Structure-based design of pyridopyrimidinediones as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.110

Dipeptidyl peptidase IV (DPP-4) inhibitors have been shown to enhance GLP-1 levels and thereby improve hyperglycemia in type II diabetes. From a small fragment hit, using structure-based design, we have discovered a new class of non-covalent, potent and s

Full text of DOI:10.1016/j.bmcl.2012.08.110

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6628–6631
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design of pyridopyrimidinediones as dipeptidyl peptidase
IV inhibitors
Betty Lam a, , Zhiyuan Zhang b, Jeffery A. Stafford c, Robert J. Skene a, Lihong Shi c, Stephen L. Gwaltney II d
⇑
a Takeda California, 10410 Science Center Drive, San Diego, CA 92121, USA
b National Institute of Biological Sciences, #7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China
c Quanticel Pharmaceuticals, 9393 Towne Center Drive, Suite 110, San Diego, CA 92121, USA
d Global Blood Therapeutics, 455 Mission Bay Blvd South, Suite 575, San Francisco, CA 94158, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Dipeptidyl peptidase IV (DPP-4) inhibitors have been shown to enhance GLP-1 levels and thereby
improve hyperglycemia in type II diabetes. From a small fragment hit, using structure-based design,
we have discovered a new class of non-covalent, potent and selective DPP-4 inhibitors.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 July 2012
Revised 21 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
DPP-4
GLP-1
Type II diabetes
Pyridopyrimidinedione
Dipeptidyl peptidase IV (DPP-4), a serine protease expressed in
most tissues as a type II membrane protein, cleaves N-terminal
dipeptides from polypeptides with L-Pro or L-Ala at the penulti-
A co-crystal structure of 2-phenylbenzylamine,9,10 in the DPP-4
active site was used as a starting point for design.11 The key inter-
actions with the protein residues that provide potency were iden-
tified as shown in Figure 2.12 The Ar1 ring occupies the
hydrophobic P1 pocket formed by Tyr666, Trp659, Tyr631,
Val656, Val711 and Tyr662. Multiple interactions are observed be-
tween the basic nitrogen of the ligand and the two carboxylic acids
mate position.1 DPP-4 regulates insulin secretion by inactivating
the incretin glucagon-like peptide 1 (GLP-1) in the blood.2 GLP-1,
a peptide released from L-cells in the gut following meals, en-
hances glucose-stimulated insulin release from beta cells in the
pancreas. GLP-1 also stimulates beta cell growth, insulin biosyn-
thesis, and inhibits glucagon secretion.3 As DPP-4 inhibitors can
enhance the duration of GLP-1 action, they are useful in the treat-
ment of type 2 diabetes. Several DPP-4 inhibitors have been FDA
approved and others are in late stage clinical trials.4–8
of Glu205 and Glu206, as well as Tyr662. In addition, there is a
p–p
stacking interaction between the Ar2 aromatic ring and Tyr547.
Shown in Figure 3 is a summary of our molecular modeling
around 2-phenylbenzylamine. By adding an additional ring in area
A, we aimed to strengthen the
p–p stacking interaction with
Supported by high-throughput protein crystallization technol-
ogy and applied structure-based drug design (SBDD), we success-
fully developed several series of DPP-4 inhibitors including the
discovery of alogliptin (NesinaÒ, Fig. 1), which has been approved
in Japan for the treatment of type 2 diabetes and is in phase 3 trials
in other countries.
Tyr547. We chose a pyrimidinedione system as the A ring fusion
because it had been used successfully in the discovery of alogliptin
and the additional carbonyl had the potential to form a hydrogen
bond with Ser630 and Tyr631. The aminopyridine modification
on Ar2 was incorporated because of synthetic accessibility13 and
literature precedence.14 In addition, the amino group on Ar2 could
restrict the orientation of the benzylamine and provided a handle
for further substitution in area B.
In this paper, we describe our efforts to utilize a small fragment
hit, 2-phenylbenzylamine (DPP-4 IC50 = 30 lM), to develop a back-
up series of pyridopyrimidinedione analogs. As we report here, this
new class of non-covalent DPP-4 inhibitors was found to be potent
and selective.
The synthesis of compounds 3a–n (Scheme 1)15 began with the
Knoevenagel condensation of substituted benzaldehydes 4a–n
with malononitrile in ethanol using a catalytic amount of aq KOH
to generate 2-benzylidenemalononitriles 5a–n. Pyridopyri-midin-
ediones 7a–n were obtained upon refluxing of 5a–n with the
aminopyrimidinedione 6 in propanol. Formation of 7a–n likely
resulted from initial Michael addition of 6 to 5a–n followed by
⇑
Corresponding author.
E-mail address: blam@takedasd.com (B. Lam).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.110

B. Lam et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6628–6631
6629
R
a
+
NC
CN
O
O
CN
4a-n
N
N
O
N
O
CN
b
N
R
NH2
+
NC
O
N
NH2
1
Figure 1. Alogliptin.
5a-n
6
R
R
O
N
O
N
CN
c
N
N
NH2
NH2
O
N
NH2
O
N
7a-n
3a-n
Scheme 1. Reagents and conditions: (a) Ethanol, 10% aq KOH; (b) Propanol, 120 °C;
(c) BH3-THF, THF, 65 °C.
F
F
O
Br
O
Br
CN
b
a
Figure 2. The co-crystal structure of 2-phenyl-benzylamine in the DPP-4 active
site.
CN
N
N
O
N
N
NH2
O
N
N
Br
cyclization.16 Nitrile reduction of 7a–n was accomplished using
borane to yield final compounds 3a–n. Compound 10a was synthe-
sized following the same sequence and conditions as described in
Scheme 1, using ethyl 2-cyanoacetate in place of malononitrile.
As shown in Scheme 2, the syntheses of compounds 10b–f be-
gan with intermediate 3n (obtained from Scheme 1). The bromo
analogue 8 was obtained via a Sandmeyer reaction, and was then
reacted with various amines to generate intermediates 9b–f. Ni-
trile reduction with borane provided final compounds 10b–f. Com-
pounds 3e–n and 10a–f were determined to exist as
diastereomeric mixtures of atropisomers by chiral SFC analysis.17
Experimental data were collected for the compound mixtures.
Our initial SAR development began with mono substitution on
the phenyl ring (compounds 3a–h, Table 1). Interestingly, a four-
fold decrease in DPP-4 potency was observed with 4–fluoro substi-
tution (compound 3b) while the 4-Br compound (3c) was two-fold
more potent. This is likely due to electronic differences. The 3-OMe
substituent was not well tolerated (compound 3d). Substitution at
3n
8
F
F
O
N
Br
O
Br
CN
c
N
N
NH2
R1
N
N
O
N
R1
O
10b-f
9b-f
Scheme 2. Reagents and conditions: (a) NaNO2, 48% HBr in formic acid, Br2; (b)
substituted amine (R1), NaHCO3, MeOH; (c) BH3-THF, THF, 65 °C.
the ortho position improved DPP-4 inhibition up to 10-fold, with a
chloro or bromogroup preferred (compound 3g and 3h, respec-
tively). After identifying the preferred ortho groups, we began
our investigation of bis-substitutions (compounds 3i–n). Both 4-
and 5-chloro substitutions were tolerated (compounds 3j and
3k), and incorporation of a 5-fluoro group (compounds 3l–n) im-
proved potency by fourfold. All of the potent compounds tested
demonstrated high selectivity (>1000-fold) for DPP-4 over DPP-8,
with good to moderate hepatic extraction ratios as determined
by incubation with rat or human liver microsomes.
Following the optimization of substitutions on the phenyl ring,
we turned our attention to optimization of the pyridine ring R1
substitutions (Table 2). Replacing 2-NH2 with OH (compound
10a) resulted in a drop in potency, likely as a result of the potential
for the OH to act as a hydrogen bond accepter for the benzylic
amine, forming an internal Hydrogen bond. The substituted amino
r1
A
1
r
NH2
A
NH2
O
N
O
2
NH2
r
B
A
2
r
N
A
A
N
2
3
Figure 3. Molecular modeling around the co-crystal structure of 2-phenyl-benzyl-
amine in the DPP-4 active site.

6630
B. Lam et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6628–6631
Table 1
Selected data for pyridopyrimidinedione analogues: phenyl ring substitution
R
O
N
NH2
NH2
O
N
N
Compounds
R
DPP-4 IC50 (nM) DPP-8 IC50 (nM) Eh (R/H)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
H
4-F
4-Br
3-OMe
2-OMe
2-CN
2-Cl
2-Br
2,3-di-Cl
2,4-di-Cl
2,5-di-Cl
2-OMe, 5-F 20
2-Cl, 5-F
2-Br, 5-F
257
1070
128
2800
57
69
33
17
371
24
NT
NT
NT
NT
NT
NT
6300
8000
NT
>10000
3200
>10000
>10000
>10000
0.65/0.38
NT
NT
NT
0.61/0.37
0.58/0.33
0.61/0.36
0.61/0.28
NT
0.61/0.54
0.55/0.38
0.40/0.22
0.68/0.41
0.53/0.36
29
7.6
4.8
Figure 4. Co-crystal structure of compound 3h in the DPP-4 active site.
NT = not tested. ⁄Hepatic extraction ratio (Eh) = Clh (unbound intrinsic.
(clearance)/Qh (hepatic blood flow).
3n were not quite comparable to that of compound 3l, with higher
clearance, somewhat lower exposure, and %F of 68. The morpho-
line compound 10e showed excellent exposure, low clearance,
and %F of 100. The considerable improvement in exposure and re-
duced clearance could be attributed to the reduced H-bond donor
count of this molecule. The piperazine compound 10f displayed
low exposure, high clearance with a very high Vdss, and a lower
%F of 31. The high clearance and Vdss could be attributed to the
addition of the basic amine in the molecule.
analogs 10b–f maintained potency similar to that of the unsubsti-
tuted compound 3n. This result suggested that a variety of groups
were tolerated in the R1 region. Based on potency, selectivity and
Eh values, we performed pharmaco-kinetic studies on 4 com-
pounds: 3l, 3n, 10e and 10f. Data for these studies is shown in Ta-
ble 3.
A co-crystal structure of compound 3h in the DPP-4 active site
Compound 3l exhibited good exposure, moderated clearance,
with %F of 83. The PK properties of the more potent compound
is shown in Figure 4.18 A single atropisomer was observed to
Table 2
Selected data for pyridopyrimidinedione analogues: R1 substitution
F
O
N
Br
NH2
R1
O
N
N
Compounds
R1
DPP-4 IC50 (nM)
DPP-8 IC50 (nM)
Eh (R/H)
3n
NH2
OH
NHMe
NHEt
N(Et)2
morpholine
1-Me-piperazine
4.8
260
2.8
6.9
2.8
4.6
9.9
158
NT
0.53/0.36
NT
10a
10b
10c
10d
10e
10f
>10,000
>10,000
>10,000
>10,000
>10,000
0.45/0.47
0.37/0.45
0.56/0.65
0.75/0.64
0.44/0.19
Table 3
Selected in vivo pharmacokinetic properties (rat) of 3l, 3n, 10e and 10f
Compounds
Dose (mg/kg)
AUC (ng⁄h/mL)
CL (mL/min/kg)
Vdss (mL/kg)
F(%)
3l
5.2
8.5
8.4
13
3389
2641
23007
1160
22
44
6.8
60
14870
3490
583
83
68
100
31
3n
10e
10f
64300

B. Lam et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6628–6631
6631
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dinedione inhibitors of DPP-4 from a small fragment hit. SAR
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properties and drug-likeness.
Acknowledgments
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The authors would like to express their appreciation to Beverly
Knight, Catherine Pham, Christopher L. Caster, Daniel B. Kassel,
Gyorgy Snell, Lisa Rahbaek, Melinda Manuel, Rongda Xu, Sridhar
Prasad and Yinong Zhang for their valuable experimental assis-
tance; and to Jun Feng, Michael B. Wallace and Stephen W. Kaldor
for their drug discovery advice. The X-ray crystallography data re-
ported here is based on research conducted at the Advanced Light
Source (ALS). ALS is supported by the Director, Office of Science, Of-
fice of Basic Energy Sciences, Material Sciences Division, of the U.S.
Department of Energy (DOE) under Contract No. DE-AC03-
76SF00098 at Lawrence Berkeley National Laboratory. We thank
the staff at ALS for their excellent support in the use of the syn-
chrotron beam lines.
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