Discovery, SAR, and X-ray structure of novel biaryl-based dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.037

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.

Full text of DOI:10.1016/j.bmcl.2005.09.037

Bioorganic & Medicinal Chemistry Letters 16 (2006) 123–128
Discovery, SAR, and X-ray structure of novel
biaryl-based dipeptidyl peptidase IV inhibitors
Lei Qiao, Christian A. Baumann, Carl S. Crysler, Nisha S. Ninan, Marta C. Abad,
John C. Spurlino, Renee L. DesJarlais, Jukka Kervinen, Mike P. Neeper,
Shariff S. Bayoumy, Robyn Williams, Ingrid C. Deckman, Malini Dasgupta,
Rolanda L. Reed, Norman D. Huebert, Bruce E. Tomczuk and Kevin J. Moriarty*
Johnson & Johnson Pharmaceutical Research and Development, 665 Stockton Drive, Exton, PA 19341, USA
Received 19 July 2005; revised 12 September 2005; accepted 13 September 2005
Available online 19 October 2005
Abstract—The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacyl-
thiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Glucagon-like peptide-1 (GLP-1) stimulates glucose-de-
pendent insulin secretion, inhibits hepatic glucose pro-
duction, lowers blood glucose levels, and promotes the
growth and differentiation of b-cells. GLP-1 may also
play a role in suppressing appetite in humans and in
modulating peripheral glucose uptake.1 The hormoneÕs
activity is rapidly abolished by the N-terminal cleavage
of the peptide by the aminopeptidase dipeptidyl pepti-
dase IV (DPP IV, CD26, EC 3.4.14.5), a sequence-spe-
cific non-classical serine protease.2 Both mice and rats
genetically deficient in DPP IV have shown enhanced
insulin secretion and accelerated clearance of blood glu-
cose partly due to increased levels of active GLP-1.3
new therapy for elevating plasma GLP-1 levels and the
treatment of Type 2 diabetes.5
Most of the small molecule inhibitors of DPP IV report-
ed in the literature have a 1ꢁ or 2ꢁ amine at the P2 site
with the P1 site occupied by a proline mimetic
(Fig. 1).6 The most active inhibitors incorporate an elec-
trophilic group (e.g., P(O)(OPh)2, CO–NH–O–COR0,
CH3
H3C
H2N
CH3
N
HO
Since Type 2 diabetes is a disease characterized by ele-
vated blood glucose levels and a relative insufficiency
of insulin, a therapeutic agent that extends the duration
of GLP-1 action may aid in controlling glucose homeo-
stasis, by enhancing b-cell glucose-stimulated insulin re-
lease and promoting insulin gene expression and its
biosynthesis.4 In addition, elevation of GLP-1 levels
may increase b-cell proliferation and survival. Thus,
DPP IV inhibition has the potential to be a promising
N
N
H
O
CN
O
CN
FE999011
NVP-LAF237
F
F
F
NH2
O
HO
H2N
N
N
N
N
N
O
CN
BMS-477118
Figure 1. Selected inhibitors of DPP IV.
CF3
Keywords: Dipeptidyl peptidase IV Inhibitors; SAR; X-ray structure.
*
MK-0431
Corresponding author. Tel.: +1 610 458 5264x6592; fax: +1 610 458
8249; e-mail: kmoriart@prdus.jnj.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.037

124
L. Qiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 123–128
B(OH)2, or CN) as a replacement for the normally
cleaved P1–P10 substrate amide on the proline at P1
and providing a reactive center to interact with the
DPP IV active site serine residue. Low nanomolar inhi-
bition and chemical stability adequate for oral adminis-
tration have been obtained with the nitrile containing
inhibitors.6c–e,7 Recently inhibitors lacking an electro-
philic group with good activity have also appeared in
the literature.6f,8
I
I
a
b
Boc
N
X
Boc
OH
N
H
N
H
60-80%
HN
X
O
O
R
R
X = C or S
R = H or CN
70-90%
Ar
Ar
Our goal was to identify non-nitrile inhibitors with good
potency and ADME characteristics. Optimizing the P2
interactions could allow for the removal of the electro-
philic group and still retain acceptable characteristics.
In this paper, we report the preparation of a series of
P2 optimized nitrile containing biaryl DPP IV inhibi-
tors. The SAR from this series was used to identify
non-nitrile inhibitors with low nanomolar potency and
good pharmacological properties. In addition, the X-
ray crystal structure of DPP IV with inhibitor 1-[2-(S)-
amino-3-biphenyl-4-yl-propionyl]-pyrrolidine-2-(S)-car-
bonitrile bound has been determined, revealing the nat-
ure of the covalent interaction of the nitrile group with
the active-site serine.
c
X
X
Boc
N
N
N
H
70-99%
H2N
O
O
R
R
Scheme 1. Synthesis of biaryl-based DPP IV inhibitors. Reagents: (a)
EDCI, HOBt, DIEA, CH2Cl2; (b) ArB(OH)2, Pd(OAc)2, Na2CO3; (c)
TFA, CH2Cl2, thioanisole.
Boc-L-4-iodophenylalanine was coupled to the proline
mimetic using EDCI and HOBt in CH2Cl2 to give the
4-iodophenylalanine derivatives in 70–90% yields. The
iodo-compounds were then converted to the biaryls
using a Pd(OAc)2 catalyzed Suzuki reaction11 (60–
80%). Removal of the Boc-protecting group with triflu-
oroacetic acid in the presence of thioanisole gave final
products in 70–99% yields.
Analysis of the published crystal structure9 of human
DPP IV with valine pyrrolidide bound in the active site
(Fig. 2, PDB ID:1N1M) revealed a linear hydrophobic
pocket at the S2 subsite formed by the side chains of
Phe357 and Arg358. We envisioned that filling this
pocket with appropriate groups would lead to more po-
tent inhibitors and potentially allow for the removal of
the nitrile moiety at P1.10
A survey of the S2 site was undertaken using the 2-
(S)-cyano-pyrrolidine at the S1 as our initial probe
with the intention of extrapolating the findings of this
study to non-nitrile analogs (Table 1). Entries 2–5
indicated that non-polar groups are favored at the
4-position of phenylalanine moiety, giving potencies
similar to the reference NVP-DPP728 (entry 1).12
Placement of a carboxylic acid moiety at the 4-posi-
tion of the phenylalanine (entry 4) resulted in a 7-fold
loss of potency compared to the known7b compound
entry 2. However, addition of a phenyl ring at the
4-position (entry 5) resulted in a 5-fold improvement
in activity. Substitution on the terminal phenyl ring
was then explored.13 Substitution with bulky or elec-
tron-donating groups, for example, 4-methyl or 3,4-di-
methoxy groups (entries 6 and 7), gave less active
compounds compared with the unsubstituted entry 5.
However, when fluorine was placed to the 4-position
(entry 8), a 4-fold improvement in activity was ob-
served. This increase in activity was attributed to a
better fit in the targeted lipophilic pocket and a stron-
ger edge-to-face p–p interaction between the middle
ring of the biphenyl group and the Phe357. Moving
the fluorine to the 2-position of the terminal phenyl
ring (entry 9) was also tolerated. Di-substitution with
fluorine at the 2- and 4-positions (entry 10) had an
additive effect to give a compound with further in-
creased activity. And lastly replacement of the termi-
nal phenyl ring with a furan (entry 11) gave a
compound with activities comparable to that of the
The general synthetic route to the biaryl-based inhibi-
tors is shown in Scheme 1. The commercially available
N
H2N
O
Ki = 2µM
Figure 2. Space filling picture of DPP IV in complex with valine
pyrrolidide. PDB ID is 1N1M. Inhibitor is shown in yellow with
nitrogen colored blue and oxygen colored red.
known10a
dine (entry 3).
4-iodophenyl-alaninyl-(S)-2-cyano-pyrroli-

L. Qiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 123–128
125
Table 1. Ki values of the 4-substituted phenylalanine cyano-(S)-
pyrrolidine derivativesa
R
NC
O
H
N
Ki (µM)
N
N
H
N
N
H2N
0.027
NC
CN
O
1
Compound
R
Ki (lM)
2
0.063
H
3
0.034
I
Figure 3. X-ray structure of DPP IV in complex with compound 5.
PDB code is 2AJL. (A) Active site residues are depicted in gray.
Inhibitor is in green and atoms are colored by element with nitrogen
blue and oxygen red. (B) Space filling picture showing the ligand-
binding cavity.
4
5
0.470
0.013
HO2C
Me
6
0.020
of Asn710 stabilizes the P2 carbonyl oxygen atom.
The biphenyl extends into the lipophilic pocket
formed by the side chains of Phe357 and methylene
groups of the Arg358 side chain. The surface represen-
tation clearly shows the biphenyl group extending into
the lipophilic pocket (Fig. 3B). The middle ring of the
biphenyl group is situated in the pocket in such a way
as to allow for an edge to face p–p interaction with
the Phe357.
OCH3
H3CO
F
7
8
9
0.026
0.0031
0.0053
Using the information gleaned from the crystal struc-
ture and the SAR generated from our study of the 4-
substituted phenylalanine cyano-(S)-pyrrolidines, we
looked to extend our results to non-nitrile inhibitors.
The nitrile group forms a reversible covalent bond
with the active site serine and yields potent inhibitors.
However, under neutral and basic aqueous conditions,
the P2 site amine could potentially undergo a nucleo-
philic attack of the carbon of the nitrile to form an
inactive cyclic amidine.16 For this reason it was of
interest to identify non-nitrile containing and revers-
ible inhibitors, which may offer an advantage over
existing inhibitors. A quick survey of the proline mim-
ics was done using 4-phenyl-phenylalanine as P2 moi-
ety (Table 2). Replacement of the (S)-cyano-
pyrrolidine with a thiazolidine ring gave compound
12 with sufficient potency (360 nM) to build on. Ex-
change of the terminal phenyl ring with a pyridyl ring
(Table 3, Entry 18) had little effect on the activity.
Placement of a carboxylic acid to the 4-position of
the biaryl gave compound 19 with only slightly in-
creased activity. However,sss addition of a carboxylic
acid at the 3-position increased the potency by 2-fold
(entry 20), indicating a potential interaction of the
acid moiety with R358. Addition of a cyano- or fluo-
rine atom to the 4-position of the biphenyl (entry 21,
22) also resulted in a 2-fold increase in potency. Inter-
estingly parallel to the SAR of the (S)-cyano-pyrroli-
dine series, disubstitution with fluorine at the 2- and
4-positions (entry 23) again demonstrated an additive
F
F
10
11
0.0022
0.036
F
O
a Potency was assessed by a kinetic method monitored at 405 nm
similar to that of Hughes et al.12 IC50 was determined from the slope
of regression lines of modified Dixon plots of uninhibited velocity/
inhibited velocity versus inhibitor concentration. Ki was calculated
using substrate concentration [S], substrate Km, and IC50 with
Ki = IC50 · (1/(1 + [S]/Km)).14 Compounds were assayed in duplicate.
The assay coefficient of variation was 10.1% (N = 5).
A crystal structure of compound 5 covalently bound
to the active site of the enzyme was determined (Figs.
3A and B).15 The structural information validated the
inhibitor design and binding hypothesis. The inhibitor
is covalently bound to active site Ser630 through the
nitrile at P1. The pyrrolidine ring fits into the hydro-
phobic S1 pocket formed by the side chains of
Arg125, Tyr547, Ser630, Tyr662, and Tyr666. The
main chain of the P2-residue interacts with the enzyme
at two anchor sites. Glu206 forms a salt bridge with
the free amino terminus of the P2 residue.9 The Nd2

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L. Qiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 123–128
Table 2. Ki values of 4-phenylphenylalaninyl proline mimetics
Table 3. Ki value of aminoacylthiazolidines
R
S
N
H2N
O
Compound
R
Ki (lM)
R
H2N
I
O
17
0.98
Compound
R
Ki (lM)
12
18
0.36
N
5
0.013
CN
0.355
12
13
14
0.36
1.16
N
S
N
OH
O
N
N
19
20
0.31
CO2H
>12.8
0.166
OH
N
N
15
16
>12.8
>12.8
NC
F
OBn
O
21
22
0.16
0.17
F
F
effect and increased the potency by about 4-fold to
96 nM and gave us our most potent inhibitor of this
series to date.
23
24
25
26
0.096
8.9
The size requirements of the S2 pocket were also probed.
Replacement of the linear biphenyl group with naphtha-
len-2-yl group resulted in a huge loss of activity (Table 3,
entry 24). A naphthalen-1-yl or a 1H-indol-3-yl group in
place of the biaryl was detrimental to activity (entry 25,
26).
>12.8
>12.8
After the discovery that the biaryl thiazolidines were
potent inhibitors of DPP IV, a study was conducted
to investigate Caco-2 permeability and microsome
stability of the thiazolidine analogs compared to the
corresponding nitrile derivatives. Five compounds
were selected and the results are shown in Table 4.
In most cases, changing from (S)-cyano-pyrrolidine
to the thiazolidine improved the Caco-2 permeability
while maintaining good human liver microsome
stability.
H
N
SAR was established. The SAR was then successfully
transferred to non-cyano containing biaryl thiazoli-
dines, and double-digit nanomolar Ki as well as im-
proved ADME characteristics were achieved.
A
In summary, a series of novel biaryl inhibitors of DPP
IV was identified using a structure-based approach.
The Ki of the biaryl (S)-cyano-pyrrolidines reached a
lower single digit nanomolar range, and a preliminary
crystal structure of human DPP IV in complex with
one of our inhibitors was obtained, and the structural
information is expected to facilitate future lead opti-
mization efforts.

L. Qiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 123–128
127
Table 4. Caco-2 permeability and human microsome stability study of selected compounds
R1
R2
H2N
O
Compound
R1
R2
Caco-2a
HLMb
A ! B
B ! A
N
5
2.66
3.82
52.3
62
CN
S
12
8
4.56
7.21
1.5
N
N
F
F
4.45
5.61
4.93
6.7
CN
S
22
16.81
4.93
78.5
73.9
N
N
F
F
10
CN
a Papp, apparent permeability (·10ꢀ6 cm/s) at 10 lM compound concentration.
b % remaining after 10 min of incubation at 1 lM compound concentration.
Sudre, B.; Broqua, P.; White, R. B.; Ashworth, D.; Evans,
M.; Haigh, R.; Junien, J.-L.; Aubert, M. L. Diabetes 2002,
51, 1461; For NVP-LAF237 see: (d) Villhauer, E. B.;
Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.; Dunning,
B. E.; Prasad, K.; Mangold, B. L.; Russell, M. E.; Hughes,
T. E. J. Med. Chem. 2003, 46, 2774; For BMS-477118 see:
(e) Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin,
D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins,
L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.;
Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan,
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Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med.
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H.; Hickey, G. J.; Kowalchick, J. E.; Leiting, B.; Lyons,
K.; Marsilio, F.; McCann, M. E.; Patel, R. A.; Petrov, A.;
Scapin, G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt,
M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N. A.; Weber,
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9. Rasmussen, H. B.; Branner, S.; Wiberg, F. C.; Wagtmann,
N. Nat. Struct. Biol. 2003, 10, 19.
phenyl-Sepharose 6FF (1.5–0 M (NH4)2SO4 gradient in
20 mM Tris–HCl, pH 8.0). Purified DPP IV (0.5 mg/mL)
was dialyzed against 20 mM Tris–HCl, pH 8.0, 25 mM
NaCl, flash-frozen in liquid nitrogen, and stored at
ꢀ80 ꢁC for enzyme assays. For crystallographic purpos-
es, DPP IV was concentrated to 18 mg/mL by filtration
(Ultracel-4, 30000 MWCO, Millipore) after dialysis. (c)
DPP IV crystallization, data collection, molecular
replacement, and structure refinement. DPP IV in
complex with compound 5 was crystallized by micro
seeding from a solution containing 25% polyethylene
glycol 4000 and 0.1 M Tris at pH 7.5. The crystals were
transferred to a cryoprotectant solution containing the
crystallization solution and 10% glycerol. X-ray diffrac-
10. During the course of this work several publications on
crystal structure of DPP IV appeared. For leading
references see: (a) Engel, M.; Hoffmann, T.; Wagner, L.;
Wermann, M.; Heiser, U.; Kiefersauer, R.; Huber, R.;
Bode, W.; Demuth, H. U.; Brandstetter, H. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 5063; (b) Oefner, C.; DÕArcy,
A.; Sweeney, A. M.; Pierau, S.; Gardiner, R.; Dale, G. E.
Acta Cryst. 2003, D59, 1206; (c) Hiramatsu, H.; Yamam-
oto, A.; Kyono, K.; Higashiyama, Y.; Fukushima, C.;
Shima, H.; Sugiyama, S.; Inaka, K.; Shimizu, R. Biol.
Chem. 2004, 385, 561.
11. Goodson, F. E.; Wallow, T. I.; Novak, B. M. Org. Synth.
1997, 75, 61.
˚
tion data to a resolution of 2.5 A were collected on a
12. (a) Hughes, T. E.; Mone, M. D.; Russell, M. E.; Weldon, S.
C.; Villhauer, E. B. Biochemistry 1999, 38, 11597; (b)
Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Dunning,
B. E.; Mangold, B. L.; Mone, M. D.; Russell, M. E.;
Weldon, S. C.; Hughes, T. E. J. Med. Chem. 2002, 45, 2362.
13. While this manuscript was in preparation, arylphenylala-
nine derivatives of fluorinated azetidine, pyrrolidine or
piperidines as DPP IV inhibitors was published Xu, J.;
Wei, L.; Mathvink, R.; He, J.; Park, Y.-J.; He, H.; Leiting,
B.; Lyons, K. A.; Marsilio, F.; Patel, R. A.; Wu, J. K.;
Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett.
2005, 15, 2533.
Bruker AXS Proteum 6000 detector. Diffraction data
were indexed, integrated, and scaled using the Proteum
Processing Program suite from Bruker AXS. The DPP
IV crystals belong to the P21 space group, with unit cell
˚
parameters a = 65.28, b = 126.88, c = 110.83 A, and
b = 99.4ꢁ. The structure was determined and refined by
molecular replacement with CNX (Brunger, A. T.;
Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.,
Nilges, M.; Pannu, N. S.; Read, R. J., Rice, L. M.;
Simonson, T.; Warren, G. L. Acta Crystallogr. 1998,
D54, 905) using the structure of DPP IV with valine-
pyrrolidide as a search model (PDB ID: 1NIM). All
model building was done using O (Jones, T. A.; Zou, J.
Y.; Cowan, S. W.; Kjeldgaard, M. Acta Crystallogr.
1991, A47, 110). The final structure was refined to an R-
factor of 22.6.
14. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
15. (a) PDB code is 2AJL; (b) DPP IV Purification. The
cDNA fragment encoding the soluble region Ser39-
Pro766 of human DPP IV was inserted into the
baculovirus vector pDEST20 (Invitrogen) and expressed
in Hi5 insect cells. Active DPP IV was purified from the
concentrated cell media on a Q-Sepharose (0–2 M NaCl
gradient in 10 mM Tris–HCl, pH 8.0) followed by
16. Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey,
B. F.; Dunning, B. E.; Prasad, K.; Mangold, B. L.;
Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46,
2774.