Potent inhibitors of lipoprotein-associated phospholipase A2: Benzaldehyde O-heterocycle-4-carbonyloxime

Article abstract of DOI:10.1016/j.bmcl.2006.08.031

A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholipase A₂ (Lp-PLA₂) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromolar potency for inhibiting Lp-PLA₂ in whole human plasma and isolated human LDL. Based on these results, structure-activity relationship was studied on modification of three parts of R¹, R², and R³ to identify a potent pharmacophore for Lp-PLA₂. In an attempt to introduce various functional groups at R² and R³, we discovered that replacement of less lipophilic groups led to an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R² and R³ had the highest potency with an IC₅₀ value of 0.05 μM in whole human plasma.

Full text of DOI:10.1016/j.bmcl.2006.08.031

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5576–5579
Potent inhibitors of lipoprotein-associated phospholipase A2:
Benzaldehyde O-heterocycle-4-carbonyloxime
Hyung Jae Jeong,a Yong-Dae Park,a,b Ho-Yong Park,a Il Yun Jeong,c
Tae-Sook Jeonga,* and Woo Song Leea,b,
*
aNational Research Laboratory of Lipid Metabolism & Atherosclerosis, Insect Resources Research Center,
Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-805, Republic of Korea
bUniversity of Science and Technology, Daejeon 305-333, Republic of Korea
cAdvanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea
Received 18 April 2006; revised 1 August 2006; accepted 4 August 2006
Available online 21 August 2006
Abstract—A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholi-
pase A2 (Lp-PLA2) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromo-
lar potency for inhibiting Lp-PLA2 in whole human plasma and isolated human LDL. Based on these results, structure–activity
relationship was studied on modification of three parts of R1, R2, and R3 to identify a potent pharmacophore for Lp-PLA2. In
an attempt to introduce various functional groups at R2 and R3, we discovered that replacement of less lipophilic groups led to
an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R2 and
R3 had the highest potency with an IC50 value of 0.05 lM in whole human plasma.
Ó 2006 Elsevier Ltd. All rights reserved.
Accumulation of LDL, particularly oxidized LDL, on
the arterial wall is known as the most important initial
step of atherosclerosis. Lp-PLA2 is able to hydrolyze
the sn-2 ester bond in phospholipids of which the fatty
acid moiety has been shortened or altered by oxidation
to yield oxidized fatty acid and lysophosphatidylcholine
(lyso-PC).1 Hydrolyzed products further accelerate
chronic inflammation related to accumulation of macro-
phage, and a positive feedback mechanism of macro-
phage forming a large amount of Lp-PLA2 further
accelerates progress of vascular disorder. The biological
role of Lp-PLA2 had been controversial with seemingly
contradictory anti- or pro-atherogenic functions being
proposed. The anti-atherogenic properties of Lp-PLA2
were suggested because of the enzymatic catabolism of
biologically active oxidized phospholipids in LDL and
degradation of the unrelated polar phospholipids,
PAF.2 In contrast, the pro-atherogenic function of Lp-
PLA2 is thought to arise from the formation of down-
stream inflammatory mediators derived from oxidized
phospholipids. This view is supported by experimental
evidence suggesting that the products of Lp-PLA2 activ-
ity on oxidized phospholipids (oxidized fatty acid and
lyso-PC) elicit potentially several pro-atherogenic ef-
fects. For example, the reported pro-atherogenic roles
of lyso-PC are impairment of endothelium-dependent
relaxation, inducement of vascular cell and intracellular
adhesion molecules, activity as chemoattractant of
monocyte and T-lymphocytes, suppressed production
and release of endothelium-derived nitric oxide, inhibi-
tion of macrophage migration, toxicity at the concentra-
tion higher than 30–50 micromole, and stimulation of
release of arachidonic acid from endothelial cells.3
Lp-PLA2 is suggested as a pro-inflammatory agent, that
is detected in macrophage of atherosclerotic lesions of
hyperlipidemic human and rabbits.4 According to Mac-
phee’s group’s researches, the formation of fatty streak
in Watanabe heritable hyperlipidemic rabbits is signifi-
cantly decreased by dosage of Lp-PLA2 inhibitor.5
Therefore, inhibition of Lp-PLA2 activity is highlighted
as
atherosclerosis.
a
target for prevention and treatment of
Keyword: Lp-PLA2 inhibitor.
*
Corresponding authors. Tel.: +82 42 860 4278; fax: +82 42 861 2675
(W.S.L.); tel.: +82 42 860 4558 (T.S.J.); e-mail addresses:
tsjeong@kribb.re.kr; wslee@kribb.re.kr
So far, the rational design of Lp-PLA2 inhibitor has
been somewhat difficult because the three-dimensional
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.031

H. J. Jeong et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5576–5579
Table 1. Synthesis of (E)- or (Z)-oximes 2a–r
5577
structure of Lp-PLA2 was not elucidateed. However, the
Compound R1
R2
Yielda (%) (E/Z)b
enzyme is known as a serine protease with a catalytic tri-
ad that was formed by a histidine and aspartic or glu-
tamic acid at the active site.6 Thirkettle et al. reported
that SB-253514 was isolated from Pseudomonas fluores-
cens DMS 11579 and has shown potent inhibitory activ-
ity against Lp-PLA2.7–9 Subsequently, Smith and his
coworker developed a novel series of pyrimidone deriv-
atives through the high-throughput screening.10 A po-
tent, orally active Lp-PLA2 inhibitor, SB-480848, has
been developed by the modification of pyrimidone deriv-
atives, which is currently in a phase II clinical study.11
Recently, we have identified a new class of Lp-PLA2
inhibitors, (E)-benzaldehyde O-benzoyloxime series,
using isolated human LDL as Lp-PLA2 enzyme sourc-
es.12 The Lp-PLA2 inhibitory effects of (E)-benzalde-
hyde O-benzoyloxime series were not quite different
between each isolated human LDL and whole human
plasma. In this article, we describe our initial optimiza-
tion studies with a series of multi-substituted oximes in
whole human plasma to afford a lead compound with
the highest inhibitory potency of Lp-PLA2.
2a
2b
2c
2d
2e
2f
Ph
4-F-Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
90 (9/1)
95 (8/1)
60 (6/1)
95 (1.7/1)
55 (1.2/1)
65 (1/2.2)
65
(3,5-Di-t-Bu-4-OMe)-Ph
PhCH=CH
Furanyl
Thiophenyl
N-4-F-Benzylpyrrole
4-CF3-Ph
4-F-Ph-Ph
2,4-(F)2-Ph
2,5-(F)2-Ph
2,6-(F)2-Ph
3,4-(F)2-Ph
3,5-(F)2-Ph
Ph
2g
2h
2i
87
78
2j
92
2k
2l
76 (4/1)
71
73
2m
2n
2o
2p
2q
2r
76
NH2 92
Ph
Ph
Cl
85
—
c
CN
Me
Ph
80 (9/1)
a Isolated yields.
b Isolated ratios or the ratios were not determined.
c Commercial reagent.
A series of (E)- or (Z)-phenyl- and -heteroaryl-substitut-
ed O-benzoyl- (or acyl)oximes 3a–n, 4a–j, and 5a–s were
synthesized according to the methods shown in Scheme
1. Treatment of various aldehydes or ketones with
hydroxylamine in the presence of Na2CO3 gave the mix-
ture of (E)- and (Z)-oximes 2a–r with a high ratio in
high yields (Table 1). Among them, (E)-N-4-fluoroben-
zylpyrrole-2-carboaldehyde oxime (2j) was obtained by
the reaction of N-fluorobenzyl-pyrrole-2-carbonylalde-
hyde with hydroxylamine. The reaction of benzonitrile
with hydroxylamine in the presence of Et3N gave (Z)-
1-amino-benzaldehyde oxime (2o). Treatment of (E)-
benzaldehyde oxime (2a) with N-chlorosuceinimide in
dichloromethane gave (Z)-1-chloro-benzaldehyde oxime
(2p). Then, pure (Z)-compounds 2o–q are obtained by
silica gel column chromatography. In order to introduce
various functional groups, multi-substituted aryl or het-
eroaryl oximes 2a–r were treated with acyl chlorides,
morpholine-4-carbonyl chloride, and heteroaryl chlo-
rides to give (E)-isomers 3a–n, 4a, 5a–s, and (Z)-isomers
4b–j. Then, (Z)-isomers 3a–n, 5a–s could be isomerized
to the (E)-isomers 3a–n and 5a–s by triethyl ammonium
hydrochloride13 (Scheme 1 and Table 2).
plasma (Table 2) as described above, the whole human
plasma was used as Lp-PLA2 enzyme sources in this
study. Then, the amount of [3H]acetate produced from
[3H] was determined to measure the Lp-PLA2 inhibitory
activity.12 The Lp-PLA2 inhibitory activities of 3a–n,
4a–j, and 5a–s were confirmed by the positive control
with SB480848 which was synthesized by us. Then,
SB480848 inhibited Lp-PLA2 with IC50 value of
0.17 lM. The strategy to explore the structure–activity
relationship (SAR) of 3a (IC50 = 5.1 lM), which was
selected as a hit, focused on examining the optimal sub-
stituents at R1, R2, and R3. The results from (E)- or (Z)-
oxime derivatives are presented in Table 2. Modification
of R1 gave the corresponding compounds 3b–n. Among
them, 4-fluoro-substituted 3b showed an enhanced activ-
ity, whereas 3,4-difluoro-substituted 3m was equipotent
to 3a. The furan-, thiophene-, and pyrrole-substituted
analogues 3e–g at R1 showed significantly lower activity.
Analogues 3c and 3d involving tert-butyl and styrene
groups resulted in a significant loss in potency. These
analogues having more lipophilic groups generally
showed more decreased potency compared with the cor-
responding analogues having less lipophilic groups. The
position of the fluorine was examined according to the
result of 4-fluoro-substituted analogue 3b. 3,4-Diflu-
oro-substituted derivative 3m showed an encouraging
inhibitory activity, whereas 2,3-, 2,4-, 2,5-, 2,6-, and
3,5-difluoro-substituted analogues 3i–l and 3n showed
an attenuated potency. The effects of substitution and
position on the phenyl ring were striking to show the
potency.
The potential of (E)- or (Z)-oxime derivatives was eval-
uated as an inhibitor of Lp-PLA2. Because the Lp-PLA2
inhibitory effects of oxime derivatives on the assay sys-
tem with [3H]PAH (1-O-hexadecyl-acetyl-3H(N)-phos-
phatidylcholine) as a substrate12 were almost same
between each isolated human LDL and whole human
R2
R2
O
Based on the above results that further simple modifica-
tion of the structure had not given further enhancements
in activity, we envisioned that more potent inhibitors
would be obtained by the substitution at R2 or R3. So,
various functional groups such as methyl, amine, chlo-
rine, N,N-diacetyl, cyanide, and ethoxy groups were
introduced at R2 to give compounds 4a–e and 4j. Then,
i, ii
R3
iii or iv
O
OH
R1
N
R1
R2
R1
N
O
1
2
3-5
Scheme 1. Reagents and condition: (i) NH2OH, Et3N, EtOH, rt; (ii)
NBS, CH2Cl2; (iii) R3COCl, Et3N, CH2Cl2; (iv) R3COCl, NaH, THF.

5578
H. J. Jeong et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5576–5579
Table 2. Lp-PLA2 inhibitory activities of multi-substituted oxime derivatives 3a–n, 4a–j, and 5a–s
R1
R2
R3
Yielda (%)
IC50 (lM)
% Inhibitionb
At 25 lM At 10 lM
b
Compound
3a
3b
3c
3d
3e
3f
Ph
4-F-Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
65
80
50
85
72
75
82
86
78
85
74
72
80
82
5.1 (3.8)c
3.2 (4.4)c
74
85
(3,5-Di-t-Bu-4-OMe)-Ph
PhCH=CH
Furanyl
4
35
16
35
Thiophenyl
N-F-Benzylpyrrole
4-CF3-Ph
3g
3h
3i
16
0
2,3-(F)2-Ph
2,4-(F)2-Ph
2,5-(F)2-Ph
2,6-(F)2-Ph
3,4-(F)2-Ph
3,5-(F)2-Ph
20
50
6
3j
3k
3l
0
3m
3n
5.0 (2.0)c
91
33
4a
4b
4c
4d
4e
4f
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Ph
Ph
80
82
90
73
78
85
75
88
73
75
38
13
31
0
NH2
Cl
Ph
Ph
75
N(Ac)2
CN
CN
Ph
0
Morpholine
Thiophenyl
Furanyl
3,4-F-Ph
Morpholine
0.051 (0.12)c
100
0
4g
4h
4i
CN
CN
15
0
CN
OEt
4j
48
5a
5b
5c
5d
5e
5f
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Morpholine
Cyclohexyl
Thiophenyl
Furanyl
90
79
83
81
88
76
79
83
85
97
81
87
72
46
24
50
65
85
84
43
20
47
37
94
73
100
49
97
61
0
Ph
Ph
4-F-Ph
4-F-Ph
Morpholine
4-F-Ph
3.5
6.1
2.2
5g
5h
5i
2,3-(F)2-Ph
2,4-(F)2-Ph
2,4-(F)2-Ph
2,5-(F)2-Ph
2,6-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,4-(F)2-Ph
3,5-(F)2-Ph
Morpholine
4-F-Ph
Morpholine
Morpholine
Morpholine
Morpholine
4-F-Ph
2.4
4.2
5j
5k
5l
98
66
5m
5n
5o
5p
5q
5r
5s
9(Z)-C17H33
9(Z),12(Z)-C17H31
C9H19
16
20
1
4-NO2-Ph
3,4-(F)2-Ph
Morpholine
9
24
58
a Isolated yields from 2.
b Using whole human plasma (1–2 mg protein).
c Using isolated human LDL (13–18 lg protein). Data are shown as mean values of two independent experiments performed in duplicate.
compounds 4a and 4b had a little effect on potency,
whereas compound 4c was proved more potent in
Lp-PLA2 inhibitory activity. More exciting was that
compound 4e was not active, but less lipophilic morpho-
line-substituted analogue 4f at R3 had dramatically
improved inhibitory activity against Lp-PLA2 with
5n–p at R3 were much less potent, even though increasing
the length of the alkyl chain increased inhibitory activi-
ty.14 Inhibitors of lipolytic enzymes are best reported in
terms of mole fraction of inhibitor in the interface (num-
ber of moles of inhibitor to the total number of moles of
lipid, detergent, and inhibitor in the micelle).15 One com-
mon critique of phospholipase inhibition assays is that
they can be subject to numerous false positives, due to
disruption of the membrane/water interface by lipophilic
compounds. It is clear from data (Table 2) that there is
no proportional correlation between the lipophilicity of
the compound and the inhibitory activity in this assay.
sub-micromolar inhibition in this series (IC50
=
0.05 lM). Therefore, we focused on variations to R3.
The inhibitory activities enhanced on substitution of
the morpholine group at R3 (4j, 5a, 5e, 5g, 5i, 5j, 5l,
5s), whereas 2,6-difluoro-substituted 5k was not active,
as shown in analogue 3l. Also, mono- or difluorophe-
nyl-substituted derivatives 5f, 5h, and 5m at R1 and R3
had somewhat increased inhibitory activity. Subsequent-
ly, the highly lipophilic C9- or C18-substituted derivatives
In conclusion, we have discovered a novel series of
human Lp-PLA2 inhibitors, (E)- or (Z)-phenyl- and

H. J. Jeong et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5576–5579
5579
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This work was supported by the Korea Research Foun-
dation Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF-
2005-042-C00099) and KRIBB Research Initiative
Program, Korea.
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