Structure-activity relationship studies on 1-(5-carboxyindol-1-yl)-propan- 2-one inhibitors of human cytosolic phospholipase A2α: Variation of the activated ketone moiety

Article abstract of DOI:10.1016/j.bmcl.2011.01.085

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be potent inhibitors of human cytosolic phospholipase A ₂α (cPLA₂α). In course of structure-activity relationship studies, we investigated the effect of the substitution of the electrophilic ketone group in the middle part of the molecule by other polar residues, such as hydroxyimino, azido, acyloxy, acylamino, urea and carbamate, on enzyme inhibition. With an IC₅₀ of 1.7 μM against cPLA ₂α from human platelets, the 4-fluorophenylcarbamate derivative 23f was the most active of the compounds tested.

Full text of DOI:10.1016/j.bmcl.2011.01.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1773–1776
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship studies on 1-(5-carboxyindol-1-yl)-
propan-2-one inhibitors of human cytosolic phospholipase A2
a:
Variation of the activated ketone moiety
⇑
Martina Kaptur, Alwine Schulze Elfringhoff, Matthias Lehr
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, 48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be
potent inhibitors of human cytosolic phospholipase A2 (cPLA2 ). In course of structure–activity relation-
ship studies, we investigated the effect of the substitution of the electrophilic ketone group in the middle
part of the molecule by other polar residues, such as hydroxyimino, azido, acyloxy, acylamino, urea and
Received 19 October 2010
Revised 18 January 2011
Accepted 18 January 2011
Available online 22 January 2011
a
a
carbamate, on enzyme inhibition. With an IC50 of 1.7
4-fluorophenylcarbamate derivative 23f was the most active of the compounds tested.
Ó 2011 Elsevier Ltd. All rights reserved.
lM against cPLA2a from human platelets, the
Keywords:
Indole
Electrophilic ketone
Carbamate
Cytosolic phospholipase A2
a
Inhibitor
Cytosolic phospholipase A2
a
(cPLA2
a
) is an esterase that selec-
In our group we have found the indole-5-carboxylic acid deriv-
ative 3, structurally related to 2, to be a potent inhibitor of
cPLA2a 13
During structure–activity relationship studies we have
already varied the heterocyclic part as well as the octylphenoxy
moiety of this lead compound.14–18 Here, we report the effect of
a modification of the activated ketone group of 3 positioned in
the middle part of the molecule on inhibitory potency against
tively cleaves the sn-2 position of arachidonoyl-glycerophospho-
lipids of biomembranes to generate free arachidonic acid and
lysophospholipids.1,2 Arachidonic acid in turn is metabolized to a
variety of inflammatory mediators including prostaglandins and
leukotrienes. Lysophospholipids with an alkyl ether moiety at the
sn-1 position can be acetylated to platelet activating factor (PAF),
another mediator of inflammation. Although several more
.
cPLA2a.
phospholipases A2
pre-eminence of cPLA2
strated especially by studies with cPLA2
a
are present in the mammalian organism, the
for lipid mediator generation was demon-
deficient mice. These ani-
The hydroxylamine-substituted carboxylic acid derivatives 7
and 8 were prepared by the route outlined in Scheme 1. The syn-
thesis started from allyl 1-oxiranylmethylindole-5-carboxylate
(3),17 which was reacted with 4-octylphenol in presence of
a
a
mals, which display a reduced eicosanoid production, are resistant
to disease in a variety of models of inflammation.3,4 Therefore,
cPLA2
diseases.4–8
First-generation cPLA2
a is considered as a target for the treatment of inflammatory
a
inhibitors were analogues of arachi-
donic acid with the COOH group replaced by COCF3 (arachidonyl
trifluoromethyl ketone, AACOCF3, 1) or CH2PO(OCH3)F (methyl
arachidonoyl fluorophosphonate, MAFP)5 (Fig. 1). While these
compounds possessed only a medium inhibitory potency, inhibi-
tors of cPLA2a discovered later on, such as thiazolidinediones from
Shionogi,9,10 benzhydrylindoles from Wyeth,11 and propan-2-ones
from AstraZeneca like ARC-70484XX (2)12 showed a high in vitro
activity.
⇑
Corresponding author. Tel.: +49251 83 33331; fax: +49251 83 32144.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
Figure 1. Described inhibitors of cPLA2a.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.085

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M. Kaptur et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1773–1776
Scheme 1. Reagents and conditions: (a) 4-Octylphenol, 4-dimethylaminopyridine, 120 °C, 40 min, 56%; (b) Dess–Martin reagent, CH2Cl2, room temp, 4 h, 86%; (c)
hydroxylamine hydrochloride, pyridine, reflux, 4 h, 96%; (d) Pd(PPh3)4, CH3COOH, THF, room temp, 24 h, 7: 22%; 8: 36%.
odinane reagent. Reaction with hydroxylamine hydrochloride in
pyridine led to a mixture of the E- and Z-isomers of oxime deriva-
tive 6. Cleavage of the allyl ester of 6 was achieved by Pd(0)-
catalyzed allyl transfer. Obtained oxime isomers 7 and 8 were
separated by reversed phase HPLC. Since NOE-experiments were
not successful, the stereochemistry of the two compounds was as-
signed on the basis of their 13C NMR spectra. It has been shown
that in the 13C NMR spectra the resonances of the carbonyl carbon
and both
a-carbons of a ketone all shift upfield on oxime forma-
tion, with the effect for the carbon in syn-position to the hydroxy
group of the oxime being greater than for the anti carbon.19–22 In
the spectrum of compound 7 the signals of the carbons standing
in
a-position to the oxime moiety appear at 66 ppm (phenoxy-C)
and 40 ppm (indolyl-C), respectively, while the chemical shifts of
the corresponding signals of 8 are 61 ppm and 46 ppm. Because
in the spectrum of compound 7 the indolyl-C signal is shifted more
upfield than in the spectrum of 8 (40 vs 46 ppm), this carbon
stands in syn-position to the oxime hydroxy group of 7. Vice versa,
the resonance of the phenoxy-C of 8 is seen at lower ppm values
than in case of compound 7 (61 vs 66 ppm), showing that the hy-
droxy moiety of 8 is syn to the phenoxy-C. The assignment of the
signals of the phenoxy-C and indolyl-C in the 13C NMR spectra of
7 and 8 was made with the help of NOE- and 1H/13C-correlation
spectra (see Supplementary data).
Scheme 2. Reagents and conditions: (a) Methoxylamine hydrochloride, pyridine,
reflux, 4 h, 88%; (b) Pd(PPh3)4, CH3COOH, THF, room temp, 24 h, 83%.
4-dimethylaminopyridine. Oxidation of the resulting alcohol inter-
mediate 4 to the ketone 5 was carried out with Dess–Martin peri-
Scheme 3. Reagents and conditions: (a) 11: acetyl chloride, pyridine, CH2Cl2, 0 °C, 6 h, 90%; 12: benzoyl chloride, pyridine, CH2Cl2, 0 °C, 6 h, 99%; 13: phenylisocyanate,
triethylamine, THF, reflux, 4 h, 66%; (b) Pd(PPh3)4, CH3COOH, THF, room temp, 5–24 h, 25–94%; (c) methanesulfonyl chloride, pyridine, room temp, 4 h, 91%; (d) trimethylsilyl
azide, tetrabutylammonium fluoride, THF, reflux, 4 h, 90%.

M. Kaptur et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1773–1776
1775
The methyloxime-substituted indole-5-carboxylic acid 10 was
synthesized by reaction of allyl ester derivative 5 with methoxyl-
amine hydrochloride followed by Pd(0) catalyzed ester hydrolysis
(Scheme 2). The target compound 10 was afforded as a 1:1 mixture
of the E- and Z-isomers.
The test substances with acetoxy-, benzoyloxy-, phenylcarba-
moyloxy- and methanesulfonyloxy-substituents at the central pro-
pyl part of the molecule (11–13 and 16) were prepared by reaction
of 4 with acetyl chloride, benzoyl chloride, phenyl isocyanate and
methanesulfonyl chloride, respectively, in presence of an amine
base and subsequent ester cleavage by palladium catalysis
(Scheme 3).
For the synthesis of the 2-azidopropyl-substituted indole-5-
carboxylic acid 17, first the methanesulfonyloxy group of com-
pound 14 was converted to an azide with trimethylsilyl azide
and tetrabutylammonium fluoride in THF (Scheme 3). Then the
Scheme 4. Reagents and conditions: (a) Methanesulfonyl chloride, pyridine, room temp, 4 h, 85%; (b) trimethylsilyl azide, tetrabutylammonium fluoride, THF, reflux, 3 h,
69%; (c) H2, Pd/C, methanol, THF, room temp, 2 h, 99%; (d) trifluoroacetic acid, CH2Cl2, room temp, 18 h, 95%; (e) 23a,b: acyl chloride, pyridine, CH2Cl2, 0 °C, 2.5–5 h, 82–84%;
23c: phenylisocyanate, triethylamine, THF, reflux, 3.5 h, 23%; 23d,e,g: substituted chloroformate, ethyl(diisopropyl)amine, CH2Cl2, DMF, room temp, 16–18 h, 57–86%; 23f: 4-
fluorophenyl chloroformate, triethylamine, 4-dimethylaminopyridine, CH2Cl2, DMF, room temp, 14 d, 12%; 23h–l: substituted chloroformate, ethyl(diisopropyl)amine, THF,
room temp, 0.5–1 h, 78–95%; (f) trifluoroacetic acid, CH2Cl2, room temp, 2–24 h, 20–97%.
Table 1
Inhibition of cPLA2a-activity
R
R
N
O
N
O
N
COOH
COOH
C8H17
7,8,10
11-13,16,17,
22, 23a-l
C8H17
a
Compd
R
Inhibition of cPLA2a IC50 (lM)
7
8
10
11
12
13
16
17
OH (E)
OH (Z)
>10 (42%b)
>10 (32%b)
>10 (22%b)
n.a.c
OCH3 (E/Z)
OCOCH3
OCOPhenyl
OCONHPhenyl
OSO2CH3
N3
NH2
NHCOCH3
NHCOPhenyl
>10 (18%b)
n.a.c
>10 (16%b)
4.3
22
>10 (37%b)
n.a.c
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
1 (AACOCF3)
3
n.a.c
NHCONHPhenyl
NHCOOCH3
n.a.c
>10 (21%b)
4.6
NHCOOPhenyl
NHCOO(4-F–Phenyl)
NHCOO(4-Cl–Phenyl)
NHCOO(4-OCH3–Phenyl)
NHCOO(3-F–Phenyl)
NHCOO(2-F–Phenyl)
NHCOO(2-Cl–Phenyl)
NHCOO(2-OCH3–Phenyl)
1.7
>10 (28%b)
>10 (23%b)
5.1
n.a.c
n.a.c
n.a.c
2.3
0.035
a
Values are the means of at least two independent determinations; errors are within 20%.
b
c
Inhibition at 10
lM.
n.a.: not active at 10
l
M.

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M. Kaptur et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1773–1776
allyl ester moiety of obtained intermediate 15 was hydrolyzed as
described above.
methoxy-substituent (23h) reduced activity. Switching the fluoro
atom from 4 into 3 or 2 position of the phenyl residue was less
favorable. While the 3-substituted derivative 23i still was as active
as the unsubstituted compound 23e, the 2-fluoro derivative 23j
had no activity any more at 10 lM. Just so the 2-chloro- and 2-
methoxy compounds 23k and 23l were inactive.
Taken together, all derivatives synthesized were less active than
the lead 3. However, with the carbamates 23e and 23f interesting
new leads have been found. Compounds with such a structural ele-
Scheme 4 outlines the chemical approach used for the prepara-
tion of the amine-, amide-, urea- and carbamate-derivatives 22 and
23a–l. The reaction sequence started from the tert-butyl indole-5-
carboxylate 18.23 The hydroxy group of this compound was con-
verted to an azide on the route described above for the synthesis
of corresponding allyl ester derivative 15 (Scheme 3). Catalytic
hydrogenation of the azide group of obtained compound 20 with
Pd on charcoal led to the amine-substituted compound 21. Reac-
tion of this intermediate with acyl halogenides, phenylisocyanate
and chloroformates, respectively, in presence of an amine base fol-
lowed by hydrolysis of the tert-butyl ester with trifluoroacetic acid
gave the desired target compounds 23a–l. The unsubstituted
amine 22 was afforded directly from 21 after ester cleavage. Be-
cause the syntheses of the target compounds 11–13, 16, 17, 22
and 23a–l were not performed under asymmetric conditions, all
these substances were afforded as racemates (see Supplementary
data).
ment have not been described as cPLA2a inhibitors yet. In further
studies structural modifications of the indole as well as the octyl-
phenyl part of the molecules will be performed in order to increase
activity of the inhibitors.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.085.
Structure–activity relationship studies on the lead compound 3
have revealed that the carboxylic acid moiety in position 5 of the
indole scaffold, the lipophilic residue bound to the phenoxy group
of the molecule, and especially the activated ketone in the central
part of the molecule are important for the high activity of the com-
References and notes
1. Kita, Y.; Ohto, T.; Uozumi, N.; Shimizu, T. Biochim. Biophys. Acta 2006, 1761,
1317.
2. Ghosh, M.; Tucker, D. E.; Burchett, S. A.; Leslie, C. C. Prog. Lipid. Res. 2006, 45,
487.
3. Sapirstein, A.; Bonventre, J. V. Biochim. Biophys. Acta 2000, 1488, 139.
4. Bonventre, J. Trends Immunol. 2004, 25, 116.
pound. On binding to cPLA2a, the electrophilic ketone moiety of 3
is supposed to form covalent binding interactions with a serine of
the active site of the enzyme.12,13 Metabolic reduction of this ke-
tone group to a secondary alcohol leads to a loss of activity.24
Experiments with rat liver microsomes have shown that such a
reduction does not occur to a high extent as far as the molecule
bears a long lipophilic residue.17,18 However, when this lipophilic
area of the molecule is reduced in order to make the compound
more drug like, the extent of metabolic keto reduction increases
significantly. Therefore, we investigated now, whether the meta-
bolically unstable activated ketone group can be replaced by other
polar groups, which are also able to interact with the serine of the
active site either by dipol–dipol interactions and formation of
H-bonds or by a covalent binding mode.
5. Connolly, S.; Robinson, D. H. Expert Opin. Ther. Patents 1995, 5, 673.
6. Clark, J. D.; Tam, S. Expert Opin. Ther. Patents 2004, 14, 937.
7. Lehr, M. Anti-Inflamm. Anti-Allergy Agents Med. Chem. 2006, 5, 149.
8. Magrioti, V.; Kokotos, G. Expert Opin. Ther. Patents 2010, 20, 1.
9. Seno, K.; Okuno, T.; Nishi, K.; Murakami, Y.; Watanabe, F.; Matsuura, T.; Wada,
M.; Fujii, Y.; Yamada, M.; Ogawa, T.; Okada, T.; Hashizume, H.; Kii, M.; Hara, S.;
Hagishita, S.; Nakamoto, S.; Yamada, K.; Chikazawa, Y.; Ueno, M.; Teshirogi, I.;
Ono, T.; Ohtani, M. J. Med. Chem. 2000, 43, 1041.
10. Seno, K.; Okuno, T.; Nishi, K.; Murakami, Y.; Yamada, K.; Nakamoto, S.; Ono, T.
Bioorg. Med. Chem. Lett. 2001, 11, 587.
11. McKew, J. C.; Foley, M. A.; Thakker, P.; Behnke, M. L.; Lovering, F. E.; Sum, F. W.;
Tam, S.; Wu, K.; Shen, M. W.; Zhang, W.; Gonzalez, M.; Liu, S.; Mahadevan, A.;
Sard, H.; Khor, S. P.; Clark, J. D. J. Med. Chem. 2006, 49, 135.
12. Connolly, S.; Bennion, C.; Botterell, S.; Croshaw, P. J.; Hallam, C.; Hardy, K.;
Hartopp, P.; Jackson, C. G.; King, S. J.; Lawrence, L.; Mete, A.; Murray, D.;
Robinson, D. H.; Smith, G. M.; Stein, L.; Walters, I.; Wells, E.; Withnall, W. J. J.
Med. Chem. 2002, 45, 1348.
Evaluation of the inhibitory activity against cPLA2a
25,26 showed
13. Ludwig, J.; Bovens, S.; Brauch, C.; Schulze Elfringhoff, A.; Lehr, M. J. Med. Chem.
2006, 49, 2611.
that the exchange of the ketone group by an oxime resulted in a
14. Hess, M.; Schulze Elfringhoff, A.; Lehr, M. Bioorg. Med. Chem. 2007, 15, 2883.
15. Bovens, S.; Kaptur, M.; Schulze Elfringhoff, A.; Lehr, M. Bioorg. Med. Chem. Lett.
2009, 19, 2107.
16. Forster, L.; Ludwig, J.; Kaptur, M.; Bovens, S.; Schulze Elfringhoff, A.;
Holtfrerich, A.; Lehr, M. Bioorg. Med. Chem. 2010, 18, 945.
17. Drews, A.; Bovens, S.; Roebrock, K.; Sunderkötter, C.; Reinhardt, D.; Schäfers,
M.; van der Velde, A.; Schulze Elfringhoff, A.; Fabian, J.; Lehr, M. J. Med. Chem.
2010, 53, 5165.
18. Bovens, S.; Schulze Elfringhoff, A.; Kaptur, M.; Reinhardt, D.; Schäfers, M.; Lehr,
M. J. Med. Chem. 2010, 53, 8298.
19. Levy, G. C.; Nelson, G. L. J. Am. Chem. Soc. 1972, 94, 4897.
20. Hawkes, G. E.; Herwig, K.; Roberts, J. D. J. Org. Chem. 1974, 39, 1017.
21. Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic
Compounds; John Wiley & Sons: New York, 1998.
drastic drop of activity. While the lead 3 inhibited the enzyme with
an IC50 of 0.035
lay above 10 M (Table 1). The E/Z-mixture of the methyloxime 10
also inhibited the enzyme to less than 50% at 10 M. At the same
lM, the IC50s of both oxime isomers 7 (E) and 8 (Z)
l
l
concentration level the acetoxy-, benzoyloxy-, phenylcarbamoyl-
oxy-, and methanesulfonyloxy-derivatives 11, 12, 13 and 16
showed no or only a marginal activity.
The first compound obtained in this series with an IC50 less than
10 lM was the azide 17. Its inhibitory potency lay in the same or-
der of magnitude as that of the reference AACOCF3 (1). Conversion
of the azide group to an amine led to a decrease of activity. Com-
pound 22 only showed an inhibition of 37% at 10 lM.
22. Kitamura, M.; Yoshida, M.; Kikuchi, T.; Narasaka, K. Synthesis 2003, 2415.
23. Lehr, M.; Ludwig, J. PCT WO2004069797, 2004.
Next several derivatives of this amine were synthesized and
tested. Acetylation (23a), benzoylation (23b) and conversion into
24. Fabian, J.; Lehr, M. J. Pharm. Biomed. Anal. 2007, 43, 601.
25. Inhibition of cPLA2
cPLA2
isolated from human platelets.26 1-Stearoyl-2-arachidonoyl-sn-
glycero-3-phosphocholine (200 M) sonicated with 1,2-dioleoyl-sn-glycerol
(100 M) in a bath sonicator at 30–35 °C was used as substrate. Enzyme
a: The target compounds were evaluated in an assay applying
a
a phenyl urea (23c) resulted in a loss of activity at 10
trast, the O-methyl carbamate 23d was slightly active at this con-
centration, and with an IC50 of 4.6 M the O-phenyl carbamate 23e
lM. In con-
l
l
reaction was terminated after 60 min by addition of a mixture of acetonitrile,
methanol and 0.1 M aqueous EDTA–Na2 solution, which contained 4-
undecyloxybenzoic acid as internal standard and nordihydroguaiaretic acid
(NDGA) as oxygen scavenger. Released product arachidonic acid was
determined with reversed phase HPLC and UV-detection at 200 nm after
cleaning up the samples with solid phase extraction. Inhibition of cPLA2
activity was calculated by comparing the arachidonic acid formed by the
enzyme in absence and presence of a test compound.
l
even possessed a considerable inhibitory potency. Like activated
ketone compounds, phenyl carbamates are known to act as serine
traps.27,28 They can form covalent bonds via the nucleophilic attack
of the hydroxyl of a serine residue of a protein, which leads to the
displacement of the leaving group phenol. Finally, this leaving
group was modified by introduction of fluoro-, chloro- and meth-
oxy-substituents. A fluoro atom in position 4 of the phenyl ring in-
a
26. Schmitt, M.; Lehr, M. J. Pharm. Biomed. Anal. 2004, 35, 135.
27. Tarzia, G.; Duranti, A.; Tontini, A.; Piersanti, G.; Mor, M.; Rivara, S.; Plazzi, P. V.;
Park, C.; Kathuria, S.; Piomelli, D. J. Med. Chem. 2003, 46, 2352.
28. Lodola, A.; Mor, M.; Rivara, S.; Christov, C.; Tarzia, G.; Piomelli, D.; Mulholland,
A. J. Chem. Commun. 2008, 14, 214.
creased activity about two- to threefold (IC50 of 23f: 1.7
lM), while
the introduction of a lipophilic 4-chloro- (23g) as well as a polar 4-