The first potent inhibitor of mammalian group X secreted phospholipase A2: Elucidation of sites for enhanced binding

Article abstract of DOI:10.1021/jm060136t

Using the X-ray structure of human group X secreted phospholipase A ₂ (hGX), we carried out structure-based design of indole-based inhibitors and prepared the compounds using a new synthetic route. The most potent compound inhibited hGX and the mouse orthologue with an IC₅₀ of 75 nM. This compound is the most potent hGX inhibitor reported to date and was also found to inhibit a subset of the other mouse and human SPLA ₂S.

Full text of DOI:10.1021/jm060136t

2858
J. Med. Chem. 2006, 49, 2858-2860
Letters
The First Potent Inhibitor of Mammalian Group
X Secreted Phospholipase A2: Elucidation of
Sites for Enhanced Binding
Brian P. Smart, Rob C. Oslund, Laura A. Walsh, and
Michael H. Gelb*
Departments of Chemistry and Biochemistry, UniVersity of
Washington, Seattle, Washington 98195
Figure 1. Representative structure of a substituted indole inhibitor of
group IIA sPLA2. Indole positions 1-7 are shown.
ReceiVed February 8, 2006
Abstract: Using the X-ray structure of human group X secreted
phospholipase A2 (hGX), we carried out structure-based design of
indole-based inhibitors and prepared the compounds using a new
synthetic route. The most potent compound inhibited hGX and the
mouse orthologue with an IC50 of 75 nM. This compound is the most
potent hGX inhibitor reported to date and was also found to inhibit a
subset of the other mouse and human sPLA2s.
Secreted phospholipases A2 (sPLA2s) are a group of enzymes
with conserved active sites and calcium-binding loops that
catalyze the hydrolysis of the sn-2 ester of glycero-phospholipids
to release free fatty acids and 2-lysophospholipids.1-4 The
sPLA2s are low molecular weight (∼14-20 kDa) enzymes with
5-8 disulfide bonds and require Ca2+ as a catalytic cofactor.1-4
To date, there are 10 distinct mammalian sPLA2s (groups IB,
IIA, IIC, IID, IIE, IIF, III, V, X, XIIA), with group IIC present
as a pseudogene in humans.1,2 Biomedical interest in this class
of enzymes stems from the observation that one or more plays
a role in the liberation of arachidonic acid from cellular
phospholipids for the biosynthesis of eicosanoids. Mammalian
cells also contain a cytosolic phospholipase (cPLA2-R).5 Recent
evidence shows that in macrophages and other cells, sPLA2s
and cPLA2-R work together to liberate arachidonic acid in
agonist-stimulated mammalian cells.6-9 The molecular basis for
the cross-talk between these two enzymes is not known. Potent
and selective inhibitors of the sPLA2 enzymes would help
resolve some of the questions surrounding the mechanism of
arachidonate release as well as probe other physiological
functions of sPLA2s.
For the past decade, the group IIA sPLA2 has received the
most attention among the sPLA2s because it was the first
nonpancreatic sPLA2 to be discovered, and it is found in high
levels in patients suffering from inflammatory diseases including
rheumatoid arthritis10 and acute pancreatitis.11 The group IIA
sPLA2 has been the focus of medicinal chemistry efforts at
several pharmaceutical companies.12-16 Workers at Lilly Labs
and Shionogi & Co. Ltd. have reported on substituted indoles,
exemplified by compound I, as potent inhibitors of group IIA
sPLA2 (Figure 1).12-15 Among the various reported inhibitors
of sPLA2,17 these compounds appear to be the most potent and
also appear to have the most drug-like properties. With the
discovery of additional sPLA2 enzymes, we have been interested
in exploring these indole analogues as inhibitors of all of the
members of the sPLA2 enzyme class.18 We are particularly
Figure 2. Compound A docked into the active site of hGX sPLA2.
As can be seen, the 2-ethyl substituent makes contact with the active
site wall and the 6-methyl group sticks out of the active site.
interested in the group X sPLA2 because it appears to have the
highest specific activity in promoting arachidonic acid release
from mammalian cells.7,19,20 To date, very few inhibitors of the
group X sPLA2 have been reported, with the general sPLA2
inhibitor YM-26734 being the most potent at 0.20 µM against
the group X enzyme.21 To this end, we recently determined the
X-ray structure of the indole-based inhibitor Me-Indoxam bound
to human group X sPLA2 (hGX).18 We now report the
development of a potent indole-based inhibitor of hGX that was
designed based on this structural information.
In our first attempt to develop potent indole-based inhibitors
of sPLA2s other than the group IIA enzyme, we made a library
of analogues in which the substituent attached to N1 of the
indole ring was varied.18 The range of IC50 values for this library
was not very broad, and potent inhibitors of hGX were not
obtained. Subsequent X-ray structural studies showed that this
substituent largely points out of the active site of the sPLA2,
toward what would be the membrane plane when the enzyme
is bound to the phospholipid bilayer.18 Thus, we turned to
chemical modifications of other areas of the indole ring.
Inspection of the X-ray structure of Me-Indoxam bound to
18
hGX sPLA2 reveals a large hydrophobic pocket near the
2-substituent that makes little interaction with the inhibitor. We
hypothesized that a larger 2-alkyl substituent would bind to the
hydrophobic pocket and increase the binding affinity of the
inhibitor. Also, the 6-position of the indole points out of the
enzyme active site and does not contribute to binding affinity.
However, modification of the 6-position may be useful for
modifying the physiochemical properties of the indole for
* Corresponding author. E-mail gelb@chem.washington.edu. Phone: 206
543 7142. Fax: 206 543 1656.
10.1021/jm060136t CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/15/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2859
Scheme 1. Synthesis of SPLA2 Inhibitor Aa
a Reagents: (a) CH3NO2, KF, 18-Crown-6, ACN, reflux; (b) (i) H2SO4, CHCl3, reflux; (ii) SOCl2, reflux; (c) AlCl3, ClCH2CH2Cl; (d) (i) NaOH, -20 °C,
MeOH; (ii) H2SO4, -20 °C, MeOH; (e) p-TsOH, toluene, HOCH2CH2OH, reflux; (f) CCl4, PPh3; (g) 12 equiv of n-BuLi, THF; (h) NaH, BnBr, DMF; (i)
n-BuLi, THF, -78 °C, Ac2O; (j) LAH, THF, reflux; (k) NaBH4, TFA, THF; (l) NaH, BnBr, DMF; (m) H2, Pd/C, MeOH; (n) NaH, BrCH2CO2t-Bu, DMF;
(o) (i) (ClCO)2, CH2Cl2; (ii) NH3; (p) TFA, CH2Cl2.
subsequent use in whole animal studies to affect pharmacoki-
netics. Docking studies were performed on the hGX enzyme in
which the indole inhibitor was modified to include a 2-ethyl
and 6-methyl, and the N1 substituent replaced with a benzyl
(Figure 2). As suspected, the larger 2-ethyl group contacts the
inner wall of the enzyme much better than a 2-methyl group,
and a 6-methyl group sticks out of the enzyme active site and
should not affect binding. As the reported synthesis12 ofsubsti-
tuted 2-ethyl indoles was unsuccessful in our laboratory and
the starting material for introduction of the 6-methyl substituent
is not commercially available, a novel synthesis for 2-ethyl-6-
methyl indoles was developed (Scheme 1). As there are few
known literature reactions to functionalize the 6-position of an
indole, the indole core had to be built up from pyrrole. Michael
addition of nitromethane to tert-butyl crotonate followed by
deprotection of this ester and subsequent treatment with thionyl
Figure 3. Structures of sPLA2 inhibitors with varying alkyl groups at
the 2- and 6-positions.
chloride produced the acyl chloride 2. This was then added to
benzenesulfonyl protected pyrrole in the presence of aluminum
trichloride to give ketone 3. Treatment of 3 with NaOH in
MeOH at low temperature followed by concentrated H2SO4
yielded dimethyl acetal 4. Ring closure to form the 4-oxyethanol
indole 5 was accomplished by addition of a catalytic amount
of acid with refluxing in toluene/ethylene glycol solvent.
Conversion to the chloride followed by addition of excess
n-butyllithium and benzyl protection yielded indole 6. Addition
of n-butyllithium and acetic anhydride produced the desired
2-acetyl compound 7 due to the ortho-lithiating director used
to protect the N1-position of the indole. Removal of the
benzenesulfonyl protecting group and reduction of the ketone
was accomplished in one step by refluxing in excess lithium
aluminum hydride. Deoxygenation at the 2-position was ac-
complished using NaBH4 and trifluoroacetic acid to produce
the 2-ethyl indole 9. N1-benzylation and 4-hydroxy deprotection
followed by addition of tert-butyl bromoacetate yielded the tert-
butyl oxyethanoate 11. Treatment of compound 11 with dilute
oxalyl chloride followed by ammonia gas and deprotection of
the tert-butyl ester with trifluoroacetic acid yielded the desired
substituted 2-ethyl-6-methyl indole (compound A). Compounds
B-D (Figure 3) were synthesized similarly (Supporting Infor-
mation).
To test the indole analogues as sPLA2 inhibitors, we used a
fluorometric assay consisting of unilamellar vesicles of 1-hexa-
decanoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphoglycer-
ol.22 The sPLA2-catalyzed liberation of 10-pyrenedecanoic acid
allows the fluorophore to dislodge from the vesicles and bind
to albumin in the buffer phase where it now undergoes monomer
fluorescent emission rather than excimer emission. The assay
results (Table 1) show the 2-ethyl substituent to have a dramatic
affect on binding to the hGX, with IC50 values of 75 nM for
compounds A and B. The 2-ethyl compounds (A and B) are
26-fold more potent than the analogous 2-methyl compounds
(C and D) against hGX, which have IC50 values of 2 µM. The
6-methyl substituent has no effect on hGX binding; compounds
A and B have identical IC50 values. The inhibitors were then
screened against a panel of recombinant human and mouse
sPLA2s (hGIB, mGIB, hGIIA, mGIIA, hGIIE, mGIIE, hGV,

2860 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Letters
Table 1. Inhibition Data against Mammalian sPLA2s for Compounds
A-Da
(9) Kuwata, H.; Nonaka, T.; Murakami, M.; Kudo, I. Search of factors
that intermediate cytokine-induced group IIA phospholipase A2
expression through the cytosolic phospholipase A2- and 12/15-
lipoxygenase-dependent pathway. J. Biol. Chem. 2005, 280, 25830-
25839.
compound IC50 (µM)
sPLA2
A
B
C
D
(10) Hara, S.; Kudo, I.; Chang, H. W.; Matsuta, K.; Miyamoto, T.; Inoue,
K. Purification and characterization of extracellular phospholipase
A2 from human synovial fluid in rheumatoid arthritis. J. Biochem.
(Tokyo) 1989, 105, 395-399.
(11) Gronroos, J. M.; Nevalainen, T. J. Increased concentrations of
synovial-type phospholipase A2 in serum and pulmonary and renal
complications in acute pancreatitis. Digestion 1992, 52, 232-236.
(12) Dillard, R. D.; Bach, N. J.; Draheim, S. E.; Berry, D. R.; Carlson,
D. G.; Chirgadze, N. Y.; Clawson, D. K.; Hartley, L. W.; Johnson,
L. M.; Jones, N. D.; McKinney, E. R.; Mihelich, E. D.; Olkowski, J.
L.; Schevitz, R. W.; Smith, A. C.; Snyder, D. W.; Sommers, C. D.;
Wery, J. P. Indole inhibitors of human nonpancreatic secretory
phospholipase A2. 1. Indole-3-acetamides. J. Med. Chem. 1996, 39,
5119-5136.
(13) Dillard, R. D.; Bach, N. J.; Draheim, S. E.; Berry, D. R.; Carlson,
D. G.; Chirgadze, N. Y.; Clawson, D. K.; Hartley, L. W.; Johnson,
L. M.; Jones, N. D.; McKinney, E. R.; Mihelich, E. D.; Olkowski, J.
L.; Schevitz, R. W.; Smith, A. C.; Snyder, D. W.; Sommers, C. D.;
Wery, J. P. Indole inhibitors of human nonpancreatic secretory
phospholipase A2. 2. Indole-3-acetamides with additional functional-
ity. J. Med. Chem. 1996, 39, 5137-5158.
(14) Draheim, S. E.; Bach, N. J.; Dillard, R. D.; Berry, D. R.; Carlson,
D. G.; Chirgadze, N. Y.; Clawson, D. K.; Hartley, L. W.; Johnson,
L. M.; Jones, N. D.; McKinney, E. R.; Mihelich, E. D.; Olkowski, J.
L.; Schevitz, R. W.; Smith, A. C.; Snyder, D. W.; Sommers, C. D.;
Wery, J. P. Indole inhibitors of human nonpancreatic secretory
phospholipase A2. 3. Indole-3-glyoxamides. J. Med. Chem. 1996,
39, 5159-5175.
(15) Schevitz, R. W.; Bach, N. J.; Carlson, D. G.; Chirgadze, N. Y.;
Clawson, D. K.; Dillard, R. D.; Draheim, S. E.; Hartley, L. W.; Jones,
N. D.; Mihelich, E. D.; Olkowski, J. L.; Snyder, D. W.; Sommers,
C.; Wery, J. P. Structure-based design of the first potent and selective
inhibitor of human nonpancreatic secretory phospholipase A2. Nat.
Struct. Biol. 1995, 2, 458-465.
(16) Beaton, H. G.; Bennion, C.; Connolly, S.; Cook, A. R.; Gensmantel,
N. P.; Hallam, C.; Hardy, K.; Hitchin, B.; Jackson, C. G.; Robinson,
D. H. Discovery of new nonphospholipid inhibitors of the secretory
phospholipases A2. J. Med. Chem. 1994, 37, 557-559.
(17) Reid, R. C. Inhibitors of secretory phospholipase A2 group IIA. Curr.
Med. Chem. 2005, 12, 3011-3026.
(18) Smart, B. P.; Pan, Y. H.; Weeks, A. K.; Bollinger, J. G.; Bahnson,
B. J.; Gelb, M. H. Inhibition of the complete set of mammalian
secreted phospholipases A(2) by indole analogues: a structure-guided
study. Bioorg. Med. Chem. 2004, 12, 1737-1749.
hGIB
mGIB
hGIIA
mGIIA 0.05 ( 0.01
hGIIE 0.05 ( 0.01
0.80 ( 0.10
0.20 ( 0.05
0.75 ( 0.15
2.00 ( 0.20
2.50 ( 0.25
2.20 ( 0.15
0.14 ( 0.075 2.00 ( 0.10
0.125 ( 0.03 0.125 ( 0.02 0.30 ( 0.05
0.275 ( 0.05
0.07 ( 0.02
0.05 ( 0.02
0.125 ( 0.02 0.125 ( 0.02
0.125 ( 0.03 0.075 ( 0.01
mGIIE 0.075 ( 0.02 0.075 ( 0.02 0.40 ( 0.05
0.40 ( 0.04
0.80 ( 0.05
1.00 ( 0.075
2.00 ( 0.15
2.50 ( 0.20
hGV
mGV
hGX
mGX
0.50 ( 0.1
0.75 ( 0.15
0.075 ( 0.01 0.075 ( 0.01 2.20 ( 0.10
0.075 ( 0.01 0.075 ( 0.01 2.50 ( 0.15
0.50 ( 0.05
0.75 ( 0.10
0.80 ( 0.05
0.85 ( 0.05
a IC50s are based on duplicate or triplicate analyses.
mGV, hGX, and mGX). In all cases the 2-ethyl compounds are
more potent than the 2-methyl derivatives, and the 6-methyl
group is tolerated (Table 1). Compounds A and B should be
useful in distinguishing the groups X and V sPLA2s based on
the ∼10- fold increased potency for the former. This is
significant because current evidence favors a role of these two
sPLA2s in arachidonate liberation in mammalian cells. Although
these compounds are also potent inhibitors of the group IIA
sPLA2s, the original lead compound Me-Indoxam is 50-fold
more potent on hGIIA and mGIIA versus hGX and mGX.18
Thus, by carrying out studies with a combination of inhibitors,
it should be possible to probe for the role of specific sPLA2s in
cellular processes.
In conclusion, the first potent inhibitor against hGX and mGX
sPLA2s has been discovered. A new chemical route to these
indole-based sPLA2 inhibitors has been developed.
Supporting Information Available: Experimental details in-
cluding the synthesis of all compounds and assay procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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