Discovery of imigliptin, a novel selective DPP-4 inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1021/ml5001905

We report our discovery of a novel series of potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors. Starting from a lead identified by scaffold-hopping approach, our discovery and development efforts were focused on exploring structure-activity relationships, optimizing pharmacokinetic profile, improving in vitro and in vivo efficacy, and evaluating safety profile. The selected candidate, Imigliptin, is now undergoing clinical trial.

Full text of DOI:10.1021/ml5001905

Letter
pubs.acs.org/acsmedchemlett
Discovery of Imigliptin, a Novel Selective DPP‑4 Inhibitor for the
Treatment of Type 2 Diabetes
Chutian Shu,† Hu Ge,‡ Michael Song,† Jyun-hong Chen,† Huimin Zhou,† Qu Qi,† Feng Wang,†
Xifeng Ma,† Xiaolei Yang,† Genyan Zhang,† Yanwei Ding,† Dapeng Zhou,† Peng Peng,†
Cheng-kon Shih,†,‡ Jun Xu,‡ and Frank Wu*,†,‡
†Department of Project Management, Medicinal Chemistry, Process, Pharmacology, Drug Metabolism and Pharmacokenetics,
Toxicology, XuanZhu Pharma, 2518 Tianchen Street, Jinan, Shandong, The People’s Republic of China
‡School of Pharmaceutical Sciences & Institute of Human Virology, Sun Yat-Sen University, 132 East Circle Road at University City,
Guangzhou, The People’s Republic of China
S
* Supporting Information
ABSTRACT: We report our discovery of a novel series of
potent and selective dipeptidyl peptidase IV (DPP-4)
inhibitors. Starting from a lead identified by scaffold-hopping
approach, our discovery and development efforts were focused
on exploring structure−activity relationships, optimizing
pharmacokinetic profile, improving in vitro and in vivo efficacy,
and evaluating safety profile. The selected candidate,
Imigliptin, is now undergoing clinical trial.
KEYWORDS: Diabetes, dipeptidyl peptidase IV (DPP-4), inhibitor, scaffold-hopping, imigliptin, OGTT, pyridinylimidazolone
he prevalence of Type 2 diabetes mellitus (T2DM) is
Tincreasing at an alarming rate, affecting 382 million people
worldwide in 2013, and China now has the largest patient
population.1,2 Dipeptidyl peptidase IV (DPP-4) is a widely
distributed physiological enzyme that can be found solubilized
in blood or membrane-anchored in tissues. The rationale for
inhibiting DPP IV activity in type 2 diabetes is that it decreases
peptide cleavage and thereby enhances endogenous incretin
hormone activity.3
unique therapeutic profile in terms of safety and efficacy. Here,
we wish to report our discovery of Imigliptin, a novel class of
DPP-4 inhibitor currently undergoing clinical trial in China.
Our discovery started with a lead compound 1, identified via
scaffold-hopping from Alogliptin structure assisted by molec-
ular modeling (Figure 2).
Since the first approval of Sitagliptin in 2006, there are
several DPP-4 inhibitors in clinical use today (Figure 1).4
However, the need for more therapeutic options is still unmet
as a significant number of patients fail to control blood sugar,
experience hypoglycemic conditions, and remain at risk from
complications such as cardiovascular and chronic kidney
diseases. We believe a novel chemical structure of inhibitor
differentiating from existing DPP-4 compounds may provide a
Figure 2. Scaffold-hopping leads to novel structure.
Our first tier assays were using DPP-4 enzyme with counter
screening enzymes DPP-8 and DPP-9. Compound 1 has DPP-4
potency of 100 nM with excellent selectivity against DPP-8 and
DPP-9 (both >100,000 nM). This is a good starting point for
our initial structure−activity relationship (SAR) studies, which
focused on pyridinyloxazolone core, and the results are
summarized in Table 1. Converting pyridinyloxazolone oxygen
of 1 with dimethylmethelene group resulted in compound 2
with a significant loss of activity, while replacing oxygen with
NH yielded a pyridinylimidazolone compound 3 with a
Received: May 10, 2014
Accepted: June 9, 2014
Figure 1. Marketed DPP-4 inhibitors.
© XXXX American Chemical Society
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(compound 9 and 10) does not appear to perturb significant
changes of inhibitory activity nor selectivity.
Table 1. SARs of Pyridinyloxazolone Core R
We next studied SARs of the cyanobenzyl portion of the
molecule. Table 2 summarizes the results. Apparently, there is a
Table 2. SAR of Cyanobenzyl Group R1
strong regio-requirement for cyano substitution at ortho-
position, as meta- or para-substitution had several orders of
magnitude loss of activity (4 vs 11 and 12). This may imply
strongly a role of cyano group participating a critical interaction
with enzyme in a regio-specific fashion. Chlorine replacing
cyano group, compound 13, was somewhat acceptable with a
loss of 10-fold, but replacement with fluorine group saw 2
orders of magnitude loss of activity (compound 14). Other
modifications included chlorine substituting at the para-
position on the phenyl ring (compound 15) or fusing an
extra ring on the phenyl ring (compound 16) of compound 4,
which all resulted in a large loss of potency. In general, this
portion of the molecule has a steep SAR and appears to lack
much room for chemistry and optimization.
DPP-4 enzyme is an exopetidase and recognizes substrates
with uncapped, protonated amino group at its N-terminal.6 The
aminopiperidine portion of the molecule appears to occupy
substrate N-terminal site. A summary of aminopiperidine ring
SAR is presented in Table 3. Studies of piperidine with
analogues of various ring sizes, where an amino group is
attached, suggested a five-membered pyrrolidine and six-
membered piperidine are optimal for potency (18 and 4 vs
17 and 19). It is worthy to notice azitidine analogue has no
activity at all at a concentration of 100 μM, and it is likely due
to the dramatic changes between the trajectory of amino group
on azetidine and that of piperidine. When aminopiperidine
group of compound 4 was replaced by a number of
heterocycles containing protonable endoamine, such as
compounds 20−22, these analogues have weak or no
appreciable potency. N-methylation of the amino group
significantly decreased potency (4 vs 23), while N-acetylation
moderate 2-fold increase of activity without any loss of
selectivity. Methylation of nitrogen on pyridinylimidazolone
(compound 4) boosted 3-fold potency, but substituting with an
isobutyl group appeared to be unfavorable (compound 5).
These results may suggest there exists a greasy but small cavity
in the enzyme pocket, and the moderate 3-fold potency
increase of 4 is a typical methyl desolvation effect.5 Because of
its good potency, compound 4 becomes a central point for the
rest of the SARs in this report. Interestingly, replacing
pyridinylimidazolone carbonyl oxygen of 4 with sulfur
(compound 6) was detrimental, implying an oxygen atom
likely involves a specific H-bond interaction with enzyme
backbone. This notion is further supported by the fact that
compound 7 has very weak potency. The carbonyl oxygen of
compound 7 appears to have different topology as its structure
was modified by a ring expansion from compound 4. Fusing an
extra aromatic group onto compound 4 provided compound 8
with a significant loss of activity, conceivably due to the steric
hindrance. Modulation of nitrogen on pyridinyl portion
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Table 3. SAR of Aminopyperidine R2
Table 4. SAR of Substituents R3
known to be very important to avoid potential off-target side
effects.7
Molecular modeling was instrumental in obtaining initial
leads and guiding our SAR work. We used the molecular
docking program GLIDE8 to dock compounds into a DPP-4
crystal structure, which is available from the RCSB Protein Data
Bank (PDB ID: 2ONC). Shown in Figure 3 is a docking pose
was detrimental (4 vs 24). Further SARs indicate the amino
group on piperidine is stereospecific, as R configuration is
strongly preferable to S configuration (4 vs 25). Combining the
above observations implies that there exists an important
interaction between protein backbone residue(s) with inhibitor
amino group, and such interaction requires a proper and strict
spatial orientation.
Finally, we explored the SAR at position 7 of the pyridinyl-
imidazolone core with a large number of diversified substituents
(compounds 26−34). The results are summarized in Table 4.
Interestingly, the SAR in the region is fairly flat, and all
analogues made have reasonably good potency despite their
large diversity in physical−chemical properties (pKa, logD,
bulkiness, etc.). It hints that this portion of the molecule is out
of enzyme active site and likely exposes to solvent region. This
is the “sweet spot” on a molecule where it is ideal to modulate
physicochemical properties for drug-like optimization.
Figure 3. Superposed binding conformations of compound 27
(brown) and Alogliptin (dark green).
of compound 27 at the enzyme active site. This model
highlights key interactions of our novel structure, reconfirming
the SAR we discussed above. Compound 27 fits in the enzyme
pocket very well topologically. The overlay of 27 against
Alogliptin shows two molecules interact with DPP-4 in a similar
fashion. The pyridinylimidazolone core of 27 lays flat in the
enzyme cavity, “sandwiched” by some hydrophobic residues,
most noticeably residues Phe357 and Tyr574. These hydro-
phobic residues may provide π−π interactions with the core.
It should further be noted that throughout the SARs around
the molecule, none of the analogues have any appreciable
activity against DPP-8 and DPP-9. The high selectivity profile is
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Table 5. Pharmacokinetics in Rat
IV
PO
compd
cLogP
Cl (L/h/kg)
Vss (L/kg)
T1/2 (h)
Cmax (ng/mL)
AUClast (ng/mL·h)
T1/2 (h)
F (%)
a
4
2.24
1.90
2.40
1.68
2.48
3.26
0.2
3.9
5.42
2.78
5.89
4.55
6.17
12.2
1.02
4.22
2.4
175
86
880
2.8
1.5
3.3
1.6
2.4
4.4
1.9
3.7
35
12
48
28
97
89
<1
b
9
3.67
5.44
5.04
2.40
2.16
1.65
4.39
0.8
249
a
10
1.2
198
291
1250
256
3.52
103
896
a
18
1.1
541
c
27
1.83
4.72
2.48
0.8
3980
1610
19.5
209
d
29
d
30
a
31
1.11
9
a
b
c
d
Dose: po at 10 mg/kg; iv at 5 mg/kg (n = 3). Dose: po at 8 mg/kg; iv at 4 mg/kg (n = 3). Dose: po at 10 mg/kg; iv at 10 mg/kg (n = 3). Dose:
po at 4 mg/kg; iv at 2 mg/kg (n = 3).
Carbonyl oxygen of imidazolone serves as a H-bond acceptor
and forms a specific H-bond with the backbone NH of Tyr631.
The flat pyridinylimidazolone core also anchors two side
moieties into their corresponding pockets well. Benzyl side
moiety fits nicely in a tight hydrophobic groove, where it forms
π−π interactions with its surrounding residues Trp659 and
Tyr666. Nitrogen atom of benzyl cyano group functions as an
H-bond acceptor to Arg125. However, the amino group from
piperidine site moiety is protonated under physiological pH
and forms an essential salt bridge with the backbone
carboxylate of Glu205 and Glu206. It should be mentioned
that this type of salt bridge interaction between enzyme and
ligand is a key feature of exopeptidase such as DPP-4. Naturally,
all marketed DPP-4 inhibitors contain such a protonable amino
group.
A large number of compounds identified with good potency
were selected for rat pharmacokinetics (PK) evaluation in our
lead optimization. Table 5 summarizes key parameters of some
examples. We had initial difficulty achieving good oral exposure
and bioavailability. As we built up a larger data set, we noticed a
reasonable connection between lipophilicity and PK properties.
Good oral compounds in this class of molecules starts to
populate around cLogP greater than 2, regardless of structural
diversity (Figure 4). We then utilized the SAR knowledge of
On the basis of its attractive in vitro and ex vivo potency (data
not shown), high selectivity over other subfamily enzymes, and
favorable pharmacokinetic profile, compound 27 was advanced
into in vivo efficacy studies. Compound 27 was first evaluated in
a proof-of-concept pharmacodynamics model, assessing its
ability to improve glucose tolerance in mice (OGTT, oral
glucose tolerance test). In the studies, a single dose of
compound 27 (0.1, 0.3, 1.0, and 3.0 mg/kg) was given orally to
C57BL/6 mice 30 min prior to the glucose challenge (3.0 g/
kg). The plasma glucose was then measured at 20, 40, 60, and
120 min after the glucose challenge, while DPP-4 activity, GLP-
1 level, and plasma drug concentration were monitored at 20
min. As shown in Table 7, compound 27 was able to reduce
plasma glucose excursion in a dose-dependent manner with a
minimum effective dose at 0.3 mg/kg (48% inhibition). The
observed glucose reduction tracked very well with drug
exposure, DPP-4 inhibition, and augmented GLP-1 level.
With regard to the comparison of Sitagliptin in the head-to-
head experiment, compound 27 showed better inhibition of
DPP-4 enzyme and comparable glucose reduction effects.9
To further characterize the effects in long-term treatment of
27 in rodent diabetic model, db/db mice were fed continuously
with food formulated-27 for 28 days. The random glucose level,
lipids, oral glucose tolerance test, GLP-1, DPP-4, and insulin
level were measured over the course of the study. Compound
27 has shown strong effects on all treated db/db mice in lowing
glucose level, reducing AUC of oral glucose tolerance test,
decreasing TG and HbA1c levels, and protecting beta-cell.
These effects correlated well with the inhibition of DPP-4 and
elevated the plasma GLP-1 level. In addition, no effects on the
body weights in db/db mice were observed under the test
conditions (data are not shown and will be published elsewhere
separately).
In order to assess the key areas for further development,
compound 27 was profiled extensively in a battery of target
independent drug-like properties and safety assays. Shown in
Table 8 are examples of these assays that include solubility,
permeability, protein binding, CYP450 inhibitions, hERG
channel inhibition/dog QT prolongation, Ames test, and in
vivo tox. These data demonstrate remarkable physicochemical
properties and good safety profile of 27 and strongly support its
developability.
The synthesis of 27 is illustrated in Scheme 1. The process
was well developed and validated at kiloscale to support early
development activities. The linear synthesis began from a
commercially available hydroxypyridone starting material 27−
01. Conversion of 27−01 under the treatment of POCl3
yielded dichloropyridine intermediate 27−02. Subsequently,
Figure 4. Bioavailability and cLogP.
the above-mentioned “sweet spot”, tuned in preferable
lipophilicity, and quickly identified good oral compound such
as 27 and 29. Of particular interest, compound 27
demonstrated a superior PK profile with a moderate clearance,
a high plasma exposure, and near 100% rat oral bioavailability.
These good PK attributes were translated into nonrodent
species of dog and monkey in our further characterizations
(Table 6).
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Table 6. Pharmacokinetics of Compound 27 in Rat, Dog, and Monkey
IV
PO
species
Cl (L/h/kg)
Vss (L/kg)
T1/2 (h)
Cmax (ng/mL)
AUClast (ng/mL·h)
T1/2 (h)
F (%)
a
rat
2.40
2.12
0.67
6.17
2.46
1.83
1.12
3.68
1250
1355
3030
3980
3630
2.4
1.3
4.8
97
89
86
b
dog
monkey
c
2.44
12900
a
b
c
Dose: po at 10 mg/kg; iv at 10 mg/kg (n = 3). Dose: po at 5 mg/kg; iv at 6.25 mg/kg (n = 3). Dose: po at 10 mg/kg; iv at 5 mg/kg (n = 3).
Table 7. OGTT Results of 27 and Sitagliptin in C57BL/6 Mice
20 min after glucose challenge
plasma glucose (mM) DPP-4 inhibition (%) plasma GLP-1 (pM) drug plasma concentration (ng/mL) inhibition (%)
a
group
N
control
20
20
20
20
20
20
15.6
11.7
14.1
12.4
11.4
10.1
0
4.0
c
b
c
c
c
Sitagliptin (3.0 mg/kg)
27 (0.1 mg/kg)
27 (0.3 mg/kg)
27 (1 mg/kg)
59.5
23.8
43.8
71.2
82.1
10.7
5.7
179
3.90
10.3
36.1
129
59
3
5.7
40
57
65
6.9
cd
,
d
c
27 (3.0 mg/kg)
10.4
a
b
c
Inhibition reflects the % decrease of plasma glucose AUC0−120min. p < 0.05 (vs vehicle control using t test). p < 0.01 (vs vehicle control using t
d
test). p < 0.01 (vs Sitagliptin using t test).
Table 8. Additional Profile of Compound 27
untouched. Further functioning at ortho-position with methyl-
amine was achieved under elevated temperature. The nitro
group of the resulting 27−04 was then reduced to amino
group. The diamino moieties of 27−05 were subsequently
treated with phosgene to give a ring closure intermediate 27−
06. Introducing cyano substituted benzyl side moiety was
completed by reacting 27−06 with the third commercially
available cyanobenzyl bromide in basic conditions in DMF.
Deprotection of Boc-group finally gave target compound 27.
The synthesis steps and conditions were optimized, and
currently, the yields for key intermediates in the illustrated
procedure are above 90%.
In summary, our fast follow-up program utilizing “scaffold−
hopping” technique identified a novel series of highly potent
and selective DPP-4 inhibitors. SARs and optimization in terms
of DMPK properties, in vivo efficacy, safety characterization,
and Chemistry Manufacturing and Controls evaluation led to
the discovery of compound 27 (common name known as
Imigliptin) as a clinical candidate. Currently, Imigliptin is under
clinical development in China after it got CFDA regulatory
approval. The profiles of Imigliptin’s in vivo evaluation, PK/
metabolism, and clinical pharmacology will be published in due
course.
Compound
27
solubility pH (1.2/4.5/6.8)
Caco2 (ab/ba)
560/560/551 mg/mL
9/28 × 10−6 cm/sec
42/74/62
PB% (rat/dog/human)
CYP450 inhibition (3A4, 2D6, 2C9, >30 μM
2C19)
hERG inhibition
QT prolongation
>25 μM
not observed at 20 mg/kg in
conscious dog
AMES
negative at maximal dose of
5000 μg/plate
tox (28 days, rat)
NOAEL 150 mg/kg
Scheme 1. Synthetic Route of Compound 27
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, characterization data of representative
compounds, description of in vitro DPP-4 enzyme assay and in
vivo OGTT studies. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(F.W.) Phone: 18615208825. E- mail: frankyongwu@yahoo.
dichloropyridine 27−02 was reacted with another readily
available intermediate Boc-protected aminopiperidine in the
presence of triethylamine under the room temperature. The
nucleophilic reaction was highly regioselective, and the
resulting 27−03 anchored aminopiperidine moiety at para-
position on pyridine ring, leaving ortho-chloro group
com.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
E
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the excellent assistance
from Ms. Linyi Qian and Ms. Yanni Teng in the preparation of
this manuscript. Hu Ge and Jun Xu acknowledge the support
by a grant from the National Natural Science Foundation of
China (No. 81173470/H2903).
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