Substituted pyrrolidine-2,4-dicarboxylic acid amides as potent dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.037

A series of substituted pyrrolidine-2,4-dicarboxylic acid amides were synthesized as potential antidiabetic agents, and many of them showed good in vitro DPP-IV inhibition (IC₅₀ = 2-250 nM) with selectivity over DPP-II, DPP8, and FAP enzymes. Selected compounds 8c and 11a showed in vivo plasma DPP-IV inhibition after oral administration in Wistar rats.

Full text of DOI:10.1016/j.bmcl.2006.03.037

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3268–3272
Substituted pyrrolidine-2,4-dicarboxylic acid amides as potent
dipeptidyl peptidase IV inhibitors
Ting-Yueh Tsai, Mohane Selvaraj Coumar, Tsu Hsu, Hsing-Pang Hsieh,
Chia-Hui Chien, Chiung-Tong Chen, Chung-Nien Chang, Yu-Kang Lo, Ssu-Hui Wu,
Chung-Yu Huang, Yu-Wen Huang, Min-Hsien Wang, Hsin-Yi Wu, Hong-Jen Lee,
Xin Chen, Yu-Sheng Chao and Weir-Torn Jiaang*
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road,
Zhunan Town, Miaoli County 350, Taiwan, ROC
Received 12 January 2006; revised 1 March 2006; accepted 13 March 2006
Available online 11 April 2006
Abstract—A series of substituted pyrrolidine-2,4-dicarboxylic acid amides were synthesized as potential antidiabetic agents, and
many of them showed good in vitro DPP-IV inhibition (IC50 = 2–250 nM) with selectivity over DPP-II, DPP8, and FAP enzymes.
Selected compounds 8c and 11a showed in vivo plasma DPP-IV inhibition after oral administration in Wistar rats.
Ó 2006 Elsevier Ltd. All rights reserved.
Dipeptidyl peptidase IV (DPP-IV; CD26; E.C. 3.4.14.5)
inhibition is a new and promising approach for the
OH
F
F
F
NH2
O
1
,2
treatment of type-II diabetes. Inhibition of DPP-IV
results in elevated circulating levels of endogenously
N
N
N
H
N
O
N
3
CN
N
N
secreted glucagon-like peptide-1 (GLP-1), which is pro-
duced by L-cells of the small intestine in response to
1
(NVP-LAF237)
2 (MK-0431)
CF3
4
food. GLP-1 stimulates the secretion of insulin in a glu-
O
N
cose dependent fashion, inhibits glucagon release, slows
gastric emptying, and induces satiety, each a benefit in
the control of glucose homeostasis in patients with
type-II diabetes. But the active form of GLP-1 is rapidly
inactivated (t1/2 ꢀ 1 min) by the plasma DPP-IV,
through the cleavage of the dipeptide from the N-termi-
HO
R1
N
N
H
H2N
R2
O
O
R1 = Cl; R2 = H
c
CN
(BMS-477118)
8
1
2
3
11a R = H; R = CN
5
,6
nus, thereby limiting its duration of action.
Thus,
Figure 1. DPP-IV inhibitors.
inhibition of DPP-IV could lead to longer-lasting
GLP-1 levels, which in turn enhance insulin secretion
and improve the glucose tolerance. In fact human
clinical trials proved unequivocally the benefits of
DPP-IV is a serine protease that preferentially hydro-
lyzes N-terminal dipeptide from proteins having pro-
line or alanine in the penultimate position. Design
of small molecule inhibitors of DPP-IV has mainly re-
volved around investigation of compounds which
resemble the P2-P1 dipeptide substrate cleavage prod-
7
DPP-IV inhibition in type-II diabetes patients. Several
DPP-IV inhibitors are under late-stage clinical develop-
8
9
ment, including NVP-LAF237 (1), MK-0431 (2), and
1
BMS-477118 (3) (Fig. 1).
0
1
1–13
uct with the P1 site occupied by a proline mimic.
Based on this strategy, various groups have success-
fully reported the use of (2S)-cyanopyrrolidine as
the P1 portion to mimic the proline in the develop-
ment of potent DPP-IV inhibitors. Some of these
workers emphasized the development of selective
Keywords: DPP-IV; Diabetes; Pyrrolidine.
*
Corresponding author. Tel.: +886 37 246166x35712; fax: +886 37
86456; e-mail: wtjiaang@nhri.org.tw
These authors contributed equally to this work.
5
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.037

T.-Y. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3268–3272
3269
DPP-IV inhibitors over other related prolyl peptidase
such as DPP-II, DPP8, DPP9, and FAP (fibroblast
The general synthetic route for the preparation of vari-
ous substituted pyrrolidine-2,4-dicarboxylic acid amides
is shown in Scheme 1. Compound 5 was prepared
12,14
activation protein).
Achieving selective inhibition
2
0,21
of DPP-IV becomes very important, as inhibition of
DPP-II results in the apoptosis of quiescent T-cells
and DPP8/9 inhibition causes toxicities in animal
according to the literature procedure
pled with the required amine (R H) using DCC and
and was cou-
1
HOBt in 1,4-dioxane to give 6. Saponification, followed
by coupling with the required amine (R H) and Cbz
1
5,16
2
studies.
deprotection with HBr/AcOH mixture, gave the desired
compounds 8a–m in 36–55% overall yields. For the syn-
thesis of compounds with (2S)-cyanopyrrolidine as the
P1 portion, the Cbz protection of 6 was changed to
Boc protection in 9. DCC coupling of 9 with L-prolin-
amide yielded carboxamide 10. Dehydration of com-
In continuation to our efforts to develop selective
1
7
DPP-IV inhibitors, we have reported compound 4,
a potent DPP-IV inhibitor (IC50 = 15 nM) with high
selectivity over DPP8 (IC >100 lM) and DPP-II
5
0
1
8
(
IC50 >100 lM). To further explore the structure–
1
0
activity relationships (SAR), and to develop potent
and selective DPP-IV inhibitors, introduction of ring-
constraint in the P2 portion of lead compound (4)
as depicted in Figure 2 was contemplated. Sakashita
et al. improved DPP-IV potency by conformationally
pound 10 using POCl gave the corresponding nitrile,
3
and then removal of Boc protection by treatment with
trifluoroacetic acid gave the desired compounds 11a,b
in 24–32% yields. Substituted isoindolines were synthe-
sized starting from corresponding phthalic anhydride
by reaction with formamide to obtain phthalimide deriv-
atives, followed by reduction with borane–THF accord-
1
9
constraining NVP-DPP728. Thus, by conformation-
ally constraining the P2 portion of 4, a better binding
with DPP-IV was envisaged. Herein, we report the
synthesis, DPP-IV inhibition, selectivity profile, and
in vivo efficacies of substituted pyrrolidine-2,4-dicar-
boxylic acid amides.
2
2,23
ing to the literature procedure.
For the SAR explorations of the ring-constrained
analogues, pyrrolidine ring without the dimethyl
S2 pocket
DPP IV
S2 pocket
DPP IV
R1
S1 pocket
Design
SAR Study
O
S1 pocket
N
4
X
2
R2
N
N
H
O
X
O
O
N
N
N
X = H or Methyl
H
N
O
CN
P1
H
R1 = 1o or 2o amine
R2 = Pyrrolidine analogues
O
CN
P2
Lead compound 4
Ring constrained analogue
Pyrrolidine-2,4-dicarboxylic acid amides
Figure 2. Design of substituted pyrrolidine-2,4-dicarboxylic acid amides as DPP-IV inhibitors.
O
O
O
HOOC
R1
R1
R1
X
X
b, c
d
a
COOCH3
R2
R2
N
X
COOP1
N
P
6
N
N
H
Cbz
X
O
O
Cbz
7
5
1
8a-m
P = Cbz; P = CH3
e, b
9
P = Boc; P1 = H
O
O
N
N
f
g, h
9
X
N
X
N
O
X = H or CH3
N
N
H
X
CONH2
X
CN
R1 = 1o or 2o amines
R2 = pyrrolidine analogues
O
Boc
1
0
11a,b
1
Scheme 1. Reagents: (a) R H, HOBt, DCC, 1,4-dioxane; (b) NaOH, THF/H
2
2
O 1:1; (c) R H, HOBt, DCC (and 4-DMAP for R H = thiazolidine); (d)
2
2 3 2 2 2 3
HBr/AcOH; (e) i—H , Pd/C, CH OH; ii—(Boc) O, CH Cl ; (f) L-prolinamide, HOBt, 4-DMAP, DCC; (g) imidazole, POCl , pyridine; (h)
CF COOH.
3

3270
T.-Y. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3268–3272
substitution was selected, as the synthesis was easy com-
pared to that of the 5,5-dimethyl pyrrolidine core. First,
the requirements of S2 pocket were explored, by keeping
pyrrolidine as the 2-position substituent and varying the
substituted compounds 8b–e. As monocyclic system,
aniline 8g, benzylamine 8h, and phenylethylamine 8i
were tested. All these modifications led to a minimum
of 20-fold decrease in DPP-IV potency. The most potent
compound 8c in this series showed similar range of
activity to that of LAF237 (1) and MK-0431 (2).
4
pare DPP-IV, DPP-II, DPP8, and FAP inhibitory prop-
-position substituent. The data shown in Table 1 com-
2
4
erties of these compounds. Use of isoindoline (bicyclic
system) as the 4-position substituent (8a) led to a potent
DPP-IV inhibition (IC50 = 109 nM), with a high selectiv-
ity over other enzymes assayed. Introduction of a mono-
substitution at the 5-position of isoindoline ring (8b–e)
only slightly increased the inhibitory activity at DPP-
IV, and most of the compounds retained their selectivity
over other enzymes. Maximum of 2-fold increase in
DPP-IV inhibition was observed for 8c analogue with
a chlorine substituent (IC50 = 50 nM), which also
showed a weak FAP inhibition. However, 4,5-dichloro
substituted compound 8f showed a drop in activity. This
compound is less selective compared to 8a and mono-
After optimizing the P2 portion, we turned our attention
to the P1 portion of the molecule. The requirements of
the S1 pocket for DPP-IV inhibition were explored by
keeping isoindoline as the 4-position substituent and
varying the 2-position substituent. Their inhibitory
properties are shown in Table 2. Changing the 2-posi-
tion substituent from pyrrolidine 8a to thiazolidine 8j
showed a slight improvement in potency. Introduction
of electron-withdrawing fluorine atom into the pyrroli-
dine ring (8k–m) did not improve the activity. When
(2S)-cyano group was introduced in the pyrrolidine ring
(11a), more than 50-fold increase in DPP-IV inhibitory
activity was observed compared to that of unsubstituted
pyrrolidine 8a. Even though 11a showed a potent DPP-
IV inhibition (IC50 = 1.7 nM), it also exhibited strong
inhibitory activity against FAP (IC50 = 175 nM) and
moderate inhibition against DPP8 (IC50 = 2.7 lM).
Next, the effect of introduction of gem-dimethyl substi-
tuent adjacent to the P2-site amine was investigated.
Compound 11b showed a 500-fold loss of DPP-IV inhib-
itory potency in comparison to its unsubstituted ana-
Table 1. Inhibition of DPP-IV, DPP8, DPP-II, and FAP by substi-
tuted 2-(pyrrolidine-1-carbonyl)-pyrrolidine-4-carboxylic acid amides
O
R
4
2
N
N
H
O
a
Compound
R
IC50 (lM)
b
DPP-IV DPP8 DPP-II FAP
Table 2. Inhibition of DPP-IV, DPP8, DPP-II, and FAP by substi-
tuted 4-(isoindolin-2-carbonyl)-pyrrolidine-2-carboxylic acid amides
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
N
0.109
0.073
0.050
0.073
0.072
0.144
4.130
1.957
2.587
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16.985
13.609
7.951
>20
O
N
F
4
X
2
N
R
N
H
X
O
Cl
a
N
Compound
X
R
IC50 (lM)
b
DPP-IV DPP8 DPP-II FAP
F C
N
8a
H
H
H
N
N
0.109
0.084
0.245
>20
>20
>20
>20
>20
>20
16.985
10.352
>20
S
8
8
j
8.803
5.129
>20
F
F
k
N
N
Cl
Cl
N
12.210 >20
8
l
H
H
0.228
0.129
>20
>20
>20
>20
F
F
g
h
i
>20
>20
>20
>20
8m
19.250 19.478
N
NH
N
>20
NH
11a
H
0.0017
2.727 >20
0.175
NC
NH
>20
>20
>20
>20
>20
N
1
4
1b
CH
3
0.984
0.015
>20
>20
>20
>20
>20
>20
4
1
2
0.015
0.051
0.030
>20
>20
>20
NC
(LAF237)
(MK-0431)
14.219 >20
>20 >20
a
a
See Ref. 24.
Values are expressed as means of three independent determinations.
See Ref. 24.
Values are expressed as means of three independent determinations.
b
b

T.-Y. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3268–3272
3271
SAR suggests that incorporation of the gem-dimethyl
substituent in the pyrrolidine ring is detrimental to
potency relative to the unsubstituted analogue. Com-
pounds 8c (DPP-IV IC50 = 50 nM) and 11a (DPP-IV
IC50 = 1.7 nM) were orally bioavailable and showed
in vivo plasma DPP-IV inhibition in Wistar rats. Fur-
ther work is in progress to improve the selectivity and
stability of 11a.
Acknowledgment
The study was financially supported by National Health
Research Institutes, Taiwan.
Figure 3. Effects of 8c and 11a on the plasma DPP-IV activity in
Wistar rat. Each compound was orally administered at a single dose of
Supplementary data
10 mg/kg to rat at 0 h. Data are expressed as means ± SEM (n = 4/
group).
LC–MS of 8a–m and 11a,b available as supplementary
data. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2006.03.037.
logue 11a. The results show that gem-dimethyl substitu-
ent adjacent to the P2-site amine is not essential for
activity in this series of compounds, but rather detrimen-
tal to the activity. In the lead compound (4) series,
gem-dimethyl substituent is essential for potent and selec-
References and notes
1
8
tive DPP-IV activity. The difference could be due to
unfavorable steric interactions by the introduction of
gem-dimethyl substituent in the P2-site pyrrolidine ring.
Thus, by introducing ring-constraint in lead compound
1
2
3
. McIntosh, C. H.; Demuth, H. U.; Pospisilik, J. A.;
Pederson, R. Regul. Pept. 2005, 128, 159.
. Deacon, C. F.; Holst, J. J. Int. J. Biochem. Cell Biol. 2006,
3
8, 831.
4
and carrying out SAR studies in a series of substituted
pyrrolidine-2,4-dicarboxylic acid amides, we obtained
1a with a 10-fold improvement in the in vitro DPP-IV
. Deacon, C. F.; Hughes, T. E.; Holst, J. J. Diabetes 1998,
47, 764.
4. Holst, J. J.; Orskov, C.; Nielsen, O. V.; Schwartz, T. W.
1
inhibition.
FEBS Lett. 1987, 211, 169.
. Mentlein, R.; Gallwitz, B.; Schmidt, W. E. Eur. J.
Biochem. 1993, 214, 829.
. Kieffer, T. J.; McIntosh, C. H.; Pederson, R. A. Endocri-
nology 1995, 136, 3585.
. Ahren, B.; Simonsson, E.; Larsson, H.; Landin-Olsson,
M.; Torgeirsson, H.; Jansson, P. A.; Sandqvist, M.;
Bavenholm, P.; Efendic, S.; Eriksson, J. W.; Dickinson,
S.; Holmes, D. Diabetes Care 2002, 25, 869.
8. 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,
5
6
7
Two of the most potent compounds 8c and 11a were
tested for in vivo DPP-IV inhibition. The compounds
were orally administered to Wistar rats at 10 mpk and
the plasma DPP-IV inhibition was assessed ex vivo,
2
5
and shown in Figure 3. Both compound showed max-
imum plasma DPP-IV inhibition around 30 min after
oral dosing, with similar inhibition onset and duration
of action. For both compounds, more than 50% of inhi-
bition lasts up to 12 h after oral dosing. Plasma DPP-IV
inhibition by the compounds could be considered as an
indicator of down stream antihyperglycemic activity and
oral bioavailability. Compound 8c is a better candidate
for further evaluation, since 11a exhibits FAP and DPP8
inhibitory activity. Moreover, in 11a, the amino func-
tion could undergo intramolecular cyclization with the
nitrile group resulting in inactive cyclic amidine or dike-
topiperazine product as reported for other DPP-IV
2
774.
9
. 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.;
Weber, A. E. J. Med. Chem. 2005, 48, 141.
1
0. 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,
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.
1. Weber, A. E. J. Med. Chem. 2004, 47, 4135.
2. Augustyns, K.; Van der Veken, P.; Senten, K.; Haemers,
A. Curr. Med. Chem. 2005, 12, 971.
8
,26
inhibitors containing 2(S)-cyanopyrrolidine portion.
An aqueous solution of 11a analyzed by LC–MS
revealed the formation of the diketopiperazine, but 8c
analogue was stable due to the absence of (2S)-cyano
function.
1
1
In summary, by introducing ring-constraint in the lead
compound 4 and carrying out SAR studies in a series
of substituted pyrrolidine-2,4-dicarboxylic acid amides,
compound 11a with 10-fold improvement in the
in vitro DPP-IV inhibition was synthesized. The
1
3. Hunziker, D.; Hennig, M.; Peters, J. U. Curr. Top. Med.
Chem. 2005, 5, 1623.
14. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.

3
272
T.-Y. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3268–3272
22. Peng, Y.; Song, G.; Qian, X. Synth. Commun. 2001, 31,
1
5. 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.
1927.
23. Gawley, R. E.; Chemburkar, S. R.; Smith, A. L.;
Anklekar, T. V. J. Org. Chem. 1988, 53, 5381.
24. DPPII, DPP-IV, and DPP8 IC50 determinations were
performed as discussed in Ref. 18. The detailed protocol
for the cloning and expression of FAP will be described
elsewhere. Briefly, cDNA of FAP was cloned into pBac-
PAC8-CD5 vector (Ref. Chien et al. J. Biol. Chem. 2004,
279, 52338–52345). The expression and purification of
FAP was carried out essentialy as described in Ref. Chen
et al. Protein Expr. Purif. 2004, 35, 142–146. The substrate
used for FAP assay was Gly-Pro-p-NA at 6 mM
concentration.
1
6. 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.
7. Lu, I. L.; Lee, S. J.; Tsu, H.; Wu, S. Y.; Kao, K. H.; Chien,
C. H.; Chang, Y. Y.; Chen, Y. S.; Cheng, J. H.; Chang, C.
N.; Chen, T. W.; Chang, S. P.; Chen, X.; Jiaang, W. T.
Bioorg. Med. Chem. Lett. 2005, 15, 3271.
1
1
8. Tsu, H.; Chen, X.; Chen, C. T.; Lee, S. J.; Chang, C. N.;
Kao, K. H.; Coumar, M. S.; Yeh, Y. T.; Chien, C. H.;
Wang, H. S.; Lin, K. T.; Chang, Y. Y.; Wu, S. H.; Chen, Y.
S.; Lu, I. L.; Wu, S. Y.; Tsai, T. Y.; Chen, W. C.; Hsieh, H.
P.; Chao, Y. S.; Jiaang, W. T. J. Med. Chem. 2006, 49, 373.
9. Sakashita, H.; Kitajima, H.; Nakamura, M.; Akahoshi,
F.; Hayashi, Y. Bioorg. Med. Chem. Lett. 2005, 15, 2441.
0. Paradisi, M. P.; Mollica, A.; Cacciatore, I.; Di Stefano, A.;
Pinnen, F.; Caccuri, A. M.; Ricci, G.; Dupre, S.; Spirito,
A.; Lucente, G. Bioorg. Med. Chem. 2003, 11, 1677.
1. Aldous, D. J.; Drew, M. G. B.; Hamelin, E. M. N.;
Harwood, L. M.; Jahans, A. B.; Thurairatnam, S. Synlett
25. The in vivo DPP-IV inhibition assay was performed as
discussed in ref. 18. Adult male rats were orally gavaged
with the test compounds at a single dose of 10 mg/kg. The
plasma DPP-IV activity was determined by cleavage rate
of Gly-Pro-AMC (H-glycyl-prolyl-7-amino-4-methyl-
coumarin; BACHEM).
26. Magnin, D. R.; Robl, J. A.; Sulsky, R. B.; Augeri, D. J.;
Huang, Y.; Simpkins, L. M.; Taunk, P. C.; Betebenner, D.
A.; Robertson, J. G.; Abboa-Offei, B. E.; Wang, A.; Cap,
M.; Xin, L.; Tao, L.; Sitkoff, D. F.; Malley, M. F.;
Gougoutas, J. Z.; Khanna, A.; Huang, Q.; Han, S. P.;
Parker, R. A.; Hamann, L. G. J. Med. Chem. 2004, 47,
2587.
1
2
2
2001, 1836.