Synthesis and biological evaluation of pyrazoline analogues with β-amino acyl group as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.11.029

A series of pyrazoline derivatives with β-amino acyl group were synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV. Several pyrazoline derivatives exhibited submicromolar inhibitory activities against DPP-IV. X-ray co-crystal structure of initial hit compound 1h was determined. Among this series, carboxylic acid substituted pyrazoline derivative 2u was the most active and greatly decreased the inhibitory activity toward CYP3A4 enzyme.

Full text of DOI:10.1016/j.ejmech.2007.11.029

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1889e1902
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of pyrazoline analogues with
b-amino acyl group as dipeptidyl peptidase IV inhibitors
Mi Ae Jun a, Woul Seong Park b, Seung Kyu Kang a, Ki Young Kim a,
Kwang Rok Kim a, Sang Dal Rhee a, Myung Ae Bae a, Nam Sook Kang a,
Sang-Kwon Sohn c, Sung Gyu Kim c, Jie Oh Lee d,
a,
Duck Hyung Lee b, Hyae Gyeong Cheon a, Sung Soo Kim a, , Jin Hee Ahn
*
*
a Drug Discovery Division, Korea Research Institute of Chemical Technology, Yuseong-Gu, Daejeon 305-600, Republic of Korea
b Department of Chemistry, Sogang University, Shinsoo-dong 1, Mapo-gu, Seoul 121-742, Republic of Korea
c Namyang Research Center, Yungjin Pharmaceutical Cooperative Limited, Musong-dong, Kyungki-do, Republic of Korea
d Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 27 September 2007; received in revised form 24 November 2007; accepted 26 November 2007
Available online 14 December 2007
Abstract
A series of pyrazoline derivatives with b-amino acyl group were synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV.
Several pyrazoline derivatives exhibited submicromolar inhibitory activities against DPP-IV. X-ray co-crystal structure of initial hit compound
1h was determined. Among this series, carboxylic acid substituted pyrazoline derivative 2u was the most active and greatly decreased the in-
hibitory activity toward CYP3A4 enzyme.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Dipeptidyl peptidase IV inhibitior; Pyrazoline; Anti-diabetic agent
1. Introduction
many tissues and body fluids, and exists as either a mem-
brane-bound or a soluble enzyme. Inhibition of DPP-IV in-
creases the level of circulating GLP-1 and thus increases
insulin secretion [5], which can ameliorate hyperglycemia in
type 2 diabetes. A number of small molecule inhibitors of
DPP-IV have been described [6] and several of these, includ-
ing Vildagliptin (LAF237) [7a], Saxagliptin (BMS477118)
[7b] and Sitagliptin (MK-0431) [8] are in late-stage of clinical
development or approved by the U.S. Food and Drug Admin-
istration (Fig. 1).
Since DPP-IV is a dipeptidase that selectively binds sub-
strates with proline at the P1 position, many of its inhibitors
investigated to date possess 5-membered heterocyclic rings
(e.g., pyrrolidine, thiazolidine, cyano-pyrrolidine, and cyano-
thiazolidine) that serve as proline mimics. A number of
DPP-IV inhibitors contain cyano-pyrrolidine ring [6]. We
have recently described several novel 5-membered proline sur-
rogates that are based on pyrazolidine [9,10] and pyrazoline
Glucagon-like peptide-1 (GLP-1) [1] is an incretin hor-
mone secreted from the L cells of the small intestine in
response to food intake. This hormone plays several biological
roles including the stimulation of insulin secretion, inhibition
of glucagon secretion, retardation of gastric emptying, induc-
tion of satiety and stimulating the regeneration and differenti-
ation of islet b cells [2]. However, GLP-1 (GLP-1[7-36]amide)
is rapidly degraded in vivo (lifetime: about 1 min) through
the action of dipeptidyl peptidase IV (DPP-IV), which cleaves
a dipeptide from the N-terminus to give the inactive GLP[9-
36]amide [3]. DPP-IV is a serine protease cleaving the N-ter-
minal dipeptide with a preference for L-proline or L-alanine at
the penultimate position [4]. This protease is expressed in
* Corresponding authors. Tel.: þ82 42 860 7076; fax: þ82 42 860 7160.
E-mail address: jhahn@krict.re.kr (J.H. Ahn).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.029

1890
M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
OH
F
H3PO4
NH2
F
O
HO
H
N
O
CN
CN
O
N
N
N
N
N
NH2
F
N
CF3
Sitagliptin (MK-0431)
Vildagliptin (LAF-237) Saxagliptin (BMS477118)
Fig. 1. Molecular structures of representative DPP-IV inhibitors.
[11]. Our compounds containing the pyrazoline template
showed a good in vivo efficacy as well as in vitro activity
against DPP-IV [11]. In the meantime, a group at Merck re-
cently described a series of structurally novel b-amino amide
derivatives that have good in vitro potency and in vivo efficacy
[8a]. Among these, MK-0431, which is approved by FDA,
shows excellent selectivity and in vivo efficacy. Also, Merck’s
researcher identified proline derived homophenylalanine de-
rivatives, which were potent and highly selective [8b].
were commercially available or synthesized according to the
procedures in Section 5. Compound 8 was deprotected by
4 M HCl to afford compound 1. Among compound 8, amide
derivatives (X ¼ NH) were hydrolyzed with LiOHꢀH2O to
give the acid (9), followed by the deprotection under acidic
condition to produce the final acid compound 2.
3. Results and discussion
Pyrazoline derivatives 1 were tested in vitro against DPP-IV
and the results are summarized in Table 1. MK-0431 was used
as a reference compound [8]. The benzyl substituted ester an-
alogue 1b showed a submicromolar activity with an IC50 value
of 0.71 mM and was 2-fold more active than the unsubstituted
acid compound 1a. But other ester derivatives (1ce1f) showed
decreases of the in vitro activities compared to benzyl ester 1b.
The N-benzyl amide analogue 1h was 3-fold more potent
than the unsubstituted amide 1g with an IC50 value of
1.6 mM. Therefore, we determined the crystal structure of
human DPP-IV complexed with 1h (Fig. 3, we ran the co-
crystallization of diastereomer mixtures of pyrazoline ring
with human DPP-IV). Chiral (S ) pyrazoline-derived co-crystal
structure was obtained from the (R) and (S ) mixtures. This
seemed that (S ) pyrazoline isomer was more effective for
binding with DPP-IV. The 2,4,5-trifluorophenyl moiety fully
occupies a catalytic pocket including S630 and (R)-b-amino
group forms an ionic interaction with carboxylate anion of
Glu205 and Glu206 residues as well known in other crystal
structures [8]. The carbonyl group of pyrazoline interacts
with side chain of Tyr547 via a water molecule. But benzyl
amide part doesn’t show a special interaction with DPP-IV,
so these areas could be more modified to increase potency.
We further modified this amide derivative 1h by adoption
of diverse substituents. Phenyl amide 1j was similar to benzyl
amide 1h. p-Methoxy carbonyl substituted phenyl amide 1k
showed a submicromolar inhibitory activity with an IC50 value
of 0.77 mM. Heteroaryl compounds (1l and 1m) exhibited
weak activities. Also, other amino, morpholino, sulfonamide,
sulfonic acid derivatives (1n, 1o, and 1s and 1t) exhibited
moderate IC50 values in the range 1.3e8.9 mM. The para-2-
ethoxyl-2-oxoethoxy substituted phenyl amide analogue 1u
showed the most potent activity (IC50 ¼ 0.69 mM), whereas,
meta or ortho substitutent showed a decrease of the in vitro ac-
tivity compared to 1u.
This observation prompted us to explore the properties of
new DPP-IV inhibitors that possess our basic pyrazoline struc-
ture and contain appended b-amino acid moiety (Fig. 2). We
now wish to report the synthesis of pyrazoline analogues
with b-amino acyl group and their biological evaluation as
DPP-IV inhibitors (Fig. 2).
2. Synthesis
Compounds selected for biological evaluation were pre-
pared as described in Scheme 1. Compound 4 was prepared
based on our previous paper [12]. Acryloyl chloride was ester-
ificated with benzyl alcohol to produce benzyl ester, followed
by cyclization with 2 M trimethylsilyl diazomethane and
removal of TMS group under acidic condition to afford the
pyrazoline ester (4). Pyrazoline ester (4) was coupled with
b-amino acid (5) in the presence of dimethylpropyl ethyl
carbodiimide hydrochloride (EDCI) to produce the coupled
product (6), which upon reaction with 10% palladium on char-
coal in MeOH yielded the corresponding acid (7). (R)-2,4,5-
Trifluorophenylbutyryl pyrazoline carboxylic acid 7 could be
converted to ester and amide derivatives (8) in the presence
of EDCI with diverse amine and alcohol derivatives which
F
HCl
O
O
NC
F
H
N
XR'
O
H2N
N
N
R
N
N
F
pyrazoline derivatives
pyrazoline derivatives
We turned our attention to replace the ester moiety with
a carboxylic acid. So, various acid derivatives were evaluated
the inhibitory activities as shown in Table 2. Notably, para
with α-amino acyl group
with β-amino acyl group
Fig. 2. The design of new DPP-IV inhibitor, b-amino pyrazoline derivatives.

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1891
F
F
Boc
O
O
NH
O
a, b, c
+
N
N
H
OBn
Cl
OH
F
5
3
4
F
F
F
F
O
F
Boc
O
F
Boc
OH
OBn
d
e
NH
O
NH
O
O
N
N
N
N
7
6
F
F
F
F
HCl
NH2 O
O
F
Boc
F
X
X
NH
O
f
d
N
N
N
N
OR
OR
O
O
1
8
X = O or NH
R = Methyl or Ethyl
g
F
F
HCl
NH2
H
N
O
F
H
N
O
F
Boc
O
NH
O
f
N
N
N
N
F
F
OH
OH
O
O
2
9
Scheme 1. Synthesis of pyrazoline derivatives. (a) Benzyl alcohol, Et3N, CH2Cl2, 83%; (b) 2 M TMSeCHN2 in Et2O, toluene; (c) TFA, CH2Cl2, 83% (for 2 steps);
(d) EDCI, Et3N, CH2Cl2, 15e99%; (e) 10% Pd/C, MeOH, H2, 95%; (f) 4 M HCl/1,4-dioxane, EtOAc, 69e99%; (g) LiOH$H2O, THF, H2O, 43e99%.
alkoxy acid substituted phenyl amide 2u showed the most ac-
tive in this series with an IC50 value of 0.51 mM.
pyrazoline derivatives 2i and 2u greatly decrease the inhibitory
activities toward CYP3A4 (62 and 59%, respectively).
We carried out the systematic study for developing new
DPP-IV inhibitor which was considered for SAR study, X-
ray structure determination, and CYP inhibition. We found
compound 2u as a pro-type of alternative DPP-IV inhibitor.
Therefore we are under further investigation to find out
more active and safe drug candidate.
Cytochrome P450 (CYP) enzymes play a major role in me-
tabolizing drug molecules. Many lead candidate molecules in
pharmaceutical development fail due to the potent inhibition
of one or more isozymic forms of CYP enzymes. Cytochrome
P450 3A4 (CYP3A4), a hemeethiolate protein, is one of the
most important P450s in human liver [13]. The ability of
CYP3A4 to metabolize >50% of administered therapeutic
agents accounts for the large number of documented druge
drug interactions associated with CYP3A4 inhibition [14]. It is
therefore of great importance to know as early as possible the
affinity of new drug candidates to CYP3A4, in order to deter-
mine their potential as inhibitors and to evaluate their influence
on the metabolism of co-administered drugs. Several pyrazoline
derivatives 1 and 2 were evaluated for their inhibitory activity to-
ward cytochrome P450 3A4 (CYP3A4) and the results are sum-
marized in Table 3. Hydrogen, amino, and ester substituted
derivatives (1h, 1n, and 1u) showed strong inhibitions toward
P450 3A4 (CYP3A4). But, carboxylic acid substituted
4. Conclusion
In conclusion, we have synthesized a series of pyrazoline
derivatives with b-amino acyl group and evaluated their ability
to inhibit dipeptidyl peptidase IV. Several pyrazoline deriva-
tives exhibited submicromolar inhibitory activities against
DPP-IV; X-ray co-crystal structure of initial hit compound
1h was determined. Among this series, carboxylic acid
substituted pyrazoline derivative 2u was the most active and
overcomed the CYP3A4 enzyme inhibition.

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M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
Table 1
In vitro activity against DPP-IV of pyrazoline derivatives
F
F
HCl
NH2
O
F
X
O
Ar
N
N
Entry
x
o
Ar
H
IC50 (mM)
Entry
x
Ar
IC50 (mM)
1a
1.4
1n
NH
NH
1.3
NH2
O
1b
1c
o
o
0.71
2.7
1o
3.1
2.1
S
NH2
O
1p
1q
NH
NH
O
O
O
O
O
O
O
1d
o
2.2
2.0
O
O
1e
1f
o
1.6
1.9
5.6
1.6
1r
1s
1t
NH
NH
NH
NH
1.6
O
N
O
O
O
S
O
o
9.1
O
NH2
OH
O
S
O
1g
1h
NH
NH
H
8.9
O
O
1u
0.69
O
O
1i
NH
3.0
1v
NH
1.9
O
O
O
O
O
1j
NH
NH
1.6
1w
1x
NH
NH
4.8
2.6
O
O
1k
0.77
O
O
O
O
O

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1893
Table 1 (continued)
Entry
x
Ar
IC50 (mM)
Entry
x
Ar
IC50 (mM)
1l
NH
7.4
1y
NH
3.6
N
O
O
O
H
N
1m
NH
3.7
MK-0431
0.11
N
IC50 values were determined from direct regression curve analysis.
5. Experimental procedure
1H), 5.85 (dd, J ¼ 1.5, 10.2 Hz, 1H), 5.20 (s, 2H). EI-MS m/z
(relative intensity) 162 (Mþ, 10), 117 (26), 105 (32), 91 (100),
77 (82), 55 (75), 51 (70).
5.1. Chemistry
To a solution of acrylic acid benzyl ester (5.0 g,
30.83 mmol) in toluene (50 ml) was added 2 M trimethylsilyl
diazomethane in Et2O (23.0 ml, 46.24 mmol). The reaction
mixture was stirred for 3 h at room temperature under N2 atmo-
sphere. The solvent was removed in vacuo and the obtained res-
idue 5 was diluted with CH2Cl2 (50 ml) and added dropwise
trifluoroacetic acid (6.9 ml, 92.48 mmol) at 0 ꢁC. The resulting
mixture was stirred for 1 h at room temperature under N2 atmo-
sphere. The solvent was removed in vacuo and the residue was
purified by flash chromatography (EtOAc:hexane ¼ 1:2) to
give 3,4-dihydro-2H-pyrazole-3-carboxylic acid benzyl ester
4 (5.2 g, 83%) as a colorless oil. 1H NMR (CDCl3,
300 MHz) d 7.36e7.27 (m, 5H), 6.86 (s, 1H), 5.18 (s, 2H),
4.28 (m, 1H), 3.03 (m, 2H). EI-MS m/z (relative intensity)
5.1.1. 3,4-Dihydro-2H-pyrazole-3-carboxylic
acid benzyl ester (4)
To a solution of acryloyl chloride 3 (12.7 ml, 123.08 mmol)
in CH2Cl2 (400 ml) was added dropwise benzyl alcohol
(10.0 ml, 123.08 mmol) and Et3N (25.7 ml, 184.62 mmol) at
0 ꢁC. The reaction mixture was stirred for 12 h at room tem-
perature under N2 atmosphere, quenched with H2O, diluted
with CH2Cl2, washed with brine and dried over anhydrous
MgSO4. The solvent was removed in vacuo and the residue
was purified by flash chromatography (EtOAc:hexane
¼ 1:19) to give acrylic acid benzyl ester (16.5 g, 83%) as a col-
1
orless oil. H NMR (CDCl3, 300 MHz) d 7.39e7.30 (m, 5H),
6.45 (dd, J ¼ 1.5, 17.4 Hz, 1H), 6.17 (dd, J ¼ 10.2, 17.4 Hz,
R358
F357
Y585
E206
2.7Å
E205
2.6 Å
R125
2.6 Å
W1
3.0 Å
Y662
Y547
S630
Fig. 3. X-ray co-crystal structure of pyrazoline derivative 1h. 1h is represented by thick stick model.

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M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
Table 3
In vitro activity against CYP of pyrazoline derivatives
Table 2
In vitro activity against DPP-IV of pyrazoline derivatives
F
HCl
NH2
H
N
O
F
F
O
HCl
H
Ar
O
F
N
NH2
O
N
Ar'
N
F
N
Entry
Ar
CYP3A4 activity at 10 mM (%)
F
N
Entry
Ar0
IC50 (mM)
1h
18.8
2i
1.4
OH
1n
2i
4.5
O
NH2
2k
5.9
62.0
OH
O
OH
O
2p
2q
2.5
2.8
1u
2u
6.9
OH
O
O
O
O
O
O
O
59.3
OH
OH
O
O
2u
2v
0.51
4.3
OH
O
O
203 (Mþ, 1), 165 (1), 149 (1), 112 (1), 91 (35), 69 (76), 58 (16),
43 (100).
O
O
OH
5.1.2. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-
3-carboxylic acid benzyl ester (6)
2w
2x
8.5
2.7
O
O
To a solution of (R)-3-tert-butoxycarbonylamino-4-(2,4,5-
5 (500 mg, 1.50 mmol) in
OH
trifluorophenyl)butyric acid
CH2Cl2 (5 ml) was added 3,4-dihydro-2H-pyrazole-3-carbox-
ylic acid benzyl ester 4 (305 mg, 4.50 mmol), EDCI
(863 mg, 4.50 mmol), and Et3N (1.5 ml, 10.50 mmol). The re-
action mixture was stirred for 12 h at room temperature,
quenched with H2O, diluted with CH2Cl2, washed with brine
and dried over anhydrous MgSO4. The solvent was removed
in vacuo and the residue was purified by flash chromatography
(EtOAc:hexane ¼ 4:1) to give an (R)-2-[3-tert-butoxycarbony-
lamino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-dihydro-2H-pyr-
azole-3-carboxylic acid benzyl ester 6 (848 mg, 54%) as
OH
OH
O
O
O
O
2y
2.2
1
a white solid. H NMR (CDCl3, 300 MHz) d 7.35e7.30 (m,
5H), 7.07e7.02 (m, 1H), 6.91e6.82 (m, 1H þ s, 1H), 5.20
(s, 2H), 4.90e4.85 (m, 1H), 4.21e4.20 (m, 1H), 3.30e3.20
(m, 1H), 2.96e2.72 (m, 5H), 1.37 (s, 9H). LCeMS m/z 520
(MHþ).
MK-0431
0.11

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1895
5.1.3. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid (7)
5.2.3. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid 4-ethoxycarbonyl phenyl ester (8e)
To
a
solution of (R)-2-[3-tert-butoxycarbonylamino-
Yield: 32%. 1H NMR (CDCl3, 300 MHz) d 8.08 (d,
J ¼ 8.7 Hz, 2H), 7.21 (d, J ¼ 8.7 Hz, 2H), 7.12e7.03 (m,
1H), 6.96 (s, 1H), 6.91e6.82 (m, 1H), 5.42 (br, 1H), 4.83
(q, J ¼ 6.0 Hz, 1H), 4.37 (q, J ¼ 7.2 Hz, 2H), 4.22 (m, 1H),
3.32e2.86 (m, 6H), 1.39 (t, J ¼ 7.2 Hz, 3H), 1.37 (s, 9H).
4-(2,4,5-trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-
3-carboxylic acid benzyl ester 6 (760 mg, 1.46 mmol) in
MeOH (5 ml) was added 10 wt.% Pd/C (142 mg). The reaction
mixture was stirred for 3 h under H2 pressure, filtered with
Celite, removed the solvent and dried in vacuo to give an
(R)-2-[3-tert-butoxycarbonylamino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carboxylic acid 7 (595 mg,
95%) as a white solid. 1H NMR (acetone-d6, 300 MHz) d 7.22e
7.15 (m, 1H), 7.13e7.00 (m, 1H), 6.96 (s, 1H), 6.00e5.80 (br s,
1H), 4.71e4.64(m, 1H), 4.17e4.15(m, 1H), 3.27e2.82(m, 6H),
1.19 (s, 9H). LCeMS m/z 429 (MHþ).
5.2.4. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid 4-methoxycarbonyl methyl phenyl ester (8f)
Yield: 31%. 1H NMR (CDCl3, 300 MHz) d 7.30 (d,
J ¼ 8.4 Hz, 2H), 7.09 (d, J ¼ 8.4 Hz, 2H), 7.08e7.01 (m,
1H), 6.95 (s, 1H), 6.91e6.82 (m, 1H), 5.38e5.35 (br, 1H),
5.01 (q, J ¼ 6.0 Hz, 1H), 4.23e4.22 (m, 1H), 3.69 (s, 3H),
3.61 (s, 2H), 3.46e2.87 (m, 6H), 1.35 (s, 9H).
5.2. General acylation procedure of 7
F
F
O
O
F
Boc
F
Boc
OH
X
5.2.5. (R)-[3-(5-Carbamoyl-4,5-dihydropyrazol-1-yl)-3-
oxo-1-(2,4,5-trifluorobenzyl)propyl]carbamic acid
tert-butyl ester (8g)
NH
O
NH O
Ar
N
N
N
N
F
F
1
Yield: 84%. H NMR (CDCl3, 300 MHz) d 7.08e7.01 (m,
7
8
1H), 7.00 (s, 1H), 6.93e6.85 (m, 1H), 5.46 (br, 1H), 5.25e
5.10 (br, 1H), 4.94e4.86 (m, 1H), 4.45e4.20 (m, 1H),
3.69e3.60 (m, 1H), 3.09e2.81 (m, 5H), 1.37 (s, 9H).
To a solution of (R)-2-[3-tert-butoxycarbonylamino-4-
(2,4,5-trifluorophenyl)-butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid 7 (30 mg, 0.07 mmol) in CH2Cl2 (1 ml) was
added various amine/alcohol (0.14 mmol), EDCI (40 mg,
0.21 mmol), Et3N (49ml, 0.35 mmol). The reaction mixture
was stirred for 12 h at room temperature, quenched with
H2O, diluted with CH2Cl2, washed with brine and dried over
anhydrous MgSO4. The solvent was removed in vacuo and
the residue was purified by flash chromatography to give the
desired product 8.
5.2.6. (R)-[3-(5-Benzylcarbamoyl-4,5-dihydropyrazol-1-yl)-
3-oxo-1-(2,4,5-trifluoro benzyl)propyl]carbamic acid tert-
butyl ester (8h)
1
Yield: 47%. H NMR (CDCl3, 300 MHz) d 7.72e7.23 (m,
5H), 7.01 (s, 1H), 7.00e6.85 (m, 2H), 5.30e5.10 (br, 1H),
4.97e4.89 (m, 1H), 4.44 (s, 2H), 4.32e4.20 (m, 1H), 3.70e
3.66 (m, 1H), 3.09e2.75 (m, 5H), 1.36 (s, 9H). LCeMS m/z
519 (MHþ).
5.2.1. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid 4-methoxycarbonyl benzyl ester (8c)
Yield: 53%. 1H NMR (CDCl3, 300 MHz) d 8.03 (d,
J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H), 7.20e7.00 (m,
1H), 6.89e6.86 (m, 1H), 6.88 (s, 1H), 5.45e5.40 (br s, 1H),
5.25 (s, 2H), 4.89 (q, J ¼ 6.0 Hz, 2H), 4.30e4.20 (m, 1H),
3.91 (s, 3H), 3.27e3.22 (m, 1H), 2.97e2.83 (m, 5H), 1.37
(s, 9H).
5.2.7. (R)-4-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]benzoic acid methyl ester (8i)
Yield: 62%. 1H NMR (CDCl3, 300 MHz) d 7.97 (d,
J ¼ 8.1 Hz, 2H), 7.85 (br s, 1H), 7.32 (d, J ¼ 8.1 Hz, 2H),
7.01 (s, 1H), 7.01e6.83 (m, 2H), 5.08 (br s, 1H), 4.95 (dd,
J ¼ 4.8, 11.7 Hz, 1H), 4.50 (d, J ¼ 5.4 Hz, 2H), 4.28e4.24
(m, 1H), 3.88 (s, 3H), 3.69e3.64 (m, 1H), 3.06 (dd,
J ¼ 11.7, 18.9 Hz, 1H), 2.90e2.81 (m, 4H), 1.34 (s, 9H).
5.2.2. (R)-2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carboxylic acid phenyl ester (8d)
Yield: 15%. 1H NMR (CDCl3, 300 MHz) d 7.39 (t,
J ¼ 7.8 Hz, 3H), 7.13 (d, J ¼ 7.8 Hz, 2H), 7.07e7.04 (m,
1H), 6.95 (s, 1H), 6.91e6.85 (m, 1H), 5.35 (br s, 1H), 5.03
(q, J ¼ 6.3 Hz, 1H), 4.24e4.22 (m, 1H), 3.41 (dd, J ¼ 13.8,
18.6 Hz, 1H), 3.21e3.12 (m, 1H), 2.99e2.88 (m, 4H), 1.35
(s, 9H).
5.2.8. (R)-[3-(5-Phenylcarbamoyl-4,5-dihydropyrazol-1-yl)-
3-oxo-1-(2,4,5-trifluoro benzyl)propyl]carbamic acid
tert-butyl ester (8j)
1
Yield: 52%. H NMR (CDCl3, 300 MHz) d 9.60 (s, 1H),
7.52 (d, J ¼ 7.8 Hz, 2H), 7.33e7.26 (m, 2H), 7.12e7.04 (m,
3H), 6.92e6.83 (m, 1H), 5.13e5.11 (m, 1H), 5.09e5.07
(m, 1H), 4.27e4.20 (m, 1H), 3.91e3.80 (m, 1H), 3.10e2.89
(m, 4H), 1.33 (s, 9H). LCeMS m/z 505 (MHþ).

1896
M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
5.2.9. (R)-4-({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)benzoic acid ethyl ester (8k)
J ¼ 6.0 Hz, 2H), 2.80 (d, J ¼ 8.1 Hz, 2H), 1.36 (s, 9H), 1.30
(t, J ¼ 7.2 Hz, 3H).
Yield: 23%. 1H NMR (CDCl3, 300 MHz) d 8.00 (d,
J ¼ 8.7 Hz, 2H), 7.61 (d, J ¼ 8.7 Hz, 2H), 7.07 (s, 1H),
7.07e7.06 (m, 1H), 6.91e6.87 (m, 1H), 5.15e5.09 (m, 2H),
4.35 (q, J ¼ 7.2 Hz, 1H), 4.40e4.20 (m, 1H), 3.90e3.80 (m,
1H), 3.11e2.89 (m, 4H), 1.38 (t, J ¼ 7.2 Hz, 3H), 1.33 (s, 9H).
5.2.15. (R)-{3-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]phenoxy}acetic acid ethyl ester (8q)
Yield: 62%. 1H NMR (CDCl3, 300 MHz) d 7.64 (br s, 1H),
7.22 (t, J ¼ 7.8 Hz, 1H), 7.05e6.99 (m, 1H), 6.99 (s,
1H), 6.92e6.87 (m, 1H), 6.88 (d, J ¼ 7.8 Hz, 1H), 6.85
(s, 1H), 6.83 (d, J ¼ 7.8 Hz, 1H), 5.10 (br, 1H), 4.92 (dd,
J ¼ 4.8, 11.2 Hz, 1H), 4.58 (s, 2H), 4.42 (s, 2H), 4.26 (q,
J ¼ 7.2 Hz, 2H), 4.25e4.23 (m, 1H), 3.72e3.62 (m, 1H),
3.10e2.80 (m, 5H), 1.35 (s, 9H), 1.26 (t, J ¼ 7.2 Hz, 3H).
5.2.10. (R)-[3-{5-(Pyridin-2-ylmethyl)carbamoyl-4,5-
dihydropyrazol-1-yl)-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamic acid tert-butyl ester (8l)
Yield: 40%. 1H NMR (CDCl3, 300 MHz) d 8.51 (d,
J ¼ 4.5 Hz, 1H), 8.07 (br s, 1H), 7.65 (t, J ¼ 7.5 Hz, 1H),
7.26e7.15 (m, 2H), 7.00e6.98 (m, 2H), 6.88e6.85 (m, 1H),
5.65e5.50 (br, 1H), 5.01e4.94 (m, 1H), 4.58 (s, 2H), 4.30e
4.15 (m, 1H), 3.70e3.55 (m, 1H), 3.14e2.77 (m, 5H), 1.35
(s, 9H). LCeMS m/z 520 (MHþ).
5.2.16. (R)-[3-[5-(4-Morpholin-4-ylphenylcarbamoyl)-4,5-
dihydropyrazol-1-yl]-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamic acid tert-butyl ester (8r)
Yield: 69%. 1H NMR (CDCl3, 300 MHz) d 7.43 (d,
J ¼ 9.0 Hz, 2H), 7.20e7.00 (m, 1H), 7.04 (s, 1H), 6.90e
6.87 (m, 1H), 6.85 (d, J ¼ 9.0 Hz, 2H), 5.08 (dd, J ¼ 4.8,
11.7 Hz, 1H), 4.35e4.20 (m, 1H), 3.87e3.79 (m, 6H),
3.12e3.09 (m, 4H), 2.94e2.88 (m, 4H), 1.30 (s, 9H).
5.2.11. (R)-[3-{5-[(1H-Benzoimidazol-2-ylmethyl)
carbamoyl]-4,5-dihydro-pyrazol-1-yl}-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamic acid tert-butyl ester (8m)
Yield: 28%. 1H NMR (CDCl3, 300 MHz) d 8.15 (br s, 3H),
7.55e7.53 (m, 2H), 7.24e7.20 (m, 2H), 7.05e6.84 (m, 3H),
5.75e5.55 (m, 1H), 4.76 (s, 2H), 4.57e4.49 (m, 1H), 4.32e
4.28 (m, 1H), 3.10e2.81 (m, 5H), 1.35 (s, 9H). LCeMS m/z
559 (MHþ).
5.2.17. (R)-[3-Oxo-3-[5-(4-sulfamoylphenylcarbamoyl)
-4,5-dihydropyrazol-1-yl]-1-(2,4,5-trifluorobenzyl)
propyl]carbamic acid tert-butyl ester (8s)
1
Yield: 83%. H NMR (CDCl3, 300 MHz) d 9.93 (br, 1H),
8.21 (br s, 3H), 7.11e7.08 (m, 1H), 6.91e9.79 (m, 2H),
6.58e6.51 (m, 4H), 5.50e5.30 (br, 1H), 5.30e5.23 (m, 1H),
4.21e4.10 (m, 1H), 4.09e3.56 (m, 1H), 3.42e3.22 (m, 2H),
2.83e2.81 (m, 1H), 1.80e1.60 (m, 2H), 1.36 (s, 9H).
5.2.12. (R)-[3-[5-(4-Amino-benzylcarbamoyl)-4,5-
dihydropyrazol-1-yl]-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamic acid tert-butyl ester (8n)
Yield: 58%. 1H NMR (CDCl3, 300 MHz) d 7.48 (br s, 1H),
7.05 (d, J ¼ 8.4 Hz, 2H), 6.99 (s, 1H), 6.99e6.85 (m, 2H),
6.61 (d, J ¼ 8.4 Hz, 2H), 5.30 (br s, 1H), 4.89 (dd, J ¼ 4.8,
11.7 Hz, 1H), 4.31 (d, J ¼ 5.7 Hz, 2H), 4.29e4.23 (m, 1H),
3.71e3.70 (m, 1H), 3.63e3.61 (br, 2H), 3.03 (dd, J ¼ 11.7,
18.9 Hz, 1H), 2.91e2.78 (m, 4H), 1.37 (s, 9H).
5.2.18. (R)-4-({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)benzenesulfonic acid (8t)
Yield: 100%. 1H NMR (CDCl3, 300 MHz) d 7.13e7.07 (m,
1H), 6.90e6.82 (m, 3H), 6.55 (s, 1H), 6.54 (d, J ¼ 5.1 Hz,
2H), 5.30e5.23 (m, 1H), 4.21e4.05 (m, 2H), 3.43e3.05 (m,
2H), 2.88e2.60 (m, 2H), 1.80e1.60 (m, 1H), 1.37 (s, 9H).
5.2.13. (R)-[3-Oxo-3-[5-(4-sulfamoyl-benzylcarbamoyl)-4,5-
dihydropyrazol-1-yl]-1-(2,4,5-trifluorobenzyl)propyl]
carbamic acid tert-butyl ester (8o)
5.2.19. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]3,4-dihydro-2H-pyrazole-3-
1
Yield: 42%. H NMR (CDCl3, 300 MHz) d 7.93 (br, 1H),
7.84 (d, J ¼ 8.1 Hz, 2H), 7.67 (br s, 1H), 7.39 (d,
J ¼ 8.1 Hz, 2H), 7.10e6.99 (m, 1H), 7.01 (s, 1H), 6.93e
6.85 (m, 1H), 5.10e5.08 (br, 1H), 4.97e4.87 (m, 1H), 4.49
(s, 2H), 4.40e4.21 (m, 2H), 3.65e3.49 (m, 1H), 2.89e2.83
(m, 1H), 1.35 (s, 9H).
carbonyl}amino]phenoxy}acetic acid ethyl ester (8u)
Yield: 76%. 1H NMR (CDCl3, 300 MHz) d 7.45 (d,
J ¼ 9.0 Hz, 2H), 7.20e7.05 (m, 1H), 7.04 (s, 1H), 6.92e
6.84 (m, 1H), 6.86 (d, J ¼ 9.0 Hz, 2H), 5.07 (dd, J ¼ 4.8,
11.7 Hz, 1H), 4.58 (s, 2H), 4.30e4.22 (m, 1H), 4.26 (q,
J ¼ 7.2 Hz, 2H), 3.90e3.80 (m, 1H), 3.11e2.89 (m, 5H),
1.33 (s, 9H), 1.26 (t, J ¼ 7.2 Hz, 3H).
5.2.14. (R)-{4-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,
5-trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]phenoxy}acetic acid ethyl ester (8p)
Yield: 33%. 1H NMR (CDCl3, 300 MHz) d 7.61 (br s, 1H),
7.19 (d, J ¼ 8.7 Hz, 2H), 7.05e6.98 (m, 1H), 6.99 (s, 1H),
6.92e6.88 (m, 1H), 6.84 (d, J ¼ 8.7 Hz, 2H), 5.18 (br,
1H), 4.90 (dd, J ¼ 4.8, 11.7 Hz, 1H), 4.57 (s, 2H), 4.37 (s,
2H), 4.26 (q, J ¼ 7.2 Hz, 2H), 4.27e4.22 (m, 1H), 3.70e
3.64 (m, 1H), 3.03 (dd, J ¼ 11.7, 20.1 Hz, 1H), 2.89 (d,
5.2.20. (R)-{3-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}acetic acid ethyl ester (8v)
1
Yield: 77%. H NMR (CDCl3, 300 MHz) d 9.63 (s, 1H),
7.34 (s, 1H), 7.21 (t, J ¼ 8.1 Hz, 1H), 7.05e7.03 (m, 3H),
6.88 (d, J ¼ 8.1 Hz, 1H), 6.67 (d, J ¼ 8.1 Hz, 1H), 5.20e
5.00 (br s, 1H), 5.08 (dd, J ¼ 4.8, 11.7 Hz, 1H), 4.60

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1897
(s, 2H), 4.30e4.23 (m, 1H), 4.26 (q, J ¼ 7.2 Hz, 2H), 3.87e
3.80 (m, 1H), 3.10e2.86 (m, 5H), 1.34 (s, 9H), 1.27 (t,
J ¼ 7.2 Hz, 3H).
MgSO4, evaporated, and solidified with pet. ether to give an
acid 9.
5.3.1. (R)-4-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]benzoic acid (9i)
5.2.21. (R)-{2-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}acetic acid ethyl ester (8w)
Yield: 32%. 1H NMR (CDCl3, 300 MHz) d 9.62 (br s, 1H),
8.33 (d, J ¼ 9.0 Hz, 1H), 7.06e7.01 (m, 3H), 7.01 (s, 1H),
6.83 (d, J ¼ 9.0 Hz, 2H), 5.30 (br, 1H), 5.11 (dd, J ¼ 4.8,
11.7 Hz, 1H), 4.71 (s, 2H), 4.26 (q, J ¼ 7.2 Hz, 2H), 3.64e
3.62 (m, 1H), 3.19e2.90 (m, 5H), 1.32 (s, 9H), 1.26 (t,
J ¼ 7.2 Hz, 3H).
1
Yield: 69%. H NMR (CDCl3, 300 MHz) d 8.73 (br, 1H),
7.86 (d, J ¼ 8.1 Hz, 2H), 7.50e7.40 (m, 1H), 7.39 (d,
J ¼ 8.1 Hz, 2H), 7.35e7.20 (m, 1H), 7.15 (s, 1H), 4.68 (dd,
J ¼ 5.7, 11.4 Hz, 1H), 4.35 (dd, J ¼ 5.7, 11.4 Hz, 2H), 4.20e
4.05 (m, 1H), 2.89e2.82 (m, 6H), 1.26 (s, 9H).
5.3.2. (R)-4-({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)benzoic acid (9k)
5.2.22. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-
(2,4,5-trifluorophenyl)butyryl]-3,4-dihydro-2H-
pyrazole-3-carbonyl}amino]phenoxy}-(S )-isopropyl
acetic acid ethyl ester (8x)
Yield: 83%. 1H NMR (CDCl3, 300 MHz) d 7.41 (d,
J ¼ 8.9 Hz, 2H), 7.10e7.03 (m, 1H), 7.04 (s, 1H), 6.92e
6.83 (m, 1H), 6.83 (d, J ¼ 8.9 Hz, 2H), 5.14 (br, 1H), 5.07
(dd, J ¼ 4.7, 11.7 Hz, 1H), 4.30 (d, J ¼ 5.5 Hz, 1H), 4.20 (q,
J ¼ 7.2 Hz, 2H), 3.86e3.80 (m, 1H), 3.10e2.88 (m, 6H),
2.26 (q, J ¼ 6.4 Hz, 1H), 1.33 (s, 9H), 1.24 (t, J ¼ 7.2 Hz,
3H), 1.06 (d, J ¼ 7.2 Hz, 6H).
1
Yield: 76%. H NMR (CDCl3, 300 MHz) d 9.93 (br, 1H),
8.21 (br s, 3H), 7.11e7.08 (m, 1H), 6.91e9.79 (m, 2H),
6.58e6.51 (m, 4H), 5.50e5.30 (br, 1H), 5.30e5.23 (m, 1H),
4.21e4.10 (m, 1H), 4.09e3.56 (m, 1H), 3.42e3.22 (m, 2H),
2.83e2.81 (m, 1H), 1.80e1.60 (m, 2H), 1.36 (s, 9H).
5.3.3. (R)-{4-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]phenoxy}acetic acid (9p)
Yield: 78%. 1H NMR (CDCl3, 300 MHz) d 7.60 (br s, 1H),
7.12 (d, J ¼ 8.7 Hz, 2H), 7.00e6.97 (m, 1H), 6.91 (s, 1H),
6.85e6.79 (m, 1H), 6.78 (d, J ¼ 8.7 Hz, 2H), 5.29 (br, 1H),
4.81 (dd, J ¼ 5.1, 11.2 Hz, 1H), 4.50 (s, 2H), 4.29 (s, 2H),
4.22e4.12 (m, 1H), 3.45e3.38 (m, 1H), 3.02 (dd, J ¼ 12.0,
20.1 Hz, 1H), 2.83e2.49 (m, 4H), 1.27 (s, 9H).
5.2.23. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-
(2,4,5-trifluorophenyl)butyryl]3,4-dihydro-2H-
pyrazole-3-carbonyl}amino]phenoxy}-(R)-isopropyl
acetic acid ethyl ester (8y)
Yield: 69%. 1H NMR (CDCl3, 300 MHz) d 7.41 (d,
J ¼ 8.9 Hz, 2H), 7.10e7.00 (m, 1H), 7.04 (s, 1H), 6.92e
6.85 (m, 1H), 6.84 (d, J ¼ 8.9 Hz, 2H), 5.14 (br, 1H), 5.07
(dd, J ¼ 4.7, 11.7 Hz, 1H), 4.30 (d, J ¼ 5.6 Hz, 1H), 4.20 (q,
J ¼ 7.2 Hz, 2H), 3.86e3.80 (m, 1H), 3.10e2.88 (m, 6H),
2.26 (q, J ¼ 6.6 Hz, 1H), 1.33 (s, 9H), 1.24 (t, J ¼ 7.2 Hz,
3H), 1.06 (d, J ¼ 7.2 Hz, 6H).
5.3.4. (R)-{3-[({2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino)methyl]phenoxy}acetic acid (9q)
Yield: 93%. 1H NMR (CDCl3, 300 MHz) d 7.53 (br s, 1H),
7.23 (t, J ¼ 7.8 Hz, 1H), 7.04e6.98 (m, 1H), 6.98 (s, 1H),
6.91e6.83 (m, 4H), 4.86 (m, 1H), 4.66 (s, 2H), 4.38 (s, 2H),
4.20e4.00 (m, 1H), 3.62e3.61 (m, 1H), 3.04e2.59 (m, 5H),
1.34 (s, 9H).
5.3. General hydrolysis procedure of ester 8
F
F
H
H
O
O
Boc
Boc
F
F
N
N
NH
O
NH
O
N
N
N
N
F
F
OR
OH
O
O
8
9
5.3.5. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}acetic acid (9u)
Yield: 100%. 1H NMR (CDCl3, 300 MHz) d 7.48 (d,
J ¼ 9.0 Hz, 2H), 7.15e7.09 (m, 1H), 7.00 (s, 1H), 6.93e
6.85 (m, 1H), 6.88 (d, J ¼ 9.0 Hz, 2H), 5.70e5.50 (br, 1H),
To a solution of ester 8 in THF/H2O (0.15 M) was added
LiOH$H2O (5 eq) at 0 ꢁC and stirred for 12 h at room temper-
ature. The solvent was removed in vacuo and the residue was
acidified with 1 N HCl to pH 3e4, extracted with EtOAc, and
washed with brine. The combined organic layer was dried over

1898
M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
5.01 (q, J ¼ 6.0 Hz, 1H), 4.56 (s, 2H), 4.25e4.24 (m, 1H),
3.50e2.86 (m, 6H), 1.34 (s, 9H).
5.4.1. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid HCl (1a)
1
Yield: 51%. H NMR (DMSO-d6, 300 MHz) d 8.05 (br s,
3H), 7.74e7.39 (m, 2H), 7.16 (s, 1H), 4.58e4.54 (m, 1H),
4.05 (m, 1H), 2.96e2.81 (m, 6H). LCeMS m/z 329 (MHþ).
5.3.6. (R)-{3-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}acetic acid (9v)
1
Yield: 92%. H NMR (CDCl3, 300 MHz) d 7.23e6.89 (m,
5.4.2. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid benzyl ester
HCl (1b)
4H), 6.86 (s, 1H), 6.74e6.70 (m, 1H), 6.58e6.56 (m, 1H),
5.10e5.06 (m, 1H), 4.64 (s, 2H), 3.82e3.74 (m, 1H), 3.06e
2.60 (m, 6H), 1.31 (s, 9H).
1
Yield: 46%. H NMR (DMSO-d6, 300 MHz) d 8.33 (br s,
3H), 7.18e7.65 (m, 2H), 7.72e7.65 (m, 2H), 7.61e7.45 (m,
5H), 7.40 (s, 1H), 5.28 (s, 2H), 4.98e4.92 (m, 1H), 3.91e
3.89 (m, 1H), 3.66e3.48 (m, 1H), 3.19e3.03 (m, 5H). LCe
MS m/z 420 (MHþ).
5.3.7. (R)-{2-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}acetic acid (9w)
Yield: 93%. 1H NMR (CD3OD, 300 MHz) d 8.97 (d,
J ¼ 9.0 Hz, 1H), 7.20e7.05 (m, 1H), 7.00e6.90 (m, 1H),
6.99 (d, J ¼ 9.0 Hz, 1H), 6.89 (s, 1H), 6.87 (t, J ¼ 9.0 Hz,
2H), 5.00e4.96 (m, 1H), 4.63 (s, 2H), 4.20e4.10 (m, 1H),
3.15e3.09 (m, 2H), 2.90e2.58 (m, 4H), 1.21 (s, 9H).
5.4.3. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid 4-methoxycarbonyl
benzyl ester HCl (1c)
Yield: 90%. 1H NMR (CD3OD, 300 MHz) d 7.88 (d,
J ¼ 8.1 Hz, 2H), 7.37 (d, J ¼ 8.1 Hz, 2H), 7.25e7.07 (m,
2H), 7.03 (s, 1H), 5.16 (s, 2H), 4.83 (q, J ¼ 6.0 Hz, 1H),
3.80 (s, 3H), 3.78e3.75 (m, 1H), 3.01e2.83 (m, 6H).
5.3.8. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}-(S )-isopropyl acetic acid (9x)
Yield: 100%. 1H NMR (CDCl3, 300 MHz) d 7.39 (d,
J ¼ 7.2 Hz, 2H), 7.03 (d, J ¼ 7.2 Hz, 2H), 6.93e6.81 (m,
2H þ s, 1H), 5.17 (br, 1H), 5.01 (m, 1H), 4.36 (d,
J ¼ 4.8 Hz, 1H), 4.29e4.19 (m, 2H), 3.08e2.86 (m, 6H),
1.33 (s, 9H), 1.08 (d, J ¼ 7.2 Hz, 6H).
5.4.4. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid phenyl ester (1d)
Yield: 99%. 1H NMR (CD3OD, 300 MHz) d 7.40 (t,
J ¼ 8.1 Hz, 3H), 7.35e7.24 (m, 2H), 7.20 (s, 1H), 7.14 (d,
J ¼ 8.1 Hz, 2H), 5.09 (q, J ¼ 6.0 Hz, 1H), 3.94e3.92 (m,
1H), 3.56e3.49 (m, 1H), 3.27e3.00 (m, 5H).
5.3.9. (R)-{4-[{2-[3-tert-Butoxycarbonylamino-4-(2,4,5-
trifluorophenyl)butyryl]-3,4-dihydro-2H-pyrazole-3-
carbonyl}amino]phenoxy}-(R)-isopropyl acetic acid (9y)
Yield: 70%. 1H NMR (CDCl3, 300 MHz) d 7.39 (d,
J ¼ 7.2 Hz, 2H), 7.03 (d, J ¼ 7.2 Hz, 2H), 6.93e6.81 (m,
2H þ s, 1H), 5.17 (br, 1H), 5.01 (m, 1H), 4.36 (d,
J ¼ 4.8 Hz, 1H), 4.29e4.19 (m, 2H), 3.08e2.86 (m, 6H),
1.33 (s, 9H), 1.08 (d, J ¼ 7.2 Hz, 6H).
5.4.5. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid 4-ethoxycarbonyl
phenyl ester HCl (1e)
Yield: 100%. 1H NMR (CD3OD, 300 MHz) d 7.96 (d,
J ¼ 8.4 Hz, 2H), 7.20 (d, J ¼ 8.4 Hz, 2H), 7.14e7.11 (m,
2H), 7.00 (s, 1H), 5.20e4.90 (m, 1H), 4.26 (q, J ¼ 6.9 Hz,
2H), 3.80e3.70 (m, 2H), 3.07e2.84 (m, 5H), 1.28 (t,
J ¼ 6.9 Hz, 3H).
5.4. General deprotection procedure of 8/9
F
F
HCl
O
O
F
F
Boc
X
X
O
NH
O
NH2
Ar
Ar
N
N
N
N
F
F
8 and 9
1 and 2
5.4.6. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid 4-methoxycarbonyl
methyl phenyl ester HCl (1f)
Yield: 100%. H NMR (CD3OD, 300 MHz) d 7.21e6.97
(m, 7H), 5.10e4.90 (m, 1H), 4.05 (m, 1H), 3.80 (m, 2H),
3.64 (s, 2H), 3.41e2.96 (m, 4H).
To a solution of 9/10 EtOAc (0.2 M) was added 4 M HCl in
1,4-dioxane (10 eq). The reaction mixture was stirred for 12 h
at room temperature under N2 atmosphere, evaporated to re-
move solvent, and recrystallized with Et2O. A recrystallized
solid was filtered, washed with pet. ether and dried in vacuo
to obtain the desired product 1/2.
1

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1899
5.4.7. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
7.42 (s, 1H), 4.95e4.91 (m, 3H), 3.90e3.80 (m, 1H), 3.51e
3.12 (m, 5H). LCeMS m/z 459 (MHþ).
dihydro-2H-pyrazole-3-carboxylic acid amide HCl (1g)
1
Yield: 74%. H NMR (DMSO-d6, 300 MHz) d 8.16e8.14
(br s, 3H), 7.62e7.50 (m, 2H), 7.16 (s, 1H), 4.60e4.52 (m,
1H), 3.85e3.75 (m, 1H), 2.98e2.79 (m, 6H).
5.4.14. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid 4-amino
benzyl amide HCl (1n)
5.4.8. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid benzyl
amide HCl (1h)
Yield: 85%. 1H NMR (CD3OD, 300 MHz) d 7.40 (d,
J ¼ 8.1 Hz, 2H), 7.35e7.32 (m, 2H), 7.26 (d, J ¼ 8.1 Hz,
2H), 7.19 (s, 1H), 4.41 (dd, J ¼ 5.4, 12.3 Hz, 1H), 3.95 (d,
J ¼ 4.8 Hz, 2H), 3.63e3.54 (m, 1H), 3.10e2.90 (m, 6H).
1
Yield: 73%. H NMR (DMSO-d6, 300 MHz) d 8.74e8.71
(br, 1H), 8.14e8.11 (br s, 3H), 7.59e7.50 (m, 2H), 7.35e
7.23 (m, 5H), 7.21 (s, 1H), 4.72e4.64 (m, 1H), 4.28 (s, 2H),
3.80e3.70 (m, 1H), 3.04e2.82 (m, 6H). LCeMS m/z 455
(MHþ).
5.4.15. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid
4-sulfamoyl benzyl amide HCl (1o)
1
Yield: 76%. H NMR (DMSO-d6, 300 MHz) d 8.14 (br s,
5.4.9. (R)-4-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino)
methyl]benzoic acid methyl ester HCl (1i)
Yield: 71%. 1H NMR (CD3OD, 300 MHz) d 8.17 (d,
J ¼ 8.1 Hz, 2H), 7.46 (d, J ¼ 8.1 Hz, 2H), 7.58e7.38 (m,
2H), 7.32 (s, 1H), 5.12e5.00 (m, 1H), 4.75, 4.60 (d,
J ¼ 15.6 Hz, 2H), 4.09 (s, 3H), 4.13e4.07 (m, 1H), 3.57e
3.37 (m, 2H), 3.24e3.07 (m, 4H).
3H), 7.76 (q, J ¼ 4.2 Hz, 1H), 7.70 (q, J ¼ 4.2 Hz, 1H),
7.54e7.42 (m, 4H), 7.22 (s, 1H), 4.68 (br s, 1H), 4.35 (d,
J ¼ 4.8 Hz, 2H), 4.15e4.12 (m, 1H), 3.78e3.76 (m, 1H),
2.96e2.84 (m, 4H).
5.4.16. (R)-{4-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino)
methyl]phenoxy}acetic acid ethyl ester HCl (1p)
Yield: 95%. 1H NMR (DMSO-d6, 300 MHz) d 8.63 (t,
J ¼ 5.7 Hz, 2H), 8.04 (br s, 1H), 7.60e7.49 (m, 2H), 7.20 (s,
1H), 7.17 (d, J ¼ 8.4 Hz, 2H), 6.85 (d, J ¼ 8.4 Hz, 2H), 4.73
(s, 2H), 4.70e4.63 (m, 1H), 4.21 (s, 2H), 4.15 (q,
J ¼ 7.2 Hz, 2H), 3.78e3.74 (m, 1H), 3.32e3.23 (m, 1H),
2.98e2.78 (m, 5H), 1.21 (t, J ¼ 7.2 Hz, 3H).
5.4.10. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid phenyl amide
HCl (1j)
1
Yield: 73%. H NMR (DMSO-d6, 300 MHz) d 8.05 (br s,
3H), 7.59e7.49 (m, 4H), 7.31 (t, J ¼ 7.8 Hz, 2H), 7.24 (s,
1H), 7.07 (t, J ¼ 7.2 Hz, 1H), 4.87e4.81 (m, 1H), 3.77e3.75
(m, 1H), 3.37e3.33 (m, 1H), 3.02e2.96 (m, 5H). LCeMS
m/z 442 (MHþ).
5.4.17. (R)-{3-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino)
methyl]phenoxy}acetic acid ethyl ester HCl (1q)
5.4.11. (R)-4-({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino)
benzoic acid ethyl ester HCl (1k)
Yield: 74%. 1H NMR (CD3OD, 300 MHz) d 7.88 (d,
J ¼ 8.7 Hz, 2H), 7.60 (d, J ¼ 8.7 Hz, 2H), 7.30e7.17 (m,
1H), 7.15e7.08 (m, 1H), 7.04 (s, 1H), 4.88e4.82 (m, 1H),
4.25 (q, J ¼ 7.2 Hz, 2H), 3.90e3.80 (m, 1H), 3.36e2.78 (m,
6H), 1.28 (t, J ¼ 7.2 Hz, 3H).
Yield: 87%. 1H NMR (DMSO-d6, 300 MHz) d 8.70 (t,
J ¼ 5.7 Hz, 2H), 8.09 (br s, 1H), 7.60e7.49 (m, 2H), 7.22
(d, J ¼ 7.8 Hz, 1H), 7.21 (s, 1H), 6.86 (d, J ¼ 7.8 Hz, 1H),
6.83 (s, 1H), 6.68 (d, J ¼ 7.8 Hz, 1H), 4.76 (s, 2H), 4.74e
4.70 (m, 1H), 4.25 (s, 2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 3.78e
3.76 (m, 1H), 3.33e3.18 (m, 1H), 2.99e2.78 (m, 5H), 1.21
(t, J ¼ 7.2 Hz, 3H).
5.4.18. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid
(4-morpholin-4-ylphenyl)amide HCl (1r)
Yield: 100%. 1H NMR (CD3OD, 300 MHz) d 7.92 (br, 1H),
7.73 (d, J ¼ 8.1 Hz, 2H), 7.59 (d, J ¼ 8.1 Hz, 2H), 7.28e7.25
(m, 1H), 7.17e7.08 (m, 1H), 7.04 (s, 1H), 4.83e4.76 (m, 1H),
4.00 (m, 4H), 3.80 (m, 1H), 3.64e3.56 (m, 6H), 3.09e2.84
(m, 4H).
5.4.12. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid
(pyridin-2-ylmethyl)amide HCl (1l)
1
Yield: 43%. H NMR (DMSO-d6, 300 MHz) d 9.10 (d,
J ¼ 5.5 Hz, 1H), 8.71 (s, 1H), 8.25 (br s, 3H), 7.70 (d,
J ¼ 7.2 Hz, 2H), 7.55 (d, J ¼ 7.7 Hz, 2H), 7.23 (s, 1H),
4.76e4.67 (m, 1H), 4.57 (s, 2H), 4.06e3.99 (m, 1H), 2.97e
2.89 (m, 6H). LCeMS m/z 420 (MHþ).
5.4.19. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carboxylic acid
(4-sulfamoyl phenyl)amide HCl (1s)
Yield: 87%. 1H NMR (CD3OD, 300 MHz) d 8.13 (d,
J ¼ 6.6 Hz, 2H), 7.43e7.40 (m, 1H), 7.25e7.23 (m, 1H),
7.12 (s, 1H), 7.02 (d, J ¼ 6.6 Hz, 2H), 5.30e5.20 (m, 1H),
5.4.13. (R)-2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-3,4-
dihydro-2H-pyrazole-3-carboxylic acid (1H-benzoimidazol-
2-ylmethyl)amide HCl (1m)
Yield: 50%. H NMR (CD3OD, 300 MHz) d 9.43 (s, 1H),
8.38 (br s, 3H), 7.96e7.93 (m, 2H), 7.70e7.68 (m, 4H),
1

1900
M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
3.95e3.68 (m, 2H), 3.54e3.47 (m, 2H), 3.19e3.04 (m, 2H),
2.14e2.04 (m, 1H).
4.36 (d, J ¼ 5.3 Hz, 1H), 4.09 (q, J ¼ 7.2 Hz, 2H), 3.90e
3.80 (m, 1H), 3.27e2.75 (m, 6H), 2.16e2.14 (m, 1H), 1.11
(t, J ¼ 7.2 Hz, 3H), 0.97 (d, J ¼ 6.8 Hz, 3H).
5.4.20. (R)-4-({2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carbonyl}amino)benzenesulfonic
acid HCl (1t)
Yield: 80%. 1H NMR (CD3OD, 300 MHz) d 8.13 (d,
J ¼ 6.8 Hz, 2H), 7.45e7.40 (m, 1H), 7.28e7.19 (m, 1H),
7.12 (s, 1H), 7.01 (d, J ¼ 6.8 Hz, 2H), 5.35 (br, 1H), 3.92e
3.67 (m, 3H), 3.20e3.00 (m, 4H).
5.4.26. (R)-4-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino)methyl]benzoic acid HCl (2i)
1
Yield: 86%. H NMR (CD3OD, 300 MHz) d 8.73 (br, 1H),
7.86 (d, J ¼ 8.1 Hz, 2H), 7.50e7.40 (m, 1H), 7.39 (d,
J ¼ 8.1 Hz, 2H), 7.35e7.20 (m, 1H), 7.15 (s, 1H), 4.68 (dd,
J ¼ 5.7, 11.4 Hz, 1H), 4.35 (dd, J ¼ 5.7, 11.4 Hz, 2H), 4.20e
4.05 (m, 1H), 2.89e2.82 (m, 6H).
5.4.21. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino]phenoxy}acetic acid ethyl ester HCl (1u)
1
Yield: 100%. H NMR (DMSO-d6, 300 MHz) d 8.16 (br s,
5.4.27. (R)-4-({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino)benzoic acid HCl (2k)
Yield: 99%. 1H NMR (CD3OD, 300 MHz) d 7.88 (d,
J ¼ 7.8 Hz, 2H), 7.24e7.18 (m, 1H), 7.14e7.09 (m, 1H),
7.02 (d, J ¼ 7.2 Hz, 2H), 7.01 (s, 1H), 4.86 (m, 1H), 3.72
(m, 1H), 3.35e3.21 (m, 2H), 3.00e2.90 (m, 3H), 2.60e2.55
(m, 1H).
1H), 7.56e7.50 (m, 2H), 7.49 (d, J ¼ 9.0 Hz, 2H), 7.23 (s,
1H), 6.88 (d, J ¼ 9.0 Hz, 2H), 4.81 (q, J ¼ 6.0 Hz, 1H), 4.73
(s, 2H), 4.16 (q, J ¼ 7.2 Hz, 2H), 3.76e3.74 (m, 1H), 3.00e
2.91 (m, 6H), 1.21 (t, J ¼ 7.2 Hz, 3H).
5.4.22. (R)-{3-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carbonyl}amino]phenoxy}
acetic acid ethyl ester HCl (1v)
Yield: 67%. 1H NMR (CD3OD, 300 MHz) d 7.24e7.21 (m,
1H), 7.21 (s, 1H), 7.16e7.11 (m, 1H), 7.12 (d, J ¼ 8.4 Hz,
1H), 7.03 (s, 1H), 7.03e7.01 (m, 1H), 6.60 (d, J ¼ 8.4 Hz,
1H), 4.81 (q, J ¼ 6.0 Hz, 1H), 4.57 (s, 2H), 4.14 (q,
J ¼ 7.2 Hz, 2H), 3.90e3.80 (m, 1H), 3.29e2.80 (m, 6H),
1.47 (t, J ¼ 7.2 Hz, 3H).
5.4.28. (R)-{4-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino)methyl]phenoxy}acetic acid HCl (2p)
1
Yield: 61%. H NMR (DMSO-d6, 300 MHz) d 8.63 (br,
1H), 8.15 (br s, 3H), 7.60e7.54 (m, 2H), 7.20 (s, 1H), 7.18
(d, J ¼ 8.7 Hz, 2H), 6.85 (d, J ¼ 8.7 Hz, 2H), 4.73 (s, 2H),
4.73e4.63 (m, 1H), 4.20 (s, 2H), 3.76e3.74 (m, 1H), 2.98e
2.78 (m, 6H).
5.4.23. (R)-{2-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino]phenoxy}acetic acid ethyl ester HCl (1w)
Yield: 74%. 1H NMR (CD3OD, 300 MHz) d 8.01 (d,
J ¼ 7.5 Hz, 1H), 7.39e7.20 (m, 2H), 7.17 (s, 1H), 7.11 (d, J ¼
7.5 Hz, 1H), 7.01 (t, J ¼ 7.5 Hz, 2H), 5.11 (q, J ¼ 6.0 Hz, 1H),
4.93e4.86 (m, 1H), 4.81 (s, 2H), 4.28 (q, J ¼ 7.2 Hz, 2H),
4.00e3.80 (m, 1H), 3.36e2.90 (m, 5H), 1.31 (t, J ¼ 7.2 Hz,
3H).
5.4.29. (R)-{3-[({2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino)methyl]phenoxy}acetic acid HCl (2q)
1
Yield: 46%. H NMR (DMSO-d6, 300 MHz) d 8.83 (br s,
1H), 8.26 (br s, 3H), 7.70e7.60 (m, 2H), 7.36e7.29 (m,
2H), 6.98e6.87 (m, 3H), 4.85 (s, 2H), 4.81e4.74 (m, 1H),
4.77 (s, 2H), 3.89e3.85 (m, 1H), 3.15e2.61 (m, 6H).
5.4.24. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)butyryl]-
3,4-dihydro-2H-pyrazole-3-carbonyl}amino]phenoxy}-(S )-
isopropyl acetic acid ethyl ester HCl (1x)
Yield: 80%. 1H NMR (CD3OD, 300 MHz) d 7.35 (d,
J ¼ 8.9 Hz, 2H), 7.30e7.21 (m, 1H), 7.17e7.08 (m, 1H),
7.02 (s, 1H), 6.75 (d, J ¼ 8.9 Hz, 2H), 4.83e4.79 (m, 1H),
4.36 (d, J ¼ 5.3 Hz, 1H), 4.09 (q, J ¼ 7.2 Hz, 2H), 3.90e
3.80 (m, 1H), 3.36e2.81 (m, 6H), 2.20e2.10 (m, 1H), 1.13
(t, J ¼ 7.2 Hz, 3H), 0.97 (d, J ¼ 6.8 Hz, 6H).
5.4.30. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino]phenoxy}acetic acid HCl (2u)
1
Yield: 61%. H NMR (DMSO-d6, 300 MHz) d 8.06 (br s,
1H), 7.83e7.47 (m, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.23 (s,
1H), 6.88 (d, J ¼ 8.4 Hz, 2H), 4.84e4.73 (m, 1H), 4.68 (s,
2H), 3.74e3.57 (m, 1H), 2.97e2.94 (m, 6H).
5.4.25. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino]phenoxy}-(R)-isopropyl acetic acid
5.4.31. (R)-{3-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}
amino]phenoxy}acetic acid HCl (2v)
Yield: 86%. 1H NMR (CD3OD, 300 MHz) d 7.21e7.05 (m,
5H), 7.03 (s, 1H), 6.61 (d, J ¼ 8.1 Hz, 1H), 4.81 (q,
J ¼ 6.0 Hz, 2H), 4.54 (s, 2H), 3.90e3.75 (m, 1H), 3.28e
2.92 (m, 6H).
ethyl ester HCl (1y)
Yield: 74%. 1H NMR (CD3OD, 300 MHz) d 7.35 (d,
J ¼ 8.9 Hz, 2H), 7.30e7.20 (m, 1H), 7.17e7.08 (m, 1H),
7.02 (s, 1H), 6.75 (d, J ¼ 8.9 Hz, 2H), 4.82e4.75 (m, 1H),

M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
1901
5.4.32. (R)-{2-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino]
phenoxy}acetic acid HCl (2w)
Yield: 57%. 1H NMR (CD3OD, 300 MHz) d 7.89 (d,
J ¼ 8.1 Hz, 1H), 7.37e7.36 (m, 1H), 7.26e7.19 (m, 1H),
7.14 (s, 1H), 7.09 (d, J ¼ 8.1 Hz, 1H), 6.97 (t, J ¼ 8.1 Hz,
2H), 5.09 (q, J ¼ 6.0 Hz, 1H), 4.70 (s, 2H), 3.90e3.80 (m,
1H), 3.78e2.88 (m, 6H).
which was added after the enzyme reaction was terminated.
The fluorescence of DBF metabolite fluorescein was measured
on a fluorescence plate reader with an excitation wavelength of
485 nm and an emission wavelength of 530 nm. The effect of
test compounds on CYP3A4 enzyme was calculated as the
percentage of the enzyme activity.
5.7. X-ray co-crystal structure determination
5.4.33. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino]
phenoxy}-(S )-isopropyl acetic acid HCl (2x)
Yield: 65%. 1H NMR (CD3OD, 300 MHz) d 7.36 (d,
J ¼ 7.2 Hz, 2H), 7.30e7.27 (m, 1H), 7.17e7.08 (m, 1H),
7.02 (s, 1H), 6.77 (d, J ¼ 7.2 Hz, 2H), 4.83e4.76 (m, 1H),
4.32 (d, J ¼ 5.0 Hz, 1H), 3.81e3.78 (m, 1H), 3.27e2.91 (m,
6H), 2.17e2.13 (m, 1H), 0.98 (d, J ¼ 6.8 Hz, 3H).
DPP-IV with a carboxy terminal 6 histidine tag was ex-
pressed in Hi5 insect cells (Invitrogen) using pAcGP67A ba-
culovirus vector (Pharmingen). The secreted DPP-IV was
purified with protein-A Sepharose affinity chromatography
(AP Biotech), ion exchange chromatography and Superdex
200 gel filtration chromatography (AP Biotech). Purified
DPP-IV was incubated with 2 mM compound 1h for overnight
and then concentrated to 10 mg/ml. The DPP-IV complex was
crystallized at 22 ꢁC using the hanging-drop vapor diffusion
method by mixing 1 ml of the protein solution and 1 ml of crys-
tallization buffer. Final crystallization condition was 0.1 M
Tris pH 8.0, 22% (w/v) PEG4000, 0.3 M Na acetate. For
data collection, the crystals were flash frozen at ꢂ170 ꢁC in
the crystallization buffer supplemented with 40% PEG4000.
The diffraction data were collected at the 4A and 6B beam
lines of the Pohang Accelerator Laboratory (PAL) and pro-
cessed using the HKL package (Table 1). The structure was
determined by molecular replacement method using the previ-
ously reported DPP-IV structure (PDB code: 1N1M) as
a search model. The program AMoRe placed the DPP-IV di-
mer in the asymmetric unit. Refinement was carried out using
the program CNS [15]. The randomly selected 5% of data
were set aside for the Rfree calculation. Rounds of refinements
were performed with manual rebuilding by using the program
O [16]. Although extra densities for compound 1h were
5.4.34. (R)-{4-[{2-[3-Amino-4-(2,4,5-trifluorophenyl)
butyryl]-3,4-dihydro-2H-pyrazole-3-carbonyl}amino]
phenoxy}-(R)-isopropyl acetic acid HCl (2y)
Yield: 79%. 1H NMR (CD3OD, 300 MHz) d 7.48 (d,
J ¼ 7.2 Hz, 2H), 7.44e7.30 (m, 1H), 7.26e7.22 (m, 1H),
7.14 (s, 1H), 6.89 (d, J ¼ 7.2 Hz, 2H), 4.94e4.86 (m, 1H),
4.43 (d, J ¼ 4.9 Hz, 1H), 3.90e3.80 (m, 1H), 3.39e2.80 (m,
6H), 2.30e3.15 (m, 1H), 1.09 (d, J ¼ 6.8 Hz, 6H).
5.5. In vitro study
Caco-2 cell lysate (10 ml) was suspended in TriseHCl (pH
7.5) and then 40 mM AlaeProeAFC (ICN Biomedicals, Inc)
was added. After treatment of compounds, the mixture was in-
cubated for 60 min at 24 ꢁC. AFC as an indicator of DPP-IV
activity was detected at 405/510 nm (Ex/Em) by Fluorometer,
Synergy HT (Biotek). IC50 was calculated by Prism 4.0 soft-
ware (GarphPad Software, Inc).
Table 4
Data collection and refinement statistics
5.6. CYP assay
Space group
Cell parameter (A)
P21
˚
a ¼ 65.8, b ¼ 126.5, c ¼ 112.1,
a ¼ r ¼ 90ꢁ, b ¼ 99.6ꢁ
2.3
The CYP3A4 enzyme assay was carried out using fluoro-
metric enzyme assays with Vivid CYP3A4 assay kit (PanVera,
USA, CA) in a 96-well microtiter plate following the manu-
facturer’s instruction with some modifications. Test com-
pounds including ketoconazole as known as CYP3A4
inhibitor were prepared in acetonitrile to give final concentra-
tions of 10 mM. Briefly, to each well of the microtiter plate was
added NADP generating solution (1.0 mM NADPþ, 3.3 mM
glucose 6-phosphate, 3.3 mM MgCl2$6H2O, and 0.4 U/ml glu-
cose 6-phosphate dehydrogenase in 10 mM KPO4, pH 8.0) fol-
lowed by the vehicle acetonitrile (control) and the test
samples. The plate was covered and then incubated at 37 ꢁC
for 20 min. Enzyme reaction was initiated by the addition of
enzyme/substrate (E/S) mixture (0.5 pmol CYP3A4 enzyme
and 5 mM dibenzylfluorescein, DBF). The plate was further in-
cubated for 20 min, followed by the addition of the stop solu-
tion to terminate the enzyme activity. Background reading was
measured in a similar manner except for the E/S mixture
˚
Highest resolution (A)
Unique reflections (total)
Completenessa (%)
63,339 (69,216)
91.5 (81.0)
8.0 (28.8)
b
Rmerge (%)
I/sI
17.6 (3.1)
Refinement
Number of reflections
Number of atoms
(Protein/inhibitor/water)
63,185
12,511
(11,912/60/538)
21.6/24.1
Rcryst/Rfree
Rms deviations
˚
Bond distances (A)
Bond angles (ꢁ)
Impropers (ꢁ)
Dihedrals (ꢁ)
a
0.015
1.8
1.07
26.0
The values in parentheses (completeness and Rmerge) are for the highest
resolution bin.
b
P
P
Rmerge
ment of an equivalent reflection with the indices h, k, l.
¼
ijIi ꢂ hIij/ jhIij, where I is the intensity for the ith measure-

1902
M.A. Jun et al. / European Journal of Medicinal Chemistry 43 (2008) 1889e1902
G.E. Welzel, D.M. Egan, J. Marcinkeviciene, S.Y. Chang, S.A. Biller,
M.S. Kirby, R.A. Parker, L.G. Hamann, J. Med. Chem. 48 (2005) 5025.
[8] (a) D. Kim, L. Wang, M. Beconi, G.J. Eiermann, M.H. Fisher, H. He,
G.J. Hickey, J.E. Kowalchick, B. Leiting, K. Lyons, F. Marsilio,
M.E. McCann, R.A. Patel, A. Petrov, G. Scapin, S.B. Patel, R.S. Roy,
J.K. Wu, M.J. Wyvratt, B.B. Zhang, L. Zhu, N.A. Thornberry,
A. Weber, J. Med. Chem. 48 (2005) 141;
apparent, the model was not included until the last stage of re-
finement. The summary of crystallographic statistics is shown
in Table 4.
Acknowledgments
(b) S.D. Edmondson, A. Mastracchio, M. Beconi, L.F. Colwell,
B. Habulihaz, H. He, S. Kumar, B. Leiting, K.A. Lyons, A. Mao,
F. Marsilio, R.A. Patel, J.K. Wu, L. Zhu, N.A. Thornberry,
A.E. Weber, E.R. Parmee, Bioorg. Med. Chem. Lett. 14 (2004) 5151.
[9] (a) J.H. Ahn, J.A. Kim, H.M. Kim, H.M. Kwon, S.C. Huh, S.D. Rhee,
K.R. Kim, S.D. Yang, S.D. Park, J.M. Lee, S.S. Kim, H.G. Cheon, Bio-
org. Med. Chem. Lett. 15 (2005) 1337;
This research was supported by the Center for Biological
Modulators of the 21st Century Frontier R&D Program, the
Ministry of Science and Technology, Korea. We thank the staff
of the 4A and 6B beam lines of Pohang Accelerator Laboratory.
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