Exploring the role of chloro and methyl substitutions in 2-phenylthiomethyl-benzoindole derivatives for 5-LOX enzyme inhibition

Article abstract of DOI:10.1016/j.ejmech.2015.11.048

Following the results we previously reported on a series of ethyl 2-phenylthiomethyl 5-hydroxyindole-3-carboxylate derivatives as 5-lipoxygenase (5-LOX) inhibitors, in order to obtain a more selective compound with respect to the previous generation of de

Full text of DOI:10.1016/j.ejmech.2015.11.048

European Journal of Medicinal Chemistry 108 (2016) 466e475
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Exploring the role of chloro and methyl substitutions in 2-
phenylthiomethyl-benzoindole derivatives for 5-LOX enzyme
inhibition
Antonella Peduto a, Verena Krauth b, Selene Collarile a, Fiederike Dehm b,
Marika Ambruosi a, Carmela Belardo a, Francesca Guida a, Antonio Massa c,
Veronica Esposito d, Sabatino Maione a, Mario de Rosa a, Oliver Werz b, Rosanna Filosa a
,
*
a Department of Experimental Medicine, Second University of Naples, Via Costantinopoli, 16 e 80138 Naples, Italy
b Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany
c Department of Chemistry, University of Salerno, Via Ponte don Melillo, 84084 Fisciano, SA, Italy
d
ꢀ
Dipartimento di Farmacia, Universita degli Studi di Napoli Federico II, Via D. Montesano 4980131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Following the results we previously reported on a series of ethyl 2-phenylthiomethyl 5-hydroxyindole-3-
carboxylate derivatives as 5-lipoxygenase (5-LOX) inhibitors, in order to obtain a more selective com-
pound with respect to the previous generation of derivatives, we decided to modify the structure of the
core ligand.
The first level of structural modification involved the annelation of benzene to the indole, yielding
corresponding benzo[g]indole derivatives, systematic optimization of methyl or chlorine groups in meta-
, ortho- and ortho/para-position of 2-phenylthiomethyl moiety were applied. The reported results show
that extension of the aromatic core led to a great enhancement of activity, especially in cell-free assay,
and the accurate structure-based design provided compounds 6f, 6g and 6l that block 5-LOX activity in
Received 16 September 2015
Received in revised form
26 November 2015
Accepted 27 November 2015
Available online 7 December 2015
Keywords:
5-lipoxygenase
Leukotriene
Indoles
cell-free assays with IC50 ranging from 0.17 to 0.22
morphonuclear leukocytes with IC50 ranging from 0.19 to 0.37
m
M, and suppress 5-LOX product synthesis in poly-
M. Moreover we have identified 6f and 6l
m
as dual 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors and
compound 6l significantly reduces inflammatory reactions in the carrageenan-induced mouse paw
oedema. The reported in vivo analysis, together with the accessible synthetic procedure, stimulate for the
generation of further potent antinflammatory benzoindoles-based agents.
© 2015 Published by Elsevier Masson SAS.
1. Introduction
numerous studies have demonstrated the overexpression of 5-LOX
in tissue samples of primary tumor cells as well as in established
In the last decades, several studies have demonstrated the key
role played by 5-lipoxygenase (5-LOX) in inflammation-related
disorders. Leukotrienes (LT), formed from AA by catalysis of 5-
LOX, are fast reacting pro-inflammatory mediators of the immune
system [1,2]; besides their physiological roles, they primarily
mediate inflammatory and allergic reactions [3] and are involved in
the onset of inflammatory diseases such as asthma, allergic rhinitis,
rheumatoid arthritis and also cardiovascular disorder [4]. However,
cancer cell lines [5]. An increasing body of evidence suggests a
crucial role for 5-LOX products in the early stage of pancreatic,
prostate and colorectal carcinogenesis [6] and glioma cell lines [7].
Recent studies showed that 5-LOX expression appears to be upre-
gulated in patients with neurodegenerative disease like AD,
demonstrating the key role of leukotrienes promoting Ab genera-
tion [8e10]. In other reports, the potent and selective 5-LOX in-
hibitor zileuton reduced the amyloid and tau pathology as well as
memory impairments in different mouse models of AD [11,12].
Continuing our studies on small molecules able to block 5-LOX
activity [13e18], we recently reported the design and synthesis of
a small collection of differently decorated 5-hydroxyindole-3-
carboxylates derivatives able to interact with 5-LOX at nanomolar
* Corresponding author. Department of Experimental Medicine, Second Univer-
sity of Naples, Via Costantinopoli, 16 e 80138 Naples, Italy.
E-mail address: rosanna.filosa@unina2.it (R. Filosa).
http://dx.doi.org/10.1016/j.ejmech.2015.11.048
0223-5234/© 2015 Published by Elsevier Masson SAS.

A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
467
O
O
concentration [14]. Our previous investigations have disclosed the
SH
influence of the substitution pattern on the phenylthiomethyl
moiety, the simultaneous presence of chlorine in ortho-positions of
the thiophenol ring as well as the introduction of three methyl
groups (ortho, para) that led to compound with marked increase of
5-LOX inhibitory activity [14]. Among all tested compounds, ethyl
5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-
S
OEt
a
b
R
4a-l
R
3a-l
O
carboxylate (1) showed good potency in inhibiting 5-LOX activity in
OEt
cell-free assays with IC50 ¼ 0.7
m
M and suppressed 5-LOX product
NH2
O
HO
synthesis in polymorphonuclear leukocytes with IC50 ¼ 0.23
m
M,
S
being equally potent to the well-recognized reference inhibitor
zileuton, used as antiasthmatic drug in the clinics. With regard to
other AA-metabolizing enzymes like COX-1, COX-2 and 12-LO,
compound 1 was rather selective for 5-LOX with minor effects also
on 15-LOX. Moreover, our previous works reported that the anne-
lation of a [g]benzene ring to the indole led to more potent in-
hibitors, exemplified by compounds 2a and 2b with IC50 of 0.086
c
OEt
N
H
S
R
5a-l
R
6a R: 3-Cl
6b R: 2-Cl
6c R: 2-CH3
6d R: 2,3-Cl
6e R: 3,4-Cl
6f R: 2,6-Cl
6g R: 3,5-Cl
6h R: 2,6-CH3
6i R: 3,5-CH3
and 0.097 mM in cell-free and 0.23 and 1.2 mM in cell-based assays,
respectively [19,20] (Fig. 1). We showed also that benzo[g]indol-3-
carboxylates potently inhibit mPGES-1 and thus represent a novel
class of dual 5-LO/mPGES-1 inhibitors.
6l R: 2,4,6-CH3
With the goal of increasing potency and establishing a better
understanding of the pharmacophore for 5-LO inhibition, we
focused our investigation on the effects of introduction one or more
chlorine and methyl substituents on the benzoindole ring. More-
over, introduction of a methoxy or ethoxy group at C5 position and
N alkylation of the indole nitrogen atom (7a-b, 8a-b and 11) were
also carried out in order to investigate the effect of OH and NH on
the potency against 5-LOX.
Here we report the synthesis and the pharmacological profile of
this second generation of benzoindole derivatives with anti-
inflammatory effectiveness in vivo. While further studies are
certainly needed to better describe the exact anti-inflammatory
mode of action, this novel focused small molecules library exerts
new interesting hits shedding further light on the structural re-
quirements needed for an optimal ligandeenzyme interaction.
Scheme 1. Synthesis of compounds 6a-l. Reagents and conditions: a) 4-
chloroacetoacetate, NEt3, CH2Cl2,
ꢀC, 30 min; b) NH4OAc, HOAc toluene, reflux, 6h;
c) 1,4-naphtoquinone, ZnI2, CH2Cl2, reflux, 40 min.
0
under basic conditions using microwave irradiation. Protection of
the hydroxy group of 6b with tertbutyl dimethyl silyl chloride
(TBDSCl), alkylation with iodomethane and final deprotection with
TBAF in THF allowed to obtain desired compound 11 in good yield
(Scheme 2) (see Scheme 3)
2.2. Evaluation of 5-LOX activity and structure-activity
Relationships
In order to assess the effects of the synthesized compounds on
5-LOX product synthesis, a cell-free assay using isolated human
recombinant 5-LOX and a cell-based assay using human neutro-
phils was applied. The cell-free assay allows identifying compounds
that directly interfere with 5-LOX catalytic activity, whereas the
cell-based test system considers cellular regulatory aspects of 5-
LOX 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-LOX-activating lipid hydroper-
oxides, protein kinases or Ca2þ mobilization, and 5-LOX trans-
location/membrane association) [21]. The reference 5-LOX
inhibitor N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (zileuton)
was used to control the 5-LOX activity assays. As shown in Table 1,
all tested compounds (6a-l) which maintain OH and NH groups in
2. Results and discussion
2.1. Chemistry
The synthesis of desired compounds 6a-l (Scheme 1) has been
accomplished in three steps of reaction. The final Nenitzescu re-
action has proven to be the simplest entry into 5-hydroxy-1H-
benzo[g]indoles. Essentially, stirring of the enaminoesters 5a-l with
1.0 equiv of 1,4-naphthoquinone in presence of catalytic amount of
ZnI2 at room temperature led to the expected 5-hydroxy-1H-benzo
[g]indoles. Nenitzescu reactants 5a-l were prepared starting from
thiophenols 3a-l, which were first reacted with ethyl 4-
chloroacetoacetate to give the b-ketoesters 4a-l. b-Ketoesters 4a-l
were converted into their appropriate enaminoesters 5a-l by
refluxing them in toluene with an excess of ammonium acetate. 7a-
b and 8a-b were prepared by alkylation of 6b with iodoalcanes
O
O
OEt
OEt
RO
HO
O
O
N
a
S
N
S
O
O
H
R1
HO
HO
Cl
R
Cl
N
N
S
H
7a R= CH3, R1= H
R1
6b
7b R= CH3, R1= CH3
8a R= CH2CH3, R1= H
8b R= CH2CH3, R1= CH2CH3
2a: R=H, R1= Cl
2b: R=Cl, R1= H
1
Scheme 2. Synthesis of compounds 7a-b and 8a-b. Reagents and conditions: a)
Fig. 1. Chemical structure of compounds 1, 2a and 2b.
Iodomethane or iodoethane K2CO3, Acetone, MW, 30 min, 130 ꢀC.

468
A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
O
imply the presence of a bulky hydrophobic pocket in the binding
O
OEt
OEt
site of enzyme which allows the arrangement of two chlorines.
Considering IC50 values in cell-based assay, we observed a similar
activity for all polychlorinated derivatives especially for 6e, 6f and
6g, while 6d is slightly less active. This could be due a supple-
mentary mechanism of inhibition of 5-LOX product synthesis, by
interaction with other targets (i.e. FLAP or PLA2).
O
HO
Si
a
N
S
b
N
S
H
H
Cl
Cl
In the case of polymethylated molecules 6h, 6i and 6l, they
showed a comparable activity in cell-based conditions with IC50 of
9
6b
O
O
OEt
0.17, 0.29 and 0.37
mM, respectively; otherwise only compound 6l
OEt
was active against isolated 5-LOX with an IC50 of 0.2
mM. The
HO
O
alkylation of the hydroxy group of compound 6b led to compounds
7a and 8a which demonstrated a significant decrease of activity,
c
Si
N
S
N
S
especially in cell-based assays (IC50 ¼ 1.15
8b vs 0.182 M for 6b). The di-alkylation of OH and NH groups is
detrimental for the activity, in fact, compounds 7b and 8b showed
an IC50 > 10 M. The N-methyl derivative of 6b, i.e. compound 11, is
completely inactive with IC50 values > 10 M in both assays
mM for 7a and 1.26 mM for
m
Cl
Cl
m
10
11
m
(Table 2). Unlike for indoles studied in our previous work [14], these
data demonstrate that in benzoindole derivatives the substitution
of hydrogen of the indole nitrogen with an alkyl residue is not
tolerated, maybe for a different positioning of molecule in the
active site.
Scheme 3. Synthesis of compound 11. Reagents and conditions: a) Imidazole, TBDSCl,
CH2Cl2, 3h, rt; b) NaH, dry THF, 2h, 0 ꢀC; c) TBAF 1M in THF, THF, 10 min, 0 ꢀC.
their structure showed a significant activity in inhibiting 5-LOX in
cell-based assay with IC50 values in the low micromolar range while
in cell-free test, the substituent plays a key role in the activity.
The corresponding 2-phenylthiomethyl benzoindoles of de-
rivatives 2a and 2b, 6a and 6b retain a good activity in cell-based
assay, especially for derivative 6b which is 6-fold more active
than 2b but they showed a decrease of potency in cell-free test. The
replacement of chlorine in position 2 with a methyl (6c vs 6b) does
2.3. Anti-inflammatory effectiveness of derivatives 1, 6f, 6g, 6l in
carrageenan-induced paw oedema
In order to verify the anti-inflammatory efficacy of the most
interesting compounds of the series, we evaluated their effect on
carrageenan-induced paw oedema, which represents a well-
established model of acute inflammation [22]. Many 5-LOX and
FLAP inhibitors that potently repressed 5-LOX product synthesis in
isolated cells failed to do so in carrageenan-induced acute inflam-
mation known as accompanied by elevated LT levels. Intraplantar
injection of carrageenan led to an increase in hind paw volume,
expressed as oedema, monitored for a period of 6 h. The increase in
paw volume (vehicle, i.p. treatment) reached a maximum at 4 h
post-carrageenan application (Fig. 2). Indole derivative 1 that pre-
viously showed potent inhibition of 5-LO in cell-based
not significantly alter the efficiency in intact cells (IC50 ¼ 0.18
and 0.26 M respectively) while an improvement of activity is
observed in the cell-free assay (0.33 vs 0.82 M). With regard to
mM
m
m
polychlorinated derivatives, the position of the substituent is
fundamental for the activity, especially in the cell-free assay. In fact,
compounds 6f (2,6-Cl) and 6g (3,5-Cl) showed the best activity in
inhibiting 5-LOX, with IC50 values of 0.17 and 0.22
mM, respectively,
while the activity of compounds 6d (2,3 Cl) and 6e (3,4 Cl) is very
low, in particular for 6d with an IC50 > 1 mM. These results could
Table 1
Inhibition of 5-LOX activity of compounds 6a-l, 2a-b and zileuton in a cell-based assay (intact PMNL) and in a cell-free assay. Data are given as mean S.E.M., n ¼ 3e4.
5-LOX activity; cell-free 5-LOX activity; intact PMNL
Cpd
6a
6b
6c
6d
6e
6f
6g
6h
6i
R
3-Cl
2-Cl
n
1
1
1
1
1
1
1
1
1
1
0
0
X
S
S
S
S
S
S
S
S
S
S
CH2
CH2
IC50
>1
0.82 0.03
0.33 0.15
>1
0.93 0.03
0.17 0.11
0.22 0.07
>1
>1
(
m
M)
Remaining activity at 1
63.8
m
M (%)
IC50
(
m
M)
Remaining activity at 1 mM (%)
5
0.257 0.1
0.182 0.09
0.255 0.05
0.657 0.07
0.237 0.09
0.19 0.09
0.24 0.12
0.17 0.12
0.29 0.07
0.37 0.07
0.23 0.0719
1.2 0.0719
0.714
2-CH3
2,3-Cl
3,4-Cl
2,6-Cl
3,5-Cl
2,6-CH3
3,5-CH3
2,4,6-CH3
3-Cl
72 14.8
65.9 5.7
63 1.7
6l
2a
2b
Zileuton
0.2 0.06
0.086 0.0219
0.097 0.0919
0.614
2-Cl

A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
469
Table 2
Inhibition of 5-LOX activity of compounds 7a-b, 8a-b, 11 vs 6b in a cell-based assay (intact PMNL) and in a cell-free assay. Data are given as mean S.E.M., n ¼ 3e4.
5-LOX activity;
Cell-free
5-LOX activity;
Intact PMNL
Cpd
R0
R00
IC50
Remaining activity at 10
98.7 2.3
mM (%)
IC50
(mM)
Remaining activity at 10
97.6 1.5
mM (%)
(
m
M)
6b
7a
7b
8a
8b
11
H
CH3
CH3
CH2CH3
CH2CH3
H
H
H
0.82 0.03
1.74 0.13
>10
2.04 0.139
>10
0.182 0.09
1.15 0.12
>10
1.26 0.084
>10
CH3
H
CH2CH3
CH3
99.4 9.3
81.4 13.2
95.5 12.2
61.9 12.3
>10
>10
(IC50 ¼ 0.23
mM) and cell-free assays (IC50 ¼ 0.7
mM) was used as
lead structure [14]. We observed that 1 (1, 2 and 4 mg/kg, i.p.)
significantly reduced paw oedema during the 2e4 h period in a
dose-dependent manner (Fig. 2). Futhermore, in agreement with
the results from intact cells, the corresponding benzoindole de-
rivative of 1, compound 6l was more efficient in the in vivo test
(Fig. 3). Among the benzo[g]indol-3-carboxylates tested (see
Table 1), 6f and 6g were the most efficient to inhibit LTB4 formation
in cell-free assays and maintained their potency in intact PMNL.
In vivo experiment 6f failed to evoke significant changes, while 6g
(1, 2 and 4 mg/kg, i.p.) decreased oedema, as compared with
vehicle-treated animals (Fig. 4). In fact, in mice treated with 4 mg/
kg 1, 6f, 6g or 6l, the peak of the response to carrageenan at 4 h was
reduced by 60.9%, 9.1%, 60%, and 77.4%, respectively, while zileuton
(20 mg/kg, i.p.), used as reference, caused only 38.2% inhibition.
Fig. 3. Effect of compounds 1 (4 mg/kg, i.p.), 6l (4 mg/kg, i.p.) and zileuton (20 mg/kg,
i.p.) on carrageenan-induced paw oedema 0e6 h after carrageenan injection. Data are
expressed as mean
SEM (n ¼ 6e8 for each group). * or *** indicate significant
(P < 0.05 or P < 0.001, respectively) differences vs carrageenan þ vehicle-treated
2.4. Screening of indole-3-carboxylates 1, 6f, 6g, 6l as direct mPGES-
1inhibitors
animals.
Based on the potent anti-inflammatory efficacy of 6l in vivo, the
compound may also interfere with other enzymes involved in the
generation of pro-inflammatory eicosanoids. Indeed, it may sup-
press other pro-inflammatory events such as the biosynthesis of
PGE2 as demonstrated for the related benzoindoles [20]. To confirm
this hypothesis it was analyzed for inhibition of PGE2 formation in
Fig. 4. Effect of compounds 6f (4 mg/kg, i.p.) and 6g (4 mg/kg, i.p.) on carrageenan-
induced paw oedema 0e6
mean
P < 0.001, respectively) differences vs carrageenan þ vehicle-treated animals.
h after carrageenan injection. Data are expressed as
SEM (n ¼ 6e8 for each group). * or *** indicate significant (P < 0.05 or
the cell-free assay together with the test compounds(Table 3);
derivatives that achieved more than 50% inhibition at 10
values were assessed.
mM, IC50
Only a moderate inhibition of mPGES-1 activity was observed at
mM (27.8 0.79%) for compound 1, enlargement of the hydro-
phobic core structure by annelation of benzene to the indole of 1,
yielding the corresponding benzo[g]indole derivate 6l, in accor-
10
Fig. 2. Effect of compounds 1 (1, 2, 4 mg/kg, i.p.) on carrageenan-induced paw oedema
0e6 h after carrageenan injection. Data are expressed as mean SEM (n ¼ 6e8 for
each group). * or *** indicate significant (P < 0.05 or P < 0.001, respectively) differences
vs carrageenan þ vehicle-treated animals.
dance with our previous results, led to an IC50 of 1.9 mM. Exchange

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A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
Table 3
Inhibition of m-PGES-1 activity of compounds l, 2a, 2b, 6f, 6g 6l. Data are given as mean S.E.M., n ¼ 3e4.
IC50
(mM)
mPGES-1
Cpd
R
n
X
IC50
(
m
M)
Remaining
activity
a
mM (%)
6f
6g
6l
2a
1
2,6-Cl
3,5-Cl
2,4,6-CH3
3-Cl
1
1
1
0
S
S
S
CH2
1.33 0.10
13.84 0.27
1.93 0.07
57 620
87.33 3.88
of the methyl by a chlorine substituents in o-position slightly
improved the potency (IC50 ¼ 1.33), while variation of the posi-
tioning of the chlorine in the thiol ring of 6g to meta (compound 6f)
4. Experimental section
4.1. Chemistry
was detrimental and led to an IC50 of 13.8 mM.
Although potent inhibition of mPGES-1 under cell-free condi-
tions must not necessarily result in efficient suppression of in vivo
prostanoid formation the high potency on isolated mPGES-1well
correlate the peak of the response to carrageenan at 4 h in vivo of 6l.
All reagents were analytical grade and purchased from Sigma-
eAldrich (Milano, Italy). Flash chromatography was performed on
Carlo Erba silica gel 60 (230e400 mesh; CarloErba, Milan, Italy). TLC
was carried out using plates coated with silica gel 60F 254 nm pur-
chased from Merck (Darmstadt, Germany). 1H 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. Mi-
croanalyses were carried out on a Carlo Erba 1106 elemental
analyzer. Melting points were performed by Stuart melting point
SMP30 and are uncorrected. All microwave experiments were per-
formed in a self-tuning single mode Biotage Initiator Microwave.
3. Conclusion
In this paper a series of new ethyl benzo[g]indole-3-carboxylate
was prepared and tested to evaluate their ability to inhibit 5-LOX
activity. Considering the in vitro biological data we suggest that
the ability to inhibit 5-LOX is closely related to the chemical
structures of the molecules.
Our studies allowed us to draw a tentative SAR profile and to
optimize this series, in particular:
4.2. General procedure for the synthesis of Ethyl 4-(thioaryloxy)
acetoacetates (4a-l)
1. Alkylation of the indole nitrogen causes loss of activity (7b, 8b
and 11).
2. Substitution of hydroxyl group in position 5 of benzoindole ring
with methoxy or ethoxy residues decreases the potency (7a and
8a).
3. Substitutions on the thiophenol ring influences 5-LOX activity.
The simultaneous presence of chlorine in ortho or meta-
positions of the thiophenol ring (6f and 6g), as well as the
introduction of three methyl groups (6l) (ortho, para), causes a
marked increase of 5-LOX inhibitory activity in cell-free assays.
A solution of 4-chloroacetoacetate (500 mg, 3.28 mmol, 1 eq.),
NEt3 (348 mg, 3.44 mmol,1.05 eq.) and appropriate thiophenols 3a-
l (1.03 eq.) in CH2Cl2 (6.5 mL) was stirred at 0 ꢀC for 30 min. The
reaction mixture was diluted with EtOAc and washed with an
aqueous solution of NaOH (1 M), hydrochloric acid (1 M) and brine.
The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed under reduced pressure. The residue was
purified by flash chromatography (silica gel, hexane/EtOAc 9:1).
4.2.1. Ethyl 4-(3-chlorophenylthio)-3-oxobutanoate (4a)
Yield: 81%. 1H NMR data are in agreement with those reported in
literature [23].
Moreover 6g and 6l are able to inhibit mPGES-1 activity in cell-
free and exhibit IC50 values of 1.33 mM and 1.93 mM respectively.
The antiflammatory properties of selected compounds (6f, 6g
and 6l) was confirmed in vivo evaluating their ability to reduce
carrageenan-induced paw oedema. The most interesting com-
pound 6l caused a reduction of oedema of 77.4%, which is superior
over zileuton and indole 1 used as reference compounds.
Future studies addressing their anti-inflammatory effectiveness
in other animal models will reveal the therapeutic potential of
these new benzo[g]indole-3-carboxylates.
4.2.2. Ethyl 4-(2-chlorophenylthio)-3-oxobutanoate (4b)
Yield: 68%. 1H NMR data are in agreement with those reported
in literature [23].
4.2.3. Ethyl 4-(o-tolylthio)-3-oxobutanoate (4c)
Yield: 79%. 1H NMR data are in agreement with those reported in
literature [24].

A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
471
4.2.4. Ethyl 4-(2,3-dichlorophenylthio)-3-oxobutanoate (4d)
Yield: 86%. 1H NMR (CDCl3, 300 MHz)
3.67 (s, 2H); 3.92 (s, 2H); 4.21 (q, 2H, J ¼ 7.34 Hz); 7.19 (m, 2H); 7.34
(d, 1H, J ¼ 7.88 Hz).
7.34 (dd, 1H, J ¼ 2.4, 6.9 Hz).
d
1.29 (t, 3H, J ¼ 7.34 Hz);
4.3.5. (Z)-ethyl 4-(3,4-dichlorophenylthio)-3-aminobut-2-enoate
(5e)
Yield: 62%. 1H NMR (CDCl3, 300 MHz)
d
1.27 (t, 3H, J ¼ 7.34 Hz);
4.2.5. Ethyl 4-(3,4-dichlorophenylthio)-3-oxobutanoate (4e)
Yield: 96%. 1H NMR data are in agreement with those reported in
literature [23].
3.59 (s, 2H); 4.14 (q, 2H, J ¼ 7.34 Hz); 4.64 (s,1H); 7.18 (dd,1H J ¼ 2.1,
8.4 Hz); 7.38 (d, 1H, J ¼ 8.4 Hz); 7.45 (s, 1H).
4.3.6. (Z)-ethyl 4-(2,6-dichlorophenylthio)-3-aminobut-2-enoate
4.2.6. Ethyl 4-(2,6-dichlorophenylthio)-3-oxobutanoate (4f)
Yield: 88%. 1H NMR data are in agreement with those reported in
literature [23].
(5f)
Yield: 73%. 1H NMR (CDCl3, 300 MHz)
d
1.22 (t, 3H, J ¼ 7.34 Hz);
3.63 (s, 2H); 4.08 (q, 2H, J ¼ 7.34 Hz); 4.50 (s, 1H); 7.37 (t, 1H,
J ¼ 7.88 Hz); 7.48 (d, 2H, J ¼ 7.88 Hz).
4.2.7. Ethyl 4-(3,5-dichlorophenylthio)-3-oxobutanoate (4g)
Yield: 62%. 1H NMR data are in agreement with those reported in
literature [23].
4.3.7. (Z)-ethyl 4-(3,5-dichlorophenylthio)-3-aminobut-2-enoate
(5g)
Yield: 59%. 1H NMR (CDCl3, 300 MHz)
d
1.24 (t, 3H, J ¼ 7.34 Hz);
4.2.8. Ethyl 4-(2,6-dimethylphenylthio)-3-oxobutanoate (4h)
Yield: 85%.1H NMR (CDCl3, 300 MHz)
d
1.29 (t, 3H, J ¼ 7.34 Hz);
3.62 (s, 2H); 4.13 (q, 2H, J ¼ 7.34 Hz); 4.70 (s, 1H); 7.21 (d, 3H,
J ¼ 1.9 Hz).
2.59 (s, 6H),; 3.53 (s, 2H); 3.55 (s, 2H); 4.18 (q, 2H, J ¼ 7.34 Hz);
7.09e7.19 (m, 3H).
4.3.8. (Z)-ethyl 4-(2,6-dimethylphenylthio)-3-aminobut-2-enoate
4.2.9. Ethyl 4-(3,5-dimethylphenylthio)-3-oxobutanoate (4i)
(5h)
Yield: 92%. 1H NMR (CDCl3, 300 MHz)
d
1.28 (t, 3H, J ¼ 7.34 Hz);
Yield: 68%. 1H NMR (CDCl3, 300 MHz)
d
1.28 (t, 3H, J ¼ 7.34 Hz);
2.29 (s, 6H); 3.65 (s, 2H); 3.80 (s, 2H); 4.20 (q, 2H, J ¼ 7.34 Hz); 6.88
2.55 (s, 6H); 3.28 (s, 2H); 4.14 (q, 2H, J ¼ 7.34 Hz); 4.43 (s, 1H);
(s 1H); 6.98 (s, 2H).
7.09e7.19 (m, 3H).
4.2.10. Ethyl 4-(mesitylthio)-3-oxobutanoate (4l)
Yield: 86%. 1H NMR (CDCl3, 300 MHz)
d
1.37 (t, 3H, J ¼ 7.34 Hz);
4.3.9. (Z)-ethyl 4-(3,5-dimethylphenylthio)-3-aminobut-2-enoate
(5i)
2.20 (s, 3H); 2.45 (s, 6H); 3.58 (s, 2H); 3.60 (s, 2H); 4.17 (q, 2H,
Yield: 53%. 1H NMR (CDCl3, 300 MHz)
d
1.27 (t, 3H, J ¼ 7.34 Hz);
J ¼ 7.34 Hz); 6.87 (s, 2H).
2.29 (s, 6H); 3.60 (s, 2H); 4.16 (q, 2H, J ¼ 7.34 Hz); 4.65 (s, 1H); 6.87
(s, 1H); 6.98 (s, 2H).
4.3. General procedure for the synthesis of Ethyl 4-(thioaryloxy)
enaminoesters (5a-l)
4.3.10. (Z)-ethyl 3-amino-4-(mesitylthio)but-2-enoate (5l)
Yield: 58%. 1H NMR (CD3OD-d4, 300 MHz)
d 1.20 (t, 3H,
The appropriate b-ketoester 4a-l (3.0 mmol) and ammonium
acetate (1.16 g, 15.0 mmol) were dissolved in dry toluene (10 mL).
After adding four drops of acetic acid, 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 pu-
rified by flash chromatography on silica gel (hexane/Ethyl acetate
9:1).
J ¼ 7.34 Hz); 2.28 (s, 3H); 2.52 (s, 6H); 3.22 (s, 2H); 4.05 (q, 2H,
J ¼ 7.34 Hz); 4.13 (s, 1H); 4.65 (br, NH2); 6.98 (s, 2H).
4.4. General procedure for the synthesis of 5-Hydroxy-1H-benzo[g]
indole-3-carboxylate (6a-l)
To a solution of 1,4-naphthoquinone (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 enaminoesters 5a-l,
respectively (1.0 mmol), in 2 mL of CH2Cl2 was added drop by drop
under stirring for 5e10 min. After refluxing for additional 20 min,
the mixture was cooled to 0e5 ꢀC for 2e3 h. The precipitated
crystals were filtered off and washed with CH2Cl2 and hexane.
4.3.1. (Z)-ethyl 4-(3-chlorophenylthio)-3-aminobut-2-enoate (5a)
Yield: 45%. 1H NMR (CDCl3, 300 MHz)
d
1.28 (t, 3H, J ¼ 7.34 Hz);
3.63 (s, 2H); 4.14 (q, 2H, J ¼ 7.34 Hz); 4.67 (s,1H); 7.20e7.25 (m, 3H);
7.34 (s, 1H).
4.3.2. (Z)-ethyl 4-(2-chlorophenylthio)-3-aminobut-2-enoate (5b)
Yield: 67%. 1H NMR (CDCl3, 300 MHz)
d
1.27 (t, 3H, J ¼ 7.34 Hz);
4.4.1. Ethyl 2-((3-chlorophenylthio)methyl)-5-hydroxy-1H-benzo
[g]indole-3-carboxylate (6a)
3.69 (s, 2H); 4.15 (q, 2H, J ¼ 7.34 Hz); 4.70 (s,1H); 7.18 (td,1H, J ¼ 1.5,
7.2 Hz); 7.24 (td,1H, J ¼ 1.5, 7.2 Hz); 7.33 (dd,1H, J ¼ 1.5, 7.2 Hz); 7.40
(dd, 1H, J ¼ 1.5, 7.2 Hz).
Rf (hexane/ethyl acetate 8:2) ¼ 0.28. Yield: 4%. M.p. 167.5 ꢀC dec.
1H NMR (DMSO-d6, 300 MHz)
d
1.35 (t, 3H, J ¼ 7.34 Hz); 4.28 (q, 2H,
J ¼ 7.34 Hz); 4.77 (s, 2H); 7.25e732 (m, 3H); 7.40e747 (m, 2H); 7.51
(s, 1H); 7.55 (t, 1H, J ¼ 6.61 Hz); 8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H,
J ¼ 7.80 Hz); 9.69 (s, 1H, OH); 12.39 (s, 1H, NH). 13C NMR (DMSO-d6,
4.3.3. (Z)-ethyl 4-(o-tolylthio)-3-aminobut-2-enoate (5c)
Yield: 87%. 1H NMR (CDCl3, 300 MHz)
d
1.23 (t, 3H, J ¼ 7.34 Hz);
2.38 (s, 3H); 3.67 (s, 2H); 4.12 (q, 2H, J ¼ 7.34 Hz); 4.67 (s, 1H);
7.08e7.22 (m, 4H).
75 MHz) d 14.4, (CH3CH2), 29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3),
121.4 (C-5a),121.7 (C-9),121.8 (C-4),122.7 (C-3a),125.1 (C-9b),126.5
(C-9a), 126.8e127.6 (C-6, C-7, C-8, C-20, C-40, C-50, C-60), 131.5 (C-10),
133.4 (C-5), 134.5 (C-30), 143.8 (C-2), 165.5 (C]O). MS-ESI (m/z):
410.06 [Mꢁ]. Anal. Calcd. for C22H18ClNO3S C, 64.15; H, 4.40; Cl,
8.61; N, 3.40; O, 11.65; S, 7.78. Found C, 63.10; H, 4.45; Cl, 8.53; N,
3.32; O, 11.54; S, 7.62.
4.3.4. (Z)-ethyl 4-(2,3-dichlorophenylthio)-3-aminobut-2-enoate
(5d)
Yield:73%. 1H NMR (CDCl3, 300 MHz)
d
1.28 (t, 3H, J ¼ 7.34 Hz);
3.70 (s, 2H); 4.13 (q, 2H, J ¼ 7.34 Hz); 4.73 (s, 1H); 7.15e7.22 (m, 2H);

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A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
4.4.2. Ethyl 2-((2-chlorophenylthio)methyl)-5-hydroxy-1H-benzo
[g]indole-3-carboxylate (6b)
(q, 2H, J ¼ 7.34 Hz); 4.70 (s, 2H); 7.32e7.55 (m, 4H); 7.64 (s,1H); 8.16
(d, 1H, J ¼ 7.88 Hz); 8.29 (d, 1H, J ¼ 7.88 Hz); 8.64 (s, 1H, OH). 13
C
Rf (hexane/ethyl acetate 8:2) ¼ 0.24. Yield: 21%. M.p. 209.0 ꢀC
NMR (DMSO-d6, 75 MHz) d 14.4, (CH3CH2), 29.3 (CH2S), 59.9,
dec. 1H NMR (DMSO-d6, 300 MHz)
d
1.33 (t, 3H, J ¼ 7.34 Hz); 4.26 (q,
(CH3CH2), 100.1 (C-3), 121.4 (C-5a), 121.7 (C-9), 121.8 (C-4), 122.7 (C-
3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6 (C-6, C-7, C-8, C-30, C-40,
C-50), 130.3 (C-20-C-60), 131.5 (C-10), 133.4 (C-5), 143.8 (C-2), 165.5
(C]O). MS-ESI (m/z): 444.02 [Mꢁ]. Anal. Calcd. for C22H17Cl2NO3S C,
59.20; H, 3.84; Cl, 15.89; N, 3.14; O, 10.75; S, 7.18 Found C, 60.27; H,
3.59; Cl, 16.07; N, 3.82; O, 11.25; S, 7.87.
2H, J ¼ 7.34 Hz); 4.71 (s, 2H); 7.22 (t, 1H, J ¼ 6.30 Hz); 7.35 (t, 1H,
J ¼ 6.30 Hz); 7.42 (d,1H, J ¼ 8.10 Hz); 7.49 (d,1H, J ¼ 8.10 Hz); 7.53 (s,
1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H,
J ¼ 7.80 Hz); 9.69 (s, 1H, OH); 12.39 (s, 1H, NH). 13C NMR (DMSO-d6,
75 MHz)
d 14.4, (CH3CH2), 29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3),
121.4 (C-5a),121.7 (C-9),121.8 (C-4),122.7 (C-3a),125.1 (C-9b),126.5
(C-9a), 126.8e127.6 (C-6, C-7, C-8, C-30, C-40, C-50, C-60), 131.5 (C-10),
132.3 (C-20), 133.4 (C-5), 143.8 (C-2), 165.5 (C]O). MS-ESI (m/z):
410.01 [Mꢁ]. Anal. Calcd. for C22H18ClNO3S C, 64.15; H, 4.40; Cl,
8.61; N, 3.40; O, 11.65; S, 7.78 Found C, 63.75; H, 3.84; Cl, 8.15; N,
3.07; O, 11.25; S, 7.48.
4.4.7. Ethyl 2-((3,5-dichlorophenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6g)
Rf (hexane/ethyl acetate 8:2) ¼ 0.26. Yield: 17%. M.p. 203.0 ꢀC
dec. 1H NMR (DMSO-d6, 300 MHz)
2H, J ¼ 7.34 Hz); 4.82 (s, 2H); 7.41e7.47 (m, 3H); 7.52 (s, 1H); 7.57 (t,
1H, J ¼ 7.88 Hz); 8.17 (d, 1H, J ¼ 7.88 Hz); 8.28 (d, 1H, J ¼ 7.88 Hz);
9.69 (s, 1H, OH); 12.39 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz)
d
1.37 (t, 3H, J ¼ 7.34 Hz); 4.30 (q,
4.4.3. Ethyl 2-((o-tolylthio)methyl)-5-hydroxy-1H-benzo[g]indole-
3-carboxylate (6c)
d
14.4, (CH3CH2), 29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3), 121.4 (C-
Rf (hexane/ethyl acetate 8:2) ¼ 0.24. Yield: 56%. M.p. 184.5 ꢀC
5a), 121.7 (C-9), 121.8 (C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a),
126.8e127.6 (C-6, C-7, C-8, C-20, C-40, C-60), 131.5 (C-10), 133.4 (C-5),
135.6 (C-30-C-50), 143.8 (C-2), 165.5 (C]O). MS-ESI (m/z): 445.92
[Mꢁ]. Anal. Calcd. for C22H17Cl2NO3S C, 59.20; H, 3.84; Cl, 15.89; N,
3.14; O, 10.75; S, 7.18 Found C, 60.21; H, 4.03; Cl, 14.84; N, 2.89; O,
10.60; S, 7.23.
dec. 1H NMR (DMSO-d6, 300 MHz)
d
1.37 (t, 3H, J ¼ 7.34 Hz); 2.22 (s,
3H); 4.26 (q, 2H, J ¼ 7.34 Hz); 4.70 (s, 2H); 6.83e6.94 (m, 4H); 7.42
(t, 1H, J ¼ 7.88 Hz); 7.52 (s, 1H); 7.57 (t, 1H, J ¼ 7.88 Hz); 8.15 (d, 1H,
J ¼ 7.88 Hz); 8.30 (d, 1H, J ¼ 7.88 Hz); 9.69 (s, 1H, OH); 12.39 (s, 1H,
NH). 13C NMR (DMSO-d6, 75 MHz)
d 14.4, (CH3CH2), 19.2, (o-CH3),
29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3), 121.4 (C-5a), 121.7 (C-9),
121.8 (C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6 (C-
6, C-7, C-8, C-20, C-30, C-40, C-50, C-60), 131.5 (C-10), 133.4 (C-5), 143.8
(C-2), 165.5 (C]O). MS-ESI (m/z): 390.13 [Mꢁ]. Anal. Calcd. for
4.4.8. Ethyl 2-((2,6-dimethylphenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6h)
Rf (hexane/ethyl acetate 8:2) ¼ 0.30. Yield: 37%. M.p. 197.5 ꢀC
C
23H21NO3S C, 70.56; H, 5.41; N, 3.58; O, 12.26; S, 8.19 Found C,
dec.1H NMR (DMSO-d6, 300 MHz)
d
1.26 (t, 3H, J ¼ 7.34 Hz); 2.29 (s,
71.48; H, 6.31; N, 4.23; O, 11.59; S, 7.37.
6H); 4.08 (q, 2H, J ¼ 7.34 Hz); 4.40 (s, 2H); 7.02e7.18 (m, 3H); 7.42 (t,
1H, J ¼ 6.61 Hz); 7.47 (s, 1H); 7.54 (t, 1H, J ¼ 6.61 Hz); 8.16 (d, 1H,
J ¼ 7.80 Hz); 8.27 (d, 1H, J ¼ 7.80 Hz); 9.64 (s, 1H, OH); 12.39 (s, 1H,
4.4.4. Ethyl 2-((2,3-dichlorophenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6d)
NH). 13C NMR (DMSO-d6, 75 MHz)
d 14.4, (CH3CH2), 19.2, (o,m-CH3),
Rf (hexane/ethyl acetate 8:2) ¼ 0.22. Yield: 29%. M.p. 184.1 ꢀC
29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3), 121.4 (C-5a), 121.7 (C-9),
121.8 (C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6 (C-
6, C-7, C-8, C-20, C-30, C-40, C-50, C-60), 131.5 (C-10), 133.4 (C-5), 143.8
(C-2), 165.5 (C]O). MS-ESI (m/z): 404.14 [Mꢁ]. Anal. Calcd. for
dec. 1H NMR (DMSO-d6, 300 MHz)
d
1.33 (t, 3H, J ¼ 7.34 Hz); 4.26 (q,
2H, J ¼ 7.34 Hz); 4.71 (s, 2H); 7.38 (t, 1H, J ¼ 6.30 Hz); 7.47 (t, 1H,
J ¼ 6.30 Hz); 7.53 (s,1H); 7.55e7.60 (m, 3H); 8.16 (d,1H, J ¼ 7.80 Hz);
8.27 (d, 1H, J ¼ 7.80 Hz); 9.69 (s, 1H, OH); 12.39 (s, 1H, NH). 13C NMR
C24H23NO3S C, 71.09; H, 5.72; N, 3.45; O, 11.84; S, 7.91 Found C,
(DMSO-d6, 75 MHz)
d
14.4, (CH3CH2), 29.3 (CH2S), 59.9, (CH3CH2),
70.89; H, 5.22; N, 3.05; O, 10.82; S, 7.37.
100.1 (C-3), 121.4 (C-5a), 121.7 (C-9), 121.8 (C-4), 122.7 (C-3a), 125.1
(C-9b), 126.5 (C-9a), 126.8e127.6 (C-6, C-7, C-8, C-40, C-50, C-60),
137.5 (C-10), 132.3 (C-20-C-30), 133.4 (C-5), 143.8 (C-2), 165.5 (C]O).
MS-ESI (m/z): 446.05 [Mꢁ]. Anal. Calcd. for C22H17Cl2NO3S C, 59.20;
H, 3.84; Cl, 15.89; N, 3.14; O,10.75; S, 7.18 Found C, 58.73; H, 3.24; Cl,
15.19; N, 2.86; O, 10.05; S, 7.03.
4.4.9. Ethyl 2-((3,5-dimethylphenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6i)
Rf (hexane/ethyl acetate 8:2) ¼ 0.28. Yield: 37%. M.p. 158.3 ꢀC
dec. 1H NMR (DMSO-d6, 300 MHz)
d
1.35 (t, 3H, J ¼ 7.34 Hz); 2.20 (s,
6H); 4.26 (q, 2H, J ¼ 7.34 Hz); 4.71 (s, 2H); 6.83 (s, 1H); 6.98 (s, 2H);
7.42 (t, 1H, J ¼ 7.88 Hz); 7.52 (s, 1H); 7.57 (t, 1H, J ¼ 7.88 Hz); 8.17 (d,
1H, J ¼ 7.88 Hz); 8.31 (d, 1H, J ¼ 7.88 Hz); 9.69 (s, 1H, OH); 12.39 (s,
4.4.5. Ethyl 2-((3,4-dichlorophenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6e)
1H, NH). 13C NMR (DMSO-d6, 75 MHz)
d14.4, (CH3CH2), 24.2 (m-
Rf (hexane/ethyl acetate 8:2) ¼ 0.26. Yield: 16%. M.p. 183.5 ꢀC
CH3), 29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3), 121.4 (C-5a), 121.7 (C-
9), 121.8 (C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6
(C-6, C-7, C-8, C-20, C-40, C-60), 131.5 (C-10), 133.4 (C-5), 138.5 (C-30,C-
50), 143.8 (C-2), 165.5 (C]O). MS-ESI (m/z): 404.10 [Mꢁ]. Anal.
Calcd. for C24H23NO3S C, 71.09; H, 5.72; N, 3.45; O, 11.84; S, 7.91
Found C, 70.89; H, 6.92; N, 2.98; O, 11.24; S, 7.37.
dec. 1H NMR (DMSO-d6, 300 MHz)
d
1.35 (t, 3H, J ¼ 7.34 Hz); 4.28 (q,
2H, J ¼ 7.34 Hz); 4.77 (s, 2H); 7.32 (d, 1H J ¼ 6.63 Hz); 7.43 (t, 1H,
J ¼ 6.61 Hz); 7.48e7.52 (m, 3H); 7.64 (s,1H); 8.16 (d,1H, J ¼ 7.80 Hz);
8.27 (d, 1H, J ¼ 7.80 Hz); 9.69 (s, 1H, OH); 12.39 (s, 1H, NH). 13C NMR
(DMSO-d6, 75 MHz)
d 14.4, (CH3CH2), 29.3 (CH2S), 59.9, (CH3CH2),
100.1 (C-3), 121.4 (C-5a), 121.7 (C-9), 121.8 (C-4), 122.7 (C-3a), 125.1
(C-9b), 126.5 (C-9a), 126.8e127.6 (C-6, C-7, C-8, C-20, C-50, C-60),
129.8 (C-40), 131.5 (C-10), 133.4 (C-5), 133.6 (C-30), 143.8 (C-2), 165.5
(C]O). MS-ESI (m/z): 445.94 [Mꢁ]. Anal. Calcd. for C22H17Cl2NO3S
C, 59.20; H, 3.84; Cl, 15.89; N, 3.14; O, 10.75; S, 7.18 Found C, 58.70;
H, 3.54; Cl, 15.37; N, 3.02; O, 10.25; S, 7.07.
4.4.10. Ethyl 5-hydroxy-2-((mesitylthio)methyl)-1H-benzo[g]
indole-3-carboxylate (6l)
Rf (hexane/ethyl acetate 8:2) ¼ 0.28. Yield: 27%. M.p. 178.5 ꢀC
dec. 1H NMR (Acetone-d6, 300 MHz)
d
1.32 (t, 3H, J ¼ 7.34 Hz); 2.20
(s, 3H); 2.25 (s, 6H); 4.17 (q, 2H, J ¼ 7.34 Hz); 4.44 (s, 2H); 6.87 (s,
2H); 7.45 (t, 1H, J ¼ 7.88 Hz); 7.53 (t, 1H, J ¼ 7.88 Hz); 7.65 (s, 1H);
4.4.6. Ethyl 2-((2,6-dichlorophenylthio)methyl)-5-hydroxy-1H-
benzo[g]indole-3-carboxylate (6f)
8.15 (d,1H, J ¼ 7.88 Hz); 8.29 (d,1H, J ¼ 7.88 Hz); 8.67 (s,1H, OH). 13
C
NMR (DMSO-d6, 75 MHz) d 14.4, (CH3CH2), 19.2, (o,m-CH3), 24.9 (p-
Rf (hexane/ethyl acetate 8:2) ¼ 0.18. Yield: 35%. M.p. 191.8 ꢀC
CH3), 29.3 (CH2S), 59.9, (CH3CH2), 100.1 (C-3), 121.4 (C-5a), 121.7 (C-
9), 121.8 (C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6
dec. 1H NMR (Acetone-d6, 300 MHz)
d
1.33 (t, 3H, J ¼ 7.34 Hz); 4.17

A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
473
(C-6, C-7, C-8, C-20, C-30, C-40, C-50, C-60), 131.5 (C-10), 133.4 (C-5),
143.8 (C-2),165.5 (C]O). MS-ESI (m/z): 418.09 [Mꢁ]. Anal. Calcd. for
25H25NO3S C, 71.57; H, 6.01; N, 3.34; O, 11.44; S, 7.64. Found C,
4.6. Ethyl 2-((2-chlorophenylthio)methyl)-5-hydroxy-1-methyl-
1H-benzo[g]indole-3-carboxylate (11)
C
70.59; H, 6.12; N, 3.25; O, 11.37; S, 7.56.
A solution of 6b (152 mg, 0.37 mmol), imidazole (93 mg,
1.48 mmol) and tert-butyl dimethyl silyl chloride (127 L,
m
0.74 mmol) in CH2Cl2 was stirred for 3 h at room temperature. The
mixture was washed with water and two times with brine. The
organic layer, dried over anhydrous Na2SO4, was evaporated under
reduced pressure and the crude was purified by flash chromatog-
raphy (eluting system hexane/ethyl acetate 8:2) affording 185 mg
of desired compound (9) as white cristals (yield: 95%). 1H NMR
4.5. General procedure for the synthesis of 7a-b and 8a-b
A mixture of 6b (34 mg, 0.08 mmol), K2CO3 (57 mg, 0.40 mmol),
and iodoalcane (3.75 eq.) in acetone (0.3 mL) was irradiated under
MW, for 30 min at 130 ꢀC. The mixture was filtered and evaporated
under vacuum. The crude was purified by flash chromatography on
silica gel (hexane/Ethyl acetate 9:1).
(CDCl3, 300 MHz)
d
0.18 (s, 6H); 1.01 (s, 9H); 1.34 (t, 3H, J ¼ 7.34 Hz);
4.28 (q, 2H, J ¼ 7.34 Hz); 4.70 (s, 2H); 7.22 (t,1H, J ¼ 6.30 Hz); 7.35 (t,
1H, J ¼ 6.30 Hz); 7.42 (d, 1H, J ¼ 8.10 Hz); 7.49 (d, 1H, J ¼ 8.10 Hz);
7.53 (s, 1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H,
J ¼ 7.80 Hz).
4.5.1. Ethyl 2-((2-chlorophenylthio)methyl)-5-methoxy-1H-benzo
[g]indole-3-carboxylate (7a)
A 25 mL three-necked round bottomed flask equipped with a
stir bar was charged with 60% NaH (21 mg, 0.53 mmol) in mineral
oil dissolved in 1 mL of dry THF, under nitrogen atmosphere. The
solution was cooled at 0 ꢀC and a solution of 9 (185 mg, 0.35 mmol)
Rf (hexane/ethyl acetate 8:2) ¼ 0.5. Yield: 27%. M.p.188.5 ꢀC dec.
1H NMR (CDCl3, 300 MHz)
d
1.50 (t, 3H, J ¼ 7.34 Hz); 4.15 (s, 3H);
4.26 (q, 2H, J ¼ 7.34 Hz); 4.81 (s, 2H); 7.22 (t,1H, J ¼ 6.30 Hz); 7.35 (t,
1H, J ¼ 6.30 Hz); 7.42 (d, 1H, J ¼ 8.10 Hz); 7.49 (d, 1H, J ¼ 8.10 Hz);
7.53 (s, 1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H,
J ¼ 7.80 Hz). Anal. Calcd. for C23H20ClNO3S C, 64.86; H, 4.73; Cl, 8.32;
N, 3.29; O, 11.27; S, 7.53. Found C, 64.88; H, 4.76; Cl, 8.35; N, 3.32; O,
11.30; S, 7.50.
in 2 mL of dry THF was added. After 30 min iodomethane (26 mL,
0.42 mmol) was added and the mixture was stirred for 2 h. The
mixture was diluted with water and extracted with CH2Cl2. Organic
layer was washed with brine, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. Desired compound (10) was
obtained after purification with flash chromatography using hex-
ane/ethyl acetate 9:1, as eluent mixture. (Yield: 50%). 1H NMR
4.5.2. Ethyl 2-((2-chlorophenylthio)methyl)-5-methoxy-1-methyl-
1H-benzo[g]indole-3-carboxylate (7b)
(CDCl3, 300 MHz)
d
0.18 (s, 6H); 1.01 (s, 9H); 1.33 (t, 3H, J ¼ 7.34 Hz);
4.10 (s, 3H); 4.28 (q, 2H, J ¼ 7.34 Hz); 4.70 (s, 2H); 7.22 (t, 1H,
J ¼ 6.30 Hz); 7.35 (t, 1H, J ¼ 6.30 Hz); 7.42 (d, 1H, J ¼ 8.10 Hz); 7.49
(d, 1H, J ¼ 8.10 Hz); 7.53 (s, 1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H,
J ¼ 7.80 Hz); 8.27 (d, 1H, J ¼ 7.80 Hz).
Rf (hexane/ethyl acetate 8:2) ¼ 0.72. Yield: 32%. M.p. 198.5 ꢀC
dec. 1H NMR (CDCl3, 300 MHz)
d
1.50 (t, 3H, J ¼ 7.34 Hz); 4.10 (s,
3H); 4.15 (s, 3H); 4.26 (q, 2H, J ¼ 7.34 Hz); 4.80 (s, 2H); 7.22 (t, 1H,
J ¼ 6.30 Hz); 7.35 (t, 1H, J ¼ 6.30 Hz); 7.42 (d, 1H, J ¼ 8.10 Hz); 7.49
(d, 1H, J ¼ 8.10 Hz); 7.53 (s, 1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H,
J ¼ 7.80 Hz); 8.27 (d, 1H, J ¼ 7.80 Hz). Anal. Calcd. for C24H22ClNO3S
C, 65.52; H, 5.04; Cl, 8.06; N, 3.18; O, 10.91; S, 7.29. Found C, 65.50;
H, 5.08; Cl, 8.09; N, 3.21; O, 10.89; S, 7.32.
A solution of TBAF 1M in THF (63
solution of 10 (35 mg, 64 mmol) in THF (420
m
L, 64 mmol) was added to a
m
L) cooled at 0 ꢀC. The
mixture was stirred for 10 min at 0 ꢀC and quenched with EtOAC
and H2O. Organic layer was washed with a saturated solution of
NaHCO3 and brine, dried over anhydrous Na2SO4 and concentrated
in vacuo affording final compound 11 (Yield: 45%). 1H NMR (CDCl3,
300 MHz)
d
(ppm) ¼ 1.50 (t, 3H, J ¼ 7.34 Hz); 4.15 (s, 3H); 4.26 (q,
4.5.3. Ethyl 2-((2-chlorophenylthio)methyl)-5-ethoxy-1H-benzo[g]
indole-3-carboxylate (8a)
2H, J ¼ 7.34 Hz); 4.80 (s, 2H); 7.22 (t, 1H, J ¼ 6.30 Hz); 7.35 (t, 1H,
J ¼ 6.30 Hz); 7.42 (d,1H, J ¼ 8.10 Hz); 7.49 (d,1H, J ¼ 8.10 Hz); 7.53 (s,
1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H,
J ¼ 7.80 Hz). Anal. Calcd. for C23H20ClNO3S C, 64.86; H, 4.73; Cl, 8.32;
N, 3.29; O, 11.27; S, 7.53. Found C, 64.98; H, 4.82; Cl, 8.29; N, 3.21; O,
11.10; S, 7.44.
Rf (hexane/ethyl acetate 8:2) ¼ 0.5. Yield: 25%. M.p.179.3 ꢀC dec.
1H NMR (CDCl3, 300 MHz)
d
1.53 (t, 3H, J ¼ 7.34 Hz); 1.63 (t, 3H,
J ¼ 7.5 Hz); 4.26 (q, 2H, J ¼ 7.34 Hz); 4.50 (q, 2H, J ¼ 7.5 Hz); 4.80 (s,
2H); 7.22 (t, 1H, J ¼ 6.30 Hz); 7.35 (t, 1H, J ¼ 6.30 Hz); 7.42 (d, 1H,
J ¼ 8.10 Hz); 7.49 (d, 1H, J ¼ 8.10 Hz); 7.53 (s, 1H), 7.55e7.60 (m, 2H);
8.16 (d, 1H, J ¼ 7.80 Hz); 8.27 (d, 1H, J ¼ 7.80 Hz). 13C NMR (CDCl3,
4.7. Biological evaluation and assay systems
75 MHz)
d 14.4, (CH3CH2), 19.2 (CH3CH2), 30.0 (CH2S), 59.9
(CH3CH2), 63.9 (CH3CH2), 99.0 (C-3), 121.4 (C-5a), 121.7 (C-9), 121.8
(C-4), 122.7 (C-3a), 125.1 (C-9b), 126.5 (C-9a), 126.8e127.6 (C-6, C-7,
C-8, C-20, C-30, C-40, C-50, C-60), 131.5 (C-10), 133.4 (C-5), 143.8 (C-2),
165.5 (C]O). Anal. Calcd. for C24H22ClNO3S C, 65.52; H, 5.04; Cl,
8.06; N, 3.18; O, 10.91; S, 7.29. Found C, 65.44; H, 5.09; Cl, 8.09; N,
3.20; O, 10.88; S, 7.32.
4.7.1. Animals
Male ICR mice, 8 weeks old (Harlan, Italy), were housed 5 per
cage under controlled illumination (12:12 h light:dark cycle; light
on 06.00 h) and standard environmental conditions (room tem-
perature 22
1
ꢀC, humidity 60 10%) for at least 1 week before
experimental use. Mouse chow and tap water were available ad
libitum. The experimental procedures were in accordance with
Italian and European regulations governing the care and treatment
of laboratory animals (Permission no. 41/2007B). Animal care was
in compliance with Ethical Guidelines of the IASP and European
Community (E.C. L358/1 18/12/86) on the use and protection of
animals in experimental research. All efforts were made to mini-
mize animal suffering and to reduce the number of animals used.
4.5.4. Ethyl 2-((2-chlorophenylthio)methyl)-5-ethoxy-1-ethyl-1H-
benzo[g]indole-3-carboxylate (8b)
Rf (hexane/ethyl acetate 8:2) ¼ 0.68. Yield: 33%. M.p. 158.5 ꢀC
dec. 1H NMR (CDCl3, 300 MHz)
d
1.53 (t, 3H, J ¼ 7.34 Hz); 1.61 (m,
6H); 4.26 (q, 2H, J ¼ 7.34 Hz); 4.50 (m, 4H); 4.80 (s, 2H); 7.22 (t, 1H,
J ¼ 6.30 Hz); 7.35 (t, 1H, J ¼ 6.30 Hz); 7.42 (d, 1H, J ¼ 8.10 Hz); 7.49
(d, 1H, J ¼ 8.10 Hz); 7.53 (s, 1H); 7.55e7.60 (m, 2H); 8.16 (d, 1H,
J ¼ 7.80 Hz); 8.27 (d, 1H, J ¼ 7.80 Hz). Anal. Calcd. for C26H26ClNO3S
C, 66.73; H, 5.60; Cl, 7.58; N, 2.99; O, 10.26; S, 6.85. Found C, 66.77;
H, 5.66; Cl, 7.55; N, 3.02; O, 10.23; S, 6.89.
4.7.2. Paw oedema
Oedema was induced by carrageenan injection (20
w/v in saline) into the plantar surface of the right hind paw. All
mL/paw, 2%

474
A. Peduto et al. / European Journal of Medicinal Chemistry 108 (2016) 466e475
derivatives were dissolved in DMSO (0.5% v/v in saline), Zileuton
were dissolved in ethanol-cromophor-saline (1:1:18). The paw
volume was measured using a hydroplethysmometer (Ugo Basile,
Varese, Italy) at 2-4-6 h following carrageenan. Mice (n ¼ 6e8 per
group) were treated with 1 or 6f (1e4 mg/kg, i.p.), 6g and 6l
(1e4 mg/kg, i.p.) 30 min before the inflammatory insult. A separate
experimental group received zileuton (20 mg/kg, i.p.) used as a
reference drug before carrageenan.
concentration of 50
final concentration of 1%) were added, and after 15 min at 4 ꢀC, the
reaction (100 l total volume) was initiated by addition of PGH2
(20
M, final concentration). After 1 min at 4 ꢀC, the reaction was
terminated using stop solution (100 l; 40 mM FeCl2, 80 mM citric
acid, and 10 M of 11 -PGE2). PGE2 was separated by solid phase
extraction on reversed phase (RP)-C18 material using acetonitrile
mg/ml. Test compounds or vehicle (DMSO at a
m
m
m
m
b
(200
ml) as eluent, and analyzed by RP-HPLC (30% acetonitrile
aq þ 0.007% TFA (v/v), Nova-Pak® C18 column, 5 ꢂ 100 mm, 4
mm
4.7.3. Determination of 5-LOX product formation in cell-based
assays
particle size, flow rate 1 ml/min) with UV detection at 195 nm.11b-
PGE2 was used as internal standard to quantify PGE2 product for-
mation by integration of the area under the peaks. IC50 values were
assessed by three determinations at compound concentrations of
PMNL were isolated from human blood of adult healthy vol-
unteers, with consent, obtained from the Institute of Transfusion
Medicine, University Hospital Jena, as described [25]. PMNL
(5 ꢂ 106 in 1 ml PG buffer) were preincubated with the test com-
pounds (15 min, 37 ꢀC) and 5-LOX product formation was started by
0.1, 1, 10 mM.
Appendix A. Supplementary data
addition of 1 mM CaCl2 and 2.5 m
M A23187. After 10 min at 37 ꢀC,
the reaction was stopped with 1 ml of methanol. Cells were placed
on ice, centrifuged at 800 ꢂ g for 5 min at 4 ꢀC and the supernatant
was added to methanol. Formed 5-LOX metabolites were extracted
and analyzed by HPLC as described [26]. 5-LOX product formation
is expressed as ng of 5-LOX 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.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.11.048.
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