Structural optimization and biological evaluation of 2-substituted 5-hydroxyindole-3-carboxylates as potent inhibitors of human 5-lipoxygenase

Article abstract of DOI:10.1021/jm900212y

Pharmacological suppression of leukotriene biosynthesis by inhibitors of 5-lipoxygenase (5-LO) is a strategy to intervene with inflammatory and allergic disorders. We recently presented 2-amino-5-hydroxy-1H-indoles as efficient 5-LO inhibitors in cell-based and cell-free assays. Structural optimization led to novel benzo[g]indole-3-carboxylates exemplified by ethyl 2-(3-chlorobenzyl)-5- hydroxy-1H-benzo[g]indole-3-carboxylate (compound 11a), which inhibits 5-LO activity in human neutrophils and recombinant human 5-LO with IC₅₀ values of 0.23 and 0.086 μM, respectively. Notably, 11a efficiently blocks 5-LO product formation in human whole blood assays (IC₅₀ = 0.83-1.6 μM) and significantly prevented leukotriene B₄ production in pleural exudates of carrageenan-treated rats, associated with reduced severity of pleurisy. Together, on the basis of their high potency against 5-LO and the marked efficacy in biological systems, these novel and straightforward benzo[g]indole-3-carboxylates may have potential as anti-inflammatory therapeutics.

Full text of DOI:10.1021/jm900212y

3474
J. Med. Chem. 2009, 52, 3474–3483
Structural Optimization and Biological Evaluation of 2-Substituted
5-Hydroxyindole-3-carboxylates as Potent Inhibitors of Human 5-Lipoxygenase
Eva-Maria Karg,† Susann Luderer,‡ Carlo Pergola,‡ Ulrike Bu¨hring,‡ Antonietta Rossi,§ Hinnak Northoff,| Lidia Sautebin,
Reinhard Troschu¨tz,† and Oliver Werz*,‡
Department of Chemistry and Pharmacy, Chair of Pharmaceutical Chemistry, Friedrich Alexander UniVersity Erlangen, Schuhstrasse 19,
D-91052 Erlangen, Germany, Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-UniVersity Tuebingen, Auf
der Morgenstelle 8, 72076 Tuebingen, Germany, IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy, Institute for Clinical and
Experimental Transfusion Medicine, UniVersity Medical Center Tuebingen, Hoppe-Seyler-Strasse 3, 72076 Tuebingen, Germany, Department of
Experimental Pharmacology, UniVersity of Naples Federico II, Naples, Italy
ReceiVed October 16, 2008
Pharmacological suppression of leukotriene biosynthesis by inhibitors of 5-lipoxygenase (5-LO) is a strategy
to intervene with inflammatory and allergic disorders. We recently presented 2-amino-5-hydroxy-1H-indoles
as efficient 5-LO inhibitors in cell-based and cell-free assays. Structural optimization led to novel
benzo[g]indole-3-carboxylates exemplified by ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-
carboxylate (compound 11a), which inhibits 5-LO activity in human neutrophils and recombinant human
5-LO with IC50 values of 0.23 and 0.086 µM, respectively. Notably, 11a efficiently blocks 5-LO product
formation in human whole blood assays (IC50 ) 0.83-1.6 µM) and significantly prevented leukotriene B4
production in pleural exudates of carrageenan-treated rats, associated with reduced severity of pleurisy.
Together, on the basis of their high potency against 5-LO and the marked efficacy in biological systems,
these novel and straightforward benzo[g]indole-3-carboxylates may have potential as anti-inflammatory
therapeutics.
Introduction
The development of selective and potent inhibitors of LT
biosynthesis for therapeutic application is a major challenge.
Many synthetic compounds and natural products have been
described as potent 5-LO inhibitors in cell-free or in cellular
assays but often markedly lost efficacy in whole blood.4,6
Moreover, most of them failed in animal studies or human
clinical trials due to lack of efficacy or due to toxic side effects.
Thus, despite intensive research, only compound 39 (zileuton,
1-(1-benzothiophen-2-ylethyl)-1-hydroxy-urea), an iron ligand-
type 5-LO inhibitor with IC50 values of 0.5-1 µM in intact
cells and whole blood,7 could enter the market in 1996 as an
antiasthmatic drug. It was presumed that, based on the poor
efficacy of LT synthesis inhibitors, pharmacological intervention
with LTs is of minor therapeutic value. However, LT antagonists
are successfully used in asthma therapy and data from 5-LO
deficient mice clearly show proof for a strong pathophysiological
role of 5-LO products in several diseases.3 Thus, the improve-
ment of the pharmacological properties of 5-LO or FLAP
inhibitors in terms of efficacy and safety is seemingly a major
challenge and requires new leads with novel modes of molecular
action.
Leukotrienes (LTsa) play established roles in the pathophysi-
ology of inflammatory and allergic disorders (i.e., asthma and
allergic rhinitis) but may also promote cancer and atheroscle-
rosis.1 LTs are formed from arachidonic acid (AA) by the initial
introduction of molecular oxygen at C5, catalyzed by the
5-lipoxygenase (5-LO) enzyme. 5-LO is a nonheme iron-
containing dioxygenase where the iron cycles between the
inactive ferrous and the active ferric state.2 Pharmacological
interference with 5-LO is one strategy in the intervention with
LT-mediated diseases.3 There are at least four classes of LT
synthesis inhibitors. Three of them are direct inhibitors of 5-LO
that act by either reducing the active-site iron or uncoupling
the redox cycle of the iron, those that chelate the iron, and finally
compounds that act by nonredox mechanisms, presumably by
competition with AA as substrate and/or with regulatory lipid
hydroperoxides.4 Besides direct 5-LO inhibitors, compounds that
block the functional interaction between 5-LO and the 5-LO-
activating protein (FLAP) are well characterized.5 Such FLAP
inhibitors block LT biosynthesis only in intact cells but do not
directly interfere with 5-LO catalytic activity itself.4
We have recently discovered 2-amino-5-hydroxyindole esters
as novel direct 5-LO inhibitors with unknown mode of 5-LO
inhibition.8 These novel structures inhibited 5-LO product
synthesis in cell-based or cell-free assays with IC50 values of
2.4 and 0.3 µM, respectively,8 and thus seem to be promising
drug candidates. Here, we explored this class of compounds in
order to optimize the structural features and to discover more
potent derivatives with high efficacy in biological test systems.
The annelation of a [g]benzene ring to the indole and replace-
ment of the 2-amino moiety by a C1 to C3 alkyl chain essentially
led to drug-like compounds that have a more than 10-fold higher
potency than the parental compound ethyl 2-[(3-chlorophenyl)-
amino]-5-hydroxy-1H-indole-3-carboxylate 37 and efficiently
* To whom correspondence should be addressed. Phone: ++49-7071-
2978793. Fax: ++49-7071-294565. E-mail: oliver.werz@uni-tuebingen.de.
†
Department of Chemistry and Pharmacy, Chair of Pharmaceutical
Chemistry, Friedrich Alexander University Erlangen.
‡
Department of Pharmaceutical Analytics, Pharmaceutical Institute,
Eberhard-Karls-University Tuebingen.
§
IRCCS Centro Neurolesi “Bonino-Pulejo”.
Institute for Clinical and Experimental Transfusion Medicine, University
|
Medical Center Tuebingen.
Department of Experimental Pharmacology, University of Naples
Federico II.
a Abbreviations: AA, arachidonic acid; FLAP, 5-lipoxygenase-activating
protein; fMLP, N-formyl-methionyl-leucyl-phenylalanine; 5-HETE, 5(S)-
hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid; LPS, lipopolysac-
charide; 5-LO, 5-lipoxygenase; LT, leukotriene.
10.1021/jm900212y CCC: $40.75
2009 American Chemical Society
Published on Web 05/07/2009

2-Substituted Indole-3-carboxylates Inhibit 5-Lipoxygenase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3475
inhibited LT biosynthesis also in human whole blood and in
pleural exudates of carrageenan-treated rats.
with methyliodide (31) in the presence of NaOH resulted in
the doubly methylated compound 32.21 Finally, we synthesized
the 5-phenyl substituted compound 36 via triflation of 11a and
subsequent Suzuki-Miyaura coupling of the generated triflate
34 with boronate 35 (Scheme 5).22
Results and Discussion
Chemistry. The Nenitzescu reaction9 has proven to be the
simplest entry into 5-hydroxyindoles. Essentially, stirring of the
nucleophilic enaminoester 7 with 1.2 equiv of 1,4-quinone in
ethanol at room temperature (RT) led to the expected 5-hy-
droxyindole derivatives (method a). We converted both 1,4-
benzoquinone (8) and 1,4-naphthoquinone (9) with a variety of
different enaminoesters 7 and the ketene aminal 13 successfully
into the desired 5-hydroxyindoles 10 or the 5-hydroxybenzo[g-
]indoles 11 and 14 (method a). Nevertheless, some of the title
compounds synthesized in this manner were difficult to isolate
due to contamination of numerous byproducts. Moreover,
method a failed when using 6,7-dimethoxy-1,4-naphthoquinone
(15), quinoline-5,8-dione (16), or the 2-aryl-1,4-benzoquinones
17 and 18 as reactants from enaminoester 7a. Due to the fact
that Lewis acid-promoted Nenitzescu reactions mostly provide
better yields, we also used ZnI2 as catalyst in a wide range of
reactions (method b).10 Moreover, the desired Nenitzescu
products of the reported 1,4-quinones 15-1811-14 could be
successfully synthesized according to method b.
Nenitzescu reactants 7 were prepared starting from carboxylic
acids 1, which were first activated to the appropriate imida-
zolides and then treated with the magnesium salt of 3-ethoxy-
3-oxopropanoic acid 3a or 3-benzyloxy-3-oxopropanoic acid 3b,
respectively. Subsequent acidic workup led both to the reported
(4a-i and k-p) and the new ꢀ-ketoesters 4j,q,r.15 ꢀ-ketoesters
4 were converted into their appropriate enaminoesters 7 by
refluxing them in toluene with an excess of ammonium acetate
(5) or with amine 6p, respectively.16 Unfortunately, the hitherto
unknown secondary enaminoester 7p could not be separated
from the remaining reactant and was thus employed as crude
product for the subsequent Nenitzescu reaction. Eight of the
enaminoesters 7 were treated with 1,4-benzoquinone (8) to form
the 5-hydroxy-1H-indole-3-carboxylates 10a-c, k-n, and s,
and 18 of them were converted into 5-hydroxy-1H-benzo[g]in-
dole-3-carboxylates 11 by using 1,4-naphthoquinone (9) as
reactant. A large part of indole-3-carboxylates 10 was still
synthesized according to method a, and preparation of the 11
series was essentially carried out under Lewis acid promotion
(method b) (Scheme 1).
Structure-Activity Relationships Regarding Inhibi-
tion of 5-Lipoxygenase. Analysis of test compounds as 5-LO
inhibitors was routinely carried out in two different test systems,
that is, a cell-based assay using human neutrophils challenged
by Ca2+ ionophore 5-methylamino-2-[[(2S,3R,5R,8S,9S)-3,5,9-
trimethyl-2-[(2S)-1-oxo-1-(1H-pyrrol-2-yl)propan-2-yl]-1,7-
dioxaspiro[5.5]undecan-8-yl]methyl]-1,3-benzooxazole-4-car-
boxylic acid (A23187) plus 20 µM exogenous AA as well as a
cell-free assay using human recombinant 5-LO enzyme in high-
speed (40000g) supernatants (S40) from lysates of transformed
Escherichia coli. The 5-LO inhibitors 38 (N-(3-phenoxycin-
namyl)-acetohydroxamic acid, BWA4C, routinely used as
reference compound23) and 397 suppressed 5-LO product
formation in the cell-based assay with IC50 values of 0.16 (
0.02 and 0.9 ( 0.1 µM, respectively, and in the cell-free assay
with 0.038 ( 0.04 and 0.8 ( 0.2 µM, respectively.
5-Hydroxyindoles were shown to possess antioxidant activi-
ties.24 Therefore, the 2-amino-5-hydroxy-1H-indole-3-carboxy-
lates could act as redox-active 5-LO inhibitors by interfering
with the redox cycle of the active-site iron of the 5-LO enzyme.
To determine the requirement of the 5-hydroxy moiety as a part
of the reducing structure, we analyzed several derivatives of
the lead compound 37 devoid of the 5-OH group and, thus,
lacking reducing properties (Table 1). The lead compound 37
potently inhibited 5-LO product synthesis with IC50 values of
2.4 ( 0.4 and 0.3 ( 0.09 µM in cell-based and cell-free assays,
respectively.8 In direct comparison to 37, the corresponding
analogue without the 5-hydroxy moiety (27a) is approximately
3-fold and about 27-fold less potent in intact cells and cell-free
assays, respectively. Hence, the strong impact of the 5-hydroxy
moiety on the potency, noticeable in cell-free assays, is only
moderate in intact cells. Repositioning of the chlorine in ortho
(27b) or para (27c) or variation of the halogen substituent
(fluorine (27d), bromine (27e)) at the 2-(N)-aniline ring caused
no substantial change in the potency versus 27a, yielding
compounds with IC50 values in the range of 3.5 ( 1.2 to 9.8 (
1.9 µM for intact cells and 8.3 ( 1.4 to 13.4 ( 2.9 µM for
cell-free assays. The replacement of the 5-hydroxy residue by
chlorine (28) impaired the efficacy about 12-fold versus 37 in
cell-free assays but only about 2-fold in intact cells. Finally,
exchange of the aniline moiety by a benzylamino group (27f),
did not significantly alter the efficacy versus 27a. As found
before8 replacement of the N-aryl group in 2-position by one
allyl moiety (27g) abrogated 5-LO inhibition, but introduction
of two N-allyl residues (27h), which resembles a phenyl ring,
led to similar potencies as obtained for 27a.
Benzo[g]indole derivative 14 was also synthesized according
method b. Ketene aminal 1317 was prepared from (E)-ethyl
3-amino-3-ethoxyacrylate (12) and 3-chloroaniline (6a) (Scheme
2).18
Using different 1,4-quinones for the Nenitzescu reaction, we
were able to introduce various substituents in positions 6 and 7
of indole-3-carboxylate 10a. Enaminoester 7a was allowed to
react with four additional 1,4-quinones 15-18 to give the
5-hydroxyindol-3-carboxylates 19-22 in moderate to good
yields (method b) (scheme 3).
Because the 5-hydroxy moiety increases the potency, we
continued with the 5-hydroxy-indole backbone in order to obtain
efficient 5-LO inhibitors. In our previous study, we found that
the nature of the substituent in 2-position of the indole
determines the efficacy. Thus, arylamine, arylethylamine, or a
4-arylpiperazin-1-yl group as well as a diallyl-amino residue
in the 2-position of indole-3-carboxylates led to active com-
pounds, whereas nonaromatic amine functions including primary
amine, dimethylamine, pyrolidine, piperidine, or morpholine
failed in this respect.8 Moreover, chlorine substitution of the
2-(N)-aniline residue in 3′-position (e.g., in 37) appeared
advantageous.
To eliminate redox activity,8 we prepared the alkyl indole-
3-carboxylates 27a-h and 28, which lack the 5-hydroxy group
in contrast to the mentioned Nenitzescu products. All derivatives
27 and compound 28 could be synthesized in analogy to
Bergman et al.19 starting from alkyl indole-3-carboxylates 23a
or 23b and the aryl-/arylalkyl or (di)allylamines 6a-h (Scheme
4).
Further reactions were carried out starting from ethyl 2-(3-
chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (11a)
shown in Scheme 5: Acylation of 11a with benzoylchloride (29)
in an alkaline medium led to compound 30.20 Refluxing 11a

3476 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Karg et al.
Scheme 1. Synthesis of Different 5-Hydroxy-1H-indole-3-carboxylates (10) and 5-Hydroxy-1H-benzo[g]indole-3-carboxylates (11)a
a Reagents and conditions: (a) N,N′-carbonyldiimidazole (2), DMF, RT; 1 h; (b) Mg-salt of 3-ethoxy-3-oxopropanoic acid (3a) or 3-benzyloxy-3-oxopropanoic
acid (3b), RT; 4 h; (d) NH4OAc (5), HOAc, toluene, reflux, 6 h; (e) benzylamine (6f), HCOOH, toluene, reflux, 6 h; (f) 1,4-benzoquinone (8), EtOH, reflux,
1 h; (g) 1,4-benzoquinone (8), CH2Cl2, ZnI2, reflux, 40 min; (h) 1,4-naphthoquinone (9), EtOH, reflux, 1 h; (i) 1,4-naphthoquinone (9), CH2Cl2, ZnI2, reflux,
40 min. All compounds 10 were synthesized via (f), 10a,c and k-n additionally via (g); all compounds of 11 (except of 11o) were synthesized via (i),
11a,c,k-m,o, and q additionally or only via (h).
Scheme 2. Synthesis of Ethyl 2-(3-Chlorophenylamino)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (14)a
a Reagents and conditions: (a) EtOH, 3-chloroaniline (6a), reflux, 48 h; (b) 1,4-naphthoquinone (9), CH2Cl2, ZnI2, reflux, 40 min.
We studied whether nitrogen in the 2-position of the indole
is actually a prerequisite for 5-LO inhibition. Compound 10a,
representing the methylene derivative of the parental com-
pound 37, was slightly more potent in neutrophils but more
than 6-fold less active in cell-free assays (Table 2), implying
that nitrogen in 2-position is actually not an absolute
requirement but may govern the 5-LO inhibitory potency, at
least in the cell-free assay. Repositioning of the chlorine in
ortho (10b) did not alter the efficacy versus 10a, whereas
chlorine in para position (10c) increased the potency in intact
cells about 2-fold. Moreover, deletion of the nitrogen bridge
of 37 yielding the 2-(3-chlorophenyl)- or 2-(4-chlorophenyl)-
indole derivatives 10k and 10l, respectively, led to active
compounds with a moderate change of potency in neutrophils
but markedly lower potency in the cell-free assay as compared
to compound 37. Finally, elongation to 1-(3-chlorophenyl)-
ethyl (10m), 1-(4-chlorophenyl)ethyl (10n), or 1-phenylethyl
(10s), essentially retained activity without substantial loss
of potency versus corresponding phenyl- or benzyl-analogues
lacking nitrogen in the 2-position.

2-Substituted Indole-3-carboxylates Inhibit 5-Lipoxygenase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3477
Scheme 3. Synthesis of the 5-Hydroxy-1H-indole-3-carboxylates 19-22a
a Reagents and conditions: 1,4-quinones 15-18, CH2Cl2, ZnI2, reflux, 40 min.
Scheme 4. Synthesis of the 2-Substituted Indole-3-carboxylates 27a-h and 28a
a Reagents and conditions: (a) N,N′-dimethylpiperazine (24), N-chlorosuccinimide (25), CH2Cl2, 0 °C, 2 h; (b) trichloroacetic acid (26), amines 6a-h,
RT, 2 h.
Scheme 5. Modifications of Ethyl 2-(3-Chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (11a)a
a Reagents and conditions: (a) 11a f 30: triethylamine, EtOAc, benzoylchloride (29), RT, 20 h. (b) 11a f 32: NaOH, methyliodide (31), THF, reflux,
10 h. (c) 11a f 36 (i) 11d f triflate 34: Tf2O (33), CH2Cl2, pyridine, 0 °C, 4 h; (ii) 34 f 36: Pd(OAc)2, PPh3, DMF, phenylboronic acid pinacol ester (35),
aq Na2CO3, reflux, 2 h.
To obtain more potent 5-LO inhibitors, further structural
variations based on the structure of the lead 37 were carried
out. Another possibility for structural variation is given by
annelation of benzene to the indole, yielding corresponding
benzo[g]indole derivatives. Benzene annelation enlarges the
hydrophobic core structure and increases the overall lipophi-
licity, a general determinant for potent 5-LO inhibitors.25
Compound 14, the benzo[g]indole analogue to 37, is a potent
inhibitor of 5-LO with IC50 values of 0.71 ( 0.25 µM (intact
neutrophils) and 0.24 ( 0.06 µM (cell-free 5-LO) being superior
over 37, particularly in intact cells (Table 3). Interestingly,
exchange of the nitrogen in 2-position of 14 by methylene,

3478 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Karg et al.
Table 1. Inhibition of 5-LO Activity in Intact Neutrophils and in a
respectively) versus the parental compound 11a (IC50 ) 0.086
( 0.02 µM). In contrast, the potency for inhibition of 5-LO
activity in intact neutrophils was slightly reduced versus 11a.
Repositioning of the chlorine into para regardless of the distance
of the aryl residue from the indole (compound 11l and 11n)
did not significantly improve the potency versus 11a. Substitu-
tion of the indole nitrogen of 11c by a benzyl moiety (yielding
11p) moderately reduced the potency. The corresponding benzyl
esters 11q and 11r of compounds 11a and 11c, respectively,
were similarly active as the ethyl esters, which indicates that
also voluminous esters are tolerated.
Cell-Free Assaya
Additional structural alterations of 11a showed that annelation
of a 2,3-dimethoxybenzene moiety (19) to the indole, instead
of benzene, is clearly detrimental. On the other hand, when a
pyridine ring was annelated to the indole (20) or when a phenyl
moiety (21) or a biphenyl (22) was substituted in 7-position,
such derivatives still blocked 5-LO activity with IC50 values
2.8 ( 0.7 to 5.7 ( 0.1 µM in intact cells and 0.33 ( 0.12 to
1.2 ( 0.2 µM in cell-free assays (Table 4). Finally, esterification
of the 5-hydroxy group of 11a with benzoic acid (leading to
30) strongly reduced the potency. Also methylation of 11a at
the 5-hydroxy moiety and of the indole nitrogen (yielding 32)
or replacement of the 5-hydroxy group by phenyl (yielding 36)
led to inactive compounds. Taken together, several variations
starting from 37 with IC50 values of 2.4 ( 0.4 (cell-based) and
0.3 ( 0.09 µM (cell-free) led to the novel benzo[g]indoles
lacking the 2-amino functionality with IC50 values of 0.23 (
0.07 (11a) in cell-based and 0.031 ( 0.01 µM (11m) in cell-
free assays, respectively.
a The IC50 values are given as mean ( SE of n ) 3-4 determinations.
Table 2. Inhibition of 5-LO Activity in Intact Neutrophils and in a
Cell-Free Assaya
Studies on the Mode of Action of Benzo[g]indole-3-
carboxylates as 5-LO Product Synthesis Inhibitors. Indole-
based structures such as 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-
5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886)
suppress 5-LO product synthesis via inhibition of the AA-
binding protein FLAP, which facilitates substrate supply toward
5-LO at the perinuclear region but does not inhibit 5-LO
directly.5 A direct interference of benzo[g]indole-3-carboxylates
with 5-LO is evident based on the inhibition of the 5-LO enzyme
in the cell-free assay. We addressed whether 11a (the most
potent derivative in intact neutrophils) shares also mechanistic
properties with FLAP inhibitors. Typically, excessive supply
of AA impairs the efficacy of FLAP inhibitors, and FLAP
inhibitors block translocation of 5-LO from the cytosol to the
perinuclear region in neutrophils. In contrast to MK-886, 11a
failed to block A23187-induced translocation of 5-LO in
neutrophils (Figure 1A). Moreover, in contrast to MK-886,26
no discrepancy in the efficacy of 11a was obvious in neutrophils
forming 5-LO products from relatively low amounts of endog-
enous AA as compared to conditions where ample exogenous
AA (20 or 50 µM) was supplemented (Figure 1B). The lack of
such a discrepancy in the efficacy of 11a in intact neutrophils
also might suggest that the compound does not act in a substrate
competitive manner. Indeed, lowering (5 µM) or increasing (50
µM) the substrate concentration did also not significantly affect
the inhibiton of 5-LO activity by 11a in the cell-free assay
(Figure 2A). Finally, wash-out experiments shown in Figure
2B support a reversible inhibition of cell-free 5-LO activity by
11a, comparable to compound 39. Together, we conclude that
the benzo[g]indole-3-carboxylates, exemplified by 11a, act as
direct and reversible inhibitors of 5-LO, and despite some
structural similarities (indole) with FLAP inhibitors, these
compounds act independently of FLAP.
a The IC50 values are given as mean ( SE of n ) 3-4 determinations.
leading to compound 11a, further increased the potency over
37 in intact cells about 10-fold (IC50 ) 0.23 ( 0.07) and in the
cell-free assay more than 3-fold (IC50 ) 0.086 ( 0.02 µM),
respectively. Therefore, we continued with compounds devoid
of the nitrogen in 2-position of the indole. Variation of the
positioning of the chlorine in the benzyl ring to the ortho
(compound 11b) or para (compound 11c) positions as well as
exchange of chlorine in 11a by fluorine (11d), bromine (11f),
or a methoxy moiety (11h) was essentially tolerated in cell-
free assays, with a small loss of potency in intact cells for 11b
and 11c. Also, substitution in the para position with fluorine
(11e), bromine (11g), or a methoxy moiety (11i) caused no
marked change of the potencies, whereas a trifluoromethyl group
(11j) was rather detrimental.
Next, the effect of the distance between the phenyl ring in
2-position and the indole was evaluated. Both direct connection
of the 3-chlorophenyl residue (11k) but also insertion of an
ethylene bridge (11m) improved the potency for inhibition of
cell-free 5-LO (IC50 ) 0.045 ( 0.01 and 0.031 ( 0.01 µM,

2-Substituted Indole-3-carboxylates Inhibit 5-Lipoxygenase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3479
Table 3. Inhibition of 5-LO Activity in Intact Neutrophils and in a Cell-Free Assaya
a The IC50 values are given as mean ( SE of n ) 3-4 determinations.
Table 4. Inhibition of 5-LO Activity in Intact Neutrophils and in a Cell-Free Assaya
a The IC50 values are given as mean ( SE of n ) 3-4 determinations.
Analysis of Benzo[g]indole-3-carboxylates in Biologi-
cal and Pharmacological Relevant Test Systems. Cell-free
assays and test systems based on isolated cells often neglect
important parameters (i.e., albumin-binding, regulatory plasma
components) that influence the bioactivity of test compounds
in vivo, and inclusion of some of these variables might better

3480 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Karg et al.
Figure 1. Effects of compound 11a on 5-LO product synthesis and 5-LO translocation in human neutrophils. Isolated human neutrophils were
preincubated with the test compounds (or DMSO as vehicle) for 15 min at 37 °C. (A) Assessment of 5-LO subcellular localization. Neutrophils
were stimulated with 2.5 µM A23187 for 5 min at 37 °C, lysed by mild detergent (0.1% NP-40), and nuclear (nuc) and non-nuclear (non-n)
fractions were separated. 5-LO protein was analyzed by Western Blot. Data are representatives for three independent experiments. (B) Assessment
of 5-LO product synthesis. A23187 (2.5 µM) was added without (-) or with (+) 20 or 50 µM AA, as indicated, and after 10 min at 37 °C, 5-LO
products were analyzed. Data are means ( SE; n ) 3-4.
Figure 2. Effects of the substrate concentration on 5-LO inhibition by compound 11a and wash-out experiments. (A) Aliquots of the supernatants
of E. coli lysates were diluted in 1 mL of PBS, pH 7.4, and 1 mM EDTA, and preincubated with the test compounds for 10 min at 4 °C. Samples
were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 and AA (5, 20, or 50 µM) was added to start the 5-LO reaction. After 10 min, 5-LO products
were analyzed. (B) Aliquots of the supernatants were incubated with 0.05 or 0.5 µM 11a, 1 or 0.1 µM 39, or vehicle (DMSO) for 10 min at RT.
Then, one aliquot of the sample containing 0.5 µM 11a and 1 µM 39 was diluted with assay buffer 10-fold (0.05 (0.5) µM 11a and 0.1 (1) µM 39,
respectively), whereas the other aliquot was not altered. Samples were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 and 20 µM AA was added
to start the 5-LO reaction. After 10 min, 5-LO products were analyzed. Data are means ( SE; n ) 3-4.
resemble the in vivo situation and anticipate in vivo outcomes.4,6
To estimate the ability to inhibit 5-LO product formation in
vivo in humans, compounds 11a and 11l were analyzed in a
human whole blood assay using A23187 (30 µM) as stimulus.
The formation of LTB4 and of 5-HETE was suppressed by 11a
in a concentration-dependent manner with IC50 ) 1.6 ( 0.3
µM (Figure 3A) and also 11l blocked 5-LO product formation
efficiently (IC50 ) 1.3 ( 0.15 µM). Next, the efficacy of
compound 11a was assessed in human whole blood primed with
lipopolysaccharide (LPS) and then challenged with N-formyl-
methionyl-leucyl-phenylalanine (fMLP). In contrast to activation
by A23187, stimulation of blood with fMLP following LPS-
priming is considered to closely mimic pathophysiological
conditions in the body.27 Compound 11a potently inhibited the
formation of 5-LO products with an IC50 ) 0.83 ( 0.07 µM
(Figure 3B).
The efficacy of compound 11a was finally assayed for its
ability to inhibit LTB4 production in vivo by the use of an animal
model of 5-LO-related inflammation, the carrageenan-induced
pleurisy in rats.28 The ip treatment of rats with 4 mg/kg of 11a,
30 min before carrageenan administration, significantly reduced
the inflammatory reaction measured as exudate volume (77%),
inflammatory cell numbers (40%), and LTB4 levels (49%) in
the pleural exudates (Table 5). The well-recognized 5-LO
inhibitor 397 (10 mg/kg) also reduced exudate formation (77%),
cell infiltration (41%), and LTB4 exudate levels (66%), and its
anti-inflammatory action was not significantly different from
compound 11a (Table 5).
Conclusions
Here we have synthesized and evaluated various series of
indole-3-carboxylates as inhibitors of human 5-LO. Structural
optimization led to 5-hydroxy-benzo[g]indole-3-carboxylates
that suppress the formation of 5-LO products in cell-free and
cellular assays, exemplified by 11a with IC50 values of 0.086
and 0.23 µM, respectively. Under more physiological relevant
conditions, e.g., in human whole blood assays, 11a potently
suppressed 5-LO product synthesis with IC50 values of 0.86 to
1.6 µM. Of interest, 11a blocked LTB4 generation in an in vivo
model of inflammation, the carrageenan-induced rat pleurisy.
This action might be responsible for the reduced inflammatory
reaction measured as decrease of exudate volume and migrating
cell numbers. Structure-activity relationships indicate that (I)
the nitrogen in 2-position of the indole of the lead 37 is not
absolutely essential but may increase the potency in cell-free
assays, (II) direct linking of a halogen-substituted phenyl moiety

2-Substituted Indole-3-carboxylates Inhibit 5-Lipoxygenase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3481
Figure 3. Concentration-response curves of compounds 11a and 11l for inhibition of 5-LO activity in human whole blood. Human whole blood
was treated with the test compounds (or DMSO as vehicle) for 10 min at 37 °C and stimulated with (A) A23187 (30 µM) or (B) primed with LPS
(1 µg/mL) for 30 min and then stimulated with fMLP (1 µM). After 15 min (LPS/fMLP) or 10 min (A23187) at 37 °C, the formation of 5-LO
products was determined. Data are means ( SE; n ) 3-5.
Table 5. Effect of Compound 11a on Carrageenan-Induced Pleurisy in
(mp): Bu¨chi melting point apparatus, uncorrected. Mass spectra:
Finnigan MAT TSQ 70 instrument. 1H NMR spectra: Bruker AM
360 spectrometer at 360 MHz. Spectra were measured in DMSO-
d6 using TMS as internal standard. Elemental analyses were
performed by the Organic Chemistry Department of the Friedrich
Alexander University Erlangen; Apparatus: Carlo Erba (model
1108) and Heraeus (CHN-Rapid). Infrared spectra were recorded
on the FTIR spectrometers Perkin-Elmer (type1740) and Jasco
(Type 410).
Procedure for the Preparation of the ꢀ-Ketoesters 4j,q,r.
To a solution of 12.0 mmol of 3-ethoxy-3-oxo-propanoic acid (for
4j) or 3-benzyloxy-3-oxo-propanoic acid (for 4q,r) in dry THF (15
mL), magnesium ethylate (687 mg, 6.0 mmol) was added and the
reaction mixture was stirred at RT for 4 h. The solvent was
evaporated, and the resulting residue was washed with ether, filtered
off, and dried again under reduced pressure to give the magnesium
salt of 3-ethoxy-3-oxo-propanoic acid (3a) or of 3-benzyloxy-3-
oxo-propanoic acid (3b), respectively. N,N′-Carbonyldiimidazole
(2, 1.78 g, 11.0 mmol) was slowly added to a solution of acid 1j,q,r
(10.0 mmol) in dry DMF (20 mL). After stirring the reaction
mixture under nitrogen atmosphere for 1 h, the appropriate
magnesium salt 3a or 3b was added and the mixture was allowed
to stir for additional 4 h. The solution was acidified with 2 N HCl
and extracted with ethylacetate. The combined organic layers were
washed with 10% aqueous NaHCO3 solution and water, dried over
Na2SO4, filtered off, and evaporated to give a colorless oil
crystallizing at <0 °C.
Procedure for the Preparation of the Unknown Enami-
noesters (7). The appropriate ꢀ-ketoester 4 (3.0 mmol) and
ammonium acetate (5, 1.16 g, 15.0 mmol) or amine 6p (6.0 mmol),
respectively, were dissolved in dry toluene (10 mL). After adding
four drops of acetic acid (or formic acid in case of enaminoester
7p), the reaction mixture was refluxed for 6 h under azeotropic
removal of water. After cooling down to RT, the mixture was
washed with saturated aqueous NaHCO3 solution. The resulting
organic layer was dried over Na2SO4, filtered, and evaporated to
give a residue, which was purified by flash chromatography on silica
gel.
(2EZ)-Ethyl 3-Amino-3-[(3-chlorophenyl)amino]acrylate
(13). (2E)-Ethyl 3-amino-3-ethoxyacrylate (12, 401.2 mg, 2.52
mmol) was dissolved in ethanol (40 mL). After addition of
3-chloroaniline (6a, 321.5 mg, 2.52 mmol), the resulting mixture
was heated under reflux for 48 h. Evaporation of the solvent resulted
in a syrupy residue, which was purified via flash chromatography.
General Procedures for the Preparation of the 5-Hydroxy-
1H-indole-3-carboxylates 10, 11, 14, and 19-22. To a solution
of enamine 7 or ketene aminal 13 (1.0 mmol) in ethanol (4 mL),
1.2 mmol of 1,4-benzoquinone (8) or 1,4-naphthoquinone (9) were
slowly added, respectively. After stirring for 1 h (under reflux using
enamines 7, at RT using ketene aminal 13), the solvent was removed
Ratsa
exudate
volume (mL)
inflammatory
LTB4
(ng/rat)
treatment
vehicle
11a (4 mg/kg)
cells × 106
0.48 ( 0.08
0.11 ( 0.0026*** 28.00 ( 6.83*
zileuton (10 mg/kg) 0.11 ( 0.065**
46.67 ( 3.53
1.17 ( 0.21
0.60 ( 0.096*
27.54 ( 4.41** 0.40 ( 0.044**
a Thirty min before intrapleural injection of carrageenan, rats (n ) 10
for each experimental group) were treated ip with 4 mg/kg 11a, 10 mg/kg
zileuton, or vehicle (DMSO 4%). Exudate volume, LTB4 levels as well as
inflammatory cell accumulation in the pleural cavity were assessed 4 h after
carrageenan injection. Data are expressed as mean ( SE, n ) 10. *p <
0.05; ** p < 0.01; ***p < 0.001 vs vehicle.
in 2-position of the indole without bridging atoms is tolerated,
(III) variations of halogen substituents regarding number, nature
(F, Cl, Br), or positioning of the halogen at the aryl residue
only slightly affect the potency, and (IV) the 5-hydroxy moiety
may govern 5-LO inhibition, particularly in cell-free assays. A
major step forward is the annelation of the benzene yielding
benzo[g]indole structures that leads to increased potency,
preferentially in intact cells. The benzo[g]indole ring system
represents a unique, basic backbone structure of eicosanoid
synthesis inhibitors. As pointed out in our previous report,8 the
free indole-3-carboxylic acids were not synthetically accessible
and no conclusion can be drawn whether or not an ester moiety
is necessary or if also the free acid may be active. The precise
mode of action of how these compounds interfere with 5-LO
activity remains unclear, and a definite assignment to a typical
class of LT synthesis inhibitors is yet not possible. Nevertheless,
our data suggest a noncompetitive and reversible mode of action
on 5-LO and exclude FLAP as critical point of attack. In
summary, on the basis of their ability to potently inhibit 5-LO
product formation in various biological assays, associated with
high efficacy in an animal model of (LT-mediated) inflammation
comparable to zileuton (39), these novel 5-hydroxybenzo[g]in-
dole carboxylates possess a high potential for therapeutic use
and deserve further pharmacological analysis in other in vivo
(animal) models.
Experimental Section
General Methods. Chromatography was carried out with
230-400 mesh silica gel (Merck, Darmstadt, Germany). The
MPLC-apparatus consisted of the following segments: MPLC pump,
¨
BUCHI (type B688); fraction collector, LKB (type 2111 Multirac
¨
and SuperFracTM); columns: BUCHI, 15 mm × 460 mm, 26 mm
× 460 mm, 36 mm × 460 mm, 49 mm × 460 mm. Melting points

3482 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Karg et al.
under reduced pressure to give a black residue, which was purified
by flash chromatography on silica gel (method a).
dimethyl product was extracted with ethylacetate, washed with
water, and dried over Na2SO4. The desired dimethylated compound
32 was isolated via flash chromatography.
To a solution of the appropriate 1,4-quinone (1.0 mmol) in 3
mL CH2Cl2, ZnI2 (32.0 mg, 0.1 mmol) was added and the resulting
mixture was heated to boiling temperature. A solution of enamine
7 or ketene aminal 13, respectively (1.0 mmol), in 2 mL of CH2Cl2
was added drop by drop under stirring for 5-10 min. After refluxing
for additional 20 min, the mixture was cooled to 0-5 °C for 2-3
h. The precipitated crystals were filtered off and washed with
CH2Cl2 and hexane. If necessary, crystallization of the compounds
was performed in solvents (method b).
Ethyl 5-[[(Trifluoromethyl)sulfonyl]oxy]-2-(3-chloroben-
zyl)-1H-benzo[g]indole-3-carboxylate (34). Ethyl 2-(3-Chlo-
robenzyl)-5-hydroxy-1H-benzo[g]indol-3-carboxylate (11a, 949.6
mg, 2.5 mmol) was dissolved in 5 mL of CH2Cl2. After addition
of triethylamine (506.0 mg; 5.0 mmol), trifluoroacetic anhydride
(33, 1.76 g, 6.25 mmol) was added under cooling and the reaction
mixture was stirred for 4 h at 0 °C. The solution was concentrated
in vacuo to give the crude product, which was purified via flash
chromatography.
Ethyl 2-(3-Chlorobenzyl)-5-phenyl-1H-benzo[g]indole-3-
carboxylate (36). A mixture of 0.55 mmol of phenylboronic acid
pinacol ester 35, ethyl 5-[[(trifluoromethyl)sulfonyl]oxy]-2-(3-
chlorobenzyl)-1H-benzo[g]indole-3-carboxylate (34), 256.0 mg,
0.50 mmol), Pd(OAc)2 (3.4 mg, 0.015 mmol), PPh3 (3.9 mg, 0.015
mmol), aqueous Na2CO3 solution, and DMF was heated under reflux
for 2 h. The mixture was poured into water and the product was
extracted with ethyl acetate. Removing the solvent in vacuo left
the crude product, which was was purified via flash chromatography.
Preparation of Human Whole Blood and Cell Isolation.
Fresh venous blood was collected in heparinized tubes (16 IE
heparin/mL blood) by venipuncture from fasted (12 h) adult healthy
volunteers, with consent (Blood Center, University Hospital,
Tuebingen, Germany). The subjects had no apparent inflammatory
conditions and had not taken anti-inflammatory drugs for at least
ten days prior to blood collection.
The method(s) applied is/are mentioned in Supporting Informa-
tion in conjunction with the appropriate compounds 10, 11, 14,
and 19-22.
Ethyl 2-(3-Chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-
carboxylate (11a). Method a: 77.7 mg (0.205 mmol, 20%); method
b: 197.2 mg (0.519 mmol, 52%); brown powder; mp 222 °C
1
(cyclohexane/ethylacetate). H NMR (DMSO-d6) δ (ppm) ) 1.34
(t, 3H, J ) 7.0 Hz, CH2CH3), 4.29 (q, 2H, J ) 7.0 Hz, CH2CH3),
4.50 (s, 2H, CH2), 7.21-7.38 (m, 4H, H-2′, H-4′, H-5′, H-6′), 7.41
(t, 1H, J ) 7.5 Hz, H-7), 7.55 (s, 1H, H-4), 7.56 (t, 1H, J ) 7.5
Hz, H-8), 8.17 (d, 1H, J ) 7.9 Hz, H-6), 8.27 (d, 1H, J ) 7.9 Hz,
H-9), 9.66 (s, 1H, OH), 12.39 (s, 1H, NH). 13C NMR (DMSO-d6)
δ (ppm) ) 14.4 (CH2CH3), 32.4 (CH2), 58.9 (CH2CH3), 101.5 (C-
4), 104.2 (C-3), 120.4 (C-9), 121.8 (C-6), 122.4 (C-5a), 123.08
(C-3a), 123.15, 123.2 (C-7, C-9a), 124.0 (C-9b), 126.0 (C-4′), 126.1
(C-8), 127.0 (C-6′), 128.1 (C-2′), 130.2 (C-5′), 132.9 (C-3′), 141.9,
142.2 (C-1′, C-2), 148.3 (C-5), 165.0 (CdO). EI-MS: m/z (%) )
379 (88) [M+], 350 (100), 270 (26), 241 (20), 121 (24). Anal. for
C22H18ClNO3: C, H, N.
For isolation of neutrophils, venous blood was subjected to
centrifugation (4000g/20 min/20 °C) for preparation of leukocyte
concentrates. Neutrophils were promptly isolated by dextran
sedimentation, centrifugation on Nycoprep cushions, and hypotonic
lysis of erythrocytes as described previously.29 Neutrophils (purity
>96-97%) were finally resuspended in ice-cold PBS plus 1 mg/
mL glucose and 1 mM CaCl2 (PGC buffer).
Ethyl 2-(3-Chlorophenylamino)-5-hydroxy-1H-benzo[g]in-
dole-3-carboxylate (14). Method b: 133.3 mg (0.350 mmol, 35%);
1
green powder; mp 204 °C (cyclohexane/ethylacetate). H NMR
(DMSO-d6) δ (ppm) ) 1.36 (t, 3H, J ) 7.2 Hz, CH2CH3), 4.30 (q,
2H, J ) 7.2 Hz, CH2CH3), 7.01 (m, 1H, Ar), 7.20 (m, 1H, Ar),
7.29 (m, 1H, Ar), 7.34 (m, 2H, Ar), 7.49 (m, 2H, Ar), 8.15 (d, 1H,
J ) 8.3 Hz, Ar), 8.31 (d, 1H, J ) 8.3 Hz, Ar), 8.82 (s, 1H, OH),
9.66 (s, 1H, NH′), 12.14 (s, 1H, NH). EI-MS: m/z (%) ) 380 (58)
[M+], 334 (84) [M+-OEt], 299 (100), 271 (21). Anal. for
C21H17ClN2O3: C, H, N.
General Procedure for the Preparation of Indole-3-
carboxylates 27a-h. First, 1.19 mmol of indole-3-carboxylate
23a (or 23b in case of indole 28) was dissolved in CH2Cl2 (5 mL)
and the solution was cooled down to 0 °C. Then, N,N′-dimeth-
ylpiperazine (24, 7.5 mg, 0.66 mmol) and N-chlorosuccinimide (25,
175 mg, 1.31 mmol) were added and the reaction mixture was
allowed to stand at 0 °C for 2 h, whereupon a solution of
trichloroacetic acid (26, 50 mg, 0.3 mmol) in CH2Cl2 (5 mL),
including 2.34 mmol of the appropriate amines 6a-h, was added.
After 2 h at RT, the reaction mixture was washed with 10% aqueous
NaHCO3 solution, with 1 M aqueous hydrochloric acid, and finally
with water. The resulting solution was dried over Na2SO4, filtered,
and evaporated to give a residue, which was purified by flash
chromatography on silica gel.
Ethyl 5-Benzoyloxy-2-(3-chlorobenzyl)-1H-benzo[g]indole-
3-carboxylate (30). Benzoylchloride (29, 421.7 mg, 3.0 mmol)
was added to a solution of ethyl 2-(3-chlorobenzyl)-5-hydroxy-
1H-benzo[g]indole-3-carboxylate (11a, 379.8 mg, 1.0 mmol) in
ethylacetate (10 mL)/triethylamine (2 mL), and the mixture was
stirred at RT for 20 h. After pouring into ice/water (100 mL) and
extraction with ethylacetate, the organic layer was dried over
Na2SO4. Removing the solvent in vacuo left the crude product,
which was crystallized from ethylacetate/CH2Cl2, yielding the
product as brown powder.
Ethyl 2-(3-Chlorobenzyl)-5-methoxy-1-methyl-1H-benzo[g]-
indole-3-carboxylate (32). Ethyl 2-(3-Chlorobenzyl)-5-hydroxy-
1H-benzo[g]indole-3-carboxylate (11a, 189.9 mg, 0.50 mmol) was
dissolved in a mixture of aqueous NaOH solution and THF. An
excess of methyliodide (31) was added, and the solution was
refluxed for 10 h. After neutralization with 2 N HCl, the residual
methyliodide was emitted. The generated mixture of mono- and
Determination of 5-LO Product Formation in Whole
Blood and in Intact Neutrophils. For assays in whole blood,
aliquots of 2 mL (A23187) or 3 mL (LPS and fMLP) were
preincubated with the test compounds or with vehicle (DMSO) for
10 min at 37 °C, as indicated, and formation of 5-LO products
was either started by addition of fMLP (1 µM) following priming
with 1 µg/mL LPS for 30 min or by addition of A23187 (30 µM).
The reaction was stopped on ice after 15 (LPS/fMLP) or 10
(A23187) min, and the samples were centrifuged (600g, 10 min, 4
°C). Aliquots of the resulting plasma (500 µL) were then mixed
with 2 mL of methanol and 200 ng of prostaglandin B1 were added
as internal standard. The samples were placed at -20 °C for 2 h
and centrifuged again (600g, 15 min, 4 °C). The supernatants were
collected and diluted with 2.5 mL of PBS and 75 µL of HCl 1 N.
Formed 5-LO metabolites were extracted and analyzed by HPLC
as described.29 5-LO product formation is expressed as ng of 5-LO
products per 106 cells, which includes LTB4 and its all-trans
isomers, and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetrae-
noic acid. Cysteinyl LTs C4, D4, and E4 (<1% of total 5-LO
products30) were not analyzed.
For assays of intact neutrophils, 107 freshly isolated neutrophils
were resuspended in 1 mL of PGC buffer. After preincubation with
the test compounds for 15 min at 37 °C, 5-LO product formation
was started by addition of 2.5 µM ionophore A23187 with or
without 20 µM AA (unless stated otherwise). After 10 min at 37
°C, the reaction was stopped with 1 mL of methanol. Then 30 µL
of 1 N HCl, 200 ng prostaglandin B1, and 500 µL of PBS were
added and 5-LO products were analyzed as described.8
Determination of 5-LO Product Formation in Cell-Free
Systems. E. coli MV1190 was transformed with pT3-5LO plasmid.
Expression of recombinant 5-LO protein and preparation of 40000g
supernatant (S40) for 5-LO activity assays was carried out as
described.31 For determination of the activity of 5-LO in S40,
aliquots of the supernatants were diluted in 1 mL of PBS, pH 7.4,
1 mM EDTA. Samples were preincubated with the test compounds
for 10 min at 4 °C, samples were prewarmed for 30 s at 37 °C,

2-Substituted Indole-3-carboxylates Inhibit 5-Lipoxygenase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3483
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carrageenan. Rats were anaesthetized and λ-carrageenan was
injected into the pleural cavity. After four hours, the exudate was
removed and the amount was measured. Infiltrated leukocytes in
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EIA according to manufacturer’s protocol. The results are expressed
as nanograms per rat and represent the mean ( SE of 10 rats. More
methodological details can be found in Supporting Information.
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obtained from measurements at 4-5 different concentrations of the
compounds, are approximations determined by graphical analysis
(linear interpolation between the points at 50% activity). The
program Graphpad Instat (Graphpad Software Inc., San Diego, CA)
was used for statistical comparisons. Statistical evaluation of the
data was performed by one-way ANOVAs for independent or
correlated samples followed by Tukey HSD post hoc tests. Where
appropriate, Student’s t test for paired and correlated samples was
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Acknowledgment. We thank Bianca Jazzar for expert
technical assistance. C.P. received a Carl-Zeiss stipend.
Supporting Information Available: Elemental analyses or
HRMS data, routine spectroscopic data (IR), general procedures
for the preparation of the 2-substituted indole-3-carboxylates and
1H NMR data, and carrageenan-induced pleurisy in rats. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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