Discovery of β-homophenylalanine based pyrrolidin-2-ylmethyl amides and sulfonamides as highly potent and selective inhibitors of dipeptidyl peptidase IV

Article abstract of DOI:10.1016/j.bmcl.2009.05.109

Modifications of DPP-4 inhibitor 5, that was discovered by structure based design, are described and structure-activity relationships discussed. With analogue 7k one of the most potent non-covalent inhibitors of DPP-4 reported to date (IC₅₀ = 0.38 nM) was discovered. X-ray structure of inhibitor 7k bound to DPP-4 revealed a hydrogen bonding interaction with Q553. First successful efforts in balancing overall properties, as demonstrated by improved metabolic stability, highlight the potential of this series.

Full text of DOI:10.1016/j.bmcl.2009.05.109

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4201–4203
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of b-homophenylalanine based pyrrolidin-2-ylmethyl amides and
sulfonamides as highly potent and selective inhibitors of dipeptidyl peptidase IV
à
§
*
Sonja Nordhoff , Silvia Cerezo-Gálvez , Holger Deppe, Oliver Hill , Meritxell López-Canet ,
Christian Rummey, Meinolf Thiemann à, Victor G. Matassa –, Paul J. Edwards ||, Achim Feurer
Santhera Pharmaceuticals (Switzerland) Ltd, Hammerstrasse 47, CH-4410 Liestal, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Modifications of DPP-4 inhibitor 5, that was discovered by structure based design, are described and
structure–activity relationships discussed. With analogue 7k one of the most potent non-covalent inhib-
itors of DPP-4 reported to date (IC50 = 0.38 nM) was discovered. X-ray structure of inhibitor 7k bound to
DPP-4 revealed a hydrogen bonding interaction with Q553. First successful efforts in balancing overall
properties, as demonstrated by improved metabolic stability, highlight the potential of this series.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 20 April 2009
Revised 25 May 2009
Accepted 27 May 2009
Available online 30 May 2009
Keywords:
Dipeptidyl peptidase-IV
DPP-4
Type 2 diabetes
Glucagon-like peptide-1
GLP-1
Inhibition of dipeptidyl peptidase IV (DPP-4) has recently
received intense interest as a novel therapeutic approach to the
treatment of type 2 diabetes.1 DPP-4 is a serine protease that
cleaves dipeptides N-terminal from its peptide substrates.2 The
incretin hormone glucagon-like peptide 1 (GLP-1) that is responsi-
ble for the secretion of insulin after food intake is rapidly inacti-
vated by DPP-4.3 Consequently inhibition of DPP-4 prolongs the
half-life of endogenous GLP-1 and enhances its beneficial effects
in glucose dependent insulin secretion and b-cell restoration.4
To date a range of structurally diverse inhibitors of DPP-4 have
been reported, some of which have now reached the market or
entered advanced stage clinical trials. Among them are Sitagliptin
(1),5 Alogliptin (4)6 and the covalent binding pyrrolidine nitriles
Vildagliptin (2)7 and Saxagliptin (3)8 (Fig. 1).
Based on our findings from the X-ray analysis of inhibitor 59
bound to DPP-4, where the fluorophenyl ring occupied the
proline-binding S1 pocket, the amino group was anchored to the
glutamates E205 and E206, the pyrrolidine located distal to S1
and the phenylamide had contacts with Y547 and R125, initial sites
attractive for SAR exploration were determined to be the phenyl
ring of the b-amino acid moiety and the pyrrolidin-2-ylmethyl
amide.
F
OH
F
NH2
O
N
N
N
N
H
CN
N
We have previously described9 our approach to the design of
DPP-4 inhibitor 5 (Fig. 2) arising from the discovery of a ‘reverse’
binding to DPP-4 when compared to classical DPP-4 inhibitors such
as 6 (P32/98).10 We now report on the elaboration of a series of
DPP-4 inhibitors, based on structure 5, and the elucidation of struc-
ture–activity relationships (SAR) therein.
O
F
N
CF3
MK-0431:
Sitagliptin (1)
NVP-LAF237:
Vildagliptin (2)
O
OH
N
CN
O
H2N
N
N
O
N
* Corresponding author. Tel.: +41 61 906 89 73; fax: +41 61 906 89 88.
E-mail address: sonja.nordhoff@santhera.com (S. Nordhoff).
Present address: Bayer CropScience AG, Monheim, Germany.
NH2
NC
à
Present address: Apogenix GmbH, Heidelberg, Germany.
Present address: Otsuka Pharma GmbH, Frankfurt, Germany.
Present address: Almirall Prodesfarma SA, Barcelona, Spain.
BMS-477118:
Saxagliptin (3)
SYR-322:
Alogliptin (4)
§
–
||
Present address: Boehringer Ingelheim (Canada) Ltd, Québec, Canada.
Figure 1. Selected DPP-4 inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.109

4202
S. Nordhoff et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4201–4203
Table 1
O
Amide-derived and sulfonamide-derived DPP-4 inhibitors
R
X
NH
NH
O
NH2
O
NH2
O
N
N
Ar
S
N
H2N
F
5
6
(P32/98)
Compound
Ar
R
X
DPP-4 IC50 (nM)9
Figure 2. DPP-4 inhibitors.
5
2-F–Ph
3-Cl–Ph
2-F–Ph
2-F–Ph
2-F–Ph
2-F–Ph
2-F–Ph
2-F–Ph
2-F–Ph
2-F–Ph
3-Cl–Ph
3-Cl–Ph
3-Cl–Ph
2,4,5-Tri-F–Ph
2-F–Ph
3-Cl–Ph
2-F–Ph
2-F–Ph
2-F–Ph
Ph
Ph
2-F–Ph
3-F–Ph
4-F–Ph
2-Py
3-Py
4-Py
4-(SO2Me)–Ph
2-(SO2Me)–Ph
2-(SO2Me)–Ph
3-(SO2Me)–Ph
Cyclopropyl
Cyclopropyl
Ph
Ph
2-F–Ph
3-F–Ph
4-F–Ph
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
90
27
49
92
92
47
87
66
77
27
14
0.38
12
9
7a
7b
7c
7d
7e
7f
7g
7h
7i
A series of analogues was prepared according to Scheme 1.11
Commercial Boc-protected (S)-2-aminomethylpyrrolidine was
coupled with a carboxylic acid giving intermediate 9a. Subse-
quently the tert-butoxycarbonyl protection group was removed
under standard conditions, then the pyrrolidine moiety coupled
with acid 10 and the resulting intermediate deprotected to yield
compounds 7a–l. Compounds 8a–i were synthesized in a similar
manner employing a sulfonylchloride to furnish the sulfonamide
intermediate 9b.
Since SAR on the b-amino acid moiety corresponds with earlier
reports for related compounds,12 only findings relevant for the work
reported here are discussed. In comparison to the initial 2-fluoro
substitution (compound 5, IC50 DPP-4 = 90 nM)9 the 3-chloro ana-
logue (7a) was found to be threefold more potent. Further improve-
ment can be achieved with a 2,4,5-trifluorophenyl moiety, which
accounts for a fivefold increase in potency or more.12
7j
7k
7l
7m
8a
8b
8c
8d
8e
8f
8g
8h
8i
34
8
53
94
27
20
24
30
8
2-F–Ph
2-F–Ph
3-Cl–Ph
2,4,5-Tri-F–Ph
4-Cl–Ph
2,5-Di-Cl–Ph
Cyclopropyl
Cyclopropyl
Exploration of the pyrrolidin-2-ylmethyl amide SAR was initi-
ated with respect to potency and basic in vitro ADME properties.
Aiming for DPP-4 inhibitors that exhibit a long plasma half-life,
we first determined the metabolic stability of 5 towards rat liver
microsomes.13 A 76% turnover of 5 after 60 min indicated meta-
bolic instability. We reasoned that the unsubstituted phenyl ring
may be one potential site for metabolism. Thus we synthesized a
range of derivatives where we introduced more metabolically
robust side-chain groups. The intention to reduce the gross lipo-
philicity of the compound (5: c Log P 2.8214) as a general concept
to increase metabolic stability directed our efforts. In addition, a
variation from amide (Table 1, 7a–m) to sulfonamide (Table 1,
8a–i), thus aiding the ‘depeptidisation’ of 5, was investigated.
Within the aryl amide derivatives explored, fluoro-phenyl or
pyridyl gave little to no improvement in potency over 5 (Table
1). Substituents of this kind in the 2-position of the aryl amide
seem to be slightly preferred over 3- or 4-substitution. However,
incorporation of a hydrogen bond acceptor motif in the 3-position
provided an order of magnitude increase in potency. The reason for
this boost in affinity was revealed by X-ray crystallography15
(Fig. 3). The sulfone group of 7k forms a hydrogen bonding interac-
tion with the N–H Atom of Q553 in DPP-4. It can be speculated that
the huge energy gain is due to the replacement of a water
molecule, which is frequently found in this position of DPP-4
R
X
NH2
Boc
N
a,b
H
N
9a, X = CO
9b, X= SO2
HN
Boc
NH
O
Ar
OH
c,d
10
R
X
NH
NH2
O
5, 7: X = CO
8: X = SO2
Ar
N
Figure 3. Co-crystal structure of compound 7k; the new hydrogen bond from one
of the sulfone oxygens to the backbone carbonyl of Q553 is highlighted in red
dashes. The figure was generated using PYMOL version 0.98 (Delano Scientific,
www.pymol.org).
Scheme 1. Synthesis of inhibitors 5, 7 and 8. Reagents: (a) RCO2H, EDC, HOBt, DIEA,
DMF or RSO2Cl, TEA, DCM; (b) TFA, DCM; (c) 10, EDC, HOBt, DIEA, DMF; (d) TFA,
DCM.

S. Nordhoff et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4201–4203
4203
Table 2
Schmitt, Tina Pfeiffer-Unckrich and Siglinde Zepter for technical
assistance.
Selectivity data for selected DPP-4 inhibitors21
Compound
DPP-4 IC50
M)
DPP-7 IC50
M)
DPP-8 IC50
M)
DPP-9 IC50
M)
(
l
(
l
(
l
(l
References and notes
7j
7k
7l
7m
8a
8b
8h
8i
0.014
0.00038
0.012
0.009
0.034
0.008
0.030
0.008
6.3
8.1
11
3.4
17
19
12
10
22
8.8
5.1
3.4
2.9
0.08
0.1
2.5
1.2
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G. J.; Kowalchick, J. E.; Leiting, B.; Lyons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.;
Petrov, A.; Scapin, G.; Patel, S. B.; Sinha Roy, R.; Wu, J. K.; Wyvratt, M. J.; Zhang, B.
B.; Zhu, L.; Thornberry, N. A.; Weber, A. E. J. Med. Chem. 2005, 48, 141.
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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, D. M.; Marcinkeviciene, J.;
Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med. Chem.
2005, 48, 5025.
9. Nordhoff, S.; Cerezo-Gálvez, S.; Feurer, A.; Hill, O.; Matassa, V. G.; Metz, G.;
Rummey, C.; Thiemann, M.; Edwards, P. J. Bioorg. Med. Chem. Lett. 2006, 16, 1744.
10. Schön, E.; Born, I.; Demuth, H.-U.; Faust, J.; Neubert, K.; Steinmetzer, T.; Barth,
A.; Ansorge, S. Biol. Chem. Hoppe-Seyler 1991, 372, 305.
5.7
4.0
5.2
0.7
0.2
4.2
7.3
co-crystal X-ray structures.16 Concomitant with this polar interac-
tion, the phenyl ring is placed favorably for a -stacking interaction
with Y547. Similar contacts with DPP-4 have been reported for an
-amino acid derived inhibitor.17 However it is worthy of note,
p
a
that the interactions of 7k with Y547 and Q553 are observed
instead of those interactions with Y547 and R125 reported recently
for the closely related compound 5.9
Compound 7k (IC50 = 380 pM, Table 1) is one of the most potent
non-covalent inhibitors reported so far. As a result of that, the
compound provided a much higher selectivity against related
enzymes in comparison with similar compounds, even though
binding affinity of 7k for DPP-8 and DPP-9 did not change (Table
2). This is in line with our recently reported findings from homol-
ogy modeling.18 It has been suggested that high selectivity, in
particular over DPP-8 and DPP-9, may be important for an optimal
safety profile.19
11. Experimental details can be found in: WO2005/056003.
12. (a) Xu, J.; Ok, H. O.; Gonzalez, E. J.; Colwell, L. F.; Habulihaz, B.; He, H.; Leiting,
B.; Lyons, K. A.; Marsilio, F.; Patel, R. A.; Wu, J. K.; Thornberry, N. A.; Weber, A.
E.; Parmee, E. R. Bioorg. Med. Chem. Lett. 2004, 14, 4759; (b) Edmondson, S. D.;
Mastracchio, A.; Beconi, M.; Colwell, L. F.; Habulihaz, B.; He, H.; Kumar, S.;
Leiting, B.; Lyons, K. A.; Mao, A.; Marsilio, F.; Patel, R. A.; Wu, J. K.; Zhu, L.;
Thornberry, N. A.; Weber, A. E.; Parmee, E. R. Bioorg. Med. Chem. Lett. 2004, 14,
5151; (c) Kim, D.; Kowalchick, J. E.; Edmondson, S. D.; Mastracchio, A.; Xu, J.;
Eiermann, G. J.; Leiting, B.; Wu, J. K.; Pryor, K.-A. D.; Patel, R. A.; He, H.; Lyons, K.
A.; Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett. 2007, 17, 3373; (d)
WO2003/000180.
13. This standard liver microsome assay was carried out at GenPharmTox BioTech
AG, Fraunhoferstr. 9, 82152 Planegg, Germany.
14. c Log P predictions were performed using Biobyte software included in the SYBYL
7.0 suite distributed by Tripos, www.tripos.com.
15. Crystal structure coordinates have been deposited in the Protein Data Bank,
accession code 3HOC.
16. Examples can be found in: (a) Rasmussen, H. B.; Branner, S.; Wiberg, F. C.;
Wagtmann, N. R. Nat. Struct. Biol. 2003, 10, 19; (b) 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.
17. (a) Xu, J.; Wei, L.; Mathvink, R. J.; Edmondson, S. D.; Eiermann, G. J.; He, H.;
Leone, J. F.; Leiting, B.; Lyons, K. A.; Marsilio, F.; Patel, R. A.; Patel, S. B.; Petrov,
A.; Scapin, G.; Wu, J. K.; Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett.
2006, 16, 5373; (b) Edmondson, S. D.; Wei, L.; Xu, J.; Shang, J.; Xu, S.; Pang, J.;
Chaudhary, A.; Dean, D. C.; He, H.; Leiting, B.; Lyons, K. A.; Patel, R. A.; Patel, S.
B.; Scapin, G.; Wu, J. K.; Beconi, M. G.; Thornberry, N. A.; Weber, A. E. Bioorg.
Med. Chem. Lett. 2008, 18, 2409.
18. Rummey, C.; Metz, G. Proteins: Struct. Funct. Bioinformatics 2007, 66, 160.
19. For a detailed discussion on DPP-8 and DPP-9 inhibition with respect to toxicity,
please see: (a) Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G. J.; Beconi, M. G.;
Biftu, T.; Chan, C.-C.; Edmondson, S.; Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.;
Lyons, K. A.; Ok, H. O.; Patel, R. A.; Petrov, A. N.; Pryor, K. A.; Qian, X.; Reigle, L.;
Woods, A.; Wu, J. K.; Zaller, D.; Zhang, X.; Zhu, L.; Weber, A. E.; Thornberry, N. A.
Diabetes 2005, 54, 2988; (b) Burkey, B. F.; Hoffmann, P. K.; Hassiepen, U.; Trappe,
J.; Juedes, M.; Foley, J. E. Diabetes Obesity Metabol. 2008, 10, 1057–1061; (c)
Connolly, B. A.; Sanford, D. G.; Chiluwal, A. K.; Healey, S. E.; Peters, D. E.; Dimare,
M. T.; Wu, W.; Liu, Y.; Maw, H.; Zhou, Y.; Li, Y.; Jin, Z.; Sudmeier, J. L.; Lai, J. H.;
Bachovchin, W. W. J. Med. Chem. 2008, 51, 6005–6013.
SAR on the corresponding sulfonamides proved to be similar
but not to run entirely parallel to that of the amides (Table 1).
To accommodate an increase in molecular weight and lipophilic-
ity accompanied by the introduction of the potent 3-Cl- or 2,4,5-tri-
fluorophenyl moieties we aimed for a reduction in size and
lipophilicity of the side chain amide or sulfonamide. Gratifyingly,
incorporation of small, metabolically robust cyclopropyl groups
(7l; 7m; 8h; 8i) provided potent and selective inhibitors of DPP-4.
Overall, balancing any increase of molecular weight and gross lipo-
philicity (7m: c Log P 1.66; 8i: c Log P 1.82) resulted in significantly
improved stability in rat liver microsomes (7m, 8i: t1/2 > 2 h).20
Table 2 provides selectivity data against DPP-7, DPP-8 and DPP-
9 for selected compounds which indicate the excellent selectivities
achievable in our DPP-4 inhibitor series. The inhibitors were also
tested against Seprase (FAP) and POP. None of the compounds
showed any relevant inhibition of these related enzymes.
In summary, a novel series of non-covalent DPP-4 inhibitors
with excellent selectivities over DPP-7, DPP-8 and DPP-9 has been
discovered. Amongst the compounds described is one of the most
potent, non-covalent inhibitors reported to date (7k), benefiting
from an unexpected polar interaction that was revealed by X-ray
analysis. First successful efforts in balancing overall properties as
demonstrated by improved metabolic stability highlight the poten-
tial of this series. The results lay the foundation for the further
optimization regarding ADME and pharmacokinetic properties.
Acknowledgments
The authors gratefully acknowledge Proteros Biostructures
GmbH, Am Klopferspitz 19, 82152, Martinsried, Germany for
co-crystallization of our inhibitors and obtaining X-ray data, and
Sabine Bostel, Nicole Di Gallo, Julia Seiler, Bettina Cardel, Ute
20. These liver microsome assays were carried out at Cyprotex PLC., 15 Beech Lane,
Macclesfield, Cheshire, SK10 2DR, UK.
21. IC50 determination was performed similar to DPP-4 inhibition as described in
Ref. 9.