Design, synthesis, and in vitro evaluation of novel aminomethyl-pyridines as DPP-4 inhibitors

Article abstract of DOI:10.1021/ml100200c

A collection of novel aminomethyl-pyridines was designed, synthesized, and investigated as potential inhibitors of DPP-4. Optimization of the screening hit afforded a number of 5-aminomethyl-pyridines with inhibitory activity in the nanomolar range. Selec

Full text of DOI:10.1021/ml100200c

pubs.acs.org/acsmedchemlett
Design, Synthesis, and in Vitro Evaluation
of Novel Aminomethyl-pyridines as DPP-4 Inhibitors
€
Katarzyna Kaczanowska, Karl-Heinz Wiesmuller, and Arnaud-Pierre Schaffner*
€
EMC microcollections GmbH, Sindelfinger Str. 3, D-72070 Tubingen, Germany
ABSTRACT A collection of novel aminomethyl-pyridines was designed, synthesized,
and investigated as potential inhibitors of DPP-4. Optimization of the screening
hit afforded a number of 5-aminomethyl-pyridines with inhibitory activity in the
nanomolar range. Selected DPP-4 inhibitors were further evaluated for their selec-
tivity over the closely related peptidase DPP-8. 5-Aminomethyl-4-(2,4-dichloro-
phenyl)-6-methyl-pyridine-2-carboxylic acid cyanomethyl-amide showed high
potency and excellent DPP-4 selectivity [IC50: 10 (DPP-4) and 6600 nM (DPP-8)]
and no toxicity in mammalian cell culture.
KEYWORDS Aminomethyl-pyridines, DPP-4, DPP-8, incretins, diabetes, structure-
activity relationship
Scheme 1
ipeptidyl peptidase IV (DPP-4) is a serine protease,
which specifically cleaves dipeptides from proteins
and oligopeptides after a penultimate N-terminal proline
D
or alanine.1-3 It is responsible for the rapid deactivation of
incretin hormones, glucose-dependent insulinotropic pep-
tide (GIP), and glucagon-like peptide 1 (GLP-1), which stimu-
lates the secretion of insulin, inhibits glucagon release,
and slows gastric emptying, which benefits in the control of
glucose homeostasis in patients with type 2 diabetes. Because
of the impressive antidiabetic actions of GLP-1, an effective
inhibition of DPP-4 became an original pathway for the treat-
ment of diabetes and, in particular, the noninsulin-dependent
diabetes and related diseases.4-8 Extensive research efforts
from both the academia and the pharmaceutical industry
have led to the launch of Sitagliptin 1 (Merck & Co. Inc.,
2006),9 Vildagliptin 2 (Novartis AG, 2008),10 and Saxagliptin
3 (Brystol-Myers Squibb Co., 2009) for the treatment of type
2 diabetes (Scheme 1).11,12
In recent years, a number of studies regarding a large
variety of scaffolds for DPP-4 inhibition show a continuing
search for small molecules as potent, selective, orally avail-
able inhibitors.13 Particular efforts were made to prolong the
stability and the long-acting potential of conventional DPP-4
inhibitors. Many DPP-4 inhibitors were developed, but the
lack of selectivity and the inhibition of other members of the
DPP family resulted in unforeseen side effects and low
tolerance.14-17 The high degree of sequence homology between,
for example, dipeptidyl peptidase 8 (DPP-8) and DPP-4,
makes the design of selective inhibitors a challenging task.14
The aim of this study was to investigate novel aminomethyl-
pyridines 4-6 (Scheme 1). The compounds are based on the
phenyl-pyridine skeleton and include structural elements
that are important for DPP-4 molecular recognition.18-21
We anticipated that the presence and the position of the
primary amine and the amide groups on the pyridine ring
might be critical for the inhibitory activity. We describe the
synthesis and in vitro evaluation of collections of the amino-
methyl-pyridines 4-6 as potential DPP-4 inhibitors (Table 1).
A novel, potent series of DPP-4 inhibitors was discovered, and
lead optimization gave clear structure-activity relationships
(SARs) and resulted in the identification of compounds 4e-2
and 4e-7 with IC50 values of 11 and 10 nM, respectively. The
most active inhibitors 4 were further investigated for the
DPP-8 activity, and a highly selective DPP-4 inhibitor 4e-7
[IC50: 10 (DPP-4) and 6600 nM (DPP-8)] was identified.22
To obtain the regioisomers 4-6 (Scheme 1), we modi-
fied 4-aryl-5-cyano-6-methyl-pyridine-2-carboxylic acids 7
(Schemes 2 and 3) and 6-aryl-3-cyano-2-methyl-isonicotinic
acids 16 (Scheme 4), which are accessible by a straight-
forward protocol that we have reported recently.23 The two
different approaches to target the 3-aminomethyl-pyridines
4 are outlined in Scheme 2.
Received Date: August 23, 2010
Accepted Date: September 29, 2010
Published on Web Date: October 05, 2010
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Table 1. Screening Results of the Aminomethyl-pyridines 4-6 for DPP-4 Inhibition
compd
R1
R2
R3
IC50 (μM)a
8a
carboxyl
4-fluoro-phenyl
aminomethyl
aminomethyl
cyano
<50
<50
>50
>50
<50
<50
<50
0.080
0.686
<50
0.937
8b
carboxyl
2,4-difluoro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2-fluoro-phenyl
2-fluoro-phenyl
2-fluoro-phenyl
4-fluoro-phenyl
4-fluoro-phenyl
4-fluoro-phenyl
4-fluoro-phenyl
11e
carboxylic acid amide
11f
carboxylic acid methylamide
carboxylic acid amide
cyano
4a-1
4a-2
4a-3
4b-1
4b-2
4b-3
4b-4
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
carbonyl-1-piperidine-4-carboxylic
acid ethyl ester
4c-1
4c-2
4c-3
4c-4
4c-5
4d
carboxylic acid cyclopropylamide
carbonyl-amino-acetic acid methyl ester
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
carboxylic acid (5-methyl-isoxazol-3-yl)-amide
carboxylic acid methylamide
carboxylic acid amide
2,4-difluoro-phenyl
2,4-difluoro-phenyl
2,4-difluoro-phenyl
2,4-difluoro-phenyl
2,4-difluoro-phenyl
4-chloro-phenyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
carboxylic acid amide
carboxylic acid amide
carboxylic acid amide
carboxylic acid amide
aminomethyl
aminomethyl
aminomethyl
aminomethyl
0.674
<50
<50
<50
0.674
<50
0.016
0.011
<50
0.044
<50
<50
0.010
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4e-1
4e-2
4e-3
4e-4
4e-5
4e-6
4e-7
4f-1
4f-2
4f-3
4f-4
4f-5
4f-6
4f-7
4f-8
5a
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
2-fluoro-phenyl
carboxylic acid methylamide
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
carboxylic acid carbamoylmethyl-amide
carboxylic acid cyanomethyl-amide
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
carboxylic acid 3,4-dichloro-benzylamide
carboxylic acid (5-methyl-isoxazol-3-yl)-amide
((r)-3-amino-pyrrolidin-1-yl)-methanone
(4-amino-piperidin-1-yl)-methanone
carboxylic acid (2-piperazin-1-yl-ethyl)-amide
aminomethyl
5b
aminomethyl
4-chloro-phenyl
5c
aminomethyl
2,4-dichloro-phenyl
4-methoxy-phenyl
pyrrolidin-1-yl-methanone
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
5d
aminomethyl
6a
4-fluoro-phenyl
6b-1
6b-2
6b-3
4-chloro-phenyl
4-chloro-phenyl
4-chloro-phenyl
carbonyl-1-piperidine-4-carboxylic
acid ethyl ester
6c-1
6c-2
6c-3
6d-1
6d-2
6d-3
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
carboxylic acid cyclopropylamide
pyrrolidin-1-yl-methanone
morpholin-4-yl-methanone
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
aminomethyl
>50
>50
>50
>50
>50
>50
a Measured in three independent experiments.
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Scheme 2a
a Reagents and conditions: (a) H2, 10% Pd/C, AcOH, 60 °C, 24 h, 52-72%. (b) Boc2O, dioxane/H2O, 60 °C, 16 h, 62-79%. (c) Amine, PyBOP, NEt3,
DCMabs, 3-16 h, 46-92%. (d) 20% TFA/DCM, 1 h, 68-99%. (e) Raney Ni, AcOH, 16 h, 34-70%. (f) Raney Ni, AcOH, 50 °C, 48 h, 60%; POCl3, 70 °C,
2 h, 70%.
Scheme 3a
a Reagents and conditions: (a) t-BuOK, H2O, 100 °C, 3 h, 65-99%. (b) Ethyl chloroformate, NEt3, THF, room temperature, 30 min; NaBH4, H2O, 30 min,
0 °C; 61-99%. (c) SOCl2, toluene, 60 °C, 16 h, 75-85%. (e) Phthalimide, t-BuOK, DMF, 60 °C, 4 h, 65-90%. (f) 30% KOH, 110 °C, 4 h, 79-91%.
Scheme 4a
Compounds 8 were protected to 9 and amidated using benzo-
triazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate
(PyBOP) as a coupling reagent to afford 10. Deprotection of
10 with TFA afforded the 3-aminomethyl-pyridines 4 in high
overall yields. Alternatively, the carboxylic acids 7 were first
amidated to afford 11 that was then selectively hydrogenated
with Raney Nickel25 to give the aminomethyl-pyridines 4
in good yields. To obtain the aminomethyl-pyridine 4e-6
(-NR1R2=-NHCH2CN), the pyridine carboxylic acid 7 was
reacted with 2-amino acetamide to afford the amide 11g
(-NR1R2 = -NHCH2CONH2). The cyano group of 11f was
then reduced to the corresponding methylamine 4e-6 with
Raney Nickel as a catalyst. Finally, the dehydration of primary
amide functionality using phosphorus oxychloride gave the
product 4e-7 in good yield.
The basic hydrolysis of the cyano group of 7 led to the corre-
sponding primary amides 12 in good to excellent yields.
Reduction of the carboxylic acids moiety to the alcohols 1325
followed by treatment with thionyl chloride afforded the corre-
sponding chlorides 14 (Scheme 3). Displacement of the chlo-
ride using phthalimide as nucleophile led to 15 that was then
directly hydrolyzed with a 30% potassium hydroxide solution
to afford 5 in good yields.
a Reagents and conditions: (a) NiCl2 6H2O, NaBH4, MeOH, 24 h, 64% or
3
H2, 10% Pd/C, AcOH, 60 °C, 24 h, 76%. (b) Boc2O, dioxane, 60 °C, 16 h,
38-43%. (c) Amine, PyBOP, NEt3, DCMabs, 3-16 h, 54-92%. (d) 20%
TFA/DCM, 1 h, 57-83%. (e) 50% NH2NH2/H2O, Raney Ni, THF, 20 min;
TFA, 46-53%.
In both pathways, the critical steps were the conversion of
the cyano group to the corresponding aminomethyl func-
tionality. The hydrogenation of 7 using palladium on char-
coal was found to be most reliable for the synthesis of 8.24
3-Aminomethyl-2-methyl-6-aryl-isonicotinamides 6 were
synthesized using two similar approaches as for the
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aminomethyl-pyridines 4 (Scheme 4). Compounds 18 were
synthesized from 3-cyano-pyridines 16 via a hydrogenation/
tert-butyloxycarbonyl (Boc) protection sequence.26 Amida-
tions of 18 were accomplished using PyBOP as a coupling
reagent followed by deprotection of the aminomethyl group
with TFA to yield 6. In the second pathway, the pyridine
carboxylic acids 16 were first amidated with a collection of
amines to give compounds 20 that were then hydrogenated
to afford aminomethyl-pyridines 6. During the catalytic
hydrogenation step with Raney Nickel as a catalyst, the use
of hydrazine hydrate as hydrogen source was more efficient
than the conditions applied for the synthesis of 4. Interest-
ingly, under basic conditions, the expected products 6 tend
to cyclize spontaneously to form the pyrrolopyridines 21 if
the reaction mixture was not acidified (Scheme 5).
The purified regioisomers 4-6 and different isolated
intermediates were then evaluated for inhibition of human
DPP-4 in an in vitro assay using Ile-thiazolidide as positive
control. The inhibition was measured by following the increase
of absorbance at 405 nm upon cleavage of the chromogenic
substrate Gly-Pro-pNA.28 The initial SAR breakthrough was
the discovery that the aminomethyl-pyridines 4 exhibited high
inhibitory activity (Table 1, entries 4a-f) contrary to the ana-
logues 5 (Table 1, e nt ri es 5a-d) and 6 (Tabl e 1, ent ri es 6a-d).
The substitution pattern appeared to be crucial for the
inhibitory activity. The pyridines 4a-e bearing an amino-
methyl moiety in the β-position to the ring nitrogen showed
an activity with IC50 values below 50 μM, in comparison
to the regioisomers 5a-d (the aminomethyl group in the
R-position) exhibiting IC50 values higher than 50 μM. Similarly,
the distance between the primary amino group and the amide
functionality was proven to be of great importance. The change
in the position of the amide group from R (5-aminomethyl-
pyridines 4) to β (3-aminomethyl-pyridines 6) results in a
loss of the inhibitory activity.
The screening results of aminomethyl-pyridines 4 and
intermediates allowed prioritization of initial analogue synthesis.
As expected, the pyridine amides 11 (Table 1, entries 11e-f)
without aminomethyl group were not active. The essential
role of hydrogen bonding between the amine groups in
ligands and the defined amino acids residues in DPP-4 have
been previously reported.18 Additional hydrogen bonds
were observed due to polar side chains in DPP-4 that interact
with the amido groups of the inhibitors. Interestingly, the
free carboxylic acids 8a and 8c presented a good inhibitory
activity (IC50<50 μM). The rigidity of DPP-4 binding pocket
appears to be highly specific to proline residues, but inhibi-
tors with substituted aromatic ring were reported to fit in the
pocket as well. Further affinity gains might be achieved by
aromatic-aromatic interactions between the biaryl system
of the inhibitors and the phenyl rings of some aromatic
residues in DPP-4 that are exposed to the ligand binding
site.18-21 The comparison of the analogues in regard to the
variations of the substitution on the aryl ring of compounds 4
indicated the importance of the halogen substitution of the
aromatic ring; for example, 4a-e (IC50 < 50 μM) was more
potent as compared to 4f (IC50 > 50 μM).
Scheme 5a
Selected compounds possessing superior potency profiles
were investigated for the effectiveness in inhibiting DPP-4 in
dose-dependent experiments. The substitution of the pri-
mary amide 4e-1 with a methyl group as in 4e-2 revealed
a Reagents and conditions: (a) 50% NH2NH2/H2O, Raney Ni, MeOH, 3 h,
66-80%.
Table 2. Selectivity of the Novel DPP-4 Inhibitors 4 over DPP-8 in Comparison to IPI
IC50/Ki (μM) a
DPP-4
0.016
compd
Ar
R1
R2
DPP-8
LC50 (μM)
4e-1
4e-2
4e-7
4e-4
4b-1
4b-2
4c-1
4c-5
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
2,4-dichloro-phenyl
4-fluoro-phenyl
H
H
H
H
33
39
>10
>10
>10
>10
>10
>10
>10
>10
Me
0.011 (0.006)
0.010
cyanomethyl
66
pyrrolidin-1-yl
cyclopropyl
0.044
0.080
0.686
0.937
0.674
3
25
H
106
137
182
179
39
4-fluoro-phenyl
pyrrolidin-1-yl
cyclopropyl
2,4-difluoro-phenyl
2,4-difluoro-phenyl
H
H
5-methyl-isoxazol-3-yl
IPI
a Measured in three independent experiments.
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a significant improvement in the potency (Table 1). It was
interesting to observe that the analogue 4e-7 with the cyano
group had a similar IC50 value as 4e-2. Several studies con-
firmed that the presence of the cyano group increases inhibitor
activity, even up to 1000-fold, due to its interaction with the
oxygen atom of the catalytic serine side chain in DPP-4.18
The increase in the size of the amide group in 4e-4 lowered
its potency in dropping the IC50 value down to 44 nM.
One of the most potent analogues, 4e-2 was then chosen
for further investigation. Measurements of the inhibitory
activity at various inhibitor and substrate concentrations
allowed data plotting,27 and a Lineweaver-Burk plot indi-
cated a competitive, reversible type of inhibition, and the
inhibitor affinity Ki =5.5 ( 2 nM was determinated.
For the investigation of the DPP-4 selectivity over DPP-8 in
dose-dependent experiments, a group of representative inhib-
itors 4 was selected (Table 2). The in vitro assay was based on
the same catalytic reaction as for DPP-4. The results from the
biological screening in comparison to the experimental data
for Ile-Pro-Ile (IPI) are summarized in Table 2. All of the tested
compounds showed high DPP-4 selectivity (2500-6600-fold)
over DPP-8. The secondary amide 4e-2 was slightly more selec-
tive in comparison to the primary amide 4e-1. Interestingly,
the aminomethyl-pyridines 4e-2 and 4e-7 with cyano groups
were similarly potent against DPP-4, but 4e-7 (with cyano
group on the side chain) was almost 2-fold less potent against
DPP-8 than 4e-2. The pyridine 4e-4 with a bulkier pyrrolidine
ring showed lower potency and selectivity.
biological experiments, and Dr. Steffen Rupp from the Fraunhofer
Institute for the toxicity test.
ABBREVIATIONS DPP-4, dipeptidyl peptidase IV; DPP-8,
dipeptidyl peptidase 8; GIP, glucose-dependent insulinotropic
peptide; GLP-1, glucagon-like peptide 1; PyBOP, benzotriazol-
1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; Boc,
tert-butyloxycarbonyl; SAR, structure-activity relationship;
IPI, Ile-Pro-Ile.
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Corresponding Author: *E-mail: emc@microcollections.de.
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DOI: 10.1021/ml100200c ACS Med. Chem. Lett. 2010, 1, 530–535
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