Glutamic acid analogues as potent dipeptidyl peptidase IV and 8 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.04.051

To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that in

Full text of DOI:10.1016/j.bmcl.2005.04.051

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3271–3275
Glutamic acid analogues as potent dipeptidyl
peptidase IV and 8 inhibitors
I-Lin Lu,a,b, Shiow-Ju Lee,a, Hsu Tsu,a, Su-Ying Wu,a Kuo-His Kao,a Chia-Hui Chien,a
Ying-Ying Chang,a Yuan-Shou Chen,a Jai-Hong Cheng,a Chung-Nien Chang,a
Tung-Wei Chen,a Sheng-Ping Chang,c Xin Chena,* and Weir-Torn Jiaanga,*
aDivision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, National Health Research
Institutes, No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, ROC
bGraduate Institute of Life Sciences, National Defense Medical Center, No.161, Section 6,
Min-Chuan East Road, Taipei 114, Taiwan, ROC
cDepartment of Obstetrics and Gynecology, Taipei Veterans General Hospital, No. 201, Sec. 2,
Shih-Pai Road, Taipei 112, Taiwan, ROC
Received 1 April 2005; revised 15 April 2005; accepted 22 April 2005
Available online 31 May 2005
Abstract—To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine
with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that
incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-
IV over both DPP8 and DPP-II. From structure–activity relationship studies, we speculate that the S2 site of DPP8 might be similar
to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic
interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.
Ó 2005 Elsevier Ltd. All rights reserved.
Dipeptidyl peptidase IV (DPP-IV, also known as CD26)
(EC 3.4.14.5) is a drug target for type II diabetes. It is a
prolyl dipeptidase involved in the in vivo degradation of
two insulin-sensing hormones, glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic polypep-
tide (GIP), by cleaving at the peptide bond of the penul-
timate position.1,2 DPP-IV knockout mice and rats have
consistently displayed healthy phenotypes.3 When chal-
lenged with a high concentration of glucose, these ani-
mals show improved glucose tolerance, enhanced
insulin secretions, and increased circulating active
GLP-1 peptide.3 By extending the duration of action
of GLP-1, DPP-IV inhibitors stimulate insulin secretion,
inhibit glucagon releases, and slow gastric emptying in
animal models, each action a benefit in the control of
glucose homeostasis.4 Recent successes in clinical trials
of several DPP-IV inhibitors have demonstrated that
selective and potent inhibitors of DPP-IV protease are
effective for the treatment of type II diabetes.5
DPP-IV belongs to the prolyl dipeptidase family, which
includes DPP-II, DPP-IV, DPP8, and DPP9.6 The in
vivo functions of these prolyl dipeptidases, other than
DPP-IV, are largely unknown. DPP-II is known as qui-
escent cell proline dipeptidase or DPP-VII (DPP7).6,7
The inhibition of DPP-II has been shown to result in
the apoptosis of quiescent T cells.8 DPP8 is a cytoplas-
mic protease with a 51% homology with DPP-IV at
the amino acid level.9 The administration of selective
DPP8/DPP9 inhibitors in animals results in severe toxic
reactions, including alopecia, thrombocytopenia, ane-
mia, enlarged spleen, multiple histological pathologies,
and increased mortality.10 In light of the physiological
importance of prolyl cleaving enzymes and the toxicity
associated with DPP8/DPP9, it is important to have
selective inhibitors targeting DPP-IV for the treatment
of type II diabetes.
Keywords: DPP-IV; DPP8.
*
Corresponding authors. Tel.: +886 37 246166x35712; fax: +886 37
586456; e-mail addresses: xchen@nhri.org.tw; wtjiaang@nhri.org.tw
These authors contributed equally.
In the current drug design of inhibitors, the selectivity of
inhibitor over other closely related enzymes must be
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.051

3272
I-Lin Lu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3271–3275
this paper, structure–activity relationship studies in this
series are discussed. Introduction of suitable amines at
the 4-position of glutamic acid as their P2 site produced
some DPP-IV inhibitors with modest selectivity in
regard to DPP8. These inhibitors were also tested against
DPP-II. Since significant inhibition (IC50 <10 lM) was
not observed, data are presented for DPP-IV and
DPP8 only. Determination of IC50 values for inhibition
of DPP-IV, DPP8, and DPP-II was carried out as
described previously.8,16
A series of (2S)-cyanopyrrolidines with aspartic acid
derivatives at the P2 site, compounds 8–11, was synthe-
sized. The synthetic strategy was as follows (Scheme 1).
Compounds 8–11 were synthesized from BOC-L-aspar-
tic acid 4-benzyl ester 5; DCC coupling of 5 with (2S)-
cyanopyrrolidine was followed by hydrogenation to give
acid 6. Compound 6 was then coupled with primary or
secondary amines, 1,2,3,4-tetrahydroisoquinoline deriva-
tives, piperazine derivatives, benzylamine derivatives, or
phenylethylamine derivatives to yield 7. Removal of the
BOC protecting group of 7 with trifluoroacetic acid
yielded the desired 4-substituted aspartic acid deriva-
tives 8–11, respectively.
Figure 1. Inhibitors of DPP-IV.
Table 2 summarizes the inhibitory properties of 2-cyano-
pyrrolidine with aspartic acid derivative substituents at
carbon 4. Generally, these inhibitors in Table 2 exhibited
a similar range of activities against DPP-IV and DPP8.
Lead compound 8a inhibited DPP-IV with an IC50 of
99 nM, and DPP8 at 120 nM. The 6,7-dimethoxyl-
substituted compound 8b had slightly higher inhibitory
activity for DPP-IV (IC50 = 63 nM), and inhibited
DPP8 with an IC50 value of 19 nM, making it one of
the most potent DPP8 inhibitors in our investigation.
Introduction of a 2-hydroxylethyl group at the first car-
bon position of isoquinoline provided compound 8c,
which is still a potent DPP-IV inhibitor with an IC50 va-
lue of 45 nM. The selectivity index of compound 8c
shows 13-fold more selectivity than that of 8b. This sug-
gests that the alkyl substituent at first carbon position in
the isoquinoline ring may confer selectivity.
determined. The structures of a number of DPP-IV
inhibitors have been published (Fig. 1). The cyanopyrr-
olidine derivatives 1 and 2 are potent DPP-IV inhibitors
developed by Novartis (Table 1), with excellent selectiv-
ity over both DPP8 and DPP-II.11,12 Compound 1 (also
known as NVP-LAF237) has progressed to mid-stage
clinical trials with positive and encouraging results for
the treatment of type II diabetes. Cyclohexylglycine-
(2S)-cyanopyrrolidine 3 is a potent DPP-IV inhibitor
with an IC50 value of 12 nM,13 but it lacks selectivity to-
ward DPP8. The IC50 value of this compound for DPP8
is 27 nM (Table 1). A related compound lacking a nitrile
group, isoleucylthiazolidide 4 (Ile-Thia, or P32/98),
shows only modest potency with an IC50 of 1.66 lM to-
ward DPP-IV inhibition,14 while it is more potent
against DPP8 with an IC50 value of 364 nM (Table 1).
This compound is effective in improving glucose toler-
ance in healthy people and in people with type 2
diabetes.15
To explore this, further modification was carried out at
the first carbon position. With a more bulky isopropyl
substituent, compound 8d preserved the high potency
of 8c, and showed some increase in selectivity with re-
spect to DPP8. However, the benzyl substituent in com-
pound 8e neither improved potency nor enhanced the
selectivity toward DPP-IV. As expected, increasing the
bulk at the first carbon position of isoquinoline resulted
in a dramatic improvement in selectivity; tert-butyl sub-
stituent 8f was a 73 nM inhibitor of DPP-IV with 30-
fold selectivity over DPP8. The 6-methoxyl analogue
8g was approximately equipotent with the 6,7-dimeth-
oxyl analogue 8f, whereas the 7-methoxyl analogue 8h
showed a slight drop in potency. Both monomethoxyl
substituted analogues 8g and 8h had reduced selectivities
relative to the 6,7-dimethoxyl analogue 8f.
From our previous study, glutamic acid derivatives 8b
and 9a are potent DPP-IV and DPP8 inhibitors.16 In
Table 1. Inhibition of DPP-IV, DPP8, and DPP-II by compounds 1–4,
8b, and 9aa
Compd
DPP-IV
IC50 (nM)
DPP8
IC50 (nM)
DPP-II
IC50 (nM)
1
51
53
14,219
4573
27
>100,000
26,520
39,873
2
3
12
4
1660
63
364
19
68,985
31,259
>100,000
8b
9a
The piperazine series of compounds 9a–c was
more potent as DPP8 inhibitors, with IC50 values
ranging from 16 to 71 nM (Table 2). Among them,
227
16
a IC50 determination was carried out as described in Refs. 8 and 16.

I-Lin Lu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3271–3275
3273
Scheme 1. Synthesis of aspartic acid derivatives: (a) DCC, HOSu, 1,4-dioxane/CH2Cl2; (b) H2, 5% Pd/C, MeOH; (c) DCC, CH2Cl2, various amines;
(d) TFA, CH2Cl2.
Table 2. Inhibition of DPP-IV and DPP8 by compounds 8–11
Compd
R1
R2
DPP-IV IC50 (nM)
DPP8 IC50 (nM)
8a
8b
H
6,7-(OMe)2
H
99
63
120
19
H
8c
8d
6,7-(OMe)2
6,7-(OMe)2
–(CH2)2OHa
Isopropyla
Benzyla
45
47
174
439
8e
6,7-(OMe)2
6,7-(OMe)2
97
73
220
8f
8g
tert-Butyla
tert-Butyla
tert-Butyla
–CH(4-FC6H5)2
Nicotinonitrile
Benzoyl
H
2196
1439
2712
16
6-OMe
7-OMe
72
8h
9a
195
227
87
H
9b
9c
H
28
71
H
123
238
252
140
212
251
116
182
305
10a
10b
10c
10d
10e
11a
11b
11c
H
389
H
Benzyla
Ethyla
2327
1412
3055
13,217
177
H
H
Isopropyla
tert-Butyla
H
H
3,4-OMe
H
H
–CH2OMea
Isopropyla
2110
1597
a The first carbon position of isoquinoline, benzylamine, or phenethylamine is racemate.
1-(4,40-difluorobenzhydryl)piperazine 9a showed the
greatest potency to DPP8 (IC50 = 16 nM), having 14-
fold selectivity over DPP-IV. A similar trend was ob-
served for the nicotinonitrile analogue 9b and the

3274
I-Lin Lu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3271–3275
benzoyl analogue 9c, which were also more potent as
DPP8 inhibitors, although selectivity over DPP-IV was
relatively low.
The P2-carbonyl oxygen forms hydrogen bonds to
Asn710 and Arg125. The free amino terminus of the
P2-residue forms hydrogen bonds with the side chain
of Glu205, Glu206, and Tyr662. In addition to hydro-
gen-bonding interactions, the phenyl ring of 1,2,3,4-tet-
rahydroisoquinoline is stacked against the side chain of
Tyr547 to produce hydrophobic interactions. Superim-
position of the modeling structure of compound 8a
with the published X-ray structure of substrate ana-
logue valine-pyrrolidine (blue) shows these predicted
interactions are consistent with the literature reports
(Fig. 2).18
Compared with the above isoquinoline series of com-
pounds 8, phenyl-substituted aliphatic amine extensions,
such as the benzylamine derivatives 10 and the pheneth-
ylamine derivatives 11, showed a slight drop in potency
(DPP-IV IC50 = 116–305 nM, Table 2). Several alkyl
substitutions at the first carbon position of benzylamine
and phenethylamine, compounds 10b–e and 11b,c, were
explored, and showed little effect in improving potency
compared with unsubstituted analogues 10a and 11a,
respectively. Nevertheless, increasing the size of the alkyl
substituent again resulted in dramatic improvements in
the selectivity profile of these compounds. Unsubsti-
tuted analogue 10a showed similar inhibition against
DPP-IV and DPP8, and ethyl substituted analogue 10c
or isopropyl substituted analogue 10d exhibited
>10-fold selectivity over DPP8. A more profound
improvement was obtained with the more bulky tert-bu-
tyl-substituent in analogue 10e, which showed 53-fold
selectivity for DPP-IV over DPP8, and exhibited no sig-
nificant DPP8 inhibition (IC50 = 13 lM), although its
IC50 value for DPP-IV was a moderate 251 nM. This
analogue represents the most selective DPP-IV inhibitor
among the 4-substituted aspartic acid series of
inhibitors.
The crystal structure of the DPP8 protein has yet to be
elucidated. An earlier report from our laboratories
speculates that the S1 site of DPP8 may be larger than
that of DPP-IV, allowing the accommodation of larger
C-terminal residues, such as isoquinoline or isoindoline,
at the P1 site.16 In this study, our results suggest that
the S2 pocket of DPP-IV might be similar to DPP8.
Thus, it is likely that some DPP-IV inhibitors may have
an inhibitory effect on DPP8. Furthermore, proline mi-
metic inhibitors, for which the P2 residues are a-carbon
substituted glycine derivatives (3, 4, 8–10), exhibited
poor selectivities. However, DPP-IV inhibitors with
N-substituted glycine in the P2 site, such as NVP
LAF-237 (1), NVP DPP-728 (2),17b or with an aromatic
moiety involving in hydrophobic interaction with the
side chain of Phe357, such as b-amino acid MK-
0431,19 provided excellent selectivity over DPP8. Never-
theless, we still found some selective 4-substituted
aspartic acid 2-cyanopyrrolidine inhibitors, such as
tert-butyl substitution at first carbon position of the iso-
quinoline 8f and the benzylamine 10e, that selectively
inhibited DPP-IV by a magnitude of >30-fold better
than DPP8.
To explore the mechanism of the inhibition, computer-
modeling study was carried out to investigate a possi-
ble interaction between these inhibitors and DPP-IV.
The predicted binding mode of compound 8a at the
active site of DPP-IV is shown in Figure 2. The (2S)-
cyanopyrrolidine moiety of inhibitor occupies the
narrow hydrophobic S1 pocket of the enzyme and
the nitrile group is correctly positioned to interact with
the active Ser630 to form an imidate intermediate.17
In summary, by introducing a series of primary or sec-
ondary amines to the 4-position of aspartic acid with
P1-site 2-cyanopyrrolidine, novel chemical compounds
that inhibit prolyl dipeptidases DPP-IV and DPP8 have
been examined. In general, this class of inhibitors shows
modest selectivity profile over DPP8. We found that
isoquinoline derivatives 8c–d are potent DPP-IV inhib-
itors (IC50 <50 nM), but less selective over DPP8 (<10-
fold). Interestingly, the bulky tert-butyl-substituted
isoquinoline derivative 8f shows 30-fold selectivity over
DPP8, and maintains excellent potency for DPP-IV
with an IC50 value of 73 nM. Phenyl-substituted piper-
azine series 9 are potent DPP8 inhibitors, with com-
pound 9a the most potent (DPP8 IC50 = 16 nM).
However, phenyl-substituted aliphatic derivatives 10
and 11 are less potent as DPP-IV and DPP8 inhibitors.
Notable among these is the tert-butyl-substituted ana-
logue 10e, which shows 53-fold selectivity for DPP-IV
over DPP8, with moderate DPP-IV inhibition
(IC50 = 251 nM). From the structure–activity relation-
ship studies, we speculate that the S2 pocket of DPP8
might be similar to that of DPP-IV, while DPP-IV
inhibitor with N-substituted glycine in the P2 site,
and/or with a moiety involving hydrophobic interaction
with the side chain of Phe357 might provide a more
selective DPP-IV inhibitor.
Figure 2. The binding mode of compound 8a is predicted by computer
model.

I-Lin Lu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3271–3275
3275
8. Leiting, B.; Pryor, K. D.; Wu, J. K.; Marsilio, F.; Patel, R.
A.; Craik, C. S.; Ellman, J. A.; Cummings, R. T.;
Thornberry, N. A. Biochem. J. 2003, 371, 525.
9. Abbott, C. A.; Yu, D. M.; Woollatt, E.; Sutherland, G.
R.; McCaughan, G. W.; Gorrell, M. D. Eur. J. Biochem.
2000, 267, 6140.
Acknowledgments
We would like to thank Dr. Yu-Sheng Chao for his sup-
port and encouragement. The study was financially sup-
ported by National Health Research Institutes, Taiwan.
10. Lankas, G.; Leiting, B.; Roy, R. S.; Eiermann, G.; Biftu,
T.; Kim, D.; Ok, H.; Weber, A.; Thornberry, N. A. The
American Diabetes Association 64th Scientific Session,
Orlando, FL, June 4–8, 2004, Abstract 7-OR.
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmcl.2005.04.051.
11. (a) 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; (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.
References and notes
12. IC50 determination was carried out as described in Refs. 8
and 16.
1. Mentlein, R. Regul. Pept. 1999, 85, 9.
2. Zhu, L.; Tamvakopoulos, C.; Xie, D.; Dragovic, J.; Shen,
X.; Fenyk-Melody, J. E.; Schmidt, K.; Bagchi, A.; Griffin,
P. R.; Thornberry, N. A.; Roy, R. S. J. Biol. Chem. 2003,
278, 22418.
13. Ashworth, D. M.; Atrash, B.; Baker, G. R.; Baxter, A. J.;
Jenkins, P. D.; Jones, D. M.; Szelke, M. Bioorg. Med.
Chem. Lett. 1996, 6, 1163.
14. Parmee, E. R.; He, J.; Mastracchio, A.; Edmondson, S.
D.; Colwell, L. E.; Eiermann, G.; Feeney, W. P.; Habu-
lihaz, B.; He, H.; Kilburn, R.; Leiting, B.; Lyons, K.;
Marsilio, F.; Patel, R.; Petrov, A.; di Salvo, J.; Wu, J. K.;
Thornberry, N.; Weber, A. E. Bioorg. Med. Chem. Lett.
2004, 14, 43.
15. (a) Pospisilik, J. A.; Staord, S. G.; Demuth, H.-U.;
Brownsey, R.; Parkhouse, W.; Finegood, D. T.; McIn-
tosh, C. H. S.; Pederson, R. A. Diabetes 2002, 51, 943; (b)
Sorbera, L. A.; Revel, L.; Castaner, J. Drugs Future 2001,
26, 859.
3. (a) Marguet, D.; Baggio, L.; Kobayashi, T.; Bernard, A.
M.; Pierres, M.; Nielsen, P. F.; Ribel, U.; Watanabe, T.;
Drucker, D. J.; Wagtmann, N. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 6874; (b) Conarello, S. L.; Li, Z.; Ronan,
J.; Roy, R. S.; Zhu, L.; Jiang, G.; Liu, F.; Woods, J.;
Zycband, E.; Moller, D. E.; Thornberry, N. A.; Zhang, B.
B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6825; (c)
Nagakura, T.; Yasuda, N.; Yamazaki, K.; Ikuta, H.;
Yoshikawa, S.; Asano, O.; Tanaka, I. Biochem. Biophys.
Res. Commun. 2001, 284, 501.
4. (a) Pospisilik, J. A.; Stafford, S. G.; Demuth, H. U.;
McIntosh, C. H.; Pederson, R. A. Diabetes 2002, 51, 2677;
(b) Ahren, B.; Holst, J. J.; Martensson, H.; Balkan, B. Eur.
J. Pharmacol. 2000, 404, 239; (c) 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; (d) Deacon,
C. F.; Danielsen, P.; Klarskov, L.; Olesen, M.; Holst, J. J.
Diabetes 2001, 50, 1588; (e) Sudre, B.; Broqua, P.; White,
R. B.; Ashworth, D.; Evans, D. M.; Haigh, R.; Junien, J.
L.; Aubert, M. L. Diabetes 2002, 51, 1461.
5. Deacon, C. F.; Ahren, B.; Holst, J. J. Expert Opin. Invest.
Drugs 2004, 13, 1091.
6. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.
7. Chiravuri, M.; Huber, B. T. Apoptosis 2000, 5, 319.
16. Jiaang, W.-T.; Chen, Y.-S.; Tsu, S.; Wu, S.-H.; Chien,
C.-H.; Chang, C.-N.; Chang, S.-P.; Lee, S. J.; Chen, X.
Bioorg. Med. Chem. Lett. 2005, 15, 687.
17. (a) Hughes, T. E.; Mone, M. D.; Russell, M. E.; Weldon,
S. C.; Villhauser, E. B. Biochemistry 1999, 38, 11597; (b)
Oefner, C.; DÕArcy, A.; Sweeney, A. M.; Pierau, S.;
Gardiner, R.; Dale, G. E. Acta Crystallogr., Sect. D 2003,
59, 1206.
18. Rasmussen, H. B.; Branner, S.; Wiberg, F. C.; Wagtmann,
N. Nat. Struct. Biol. 2003, 10, 19.
19. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher,
M. H.; He, 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.;
Webe, A. E. J. Med. Chem. 2005, 48, 141.