Synthesis and evaluation of pyrazolidine derivatives as dipeptidyl peptidase IV (DP-IV) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.01.020

A new series of pyrazolidine derivatives was synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV (DP-IV). Compound 9i was the most active in this series, exhibited IC₅₀ value of 1.56 μM and ED₅₀ value of 80

Full text of DOI:10.1016/j.bmcl.2005.01.020

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1337–1340
Synthesis and evaluation of pyrazolidine derivatives as
dipeptidyl peptidase IV (DP-IV) inhibitors
Jin Hee Ahn,a,* Jin Ah Kim,a Hye-Min Kim,a Hyuk-Man Kwon,b Sun-Chul Huh,b
Sang Dal Rhee,a Kwang Rok Kim,a Sung-Don Yang,a Sung-Dae Park,b,* Jae Mok Lee,c
Sung Soo Kima and Hyae Gyeong Cheona
aMedicinal Science Division, Korea Research Institute of Chemical Technology, Taejon 305-600, Republic of Korea
bJEIL Pharmaceutical Co., Ltd, R&D Center, Yongin 449-861, Republic of Korea
cR&D Center of Pharmaceuticals, CJ Corporation, Ichon 467-810, Republic of Korea
Received 27 October 2004; revised 7 January 2005; accepted 11 January 2005
Abstract—A new series of pyrazolidine derivatives was synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV
(DP-IV). Compound 9i was the most active in this series, exhibited IC50 value of 1.56 lM and ED50 value of 80 mg/kg (in vivo DP-
IV inhibition; po).
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
O
CN
H
N
R
N
N
The serine peptidase dipeptidyl peptidase IV (DP-IV)
modulates the biological activity of several peptide hor-
mones, chemokines and neuropeptides by specifically
cleaving after a proline or alanine at amino acid position
2 from the N-terminus.1 DP-IV cleaves and inactivates
glucagon-like peptide 1 (GLP-1),2 which is an important
stimulator of insulin secretion.3 Inhibition of DP-IV in-
creases the level of circulating GLP-1 and thus increases
insulin secretion,4 which could ameliorate hyperglyce-
mia in type 2 diabetes. Consequently, DP-IV inhibition
has been proposed as a new treatment of type 2 diabetes.
Small molecule inhibitors of DP-IV have been reported
in the literatures and progressed into clinical trials with
positive results.5
Figure 1.
trophilic nitrile. This prompted us to synthesize a new
series of pyrazolidine derivatives lacking an electrophile.
O
O
CN
R
N
N
R
N
N
R'
Cyano-pyrazoline
Pyrazolidine
Recently, we have reported the synthesis and biological
evaluation of a new series of cyano-pyrazoline deriva-
tives with the secondary amine at P-2 position6 (Fig. 1).
The structural modification of cyano-pyrazoline to pyr-
azolidine would be an effective approach to increase
compound stability. We now report the synthesis and
biological evaluation of pyrazolidine derivatives as
DP-IV inhibitors.
This compound (Fig. 1) showed a good in vitro and in
vivo DP-IV inhibitory activity, but it contained an elec-
2. Chemistry
Keywords: Dipeptidyl peptidase IV; DP-IV; Pyrazolidine; Anti-diabetic
agent.
*
A series of pyrazolidine derivatives were synthe-
sized according to the synthetic Schemes 1–3.
Corresponding authors. Tel.: +82 42 860 7076; fax: +82 42 860
7160; e-mail: jhahn@krict.re.kr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.020

1338
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1337–1340
Di-tert-butyldihydrazodiformate (1) was treated with
1,3-dibromopropane in the presence of Et4NBr in tolu-
ene under reflux, followed by deprotection of Boc using
4 M HCl to afford the pyrazolidine 2HCl (3) in 87%
yield from 1 by BorosÕs procedure.7 Compound 3 was
acylated by Boc–isoleucine and EDCI to produce key
intermediate 4 (Boc–isoleucine–pyrazolidide) in 78%
yield. Compound 4 was deprotected by HCl to afford
the desired isoleucine–pyrazolidide (5).
produce the desired 7a–c. Also, 4 was coupled with
ethyl, benzyl and phenyl isocyanate to afford 9a–c.
Introduction of tosyl group at NH of Boc–isoleucine–
pyrazolidide (4) was achieved using TsCl as an electro-
phile to give the corresponding compound (11).
In order to cover possible substituents at NH of isoleu-
cine–pyrazolidide (4), benzyloxycarbonyl and benzyl
group were also prepared as shown in Scheme 3. Com-
pound 127 was deprotected by HCl to produce 13, fol-
lowed by acylation using N-Boc–isoleucine and EDCI,
deprotection of NHBoc, and then converted to the fi-
nal compound 15. Hydrogenation of 12 with Pd/C un-
der H2 produced the monosubstituted pyrazolidine
(16), which upon treatment with benzyl bromide
Boc–isoleucine pyrazolidide (4) reacted with diverse
electrophiles to form the corresponding N-substituted
isoleucine pyrazolidides as shown in Scheme 2. Com-
pound 4 was acylated by activated formyl, acetyl and
benzoyl group, followed by deprotection using HCl to
O
O
d
c
a
b
N
HN NH
Boc Boc
N
HN
N
N
Boc
HN NH
2HCl
NH HN
NH2
Boc
2HCl
Boc
4
5
3
1
2
Scheme 1. Reagents and conditions: (a) 50% NaOH, dibromopropane, Et4NBr, toluene, reflux, 5 h, 87%; (b) 4 M HCl, dioxane, 100%; (c) Boc–
isoleucine, EDCI, TEA, CH2Cl2, 78%; (d) 4 M HCl, dioxane–EtOAc, rt, 12 h, 87%.
O
O
O
O
O
b
a
c
N
b
N
N
N
N
HN
N
N
NH
NH2
N
NH2
N
NH
Boc
NH
HCl
O
O
Boc
HCl
Boc
O
O
6
NH
NH
R
R
9
4
7
8
R
R
d
O
O
b
N
N
N
N
NH2
HCl
NH
Boc
Ts
Ts
10
11
Scheme 2. Reagents and conditions: (a) HCOOH, EDCI, CH2Cl2, 0 °C–rt, 45%; Ac2O, TEA, CH2Cl2, 90%; BzCl, TEA, CH2Cl2, 67%; (b) 4 M HCl,
dioxane–EtOAc, rt, 12 h, 70–95%; (c) RNCO, CH2Cl2; rt, 12 h, 70–80%; (d) TsCl, Py, CH2Cl2, rt, 15 h, 33%.
O
O
b
c
a
N
N
N
N
HN
HCl
N
N
N
NH2
O
Cbz
Boc
Cbz
NH
O
HCl
Boc
13
12
O
O
15
14
d
O
O
e
f
g
h
N
N
HN
N
N
N
NH
NH2
N
N
N
NH
Boc
2HCl
20
Boc
Boc
19
16
17
18
Scheme 3. Reagents and conditions: (a) 4 M HCl, dioxane, rt, 80%; (b) Boc–isoleucine, EDCI, CH2Cl2, rt, 40%; (c) 4 M HCl, dioxane–EtOAc, rt,
93%; (d) Pd/C, H2, MeOH, rt, 93%; (e) benzyl bromide, K2CO3, acetone, reflux, 52%; (f) trifluoroacetic acid, CH2Cl2, 77%; (g) Boc–isoleucine, TEA,
EDCI, CH2Cl2, rt, 30%; (h) 4 M HCl, dioxane–EtOAc, rt, 92%.

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1337–1340
1339
Table 2. Inhibitory activities of urea derivatives against DP-IV
afforded 17. Compound 17 was deprotected, acylated
and deprotected to give the benzylated isoleucine–pyr-
azolidide (20).
O
N
N
NH2
O
HCl
3. Results
NH
DP-IV enzyme assay was carried out using rat plasma
by
measuring
7-amino-4-trifluoromethylcoumarin
R
(AFC) liberated from Ala-Pro-AFC in the presence or
absence of a test compound.9,10 Rat plasma preparation
(20 lL) was incubated with Ala-Pro-AFC (40 lM) at
room temperature, pH 7.8 for 1 h in the presence or ab-
sence of test compounds (20 lM). Test compounds were
dissolved in DMSO. DMSO concentration in the assay
mixture was 5%, which did not affect enzyme activity.
After 1 h incubation, the fluorescence of AFC released
by the reaction was measured at 360 nm (excitation
wavelength) and at 485 nm (emission wavelength).
P32/98 was used as a reference compound.8
a
Compd
R
IC50, lM
9d
9e
4-CF3
2-CF3
25.0
na
9f
4-CN
3-CN
9.20
4.04
7.06
1.56
9g
9h
9i
4-NO2
3-NO2
2-NO2
9j
na
9k
9l
3-Cl-4-NO2
3-F-4-NO2
4-CO2Et
3-CO2Et
2-CO2Et
4.71
2.27
3.90
8.80
9m
9n
9o
9p
P32/98
Various isoleucine–pyrazolidide derivatives were evalu-
ated the biological activities as shown in Tables 1 and
2. N-Boc–isoleucine–pyrazolidide (4) and isoleucine–
pyrazolidide (5) were inactive. Also, amide substituents
such as formyl (7a), acetyl (7b) and benzoyl (7c) showed
no inhibitory activity. Ethyl (9a) and benzyl urea (9b)
were inactive, but phenyl substituent (9c) was modestly
potent with an IC50 of 15 lM. So, we further modified
this phenyl urea structure by adoption of diverse substitu-
ents as shown in Table 2.
na
3,4-Dichloro
5.60
2.7
a IC50 values were determined by curve analysis software (GraphPad
Prism).
In Table 2, the effects of the substituents (R) on phenyl
urea group were investigated. para or ortho CF3–phenyl
substituents (9d and 9e) were less active than phenyl (9b)
or not active. CN–phenyl groups (9f and 9g) showed
better in vitro activities than 9c, particularly, CN-3-phe-
nyl (9g) exhibited IC50 value of 4.04 lM. Phenyl deriva-
tives having NO2 (9h,i) except for NO2 at ortho position
(9j) were found to be more potent. Compound 9i having
NO2 at meta position was the most active in this series
with an IC50 value of 1.56 lM, and more potent than
reference P32/98 in vitro.
Meanwhile, tosyl (11) derivative resulted in loss of inhibi-
tory activity. Carbamate compound (15) was also mod-
erately active with IC50 of 23.3 lM. Benzyl group (20)
was not active.
Table 1. Inhibitory activities of pyrazolidine derivatives against DP-IV
O
N
From these results, we turned our attention to replacing
the amino acid group of compound 9i with other various
amino acid as shown in Table 3. Although valine and
N
NH2
HCl
R
a
Compd
R
IC50, lM
Table 3. Inhibitory activities of amino acid derivatives against DP-IV
5
H
CHO
na
na
Amino acid
N
7a
7b
7c
9a
9b
9c
11
15
20
CH3CO
C6H5CO
EtNHCO
na
na
N
O
na
na
NH
C6H5CH2NHCO–
C6H5NHCO
Tosyl
15.0
na
O2N
C6H5CH2OCO
C6H5CH2
23.3
na
a
Compd
Amino acid
IC50
,
lM
9i
21
Isoleucine
Valine
Leucine
1.56
2.1
O
2.7b
22
23
20.7
na
4.6
2.7
N
P32/98
S
Proline
Cyclohexylglycine
NH2
HCl
24
P32/98
a IC50 values were determined by curve analysis software (GraphPad
Prism).
a IC50 values were determined by curve analysis software (GraphPad
Prism).
b Lit. 2.8 lM.

1340
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1337–1340
Table 4. DP-IV inhibition of selected compound in vitro and in vivo
3. (a) Holst, J. J. Gastroenterology 1994, 107, 1048; (b)
Orsakov, C. Diabetologia 1992, 35, 701; (c) Drucker, D. J.
Diabetes 1998, 47, 159.
Compd Structure
IC50, lM
ED50
(rat plasma) (po, 1 h)a,b
´
4. (a) Ahren, B.; Holst, J. J.; Martensson, H.; Balkan, B. Eur.
O
J. Pharmacol. 2000, 404, 239; (b) Deacon, C. F.; Hughes,
T. E.; Holst, J. J. Diabetes 1998, 47, 764; (c) Pederson, R.
P.; White, H. A.; Schlenzig, D.; Pauly, R. P.; McIntosh, C.
R. P.; Demuth, H. U. Diabetes 1998, 47, 1253; (d) Pauly,
R. P.; Rosche, R.; Schmidt, J.; White, H. A.; Lynn, F.;
McIntosh, C. H. S.; Pederson, R. A. Metabolism 1999, 3,
385; (e) Pospisilik, J. A.; Stafford, S. G.; Demuth, H. U.;
Brownsey, R.; Parkhouse, W.; Finegood, D. T.; McIn-
tosh, C. H. S.; Pederson, R. A. Diabetes 2002, 51, 943; (f)
Balkan, B.; Kwasnik, L.; Miserendino, R.; Holst, J. J.; Li,
X. Diabetologia 1999, 42, 1324; (g) Mitani, H.; Takimoto,
M.; Hughes, T. E.; Kimura, M. Jpn. J. Pharmacol. 2002,
88, 442; (h) Mitani, H.; Takimoto, K. M. Jpn. J.
Pharmacol. 2002, 88, 451; (i) Sudre, B.; Broqua, P.;
Ashworth, D.; Evans, E. M.; Haigh, R.; Junien, J. L.;
Aubert, M. L. Diabetes 2002, 51, 1461.
N
N
9i
1.56
80 mg/kg
NH2
O
NO2
HCl
HN
a ED50 values were determined by curve analysis software (GraphPad
Prism).
b n = 6.
cyclohexylglycine derivatives (21 and 24) exhibited good
in vitro activities, isoleucine 9i was still the most active
in vitro.
As a proof of concept, these compounds were evaluated
in vivo for their ability to reduce DP-IV activity in nor-
mal C57BL/6J mice. As shown in Table 4, compound 9i,
which was the most active in vitro showed in vivo effi-
cacy with an ED50 value of 80 mg/kg, (po at 1 h
postdose).
5. For a review, see: (a) Wiedeman, P. E.; Trevilyan, J. M.
Curr. Opin. Invest. Drugs 2003, 4, 412; (b) Peters, J. U.;
Weber, S.; Kritter, S.; Weiss, P.; Wallier, A.; Boehringer,
M.; Hennig, M.; Kuhn, B.; Loeffler, B. M. Bioorg. Med.
Chem. Lett. 2004, 14, 1491; (c) Caldwell, C. G.; Chen, P.;
He, J.; Parmee, E. R.; Leiting, B.; Marsilio, F.; Patel, R.
A.; Wu, J. K.; Eiermann, G. J.; Petrov, A.; He, H.; Lyons,
K. A.; Thornberry, N. A.; Weber, A. E. Bioorg. Med.
Chem. Lett. 2004, 14, 1265; (d) Ashton, W. T.; Dong, H.;
Sisco, R. M.; Doss, G. A.; Leiting, B.; Patel, R. A.; Wu, J.
K.; Marsilio, F.; Thornberry, N. A.; Weber, A. E. Bioorg.
Med. Chem. Lett. 2004, 14, 859; (e) Parmee, E. R.; He, J.;
Mastracchio, A.; Edmondson, S. D.; Colwell, L.; Eier-
mann, G.; Feeney, W. P.; Habulihaz, B.; He, H.; Kilburn,
R.; Leiting, B.; Lyons, K.; Marsilio, F.; Patel, R. A.;
Petrov, A.; Di Salvo, J.; Wu, J. K.; Thornberry, N. A.;
Weber, A. E. Bioorg. Med. Chem. Lett. 2004, 14, 43; (f)
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.
In conclusion, a new series of pyrazolidine derivatives
was synthesized and evaluated for their ability to inhibit
dipeptidyl peptidase IV (DP-IV). Compound 9i was the
most active in this series, showed a potent inhibitory
activity and in vivo efficacy. Further studies aimed at
improving efficacy are in progress and will be reported
in due course.
Acknowledgements
This research was supported by the Center for Biologi-
cal Modulators of the 21st Century Frontier R&D Pro-
gram, the Ministry of Science and Technology, Korea.
Authors appreciate the cooperation of Korea Chemical
Bank.
6. Ahn, J. H.; Kim, H.-M.; Jung, S. H.; Kang, S. K.; Kim, K.
R.; Rhee, S. D.; Yang, S.-D.; Cheon, H. G.; Kim, S. S.
Bioorg. Med. Chem. Lett. 2004, 14, 4461.
References and notes
7. Boros, E. E.; Bouvier, F.; Randhawa, S.; Rabinowitz, M.
H. J. Heterocycl. Chem. 2001, 38, 613.
1. Mentlein, R. Regul. Pept. 1999, 85, 9.
8. Sorbera, L. A.; Revel, L.; Castaner, J. Drugs Future 2001,
26, 859.
9. 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.
2. (a) Mentlein, R.; Gallwitz, B.; Schmidt, W. E. Eur. J.
Biochem. 1993, 214, 829; (b) Kieffer, T. J.; McIntosh, C.
H. S.; Pederson, R. A. Endocrinology 1995, 136, 3585; (c)
Lene, H.; Deacon, C. F.; Orskov, C.; Holst, J. J.
Endocrinology 1999, 140, 5356; For a review, see: (d)
Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.
10. Balkan, B.; Kwasnik, L.; Milserendino, R.; Holst, J. J.; Li,
X. Diabetologia 1999, 42, 1324.