Novel 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A2

Article abstract of DOI:10.1016/S0960-894X(01)00472-3

Derivatives of 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) with modified substituents at the indole-1-position were synthesized and evaluated for their ability to inhibit the arachidonic acid release in human platelets mediated by the cytosolic phospholipase A₂. One of the most active compounds obtained was 26 with an IC₅₀ of 0.44 μM.

Full text of DOI:10.1016/S0960-894X(01)00472-3

Bioorganic & Medicinal ChemistryLetters 11 (2001) 2569–2572
Novel 3-Dodecanoylindole-2-carboxylic Acid Inhibitors of
Cytosolic Phospholipase A2
Matthias Lehr,* Monika Klimt and Alwine Schulze Elfringhoff
Institute of Pharmaceutical Chemistry, University of Munster, Hittorfstrasse 58–62, D-48149 Munster, Germany
¨
¨
Received 8 May2001; revised 3 July2001; accepted 5 July2001
Abstract—Derivatives of 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) with modified substituents at the
indole-1-position were synthesized and evaluated for their ability to inhibit the arachidonic acid release in human platelets mediated
bythe cytosolic phospholipase A 2. One of the most active compounds obtained was 26 with an IC50 of 0.44 mM. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
these PLA2s the a-subtype of cPLA2 seems to playthe
central role in the arachidonic acid cascade and during
the inflammatoryresponse as supported byexperiments
with animals which overexpressed cPLA2 and with
cPLA2 knockout animals.10À12
Phospholipase A2 (PLA2) constitutes a large and diverse
family of enzymes that catalyze the hydrolysis of mem-
brane phospholipids at the sn-2 position to release fatty
acids and lysophospholipids. When the liberated fatty
acid is arachidonic acid, subsequent metabolism
through the cyclooxygenase or the lipoxygenase path-
ways leads to the formation of eicosanoids, including
prostaglandins and leukotrienes. Distinct lysophospho-
lipids (1-O-alkyl-substituted choline glycerolysophos-
pholipids) can be converted to the platelet-activating
factor (PAF). Prostaglandins, leukotrienes, lysophos-
pholipids and the PAF are potent mediators of inflam-
mation.1À3 Thus, inhibition of PLA2 is considered as an
attractive target for the design of new anti-inflammatory
drugs.4À7
9
Recently, we have found that several 3-acylindole-2-
carboxylic acid derivatives are inhibitors of the cPLA2-
mediated arachidonic acid release induced bycalcium
ionophore A23187 in bovine platelets.13 One of these
compounds was the indole derivative 1, which showed
an IC50 of 8 mM in this assay(Fig. 1). An increase of
inhibitorypotencycould be achieved byreplacement of
the N-methyl group of 1 bya longer carboxylic acid
moiety. For the compounds 2 and 3 IC50 values of 1.6
mM were evaluated. The indoles 2 and 3 have also
One problem associated with the in vitro search for
PLA2 inhibitors is the selection of the appropriate
enzyme, since many different PLA2s are present in the
mammalian organism.8 Theycan be divided in PLA s
2
utilizing a catalytic histidine and in PLA2s having a
serine in the active site. The small molecular weight
secretoryPLA 2s (sPLA2) are members of the first
group. The second group consists of the cytosolic
PLA2s (cPLA2), the calcium-independent PLA2 (iPLA2)
and the lipoprotein-associated PLA2s, which have
higher molecular weights than the sPLA2s. From all
*Corresponding author. Tel.: +49-251-83-33331; fax: +49-251-83-
32144; e-mail: lehrm@uni-muenster.de
Figure 1. Structures of indole-2-carboxylic acid inhibitors of cPLA2.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00472-3

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M. Lehr et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2569–2572
shown activityin vivo in the carrageenan-induced rat
paw edema model and in the phorbol ester-induced and
the croton oil-induced ear edema models of the
mouse.14,15
trifluoroacetic anhydride in dichloromethane13 and at
least heated in 10% aqueous NaOH to hydrolyze the
ethyl ester groups and to cleave the acetyl group pro-
tecting the amine moiety. The target compound 15 was
obtained byFriedel–Crafts acylation of 12 with dode-
canoyl chloride and AlCl3 followed bysaponification
with KOH.
The replacement of the carboxylic acid side chain of 2
or 3 in position 1 of the indole bya 4-ethoxybenzoic
acid residue led to a further increase of in vitro activ-
ity.13 With an IC50 of 0.5 mM compound 4 was about 3-
fold more active than 2 and 3 in the assayusing bovine
The remaining indoles (17–31) were prepared byreact-
ing ethyl 3-dodecanoylindole-2-carboxylate (5) with
ethyl 4-(3-bromopropyl)benzoate19 (16a) and 4-(o-bro-
moalkoxy)benzoates (16b–16n), respectively, either in
DMSO with t-BuOK as base or with powdered KOH,
18-crown-6 ether in refluxing benzene20 followed by
saponification in each case (Scheme 3). The procedure
using crown ether usuallygave better yields. The deri-
vative with a hydroxy group in position 2 of the phenyl
ring (24) was obtained bycleaving the ethoxygroup of
23 with BBr3 in hexane/CH2Cl2 allowing the mixture to
warm up from 0 ꢀC to room temperature. The 4-(o-
bromoalkoxy)benzoic acid esters necessary for the
synthesis of 18, 19, 22–26, 28, 29 and 31 could be syn-
thesized by refluxing the substituted 4-hydroxybenzoic
acid esters with an excess of 1,3-dibromopropane,
cells. Applying human platelets the same discrepancy in
16
activitywas assessed.
For 2 and 3 IC50 values of 2.5
and 2.6 mM, respectively, were evaluated, while the IC50
of 4 was 0.86 mM.
Here, we report the effects of the systematic variation of
the (4-carboxyphenoxy)ethyl moiety of 4 on its cPLA2-
inhibitoryactivityin human platelets.
Chemistry
The synthesis of 7, in which the oxygen of the phenoxy
moietyof 4 was replaced bysulfur, started from ethyl 3-
dodecanoylindole-2-carboxylate (5).13 This was con-
verted to 6 bytreatment with t-BuOK and 1,2-dibro-
moethane in DMSO at 110–120 ꢀC (Scheme 1).
Reaction of 6 with ethyl 3-sulfanylbenzoate17 and t-
BuOK in ethanol/THF led to the diethylester of 7,
which was saponified byKOH to yield the desired test
compound.
The indole derivatives 14 and 15, in which the benzoic
acid moietywas attached to the indole via a 2-ami-
noethyl or 2-(methylamino)ethyl spacer, were prepared
bythe routes shown in Scheme 2. Ethyl indole-2-car-
boxylate (8) was reacted in DMSO at 110–120 ꢀC with
the appropriate ethyl 4-[(chloroacetyl)amino]benzoates18
in the presence of t-BuOK. The carbamoyl groups of
the resulting compounds 9 and 10 were reduced to the
amines 11 and 12 byNaBH
and BF3–etherate in
4
refluxing THF. For the synthesis of 14 the intermediate
11 was acetylated at the aniline nitrogen with acetyl-
chloride in pyridine, then acylated in position 3 of the
indole with dodecanoic acid, polyphosphoric acid and
Scheme 2. (i) t-BuOK, DMSO, ethyl 4-[(chloroacetyl)amino]benzoate
or ethyl 4-[(chloroacetyl)(methyl)amino]benzoate, 110–120 ꢀC, 5 min;
(ii) NaBH4, BF3–etherate, THF, reflux, 1 h; (iii) pyridine, acetyl
chloride, rt, 15 min; (iv) dodecanoic acid, polyphosphoric acid, tri-
fluoroacetic anhydride, CH2Cl2, rt, 5 days; (v) 10% aq NaOH, reflux,
2 h; (vi) dodecanoyl chloride, AlCl3, CH2Cl2, rt, 20 h; (vii) EtOH, 10%
aq KOH, reflux, 1 h.
Scheme 1. (i) t-BuOK, DMSO, 1,2-dibromoethane, 110–120 ꢀC, 10
min; (ii) t-BuOK, EtOH, THF, ethyl 4-sulfanylbenzoate, reflux, 1 h;
(iii) EtOH, 10% aq KOH, reflux, 1 h.

M. Lehr et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2569–2572
2571
1,4-dibromobutane and 1,2-dibromoethane, respec-
tively, in an ethanolic sodium ethoxide solution for 5 h
to 3 days.13 Since the yield of the nitro compound 16k
was verylow (3%), the reaction was repeated with the
recovered starting material in this case.
oxidized to a carboxylic acid moiety in a haloform
reaction with Br2 and NaOH. The obtained acid was
esterified with thionyl chloride/ethanol to yield the
benzoate components necessaryfor the synthesis of 27
and 30.
Deviating, the 4-(o-bromoalkoxy)benzoates used for
the preparation of 20 and 21 were synthesized as shown
in Scheme 4 starting from ethyl 2,4-dihydroxybenzoate.
This was converted to ethyl 4-(2-bromoethoxy)-2-
hydroxybenzoate by reaction with 1,2-dibromoethane.
The remaining free hydroxy group in ortho-position of
the ester moiety was then methylated with methyl p-
toluenesulfonate or acetylated with acetyl chloride to
give 16d and 16e (Scheme 4). The synthesis of the 4-(2-
bromoethoxy)benzoate intermediates 16j and 16m star-
ted from 4-hydroxyacetophenone derivatives (Scheme 5).
These were reacted with 1,2-dibromoethane and sodium
ethoxide in ethanol. Subsequentlythe acetyl group was
Scheme 5. (i) NaOC2H5, EtOH, 1,2-dibromoethane, reflux, 7–14 h;
(ii) Br2, 12% aq NaOH, dioxane, 0 ꢀC, 1.5 h; (iii) SOCl2, benzene,
reflux, 3h; (iv) EtOH, reflux, 15 min.
Biological Evaluation
The cPLA2-inhibitorypotencyof the test compounds
was evaluated bymeasuring the calcium ionophore
A23187-induced arachidonic acid release from human
platelets with HPLC and UV-detection at 200 nm.16,21
To avoid metabolism of arachidonic acid via the cyclo-
oxygenase-1 and the 12-lipoxygenase pathways, the dual
cyclooxygenase-1/12-lipoxygenase inhibitor 5,8,11,14-
eicosatetraynoic acid (ETYA) was added to the platelets
in these experiments. Since lysis of the platelets by a test
compound maymisleadinglyindicate enzmy e inhibi-
tion, we also measured the cell lytic potency of each
22
compound byturbidimetry.
In these experiments, it
was found that none of the compounds showed cell lytic
properties at concentrations near its IC50 against cPLA2.
Results and Discussion
To explore the importance of the ether oxygen of the (4-
carboxyphenoxy)ethyl substituent for the cPLA2-inhi-
bitoryactivityof the indole 4, we replaced this atom by
sulfur (7), nitrogen (14), methylated nitrogen (15) and
methylene (17). As shown in Table 1, all these variations
led to a decrease of activity. The IC50-values of the
compounds 7, 15 and 17 laybetween 2 and 3 mM. The
greatest loss of inhibitorypotencyoccurred when the
oxygen was exchanged by nitrogen. With an IC50 of 5.4
mM 14 was about 6-fold less active than 4.
Scheme 3. (i) t-BuOK, DMSO, 110–120 ꢀC, 5 min; (ii) powdered
KOH, 18-crown-6 ether, benzene, reflux, 5–12 h; (iii) EtOH, 10% aq
KOH, reflux, 1 h.
An elongation of the ethoxymoietyof the (4-carboxy-
phenoxy)ethyl substituent by one (18) or two (19)
carbons also reduced in vitro potency.
Modifications on the 4-carboxyphenoxy residue had
different effects. While a methoxy( 20), hydroxy (21) and
chloro (22) substituent in ortho-position or a methoxy
(23), hydroxy (24) and nitro (28) group in meta-position
to the carboxylic acid functionality lowered activity, a
chloro (25), fluoro (26) or methyl moiety (27) in meta-
position slighthlyincreased it. Introduction of an addi-
tional chloro (29), fluoro (30) or methyl substituent (31)
Scheme 4. (i) NaOC2H5, EtOH, 1,2-dibromoethane, reflux, 2 h; (ii) t-
BuOK, DMSO, methyl p-toluenesulfonate, rt, 2 h; (iii) pyridine, acetyl
chloride, rt, 30 min.

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M. Lehr et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2569–2572
Table 1. Inhibition of the cPLA2-mediated arachidonic acid release
from human platelets stimulated with calcium ionophore A23187
3. Ryborg, A. K.; Deleuran, B.; Sogaard, H. R.; Kragballe,
K. Acta Derm. Venereol. 2000, 80, 242.
4. Connolly, S.; Robinson, D. H. Expert Opin. Ther. Pat.
1993, 3, 1141.
5. Connolly, S.; Robinson, D. H. Expert Opin. Ther. Pat.
1995, 5, 673.
6. Tibes, U.; Friebe, W. G. Expert Opin. Invest. Drugs 1997, 6,
279.
7. Mayer, R. J.; Marshall, L. A. Emerging Drugs 1998, 3, 333.
8. Six, D. A.; Dennis, E. A. Biochim. Biophys. Acta 2000,
1488, 1.
Compd
R
IC50
(mM)a
2
3
4
7
–(CH2)7COOH
2.5
2.6
0.86
2.5
5.4
2.9
2.1
3.3
1.5
2.8
2.3
2.9
4.9
5.2
0.52
0.44
0.57
3.0
1.8
0.64
9. Lin, L. L.; Lin, A. Y.; Knopf, J. L. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 6147.
–CH2CH2OPhenyl(4-CH2COOH)
–CH2CH2OPhenyl(4-COOH)
–CH2CH2SPhenyl(4-COOH)
10. Uozumi, N.; Kume, K.; Nagase, T.; Nakatani, N.; Ishii,
S.; Tashiro, F.; Komagata, Y.; Maki, K.; Ikuta, K.; Ouchi, Y.;
Miyazaki, J.; Shimizu, T. Nature 1997, 390, 618.
11. Bonventre, J. V.; Huang, Z.; Taheri, M. R.; O’Leary, E.;
Li, E.; Moskowitz, M. A.; Sapirstein, A. Nature 1997, 390,
622.
12. Gijon, M. A.; Spencer, D. M.; Siddiqi, A. R.; Bonventre,
J. V.; Leslie, C. C. J. Biol. Chem. 2000, 275, 20146.
13. Lehr, M. J. Med. Chem. 1997, 40, 2694.
14
15
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
–CH2CH2NHPhenyl(4-COOH)
–CH2CH2N(CH3)Phenyl(4-COOH)
–CH2CH2CH2Phenyl(4-COOH)
–CH2CH2CH2OPhenyl(4-COOH)
–CH2CH2CH2CH2OPhenyl(4-COOH)
–CH2CH2OPhenyl(3-OCH3)(4-COOH)
–CH2CH2OPhenyl(3-OH)(4-COOH)
–CH2CH2OPhenyl(3-Cl)(4-COOH)
–CH2CH2OPhenyl(2-OCH3)(4-COOH)
–CH2CH2OPhenyl(2-OH)(4-COOH)
–CH2CH2OPhenyl(2-Cl)(4-COOH)
–CH2CH2OPhenyl(2-F)(4-COOH)
–CH2CH2OPhenyl(2-CH3)(4-COOH)
–CH2CH2OPhenyl(2-NO2)(4-COOH)
–CH2CH2OPhenyl(2-Cl,6-Cl)(4-COOH)
–CH2CH2OPhenyl(2-F,6-F)(4-COOH)
14. Tries, S.; Neher, K.; Laufer, S.; Abraham, W. M.; Lehr,
M. Mediat. Inflamm. 1999, 8 (Suppl. 1), S123.
15. Tries, S.; Neupert, W.; Albrecht, W.; Lehr, M.; Laufer, S.
Abstract of Papers, 10th National Meeting of the Inflamma-
tion Research Association, Hot Springs, USA, 2000.
16. Lehr, M.; Schulze Elfringhoff, A. Arch. Pharm. Pharm.
Med. Chem. 2000, 333, 312.
–CH2CH2OPhenyl(2-CH3,6-CH3)(4-COOH) 3.6
17. Horner, L.; Lindel, H. Phosphorus Sulfur 1983, 15, 1.
18. Dave, M. P.; Patel, J. M.; Langalia, N. A.; Shah, S. R.;
Thaker, K. A. J. Indian Chem. Soc. 1985, 62, 386.
19. Bicking, J. B.; Robb, C. M.; Cragoe, E. J.; Blaine, E. H.;
Watson, L. S.; Dunlay, M. C. J. Med. Chem. 1983, 26, 335.
20. General procedure for the alkylation of 3-dodecanoyl-
indole-2-carboxylic acid (5) using KOH/crown ether: To the
mixture of 5 (372 mg, 1 mmol), powdered KOH (85%) (66 mg,
1 mmol) and 18-crown-6 ether (0.15 mmol, 40 mg) was added
drybenzene (10 mL). After removing about half of the volume
of the benzene byevaporation the mixture was refluxed for 30
min. The solution of the appropriate 4-(2-bromoethoxy)
benzoic acid ester (1.2 mmol) in drybenzene (5 mL) was
added and heating at reflux was continued for 5–12 h. To the
cooled reaction mixture kieselguhr was added. After filtration,
the solution was concentrated and chromatographed on silica
gel using petroleum ether–ethyl acetate as eluent. The ester
intermediate was hydrolyzed with KOH according to pub-
lished procedures.
21. HPLC analysis of the arachidonic acid: internal standard
3-(4-decyloxyphenyl)propanoic acid (Diczfalusy, E.; Ferno,
O.; Fex, H.; Hogberg, B. Acta Chem. Scand. 1963, 17, 2536);
column: multospher 100 RP18—3 mM (125Â3.0 mm) with
a pre-column multospher 100 RP18—5 mM (20Â3.0 mm) (CS-
chromatographie service, Langerwehe, Germany); injection
volume: 300 mL; mobile phase: acetonitrile/10 mM
(NH4)2HPO4 buffer adjusted to pH 7.4 with ortho-phosphoric
acid (50:50, v/v); flow rate: 0.33 mL/min, detection wavelength
200 nm applying a Waters 2487 UV detector.
AR-C73346XX
0.24
aValues are the means of at least two independent determinations;
errors are within Æ20%; in case of 4: n=5, standard deviation Æ0.1
mM.
in the second meta-position (position 6 of the phenyl
ring) did not have an additive effect, but resulted in a
decrease of inhibition.
In summary, the structural variations performed led to
a reduction of enzyme inhibition in most cases. How-
ever, none of the synthesized compounds was found to
be inactive. Beneficial effects could be obtained with the
introduction of a lipophilic methyl or chloro substituent
or a fluoro moietyin meta-position to the carboxylic
acid moietyof the benzoic acid residue. With an IC 50 of
0.44 mM the fluoro derivative 26 was about twice as
potent as the starting compound 4 and half as potent as
the reference AR-C73346XX (4-{2-oxo-3-[4-(4-phe-
nylbutylthio)phenoxy]propoxy}benzoic acid),23 which
belongs to the most potent cPLA2 inhibitors known
today.
22. Lehr, M. Arch. Pharm. Pharm. Med. Chem. 1996, 329,
386.
References and Notes
23. Mete, A.; Austin, R.; Bennion, C.; Bernstein, M.; Con-
nolly, S.; Jackson, C. G.; King, S.; Lawrence, L.; Lewis, R.;
Robinson, D. H.; Stein, L.; Walters, I.; Withnall, W. J.
Abstract of Papers, 15th EFMC Int. Symp. Med. Chem.,
Edinburgh, 1998; P250.
1. Serhan, C. N.; Haeggstrom, J. Z.; Leslie, C. C. FASEB J.
1996, 10, 1147.
2. Huang, Y. H.; Schafer-Elinder, L.; Wu, R.; Lesson, H. E.;
Frostegard, J. Clin. Exp. Immunol. 1999, 116, 326.