Novel 3,5-diaryl pyrazolines and pyrazole as low-density lipoprotein (LDL) oxidation inhibitor

Article abstract of DOI:10.1016/j.bmcl.2004.03.072

Compounds 4a-j and 5 were synthesized by cyclocondensation of 3a-j and hydrazine and showed significant LDL-antioxidant activities in the TBARS assay, the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation, and the macrophage-mediated LDL oxidation. Among compounds 4a-j and 5, 4a was found to be the most active compound as an inhibitor of LDL oxidation and 4a (IC ₅₀=0.1μM) was 6-fold more potent than probucol (IC ₅₀=0.6μM) in the TBARS assay.

Full text of DOI:10.1016/j.bmcl.2004.03.072

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2719–2723
Novel 3,5-diaryl pyrazolines and pyrazole as low-density
lipoprotein (LDL) oxidation inhibitor
Tae-Sook Jeong, Kyung Soon Kim, Ju-Ryoung Kim, Kyung-Hyun Cho,
Sangku Lee and Woo Song Lee*
National Research Laboratory of Lipid Metabolism & Atherosclerosis,
Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, South Korea
Received 27 February 2004; revised 25 March 2004; accepted 25 March 2004
Abstract—Compounds 4a–j and 5 were synthesized by cyclocondensation of 3a–j and hydrazine and showed significant LDL-
antioxidant activities in the TBARS assay, the lag time of conjugated diene production, the relative electrophoretic mobility (REM)
of ox-LDL, the apoB-100 fragmentation, and the macrophage-mediated LDL oxidation. Among compounds 4a–j and 5, 4a was
found to be the most active compound as an inhibitor of LDL oxidation and 4a (IC50 ¼ 0.1 lM) was 6-fold more potent than
probucol (IC50 ¼ 0.6 lM) in the TBARS assay.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Since it has been reported that oxidized LDLs (ox-
LDLs) play a key role in the early stages of atheroscle-
rosis,1 many antioxidants have been developed to
HO
exhibit the antiatherogenic activities by inhibiting the
foam cell formation in animal model.2 Among these
antioxidants, natural, or synthetic antioxidants (vitamin
E, probucol) are well known to lower lipid levels and
coronary heart disease incidence.3 However, both vita-
min E and probucol as antioxidants have an untoward
effect of lowering serum high-density lipoprotein
(HDL)-cholesterol levels. By analysis of their molecular
structures, we realized that these two antioxidants have
a structural characteristic of hindered phenol as shown
in Figure 1. It has been revealed that bulky substituents
on phenol ring stabilize the phenoxy radical formed at
the ortho-positions of the phenolic hydroxyl group.4 In
general, various heterocyclic compounds including pyr-
azole moiety have been reported to show potent bio-
logical activities.5 Therefore, we introduced pyrazoline
and pyrazole moieties between two phenol rings instead
of the central disulfide carbon of probucol and bulky
substituents at ortho-positions of each phenolic ring.
This paper deals with the study of biological activity of
4a–j and 5. The antioxidant activity of LDL was moni-
O
C16H33
α-Tocopherol
(Vitamin E)
t-Bu
t-Bu
t-Bu
t-Bu
HO
S
S
OH
Probucol
Figure 1. Natural and synthetic antioxidants.
tored by various tools, such as conjugated diene for-
mation, relative electrophoretic mobility (REM), frag-
mentation of apoB-100, and macrophage-mediated
LDL oxidation.
Pyrazolines 4a–j were prepared according to the meth-
ods shown in Scheme 1. Treatment of 1-(3,5-di-tert-
butyl-4-hydroxyphenyl)ethanone (1) with 3,5-di- or
2,3,5-tri-substituted 4-hydroxybenzaldehydes 2a–j gave
a,b-unsaturated ketones 3a–j by a typical acid-catalyzed
aldol condensation in 28–86% yields, as shown in
Scheme 1. The reaction of 3a–j and hydrazine mono-
hydrate afforded 1,3-pyrazolines 4a–j via a one-pot
addition–cyclocondensation process in 40–96% yields
Keywords: LDL-antioxidant.
* Corresponding author. Tel.: +82-42-860-4278; fax: +82-42-861-2675;
e-mail: wslee@kribb.re.kr
Both authors contributed equally to the work.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.072

2720
T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2719–2723
R1
O
R1
O
O
R2
t-Bu
R2
t-Bu
CH3
H
i
HO
OH
HO
OH
R3
t-Bu
R3
t-Bu
1
2a-j
3a-j
3a R1 = H, R2 = R3 = t-Bu, 86% 3b R1 = H, R2 = R3 = i-Pr, 54%
3c R1 = H, R2 = R3 = Me, 66% 3d R1= R2 = R3 = Me, 52%
3e R1 = H, R2 = R3 = F, 58%
3f R1 = H, R2 = R3 = Ph, 28%
3g R1 = R3 = H, R2 = OMe, 71% 3h R1 = R3 = H, R2 = NO2, 48%
3i R1 = R3 = H, R3 = OH, 74% 3j R1 = R2 = R3 = H, 67%
R1
R2
t-Bu
NH
NH
N
N
t-Bu
t-Bu
OH
OH
4a, iii
ii
R3
t-Bu
HO
HO
4a-j
t-Bu
t-Bu
5
Scheme 1. Reagents and conditions: (i) H2SO4, MeOH, reflux; (ii) N2H4, EtOH, rt––reflux; (iii) MnO2, CH2Cl2, rt.
(Table 1). The compound 4a was oxidized by manganese
dioxide to give the corresponding pyrazole 5 in good
yield.6 The structures of 4a–j and 5 were determined by
their spectroscopic analyses.7
preliminary evaluation to select the best candidate is
based on a malondialdehyde (MDA) dosage, and the
results are expressed as IC50 (concentration inhibiting
50% of the Cu2þ-induced lipid peroxidation), deter-
mined by the thiobarbituric acid reactive substances
(TBARS),9 and expressed as a MDA equivalent. The
ability of compounds 4a–j and 5 to attenuate LDL
oxidation was measured by measuring the amount of
TBARS, as shown in Table 1. Effects of 4a–j and 5 on
production of TBARS were examined by incubating
human LDL (120 lg/mL) in the presence of 10 lM
CuSO4 as an oxidation initiator for 4 h. Compound 4a
proved to be 6-fold more active than probucol (0.6 lM),
with IC50 values of 0.1 lM. In addition, human plasma
(final concn 38.8 mg/mL) was subjected to a radical
reaction initiated by 400 lM of Cu2þ for 4 h at 37 ꢁC in
the presence or absence of 4a or probucol.10 The IC50
value (0.8 lM) of 4a was significantly lower than pro-
bucol (2.5 lM) in the radical reaction of human plasma.
The various substituted compounds, 4c, 4e, and 4g–j,
were less effective than 4a but more effective than pro-
bucol to be a positive control. The relative efficacy of
compounds 4b, 4d, and 4f was low, but with similar
LDL-antioxidant activity of probucol. In addition, the
compound 4a was converted to pyrazole 5 by oxidation
using MnO2 with expecting more antioxidant activity.
However, the compound 5 having a conjugated system
showed a similar activity as compared to the probucol.
These results demonstrated that bulky di-tert-butyl
groups contributed to more potent activity, which agrees
with earlier work.11 Also it has been demonstrated that
2,6-di-tert-butylphenol is more reactive toward radical
source to afford 2,6-di-tert-butylphenoxy radical in
inhibition of free radical reaction.12 Furthermore, the
Various substituted pyrazolines 4a–j and pyrazole 5
were synthesized and evaluated. All were examined
in vitro for their abilities to protect human LDL against
Cu2þ-induced peroxidation.8 The prescreening test as
Table 1. Inhibition of copper-induced lipid peroxidation by pyrazo-
lines 4a–j and pyrazole 5
R1
R2
NH
N
t-Bu
OH
R3
HO
t-Bu
Compd
R1
R2
R3
Inhibition of lipid
peroxidation
Yield
(%)a
IC50
(lM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
Me
H
H
H
H
H
H
H
––
t-Bu
i-Pr
Me
Me
F
t-Bu .1
i-Pr .7
Me
500
500
84
0
.2
.2
Me
79
95
0.6
0
F
Ph
Ph
H
40
96
0.6
0.2
0.3
0
OMe
NO2
OH
H
H
96
65
H
.3
4j
H
96
0
.2 steric and electronic factors of substituents to stabilize
phenoxy radical formed from phenolic hydroxy group
may influence antioxidant activities for human low-
density lipoprotein.4 Therefore, 4a was selected for
further studies on the development of a potent antioxi-
dant, using the following tools, such as detection of
5
Pc
t-Bu
––
t-Bu
––
78b
––
0.7
0.6
a Isolated yield from 3a–j.
b Isolated yield from 4a.
c Probucol was used as a reference antioxidant.

T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2719–2723
2721
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
control
4a (1µM)
4a (3 µM)
probucol (3 µM)
probucol (5 µM)
Figure 3. Antioxidant effect of 4a on the Cu2þ-mediated oxidation and
electrophoretic mobility of LDL. LDL (100 lg/mL in PBS) was incu-
bated for 12 h at 37 ꢁC with 5 lM CuSO4. After incubation, approxi-
mately 3.0 lg of LDL protein was loaded onto 0.7% agarose gel for
electrophoresis. The gel was dried and stained with Coomassie brilliant
blue R-250. Lane 1: native LDL (absence of CuSO4), Lane 2: ox-LDL,
Lane 3: 4a (20 lM), Lane 4: 4a (10 lM), Lane 5: 4a (5 lM), Lane 6: 4a
(2 lM), Lane 7: Probucol (20 lM), Lane 8: Probucol (10 lM), Lane 9:
Probucol (5 lM), Lane 10: Probucol (2 lM). Probucol is positive
control.
0
20 40 60 80 100 120 140 160 180 200 220 240
Time (min)
Figure 2. Effect of 4a on the oxidation of LDL copper ion. LDL
(100 lg/mL) in phosphate-buffered saline, at pH 7.4, was incubated
with 5 lM CuSO4 at 37 ꢁC in the presence or absence of antioxidant,
4a. Conjugated diene formation was measured by determining the
absorbance at 234 nm every 10min for 4 h. Probucol was used as ref-
erence antioxidant.
Table 2. Antioxidant effect of 4a on the Cu2þ-mediated oxidation and
apoB-100 fragmentation in LDLa
Compounds (lM)
Areab(AU/mm)
18.95
Native LDL
conjugated diene formation, REM of ox-LDL, frag-
mentation of apoB-100, and macrophage-mediated
LDL oxidation.
Ox-LDL
4a (20)
0
9.97
9.26
5.63
9.22
4.73
0
4a (10)
4a (5)
The extent of lag time was interpreted as the antioxidant
resistant capacity of LDL.13 The formation of the con-
jugated diene during LDL oxidation represents the early
peroxidation of the LDL. The LDL (100 lg/mL) was
incubated with 5 lM CuSO4 in the absence or presence
of 4a. The oxidation of LDL was determined by mea-
suring conjugated diene formation at 234 nm for
240min. The results show a typical effect of the com-
pound 4a (1.0and 3.0 lM) on the prolonging of lag
time, as shown in Figure 2. First, the LDL was incu-
bated with CuSO4 alone to have a lag time of 66 min.
The addition of 4a at 1.0 lM appeared to have a mod-
erate effect on the lag time (163 min) delay, whereas the
addition of 3.0 lM of 4a did not detect the lag time
because of too prolong. Whereas, probucol (3.0and
5.0 lM) extended lag time 74 and 85 min, respectively.
Thus, 4a delayed LDL oxidation to a much greater
extent than did probucol.
Probucol (20)
Probucol (10)
Probucol (5)
a LDL (240 lg/mL in PBS) was incubated for 12 h at 37 ꢁC with 5 lM
CuSO4 in the absence or presence of 5–20 lM of 4a or probucol.
After incubation, approximately 2.0 lg of LDL protein was applied
to SDS-PAGE (4%). After the electrophoresis, the gel was stained
with Coomassie Brilliant blue R250and subjected to densitometric
scanning by Bio Rad Model GS-800 with Bio Rad Quantity One-
4.4.0software.
b Areas of the peaks of the apoB-100 expressed as absorbance units per
millimeter.
the presence of sodium dodecylsulfate (SDS-PAGE),
because apoB-100 is a major component of the LDL.15
As shown in Table 2, the densitometric values related to
the areas of the peaks of the apoB-10016 were expressed
as absorbance units per millimeter for the compound 4a
and probucol at dose-dependent concentration ranging
from 20to 5 lM. The band of apoB-100 was observed
on the native LDL (100 lg/mL in PBS), which was
incubated without CuSO4 for 12 h at 37 ꢁC, but the band
completely disappeared when the LDL was incubated
with 5 lM CuSO4. In the presence of 20, 10, and 5 lM of
4a, the percent of remaining apoB-100 against intact
apoB-100 of native LDL was 52.6%, 48.9%, and 29.7%,
respectively. Whereas, in the presence of 20and 10 lM
of probucol, it was 48.7% and 25.0%, respectively, and
the band of apoB-100 did not preserve in the presence of
5 lM probucol. As a result, 4a was more potent than
probucol in the protection of human LDL against
copper-mediated oxidation.
The effects of various concentration of 4a on Cu2þ
-
mediated oxidation of LDL were determined by REM
assay.14 The 5 lM CuSO4 incubated with LDL for 12 h
was used to induce the oxidation of LDL. When treating
with 20and 10 lM of 4a, the REM was reduced com-
pletely, whereas REM was lowered to 68% and 26% at 5
and 2 lM of 4a, respectively, compared to that of oxi-
dized LDL. On the other hand, the mobility of LDL in
probucol used as a standard was reduced to 100
(20 lM), 84 (10 lM), 37% (5 lM), and 16% (2 lM),
respectively, as shown in Figure 3.
The inhibition of the oxidative process of 4a or probu-
col, as a positive control, was evaluated also by the
study of the fragmentation of the apoB-100 through
the electrophoretic analysis on 4% polyacrylamide gel in
Next, we were interested in antioxidant activity of 4a in
macrophage-mediated oxidation of LDL.17 The cellular

2722
T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2719–2723
Table 3. Effects of 4a on macrophage-mediated LDL oxidation
References and notes
Incubation conditionsa
MDA nmol/mg LDL proteinb
1. Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J.
C.; Witztum, J. L. N. Engl. J. Med. 1989, 320, 915.
2. Bjorkhem, I.; Henriksson-Freyschuss, A.; Breuer, O.;
Diczfalusy, U.; Berglund, L.; Henriksson, P. Arterioscler.
Thromb. 1991, 11, 15.
LDL+Cu2þ
40.15 5.35
210.61 10.72
43.64 1.71ꢀ
74.09 4.49ꢀ
153.79 6.64ꢀ
199.09 10.71ꢀ
209.09 3.85ꢀ
LDL+Cell+Cu2þ (control)
LDL+Cell+Cu2þ+2.0 lM 4a
LDL+Cell+Cu2þ+1.0 lM 4a
LDL+Cell+Cu2þ+0.4 lM 4a
LDL+Cell+Cu2þ+2 lM probucol
LDL+Cell+Cu2þ+1 lM probucol
3. (a) Kita, T.; Nagano, Y.; Yokode, M.; Ishii, K.; Kume,
N.; Ooshima, A.; Yoshida, H.; Kawai, C. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 5928; (b) Carew, T. E.;
Schwenke, D. C.; Steinberg, D. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 7725; (c) Nagano, Y.; Nakamura, T.;
Matsuzawa, Y.; Cho, M.; Ueda, Y.; Kita, T. Atheroscle-
rosis 1992, 92, 131; (d) Daugherty, A.; Zweifel, B. S.;
Schonfeld, G. Br. J. Pharmacol. 1989, 98, 612; (e)
Sasahara, M.; Raines, E. W.; Chait, A.; Carew, T. E.;
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ꢀP < 0:01 versus control.
a LDL (120 lg/mL) was incubated for 24 h at 37 ꢁC in serum-free
RPMI 1640medium with 2 lM of Cu2þ in 12-well plate containing
macrophages, in the absence (control) or presence of increasing
concentrations of the compound 4a tested (0.4–2.0 lM).
b The extent of LDL oxidation was determined directly in the harvested
medium using the TBARS assay. Data are shown as means SD
(n ¼ 3).
oxidative modification of LDL to a form recognized by
the scavenger receptor requires the presence of transi-
tion metal ions in the medium.18 And so, the LDL
(120 lg/mL) was incubated for 24 h in serum-free RPMI
1640medium with 2 lM CuSO4 at 37 ꢁC in a 12-well
plate containing macrophages, in the presence or
absence of 4a or probucol. In macrophage-mediated
LDL oxidation, the TBARS formation in the harvested
medium was also inhibited by 4a at a similar order of
activity to that obtained in Cu2þ-induced LDL oxida-
tion. The LDL oxidation (210.61 10.72 MDA nmol/
mg LDL protein) measured by TBARS production was
five-fold higher in the presence of THP-1 macrophages
compared with incubations in the absence of cells
(40.15 5.35 MDA nmol/mg LDL protein) (Table 3).
This result coincides with the previous reports.19
Therefore, antioxidant activities of 4a and probucol
were tested by macrophage-mediated LDL oxidation at
dose-dependent concentration ranging from 2 to 0.4 lM.
In the concentration of 2, 1, and 0.4 lM of 4a, the
content of ox-LDL was 43.64 1.71, 74.09 4.49, and
153.79 6.64 MDA nmol/mg LDL protein, respectively.
At the concentration of probucol (2 and 1 lM), the
content of ox-LDL was 199.09 10.71 and 209.09 3.85
MDA nmol/mg LDL protein, respectively. As a result,
antioxidant activity of 4a was about five-fold higher
than that of probucol in macrophage-mediated LDL
oxidation (Table 3).
4. Paul, C. U.; David, T. C.; Wiaczeslaw, A. C.; Roderick, J.
S.; Catherine, R. K.; Jagadish, C. S.; Clifford, D. W.;
Denis, J. S.; Richard, D. D. J. Med. Chem. 1994, 37, 322,
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5. Kost, A. N.; Grandberg, I. I. In Advances in Heterocyclic
Chemistry; Katrizky, A. R., Boulton, A. J., Eds.; Aca-
demic: New York, 1996; Vol. 6, p 347.
6. Agrawal, K. J. Indian Chem. Soc. 1987, 408.
7. Physical and spectroscopic data 4a: colorless prisms, mp
243–244 ꢁC, 1H NMR (300 MHz, CDCl3): d 1.45 (18H, s),
1.46 (18H, s), 3.00 (1H, dd, J ¼ 10:5, 16.2 Hz), 3.43 (1H,
dd, J ¼ 10:2, 15.9 Hz), 4.83 (1H, t-like, J ¼ 10:5 Hz), 5.20
(1H, s, –OH), 5.37 (1H, s, –OH), 5.84 (1H, br, –NH), 7.23
(2H, s), 7.53 (2H, s); 13C NMR (75 MHz, CDCl3): d 30.2,
30.3, 34.37, 34.4, 42.2, 64.9, 123.1, 123.3, 124.4, 133.1,
136.0, 136.1, 152.6, 153.3, 154.7.
8. Liu, F.; Ng, T. B. Life Sci. 2000, 66, 725.
9. Ahn, B. T.; Lee, S.; Lee, S. B.; Lee, E. S.; Kim, J. G.; Bok,
S. H.; Jeong, T. S. J. Nat. Prod. 2001, 64, 1562. Procedure
for isolation of LDL: Blood was collected from normal-
ipidemic volunteers. EDTA was used as anticoagulant
(1.5 mg/mL of blood). After low-speed centrifugation of
the whole blood to obtain plasma and to prevent
lipoprotein modification, EDTA (0.1%), NaN3 (0.05%),
and PMSF (0.015%) were added. LDL was isolated from
the plasma by discontinuous density gradient ultra centri-
fugation.20 Briefly, the plasma was centrifuged at 100,000g
at 4 ꢁC for 20h. After the top layers containing chylo-
micron and very low-density lipoprotein (VLDL) were
removed, the density of remaining plasma fractions was
increased to 1.064 with NaBr solution and they were
recentrifuged at 100,000g for an additional 24 h. The LDL
fraction in the top of the tube was collected and dialyzed
overnight against three changes of phosphate buffer
(pH 7.4), containing NaCl (150mM), in the dark at 4 ꢁC
to remove NaBr and EDTA. The LDL in PBS was stored
at 4 ꢁC and used within 4 weeks. The purity of the fraction
was confirmed by agarose gel electrophoresis and SDS-
PAGE as described elsewhere.21 Concentration of LDL
protein was determined using bovine serum albumin
(BSA) as a standard.
In this study, we demonstrated that 3-(3,5-di-tert-butyl-
4-hydroxyphenyl)-5-(multi-substituted-4-hydroxyphenyl)-
2-pyrazolines 4a–j and pyrazole 5 were synthesized and
evaluated for antioxidant activity. Among compounds
4a–j and 5, 4a was found to be the most active com-
pound as an inhibitor of LDL oxidation. These findings
need further study to clarify the mechanism of antioxi-
dant action of the pyrazolines and pyrazoles in LDL
system.
10. Hashimoto, R.; Yaita, M.; Tanaka, K.; Hara, Y.; Kojo, S.
J. Agric. Food Chem. 2000, 48, 6380.
11. Lazer, E. S.; Wong, H. C.; Possanza, G. J.; Graham, A.
G.; Farina, P. R. J. Med. Chem. 1989, 32, 100.
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This work is supported by a grant (National Research
Laboratory Program) from the Ministry of Science and
Technology (M1-0302-00-0033), Korea.

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2723
14. Reid, V. C.; Mitchinson, M. J. Atherosclerosis 1993, 98,
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17. Cell culture and oxidative modification of LDL: Human
monocytic THP-1 cells (ATCC) were cultured in RPMI
1640medium (Gibco/BRL) with phenol red containing
10% fetal bovine serum (Gibco/BRL), 100 U/mL penicil-
lin, and 100 mg/mL streptomycin at 37 ꢁC under 5% CO2
in air. Cells in RPMI 1640medium with serum and
antibiotics were plated in 12-well plates (1 · 106 cell/well in
1 mL). Differentiation of THP-1 cells to macrophage was
induced with phobal 12-myristrate 13-acetate (PMA, 150
ng/mL, Sigma) for 3 days.22 THP-1 macrophages were
washed three times with serum-free RPMI 1640media. To
examine the effect of the compounds on macrophage-
mediated LDL oxidation, cells were incubated with LDL
(120 lg/mL) in the culture medium with or without
compounds, supplemented with 2 lM CuSO4 for 24 h at
37 ꢁC. The extent of LDL oxidation was determined