Design, synthesis, and pharmacological evaluation of highly potent and selective dipeptidyl peptidase-4 inhibitors

Article abstract of DOI:10.1002/ardp.201500082

The optimization of a series of fused β-homophenylalanine inhibitors of dipeptidyl peptidase-4 (DPP-4) is described. Modification on the P2-binding moiety of 6 (IC₅₀ = 10 nM) led to the discovery of β-homophenylalanine derivatives containing pyrrolidin-2-ylmethyl amides. The introduction of a sulfamine in the meta position of the phenyl ring improved the potency against DPP-4 (6-12-fold increase). Compound 14k showed DPP-4 inhibitory activity with an IC₅₀ value of 0.87 nM. Meanwhile, in vivo experiments exhibited that 14h had an efficiency comparable to sitagliptin at the dose of 10 mg/kg.

Full text of DOI:10.1002/ardp.201500082

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
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Full Paper
Design, Synthesis, and Pharmacological Evaluation of Highly
Potent and Selective Dipeptidyl Peptidase-4 Inhibitors
Tao Jiang, Yuren Zhou, Jianming Zhu, Zhuxi Chen, Peng Sun, Qiang Zhang, Zhen Wang, Qiang Shao,
Xiangrui Jiang, Bo Li*, Heyao Wang*, Weiliang Zhu, and Jingshan Shen
Drug Discovery and Design Center, Key Laboratory for Receptor Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai, China
The optimization of a series of fused b-homophenylalanine inhibitors of dipeptidyl peptidase-4 (DPP-4) is
described. Modification on the P2-binding moiety of 6 (IC50 ¼ 10 nM) led to the discovery of b-
homophenylalanine derivatives containing pyrrolidin-2-ylmethyl amides. The introduction of a sulfamine
in the meta position of the phenyl ring improved the potency against DPP-4 (6–12-fold increase).
Compound 14k showed DPP-4 inhibitory activity with an IC50 value of 0.87 nM. Meanwhile, in vivo
experiments exhibited that 14h had an efficiency comparable to sitagliptin at the dose of 10 mg/kg.
Keywords: b-Homophenylalanine / DPP-4 / Drug design / Inhibitors / P2-binding moiety
Received: March 2, 2015; Accepted: March 6, 2015
DOI 10.1002/ardp.201500082
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Compound 6, a DPP-4 inhibitor which was discovered by
structure-based drug design in our laboratory, showed potent
Introduction
Secreted from the L cells of the small intestine [1], GLP-1
increases glucose-induced insulin secretion while it decreases
glucagon secretion [2]. In addition, it also inhibits acid
secretion and the gastric emptying in the stomach, together
effects provide benefits for glycemic control [3]. However,
GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) in
vivo, which cleaves a dipeptide from the N-terminus of GLP-1
to give the inactive form of GLP-1 [4]. Therefore, inhibitors of
DPP-4 would prolong the half-life of active GLP-1 and
enhance the glucose-lowering effects [5], contributing to
their potential to be anti-diabetic agents.
To date, a range of structurally diverse DPP-4 inhibitors
have been reported, some of which have now been launched
onto the market. Among them are sitagliptin (1) [5],
vildagliptin (2) [6], saxagliptin (3) [7], alogliptin (4) [8], and
linagliptin (5) [9] (Fig. 1).
inhibitory activity, excellent selectivity, and good efficacy in
an oral glucose tolerance test (OGTT) in C57BL/6 mice. The
three-dimensional (3D) structural mode of inhibitors 6 to DPP-
4 (Fig. 2) showed that the fused phenyl subunit which
occupied the S1 hydrophobic pocket and the b-amino group
which stabilized the binding site of DPP-4 by forming four
hydrogen bonding interactions are essential for their
inhibitory activity against DPP-4 [1]. However, there is a large
unoccupied space around the P2-binding moiety (the fused
heterocyclic ring). We expected that modification on these
parts could generate a new series of DPP-4 inhibitors. We
initially performed a replacement of the fused heterocyclic
ring with
a chainlike N-substituted ethylenediamine to
quickly explore the feasibility of modifying the P2-
binding moiety of 6. The inhibitory activity of this series
was moderate, which indicated a high tolerance for various
functional groups at that range of the binding site of DPP-4.
Reducing the number of rotatable bonds and increasing the
rigidity of the molecules would probably improve the PK/PD
Correspondence: Dr. Xiangrui Jiang, Drug Discovery and Design
Center, Key Laboratory for Receptor Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China.
E-mail: jiangxiangrui@simm.ac.cn
Fax: þ86-21-20231000-2407
*Additional correspondence: Dr. Bo Li, E-mail: boli@simm.ac.cn;
Heyao Wang, E-mail: hywang@simm.ac.cn
Tao Jiang, Yuren Zhou, and Jianming Zhu contributed equally to
this work.
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Scheme 1. Synthesis of intermediates 9a–c. Reagents and
conditions: (i) pyridine, MsCl, and CH2Cl2.
Figure 1. DPP-4 inhibitors.
profiles of molecules [10–12]. Meanwhile, inspired by the N-
acylpyrrolidine moiety in the structure of vildagliptin (2) and
saxagliptin (3), the chainlike linker was replaced with a
pyrrolidine. Furthermore, to explore the substitution effects
on the aromatic ring, the 3D binding modes of compounds
14d and 14h to DPP-4 (from 2AJL) were generated based on
docking simulations. According to the information from 3D
binding mode, various substituents were introduced on the
aromatic ring. After optimization of this series, most of them
emerged as showing excellent enzyme inhibitory activity and
selectivity for DPP-4 over two dipeptidyl peptidase family
homologues: DPP-8 and DPP-9. Inhibitors were then advanced
to in vivo studies to assess the utility of series; 14h and 14o
showed efficacy similar to that of sitagliptin in the OGTT.
giving sulphonamides 8a–c, which were respectively con-
verted to 9a–c by hydrolysis [13].
As shown in Scheme 2, sulfonylation of benzoic acids 7i–k
produced 8i–k, respectively, which were coupled with
according amines to give the corresponding sulfonamide
derivatives 9i–r [14].
Compounds 14a–r were synthesized according to the routes
described in Scheme 3. Corresponding amines were coupled
with carboxylic acid derivatives 9a–r which were commercially
available or synthesized (Schemes 1 and 2) giving intermedi-
ates 10a–r, respectively. Subsequently, removal of the tert-
butoxycarbonyl group under standard conditions produced
11a–r. Compound 12 coupled with 11a–r gives 13a–r, which
was de-protected to yield target compounds 14a–r.
In vitro DPP-4 inhibitory activity and selectivity
The inhibitory activity of compounds 14a–r on DPP-4 was
evaluated in vitro (Table 1). The chain-like ethylenediamine
derivatives (14a–c) showed moderate inhibitory activity
against DPP-4. When the chain-like linker was replaced with
2-aminomethyl pyrrolidine, 14d and 14e also exhibited
moderate activity (IC50 ¼ 34 and 26 nM, respectively). Within
the 2-aminomethyl pyrrolidine analogs, replacing the phenyl
ring with thiazolyl ring produced compound 14f, which
showed a slight increase in inhibitory activity over 14d or 14e;
while this increase was remarkable in the pyridyl analog (14g).
Such increase was also seen in compounds 14h and 14i, in
which the aminosulfonyl was replaced by sulfone group or
sulfonamide group. To explain this result, the 3D binding
modes of 14d and 14h to DPP-4 were generated. The binding
modes showed that the sulfone group of 14h forms a
hydrogen bonding interaction with the N–H atom of Q553 in
DPP-4, while such hydrogen bond interaction cannot
formed between the sulfone group of 14d and the backbone
N–H atom of Q553 (Figs. 3 and 4). As the sulfonyl group on
the phenyl ring can form a hydrogen bonding interaction
with DPP-4 (Fig. 4), we select sulfonyl group as our
preferred substituent of the phenyl ring. In light of the
findings noted above, a series of sulfonamide derivatives
were synthesized according to Scheme 3. From comparison
of 14i versus 14j versus 14k, 14l versus 14m versus 14n,
Results and discussion
Chemistry
As shown in Scheme 1, commercially available compounds 7a–
c reacted with methanesulfonyl chloride in dichloromethane
Figure 2. Three-dimensional structural mode of inhibitor 6 to
DPP-4 derived from the docking simulations. This image was
generated using the PyMOL program.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
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Scheme 2. Synthesis of intermediates 9i–r. Reagents and conditions: (i) ClSO3H, 150°C; (ii) for 8i–k, NH4OH, 0°C; for 8l–n,
diethylamine, EtOAc, 0°C; for 8o, N-methyl piperazine, acetone, 0°C; for 8p, piperidine, NEt3, CH2Cl2, rt; for 8q, morpholine, NEt3,
CH2Cl2, rt; for 8r, cyclopropylamine, NEt3, CH2Cl2, rt.
introduction of fluorine atom on the phenyl moiety was of
influence on improving inhibitory activities. From comparison
of 14i versus 14l, 14j versus 14m, and 14k versus 14n,
ethylation of the sulfonamide decreases the inhibitory
activities. Even so, all the sulfonamide derivatives exhibited
excellent DPP4 inhibitory activity, with IC50 values ranging
from sub- to single-digit nanomolar. Steric and electronic
effects did not appear to affect the binding activity to any
appreciable extent, as small alkyl amides (14i–n), cyclicamides
(14o–r) represented in this group all displayed comparable
activity.
From the selectivity profiles (Table 2), most of the compounds
had more than 2500-fold selectivity for DPP-4 over DPP-8 and
DPP-9. Meanwhile, compounds 14h–k and 14o had minimal
hERG liabilities (IC50 > 20 mM). High selectivity and good
safety profiles improve the suitability of these compounds
for further assessment in animal models.
In vivo evaluations
Based on in vitro potency, selectivity, and hERG liability
analysis, compounds 14h, 14k, and 14o were selected to
evaluate the in vivo efficacy by acute OGTT in C57BL/6 mice.
Scheme 3. Synthesis of compounds 14a–r. Reagents and conditions: (i) for 9a–c, N-Boc-ethylenediamine, HOBT, EDC, DIPEA, DMF, rt;
for 9d–r, (S)-2-(aminomethyl)-1-(tert-butoxycarbonyl)pyrrolidine, HOBT, EDC, DIPEA, DMF, rt; (ii) CF3COOH, CH2Cl2, rt; (iii) HATU,
NEt3, DMF, rt; (iv) CF3COOH, CH2Cl2, rt.
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Table 1. DPP-4 inhibitory activity of compounds 14a–r.
Compounds
Ar
IC50 (nM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
14q
14r
3-(NHSO2CH3)-ph
2-F-5-(NHSO2CH3)-ph
3-(NHSO2CH3)-4-Cl-ph
2-F-5-[NHSO2(CH2)2CH3]-ph
2,6-di-F-5-[NHSO2(CH2)2CH3]-ph
5-thiazole
41.1 ꢀ 3.0%a)
52.4 ꢀ 3.4%a)
57.4 ꢀ 1.5%a)
33.9 ꢀ 7.5
26.3 ꢀ 3.5
50.5 ꢀ 13%a)
5.13 ꢀ 1.26
1.06 ꢀ 0.37
1.72 ꢀ 0.22
1.40 ꢀ 0.18
0.87 ꢀ 0.14
8.20 ꢀ 1.34
6.99 ꢀ 1.30
3.48 ꢀ 0.99
1.10 ꢀ 0.24
4.26 ꢀ 2.20
3.95 ꢀ 1.78
5.52 ꢀ 3.03
19.0
3-pyridine
3-(SO2Me)-ph
3-(SO2NH2)-ph
3-(SO2NH2)-4-F-ph
2,4-di-F-5-(SO2NH2)-ph
3-(SO2NEt2)-ph
3-(SO2NEt2)-4-F-ph
2,4-di-F-5-(SO2NEt2)-ph
3-[SO2N(CH2CH2)2NCH3]-ph
2,4-di-F-5-[SO2N(CH2)5]-ph
2,4-di-F-5-[SO2N(CH2CH2)2O]-ph
2,4-di-F-5-[SO2NH(CH2)3]-ph
–
Sitagliptin
a) Inhibition ratio (%) at 100 nM.
The compounds were administrated at a dose of 10 mg/kg in
water 1 h prior to a glucose challenge (5 g/kg). Blood glucose
was monitored at different time intervals from 0 to 80 min.
Compared to sitagliptin, compound 14k showed almost no
effect on glucose excursion, while compounds 14h and 14o
showed comparable glucose lowering effect at 10 mg/kg
dose: 14k (12%), 14h (40%), 14o (34%) versus sitagliptin (41%,
Fig. 5).
Conclusions
In summary, based on the analysis of large volume of crystal
structure data available in the protein data bank (PDB), we
designed, synthesized, and evaluated a series of novel fused
b-homophenylalanine derivatives containing pyrrolidin-2-
ylmethyl amides as potent and selective DPP-4 inhibitors.
Most of them showed excellent inhibitory activity against
DPP-4 and good selectivity over DPP-8 and DPP-9. Moreover,
14h exhibited comparable in vivo efficiency with sitagliptin at
the dose of 10 mg/kg.
Figure 3. Three-dimensional structural modes of inhibitor 14d
to DPP-4 derived from the docking simulations. This image was
generated using the PyMOL program.
Figure 4. Three-dimensional structural modes of inhibitor 14h
to DPP-4 derived from the docking simulations. This image was
generated using the PyMOL program.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
Dipeptidyl Peptidase-4 Inhibitors
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Table 2. Inhibitory activities of selected compounds against DPP-4, DPP-8, DPP-9, and hERG.
Compounds
DPP-4 (IC50, nM)
DPP-8a) (%)
DPP-9a) (%)
hERG (IC50, mM)
14g
14h
14i
14j
14k
5.13 ꢀ 1.26
1.06 ꢀ 0.37
1.72 ꢀ 0.22
1.40 ꢀ 0.18
0.87 ꢀ 0.14
8.20 ꢀ 1.34
6.99 ꢀ 1.30
3.48 ꢀ 0.99
1.10 ꢀ 0.24
4.26 ꢀ 2.20
3.95 ꢀ 1.78
5.52 ꢀ 3.03
32.7
24.3
34.3
31.1
28.2
47.0
36.4
68.7
9.97
2.19
2.92
NA
36.2
25.6
50.5
60.6
50.2
37.5
49.8
59.9
36.1
30.1
46.9
NA
NA
>20
>20
>20
>20
4.61
2.60
3.46
>20
NA
14l
14m
14n
14o
14p
14q
14r
NA
NA
NA, not available.
a)Inhibition ratio (%) at 10 mM.
Experimental
Chemistry
Compound synthesis
All reactions were carried out under a static atmosphere of
nitrogen, and stirred magnetically unless otherwise noted. All
solvents and reagents were obtained from commercial
suppliers and used without further drying or purification.
All reaction mixtures were monitored using thin-
layer chromatography (TLC) on silica gel F-254 TLC plates.
Nuclear magnetic resonance (NMR) spectroscopy was
recorded on a Bruker AMX-300 or AMX-400 or AMX-500
NMR using Me4Si as an internal standard. Chemical shifts (d)
are given in parts per million (ppm). Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). EI-MS spectra were obtained
on a Finnigan MAT95 spectrometer, and ESI-MS spectra were
obtained on a Krats MS 80 mass spectrometer. All final
compounds were purified to >95% purity by HPLC analysis.
All the final compounds exist as a mixture of amide
rotamers (hindered rotation around the nitrogen–carbonyl
bond). Severe overlap of signals does not permit unequivocal
assignment of each rotamer. Complexity due to 19F spin–spin
coupling does not permit assignment of all 13C resonances,
therefore, 13C data are not presented.
General procedure for the synthesis of sulphonamides 9a–c
Example: Synthesis of 3-(methylsulfonamido)benzoic acid
(9a): To
a solution of methyl 3-amino-benzoate (1.0 g,
Figure 5. Effect of compounds on plasma glucose (a) and
baseline (0 min)-adjusted AUC0–80 min of plasma glucose (b) after
an oral glucose load in glucose tolerance test for C57BL/6 mice.
Compounds or water (control) was administered 1 h prior to
an oral dextrose challenge (5 g/kg) after an overnight fast.
The glucose AUC was determined from 0 to 80 min. Data are
represented as mean ꢀ SEM (n ¼ 6–8), ***p < 0.001, Student’s
t-test.
6.6 mmol) and pyridine (1.15 g, 14.5 mmol) in dichlorome-
thane (20 mL) at 0°C under a nitrogen atmosphere, meth-
anesulfonyl chloride (1.1 mL, 14.5 mmol) was slowly added.
After completion of the reaction as confirmed by TLC, the
solution was diluted with dichloromethane, washed sequen-
tially with 1 M hydrochloric acid aqueous solution and brine,
dried over Na2SO4, and the solvent was removed under
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vacuum. The crude product was used in the next step without
further purification. A solution of the residue and lithium
hydroxide (0.5 g, 12 mmol) in methanol (20 mL) and water
(20 mL) was stirred for 18 h. The residue after removal of
methanol was dissolved in 1 N hydrochloric acid and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over Na2SO4, and removed under vacuum to get 8a as an oil
(0.8 g, 56%). 1H NMR (300 MHz, CDCl3): d 2.95 (s, 3H), 7.35–7.42
(m, 1H), 7.47–7.52 (m, 1H), 7.75–7.82 (m, 2H). MS m/e 214 [MꢁH].
3-(N,N-Diethylsulfamoyl)benzoic acid (9l)
1H NMR (300 MHz, CDCl3): d 1.15 (t, 6H), 3.29 (q, 4H), 7.64 (t,
1H), 8.07 (dt, 1H), 8.29 (dt, 1H), 8.54 (t, 1H). MS m/e 256 [M–H].
3-(N,N-Diethylsulfamoyl)-4-fluorobenzoic acid (9m)
1H NMR (300 MHz, CDCl3): d 1.16 (t, 6H), 3.38 (q, 4H), 7.29 (t,
1H), 8.26–8.32 (m, 1H), 8.66 (dd, 1H). MS m/e 274 [MꢁH].
3-((4-Methylpiperazin-1-yl)sulfonyl)benzoic acid (9o)
1H NMR (300 MHz, CDCl3): d 2.20 (s, 3H), 2.43–2.48 (m, 4H),
2.90–2.98 (m, 4H), 7.79 (t, 1H), 7.96 (dt, 1H), 8.17 (t, 1H), 8.23
(dt, 1H). MS m/e 283 [MꢁH].
2-Fluoro-5-(methylsulfonamido)benzoic acid (9b)
1H NMR (300 MHz, CDCl3): d 2.90 (s, 3H), 7.03–7.12 (m, 1H),
7.41–7.51 (m, 1H), 7.65–7.72 (m, 1H). MS m/e 232 [MꢁH].
2,4-Difluoro-5-(piperidin-1-ylsulfonyl)benzoic acid (9p)
1H NMR (300 MHz, DMSO-d6): d 1.35–1.52 (m, 6H), 3.00–3.08
(m, 4H), 7.73 (t, 1H), 8.21 (t, 1H). MS m/e 304 [MꢁH].
4-Chloro-3-(methylsulfonamido)benzoic acid (9c)
1H NMR (300 MHz, CDCl3): d 3.02 (s, 3H), 7.42–7.53 (m, 1H),
7.75–7.81 (m, 1H), 7.91–7.96 (m, 1H). MS m/e 250 [MꢁH].
2,4-Difluoro-5-(morpholinosulfonyl)benzoic acid (9q)
1H NMR (300 MHz, DMSO-d6): d 3.01–3.09 (m, 4H), 3.58–3.66
(m, 4H), 7.76 (t, 1H), 8.21 (t, 1H). MS m/e 306 [MꢁH].
General procedure for the synthesis of intermediates 8i–k
Example: Synthesis of 3-(chlorosulfonyl)benzoic acid (8i): A
mixture of benzoic acid (2 g, 16.4 mmol) in chlorosulfonic acid
(5 mL) was heated to 150°C in an oil bath for 2 h. After
completion of the reaction as confirmed by TLC, the reaction
mixture was slowly poured over ice and filtered. The solid was
dried in vacuo to yield intermediates 8i as white solid (3 g,
83%). 1H NMR (300 MHz, CDCl3): d 7.80 (t, 1H), 8.30 (dt, 1H),
8.49 (dt, 1H), 8.78 (t, 1H). MS m/e 219 [MꢁH].
5-(N-Cyclopropylsulfamoyl)-2,4-difluorobenzoic acid (9r)
1H NMR (300 MHz, DMSO-d6): d 0.29–0.56 (m, 4H), 2.18–2.31
(m, 1H), 7.70 (t, 1H), 8.30 (t, 1H). MS m/e 276 [MꢁH].
General procedure for the synthesis of compounds 10a–r
A
mixture of corresponding acid (9a–r, 1 mmol), EDCI
(1.5 mmol), HOBT (1.5 mmol), and 4-methylmorpholine
(3 mmol) in DMF was stirred at room temperature for 10 min,
then a solution of corresponding amine (1 mmol) in DMF was
added, and stirring was continued overnight. The solution was
diluted with of ethyl acetate, washed sequentially with 1 M
hydrochloric acid aqueous solution, saturated aqueous sodium
bicarbonate solution and brine, dried over Na2SO4, and the
solvent was removed under vacuum. The crude product was
used in the next step without further purification.
3-(Chlorosulfonyl)-4-fluorobenzoic acid (8j)
1H NMR (300 MHz, CDCl3): d 7.48 (t, 1H), 8.47–8.54 (m, 1H), 8.74
(dd, 1H), 9.99 (br, 1H). MS m/e 237 [MꢁH].
5-(Chlorosulfonyl)-2,4-difluorobenzoic acid (8k)
1H NMR (300 MHz, CDCl3): d 7.17–7.29 (m, 1H), 8.69–8.80 (m,
1H). MS m/e 255 [MꢁH].
General procedure for the synthesis of intermediates 9i–r
Sulfonyl chloride derivative 8i or 8j or 8k (3.99 mmol) was
added gradually to a mixture of amine (12 mmol) in EtOAc
with stirring at 0°C. The reaction mixture was stirred at room
temperature. After completion of the reaction as confirmed
by TLC, the reaction mixture was dissolved in dichloro-
methane and extracted with 10% NaOH. After the aqueous
layer was acidified with 2 N HCl, the precipitate was collected
by filtration, washed with H2O, and dried in vacuo to give the
desired products (9i–r), which were carried forward without
further purification.
General procedure for the synthesis of compounds 11a–r
TFA was added to a stirred solution of 10a–r in CH2Cl2. The
solution was stirred at room temperature for 0.5 h and
the CH2Cl2/TFA solvent was removed under reduced pressure.
The residue was diluted with CH2Cl2 and the solvent
was removed once again under reduced pressure. The
resultant amine was used in the next step without further
purification.
General procedure for the synthesis of compounds 13a–r
To a solution of the intermediate 12 (0.35 mmol) in DMF, TEA
(1.75 mmol) and HATU (0.53 mmol) were added. After 2 min, a
solution of the resultant amine (11a–r, 0.42 mmol) obtained in
the previous step in DMF was added. After the completion of
the reaction as confirmed by TLC, water was added to the
reaction mixture. The crude product was extracted with ethyl
acetate. The aqueous layer was washed with ethyl acetate.
The combined organic extract was dried over Na2SO4 and the
solvents were removed in vacuo to afford the crude
3-Sulfamoylbenzoic acid (9i)
1H NMR (300 MHz, DMSO-d6): d 7.50 (s, 2H), 7.71 (t, 1H), 8.04
(dt, 1H), 8.13 (dt, 1H), 8.38 (t, 1H), 13.44 (br, 1H). MS m/e 200
[MꢁH].
4-Fluoro-3-sulfamoylbenzoic acid (9j)
1H NMR (300 MHz, DMSO-d6): d 7.21–7.24 (m, 1H), 7.37–7.41
(m, 1H), 7.55–7.59 (m, 1H). MS m/e 218 [M–H].
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compound, which was used in the next step without further
purification.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)thiazole-5-
carboxamide (14f)
General procedure for the synthesis of compounds 14a–r
To a stirred solution of 13a–r in CH2Cl2, TFA was added. The
solution was stirred at room temperature. After completion
of the reaction, the CH2Cl2/TFA solvent was removed under
reduced pressure. The residue was purified by column
chromatography to afford target compounds.
1H NMR (300 MHz, CD3OD) d 1.88–2.15 (m, 6H), 2.26–2.43 (m,
1H), 2.60–3.12 (m, 4H), 3.42–3.67 (m, 5H), 3.99–4.42 (m, 1H),
7.06–7.30 (m, 2H), 8.38 (d, 1H), 9.13 (d, 1H). HRMS (ESIþ) calcd.
for C21H25F2N4O2S (MþH)þ m/e, 435.1661; found 435.1663.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)nicotinamide
(14g)
N-(2-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanamido)ethyl)-3-(methylsulfonamido)-
benzamide (14a)
1H NMR (300 MHz, CD3OD) d 1.75–2.26 (m, 7H), 2.33–2.48 (m,
1H), 2.66–2.92 (m, 2H), 3.11–3.20 (m, 1H), 3.38–3.60 (m, 5H),
4.02–4.25 (m, 1H), 7.19–7.36 (m, 2H), 7.45–7.54 (m, 1H), 8.13–
8.20 (m, 1H), 8.65–8.79 (m, 1H), 8.94–9.03 (m, 1H). HRMS (ESIþ)
1H NMR (300 MHz, DMSO-d6) d 1.98–2.19 (m, 2H), 2.65–2.92
(m, 4H), 2.98 (s, 3H), 3.18–3.44 (m, 5H), 3.86 (br, 1H), 7.23–7.43
(m, 4H), 7.56 (d, 1H), 7.65 (s, 1H), 7.98 (br, 3H), 8.40 (br, 1H),
8.59 (br, 1H), 9.89 (s, 1H). HRMS (ESIþ) calcd. for C22H27F2N4O4S
(MþH)þ m/e, 481.1716; found 481.1723.
calcd. for
429.2103.
C
23H27F2N4O2 (MþH)þ m/e, 429.2097; found
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-
(methylsulfonyl)benzamide (14h)
N-(2-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanamido)ethyl)-2-fluoro-5-
(methylsulfonamido)benzamide (14b)
1H NMR (300 MHz, CD3OD) d 1.88–2.22 (m, 5H), 2.26–2.43 (m,
1H), 2.60–2.76 (m, 1H), 2.79–3.09 (m, 3H), 3.16 (s, 3H), 3.20 (s,
1H), 3.41–3.70 (m, 4H), 4.02–4.25 (m, 1H), 4.42 (d, 1H), 7.10 (t,
1H), 7.27 (t, 1H), 7.68–7.80 (m, 1H), 8.07–8.21 (m, 2H), 8.39 (br,
1H). HRMS (ESIþ) calcd. for C25H30F2N3O4S (MþH)þ m/e,
506.1920; found 506.1927.
1H NMR (300 MHz, CD3OD) d 1.95–2.08 (m, 2H), 2.26–2.38 (m,
1H), 2.50–2.59 (m, 1H), 2.79–2.92 (m, 2H), 2.96 (s, 3H), 3.32–
3.56 (m, 5H), 3.92–3.99 (m, 1H), 7.10–7.26 (m, 3H), 7.31–7.38
(m, 1H), 7.60–7.65 (m, 1H). HRMS (ESIþ) calcd. for
C
22H26F3N4O4S (MþH)þ m/e, 499.1621; found 499.1623.
N-(2-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanamido)ethyl)-4-chloro-3-
(methylsulfonamido)benzamide (14c)
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-
sulfamoylbenzamide (14i)
1H NMR (300 MHz, DMSO-d6) d 1.98–2.19 (m, 2H), 2.65–2.92
(m, 3H), 2.98–3.05 (m, 1H), 3.06 (s, 3H), 3.18–3.44 (m, 5H), 3.86
(br, 1H), 7.27 (td, 1H), 7.37 (td, 1H), 7.57(d, 1H), 7.72 (dd, 1H),
7.88 (d, 1H), 7.98 (br, 3H), 8.41 (br, 1H), 8.74 (br, 1H), 9.59 (s,
1H). HRMS (ESIþ) calcd. for C22H26ClF2N4O4S (MþH)þ m/e,
515.1326; found 515.1310.
1H NMR (300 MHz, DMSO-d6) d 1.76–2.06 (m, 6H), 2.15–2.27
(m, 1H), 2.31–2.42 (m, 1H), 2.65–2.90 (m, 2H), 3.18 (br, 1H),
3.38–3.61 (m, 5H), 4.18 (d, 1H), 7.23 (t, 1H), 7.33 (t, 1H), 7.62–
7.72 (m, 1H), 7.91–8.11 (m, 2H), 8.31 (d, 1H), 8.92 (d, 1H). HRMS
(ESIþ) calcd. for C24H29F2N4O4S (MþH)þ m/e, 507.1872; found
507.1863.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-2-fluoro-5-
(propylsulfonamido)benzamide (14d)
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-4-fluoro-3-
sulfamoylbenzamide (14j)
1H NMR (300 MHz, DMSO-d6) d 0.96 (t, 3H), 1.68–1.78 (m, 2H),
1.82–2.16 (m, 6H), 2.31–2.42 (m, 1H), 2.65–2.96 (m, 4H), 3.08 (t,
2H), 3.19–3.56 (m, 4H), 3.73 (br, 1H), 4.15 (br, 1H), 7.16–7.55
(m, 5H), 8.59 (d, 1H). HRMS (ESIþ) calcd. for C27H34F3N4O4S
(MþH)þ m/e, 567.2247; found 567.2247.
1H NMR (300 MHz, DMSO-d6) d 1.76–2.15 (m, 6H), 2.19–2.27 (m,
1H),2.34–2.42(m,1H), 2.68–2.90(m,2H), 3.18(br,1H),3.38–3.65
(m, 5H), 4.15 (d, 1H), 7.24 (t, 1H), 7.34 (t, 1H), 7.50–7.63 (m, 1H),
8.12 (d, 1H), 8.31 (dd, 1H), 8.97 (d, 1H). HRMS (ESIþ) calcd. for
C
24H28F3N4O4S (MþH)þ m/e, 525.1778; found 525.1779.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-2,6-difluoro-
3-(propylsulfonamido)benzamide (14e)
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-2,4-difluoro-
5-sulfamoylbenzamide (14k)
1H NMR (300 MHz, CD3OD) d 1.03 (t, 3H), 1.78–1.89 (m, 2H),
1.92–2.16 (m, 6H), 2.26–2.42 (m, 1H), 2.57–2.67 (m, 1H), 2.74–
2.82 (m, 1H), 2.82–3.04 (m, 2H), 3.08 (t, 2H), 3.45–3.56 (m, 4H),
3.95–4.03 (m, 1H), 4.25 (d, 1H), 6.99–7.35 (m, 3H), 7.50–7.84 (m,
1H). HRMS (ESIþ) calcd. for C27H33F4N4O4S (MþH)þ m/e,
585.2153; found 585.2162.
1H NMR (300 MHz, DMSO-d6) d 1.76–2.15 (m, 6H), 2.23–2.33
(m, 1H), 2.37–2.42 (m, 1H), 2.68–2.90 (m, 2H), 3.23 (br, 1H),
3.38–3.64 (m, 4H), 3.65 (br, 1H), 4.19 (d, 1H), 7.25 (t, 1H), 7.35
(t, 1H), 7.62–7.70 (d, 1H), 8.00–8.12 (m, 1H), 8.73 (d, 1H). HRMS
(ESIþ) calcd. for C24H27F4N4O4S (MþH)þ m/e, 543.1684; found
543.1692.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. Jiang et al.
Archiv der Pharmazie
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-(N,N-
diethylsulfamoyl)benzamide (14l)
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-(N-
cyclopropylsulfamoyl)-4-fluorobenzamide (14r)
1H NMR (300 MHz, CD3OD) d 0.46–0.62 (m, 4H), 0.83–0.93 (m,
1H), 1.84–2.46 (m, 8H), 2.83–3.11 (m, 3H), 3.46–3.67 (m, 4H),
4.18 (br, 1H), 4.42 (br, 1H), 7.14 (t, 1H), 7.26–7.42 (m, 2H), 8.30
(t, 1H). HRMS (ESIþ) calcd. for C27H31F4N4O4S (MþH)þ m/e,
583.1997; found 583.1988.
1H NMR (300 MHz, DMSO-d6) d 1.04 (t, 6H), 1.76–2.06 (m, 6H),
2.15–2.27 (m, 1H), 2.31–2.42 (m, 1H), 2.65–2.90 (m, 2H), 3.14–
3.22 (m, 5H), 3.38–3.54 (m, 4H), 3.60 (br, 1H), 4.15 (d, 1H), 7.23
(t, 1H), 7.33 (t, 1H), 7.72 (dt, 1H), 7.95 (dd, 1H), 8.11 (dd, 1H),
8.25 (d, 1H), 8.99 (d, 1H). HRMS (ESIþ) calcd. for C28H37F2N4O4S
(MþH)þ m/e, 563.2498; found 563.2503.
In vitro studies
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-(N,N-
diethylsulfamoyl)-4-fluorobenzamide (14m)
DPP-IV inhibition measurement in vitro: Caco-2 assay
DPP-4 was extracted from confluent Caco-2 cells as described
previously [15]. Assays were performed by mixing 10 mL of
appropriate compound dilutions with 40 mL of the substrate
for the DPP-4 enzyme, H-Gly-Pro-AMC (AnaSpec; final
concentration in the assay, 303 mM), and 50 mL of the Caco-
2 cell extract (diluted 5ꢂ with 100 mM Tris-HCl, 100 mM
NaCl, pH 7.8). Plates were incubated at room temperature for
10 min, and fluorescent absorbance was measured at excita-
tion/emission wavelengths of 380/460 nm using FlexStation
3 microplate reader (Molecular Devices). Maximal reaction
rates (fluorescence units/seconds 1000) in 3 min intervals
were calculated using the SoftMax software of the FlexStation
3 and modified by the rate of an uninhibited reaction
[(vcontrol ꢁ vinhibitor)/vcontrol]. All serial inhibitor dilutions
were in DMSO and final solvent concentration did not
exceed 0.1%.
1H NMR (300 MHz, DMSO-d6) d 1.04 (t, 6H), 1.76–2.15 (m, 6H),
2.19–2.27 (m, 1H), 2.34–2.42 (m, 1H), 2.68–2.90 (m, 2H), 3.18
(br, 1H), 3.38–3.65 (m, 8H), 3.73–3.82 (m, 1H), 4.15 (d, 1H), 7.24
(t, 1H), 7.34 (t, 1H), 7.54–7.63 (m, 1H), 8.17 (dd, 1H), 8.31 (dd,
1H), 8.99 (d, 1H). HRMS (ESIþ) calcd. for C28H36F3N4O4S
(MþH)þ m/e, 581.2404; found 581.2421.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanyl)pyrrolidin-2-yl)methyl)-5-(N,N-
diethylsulfamoyl)-2,4-difluorobenzamide (14n)
1H NMR (300 MHz, DMSO-d6) d 1.04 (t, 6H), 1.76–2.15 (m, 6H),
2.19–2.27 (m, 1H), 2.34–2.42 (m, 1H), 2.68–2.90 (m, 2H), 3.18
(br, 1H), 3.38–3.65 (m, 8H), 3.73–3.81 (m, 1H), 4.01–4.25 (m,
1H), 7.26 (t, 1H), 7.36 (t, 1H), 7.64–7.77 (m, 1H), 8.01–8.11 (m,
1H), 8.74 (d, 1H). HRMS (ESIþ) calcd. for C28H35F4N4O4S
(MþH)þ m/e, 599.2310; found 599.2312.
DPP-8 and DPP-9 inhibition measurement in vitro
Recombinant human DPP8 (Abcam) and DPP9 (R&D Systems)
enzyme activity were performed according to product activity
assay protocols. Enzymes were diluted 1250ꢂ to a final
volume of 50 mL in assay buffer (100 mM Tris-HCl, 100 mM
NaCl, pH 7.8) and added to 96-well flat-bottom microtiter
plates, followed by the addition of 10 mL inhibitor and 40 mL
substrate (H-Gly-Pro-AMC, final concentration in the assay,
303 mM). The plates were incubated at 37°C for 10 min. After
incubation, fluorescence was measured using FlexStation 3
microplate reader (Molecular Devices) (excitation 380 nm/
emission 460 nm).
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-((4-
methylpiperazin-1-yl)sulfonyl)benzamide (14o)
1H NMR (300 MHz, DMSO-d6) d 1.75–2.08 (m, 6H), 2.11 (s, 3H),
2.18–2.28 (m, 1H), 2.30–2.39 (m, 5H), 2.53–3.00 (m, 6H), 3.15 (br,
1H), 3.37–3.63 (m, 5H), 4.15 (d, 1H), 7.17–7.38 (m, 2H), 7.71–7.91
(m, 2H), 8.11–8.24 (m, 2H), 8.99 (d, 1H). HRMS (ESIþ) calcd. for
C
29H38F2N5O4S (MþH)þ m/e, 590.2607; found 590.2607.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-2,4-difluoro-
5-(piperidin-1-ylsulfonyl)benzamide (14p)
Oral glucose tolerance test in C57BL/6 mice
C57BL/6 male mice of 4–8 weeks-old were maintained under
constant temperature and humidity conditions: a 12 h light–
dark cycle, and free access to water and regular chow diet.
After an overnight fasting period, the mice were orally
administrated with vehicle (water) or DPP4 inhibitors. Blood
samples were taken at ꢁ60 and 0 min as baselines. Glucose
(5 g/kg) was then administered orally (at 0 min). Serial blood
samples were collected at 20, 40, and 80 min for glucose
determinations. Data represent the mean of 7–8 mice/group.
Statistical analysis was performed using Student’s t-test.
1H NMR (300 MHz, CD3OD) d 1.47–1.66 (m, 6H), 1.85–2.11 (m,
5H), 2.29–2.41 (m, 1H), 2.64–2.75 (m, 1H), 2.79–3.06 (m, 3H),
3.12–3.20 (m, 5H), 3.37–3.64 (m, 4H), 4.15 (br, 1H), 4.42 (br, 1H),
7.12(t, 1H), 7.24–7.44(m, 2H), 8.19(t, 1H). HRMS (ESIþ) calcd. for
C
29H35F4N4O4S (MþH)þ m/e, 611.2310; found 611.2323.
N-(((S)-1-((S)-3-Amino-3-((S)-5,6-difluoro-2,3-dihydro-1H-
inden-1-yl)propanoyl)pyrrolidin-2-yl)methyl)-3-
(morpholinosulfonyl)benzamide (14q)
1H NMR (300 MHz, CD3OD) d 1.85–2.21 (m, 6H), 2.29–2.41 (m,
1H), 2.62–2.72 (m, 1H), 2.84–3.06 (m, 3H), 3.16 (t, 4H), 3.37–
3.62 (m, 4H), 3.71 (t, 4H), 4.11 (br, 1H), 4.42 (br, 1H), 7.15 (t,
1H), 7.24–7.44 (m, 2H), 8.22 (t, 1H). HRMS (ESIþ) calcd. for
The authors gratefully acknowledge Zhaobing Gao for his
help in hERG safety assay and Topharman Shanghai Co., Ltd.
for providing intermediates. We are grateful for financial
C
28H33F4N4O5S (MþH)þ m/e, 613.2102; found 613.2110.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–9
Dipeptidyl Peptidase-4 Inhibitors
Archiv der Pharmazie
support from the National Natural Science Foundation of
China (grant no. 21373258).
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.
The authors declare no competing financial interest.
[8] 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, J. Med. Chem. 2007, 50, 2297–2300.
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L. Thomas, H. Nar, W. Pfrengle, B. Guth, R. Lotz,
P. Sieger, H. Fuchs, F. Himmelsbach, J. Med. Chem.
2007, 50, 6450–6453.
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ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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