Design, synthesis, and pharmacological evaluation of fused β-homophenylalanine derivatives as potent DPP-4 inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00074

Dipeptidyl peptidase-4 (DPP-4) inhibitors are accepted as a favorable class of agents for the treatment of type 2 diabetes. Herein, a series of fused β-homophenylalanine derivatives as novel DPP-4 inhibitors were designed, synthesized, and evaluated for their inhibitory activities against DPP-4. Most of them displayed excellent DPP-4 inhibitory activities and good selectivity. Among them, 9aa, 18a, and 18m also showed good efficacy in an oral glucose tolerance test (OGTT) in ICR mice. Moreover, when dosed 8 h prior to glucose challenge, 18m showed significantly greater potency than sitagliptin. It thus provides potential candidates for the further development into potent drugs targeting DPP-4.

Full text of DOI:10.1021/acsmedchemlett.5b00074

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Pharmacological Evaluation of Fused
β‑Homophenylalanine Derivatives as Potent DPP‑4 Inhibitors
Tao Jiang,† Yuren Zhou,† Zhuxi Chen,† Peng Sun, Jianming Zhu, Qiang Zhang, Zhen Wang, Qiang Shao,
Xiangrui Jiang,* Bo Li,* Kaixian Chen, Hualiang Jiang, 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, 555 Zuchongzhi Road, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: Dipeptidyl peptidase-4 (DPP-4) inhibitors are
accepted as a favorable class of agents for the treatment of type 2
diabetes. Herein, a series of fused β-homophenylalanine derivatives
as novel DPP-4 inhibitors were designed, synthesized, and evaluated
for their inhibitory activities against DPP-4. Most of them displayed
excellent DPP-4 inhibitory activities and good selectivity. Among
them, 9aa, 18a, and 18m also showed good efficacy in an oral
glucose tolerance test (OGTT) in ICR mice. Moreover, when dosed
8 h prior to glucose challenge, 18m showed significantly greater
potency than sitagliptin. It thus provides potential candidates for the
further development into potent drugs targeting DPP-4.
KEYWORDS: Diabetes, DPP-4 inhibitor, β-homophenylalanine derivatives, sitagliptin
ype 2 diabetes mellitus (T2DM) is a chronic, progressive
Tdisease that affects millions of people worldwide. The
Figure 2. Three-dimensional structural modes of inhibitors 9aa
(green) and sitagliptin (blue) to DPP-4 derived from the docking
simulations.
Figure 1. Approved DPP-4 inhibitors.
Recently, a number of small molecule DPP-4 inhibitors,
death toll and economic costs associated with diabetes and its
complications are enormously high.1 The incretin hormone
including sitagliptin, vildagliptin, saxagliptin, alogliptin, and
linagliptin (Figure 1) have emerged as a favorable class of
glucagon-like peptide 1 (GLP-1) has been the subject of
agents for the treatment of type 2 diabetes. The glucose-
intense research efforts related to the treatment of type 2
diabetes.2 Secreted from the L cells of the small intestine,3
lowering effect of DPP-4 inhibitors is glucose-dependent, thus
these agents could provide a lower risk of hypoglycemia.7
GLP-1 stimulates glucose-induced insulin secretion, inhibits
glucagon secretion,4 and delays the gastric emptying, all of
which are beneficial in controlling the blood glucose.5 GLP-1 is
one of the substrates of dipeptidyl peptidase-4 (DPP-4, also
known as CD26).3 By cleaving a dipeptide from the N
terminus, DPP-4 efficiently inactivates the active form of GLP-
1.6 Therefore, inhibitors of DPP-4 would increase the half-life
of active GLP-1 and prolong the beneficial effects of this
incretin hormone on balancing the blood glucose level, leading
to their potential to be antidiabetic agents.
Previous study reported the structure−activity relationship
(SAR) of the DPP-4 inhibitors derived from sitagliptin, which
showed that the trifluorophenyl subunit occupied the S1
hydrophobic pocket and the β-amino group, which forms
hydrogen bonding interactions with the side chains of a
Received: February 15, 2015
Accepted: April 8, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00074
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a
Table 1. DPP-4 Inhibitory Activity
Scheme 1. Synthesis of Compound 9aa
a
Reagents and conditions: (i) malonic acid, piperidine, pyridine,
reflux; (ii) Pd/C, H2, EtOH, rt; (iii) oxalyl chloride, CH2Cl2, rt, then
AlCl3, CH2Cl2, reflux; (iv) ZnI2, cyanotrimethylsilane, CH2Cl2, rt, then
SnCl2, HCl(conc.), AcOH, reflux, then KOH, 1,4-dioxane/H2O,
reflux; (v) carbonyl diimidazole, potassium methylmalonate, MgCl2,
CH3CN, rt; (vi) HCOONH4, CH3OH, 60 °C, then NaBH3CN, rt;
(vii) Boc2O, CH2Cl2, rt; (viii) LiOH, CH3OH/H2O, rt; (ix) Chiralpak
AD-H 250 mm × 5μm × 20 mm; hexane/ethanol (85:15); flow rate
8.0 mL/min; (x) HATU, NEt3, DMF, rt; (xi) CF3COOH, CH2Cl, rt.
a
Scheme 2. Synthesis of Compound 18i
a
tyrosine (Tyr662) and two glutamate residues (Glu205 and
Glu206), are essential for their inhibitory activity against DPP-4
(Figure 2). Recently, most of the reported modifications focus
on the P2 binding moiety (the fused heterocyclic ring) and
little effort has been made to improve the trifluorophenyl
moiety. We present our hypothesis and validation that fusing
the aromatic S1-binding moiety could increase the molecular
rigidity, lower the entropy loss of binding, and eventually lead
to higher potency. To fuse the fluorine-substituted phenyl ring,
we first proposed potential strategies using five- and six-
membered saturated carboatomics. Among them, compound
Reagents and conditions: (i) glycine methyl ester HCl salt, DMF, rt;
(ii) benzyl chloroformate, NaHCO3, 1,4-dioxane/H2O, 0 °C; (iii) Fe,
NH4Cl, CH3OH/H2O, 70 °C; (iv) LiOH, THF/H2O and then
HOBT, EDCI, DIPEA, CH2Cl2, rt; (v) H2, Pd/C, rt; (vi) 7aa, HATU,
10, NEt3, DMF; (vii) CF3COOH, CH2Cl.
9aa is able to adopt very similar binding conformation
compared to sitagliptin (Figure 2).
Encouraged by these docking studies,8,9 we selected
compound 9aa to verify our hypothesis. To our delight,
compound 9aa exhibited higher DPP-4 inhibitory activity than
a
Table 2. Pharmacokinetic Properties of Compound 9aa (Mean) in Male SD Rats
cpd
route
dose (mg/kg)
Cmax (μg/L)
Tmax (h)
T1/2 (h)
AUC0‑∞ (μg/L·h)
CLp (L/h/kg)
Vss (L/kg)
F (%)
9aa
p.o.
i.v.
10
5
466
0.55
5.11
4.39
2848
2266
3.545
2.223
25.792
14.182
61
1493
a
Abbreviations: Cmax, peak plasma concentration of a drug after administration; Tmax, time to reach Cmax; T1/2, elimination half-life; AUC, area under
the concentration−time curve; CLp, plasma clearance; Vss, volume of distribution at steady state; F, bioavailability; p.o., per oral; i.v., intravenous.
B
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Table 3. Inhibitory Activities of Selected Compounds against
DPP-8, DPP-9, and hERG and Rat Liver Microsome
Stability
IC50 (μM)
b
cpd
DPP-8
DPP-9
hERG
T1/2 (min)
sitagliptin
9aa
32
88
>10
>10
>10
>10
>20
>20
353
84
18a
18d
111
a
18i
>10
>10
>10
>10
>10
>10
NT
a
18m
18n
NT
a
NT
a
b
NT means “not tested”. Elimination half-life toward rat liver
microsomes.
Figure 4. Effect of compounds on plasma glucose (a) and baseline (0
min)-adjusted AUC0−120 min of plasma glucose (b) after an oral glucose
load in glucose tolerance test for ICR mice. Data are represented as
mean
Student’s t test.
SEM (n = 6−8); *p = 0.05, **p = 0.01, ***p ≤ 0.001,
(Scheme 1). The absolute configuration of 7bb was confirmed
by X-ray single crystallography of compound (3R)(12S)(29R)-
19 (see Supporting Information).
Figure 3. Effect of compounds on plasma glucose (a) and baseline (0
min)-adjusted AUC0−120 min of plasma glucose (b) after an oral glucose
load in glucose tolerance test for ICR mice. Data are represented as
Further modification on the P2-binding moiety (the fused
heterocyclic ring) was also done, we expected that modification
on these parts could increase the potency of the compounds.
With this rationale, a combinatorial approach was then taken to
prepare different amides by using a wide variety of differ-
entiated amines coupled with the intermediate 7aa, which led
to the discovery of inhibitors 18a with an IC50 value of 4.9 nM.
Then a number of analogues were synthesized and evaluated.
Among them, replacement of phenyl moiety with a
heteroaromatic ring resulted in a significant decrease in
potency. Analogues having pyridine or pyridazine ring, 18b
and 18c, showed a 30- and 18-fold decrease in the DPP-4
potency over 18a, respectively; when the metabolic stability of
18a toward rat liver microsomes was determined, we found a
50% turnover of 18a after 84 min, which indicated metabolic
instability. We reasoned that the unsubstituted phenyl ring and
the lactam may be the potential sites for metabolism. Aiming
for DPP-4 inhibitors that exhibit a long plasma half-life, various
substituents on the phenyl ring were introduced to explore the
substitution effects in the region. Our initial SAR development
began with monosubstitution on the phenyl ring. Addition of a
fluorine at the 8-position of the phenyl ring had a slight effect in
increasing potency (18d). However, when other substitutions
with electron-rich (OMe) and electron-poor (CF3, CN, Br)
residues were introduced on the phenyl moiety, the potency for
both was decreased compared to that of compound 18a. From
the above results, it appears that substitution except fluorine on
mean
Student’s t test.
SEM (n = 6−8); *p = 0.05, **p = 0.01, ***p ≤ 0.001,
sitagliptin with an IC50 value of 10.8 nM (Table 1). Of
particular interest, compound 9aa also demonstrated an
excellent PK profile (Table 2).
Compound 9aa was synthesized starting from commercially
available 3,4-difluorobenzaldehyde, which was converted to
compound 3 via Knoevenagel condensation, reduction, and
Friedel−Crafts reaction. Compound 4 was obtained in high
yields by converting compound 3 to the cyanohydrin
intermediate and subsequent acidic hydrolysis. Compound 4
was then converted to compound 5 by activation with carbonyl
diimidazole, followed by treatment with potassium ethyl-
malonate. Reductive amination of intermediate 5 gives
compound 6, the mixture of four optical isomers. Compound
6aa with its enantiomer 6bb was separated from the mixture by
flash column chromatography. After the protection of amino
with t-butyloxycarbonyl, hydrolysis, and following resolution on
a chiral column chromatography, intermediates 7aa (ee: 97.9%)
and 7bb (ee: 99.9%) were obtained. The key intermediate 7aa
was then condensed with 10 in the presence of HATU to
produce compound 8aa, which was then deprotected with TFA
in dichloromethane to produce the target compound 9aa
C
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Figure 5. Effect of compounds on plasma glucose (a) and baseline (0
min)-adjusted AUC0−120 min of plasma glucose (b) after an oral glucose
load in glucose tolerance test for ICR mice. Data are represented as
Figure 6. Effect of compounds on plasma glucose (a) and baseline (0
min)-adjusted AUC0−120 min of plasma glucose (b) after an oral glucose
load in glucose tolerance test for ICR mice. Data are represented as
mean
SEM (n = 6−8); *p = 0.05, **p = 0.01, ***p ≤ 0.001,
Student’s t test.
mean
Student’s t test.
SEM (n = 6−8); *p = 0.05, **p = 0.01, ***p ≤ 0.001,
the phenyl ring was somewhat detrimental to activity. Once this
was established, we focused to study the SAR of the diazepin-2-
one moiety. In order to improve the metabolic stability of
compounds, the lactam was reduced. Unfortunately, com-
pounds 18j, 18k, and 18l showed a significantly decrease in
potency, which indicated that the lactam was essential to their
inhibitory activity. When the lactam was alkylated, the
inhibitory activity showed a slight change: The methylated
reaction product of 18a (18m) showed a slight increase in
DPP-4 potency. Such increase was also seen when the lactam of
compound 18a was introduced to an ethyl or a 2-ethoxy-2-
oxoethyl, while isopropylation of 18a slightly decreased
inhibitory activity.
introduced showed a slight increase in metabolic stability
toward rat liver microsomes (Table 3).
Based on in vitro potency and selectivity analysis, compounds
9aa, 18a, 18m, and 18n were selected for acute efficacy
evaluation by oral glucose tolerance test (OGTT) in ICR mice.
Compounds dissolved in water were orally administrated at a
dose of 10 mg/kg 1 h prior to glucose challenge (2.5 g/kg).
The blood glucose was monitored at different time intervals
from 0 to 2 h.
Compared to sitagliptin, compounds 9aa, 18a, and 18n
showed comparable glucose lowering effect at 10 mg/kg dose:
18a (34%), 18m (29%) vs sitagliptin (40%); 9aa (57%), 18n
(48%) vs sitagliptin (55%), respectively (Figures 3 and 4).
To determine whether these compounds have long effective
duration in vivo, the most potent compounds 9aa, 18a, and 18n
were selected for assessment of their efficacy at 8 h after
administration. At a dose of 10 mg/kg, compounds 9aa and 18a
were as effective as sitagliptin (24% vs 24%, 35% vs 36%),
whereas 18n was less potent (30% vs 36%, Figures 5 and 6).
Considering that 18m might be metabolized into 18a in vivo,
the long duration efficacy of 18m was also investigated. It was
found that, after 8 h administration, 18m also had greater
potency than sitagliptin in glucose challenge (Figure 6).
In summary, based on the analysis of a large volume of
crystal structure data available in the protein data bank (PDB),
we designed, synthesized, and evaluated a series of novel fused
β-homophenylalanine derivatives as potent and selective DPP-4
inhibitors. Compounds 9aa, 18a, and 18m possessed an
The benzo[e][1,4]diazepine-2-one derivatives10 (represented
as example 18i) were synthesized starting from 1-(bromo-
methyl)-4,5-difluoro-2-nitrobenzene (11i), which was con-
verted to compound 12i via replacement reaction with glycine
methyl ester, and the later was protected by benzyl
chloroformate to furnish 13i. Compound 13i was then reduced
to give 14i, which was converted to 15i via hydrolysis and
subsequent intramolecular cyclization. Deprotection of 15i
produced 16i, which was condensed with 7aa in the present of
HATU to produce compound 17i. Deprotection of 17i with
TFA in dichloromethane produced the target compound 18i
(Scheme 2).
As a result of their high potency against DPP-4, 9aa, 18a, 18i,
18m, and 18n were selected to investigate their selectivity
versus other related serine proteases. Differing from their
excellent DPP-4 inhibitory potency, all test compounds showed
weak inhibitory activity against DPP-8, DPP-9, and hERG.
Compared to compound 18a, 18d in which a fluorine was
D
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Trifluorophenyl)butane-1,3-diamines as Dipeptidyl Peptidase IV
Inhibitors. ChemMedChem 2013, 8, 1104−1116.
excellent DPP-4 inhibitory activity, high selectivity, and good in
vivo efficacy in an OGTT in ICR mice. Moreover, 18m had
significantly greater potency than sitagliptin when dosed 8 h
prior to a glucose challenge, thus providing potential candidates
for the further development into potent drugs targeting DPP-4.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and spectroscopic data for the compounds
described in this Letter. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: jiangxiangrui@simm.ac.cn.
*E-mail: boli@simm.ac.cn.
*E-mail: hywang@simm.ac.cn.
(7) Ji, X.; Su, M.; Wang, J.; Deng, G.; Deng, S.; Li, Z.; Tang, C.; Li, J.;
Li, J.; Zhao, L.; Jiang, H.; Liu, H. Design, Synthesis and Biological
Evaluation of Hetero-Aromatic Moieties Substituted Pyrrole-2-carbon-
itrile Derivatives as Dipeptidyl Peptidase IV Inhibitors. Eur. J. Med.
Chem. 2014, 75, 111−122.
Author Contributions
†These authors contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
(8) OMEGA, version 2.4.6; OpenEye Scientific Software: Santa Fe,
NM. http://www.eyesopen.com.
Funding
(9) Hawkins, P. C. D.; Nicholls, A. Conformer Generation with
OMEGA: Learning from the Data Set and the Analysis of Failures. J.
Chem. Inf. Model. 2012, 52, 2919−2936.
(10) Jain, R.; Trehan, S.; Singh, N.; Nanda, G. K.; Magadi, S. K.;
Sharma, S. K.; Das, J. WO 2009/093269.
We are grateful for financial support from the National Natural
Science Foundation of China (grant 21373258).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge Dr. Jie Sun for obtaining X-
ray data, and Topharman Shanghai Co., Ltd for providing
intermediates.
ABBREVIATIONS
■
ICR, Institute of Cancer Research; HbA1c, glycated hemoglo-
bin; SAR, structure−activity relationship; PDB, protein data
bank; Cbz, benzyloxycarbonyl; rt, room temperature; THF,
tetrahydrofuran; DMAP, 4-(N,N-dimethylamino) pyridine;
HOBT, hydroxybenzotriazole; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl) carbodiimide; DIPEA, N,N-diisopro-
pylethylamine; TFA, trifluoroacetic acid; HATU, o-(7-azaben-
zotriazol-1-yl)-N,N,N,N′-tetramethyluroniumhex-afluorophos-
phate
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