2. However, the
specific roles of DPPIV derived from different tissues or cells in
many physiological and pathological processes remain inadequately
understood. Even for the deeply studied hypoglycemic effect, the
precise roles of DPPIV-containing cells and tissues were only
identified by Mulvihill and coworkers in 2017, eleven years after the
first DPPIV inhibitor was marketed. They found that DPPIV from
endothelial and hematopoietic cells contributed to glucose-lowering
actions, while enterocyte DPPIV, although representing substantial
intestinal DPPIV activity, did not produce significant effects on the
plasma DPPIV activity and incretin hormone levels11. Furthermore,
the precise role of enterocyte DPPIV in other physiological
processes is also unknown and previous studies on DPPIV usually
employed systemic inhibitors or conventional gene knockout and
therefore could not accurately reflect the consequences of DPPIV
inhibition in specific tissues. In view of the intensive distribution of
the DPPIV enzyme in the intestine and the high involvement of the
Therefore, because of the variety of its enzymatic and non-
enzymatic functions, the panoply of its bioactive substrates and
binding partners and the universality of its distribution, changes in
DPPIV expression and/or activity are closely associated with a great
number of pathological conditions, for example, metabolic
disorders1, tumors4, autoimmune5 and inflammatory diseases2,
6
.
12,
intestine in a large number of physiological processes3, 13, we
Among them, the relationship between DPPIV and glucose
homeostasis is extensively studied and more than 12 DPPIV
expect to identify a series of non-systemic intestine-targeted (NSIT)
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DPPIV inhibitors that could be used as chemical tools to determine
Page 2 of 7
large enough to tolerate multifarious changes in its substituents
without inducing significantly adverse effect on the pharmacological
activity 16. Therefore, we selected a β-homophenylalanine derivative,
compound 317, as the starting point for the NSIT DPPIV inhibitors
discovery program (Figure 1B), and focused exploration on the
benzothiazole moiety that is in the S2 pocket (Figure 1C)16. There
are three reasons for this choice: first, β-homophenylalanine is a
reliable and widely studied scaffold among DPPIV inhibitors, as
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the biological functions of intestinal DPPIV.
MOLECULAR DESIGN
Currently, NSIT drugs have been designed for various diseases
including inflammatory bowel disease, diabetes mellitus and
obesity 14. A number of approaches have been successively
developed to obtain NSIT drug candidates, resulting in three
categories of NSIT drugs: non-absorbable prodrugs, rapidly
metabolized soft drugs and compounds with physicochemical
properties outside “Lipinskis Rule of Five”14. For instance,
compound 2, an apical sodium-dependent bile acid transporter
(ASBT) inhibitor, is an example of the last class (Figure 1A)15. By
introducing a highly polar and zwitterionic aminodiacid moiety to
the pharmacophores (compound 1), the structural units accounting
for nearly all of the pharmacological activity, the resulting
compound 2 generated moderately increased potency (4-fold) but
substantially decreased the cLogP value, cellular permeability and
portal vein and systemic drug levels after oral administration (Figure
1A)15. Consequently, the introduction of highly polar moieties, also
known as kinetophores that minimally influence the intrinsic
pharmacological activity of pharmacophores but significantly alter
specific physicochemical characteristics, facilitate the non-systemic
targeting of the intestine14.
sitagliptin, the first marketed DPPIV inhibitor, is
a β-
homophenylalanine derivative7; second, these derivatives are
convenient to prepare and the β-homophenylalanine moiety is
commercially available; finally, position 6 of the benzothiazole
moiety seems to be a preferable choice to introduce the
kinetophores (Figure 1B), and a previous study revealed that this
position could tolerate relatively large substituents such as a 2-
morpholinoethyoxyl group17. Collectively, we adjusted the
pharmacokinetic behavior of compound 3 to meet the need of high
intestinal retention by introducing highly polar kinetophores to
position 6 of the benzothiazole moiety (labeled by a blue box in
Figure 1B).
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CHEMISTRY
Scheme 1. Synthesis of Compounds 4-12a.
S
S
S
a
O
N
N
O
S
OH
O
S
S
3a
c
N
O
H
N
O
S
NH2
b
S
3c
+
N
O
S
3b
F
O
O
S
F
F
S
d
S
e
f
O
H3N
Cl
S
N
OH
Br
N
H
N
OH
3d
3e
O
O
O
F
F
O
F
F
O
S
S
n
F
F
O
g
O
O
O
O
S
S
N
H
N
H
N
N
O
n
OH
3f
3g-3i
O
F
F
O
O
F
O
H2N
e
R
S
S
CF3COO
NH3
i
F
h
F
O
O
OH
O
S
HN
R
S
N
N
H
H
n
N
O
F
N
n
O
O
3j-3l
4-12
; 3i, 3l, 6, 9, 12:
n=5
3g, 3j, 4, 7, 10:
;
3h, 3k, 5, 8, 11:
n=1
n=11
OH OH
CF3COO
N
4, 5, 6:
OH
7, 8, 9: R=
R=
R=
10, 11, 12:
SO3
H
OH OH
a Reagents and conditions：(a) NaH, dry THF, rt; (b) dry THF, rt;
(c) nBu-Li, dry THF, -78℃; (d) acetylchloride, EtOH, rt; (e)
HATU, DIPEA, DMF, rt; (f) mCPBA, DCM; (g)K2CO3, DMF, rt;
(h) Pd/C, H2, MeOH, rt; (i) DCM/TFA (v/v = 5:1), rt.
The designed compounds 4−12 were synthesized by using the route
outlined in Scheme 1. Briefly, the attack of 6-(ethoxymethoxy)
benzo[d]thiazole (3a) to the sulfinamide intermediate (3b)
generated the intermediate 3c. After the deprotection of 3c and
condensation with β-homophenylalanine, the pharmacophore (3f)
was obtained from the oxidization of the above condensation
product, 3e. Subsequently, the intermediates 3g-3i were obtained by
a nucleophilic substitution reaction between 3f and benzyl aliphatic
esters containing bromide substitution. The carboxyl group was
exposed under reductive conditions, and then condensed with
different highly polar moieties to obtain compounds 4-12. Detailed
Figure 1. (A) The design of NSIT compounds with high polar
moieties. (B) The design of the NSIT DPPIV inhibitors. The site
where the highly polar moieties were introduced was labeled with
a blue box. (C) The binding mode of β-homophenylalanine
derivatives with DPPIV.
Inspired by the design of compound 2, we attempted to obtain
NSIT DPPIV inhibitors by introducing highly polar kinetophores to
a known systemic DPPIV inhibitor, namely, pharmacophore.
According to the binding modes of DPPIV with known inhibitors,
the S2 binding pocket, formed by Phe357, Ser209, and Arg358, is
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synthetic procedures and structural characterization are provided in
(Papp A to B less than 0.4 nm/s) compared to sitagliptin with
moderate permeability, suggesting a poor ability to penetrate the
membrane of Caco-2 cells (Table 2). A longer linker might facilitate
the corresponding compound become a substrate of efflux
transporters, resulting in a high efflux ratio (compounds 5, 8, and 11).
On the contrary, compound 10 showed the lowest efflux ratio
among tested compounds, indicating that it might be a suitable
substrate of uptake transporters, which might be easy to be absorbed
into the blood. Taken together, these results showed that
compounds 4 and 7 owned excellent potency, decent aqueous
solubility and poor membrane permeability, preferably meeting the
designing goals. Then, they were selected for further study.
Table 2. The result of Caco-2 permeability
1
2
3
4
5
6
7
8
the Supporting Information.
RESULTS
In vitro DPPIV inhibitory potency, aqueous solubility and Caco-2
permeability assays. To cover multiple structural types, we
characterized the kinetophores with sulfonic acid, quaternary
ammonium salt and saccharide fragments, which will present anion,
cation and neutral forms, respectively, under the physiological
conditions. In addition, to minimize the potential adverse effect on
the pre-existing inhibitory activity, these three hydrophilic
fragments were extended far from the benzothiazole moiety by an
aliphatic chain containing 2, 6, or 12 carbon atoms. Consequently,
we synthesized nine compounds in these three categories and tested
their ability to inhibit the DPPIV activity of mouse plasma in vitro.
Table 1. The results of the inhibitory potency of compounds on
mouse plasma DPPIV in vitro.
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10
11
12
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Mean Papp
(nm/s)
Solubility
Efflu
x
Rati
o
Comp
ds
Apical
to
Basal
to
pH 6.8 pH 7.4 pH 7.0
(µM)
(µM)
(μM)
O
O
F
Basal
Apical
CF3COO
NH3
S
F
O
4
5
7
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
0.2
0.2
0.4
0.04
1.1
0.2
6
S
N
O
n
H
F
N
O
R
0.1
0.3
N
H
20 ~
50
8
> 50
> 50
0.02
0.2
5.9
0.01
2.7
295
0.05
13
IC50  SD
Compds
R
n
ClogPa
(nM)
10
11
> 50
> 50
> 50
4
5
1
5
-1.9
-0.8
2.4
1.4  0.5
3.0  0.8
115.7  32.7
1.1  0.3
CF3COO
N
20 ~
50
20 ~
50
> 50
0.2
Sitagli
ptin
6
11
1
-
-
-
2.5
122.4 48.7
7
-2.4
-1.3
1.9
8
5
2.0  0.9
81.8  22.3
2.2  0.4
In vitro stability evaluation in human and mouse intestinal S9
fraction. To explore whether there are the metabolites that
generated within the intestine and could enter systemic circulation
and inhibit systemic DPPIV, we evaluated the intestinal stability of
compounds 4 and 7 in by commercially human and mouse intestinal
S9 fraction. Compared to the positive controls, testosterone and 7-
hydroxycoumarin (7-HC), we were delighted to find that
compounds 4 and 7 showed decent metabolic stability against both
phase I and phase II enzymes in human and mouse intestinal S9
fractions (Table S1), indicating the low possibility of generating
systemic metabolites.
SO3H
9
11
1
10
11
0.7
OH OH
5
1.7
3.5  1.4
OH OH OH
12
11
4.9
0.4
168.1  50.4
Sitagliptin
-
-
14.7  1.8
aCalculated by ChemDraw Professional 16.0.
As shown in Table 1, compounds with the sulfonic acid
substituent (7-9) showed the best inhibitory potency of mouse
plasma DPPIV compared with the compounds containing the other
two high polar substituents (compounds 4-6 and 10-12) when
linked by an aliphatic chain of the same length. The 2 carbon
atom-length aliphatic chain (compounds 4, 7, and 10) might be
the optimal linker, as the inhibitory activity decreased as the
length of the chain increased. In addition, the compounds with
short linkers exhibited negative ClogP values (compounds 4, 5,
7, and 8), suggesting a high hydrophilcity (Table 1). We then
determined the aqueous solubility of the six compounds with
good inhibitory activity under three different conditions (Table
2). The results show that compounds 4, 7, and 10, whose highly
polar moiety was linked by 2 carbon atoms, exhibited decent
aqueous solubility under all three conditions, while a longer
aliphatic chain would interfere with the solubility to some extent
(compounds 8 and 11). Collectively, the compounds linked a polar
moiety by 2 carbon atoms possessed preferable DPPIV inhibitory
potency and aqueous solubility.
In vivo pharmacokinetic study and plasmic and intestinal DPPIV
activity determination. We subsequently determined the plasma
exposure of compounds 4 and 7 after oral administration at 30
mg/kg. Surprisingly, although compounds 4 and 7 had similar Caco-
2 cell permeability, they showed great difference in plasma
concentrations after oral administration. As shown in Figure S1,
compound 4 possessed 45-fold and 38-fold higher plasma
concentration than that of compound 7 at the 2 h and 4 h time
points, respectively.
Furthermore, we characterized the alteration of plasmic and
intestinal DPPIV activity after oral administration of compounds 4
and 7, taking sitagliptin as a positive comparison. Although the
dosage was as high as 30 mg/kg, the plasmic DPPIV activity of the
compound 7-treated group showed a trend similar to that of the
control group, indicating a negligible impact on the plasma DPPIV
enzyme (Figure 2A). However, compound 4 and sitagliptin could
significantly inhibit the plasma DPPIV activity even at the 8 h time
point after oral administration (Figure S3A). Based on the results of
oral plasma exposure, we supposed that the significant plasma
A further Caco-2 permeability evaluation found that the above six
compounds had a much low apparent permeability coefficient
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DPPIV inhibition of compound 4 might result from its high
concentration in the plasma. In the intestine, both the compounds
4-treated, 7-treated and sitagliptin-treated groups showed intensive
DPPIV inhibition in all three selected parts of the intestine (Figure
2B and Figure S3B). Notably, the significant intestinal DPPIV
inhibition of compound 7 could also be reflected by its high
concentration within the intestine (Figure S2).
Page 4 of 7
molecules might harbor low toxicity risks as a result of their
extremely poor systemic exposure. Therefore, coupled with the
good performance of compound 7 in isozyme selectivity and
preliminary toxicity investigations, we believed that it could serve as
a qualified NSIT DPPIV inhibitor to determine the precise roles of
intestinal DPPIV in various physiological and pathological
processes.
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4
5
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For example, the relationship between DPPIV and inflammatory
bowel disease (IBD) has been determined in a vast number of
publications2, 19-22. However, the defined role of DPPIV in the
process of IBD remains unclear, and several mechanisms have been
proposed including the changing of the immune response at
systemic and local levels20, 23 and the degradation of glucagon-like
peptide-2 (GLP-2)19, 24, a potent and specific gastrointestinal growth
factor. Although there are still some inconsistencies, most studies
observed that DPPIV inhibition could lead to the alleviation of IBD6,
24-26, suggesting a potential and novel approach to treat IBD.
However, DPPIV inhibition in all of these studies was
accomplished by a systemic DPPIV inhibitor or nonspecific
DPPIV gene knockout, and, as far as we know, no selective
intestinal DPPIV inhibitor was applied. Because the nidus of IBD
is located within the intestine, illuminating the role of intestinal
DPPIV in the progress of IBD is beneficial for understanding the
pathological process or even for developing a new treatment for
IBD.
A NSIT DPPIV inhibitor could also be used to comprehensively
understand the impact of the DPPIV inhibitor on the gut
microbiota. Recently, Olivares et al. found that vildagliptin, a
systemic DPPIV inhibitor, could prevent the disruption of intestinal
homeostasis in association with modulation of the gut microbiota27.
Very recently, Liao et al. revealed an important alteration of gut
microbiota induced by sitagliptin treatment, indicating a new
hypoglycemic mechanism and an additional benefit of DPPIV
inhibitors28. Considering that sitagliptin and vildagliptin decreased
both systemic and intestinal DPPIV activity27, we could not exclude
that this beneficial effects or the alteration in the composition of the
gut microbiota resulted from the changes of the systemic
environment induced by vildagliptin. In this context, comparing the
effects of systemic and intestinal DPPIV inhibitors on the gut
microbiota is conducive for determing the precise regulatory
mechanisms for gut dysfunctions. Furthermore, with increasing
knowledge of the relationships between intestinal microbiota and
numerous human pathologies, managing the microbial composition
will gradually become an attractive therapeutic approach to different
diseases29-32. Therefore, clarifying the effects of the NSIT DPPIV
inhibitor on the gut microbiota might provide a new method to
regulate the gut microbiota, which may opened new therapeutic
approaches to different dysfunctions.
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Figure 2. The relative DPPIV activity in plasma (A) and the intestine
(B) after oral administration 30 mg/kg of sitagliptin and compound
7 to mice. The intestinal DPPIV activity was tested at 8h after oral
administration. (n = 6). The data are expressed as the mean ± SEM.
**P < 0.01, compared with control groups. Student’s t-test statistical
analysis was used.
Selectivity and preliminary toxicity investigations. As a qualified
chemical tool, isozyme selectivity is essential. After determining the
inhibitory effects of compound 7 on DPP8 and DPP9, we were
delighted to find that compound 7 showed a greater than 2000-fold
selectivity towards these two isozymes (Table S2).
To evaluate the cytotoxic effect of compound 7, human
hepatocellular carcinoma cell line (HepG2), human embryonic
kidney 293 cell line (HEK293) and mouse primary hepatocytes
were treated with compound 7 for 24 h, and cell viability was
measured using the MTT assay. As shown in Figure S4, compound
7 exhibited no obvious cytotoxicity after 24 h of treatment up to a
concentration of 200 µM. For the acute toxicity study, ICR mice
were treated with single doses of compound 7 (1000 mg/kg). All of
the animals survived the 4 days of the experimental period (Figure
S5A). General conditions, such as body weight and clinical and
behavioral symptoms, were recorded during this period, and no
obvious change or death attributable to the toxicity of compound 7
was observed (Figure S5B). Compound 7 was found to be safe at the
dosage of 1000 mg/kg employed in the present study.
CONCLUSION AND DISCUSSION
DPPIV possesses multiple physiological functions and is widely
distributed in body tissues9, 11, 18. The discriminatory contribution of
DPPIV with different cellular sites on glucose homeostasis indicates
that the biological roles of DPPIV are cell or tissues specific11.
However, studies regarding DPPIV were rarely performed
depending on its cellular sites or originating tissues, probably due to
the lack of tissue-selective DPPIV inhibitors. Here, by introducing
high polar moieties to a systemic DPPIV inhibitor, we discovered a
NSIT DPPIV inhibitor, compound 7, which was distributed
throughout the intestine and could selectively inhibit intestinal
DPPIV activity while having no significant effect on the plasmatic
DPPIV activity after oral administration at a dose as high as 30
mg/kg. In addition, compared with systemic compounds, NSIT
Finally, whether the marketed DPPIV inhibitors could reduce the
cardiovascular (CV) events and mortality in type 2 diabetic patients
is a pending question33-35. Except for linagliptin, most marketed
DPPIV inhibitors could not produce significantly beneficial effects
on the risk of CV events, and even worse, saxagliptin was found to be
responsible for an increased risk of heart failure36, 37. Since CV
complications are the main cause of death in type 2 diabetic patients,
it is worthwhile to study the potential risks of DPPIV inhibitors
38
thoroughly . Currently, several mechanisms have been suggested
but all are focused on the consequences of systemic DPPIV
inhibition, such as the increased level of stromal cell-derived factor-
1 (SDF-1) in circulatory system38, the sympathetic activation39 and
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the inhibition of luminal sodium-hydrogen exchanger 3, (NHE3)40.
135.75 , 123.37 , 119.91 (d, J = 5.9 Hz), 119.64 (dd, J = 19.4, 5.1 Hz),
117.09 (q, J = 299.3 Hz), 115.89 , 106.18 (dd, J = 29.0, 20.2 Hz),
106.16 , 67.52 , 63.68 , 56.73 , 52.61 , 52.58 , 52.56 , 47.28 , 46.50 ,
46.46 , 36.72 , 35.12 , 33.60 , 33.31 , 32.99 . HRMS (ESI): m/z [M-
1
2
3
4
5
6
7
8
However, as shown in Figure 2, the systemic DPPIV inhibitor
simultaneously inhibited plasmic and intestinal DPPIV activity even
at the 8 h time point after oral administration, indicating that it is
hard to distinguish the effects of plasmic and intestinal DPPIV on
the risk of CV. Therefore, compound 7, a NSIT DPPIV inhibitor,
might contribute to the determination of the specific roles of plasmic
and intestinal DPPIV in the pathology of CV or other disorders.
Collectively, by adjusting the physicochemical properties of a
systemic DPPIV inhibitor out of the Lipinski's rule of five, we firstly
reported a NSIT DPPIV inhibitor, compound 7. And, the studies
regarding the application of this compound are undergoing.
+
CF3COOH-CF3COO-]+ calculated for C29H37F3N5O5S2 ,
656.2183; found, 656.2192. HPLC purity, 96%; tR, 4.86 min.
(R)-4-((1,1-dioxido-4-(6-(2-oxo-2-((2-
sulfoethyl)amino)ethoxy)benzo[d]thiazol-2-yl)tetrahydro-2H-
thiopyran-4-yl)amino)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-
aminium trifluoroacetate (7). Starting with 3j (1 g, 1.5 mmol) and
2-aminoethane-1-sulfonic acid (288 mg, 2.3 mmol), 7 was obtained
by using the process of the preparation of compound 4 (white solid,
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1
41%). H NMR (500 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.41 – 8.30
EXPERIMENTAL SECTION
(m, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.69 – 7.25 (m, 6H), 7.14 (d, J =
8.7 Hz, 1H), 4.53 (s, 2H), 3.69 – 3.63 (m, 1H), 3.46 – 3.34 (m, 6H),
3.18 (d, J = 11.7 Hz, 2H), 3.01 – 2.78 (m, 4H), 2.67 – 2.57 (m, 4H).
13C NMR (126 MHz, DMSO-d6) δ 174.71 , 170.83 , 167.08 , 158.47
(q, J = 31.1 Hz), 156.17 (dd, J = 243.3, 9.9 Hz), 155.59 , 148.50 (dt,
J = 248.2, 13.9 Hz), 147.34 , 146.04 (dd, J = 242.0, 11.8 Hz), 135.79
, 123.36 , 120.11 (d, J = 18.4 Hz), 119.71 (dd, J = 19.3, 5.2 Hz),
117.21 (d, J = 299.8 Hz), 116.06 , 106.24 , 106.01 (dd, J = 29.0, 21.4
Hz), 67.66 , 56.79 , 50.11 , 47.55 , 46.60 , 46.56 , 37.11 , 35.23 , 33.59
, 33.42 , 31.36 . HRMS (ESI): m/z [M-CF3COO-]+ calculated for
General Chemistry. All reagents were purchased from commercial
suppliers and used without further drying or purification unless
otherwise stated. Yields were not optimized. 1H NMR and 13C NMR
spectra were recorded on a Bruker AC400 or a Bruker AC500 NMR
spectrometer using tetramethylsilane (CDCl3) or deuterium
reagent itself (DMSO-d6) as an internal reference. Low-resolution
mass spectra were determined on an Agilent liquid-chromatography
mass spectrometer system that consisted of an Agilent 1260 infinity
LC coupled to Agilent 6120 Quadrupole mass spectrometer (ESI).
High-resolution mass spectra were conducted on a triple TOF
5600+ MS/MS system (AB Sciex, Concord, Ontario, Canada) in the
negative or positive ESI mode. The purity of test compounds are
determined by HPLC (Agilent ChemStation, Purospher® STAR RP-
18 endcapped (2 µm), 2.1×50 mm, 30 °C, UV 240 nM) and the
mobile phase was method A in Table S3. All the assayed compounds
are purified by preparative liquid chromatograph (Instrument:
Unimicro Easysep-1010 series LC, UV 254 nm, 25 °C; Column:
Agilent Prep-C18 10 μm, 21.2 × 250 mm, the mobile phase was
methods B-D in Table S3) and possess a more than 95% purity.
Column chromatography was performed on silica gel (200−300
mesh) or with pre-packed silica cartridges (4-40g) from Bonna-
Agela Technologies Inc. (Tianjin, China) and eluted with a
CombiFlash@ Rf 200 from Teledyne Isco, and preparative TLC was
performed on HSGF 254 (0.4−0.5 mm thickness; Yantai Jiangyou
Company, Yantai, Shangdong, China).
+
C26H30F3N4O8S3 , 679.1172; found, 679.1179. HPLC purity, 98%;
tR, 5.37 min.
The detailed experimental experiment procedures and other
designed compounds were synthesized by following a similar
procedure (Supporting Information).
ASSOCIATED CONTENTS
Supporting Information
The materials are available free of charge via the Internet at
http://pubs.acs.org.
DPPIV inhibitory effects of compounds in vitro, the solubility test,
Caco-2 permeability assay, the stability test in human and mouse
intestinal S9 fraction, in vivo pharmacokinetic study, determination
of plasma and intestinal DPPIV activity in vivo, DPP8 and DPP9
inhibitory effect of compounds in vitro, in vitro cytotoxicity test by
MTT assay, acute toxicity study of compound 7 in mice, synthetic
chemistry and abbreviations. (PDF)
(R)-4-((1,1-dioxido-4-(6-(2-oxo-2-((2-
(trimethylammonio)ethyl)amino)ethoxy)benzo[d]thiazol-2-
yl)tetrahydro-2H-thiopyran-4-yl)amino)-4-oxo-1-(2,4,5-
Molecular formula strings (CSV)
trifluorophenyl)butan-2-aminium trifluoroacetate (4). To
a
AUTHOR INFORMATION
solution of 3j (1 g, 1.5 mmol) in DMF was successively added the
HATU (1.1 g, 3.0 mmol), DIPEA (784 μL, 4.5 mmol) and the
trifluoroacetate of 2-amino-N,N,N-trimethylethan-1-aminium
trifluoroacetate (759 mg, 2.3 mmol). The mixture was stirred for 2h
at room temperature. The solvent was directly evaporated in vacuo
and the residue was redissolved by the mixed solution of DCM and
TFA (V/V = 5:1). After stirring overnight, the mixture was directly
evaporated in vacuo and the residue was purified by preparative
liquid chromatograph to give 4 (white solid, 464 mg, yield 35%). 1H
NMR (500 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.52 (t, J = 5.9 Hz, 1H),
8.06 – 7.95 (m, 3H), 7.88 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 2.6 Hz,
1H), 7.58 (td, J = 10.3, 7.0 Hz, 1H), 7.53 – 7.46 (m, 1H), 7.18 (dd,
J = 9.0, 2.6 Hz, 1H), 4.60 (s, 2H), 3.69 (dq, J = 12.7, 6.9 Hz, 1H),
3.58 (q, J = 6.1 Hz, 2H), 3.52 – 3.41 (m, 4H), 3.24 – 3.16 (m, 2H),
Corresponding author
*(Y.L.) Phone: +86-21-50806059. E-mail: yleng@simm.ac.cn
*(J.S.) Phone: +86-21-20231969. E-mail: jhshen@simm.ac.cn
Author contribution
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOELEDGEMENTS
This work was supported by State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM1903ZZ-02).
13
3.09 (s, 9H), 2.99 – 2.74 (m, 4H), 2.70 – 2.52 (m, 4H). C NMR
(126 MHz, DMSO-d6) δ 174.60 , 170.45 , 168.18 , 157.99 (q, J =
31.5 Hz), 156.14 (dd, J = 243.1, 9.3 Hz), 155.50 , 148.33 (dt, J =
248.2, 14.5 Hz), 147.31 , 145.98 (ddd, J = 242.7, 13.6, 3.9 Hz),
ABBREVIATIONS
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Journal of Medicinal Chemistry
Page 6 of 7
(GSK2330672) for treatment of type 2 diabetes. J. Med. Chem. 2013, 56,
5094-5114.
IC50, half maximal inhibitory concentration; DCM,
1
2
3
4
5
6
7
8
dichloromethane; EA, ethyl acetate; THF, tetrahydrofuran; DMF,
N,N-dimethyl formamide; NMR, nuclear magnetic resonance;
HPLC, high performance liquid chromatography; ESI, electron
spray ionization; CH3CN, acetonitrile; UV, under voltage; FA,
formic acid; TLC, thin-layer chromatography; NaH, sodium
hydride; THF, tetrahydrofuran; rt, room temperature; nBu-Li, n-
butyllithium; EtOH, ethanol; HATU, 2-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA,
N,N-Diisopropylethylamine; mCPBA, 3-chloroperoxybenzoic acid;
K2CO3, potassium carbonate; MeOH, methanol; TFA,
trifluoroacetic acid; NH4Cl, ammonium chloride; MgSO4,
magnesium sulfate; EDCI, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; DMAP, dimethylaminopyridine.
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