Further studies on ethyl 5-hydroxy-indole-3-carboxylate scaffold: Design, synthesis and evaluation of 2-phenylthiomethyl-indole derivatives as efficient inhibitors of human 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2014.05.033

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the design, synthesis and

Full text of DOI:10.1016/j.ejmech.2014.05.033

Accepted Manuscript
Further Studies on Ethyl 5-hydroxy-indole-3-carboxylate scaffold: Design, Synthesis
and Evaluation of 2-phenylthiomethyl-Indole Derivatives as Efficient Inhibitors of
Human 5-Lipoxygenase
Antonella Peduto , Ferdinando Bruno , Friedrike Dehm , Verena Krauth , Paolo de
Caprariis , Christina Weinigel , Dagmar Barz , Antonio Massa , Mario De Rosa , Oliver
Werz , Rosanna Filosa , Dr.
PII:
S0223-5234(14)00447-4
10.1016/j.ejmech.2014.05.033
EJMECH 6989
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2014
Revised Date: 7 May 2014
Accepted Date: 11 May 2014
Please cite this article as: A. Peduto, F. Bruno, F. Dehm, V. Krauth, P. de Caprariis, C. Weinigel,
D. Barz, A. Massa, M. De Rosa, O. Werz, R. Filosa, Further Studies on Ethyl 5-hydroxy-indole-3-
carboxylate scaffold: Design, Synthesis and Evaluation of 2-phenylthiomethyl-Indole Derivatives as
Efficient Inhibitors of Human 5-Lipoxygenase, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.05.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Further Studies on Ethyl 5-hydroxy-indole-
3-carboxylate scaffold: Design, Synthesis and
Evaluation of 2-phenylthiomethyl-Indole
derivatives as Efficient Inhibitors of Human
5-Lipoxygenase
Leave this area blank for abstract info.
Antonella Pedutoa,f, Ferdinando Brunob, Friedrike Dehmc, Verena Krauthc, Paolo de Caprariisb, Mario
De Rosaa, Christina Weinigeld, Dagmar Barzd, Antonio Massae, Oliver Werzc, Rosanna Filosa*a

ACCEPTED MANUSCRIPT
Further Studies on Ethyl 5-hydroxy-indole-3-carboxylate
scaffold:
Design, Synthesis and Evaluation of 2-phenylthiomethyl-
Indole Derivatives as Efficient Inhibitors of Human 5-
Lipoxygenase
Antonella Pedutoa,f, Ferdinando Brunob, Friedrike Dehmc, Verena Krauthc, Paolo de Caprariisb,
Christina Weinigeld, Dagmar Barzd, Antonio Massae, Mario De Rosaa, Oliver Werzc, Rosanna
Filosa*a
aDepartment of Experimental Medicine, University of Naples, Via Costantinopoli, 16 - 80138 Naples, Italy;
bDepartment of Pharmaceutical and Biomedical Sciences, Via Giovanni Paolo II, - Fisciano (SA), Italy;
cChair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University;
dInstitute of Transfusion Medicine, University Hospital Jena, 07743 Jena, Germany;
eDepartment of Chemistry, University of SalernoVia Ponte don Melillo, 84084 Fisciano (SA) Italy
f Dermofarma Italia, Via Cortenocera,82030 S. Salvatore Telesino (BN) Italy
Corresponding author: Dr. Rosanna Filosa, Department of Experimental Medicine, University of Naples, Via
Costantinopoli, 16 - 80138 Naples, Italy; Phone:0815667663; e-mail: rosanna.filosa@unina2.it

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Abstract
5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-
inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and
allergic diseases. Here, we present the design, synthesis and biological evaluation of novel series of
ethyl 5-hydroxyindole-3-carboxylate derivatives that efficiently inhibit human 5-LO. SAR analysis
revealed that the potency of compounds is closely related to the positioning of the substituents at the
phenylthiomethyl ring. The introduction of methyl or chlorine groups in ortho- and ortho/para-
position of thiophenol represent the most favourable modifications. Among all tested compounds,
ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19) is the most potent
derivative which blocks 5-LO activity in cell-free assays with IC50 = 0.7 µM, and suppressed 5-LO
product synthesis in polymorphonuclear leukocytes with IC50 = 0.23 µM.
Keywords: 5-lipoxygenase, leukotriene, indoles.
Corresponding author: Dr. Rosanna Filosa, Department of Experimental Medicine, University of Naples, Via
Costantinopoli, 16 - 80138 Naples, Italy; Phone:0815667663; e-mail: rosanna.filosa@unina2.it

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1.1 Introduction
Leukotrienes (LTs) that are formed from arachidonic acid (AA) by the 5-lipoxygenase (5-LO)
pathway play important roles in various biological processes including bronchoconstriction,
increased vascular permeability, vasoconstriction of coronary arteries, as well as in cell survival and
proliferation [1]. 5-LO is the key enzyme in the conversion of AA to LTA4 in two successive steps:
oxygenation of AA forms 5(S)-hydroperoxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-HPETE),
and then dehydration of 5-HPETE leads to the unstable epoxide LTA4 which is subsequently
converted to either LTB4 by LTA4 hydrolase or to cysteinyl-LTs (LTC4, D4 and E4) through
conjugation with glutathione, catalyzed by LTC4 synthases [2]. LTB4 acts as a high affinity ligand
for G protein-coupled receptors (GPCR), namely BLT1 and BLT2 and stimulates the inflammatory
response as well as neutrophil and eosinophil chemotaxis [3]. The cysteinyl-LTs also activate
GPCRs such as CysLT1 and CysLT2 causing bronchoconstriction, airway edema and mucus
secretion [3]. Therefore, inhibition of 5-LO results in the reduction of LT formation and represents
a valuable target for the pharmacotherapy of inflammatory ailments like asthma, allergic rhinitis,
atherosclerosis as well as certain types of cancers. Today, while cysteinyl-LT receptor antagonists
(i.e. montelukast and zafirlukast) are successfully applied for asthma therapy, the development of
LT biosynthesis inhibitors is little advanced and thus far zileuton (1) is the only 5-LO inhibitor
currently in clinical use [4].
Recently, a series of 5-hydroxyindole-3-carboxylates were identified as potent inhibitors of isolated
human recombinant 5-LO as well as 5-LO in human neutrophils [5]. The lead compound of the
series is compound 2 (IC50 = 2.4 µM in PMNL and 0.3 µM in cell-free based assay) (Fig. 1).
Structure-activity relationships (SAR) studies indicate that (I) the nitrogen in 2-position of the
indole is not absolutely essential but may increase the potency in cell-free assays, (II) direct linking
of a halogen-substituted phenyl moiety 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 favor 5-LO inhibition, particularly in cell-free assays [5, 6].

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From these known indole bioactive ligands, in order to expand the hit matter identified, extensive
exploration focused on the core scaffold was utilized to improve biological activity in our medicinal
chemistry programs [7-11] focused on optimizing small molecules as anti-inflammatory candidates.
Here we explored the SARs and present the design and synthesis of novel ethyl 5-hydroxyindole-3-
carboxylates, this scaffold-focused screen identifies active compounds that proved to be efficient
inhibitors of human 5-LO in cell-free assays as well as in intact cells. Structurally, these compounds
exhibit differently substituted phenylthiomethyl rings in position 2 of the indole and indole N-
methylation.
2. Results and Discussion
2.1. Chemistry
The title compounds were obtained as described in Scheme 1. The synthesis of ethyl-5-hydroxy-2-
methyl-1H-indole-3-carboxylate derivatives (7-19) started from commercially available ethyl 5-
hydroxy-2-methylindole-3-carboxylate (3), which underwent protection reaction using acetic
anhydride and pyridine affording intermediate 4 in good yields. Methylation with iodomethane
under basic condition in DMF provided compound 5 which was submitted to radicalic bromination
yielding a mixture of mono (6)- and dibromo-derivatives [12] easily separated by chromatography
on silica gel. Final nucleophilic displacement by differently substituted thiophenols gave desired
compounds 7 [13]-19.
2.2. Evaluation of 5-LO Activity and Structure-Activity Relationships
In order to assess the effects of the synthesized compounds on 5-LO product synthesis, a cell-free
assay using isolated human recombinant 5-LO and a cell-based assay using human neutrophils were
applied. The cell-free assay allows identifying compounds that directly interfere with 5-LO catalytic
activity, whereas the cell-based test system considers cellular regulatory aspects of 5-LO product
synthesis, and as such offers several points of attack of a given compound (e.g., inhibition of FLAP
or coactosine-like protein (CLP), interference with 5-LO-activating lipid hydroperoxides, protein
kinases or Ca2+ mobilization, and 5-LO translocation/membrane association) [14]. The reference 5-
LO inhibitor N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (zileuton, 1) [15] was used to control
the 5-LO activity assays. As shown in table 1, all tested compounds inhibited 5-LO activity both in

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cell-free and in cell-based assay with IC50 values in the low micromolar range. These results imply
that the insertion of a phenylthiomethyl group and the methylation of N1 of indole ring could
represent favorable modifications to interfere with 5-LO activity. Moreover, the obtained data
reveal important details about the role of the substituents on the thiophenol towards the biological
activity. Compound 5, which has no substituent at the thiophenol group, shows a moderate potency
against 5-LO both in the cell-free (IC50 = 5.7 µM) and in the intact cell assay (IC50 = 2.4 µM). The
insertion of an electron donor group (i.e., methoxy or trifluoroacetoxy) in position 4 at the
phenylthiomethyl ring (9 and 10) does not significantly alter the efficiency in intact cells, (IC50
=
3.1 µM and 2.1 µM respectively) while an improvement of activity is observed in the cell-free
assay, especially for 10, with an IC50 of 0.9 µM. The shift of the methoxy group in 10 from position
4 to position 2 of the ring (8) retains the potency in the cell-free assay with a small loss of potency
in intact cells. Compounds 12, 13 and 14, carrying electron withdrawing groups, such as halogens -
CF3, -F, or -Cl in position 4 and 3 respectively, are about equipotent to 7 in the cell-free assay, with
IC50 values ranging from 3.8 to 5.0 µM, but markedly loose potency in the cell-based system. When
the trifluoromethyl group is shifted from position 4 to position 2 (11), the potency in cell-free assay
(IC50 = 0.7 µM) versus the parental compound 12 (IC50 = 0.9 µM) was hardly altered. The
introduction of a second fluorine in ortho-position of compound 13 leads to compound 15 which is
more potent both in cell-free and in cell-based assays (IC50 = 3.2 µM and 6.8 µM vs 5 µM and 9.1
µM). When two chlorine or methyl occupy the ortho-positions of the thiophenol ring (16-17) we
observed a substantial increase of 5-LO inhibitory activity against purified 5-LO and also in PMNL
for 16 (IC50 = 0.8 µM and 0.9 µM). The introduction of two methyl groups in meta-positions (18)
increases the potency over 7 in cell-free assay about 3-fold (IC50 = 1.8 µM), while no substantial
modifications in cell-based assay are observed. Interestingly, the presence of three methyl groups in
ortho- and para-positions, leading to compound 19, causes a marked increase of 5-LO inhibitory
activity with IC50 values of 0.23 µM (cell-based) and 0.7 µM (cell-free). Variation of the substrate
supply to higher (50 µM) or lower (5 µM) AA concentrations had no significant impact on the
concentration-dependent 5-LO inhibitory potency of 19 (Fig. 2).

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Besides 5-LO, other human LOs such as platelet-type 12-LO and 12/15-LO (also termed 15-LO-1)
catalyze the incorporation of molecular oxygen into AA as substrate [16]. In order to determine the
selectivity of the compounds, we analyzed their effects on the formation of 12-H(P)ETE and 15-
H(P)ETE that are produced by 12- and 12/15-LOs (Table 2). None of the test compounds
significantly suppressed the synthesis of 12-H(P)ETE at a concentration range up to 10 µM. For 15-
LO inhibition, a slightly activity is evident for compound 19 (IC50 = 8.2 µM)), indicating that active
compounds are rather selective for 5-LO. In contrast, cinnamyl-3,4-dihydroxy-α-cyanocinnamate
(CDC, 10 µM), an inhibitor of 5-, 12- and 15-LO [17]{Pergola C et al J Pharmacol Exp Ther. 2011
338(1):205-213}, efficiently suppressed 12- and 15-LO activities as expected. Moreover, we
analyzed the effects of the most potent compounds 19 and 16 on the activity of the related COX-1/2
enzymes that in analogy to LOs convert AA to oxygenated products (i.e. prostanoids). As shown in
figure 3, in a cell-free assay, 16 and 19 failed to inhibit COX-1 and COX-2, whereas the reference
inhibitor indomethacin and celecoxib suppressed the activities of these enzymes, respectively.
3. Conclusions
According to the above results, the following conclusions can be made:
1. The amino moiety linking the phenyl ring with the indole core can be replaced by a
phenylthiomethyl group;
2. Hydrogen of the indole nitrogen can be substituted with a methyl residue without
significant loss of activity;
Substitutions of the thiophenol ring can improve anti-LO activities. The simultaneous
presence of chlorine in ortho-positions of the thiophenol ring as well as the introduction
of three methyl groups (ortho, para) causes a marked increase of 5-LO inhibitory activity.
Compounds 16 and 19 are the most active of the series and they show a greater potency than lead
2 in PMNL assay.
Compound 19, carrying three methyl groups at the thiophenol is the most potent derivative which
blocks 5-LO activity in cell-free assays with IC50 = 0.7 µM, and suppressed 5-LO product synthesis
in intact cells with IC50 = 0.23 µM, being equally potent to the well-recognized reference inhibitor
zileuton, used as anti-asthmatic drug in the clinics. With regard to other AA-metabolizing enzymes

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like COX-1, COX-2 and 12-LO, compound 19 is rather selective for 5-LO with minor effects also
on 15-LO.
4. Experimental section
4.1 Chemistry
All reagents were analytical grade and purchased from Sigma–Aldrich (Milano, Italy). Flash
chromatography was performed on Carlo Erba silica gel 60 (230–400 mesh; CarloErba, Milan,
Italy). TLC was carried out using plates coated with silica gel 60F 254 nm purchased from Merck
1
(Darmstadt, Germany). H and 13C NMR spectra were registered on a Brucker AC 300. Chemical
shifts are reported in ppm. The abbreviations used are follows: s, singlet; d, doublet; dd double
doublet; bs, broad signal. MS spectrometry analysis ESI-MS was carried out on a Finnigan LCQ
Deca ion trap instrument. Microanalyses were carried out on a Carlo Erba 1106 elemental analyzer.
Melting points were performed by Stuart melting point SMP30 and are uncorrected.
Ethyl 5-acetoxy-2-(bromomethyl)-1-methyl-1H-indole-3-carboxylate (6)
The synthesis of this compound is reported in an our previous work [14].
4.2 General procedures for synthesis of 7-19
To a solution containing KOH (3.0 eq.) in 1 mL of methanol at room temperature different
thiophenols were added (1 eq). After 15 minutes, 6 (30 mg, 0.083 mmol) was also added. The
mixture was allowed to stay to room temperature for 3 h followed by neutralizing with a diluted
acetic acid. The mixture was evaporated and the product was purified by flash chromatography.
4.2.1 Ethyl 5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3-carboxylate (7)
Elution with hexane/EtOAc (7.5:2.5) afforded 7 (93 %). 1H NMR data are in agreement with those
reported in literature[15].
4.2.2 Ethyl 5-hydroxy-2-((2-methoxyphenylthio)methyl)-1-methyl-1H-indole-3-carboxylate (8)
Elution with toluene/EtOAc (9:1) afforded 8 (84 %). 1H NMR (d6-DMSO, 300MHz): δ 1.29 (t, 3 H,
J = 7.1 Hz); 3.69 (s, 3 H); 3.74 (s, 3 H); 4.19 (q, 2H, J = 7.1 Hz); 4.71 (s, 2H); 6.74 (dd, 1H, J = 8.5,
2.3 Hz), 6.89 (td, 1 H, J = 7.5, 1.2 Hz); 6.98 (dd, 1 H, J=1.2, 8.5 Hz); 7.27(td, 1 H J=1.9, 7.9 Hz);
13
7.30-7.33 (m, 1H); 7.33-7.35(m, 1H); 7.36 (d, J=2.2 Hz); 9.02 (s, OH). C (d6-DMSO, 75 MHz):δ
14.8, 28.7, 30.5, 56.3, 59.5, 103.7, 105.9, 111.2, 111.5, 121.3, 123.2, 127.1, 128.9, 131.6, 132.5,

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134.1, 142.5, 153.6, 155.2, 165.0. M.p. 169°C. ESI-MS [M + Na+]: 394.45 Anal. calcd for
C20H21NO4S: C, 64.67; H, 5.70; N, 3.77; O, 17.23; S, 8.63, Found:C, 64.21; H, 6.05; N, 3.12; O,
16.58; S, 8.12.
4.2.3 Ethyl 5-hydroxy-2-((4-methoxyphenylthio)methyl)-1-methyl-1H-indole-3-carboxylate (9)
Elution with hexane/EtOAc (75:25) afforded 9 (99 %). 1H NMR (d6-DMSO, 300MHz): δ 1.28 (t, 3
H, J = 7.1 Hz); 3.61 (s, 3 H); 3.72 (s, 3 H); 4.16 (q, 2H, J = 7.1 Hz); 4.65 (s, 2H); 6.71 (dd, 1H, J =
8.5, 2.1 Hz), 6.83 (d, 2 H, J = 8.5 Hz); 7.24 (d, 2 H, J = 8.5 Hz); 7.31, (d, 1 H, J = 8.5 Hz); 7.33 (d,
1 H, J = 2.1 Hz); 9.03 (s, OH). 13C (d6-DMSO, 75 MHz):δ 14.7, 30.3, 55.7, 59.2, 103.7, 106.1,
111.3, 113.0, 115.1, 124.6, 127.1, 131.6, 135.2, 143.6, 153.4, 159.9, 165.0. M.p. 173°C. ESI-MS
[M + Na+]: 394.45 Anal. calcd for C20H21NO4S: C, 64.67; H, 5.70; N, 3.77; O, 17.23; S, 8.63,
Found: C, 64.12; H, 5.03; N, 3.41; O, 17.96; S, 7.98.
4.2.4 Ethyl 5-hydroxy-1-methyl-2-((4-(trifluoromethoxy)phenylthio)methyl)-1H-indole-3-
carboxylate (10)
1
Elution with hexane/EtOAc (8:2) afforded 10 (86 %). H NMR (d6-DMSO, 300MHz): δ 1.28 (t, 3
H, J = 6.8 Hz); 3.74 (s, 3 H); 4.17 (q, 2H, J = 6.8 Hz); 4.74 (s, 2H); 6.75 (dd, 1H, J = 8.9, 2.5 Hz),
7.26-7.37, (m, 4H), 7.47 (d, 2H, J = 8.9Hz). 13C (d6-DMSO, 75 MHz):δ 14.7, 28.8, 30.5, 59.4,
103.7, 105.9, 111.3, 113.1, 122.0, 127.0, 131.5, 133.2, 134.4, 142.4, 148.3, 153.4, 164.9.
M.p.174°C. ESI-MS [M + Na+]: 448.42 Anal. calcd for C20H18F3NO4S: C, 56.46; H, 4.26; F, 13.40;
N, 3.29; O, 15.04; S, 7.54, Found: C, 56.87; H, 4.65; F, 13.95; N, 3.01; O, 14.96; S, 7.03.
4.2.5
Ethyl
5-hydroxy-1-methyl-2-((2-(trifluoromethyl)phenylthio)methyl)-1H-indole-3-
carboxylate (11)
1
Elution with hexane/EtOAc (8:2) afforded 11 (91 %). H NMR (d6-DMSO, 300MHz): δ 1.25 (t, 3
H, J = 6.9 Hz); 3.73 (s, 3 H); 4.16 (q, 2H, J = 6.9 Hz); 4.91 (s, 2H); 6.75 (dd, 1H, J = 8.8, 2.3 Hz),
7.35 (dd, 2 H, J = 3.5, 5.6 Hz); 7.43-7.50 (m, 1 H); 7.59, (d, 2H, J = 3.5 Hz); 7.74 (d, 1H, J=7.8 Hz);
9.05 (s, OH). 13C (d6-DMSO, 75 MHz):δ 14.6, 29.1, 30.3, 59.6, 103.9, 105.8, 111.4, 113.2, 118.7,

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122.3, 125.9, 126.9, 127.1 (q, J= 5.3 Hz), 128.0, 129.6 (q, J=29.3 Hz), 131.6, 133.1, 133.9, 134.2,
141.7, 153.2, 165.0. M.p. 174°C. ESI-MS [M + Na+]: 432.42 Anal. calcd for C20H18F3NO3S: C,
58.67; H, 4.43; F, 13.92; N, 3.42; O, 11.72; S, 7.83, Found: C, 58.17; H, 4.32; F, 13.08; N, 3.58; O,
11.62; S, 7.52.
4.2.6
Ethyl
5-hydroxy-1-methyl-2-((4-(trifluoromethyl)phenylthio)methyl)-1H-indole-3-
carboxylate (12)
1
Elution with hexane/EtOAc (8:2) afforded 12 (94 %). H NMR (d6-DMSO, 300MHz): δ 1.40 (t, 3
H, J = 7.1 Hz); 3.76 (s, 3 H); 4.21 (q, 2H, J = 7.1 Hz); 4.96 (s, 2H); 6.75 (dd, 1H, J = 8.7, 2.4 Hz),
7.22 (d, 1 H, J = 8.7 Hz); 7.37 (s, 1 H); 7.57, (d, 1 H, J = 7.1 Hz), 7.67 (d, 1 H, J= 7.1 Hz), 9.07 (s,
13
OH). C (d6-DMSO, 75 MHz):δ 14.9, 27.7, 30.9, 59.3, 70.1, 105.9, 111. 3, 113.2, 126.0, 126.1 (d,
J=3.7Hz), 129.7, 131.7, , 141.7 (d, J=11.8 Hz), 153.5, 165.0. M.p. 176°C. ESI-MS [M + Na+]:
432.33 Anal. calcd for C20H18F3NO3S: C, 58.67; H, 4.43; F, 13.92; N, 3.42; O, 11.72; S, 7.83,
Found: C, 58.01; H, 3.96; F, 12.02; N, 3.97; O, 11.57; S, 7.12.
4.2.7 Ethyl 2-((4-fluorophenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate (13)
1
Elution with hexane/EtOAc (8:2) afforded 13 (83 %). H NMR (d6-DMSO, 300MHz): δ 1.28 (t, 3
H, J = 6.8 Hz); 3.68 (s, 3 H); 4.16 (q, 2H, J = 6.8 Hz); 4.77 (s, 2H); 6.73 (dd, 1H, J = 8.68, 2.4 Hz),
7.13 t (2H, J=8.4 Hz); 7.30-7.40 (m, 4H); 9.03 (s, OH). 13C (d6-DMSO, 75 MHz):δ 14.7, 29.4, 30.4,
59.4, 103.7, 105.9, 111.3, 113.0, 116.2, 116.5, 127.0, 129.4, 131.5, 134.8, 134.9, 142.9, 153.4,
163.2, 165.0. M.p. 194°C. ESI-MS [M + Na+]: 384.21 Anal. calcd for C19H18FNO3S: C, 63.49; H,
5.05; F, 5.29; N, 3.90; O, 13.35; S, 8.92, Found: C, 63.11; H, 4.96; F, 5.12; N, 3.52; O, 13.09; S,
8.43.
4.2.8 Ethyl 2-((3-chlorophenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate (14)
Elution with hexane/EtOAc (8:2) afforded 14 (90 %). 1H NMR (d6-DMSO, 300 MHz): δ 1.29 (t, 3
H, J = 6.98 Hz); 3.70 (s, 3 H); 4.10 (q, 2H, J = 6.98 Hz); 4.82 (s, 2H); 6.75 (dd, 1H, J = 8.6, 2.4 Hz),
7.33 (d, 2H, J= 8.8 Hz) 7.35-7.37 (br, 4H) 9.05 (s, OH). 13C (d6-DMSO, 75 MHz):δ 14.8, 28.7,

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30.5, 59.5, 103.7, 105.9, 111.3, 113.2, 127.0, 129.4, 131.6, 132.5, 133.1, 134.1, 142.5, 153.6, 165.0.
M.p. 195°C. ESI-MS [M + Na+]: 398.87 Anal. calcd for C19H18ClNO3S: C, 60.71; H, 4.83; Cl, 9.43;
N, 3.73; O, 12.77; S, 8.53, Found: C, 60.12; H, 5.32; Cl, 8.71; N, 3.08; O, 12.02; S, 8.27.
4.2.9 Ethyl 2-((2,4-difluorophenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate
(15)
1
Elution with hexane/EtOAc (8:2) afforded 15 (87 %). H NMR (d6-DMSO, 300MHz): δ 1.26 (t, 3
H, J = 6.9 Hz); 3.72 (s, 3 H); 4.10 (q, 2H, J = 6.9 Hz); 4.72 (s, 2H); 6.73- (dd, 1H, J= 2.3, 8.8 Hz),
6.98 (dt, 1 H, J = 2.3, 8.4 Hz); 7.23-7.38 (m, 4 H), 9.01 (s, 1H).. 13C (d6-DMSO, 75 MHz):δ 14.7,
28.8, 30.3, 59.3, 103.7, 104.8 (t, J=26Hz), 105.9, 111.3, 112.4 (dd, J=3.8, 22 Hz), 113.1, 127.0,
131.6, 137.8,(d, J=10Hz), 142.4, 153.3, 163.0 (d, J=230Hz), 164.8. M.p. 173 °C. ESI-MS [M +
Na+]: 400.40 Anal. calcd for C19H17 F2NO3S: C, 60.47; H, 4.54; F, 10.07; N, 3.71; O, 12.72; S, 8.50
Found: C, 59.97; H, 4.03; F, 9.87; N, 3.01; O, 12.55; S, 8.78.
4.2.10 Ethyl 2-((2,6-dichlorophenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate
(16)
1
Elution with hexane/EtOAc (8:2) afforded 16 (74 %). H NMR (d6-DMSO, 300MHz): δ 1.23 (t, 3
H, J = 6.9 Hz); 3.75 (s, 3 H); 4.01 (q, 2H, J = 6.9 Hz); 4.75 (s, 2H); 6.72 (dd, 1H, J = 88, 2.6 Hz),
7.28 (d, 1H, J=2.4 Hz); 7.30-7.38 (m, 2 H); 7.45 (s, 1H); 7.48 (d, 1 H, J = 2.4 Hz); 9.05 (s, OH)..
13C (d6-DMSO, 75 MHz):δ 14.8, 28.7, 30.5, 59.5, 103.7, 105.9, 111.3, 113.2, 127.0, 129.4, 131.6,
132.5, 133.1, 134.1, 142.5, 153.6, 165.0. M.p. 198 °C. ESI-MS [M + Na+]: 410.32 Anal. calcd for
C19H17Cl2NO3S: C, 55.62; H, 4.18; Cl, 17.28; N, 3.41; O, 11.70; S, 7.81, Found: C, 55.22; H, 4.48;
Cl, 17.18; N, 3.91; O, 12.05; S, 7.03.
4.2.11 Ethyl 2-((2,6-dimethylphenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate
(17)
Elution with hexane/EtOAc (8:2) afforded 17 (74 %). 1H NMR (d6-DMSO, 300MHz): δ 1.39(t, 3 H,
J = 7.1 Hz); 2.26 (s, 6H); 3.81 (s, 3 H); 4.22 (q, 2H, J = 7.1 Hz); 4.72 (s, 2H); 6.88 (dd, 1H, J = 8.7,

ACCEPTED MANUSCRIPT
2.4 Hz), 7.20-7.38 (m, 4 H); 7.57 (d, 1 H, J = 2.4 Hz); 9.05 (s, OH). 13C (d6-DMSO, 75 MHz):δ
15.99, 22.34, 29.46, 31.71, 60.63, 104.82, 107.12, 112.49, 114.20, 128.31, 129.52, 130.09, 132.68,
135.80, 144.25, 154.61, 166.28. M.p. 158 °C. ESI-MS [M + Na+]: 392.32 Anal. calcd for
C21H23NO3S: C, 67.52; H, 6.18; N, 3.41; O, 11.90; S, 7.89, Found: C, 68.27; H, 6.27; N, 3.79; O,
12.09; S, 7.06.
4.2.12 Ethyl 2-((3,5-dimethylphenylthio)methyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate
(18)
Elution with hexane/EtOAc (8:2) afforded 18 (99 %). Elution with hexane/EtOAc (8:2) afforded 19
(91 %). 1H NMR (CDCl3, 300MHz): δ 1.39 (t, 3 H, J = 7.1 Hz); 2.23 (s, 6H); 3.66 (s, 3 H); 4.22 (q,
2H, J = 7.1 Hz); 4.70 (s, 2H); 6.97- (s, 2H), 7.20 (d, 1 H, J = 9 Hz); 7 53 (d, 1 H, J = 2.1 Hz).13C
(CDCl3,, 75 MHz):δ: 15.99, 22.34, 29.46, 31.71, 60.63, 104.82, 107.12, 112.49, 114.20, 128.31,
129.52, 130.09, 132.68, 135.80, 144.25, 154.61, 166.28. M.p. 170°C. ESI-MS [M + Na+]: 392.48
Anal. calcd for C21H23NO3S: C, 68.27; H, 6.27; N, 3.79; O, 12.99; S, 8.68, Found: C, 68.01; H,
6.08; N, 3.61; O, 12.01; S, 8.55.
4.2.13 Ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19)
Elution with hexane/EtOAc (8:2) afforded 19 (91 %). 1H NMR (d6-DMSO, 300MHz): δ 1.39(t, 3 H,
J = 7.1 Hz); 2.22 (s, 3H); 2.26 (s, 6H); 3.58 (s, 3 H); 4.22 (q, 2H, J = 7.1 Hz); 4.42 (s, 2H); 6.82-
6.88 (m, 2H), 7.20-7.38 (m, 4 H, J = 0.9, 8.7 Hz); 7 53 (d, 1 H, J = 0.9 Hz), 9.05 (s, OH). 13C (d6-
DMSO, 75 MHz):δ 15.99, 22.34, 23.6, 29.46, 31.71, 60.63, 104.82, 107.12, 112.49, 114.20, 128.31,
129.52, 130.09, 132.68, 135.80, 144.25, 154.61, 166.28. M.p. 169°C. ESI-MS [M + Na+]: 406.50
Anal. calcd for C22H25NO3S: C, 68.90; H, 6.57; N, 3.65; O, 12.52; S, 8.36, Found: C, 68.16; H,
6.15; N, 3.99; O, 12.34; S, 8.12.
4.3. Biological evaluation and assay systems
4.3.1. Materials

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CDC was from Enzo Life Sciences (Lörrach, Germany). Zileuton was from Sequoia Research
Products (Oxford, UK). Indomethacin, celecoxib and all other fine chemicals were from Sigma-
Aldrich (Deisenhofen, Germany) unless stated otherwise.
4.3.2. Cells and isolation
PMNL were freshly isolated from buffy coats obtained from the Institute of Transfusion Medicine,
University Hospital Jena as described [18]. Briefly, human peripheral blood was collected in
heparinized tubes (16 I.E. heparin/ml blood) by venipuncture from fasted (12 h) adult healthy
volunteers, with consent, and leukocyte concentrates were prepared by centrifugation (4000 × g, 20
min, 20 °C). The subjects had no apparent inflammatory conditions and had not taken anti-
inflammatory drugs for at least ten days prior to blood collection. PMNL were immediately isolated
by dextran sedimentation and centrifugation on Nycoprep cushions (PAA Laboratories, Linz,
Austria) and hypotonic lysis of erythrocytes was performed as described [19]. PMNL were finally
resuspended in PBS pH 7.4 containing 1 mg/ml glucose and 1 mM CaCl2 (PGC buffer) (purity >
96-97%).
4.3.3. Determination of 5-LO, 12-LO, and 15-LO product formation in cell-based assays.
For assays of 5-LO, 12-LO and 15-LO, freshly isolated PMNL (5 × 106) were resuspended in 1 ml
PGC buffer. After pre-incubation with the test compounds (15 min, 37 °C), 5-LO product formation
was started by addition of 2.5 µM A23187 plus 20 µM AA. After 10 min at 37 °C, the reaction was
stopped with 1 ml of methanol and 30 µl of 1 N HCl, 200 ng PGB1 and 500 µl of PBS were added.
Formed LO metabolites were extracted and analyzed by HPLC as described [16]. 5-LO product
formation is expressed as ng of 5-LO products per 106 cells, which includes LTB4 and its all-trans
isomers, 5(S),12(S)-di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-DiHETE), and
5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-H(p)ETE). Cysteinyl LTs C4, D4
and E4 were not detected, and oxidation products of LTB4 were not determined. The biosynthesis of
the 12-LO product 12(S)-hydro(pero)xy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid (12-H(P)ETE)
and of the 15-LO product 15(S)-hydro(pero)xy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-
H(P)ETE) is expressed as ng per 106 cells.

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4.3.4. Expression and purification of human recombinant 5-LO from E. coli, and
determination of 5-LO activity in cell-free systems.
Transformation of E.coli BL21 with pT3-5LO plasmid as well as expression and purification of
human recombinant 5-LO protein was performed as described [19]. Purified 5-LO was immediately
used for 5-LO activity assays. Thus, 0.5 µg purified 5-LO were diluted with PBS/EDTA (final
volume 1 ml) and pre-incubated with the test compounds. After 5 – 10 min at 4 °C, samples were
pre-warmed for 30 s at 37 °C, and 2 mM CaCl2 plus the indicated amounts of AA were added to
start 5-LO product formation. After 10 min at 37 °C the reaction was stopped by addition of 1 ml
ice-cold methanol and the formed metabolites were analyzed by HPLC as described above for intact
cells.
4.3.5 Activity assays of isolated COX-1 and COX-2
Inhibition of the activities of isolated ovine COX-1 and human COX-2 was performed as described
[20]. Briefly, purified COX-1 (ovine, 50 units) or COX-2 (human recombinant, 20 units) were
diluted in 1 ml reaction mixture containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5 µM
haemoglobin, and 100 µM EDTA at 4 °C and pre-incubated with the test compound for 5 min.
Samples were pre-warmed for 60 s at 37 °C, and AA (5 µM for COX-1, 2 µM for COX-2) was
added to start the reaction. After 5 min at 37 °C, the reaction was stopped, PGB1 as standard added
and 12-HHT was extracted and then analyzed by HPLC. Indomethacin (10 µM) and celecoxib (5
µM) were used as well-recognized reference inhibitors of COX-1 and of COX-2, respectively, to
control the assays.
4.4. Statistics Data are expressed as mean ± S.E.M. IC50 values were calculated by nonlinear
regression using SigmaPlot 9.0 (Systat Software Inc., San Jose, USA) one site binding competition.

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References
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[2]O. Radmark, O. Werz, D., B. Steinhilber, Samuelsson. 5-Lipoxygenase: regulation of expression and enzyme
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[3]M. Back, S.E. Dahlen, J.M. Drazen, J.F. Evans, C.N. Serhan, T. Shimizu, T. Yokomizo, G.E. Rovati.
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[6]E.M. Karg, S. Luderer, C. Pergola, U. Buhring, A. Rossi, H. Northoff, L. Sautebin, R. Troschutz, O. Werz.
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inhibitors of human 5-lipoxygenase, J Med Chem 52 (2009) 3474-3483.
[7]R. Filosa, A. Peduto, P. Aparoy, AM Schaible, S. Luderer, V. Krauth, C. Petronzi, A. Massa, M. de Rosa, P.
Reddanna, O. Werz. Discovery and biological evaluation of novel 1,4-benzoquinone and related resorcinol
derivatives that inhibit 5-lipoxygenase. Eur J Med Chem 67, (2013) Sep;269-79.
[8]A.M. Schaible, H. Traber, V. Temml, SM Noha, R. Filosa, A. Peduto, C. Weinigel, D. Barz, D. Schuster, O.
Werz. Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E₂ synthase-1 by the anti-
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[9]C. Petronzi, R. Filosa, A. Peduto, MC Monti, L. Margarucci, A. Massa, S.F. Ercolino, V. Bizzarro, L. Parente, R.
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[13]G. W. Carter, P. R. Young, D. H. Albert, J. Bouska, R. Dyer, R. L. Bell, J. B. Summers, D.W. Brooks. 5-
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3 (2008) 975-982

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O
O
HO
NH
N
H
Cl
Figure 1
Figure 2

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Figure 3

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O
O
HO
R
n
N
X
5-LO activity;
cell-free
5-LO activity;
intact PMNL
Cpd
R:
n
X
Remaining
Remaining activity
IC50 (µM)
activity
IC50 (µM)
at 10 µM (%)
at 10 µM (%)
HO
NH2
O
Zileuton
1
N
6.3 ± 2.7
0.6
18.1 ± 2.9
0.7
S
3-Cl
-H
0
1
1
1
1
1
1
1
1
1
1
1
1
1
NH
S
4.9 ± 2.7
40.7 ± 7.6
21.9 ± 5.8
18.4 ± 3.3
33.9 ± 6.3
35.9 ± 5.6
34.4 ± 6.1
41.2 ± 7.6
45.3 ± 7.1
37.4 ± 6.2
33.9 ± 7.4
35.9 ± 7.4
10.5 ± 2.1
14.7 ± 6.2
0.3
5.7
2.9
2.3
0.9
0.8
4.2
5.0
3.8
3.2
0.8
0.8
1.8
0.7
12.5 ± 3.9
25.6 ± 9.7
30.6 ± 12.0
16.2 ± 5.7
36.6 ± 5.6
15.2 ± 7.7
53.2 ± 8.0
48.5 ± 6.2
71.0 ± 1.7
45.7 ± 3.0
20.8 ± 8.4
9.3 ± 1.4
2.4
2.4
5.1
3.1
2.1
1.2
>10
9.1
>10
6.8
0.9
1.2
2.7
0.23
2
7
2-OCH3
4-OCH3
4-OCF3
2-CF3
S
8
S
9
S
10
11
12
13
14
15
16
17
18
19
S
4-CF3
S
4-F
S
3-Cl
S
2,4-F
S
2,6-Cl
2,6-CH3
3,5-CH3
2,4,6-CH3
S
S
S
11.8 ± 4.4
5.9 ± 1.8
S
Table 1

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Compound 12-LO activity;
intact PMNL
15-LO activity;
intact PMNL
Remaining activity Remaining activity
at 10 µM (%)
at 10 µM (%)
92.4 ±4.3
130.0 ± 20.3
7
107.6 ± 17.1
92.1 ± 12.3
94.1 ± 5.4
136.2 ± 21.7
128.3 ± 20.3
107.1 ± 11.0
125.8 ± 29.0
113.9 ± 10.6
120.4 ± 13.8
114.0 ± 5.8
101.8 ± 6.0
135.8 ± 24.6
125.3 ± 20.3
149.7 ± 25.6
33.0 ± 5.1
8
9
10
11
12
13
14
15
16
17
18
19
103.5 ± 17.6
102.4 ± 12.1
100.0 ± 9.9
92.9 ± 6.3
91.4 ± 4.1
102.6 ± 10.0
94.1 ± 12.3
107.4 ± 11.4
86.5 ± 12.8
(IC50 = 8.2 µM)
11.1 ± 3.7
5.6 ± 0.9
CDC
Table2

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O
O
O
O
O
O
O
O
HO
b
a
O
N
O
N
H
N
H
5
3
4
O
O
O
O
O
Br
HO
d
c
R
O
N
N
S
6
7
R=H
8
9
R= 2-OCH3
R= 4-OCH3
10 R= 4-OCF3
11 R= 2-CF3
12 R= 4-CF3
13 R= 4-F
14 R= 3-Cl
15 R= 2,4-F
16 R= 2,6-Cl
17 R= 2,6-CH3
18 R= 3,5-CH3
19 R= 2,4,6-CH3
Scheme 1

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Figure and Scheme legends
Figure 1: Chemical Structure of compound 2 [ethyl 2-[(3-chlorophenyl)-amino]-5-hydroxy-1H-
indole-3-carboxylate].
Figure 2: Inhibition of the activity of isolated 5-LO by 19 at low (5 µM) and high (50 µM) AA
concentration. Data are given as mean +/- SEM, n=3.
Figure 3: Inhibition of the activity of isolated COX-1 and COX-2 by 16 and 19. Data are given as
mean +/- SEM, n=3. Reference inhibitor COX-1: indomethacin, 10 µM: 6 +/- 2% residual activity;
reference inhibitor COX-2: celecoxib, 5µM: 22 +/- 8% residual activity.
Table 1. Inhibition of 5-LO activity in cell-free assays and in PMNL incubations. Data are given as
mean ± S.E.M., n = 3-4.
Table 2. Inhibition of 12-LO and 15-LO activity in PMNL incubations by test compounds at 10
µM. Data are given as mean ± S.E.M., n = 3-4. Cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC),
10 µM, was used as reference.
Scheme 1: Synthesis of compounds 7-19. Reagents and conditions: a) acetic anhydride, pyridine,
1h, reflux, 74%; b) CH3I, NaH 60%, DMF, 1h, rt, 70%, c) NBS, benzoyl peroxide, CCl4, o.n. ,
reflux, 46%; d) opportune tiophenol, KOH, CH3OH, 3h, rt.

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Highlights
We described SAR analysis of novel series of ethyl 5-hydroxyindole-3-carboxylate,
We examined changes in positioning of the substituents at the phenylthiomethyl ring,
We studied the inhibitory activities on 5-LO product synthesis
We determined the inhibitory activities of the major AA-metabolizing enzymes with the most
active derivative.

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Supporting Information
Further Studies on Ethyl 5-hydroxy-indole-3-carboxylate
scaffold: Design, Synthesis and Evaluation of 2-
phenylthiomethyl-Indole Derivatives as Efficient Inhibitors
of Human 5-Lipoxygenase
Antonella Peduto, Ferdinando Bruno, Friedrike Dehm, Verena Krauth, Paolo de Caprariis, Mario De Rosa,
Christina Weinigel, Dagmar Barz, Antonio Massa, Oliver Werz, Rosanna Filosa
Contents:
I
1H NMR and 13C NMR spectra of selected compounds

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Ethyl 5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3-carboxylate (7)

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Ethyl 5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3-carboxylate (7)

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Ethyl 5-hydroxy-2-((2-methoxyphenylthio)methyl)-1-methyl-1H-indole-3-carboxylate (8)

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Ethyl 5-hydroxy-2-((2-methoxyphenylthio)methyl)-1-methyl-1H-indole-3-carboxylate (8)

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Ethyl 5-hydroxy-2-((4-methoxyphenylthio)methyl)-1-methyl-1H-indole-3-carboxylate (9)