Pyrrolidine inhibitors of human cytosolic phospholipase A2 [1]

Full text of DOI:10.1021/jm9905155

© Copyright 2000 by the American Chemical Society
Volume 43, Number 6
March 23, 2000
Communications to the Editor
Ch a r t 1
P yr r olid in e In h ibitor s of Hu m a n
Cytosolic P h osp h olip a se A2
Kaoru Seno,* Takayuki Okuno, Koichi Nishi,
Yasushi Murakami, Fumihiko Watanabe,
Takaharu Matsuura, Masaaki Wada, Yasuhiko Fujii,
Masaaki Yamada, Tomoyuki Ogawa, Tetsuo Okada,
Hiroshi Hashizume, Makoto Kii, Shin-ichiro Hara,
Sanji Hagishita, Shozo Nakamoto,
Katsutoshi Yamada, Yukiko Chikazawa,
Masahiko Ueno, Isao Teshirogi, Takashi Ono,* and
Mitsuaki Ohtani*
Shionogi Research Laboratories, Shionogi & Company, Ltd.,
12-4, Sagisu 5-chome, Fukushima-ku,
Osaka 553-0002, J apan
deficient in cPLA2R showed a marked decrease in
eicosanoid biosynthesis,4 allergic symptoms,4a and pos-
tischemic brain injury,4b indicating a direct contribution
of cPLA2R to the production of eicosanoids and the
importance of cPLA2R in inflammation and reperfusion
injury. Therefore, cPLA2R inhibitors are very attractive
targets as agents to treat inflammatory diseases and
stroke. Although some compounds have been reported
as cPLA2R inhibitors,5 none have been worthy of further
pharmacological and clinical study in terms of potency,
specificity, and material characteristics.6 In searching
for PLA2 inhibitors, kinetic analysis alone is not suf-
ficient to determine whether compounds can inhibit
PLA2 activity by affecting the interfacial quality of the
substrate or by directly inhibiting the interaction be-
tween the substrate and the active site of the enzyme.
To screen sPLA2 inhibitors identified as clinical candi-
dates,7 in addition to enzyme assays, a tissue assay
system was introduced as a secondary in vitro assay to
eliminate the false positive compounds such as those
inhibiting the interaction between the substrate inter-
face and enzyme, but not its catalytic site. The most
useful were the X-ray cocrystallographic data8 of the
enzyme and its lead compound which were valuable for
improving the true inhibitory activity at the active site.
The X-ray crystal structure of cPLA2R has recently been
solved;9 however, the complexity of the enzyme kinetics
and the fact that X-ray cocrystallography of cPLA2R and
Received October 13, 1999
In tr od u ction . Phospholipase A2 (PLA2) is a group
of lipolytic enzymes that catalyze the hydrolysis of fatty
acid ester bonds at the sn-2 position of phospholipids.
This enzyme is thought to play an important role in the
biosynthesis of eicosanoids via the release of arachidonic
acid from biomembranes. Another product from biomem-
branes, a lysophospholipid, is converted to platelet-
activating factor (PAF) known as an inflammatory
mediator. PLA2s have been generally classified into
secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and
Ca2+-independent PLA2 (iPLA2) by their molecular
weights, amino acid sequences, and calcium require-
ments.1
cPLA2 comprises three distinct types of enzymes: R,
â, and γ.2 cPLA2R, an 85-kDa protein, contains a
calcium-dependent lipid binding domain and a catalytic
domain, requires micromolar levels of Ca2+ for mem-
brane translocation, and has a specificity for arachidonic
acid bound to the sn-2 position of phospholipids3 in
contrast with sPLA2 and iPLA2 which have broad
substrate specificities, suggesting that cPLA2R is in-
volved in the production of eicosanoids. In fact, mice
* To whom correspondence should be addressed. Phone: +81-6-
6458-5861. Fax: +81-6-6458-0987. E-mail: kaoru.seno@shionogi.co.jp,
takashi.ono@shionogi.co.jp, mitsuaki.ohtani@shionogi.co.jp.
10.1021/jm9905155 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/02/2000

1042 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Ch a r t 2. Synthesized Compounds
Communications to the Editor
Ta ble 1. Inhibitory Activity of Pyrrolidine Derivatives against
Human cPLA2 and Effect on Production of Arachidonic Acid
from THP-1 Cells Stimulated with A23187a
IC50 (µM)
compd
1a
1b
1c
1d
1e
1f
2a
2b
3a
3b
3c
3d
3e
3f
4a
PC/DOGb
THP-1c
0.165 ( 0.020
0.078 ( 0.009
0.049 ( 0.018
0.090 ( 0.004
>10
0.67 ( 0.07
0.32 ( 0.03
0.23 ( 0.03
0.70 ( 0.23
5.9 ( 0.5
>10
5.5 ( 3.2
0.034 ( 0.007
0.072 ( 0.033
0.069 ( 0.016
0.083 ( 0.026
0.038 ( 0.002
0.022 ( 0.003
0.0053 ( 0.0018
0.0021 ( 0.0004
0.021 ( 0.001
0.0062 ( 0.0026
0.0031 ( 0.0006
0.0018 ( 0.0005
0.42 ( 0.28
0.25 ( 0.01
0.40 ( 0.02
0.31 ( 0.04
0.66 ( 0.24
0.30 ( 0.03
0.22 ( 0.02
0.092 ( 0.011
0.055 ( 0.011
0.18 ( 0.06
0.047 ( 0.008
0.052 ( 0.009
0.022 ( 0.001
86 ( 15
4b
4c
4d
AACOCF3
a
Data are expressed as the mean ( SD of three independent
determinations. b Enzyme assay (PC/DOG assay): the PLA2 activ-
ity was measured using the liposome containing 2.5 µM 1-palmi-
toyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine (50 mCi/
mmol) and 1.25 µM sn-1,2-dioleoylglycerol according to the method
described previously.3c c Cellular assay: human THP-1 cells were
grown in RPMI 1640 containing 10% fetal calf serum and pre-
treated with 1.3% dimethyl sulfoxide for 2 days. Following washing
with phosphate-buffered saline (PBS), the cells were suspended
in Hanks’ balanced salt saline (pH 7.6) containing 0.1% bovine
serum albumin. The cell suspension was preincubated with
inhibitor at 37 °C for 15 min and then incubated with 3 µM A23187
(calcium ionophore) at 37 °C for 20 min. The reaction was
terminated using Dole’s solution (isopropyl alcohol:heptane:2 N
sulfuric acid ) 40:10:1). Arachidonic acid was labeled with
9-anthryldiazomethane and quantified by high-performance liquid
chromatography.14
one of its inhibitors has not been successful make it
difficult to synthesize more potent and useful cPLA2R
inhibitors.
Biologica l Resu lts a n d Discu ssion . Although the
X-ray crystal structure of human cPLA2R has recently
been solved,9 there were no available data on the three-
dimensional structure of cPLA2R when we started this
structure-activity relationship study. Since it was
reported at that time that Ser228 was the most important
amino acid residue in the active site of cPLA2R and both
Arg200 and Asp549 were essential for the activity from
the result of site-directed mutagenesis,12,13 we expected
that Arg200 could be trapped by the thiazolidinedione
part with the acid-base interaction and Asp549 might
exist near this active center of the enzyme. Further-
more, we supposed that Ser228 would be trapped by a
functional group of the inhibitor which could interact
with the hydroxyl group of the serine residue. We
therefore focused our synthetic efforts on searching for
groups which might interact with the hydroxyl group
of Ser228 and found the carbonyl group of the o-(benzoyl)-
benzoyl group of compound 1a to be suitable for this
function. To confirm this, we synthesized meta- and
para-substituted isomers of compound 1a (see com-
pounds 1e,f of Table 1). The inhibitory activity of
cPLA2R was lost in both of these isomers. Introduction
of fluorine atom(s) at the para and/or ortho position of
the terminal benzoyl group of 1a led to increased
inhibitory activity (1b-d in Table 1). Replacement of
the oxygen atom of the ether part at the 4-position of
pyrrolidine to a sulfur atom (2a ,b) led to increased
inhibition, and substitution of thiazolidinedione to
We searched for nonpeptide and low-molecular-weight
inhibitors of cPLA2R and identified two compounds (I
and II) as lead compounds for cPLA2R inhibitors from
our compound library (Chart 1). These compounds
showed cPLA2R inhibitory activities with IC50 values of
1.5 µM (I) and 1.7 µM (II) with the enzyme assay. By
combining these two compounds and conducting struc-
ture-activity relationship studies using both enzyme
assay and secondary-cell-based assay established to
select the inhibitor specific for arachidonic acid release
and penetrating the cell membrane, we found a com-
pound having inhibitory activity about 900-fold more
than the lead compounds.
In this communication, we report on these pyrrolidine
cPLA2R inhibitors having structures 1-4 (Chart 2),
which possess very potent cPLA2R inhibitory activity.
Ch em istr y. We synthesized four types of pyrrolidine
derivatives (1-4) using known synthetic methods.
Compounds (1) having substituted benzyloxy groups at
the 4-position of pyrrolidine were synthesized from
trans-N-Boc-4-hydroxy-L-proline methyl ester.10 Pyrro-
lidine derivatives (2) having substituted benzylthio
groups were synthesized from cis-N-Boc-4-hydroxy-L-
proline methyl ester.11 4-Aryloxypyrrolidine derivatives
(3) and N,N-disubstituted 4-aminopyrrolidines (4) were
also synthesized from the same cis-N-Boc-4-hydroxy-L-
proline methyl ester.

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1043
Ch a r t 3
was more active by about 230-fold in the enzyme assay
and about 3900-fold in the cellular assay than arachi-
donyl trifluoromethyl ketone (AACOCF3), which is
generally known as a potent cPLA2R inhibitor.5a,15
Compound 4d at 1 µM almost completely inhibited
arachidonic acid release from THP-1 cells.
In addition to the arachidonic acid release, we deter-
mined whether the pyrrolidine derivatives could inhibit
eicosanoid production in A23187-stimulated THP-1 cells
(Table 2). Compounds 1a ,b, 3a , and 4d inhibited the
formation of PGE2 and LTC4 with IC50 values compa-
rable to those of the arachidonic acid release. Because
none of these compounds induced the release of lactate
dehydrogenase as an indicator of cell viability even at
the concentration of 100 µM, the observed inhibitions
were not due to a cytotoxic effect.
To determine whether the observed inhibition of the
pyrrolidine derivatives for eicosanoid production in-
volves action against a step-down arachidonic acid
release, the effect of compound 4d on the production of
PGE2 and LTC4 by direct addition of arachidonic acid
to THP-1 cells was examined (Table 3). On addition of
arachidonic acid to the cells, the standard cyclooxyge-
nase inhibitor indomethacin and the standard 5-lipoxy-
genase inhibitor 2,3,5-trimethyl-6-(12-hydroxy-5,10-
dodecadiynyl)-1,4-benzoquinone (AA-861)16 inhibited the
production of PGE2 and LTC4, respectively, as in A23187
stimulation, whereas compound 4d did not inhibit either
production. Therefore, the inhibition of PGE2 and LTC4
biosynthesis by compound 4d was apparently due to
inhibition of only the step of arachidonic acid release.
In contrast to the pyrrolidine derivatives, AACOCF3
inhibited not only the step of arachidonic acid release
but also the thromboxane biosynthetic step located
further downstream, probably because its fundamental
structure is arachidonic acid.15a
Recently, cPLA2â and cPLA2γ have been identified as
new members of cPLA2 and characterized.2 The molec-
ular weights of cPLA2â and cPLA2γ were about 114 and
61 kDa, respectively, and the sequences of both PLA2s
revealed about 30% identity with that of cPLA2R, but
the physiological roles of cPLA2â and cPLA2γ have not
yet been clarified. Although the inhibitory activity of
pyrrolidine derivatives for cPLA2â and cPLA2γ have not
been examined, it is possible that these compounds also
inhibit cPLA2â and cPLA2γ, because amino acids es-
sential for catalysis were strictly conserved in cPLA2R,
cPLA2â, and cPLA2γ.2
Ta ble 2. Effect of Pyrrolidine Derivatives on Production of
Eicosanoids by Cultured THP-1 Cells Stimulated with A23187a
THP-1 cellular assay IC50 (µM)
c
c
compd
AAb
PGE2
LTC4
1a
1b
3a
4d
0.67 ( 0.07
0.32 ( 0.03
0.31 ( 0.04
0.41 ( 0.07
0.26 ( 0.10
0.21 ( 0.11
0.37 ( 0.07
0.26 ( 0.06
0.14 ( 0.06
0.013 ( 0.001
0.022 ( 0.001 0.031 ( 0.043
indomethacin >10
0.0036 ( 0.0043 >10
a
Data are expressed as the mean ( SD of three independent
determinations. See footnote c of Table 1. c After incubation with
b
A23187 at 37 °C for 20 min as shown in footnote c of Table 1, the
reaction was stopped by cooling on ice, and LTC4 and PGE2 levels
in the supernatant were measured by radio immunoassay and
enzyme immunoassay.
rhodanine (1d , 2b, 3b) led to a slight decrease of the
inhibition (Table 1). The inhibitory activity was not
affected by the substituents at the 4-position being
substituted benzyl groups (1b,d ) or substituted aryl
groups (3a ,b). In addition to the hydrophobic interaction
between the lipophilic substituent at the 4-position of
pyrrolidine and the lipophilic part of the enzyme, the
sterically crowded environment also seemed to be
needed (3c-f). Introduction of a voluminous tertiary
amino group to the 4-position offered a new way of
crowding the environment at this position without loss
of lipophilicity to increase the activity (4a ,b). Also,
elongation of the substituent at the 2-position with
insertion of a double bond between the amide carbonyl
group and the phenyl group led to a great increase in
the activity (4c,d ). The inhibition of arachidonic acid
release in human monocytic leukemia cells (THP-1 cells)
stimulated with A23187 was enhanced in parallel with
the increase of inhibitory activity against cPLA2R by the
chemical modification as shown in Table 1. The most
active compound 4d (Chart 3) among those listed in
Table 1 inhibited the cPLA2R enzyme with an IC50 value
of 1.8 nM and arachidonic acid release in A23187-
stimulated THP-1 cells with an IC50 value of 22 nM and
Taken together, these results indicate that the pyr-
Ta ble 3. Effect of Various Inhibitors on Production of PGE2 and LTC4 by Cultured THP-1 Cells Stimulated with Arachidonic Acida
THP-1 cellular assay IC50 (µM)
A23187
AA
AAb
PGE2
LTC4
PGE2
LTC4
c
c
d
e
compd
4d
0.022 ( 0.001
>10
>10
0.031 ( 0.043
0.0036 ( 0.0043
ndf
0.013 ( 0.001
>10
0.0047 ( 0.0010
>10
>10
>10
0.068 ( 0.026
indomethacin
AA861
0.013 ( 0.007
ndf
a
b
Data are expressed as the mean ( SD of three independent determinations. See footnote c of Table 1. c See footnote c of Table 2.
d
DMSO differentiated THP-1 cells, suspended in Hanks’ balanced salt saline (pH 7.6) containing 0.1% bovine serum albumin, were
preincubated with the inhibitor at 37 °C for 15 min, 50 µM arachidonic acid was added, and the reaction mixture was incubated at 37 °C
for 20 min. The reaction was stopped by cooling on ice, and PGE2 levels in the supernatant were measured by radio immunoassay.
e DMSO differentiated THP-1 cells, suspended in Hanks’ balanced salt saline (pH 7.6) containing 0.1% bovine serum albumin, were
preincubated with 200 µM aspirin at 37 °C for 60 min; the cells were washed with PBS. Following preincubation with the inhibitors at
37 °C for 15 min, 250 µM arachidonic acid was added and the reaction mixture was incubated at 37 °C for 20 min. The reaction was
stopped by cooling on ice, and LTC4 levels in the supernatant were measured by enzyme immunoassay. f nd, not done.

1044 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Communications to the Editor
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rolidine derivatives inhibit PGE2 and LTC4 production
by inhibiting cPLA2R without any effect on enzymes
located downstream of the arachidonic acid cascade, i.e.,
cyclooxygenase and 5-lipoxygenase. The effect of pyr-
rolidine derivatives on eicosanoid synthesis observed in
this study was quite consistent with the findings that
the syntheses of PGE2 and cysteinyl leukotrienes were
significantly decreased in stimulated peritoneal mac-
rophages from cPLA2R-deficient mice,4 demonstrating
that cPLA2R is important for eicosanoid biosynthesis
and that the pyrrolidine derivatives are of great value
for elucidating the physiological role of cPLA2R as well
as the role of cPLA2R in the process of eicosanoid
biosynthesis. We are now examining the pharmacologi-
cal effect of the pyrrolidine derivatives.
We have described here the discovery of potent
inhibitors of cPLA2R having 1,2,4-trisubstituted pyrro-
lidine frameworks. Their structural features include the
ortho-substituted (benzoyl)benzoyl group and 2,4-dioxo-
(or 2-oxo-4-thioxo)thiazolidin-5-ylidenemethylphenyl
group. Our newly synthesized pyrrolidine compounds
are extremely potent cPLA2R inhibitors compared to
others reported to date. Detailed data from the synthe-
ses and biological evaluations will be published in full
papers soon.
(6) Tibes, U.; Friebe, W.-G. Phospholipase A2 inhibitors in develop-
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Ack n ow led gm en t. We thank our colleagues at Lilly
Research Laboratories, Indianapolis, IN, who have
collaborated with us during the course of this research
project and provided us with purified human recombi-
nant cPLA2R. We also thank Drs. Hitoshi Arita, Kenji
Kawada, and Yozo Hori for their encouragement and
helpful discussions throughout this study.
(10) This starting material is commercially available and easily
prepared from trans-4-hydroxy-L-proline by Boc protection fol-
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Su p p or tin g In for m a tion Ava ila ble: Synthetic schemes
and physical data of final compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
(11) This starting material is also commercially available and easily
prepared from N-Boc-trans-4-hydroxy-L-proline methyl ester by
inversion of the hydroxyl group by the Mitsunobu reaction.
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