Discovery of new binding elements in DPP-4 inhibition and their applications in novel DPP-4 inhibitor design

Article abstract of DOI:10.1016/j.bmcl.2008.05.061

Probing with tool molecules, and by modeling and X-ray crystallography the binding modes of two structurally distinct series of DPP-4 inhibitors led to the discovery of a rare aromatic fluorine H-bond and the spatial requirement for better biaryl binding in the DPP-4 enzyme active site. These newly found binding elements were successfully incorporated into novel DPP-4 inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.05.061

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3706–3710
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new binding elements in DPP-4 inhibition and their applications
in novel DPP-4 inhibitor design
a
a
a
a
b
Gui-Bai Liang a, , Xiaoxia Qian , Tesfaye Biftu , Suresh Singh , Ying-Duo Gao , Giovanna Scapin ,
Sangita Patel b, Barbara Leiting c, Reshma Patel c, Joseph Wu c, Xiaoping Zhang c, Nancy A. Thornberry c,
Ann E. Weber a
*
a Merck Research Laboratories, Department of Medicinal Chemistry, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
b Merck Research Laboratories, Global Structural Biology, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
c Merck Research Laboratories, Department of Metabolic Disorders, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Probing with tool molecules, and by modeling and X-ray crystallography the binding modes of two struc-
turally distinct series of DPP-4 inhibitors led to the discovery of a rare aromatic fluorine H-bond and the
spatial requirement for better biaryl binding in the DPP-4 enzyme active site. These newly found binding
elements were successfully incorporated into novel DPP-4 inhibitors.
Received 23 April 2008
Revised 12 May 2008
Accepted 15 May 2008
Available online 20 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Diabetes
DPP-4 inhibitors
Fluorine H-bond
Incretin hormones, primarily glucagon-like peptide 1 (GLP-
1)1,2 and glucose dependent insulinotropic polypeptide (GIP),3
are known to stimulate glucose dependent insulin biosynthesis
and secretion.4 Inhibition of dipeptidyl peptidase IV (DPP-4)
has been shown to prolong the beneficial effects of these incretin
hormones by stabilizing the intact (active) form of the hor-
mones.5 Human clinical studies of several small molecule DPP-
4 inhibitors, such as sitagliptin,6 have shown improvement in
glycemic control and improvement in b-cell function in patients
with type 2 diabetes.7 At an early stage of the DPP-4 project,
which ultimately led to the discovery of sitagliptin (16), two
structurally distinctive lead series had been studied (Fig. 1).8,9
potency,9b but also the pyrrolidine itself could be replaced by
piperazine and other cyclic amines.6 In addition, in series 1 the
cyclohexyl ring could be substituted at the 4-position with a vari-
ety of aryl sulfonamides providing compounds with enhanced po-
tency;8a however, only small substituents such as fluorine,
chlorine, and methyl group are tolerated on the benzyl group in
series 2.9a Before the X-ray structure of DPP-4 was published in
2003,11 these distinct SARs provided an opportunity for us to probe
the binding modes of these inhibitors in the DPP-4 enzyme active
site, leading to the design of novel inhibitors.
We speculated that the fluoropyrrolidine amide moiety in the
a-series and fluorobenzyl group of the b-series occupied the same
The glycine-based DPP-4 inhibitors (
a
-series 1) were developed
hydrophobic pocket in the DPP-4 active site. To test this hypothe-
sis, we turned our attention to aspartic acid-based analogs which
embedded both a glycine unit and a b-alanine unit (Fig. 2). Starting
from N-Boc protected aspartate 3, these compounds were readily
made through a reaction sequence of amide coupling, ester hydro-
lysis, second amide coupling, and finally deprotection (Scheme 1).
However, when these compounds (6a–c)12 were compared to their
b-series analogs (2a–c),9a significantly reduced activities were ob-
served (Table 1). All reported compounds were evaluated in vitro
for their inhibition of DPP-4 activity13 and selectivity against
DPP-8 and DPP-9. Although disputed recently,14 inhibition of
DPP-8 and DPP-9 is very likely associated with toxicity in preclin-
ical species.15 DPP-8 and DPP-9 activities are similar, thus only
DPP-8 data are reported. All new compounds were inactive
from compounds described in earlier publications in the field,10
whereas the b-alanine-based DPP-4 inhibitors (b-series 2) were
developed from a high throughput screening lead.9 Structure-
activity relationship studies suggested that the pyrrolidine amide
moieties of series 1 and 2 did not occupy the same pocket in the
enzyme active site.
For
a-series 1, only small substituents (e.g., fluorine, nitrile)
were tolerated on the pyrrolidine ring,8b whereas in b-series 2,
not only could the pyrrolidine amide be substituted with large
groups at either the 2 and 3 positions leading to increased
* Corresponding author. Tel.: +1 732 594 3543; fax: +1 732 594 3007.
E-mail address: gui-bai_liang@merck.com (G.-B. Liang).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.061

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3706–3710
3707
Table 1
F
F
H
Activities of aspartic acid-based DPP-4 inhibitors 6 and their b-series analogs 2
N
O2S
Ar
O
NH2
O
a
a
Compound
X, R
DPP-4 IC50 (nM)
DPP-8 IC50 (nM)
R
N
N
X
6a
2a
6b
2b
6c
2c
X@S; R@H
9800
270
9200
200
8800
55
17,000
2100
18,000
27,000
47,000
>100,000
NH2
F
X@CH2; R@CH2OH
β-Series, 2
α-Series, 1
N
O
Figure 1.
N
a
Values reported are the mean of a minimum of two experiments with a stan-
dard deviation <25% of the mean, and the same for data in other tables.
F
β-Alanine Unit
NH2
O
N
X
N
F
F
F
Glycine Unit
Boc
Boc
O
O
4 Steps
Ref. 17
O
R
LDA
MeI
R
N
Aspartic Acid Based Design
N
Figure 2.
F
8
9
HO
H2N
N
X
F
F
(IC50 > 50 lM) against fibroblast activation protein and quiescent
Boc
NH
cell proline dipeptidase (QPP, also known as DPP-7).16
R
O
Y
Although the simple replacement of the fluorobenzyl group
with a fluoropyrrolidine amide moiety was not successful, contin-
OH
EDC, MeCN, reflux
ued SAR studies in the
a-series had led to a group of oxadiazole
10
containing compounds (7a–c),17 and a completely different ap-
proach (amide bioisostere replacement) in the b-series had led to
a group of very similar oxadiazole containing compounds (12a–
c). The synthesis of the latter is outlined in Scheme 2, starting from
the known b-lactam intermediate 8.18 Methylation yielded mostly
the trans isomer 9, which was converted to acid 10 following a
route previously reported.19 Acid 10 was then treated with corre-
sponding amidoximes in the presence of EDC to yield oxadiazole
11, which were then deprotected to give 12. The similarity be-
tween their structures, as well as their DPP-4 inhibitory activities
(summarized in Table 2), strongly suggest that the fluoropyrroli-
F
Boc
NH
R
O
N
X
N
11
F
Y
4N HCl
Dioxane
F
R
R
N
NH2
X
O
NH2
O
N
X
N
N
N
O
F
Y
Y
dine amide moiety of the
a-series and the fluorobenzyl group of
7a-c
12a-c
the b-series occupy the same pocket in the enzyme active site.19
To seek supporting evidence for this working hypothesis, com-
puter modeling was carried out after the publication of the DPP-4
X-ray structure.11 In the original structure of the enzyme bound to
L-valine pyrrolidine amide 13, two key interactions were identi-
fied: a salt bridge between the amino group of the valine amide
and Glu205 and/or Glu206 and a hydrogen bond between the va-
line carbonyl oxygen and Arg125. When b-alanine-based DPP-4
inhibitors were docked in a similar orientation (A), many highly
potent inhibitors with large substituents on the amide ring, e.g.
compound 14,20 could not be accommodated (Fig. 3). This result
prompted a computer search for new orientations that would fit
a large substituent on the amide ring into the active site. The
Scheme 2.
Table 2
Activities of oxadiazole containing DPP-4 inhibitors 7 and their b-series analogs 12
Compound
R, X, Y
DPP-4 IC50 (nM)
DPP-8 IC50 (nM)
7a
12a
7b
12b
7c
12c
R@H; X@H, Y@OCF3
1800
4400
615
600
40
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
R@F; X@Cl; Y@Cl
R@F; X@Cl, Y@SO2Me
46
Glu206
Arg125
NH2
O
Salt Bridge
F
H-Bond
Boc
Boc
NH
O
EDC, HOBT
F-Pyrrolidine
NH
O
N
HO
OMe
N
13
OMe
O
Salt Bridge
Arg125
Salt Bridge
Glu206
F
O
H-Bond
Glu206
3
4
F
Boc
NH
New H-Bond?
Ph
No Fit!
1) LiOH, THF
4N HCl
O
R
F
Fit!
Ph
N
Dioxane
2) EDC, Amine
F
F
N
O
O
H2N
O
O
NH2
X
O
5
N
NH
HN
N
F
F
F
F
F
14 A
14 B
NH2
N
O
NH2
O
R
R
Figure 3.
N
N
X
X
O
6a-c
2a-c
search resulted in a new orientation (B) consistent with our work-
ing hypothesis that the fluoropyrrolidine amide moiety of the
a-
Scheme 1.

3708
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3706–3710
series and fluorobenzyl group of the b-series occupy the same
hydrophobic pocket in DPP-4 active site, while the free amino
group keeps the same salt bridge with Glu205 and/or Glu206.
However, a hydrogen bond to the carbonyl oxygen was not well
defined, and no conclusions were drawn on detailed alignment
and interactions until X-ray structures of the b-series of inhibitors
bound DPP-4 became available.
O
Cs2CO3
DMF
O
Br
+
OEt
N
NH
OEt
O
O
17
Ph
1) LiOH
2) H2, Pd-C
NH2 O
NH
O
Ph
NH2
neat
N
OH
N
The overlay of X-ray structures of compound 1521
(a-series) and
OEt
O
N
sitagliptin6 (16, b-series) bound to the active site of DPP-4 is shown
in Figure 4. As it was proposed, the fluoropyrrolidine amide moiety
of the a-series and the fluorobenzyl group of the b-series do occu-
py the same pocket in the enzyme active site, even though sitaglip-
tin 16 lacks the sterically demanding benzyl substitution. In this
new orientation, the carbonyl oxygen of 16 forms a hydrogen bond
to Tyr547 through a bridging water molecule in the active site.6 In
the complex with compound 15, the water molecule was displaced
by the dimethyl amide moiety which forms a hydrogen bond di-
rectly with Tyr547 (in green), which was re-oriented to have a bet-
ter alignment for hydrogen bonding.21
O
18
19
O
NH2
O
1) tBoc
2) EDC
1) Chiral Separation
2) Deportection
N
NH
O
20a
Scheme 3.
Table 3
Activities of valerolactam containing DPP-4 inhibitors 20 and their b-series analogs 21
An unexpected discovery was that the fluorine (Fig. 4, X) at the
2 position of the benzyl group in 16 occupied the same space as the
pyrrolidine amide oxygen (Fig. 4, X) in 15 and formed a hydrogen
bond to Asn710. The distance between the fluorine and the NH
nitrogen is measured at 3.2 Å, in the range of a typical hydrogen
bond. Although rarely observed, aromatic carbon-bound fluorine
is known to participate in significant hydrogen bonds and can also
interact with charged molecules.22 This newly discovered binding
element became a focal point in our continued search for structur-
ally diverse new leads. We envisaged that a carbonyl oxygen could
be used to replace the fluorine, form the same hydrogen bond, and
therefore, retain the binding energy. Thus, valerolactam-based
DPP-4 inhibitors 20 were designed and synthesized (Scheme 3).
Using cesium carbonate as a base, alkylation of pyridone went
exclusively on nitrogen to yield 17. Michael addition was carried
NH2
O
NH2
O
N
X
X
O
F
20a-c
21a-c
Compound
X
DPP-4 IC50 (nM)
DPP-8 IC50 (nM)
O
20a
21a
20
40
41,000
87,000
N
NH
F3C
O
20b
21b
120
85
15,000
27,000
N
NH
N
N
20c
21c
120
98
43,000
75,000
N
N
out in neat
a-methylbenzylamine. Diastereomers of 18 were not
CF3
easily separated, so the mixture was carried on, and the two dias-
teromers of the t-Boc protected final product were separated by
HPLC using a Chiral-Cell OJ column. The absolute configuration of
20a was confirmed by X-ray crystal structure, and others were as-
signed analogously, based on their DPP-4 inhibitory activities.
When these valerolactam DPP-4 inhibitors (20) were compared
to their 2-fluorophenyl analogs 21,19 very similar potencies were
observed (Table 3), indicating the arylfluorine to Asn710 H-bond
was successfully replaced with a carbonyl to Asn710 H-bond.
These new DPP-4 inhibitors are potent and selective against DPP-
8. Unfortunately, preliminary rat PK studies on 20a showed poor
oral bioavailability, much lower than that of fluorophenyl analogs
reported earlier.20
Additional analysis of the X-ray structure overlay indicated an
interesting alignment of C1 of the biaryl moiety in compound 15
(Fig. 4, C) with the
a-carbon of the b-alanine unit in compound
16 (Fig. 4, C), suggesting another possible hybrid structure:
replacement of the amidomethylene moiety in the b-series with
the biaryl unit in the a-series to provide compounds 24.
The synthesis of this new series is shown in Scheme 4. The lith-
ium enolate of methyl phenylacetate was alkylated with 2,5-dif-
luorobenzylbromide, followed by ester hydrolysis. The resulting
carboxylic acid 22 was subjected to Curtius rearrangement with
diphenylphosphoryl azide under basic condition, and the product
was captured in situ by tert-butanol to give the t-Boc protected
amino aryl bromide 23. Suzuki coupling with various aromatic
boronic acids, followed by chiral separation and acid deprotection
of the t-Boc group yielded biaryl compounds 24. Again, the abso-
lute configuration of 24d was confirmed by X-ray crystal structure
(Fig. 5) and the others by analogy based on DPP-4 inhibitory
activities.
F
N
F
F
NH2
O
C
N
N
NH2
O
N
N
N
C
N
X
F
N
X
O
NMe2
15
16
CF3
Comparison of DPP-4 inhibition of the new biaryl compounds
Figure 4. Overlay of X-ray structures of 15 (yellow) and 16 (magenta) bound to the
active site of DPP-4.
24 and their a
-series analogs 2523 is summarized in Table 4. Biaryl

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3706–3710
3709
F
F
inhibitor 24d, in particular, showed good potency (DPP-4
a) DPPA, Et3N
CO2Me
IC50 = 32 nM) and selective against DPP8. More importantly, its
1) LDA, ArCH2Br
2) LiOH, THF
CO2H
pharmacokinetic properties24 (Clp, 13 mL/min/kg; t1/2, 3.8 h; Cmax
,
b) tBuOH
TMSCl
2.1
l
M; F%, 130) compare favorably to the
a-series analogs.
Br
The X-ray structure of 24d,25 shown in Fig. 5, revealed that it
Br
22
overlapped with Sitaglitin 16 almost perfectly in the DPP-4 active
site. Not only the aromatic fluorine is in the same position for H-
bonding, but also the carboxamide group of 24d is overlapping
with the trifluoromethyl group of 16, and engaged in favorable
interactions with the side chains of residue F208, S209, and R358
(Fig. 5). This observation is consistent with our working hypothesis
mentioned earlier that this new orientation of the biaryl moiety
would result in better inhibitors of DPP-4.
F
F
F
Boc
1) ArB(OH)2, dppf
NH
NH2
2) Chiral Separation
3) HCl, Dioxane
F
Br
Ar
23
24
Scheme 4.
In summary, our efforts to seek common features of two struc-
turally distinct series of DPP-4 inhibitors led to compounds which
provided insights into the binding modes of these inhibitors in the
DPP-4 active site. Computer modeling based on the DPP-4 X-ray
structure provided credible supporting evidence of the possible
alignment of the two inhibitor classes. Finally the availability of
the X-ray structures of enzyme-inhibitor complexes allowed de-
tailed analysis of specific interactions and structural unit align-
ment. With newly discovered binding elements, such as the
aromatic fluorine H-bond, we were able to design structurally dis-
tinct, potent and selective inhibitors of DPP-4. These studies fur-
ther demonstrated the value of structural biology and computer
modeling in medicinal chemistry programs.
Acknowledgments
We thank Dr. Phil Eskola, Regina Black, Mark Levorse, Joe Leone,
Bob Frankshun, Amanda Makarewicz, Daniel Kim and Dr. Derek
Von Langen of Synthetic Services Group for large scale synthetic
support; Ms. Kathy Lyons and Huaibing He for PK support; and
Dr. Bernard Choi and Eric Streckfuss of Analytical Support Group
for open access LC-MS service. Use of the IMCA-CAT beamline
17-ID and of beamline 32-ID with beamline management and sup-
port provided by the IMCA-CAT staff at the Advanced Photon
Source was supported by the companies of the Industrial Macro-
molecular Crystallography Association through a contract with
the Center for Advanced Radiation Sources at the University of Chi-
cago. Use of the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy Sci-
ences, under Contract No. W-31-109-Eng-38.
F
F
F
24d
NH2
F
NH2
NH2 O
C
N
N
N
X
F
N
X
O
C
16
CF3
Figure 5. Overlay of X-ray structures of 24d (yellow) and 16 (cyan) bound to the
active site of DPP-4.
References and notes
Table 4
Activities of biaryl containing DPP-4 inhibitors 24 and their
a
-series analogs 25
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25a-d
24a-d
Compound
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DPP-4 IC50 (nM)
DPP-8 IC50 (nM)
24a
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1300
245
>100,000
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24b
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110
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24c
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24d
25d
32
100
>100,000
39,000

3710
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3706–3710
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7
nor 12
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25. The coordinates for the structure of DPP-4 in complex with 24d were deposited
with the Protein Data Bank, Accession code 3D4L.