Synthesis and DP-IV inhibition of cyano-pyrazoline derivatives as potent anti-diabetic agents

Article abstract of DOI:10.1016/j.bmcl.2004.06.046

A new series of cyano-pyrazoline derivatives with secondary amine at P-2 site was synthesized through achiral and chiral synthetic methods and evaluated for their ability to inhibit dipeptidyl peptidase IV (DP-IV). Compound 5i revealed good in vivo efficacy (ED₅₀: 4.1mg/kg; in vivo DP-IV inhibition). Also chiral derivative (11b) having (S)-configuration of compound 5i was found to be more potent.

Full text of DOI:10.1016/j.bmcl.2004.06.046

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4461–4465
Synthesis and DP-IV inhibition of cyano-pyrazoline
derivatives as potent anti-diabetic agents
Jin Hee Ahn,* Hye-Min Kim, Sun Ho Jung, Seung Kyu Kang, Kwang Rok Kim,
Sang Dal Rhee, Sung-Don Yang, Hyae Gyeong Cheon and Sung Soo Kim
Medicinal Science Division, Korea Research Institute of Chemical Technology, Yusung, Taejon 305-600, South Korea
Received 1 June 2004; revised 16 June 2004; accepted 16 June 2004
Available online 19 July 2004
Abstract—A new series of cyano-pyrazoline derivatives with a secondary amine at P-2site was synthesized through achiral and
chiral synthetic methods and evaluated for their ability to inhibit dipeptidyl peptidase IV (DP-IV). Compound 5i revealed good in
vivo efficacy (ED50: 4.1 mg/kg; in vivo DP-IV inhibition). Also chiral derivative (11b) having (S)-configuration of compound 5i was
found to be more potent.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of the search for DP-IV inhibitor through
high throughput screening (HTS) using chemical library
of Korea Chemical Bank, cyano-pyrazoline skeleton
was discovered as a hit toward DP-IV inhibitor.
The serine peptidase dipeptidyl peptidase IV (DP-IV)
modulates the biological activity of several peptide
hormones, chemokines, and neuropeptides by specifi-
cally cleaving after a proline or alanine at amino acid
Recently, compound 1a with cyano-pyrazoline moiety
was reported by WO020831286 as a DP-IV inhibitor.
Since under neutral and basic aqueous conditions the
P-2site primary amine of the compound 1a can
nucleophilically attack the carbon of the nitrile to form
an inactive cyclic amidine,7 the modification of the
amino acid part of the compound 1a would be an
effective approach to increase in vivo stability.
1
position 2from the N-terminus. 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 increases the level of circulating GLP-1 and
thus increases insulin secretion,4 which could ameliorate
hyperglycemia in type 2diabetes. Consequently, DP-IV
inhibition has been proposed as a new treatment of type
2diabetes. Small molecule inhibitors of DP-IV have
been reported in the literatures and progressed into
clinical trials with positive results.5
CN
CN
O
O
H
N
Structure Modification
R
N
N
R
N
N
NH2
O
CN
CN
P2 site
P2 site
N
N
N
N
NH2
We now report the synthesis and biological evaluation
of cyano-pyrazoline derivatives with the secondary
amine at P-2site as DP-IV inhibitors.
P2 site
P1 site
1a
WO02083128
Cyano-pyrazoline skeleton
2. Chemistry
Keywords: Dipeptidyl peptidase IV; DP-IV; Pyrazoline; Anti-diabetic
agent.
The general pathway outlined in Scheme 1 yielded the
desired compounds. Acrylonitrile (2) was treated with
trimethylsilyldiazomethane in toluene–hexane at room
* Corresponding author. Tel.: +82-42-860-7076; fax: +82-42-860-7160;
e-mail: jhahn@krict.re.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.046

4462
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4461–4465
CN
O
CN
H
a
b
c
N
N
CN
N
CN
R
N
O
N
N
N
H
Cl
2
3
4
5a-l
Scheme 1. Reagents and conditions: (a) (i) Me3SiCHN2, toluene–hexane, rt; (ii) trifluoroacetic acid, CH2Cl2; (b) chloroacetyl chloride, CH2Cl2, 0 °C;
(c) RNH2, K2CO3, KI, THF, rt.
temperature, followed by desilyation using trifluoro-
acetic acid to afford the cyano-pyrazoline (3), which was
acylated by chloroacetyl chloride to afford 4 in 45%
yield from 2. Nucleophilic substitution of compound 4
by diverse primary amines was performed in the pres-
ence of K2CO3, and KI in THF to produce the desired
secondary amine-substituted cyano-pyrazoline deriva-
tives in 40–90% (5a–l).
CN
CN
a
b
CN
BocNH-AA
NH2-AA
N
N
N
N
N
N
H
3
1
1a- g
L-AA p-nitrophenyl
Scheme 3. Reagents and conditions: (a) N-t-Boc-
ester, TEA, CH2Cl2, rt or reflux; (b) TFA, CH2Cl2, rt.
Also, chiral cyano-pyrazoline derivatives were synthe-
sized as shown in Scheme 2. Chiral 2,10-camphorsultam
derived dipolarphile (6) was reacted with trimethylsilyl
diazomethane to form the pyrazoline (7) by Carreira’s
procedure,8 which showed 9:1 diastereoselectivity and
was readily separated by chromatography on silica gel.
Aminolysis of the sultam moiety produced a chiral 3,4-
dihydro-2H-pyrazole-3-carboxylic acid amide (8) in 60%
yield, followed by acylation using chloroacetyl chloride,
and dehydration with POCl3 to afford the chiral pre-
cursor (10) in 56% yield from 8. Coupling of 10 with
primary amines furnished chiral cyano-pyrazoline
derivatives (11b) in 70% yield.
3. Results
DP-IV enzyme assay was carried out using rat plasma,
caco-2, and porcine kidney by measuring 7-amino-4-
trifluoromethylcoumarin (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 absence 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 mea-
sured at 360 nm (excitation wavelength) and at 485 nm
(emission wavelength). NVP-728 was used as a reference
compound.7
Compound 1a and its derivatives were also prepared for
comparison to our compounds as shown in Scheme 3.
Compound 3 reacted with N-Boc-protected amino acid
activated ester such as p-nitrophenyl or N-hydroxy
succinimide to give the coupling product (12) in 10–30%
yields, which was deprotected by trifluoroacetic acid to
afford cyano-pyrazoline derivatives with primary amine
at P-2position ( 1a–g).
Because compound 1a reported by WO02083128 did not
show any biological activity, compound 1a and its
derivatives (1b–g) were synthesized and evaluated bio-
logical activities in our hand as shown in Table 1. Iso-
O
O
NH2
O
NH2
O
a
O
b
c
N
Cl
N
HN
N
S
N
N
O2
S
HN
N
O2
6
7
8
9
d
O
O
CN
CN
H
N
e
Cl
R
N
N
N
N
11b
10
Scheme 2. Reagents and conditions: (a) (i) Me3SiCHN2, toluene–hexane, rt; (ii) trifluoroacetic acid, CH2Cl2; (b) ammonia 2M methanol solution;
(c) chloroacetyl chloride, CH2Cl2, 0 °C; (d) POCl3, pyridine, rt; (e) RNH2, K2CO3, KI, THF, rt.

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4461–4465
4463
Table 1. Inhibitory activity of cyano-pyrazoline derivatives with primary amine at P-2position against DP-IV
CN
Xaa
N
N
Compound
Xaa
IC50, lMa (rat plasma)
IC50, lM
1a
Isoleucine
0.22
0.13 (caco-2)
0.08 (Porcine)
1b
1c
1d
1e
1f
Alanine
Valine
Leucine
1.2
1.9
4.3
2.1
0.66
2.3
tert-Butylglycine
Cbz-lycine
Cyclohexylglycine
1g
NC
O
CN
H
N
NVP-728
0.06b;7
N
N
H
N
a IC50 values were determined by curve analysis software (GRAPHPAD PRISM).
b Lit. 0.022 lM.
leucine derivative (1a) was the best compound of this
series in vitro with an IC50 value of 0.22, 0.13, and
0.08 lM (rat plasma, caco-2, and porcine kidney,
respectively). Alanine and valine analogues (1b and 1c)
exhibited IC50 values in the range of 1.2–1.9 lM. Other
compounds (1d–g) showed micro or submicromolar
inhibitory activities (0.66–4.3 lM).
of 6-{2-[2-(5-cyano-4,5-dihydropyrazol-1-yl)-2-oxoeth-
ylamino]ethylamino}nicotinonitrile (11a and 11b) syn-
thesized using a chiral auxiliary evaluated the biological
data as shown in Table 4. The (R) configuration com-
Table 2. Inhibitory activity of cyano-pyrazoline derivatives with sec-
ondary amine at P-2position against DP-IV
O
CN
For finding out the candidate of showing good in vivo
stability and efficacy, we have prepared and evaluated a
new series of cyano-pyrazoline derivatives with second-
ary amine at P-2. In vitro activities were summarized in
Table 2. A t-butyl substituent exhibited a weak inhibi-
tory activity against DP-IV (5a). Five- to seven-mem-
bered cyclic moieties resulted in better than t-butyl
group with the range of 1.3–2.6 lM (5b–d). Bi- or tri-
cyclic-substituents showed similar activities to cyclic
ones (5e–h). Heteroarylamino ethyl groups were found
to be more potent than alkyl groups (5i–k). Compound
5i having the same P2structure with NVP-728 was the
most active in this series with an IC50 value of 0.8, 0.41,
and 0.96 lM in rat plasma, caco-2, and porcine kidney,
respectively.
H
N
R
N
N
Com-
pound
R
IC50, lMa
IC50,
(rat plasma) lM
5a
5b
5c
5d
5e
5f
tert-Butyl
24.4
1.3
2.8
2.6
2.1
1.5
3.2
6.4
Cyclopentyl
Cyclohexyl
Cyclooctyl
Bicyclo[2.2.1]hept-2-yl
Adamantan-1-yl
Adamantan-2-yl
5g
5h
3-Hydroxyadamantan-1-yl
NC
5i
0.8
0.41
(caco-2)
N
N
H
As a proof of concept, the compounds were evaluated in
vivo for their ability to reduce DP-IV activity in normal
C57BL/6J mouse as shown in Table 3. Although 2-(2-
amino-3-methylpentanoyl)-3,4-dihydro-2H-pyrazole-3-
carbonitrile (1a) showed most active in vitro inhibitory
activity, 6-{2-[2-(5-cyano-4,5-dihydropyrazol-1-yl)-2-oxo-
ethylamino]ethylamino}nicotinonitrile (5i) was found
to be more potent in vivo (4-fold better, ED50: 4.1 mg/
kg), thus these data suggest that the cyano-pyrazoline
derivative with secondary amine has better in vivo
efficacy than that of primary amine at P-2position.
0.96
(Porcine)
O2N
O2N
5j
1.2
1.2
N
N
H
5k
N
N
H
O
5l
4.4
N
H
From this interesting result, we turned our attention
to obtaining optically pure 6-{2-[2-(5-cyano-4,5-
dihydropyrazol-1-yl)-2-oxoethylamino]ethylamino}nico-
tinonitrile (11a and 11b). Both chiral isomers (R and S)
NVP-728
0.067
a IC50 values were determined by curve analysis software (GRAPHPAD
PRISM).

4464
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4461–4465
Table 3. DP-IV inhibition of selected compounds in vitro and in vivo
Compound
IC50, lM (rat plasma)
ED50 (sc, 1 h)a;b
16.1 mg/kg
O
CN
1a
5i
0.22
N
N
NH2
NC
O
CN
H
0.8
4.1 mg/kg
N
N
N
N
N
H
a ED50 values were determined by curve analysis software (GRAPHPAD PRISM).
b n ¼ 6.
Table 4. Inhibitory activity of chiral cyano-pyrazoline derivatives against DP-IV
Compound
IC50, lMa (rat plasma)
IC50, lM
NC
NC
NC
O
O
O
CN
CN
CN
H
N
5i
0.8
0.41 (caco-2)
0.96 (Porcine)
N
N
N
N
H
N
N
H
N
11ab
11bc
5.20
2.62 (caco-2)
6.82(Porcine)
N
H
N
N
H
N
0.36
0.20 (caco-2)
0.44 (Porcine)
N
H
N
N
NVP-728
0.067
a IC50 values were determined by curve analysis software (GRAPHPAD PRISM).
b 95% Ee determined by HPLC with chiral column (Chiralcel OD).
c 94% Ee.
pound (11a) had a diminished inhibitory activity
in comparison to the racemic compound (5i). (S)-6-{2-
[2-(5-Cyano-4,5-dihydropyrazol-1-yl)-2-oxoethylamino]-
ethylamino}nicotinonitrile (11b) exhibited 2-fold more
potent with an IC50 value of 0.36, 0.20, and 0.44 lM (rat
plasma, caco-2, and porcine kidney, respectively), also
inhibited DP-IV activity in ob/ob mouse (30 min; 87%,
60 min; 84% inhibition, postdose, 50 mg/kg dose, n ¼ 7).
Further investigation of the pharmacological profile of
this isomer is in progress.
Program, the Ministry of Science and Technology,
Korea. Authors appreciate the cooperation of Korea
Chemical Bank to use chemical library for HTS.
References and notes
1. Mentlein, R. Regul. Pept. 1999, 85, 9.
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.
3. (a) Holst, J. J. Gastroenterology 1994, 107, 1048; (b)
Orsakov, C. Diabetologia 1992, 35, 701; (c) Drucker, D. J.
Diabetes 1998, 47, 159.
In conclusion, a new series of cyano-pyrazoline deriva-
tives with secondary amine at P-2position was synthe-
sized through achiral and chiral synthetic methods and
evaluated for their ability to inhibit dipeptidyl peptidase
IV (DP-IV). N-Alkyl derivative at P-2position showed
moderate in vitro activities. Heteroaryls (5i–k) exhibited
better biological activities. Among them, compound 5i
revealed good in vivo efficacy (ED50: 4.1 mg/kg: in vivo
DP-IV inhibition). Also chiral compound (11b) having
(S)-configuration of compound 5i was found to be more
potent.
ꢀ
4. (a) Ahren, B.; Holst, J. J.; Martensson, H.; Balkan, B. Eur.
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.; McIntosh,
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,
Acknowledgements
This research was supported by the Center for Biologi-
cal Modulators of the 21st Century Frontier R&D

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4461–4465
4465
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.
Webeer, A. E. Bioorg. Med. Chem. Lett. 2004, 14, 43;
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.
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.; Pater, 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.; Pater, R. A.;
Petrov, A.; Salvo, J. D.; Wu, J. K.; Thornberry, N. A.;
6. Sulsky, R. B.; Robl, J. A. WO02083128, 2002.
7. 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.
8. Mish, M. R.; Guerra, F. M.; Carreira, E. M. J. Am. Chem.
Soc. 1997, 119, 8379.
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.
10. Balkan, B.; Kwasnik, L.; Milserendino, R.; Holst, J. J.
Diabetologia 1999, 42, 1324.