Structural insight into the optimization of ethyl 5-hydroxybenzo[g]indol-3-carboxylates and their bioisosteric analogues as 5-LO/m-PGES-1 dual inhibitors able to suppress inflammation

Article abstract of DOI:10.1016/j.ejmech.2018.05.041

The release of pro-inflammatory mediators, such as prostaglandines (PGs) and leukotrienes (LTs), arising from the arachidonic acid (AA) cascade, play a crucial role in initiating, maintaining, and regulating inflammatory processes. New dual inhibitors of 5-lipoxygenase (5-LO) and microsomal prostaglandin E₂ synthase-1 (mPGES-1), that block, at the same time, the formation of PGE₂ and LTs, are currently emerged as a highly interesting drug candidates for better pharmacotherapie of inflammation-related disorders. Following our previous studies, we here performed a detailed structure-based design of benzo[g]indol-3-carboxylate derivatives, disclosing several new key factors that affect both enzyme activity. Ethyl 2-(3,4-dichlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4b, RAF-01) and ethyl 2-(3,4-dichlorophenyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (7h, RAF-02) emerged as the most active compounds of the series. Additionally, together with selected structure based analogues, both derivatives displayed significant in vivo anti-inflammatory properties. In conclusion, modeling and experimental studies lead to the discovery of new candidate compounds prone to further developments as multi-target inhibitors of the inflammatory pathway.

Full text of DOI:10.1016/j.ejmech.2018.05.041

Accepted Manuscript
Structural insight into the optimization of ethyl 5-hydroxybenzo[g]indol-3-carboxylates
and their bioisosteric analogues as 5-LO/m-PGES-1 dual inhibitors able to suppress
inflammation
Ferdinando Bruno, Suann Errico, Simona Pace, Maxim B. Nawrozkij, Arthur S.
Mkrtchyan, Francesca Guida, Rosa Maisto, Abdurrahman Olgaç, Michele D'Amico,
Sabatino Maione, Mario De Rosa, Erden Banoglu, Oliver Werz, Antonio Fiorentino,
Rosanna Filosa
PII:
S0223-5234(18)30454-9
10.1016/j.ejmech.2018.05.041
EJMECH 10452
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 February 2018
Revised Date: 23 May 2018
Accepted Date: 25 May 2018
Please cite this article as: F. Bruno, S. Errico, S. Pace, M.B. Nawrozkij, A.S. Mkrtchyan, F. Guida,
R. Maisto, A. Olgaç, M. D'Amico, S. Maione, M. De Rosa, E. Banoglu, O. Werz, A. Fiorentino, R.
Filosa, Structural insight into the optimization of ethyl 5-hydroxybenzo[g]indol-3-carboxylates and their
bioisosteric analogues as 5-LO/m-PGES-1 dual inhibitors able to suppress inflammation, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.041.
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ACCEPTED MANUSCRIPT
Structural Insight into the Optimization of Ethyl
5
-Hydroxybenzo[g]indol-3-carboxylates
and their Bioisosteric Analogues as 5-LO/m-PGES-1
Dual Inhibitors Able to Suppress Inflammation
a#
,a,d#
d
b
b
Ferdinando Bruno , Suann Errico , Simona Pace , Maxim B. Nawrozkij ,Arthur S.Mkrtchyan ,
a
a
c
Francesca Guida , Rosa Maisto , Abdurrahman Olgaç ,
a
a
a
c
Michele D’Amico , Sabatino Maione , Mario De Rosa , Erden Banoglu ,
d
fe
a,f,g
Oliver Werz , Antonio Fiorentino and Rosanna Filosa
a
b
c
Università degli Studi della Campania Luigi Vanvitelli, Department of Experimental Medicine, Naples, Italy
Volgograd State Technical University, Organic chemistry Department, Lenin avenue 28, Russian Federation
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Yenimahalle, 06330 Ankara, Turkey
d
Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University of Jena,
Philosophenweg 14, Jena, Germany
e
Università della Campania Luigi Vanvitelli, Department of Environmental Biological and Pharmaceutical Sciences
and Technologies, Naples, Italy
f
Consorzio Sannio Tech, Appia str., 82030 Apollosa (BN), Italy
g
Institute of Food Sciences, National Research Council Roma str. 64, 83100 Avellino, Italy

ACCEPTED MANUSCRIPT
Abstract: The release of pro-inflammatory mediators, such as prostaglandines (PGs) and
leukotrienes (LTs), arising from the arachidonic acid (AA) cascade, play a crucial role in initiating,
maintaining, and regulating inflammatory processes.
New dual inhibitors of 5-lipoxygenase (5-LO) and microsomal prostaglandin E synthase-1
2
(
mPGES-1), that block, at the same time, the formation of PGE and LTs, are currently emerged as
2
a highly interesting drug candidates for better pharmacotherapie of inflammation-related disorders.
Following our previous studies, we here performed a detailed structure-based design of
benzo[g]indol-3-carboxylate derivatives, disclosing several new key factors that affect both enzyme
activity. Ethyl 2-(3,4-dichlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4b, RAF-01)
and ethyl 2-(3,4-dichlorophenyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (7h, RAF-02)
emerged as the most active compounds of the series. Additionally, together with selected structure
based analogues, both derivatives displayed significant in vivo anti-inflammatory properties.
In conclusion, modeling and experimental studies lead to the discovery of new candidate
compounds prone to further developments as multi-target inhibitors of the inflammatory pathway.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Introduction
5
-Lipoxygenase (5-LO) is defined as a non-heme iron-containing dioxygenase involved in
the biosynthesis of leukotrienes (LTs) from arachidonic acid (AA) [1,2]. Upon cell stimulation, the
cytosolic PLA (cPLA ) releases AA that is converted by 5-LO with the help of the 5-LO-activating
2
2
protein (FLAP) into an unstable intermediate LTA that is further transformed, in enzymatic
4
reactions, to LTB or so-called cysteinyl (cys)-LTs.
4
LTB acts as a potent pro-inflammatory agent by inducing chemotaxis and activation of leukocytes,
4
whereas the cys-LTs essentially act on smooth muscles causing vaso and bronchoconstriction [3,4].
In view of the significant pathophysiological role of LTs, pharmacological intervention strategies
have been developed to either block the action of LTs or to inhibit their biosynthesis, defining 5-LO
and/or FLAP as valid drug targets for pharmacotherapy of inflammatory disorders [5,6].
Furthermore, recent studies have described age-dependent increase in 5-LO expression and
oxidative stress in rat retina [7-10], it is described that light and trauma activate 5-LO, suggesting its
involvement also in the pathogenesis of retinal diseases due to light damage [11,12].
Until now, in order to modulate cellular 5-LO product synthesis, three concrete
pharmacological strategies can be basically pursued: inhibition of cPLA , inhibition of FLAP
2
activity and direct inhibition of 5-LO [13].
Even if 5-LO is a reasonable target for the discover of antinflammatory drugs, taking in mind the
multitude of pro-inflammatory and pro-resolving mediators produced from AA, the block of a
single branch by a selective drug may cause redirection/shunting and even amplification of
alternative pathways The overcome of the “one-drug-hits-one-target” approach paved the way to
.
the discover of drugs with polypharmacological modes of action able to modulate also the
cyclooxygenase (COX) mediated formation of prostanoids [14,15]. Unfortunately, over the last
years, several clinical evidences indicated that selective COX-2 inhibitors may be involved in
serious cardiovascular accidents [16-19].

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Deduced from previous studies, a novel pharmacological strategy was suggested by targeting
mPGES-1 [20-22] having, this isoform, a considerable implications in inflammation and a pivotal
role in the production of PGE that mediates acute pain and fever during an inflammatory response
2
[
23-25]. Therefore, the rational development of chemical structures that contain fragments able to
inhibit 5-LO and mPGES-1 is now day a highly interesting field of research with promise for better
pharmacotherapies also over dual 5-LO/COX-2 inhibition in terms of higher anti-inflammatory
efficacy and lower incidences of side effects.[26]
Our groups have been interested for a long time in the synthesis and biological evaluation
of anti-cancer and anti-inflammatory agents [27-39]; in our drug discovery projects we have
considered lead structures chemical compounds, either from medicinal plants [40, 41] or
synthesized drugs, [42-45] which display pharmacological or biological activity likely to be
therapeutically useful, but that may still have suboptimal structure modification to fit better to the
respective target(s).
For instance, novel series of ethyl 5-hydroxyindole-3-carboxylate has led to the discovery and
development of two potent inhibitors of human 5-LO (Fig. 1). Among all tested compounds, ethyl
5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (Compound 1) was the most
potent derivative which blocks 5-LO activity in cell-free assays with an IC50 of 0.7 µM, and
suppressed 5-LO product synthesis in polymorphonuclear leukocytes with an IC50 of 0.23 µM,
being equally potent to the well-recognized reference 5-LO inhibitor Zileuton, used as antiasthmatic
drug in the clinics [46]. Furthermore, we have confirmed that the annelation of a [g]benzene ring to
the indole ring in the series led to more potent inhibitors, exemplified by compounds 6f and 6l with
IC of 0.17 and 0.20 µM in cell-free and 0.19 and 0.37 µM in cell-based 5-LO activity assays,
5
0
respectively [47]. We also demonstrated that both derivatives directly inhibit mPGES-1 (1.33 µM
and 1.93 µM) representing a novel class of dual 5-LO/mPGES-1 inhibitors. The anti-inflammatory
property of the selected compounds 6g and 6l was also confirmed in vivo evaluating their ability to
reduce carrageenan-induced paw edema in mice [47-49].

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In our ongoing efforts to further elucidate the structural requirements of this class of
compounds we focused our investigations on the effect caused by:
(
(
(
i) Introduction of one or more halogens and methyl substituents on the phenyl ring;
ii) Presence of oxygen or nitrogen in the heterocycle scaffold;
iii) Change of the distance between the substituted-phenyl and the heterocycle rings.
In this study, we report the MW (MicroWaves) catalyzed synthesis of this third generation of
benzo[g]indole derivatives together with their benzofuran and naphthofuran bioisosters, and their
biological evaluation, leading to potentially novel, powerful compounds as multi-target inhibitors of
the inflammatory pathway.

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R
First focusedlibrary
1
IC 5 (5-LOX-PMNL)= 0.70 µM
0
Ring Expansion
6l
6f
IC 50 (5-LOX-PMNL)= 0.19±0.09 µM
IC 50 (m-PGES-1)= 1.33 ± 0.10 µM
IC 5 (5-LOX-PMNL)= 0.37±0.07 µM
0
IC 5 (m-PGES-1)= 1.93 ± 0.07 µM
0
Fig.1

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2
2
. Results and discussion
.1. Chemistry
Preparation of 2-substituted 5-hydroxybenzo[g]indole-3-carboxylic acid ethyl esters and
their naphthofuran bioisosters is a good example of diversity-oriented synthesis (DOS) of
exploratory libraries of “drug-like” compounds. “Synthetic trees” lying behind the target
compounds are summarized in Schemes 1 and 2. First of them shows the synthesis of the
corresponding 2-benzyl-5-hydroxybenzo[g]indole carboxylic acids ethyl esters, starting from the
commercially available substituted arylacetic acids (1 a-d). The latter have been converted into the
corresponding arylacetylchlorides, which were purified by distillation in vacuo, yielding the pure
products in 86-99% yield. These were used in the synthesis of 4-aryl-3-oxobutyric acid ethyl esters
according to the Moody’s variation of Oikawa and Yonemitsu synthesis. Namely, 2,2-dimethyl-1,3-
dioxan-4,6-dione (Meldrum’s acid) solution in a mixture of anhydrous DCM and Hunig’s base
(N,N-diisopropyl-N-ethylamine) was treated with one of the above mentioned arylacetylchlorides,
followed by subsequent work-up procedure and ethanolysis of the reaction product, yielding the
desired technical grade 3-oxoesters (2 a-d). Purification of the intermedia has been achieved via
flash-chromatography and/or fractional distillation under diminished pressure. It is necessary to
mention, that synthesis of compounds 2a and 2c has been previously described in literature and
carried out by a different method. Nevertheless, we preferred a Meldrum’s acid based strategy, as it
may widen the scope of the title compounds simply by varying the nature of the alcohol, used for
solvolysis of an acylated 2,2-dimethyl-1,3-dioxane-4,6-dione. The thus obtained pure 3-oxoesters
undergone successive transformation into the corresponding 3-amino-3-arylcrotonates upon
treatment with NH OAc in PhMe in the presence of molecular sieves under microwave irradiation.
4
The resulting 3-aminocrotonates have been purified by column chromatography and involved in
Nenitzescu indole synthesis with 1,4-naphthoquinone in anhydrous DCM, catalyzed by ZnI2
according to the method of Velezheva and co-authors.

ACCEPTED MANUSCRIPT
A variety of ethyl 3-aryl-3-oxopropionates (5a-n), prepared according to the known
procedures, served as starting material in the synthesis of the congeners of the above mentioned 2-
benzylbenzo[g]indoles, containing an aryl-substituent at the C-2 position of the benzo[g]indole
nucleus. As well, their benzo and naphthofuran bioisosters have been prepared. The total synthesis
has much in common with the protocol, described above, but with several important peculiarities.
As in the case of 4-aryl-3-oxobutyric acid ethyl esters, the corresponding ethyl 3-aryl-3-
oxopropanoates undergone transformation into the desired 3-amino-3-arylacrylic acid ethyl esters
upon treatment with NH Oac in PhMe under microwave irradiation and in the presence of
4
molecular sieves. Their conversion into the target benzo[g]indoles was conducted according to the
method of Velezheva and co-authors, as well. On the other side, the Nenitzescu synthesis of the
target benzofurans and naphthofurans from ethyl 3-aryl-3-oxopropionates in the presence of ZnI in
2
DCM, required microwave irradiation. After the extractive work-up procedure the title compounds
were purified via flash column chromatography. This modification of the literature procedure,
allowed a significantly decreased reaction time, accompanied by higher yields of the target
compounds (from about 60% to ~90%). In a number of entries, the same synthetic approach
appeared to benefit with the preparation of benzo[g]indoles, too.

ACCEPTED MANUSCRIPT
Scheme 1
OH
O
O
O
O
O
a
Ar
c
d
b
Ar
Ar
O
Ar
OH
Cl
OEt
Me
1
a-d
O
O
2 a-d
Me
O
NH2
O
e
HO
OEt
Ar
Ar
OEt
NH
3
a-d
4
a-d
Ar = 2,6-Cl C H (a), 3,4-Cl C H (b), 2,6-F C H (c), 2,4,6-Me C H (d)
2
6
3
2
6
3
2
6
3
3
6
2
Reagents and conditions: a) PCl , PhMe; b) Meldrum’s acid, i-Pr NEt, DCM, 0-5°C, overnight; c) EtOH, reflux
5
2
overnight; d) NH OAc , AcOH, dry PhMe, molecularsieves, 140 °C, 20 min, MW ; e) 1,4-Naphtoquinone , ZnI , DCM
4
2
reflux 30 min. and then 0 °C overnight.
,

ACCEPTED MANUSCRIPT
Scheme 2
b
b
a
5
a-n
6
a-n
7
a-n
8
a-k
d
9
a-k
R = Me (a), 2-FC H (b), 2-BrC H (c), 2-ClC H (d), 3-BrC H (e), 3-ClC H (f), 4-ClC H (g), 3,4-Cl C H (h), 4-Br-
6
4
6
4
6
4
6
4
6
4
6
4
2
6
3
2
-FC H (i), 5-Br-2-FC H (j), 2-MeOC H (k), 2-MeC H (l), 3-MeC H (m), 3,5-Me C H (n)
6 3 6 3 6 4 6 4 6 4 2 6 3
Reagent and conditions: a) NH OAc, PhMe, AcOH, molecularsieves, MW, 140 °C, 20min; b) 1,4-Naphtoquinone,
4
ZnI , DCM, reflux 30 min then 0-5°C overnight; c) 1,4-Naphtoquinone, ZnI , DCM, MW, 110 °C, 30min, c) 1,4-
2
2
Naphtoquinone , ZnI , DCM, MW, 110°C, 30 min; d) Benzoquinone, ZnI , DCM, MW, 110°C, 30 min
2
2

ACCEPTED MANUSCRIPT
2.2 Evaluation of 5-LO and mPGES-1 activity and structure-activity relationships
Analysis of test compounds as 5-LO inhibitors was routinely carried out in two different test
systems, a cell-free assay using isolated human recombinant 5-LO and a cell-based assay using
human neutrophils. 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.,
2
+
inhibition of FLAP, interference with 5-LO-activating lipid hydroperoxides, protein kinases or Ca
mobilization, and 5-LO translocation/membrane association). The reference 5-LO inhibitor N-[1-(1-
benzothien-2-yl)ethyl]-N-hydroxyurea (Zileuton) was used as reference drug. For analysis of
mPGES-1 inhibition, a well-established cell-free assay using microsomes of IL-1β-stimulated A549
cells as enzyme source and 20 µM PGH as substrate was used [20].
2
In direct comparison to 16 and 19, the corresponding analogues with a methylene bridge
(4d and 4a) were less potent against 5-LO in intact cells and cell-free assays, respectively. The
strong impact of the thiomethylene moiety on the potency was also evident in the case of mPGES-1
evaluation. In contrast, variation of the halogen substituent chlorine vs fluorine (4c), leads to
compound which is more potent both in cell-free and in cell-based 5-LO activity assays (IC 0.17
5
0
µM each vs 0.78 µM and 1.15 µM). Moreover, repositioning of the chlorine in meta- and para-
positions of the C-2 phenyl ring, (4b) caused a remarkable increase in the potency versus 16,
yielding compounds with IC values in the range of 45 nM for intact cells and 54 nM for cell-free
5
0
assays (Table 1). Of note, both 4c and 4b were most potent direct mPGES-1 inhibitors with IC50
values of 4.2 µM. Regarding the second series in which the phenyl moiety is directly attached to the
benzoindoles core, the insertion of an activating group (i.e., methoxy) in ortho-position of the
phenyl ring (7k) does not significantly alter the efficiency against 5-LO in intact cells and in the
cell-free assay vs our previous lead 7d. Compounds carrying deactivating groups, such as halogens -
F, -Cl or -Br in ortho- and para-positions, are about equipotent to 7k in the cell-free assay, with

ACCEPTED MANUSCRIPT
IC50 values ranging from 0.13 to 0.16, respectively (except for 7f) in the cell-based 5-LO test
system, but markedly loose potency for mPGES-1 inhibition (Table 2).
In the case of polyhalogenated derivatives, the position of the substituents in an aromatic
ring of the C-2 side chain is fundamental for the activity, especially in the cell-free assay for
mPGES-1. In fact, compounds 7h (3,4-Cl) and 7j (5-Br, 2-F) showed the best activity, with IC50
values of 0.48 and 2.13 µM, respectively. In the case of polymethylated molecules 7l-n, all
derivatives were comparably active against 5-LO under cell-based conditions with IC of 0.42,
5
0
0.12 and 0.30 µM, respectively; otherwise only compound 7n was active against mPGES-1 in the
low micromolar range with an IC of 2.6 µM.
5
0
Finally, we replaced the indole by benzofurane. The respective derivatives moderately
impaired potency against 5-LO in cell-free assays but substantial decrease 5-LO inhibitory activity
in PMNL, implying that nitrogen is actually an important requirement in the aromatic system that
may govern the 5-LO inhibitory potency (Table 3). Also, the annelation of benzene to the
benzofuran, yielding corresponding dibenzofuran derivatives were less active as compared to the
respective benzoindoles.

ACCEPTED MANUSCRIPT
2.3 Docking studies and molecular dynamic (MD) simulations with mPGES-1 and 5-LO
For rationalizing the inhibitory activities of 4b and 7h by means of molecular docking and
MD simulations (10 ns) to 5-LO, we used the recently reported apo form of the human 5-LO crystal
structure (PDB code: 3O8Y) [50]. We combined docking studies with four copies of MD
simulations to investigate the binding modes of 4b and 7h taking into account ligand-induced
conformational changes of 5-LO. The rationalization of the 5-LO binding mode was obtained
considering the fundamental amino acids in the active site of the enzyme as described previously
[
51-53]. As a result of docking/MD studies for both molecules, we observe that 4b and 7h perfectly
fits into the active site (Figure 4A and 4B, respectively), and the ester carbonyl at 3-position and
hydroxyl at 5-positon of benzoindole nucleus makes H-bonding interactions to Asn425 and Gln363,
respectively, positioning the benzoindole nitrogen for H-bonding to Tyr181 in the upper part of the
main binding cleft, which is a gatekeeper amino acid along with Phe177 at the active site entrance
of 5-LO [54]. Additionally, annealed benzene ring lies in close proximity to His367 and His372,
which engages through π-π interactions.
Luz et al. recently published co-crystal structures of mPGES-1 in complex with four diverse
inhibitors provide important insights into inhibitor binding sites [55-56]. Based on the reported
interaction network of these known inhibitors and to provide further insights into the interaction of
4b and 7h with mPGES-1, we performed docking studies in combination with four copies of MD
simulations (10 ns) using the mPGES-1 crystal structure (PDB code: 4YL3) [56]. Since the
available mPGES-1 x-ray structure does not include membrane coordinates, we analyzed the
binding modes of mPGES-1/4b and mPGES-1/7h complex by means of MD simulations including
membrane insertion (taken from OPM Database [57]) in the simulation systems (Figure 4C and 4D,
respectively). By inclusion of the membrane bilayer, it was possible to observe its effect on
the binding in which the adopted techniques have been widely used for the analysis of other
membrane proteins [58].

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Taking into account the considerations reported for these co-crystal structures, the binding
specificities of 4b and 7h to mPGES-1 were conferred by specific polar interactions at the
cytoplasmic part of the binding groove. For example, the C5-hydroxyl functionality of 4b and 7h
forms direct or water-mediated H-bonds with the Arg52 and His53 at the cytoplasmic entrance
orienting the rest of the molecule into the hydrophobic binding cavity for additional π-π contacts
and hydrophobic interactions, i.e., the 3,4-dichlorophenyl group engages π-π contacts with Tyr130
while the ethyl carboxylate group at 3-position of the benzoindole lies in close proximity of Leu39
and Phe44 for hydrophobic interactions. Another stable H-bonding interaction is also observed
between the side-chain of Gln36 and the NH of benzoindole nucleus (Figure 4C and 4D).
As can be seen from the Figure 4A, 4B, 4C and 4D, both molecules, 4b and 7h, show
similar interaction network for both enzymes, although the inhibitory potency of the compound 4b
on mPGES-1 in comparison to 7h was about 9-fold less potent (IC =4.2 vs. 0.48 µM). Based on
5
0
these docking/MD simulations, it is still hard to make a conclusive explanation of this difference in
activity, this may be related to the entropic cost upon binding, which may arise by the presence of a
methylene bridge in 4b causing instability inside the active site, as can be seen by the increased
standard deviation trend of mPGES-1/4b complex’s RMSD values given at Figure S1 (see
supporting information).

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Table 1 Inhibition of 5-LOX and inhibition of m-PGES-1 activity of compounds 4a-d, 11 and Zileuton in a cell-based assay (intact PMNL) and in a cell-free assay. Data are given
as mean ± S.E.M., n = 3–4.
Inhibition of 5LO activity
Inhibition of m-PGES1
COOEt
(
IC values [µM] )
50
activity(IC values [µM] )
HO
50
N
H
R
Cpd
R
Intact
Cell-free
Cell-free
4
a
2,6-Cl
3,4-Cl
2,6-F
1.158±0.051
0.045±0.018
0.094±0.026
0.130±0.142
0.301±0.029
0.782±0.08
0.054±0.025
0.169±0.021
0.298±0.029
0.317±0.052
Remaining activity: 78.12 ± 13.7%
4
b
4.2 ± 1.4
4.2 ± 2.2
58.8 ± 4.9
0.96 ± 0.09
-
4
c
4
d
2,4,6-CH3
2-Cl
1
1
[46]
[46]
Zileuton
0.7
0.6

ACCEPTED MANUSCRIPT
Table 2 Inhibition of 5-LOX and inhibition of m-PGES-1 activity of compounds 7a-n in a cell-based assay (intact PMNL) and in a cell-free assay. Data are given as mean ± S.E.M.,
n = 3–4.
COOEt
Inhibition of 5LOX activity
IC values [µM] )
Inhibition of m-PGES1 activity
HO
(
(IC values [µM])
50
50
N
H
R
1
Cpd
R
Intact
Cell-free
Cell-free
7
7
a
CH3
2-F
0.42±0.01
0.15±0.01
0.20 ± 0.04
0.24 ± 0.25
0.21 ± 0.06
1.07±0.26
0.32±0.11
0.13 ± 0.03
0.86 ± 0.11
0.23± 0.06
0.21± 0.12
0.42 ± 0.1
0.12 ± 0.02
0.3 ± 0.03
0.48±0.02
0.10±0.04
n.d.
b
50.58 ± 4,9%
2.54 ± 0.41
No values
1.19 ± 0.1
6.01± 0.1
3.4*
7
c
2-Br
0.16 ± 0.02
0.13 ± 0.07
0.13 ± 0.02
2.12 ± 0.65
0.07 ± 0.02
0.13 ± 0.01
0.27 ± 0.05
0.11 ± 0.07
0.19 ± 0.05
0.11 ± 0.05
0.14 ± 0.02
0.12 ± 0.03
7
d
2-Cl
7
e
3-Br
7
f
3- Cl
7
7
g
4-Cl
h
3,4-Cl
4-Br,2-F
5-Br, 2-F
2-OCH3
2-CH3
3-CH3
3,5-CH3
0.48 ± 0.13
3.43 ± 2.41
2.13 ± 1.09
8.6 ± 0.21
12.7 ± 0.24
2.56 ± 0.31
7.06 ± 0.53
7
i
7
j
7
k
7
l
7
m
7
n

ACCEPTED MANUSCRIPT
Table 3 Inhibition of 5-LOX activity of compounds 8b-k vs 9b-k in a cell-based assay (intact PMNL) and in a cell-free assay. Data are given as mean ± S.E.M., n =
3–4.
Inhibition of 5LO activity
IC values [µM] )
Inhibition of 5LO activity
(
(IC values [µM])
50
50
COOEt
COOEt
HO
HO
O
O
R
1
R1
Cpd
R
Intact
Cell-free
Cpd
9b
Intact
Cell-free
8
b
2-F
0.67 ± 0.17
0.37 ± 0.12
116.9 ± 17.4
97.5 ± 7.6
8
c
2-Br
2-Cl
0.79 ± 0.46
1.86 ± 0.14
1.64 ± 0.01
3.92 ± 0.11
3.74 ± 0.75
1.24 ± 0.66
2.32 ± 0.23
4.49 ± 1.49
1.36 ± 0.31
0.16 ± 0.04
0.37 ± 0.09
0.86 ± 0.04
0.46 ± 0.06
0.13 ± 0.01
1.70 ± 0.09
1.06 ± 0.24
2.03 ± 0.07
0.22 ± 0.04
9c
9d
9e
9f
1.4 ± 0.11
0.78 ± 0.14
3.3 ± 0.04
1.79 ± 0.15
2.49 ± 0.45
4.76 ± 0.22
1.86 ± 024
2.19 ± 0.05
4.71 ± 0.79
6.39 ± 0.16
59.5 ± 5.5
2.76 ± 0.06
3.55 ± 1.35
9.52 ± 0.6
54.3 ± 6.0
4.49 ± 0.8
3.79 ± 0.08
8.36 ± 1.53
8
d
8
e
3-Br
8
f
3- Cl
8
8
g
4-Cl
9g
9h
9i
h
3,4-Cl
4-Br, 2-F
5-Br, 2-F
2-OCH3
8
i
8
j
9j
8
k
9k

ACCEPTED MANUSCRIPT
A
B
C
D
Figure 4. (A, B) Binding mode analysis of 4b and 7h, depicted with interacting 5-LO (PDB code 3O8Y) residues. (C,
D) Binding mode analysis of 4b and 7h during interaction with the mPGES-1 (PDB code 4YL3) considering membrane
residues. Main interactions are represented schematically with their occupancies calculated in the time window 0-10 ns.

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2.3 Anti-inflammatory effectiveness of derivatives 4 b-c, 7c, 7e, 7 h-j, 7m in carrageenan-
induced paw oedema
In order to verify the anti-inflammatory efficacy, we evaluated the most interesting
compounds of the current series against carrageenan-induced paw oedema, which represents a well-
established model of acute inflammation. [48] 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 its maximum at 4 h post-carrageenan application (Fig.
6-7). The vast majority of compounds (4 mg/kg, i.p.) significantly reduced paw oedema, in
agreement with the results from in vitro studies.
In details, among the benzo[g]indol-3-carboxylates tested with C-2 benzylic branch (see
Table 1), 4b and 4c were the most effective, in the in vivo experiment both compounds also
significant reduced oedema formation by 73.0 and 81%, respectively, as compared with vehicle-
treated animals, and they maintained their potency after 6 h of treatment (Fig. 6).
Regarding the second series, that is, the polyalogenated derivatives 7h, 7i and 7j (4 mg/kg,
i.p., each) the peak of the response to carrageenan at 4 h was reduced by 47.3, 69.35, and 73.95%,
respectively (Fig. 7 A), while Zileuton (20 mg/kg, i.p.) used as reference, as previously reported by
us, caused only 38.2% inhibition (data not shown).
Finally, the ortho-brominated derivatives 7c and 7e demonstrated the lowest anti-inflammatory
effects with 47.0 and 18.1% inhibition, respectively. Of interest, compound 7m was most effective,
with a reduction of the response to carrageenan at 4 h by 75% (Fig. 7 B).

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Fig.6 Effect of compounds 4b and 4c (4 mg/kg, i.p.) on carrageenan-induced paw oedema 0–6 h after carrageenan
injection. Data are expressed as mean ± SEM (n = 6 for each group). *** indicate significant (P < 0.001) differences vs
carrageenan+ vehicle-treated animals.

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Fig.7 Effect of compounds 7i, 7h and 7j (A) or 7m, 7c and 7e (B) (4 mg/kg, i.p.) on carrageenan-induced paw oedema
0
–6 h after carrageenan injection. Data are expressed as mean ± SEM (n= 6 for each group). * or *** indicate
significant (P < 0.05 or P < 0.001, respectively) differences vs carrageenan + vehicle-treated animals.

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Conclusion
A series of new benzoindole, benzofuran and naphthofuran derivatives were prepared and
evaluated for their ability to inhibit both 5-LO and m-PGES-1 activity. Considering the biological
data, we were able to perform a detailed structure based design of a revisited version of
benzo[g]indol-3-carboxylic acid derivatives. Moreover, we disclosed new key factors that may govern
the 5-LO/m-PGES-1 inhibitory potency of the target structures.
Our studies allowed us to complete SAR profile and to optimize chemical structures of the title
compounds of the series. In particular, according to the above mentioned results, it is possible to
conclude that:
•
the thiomethylene linker between the phenyl ring and C-2 position of benzo[g]indole
heterocyclic system may be omitted, allowing the direct connection of 3,4-chlorophenyl-
substituent to heterocycle, leading to 7h with an increased inhibitory activity vs previous
hits (16 and 19) in the nanomolar range for both enzymes;
•
the presence of nitrogen of the indole or benzo[g]indole-3-carboxylic acid derivatives
remains an indispensable requirement for the activity, in fact, its substitution for benzofuran
or naphthofuran bioisoster reduces the inhibitory activity of the target compounds (7h vs 8h)
the substitution of the phenyl ring radical influences the inhibitory activity. The
simultaneous presence of chlorine in meta- or para-positions of the C-2 benzylic aromatic
ring (4b, and 7h), as well as the introduction of methyl groups (7m) (in meta- position on
the phenyl ring), causes a marked increase of 5-LOX and mPGES-1 inhibitory activity in
cell-free assay.
•
The antiflammatory properties of selected compounds were confirmed by in vivo evaluation of their
ability to reduce carrageenan-induced paw oedema. In particular, methylated compound 7m and its
dichloro-substituted congener 4b exhibit antinflammatory activity in lower doses than our previous
hit compounds.

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4
4
.Experimental section
.1 Chemistry
All reagents were analytical grade and purchased from Sigma-Aldrich (Milano-Italy). All
microwave irradiation experiments were carried out in a Biotage® Initiator+ Microwave
synthesizer (Biotage, Sweden AB, Uppsala, Sweden). The reactions were carried out in 10 mL glass
tubes, sealed with aluminium/Teflon crimp tops, which can be exposed to 300 °C and 30 bar
internal pressure. After the irradiation period, the reaction vessel was cooled rapidly(60-120s) to
ambient temperature by gas jet cooling. 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 60 F254 nm purchased from Merck (Darmstadt, Germany). Melting points were determined in
open capillary tubes on Stuart®SMP30 Melting Point Apparatus and are uncorrected. Reaction
1
13
yields refer to chromatographically and spectroscopically pure products. H and C NMR spectra
were registered on a Bruker AC 300. Chemical shifts are reported in ppm relative to
tetramethylsilane 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.
The following specific procedures illustrate the general methods for the preparation of key
intermediates and title compounds ethyl 4-aryl-3-oxobutanoates
4.2 General procedure for the synthesis of Ethyl 4-aryl-3-oxobutanoates (2a-d)
Step-1
A mixture of 10 g (48.7 mmol) of 2-substituted phenyl acetic acid, 40 ml of anhydrous PhMe was
treated with 11.2 g (53.8 mmol) PCl at ambient temperature under protection of moisture. After the
5
effervescence ceased, the resulting clear yellow solution was heated at reflux for 2 hours, only in
the case of 2-(2,4,6-trimethylphenyl)acetyl chloride the reaction was conducted at room

ACCEPTED MANUSCRIPT
temperature overnight. Due to their hydrolytic lability, all intermedia were not analyzed, but used
directly in the next step.
Step-2
A stirred solution of 7.7 g (53.5 mmol) 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid) in
1
50 ml of anhydrous CH Cl was chilled in an ice chest and treated with 25 ml (~18.6 g, 143.5
2 2
o
mmol) i-Pr NEt (Hunig’s base). The resulting mixture was cooled to – 8 C in an ice-salt bath with
2
stirring, followed by dropwise addition of dichloromethane solution of 2-substituted phenyl acetyl
chloride (50.9 mmol). During the addition of the acylating agent, the inner temperature was kept
o
below 0 C. After the addition of chloroanhydride was complete, the whole reaction mixture was
stirred for 2 hours more at the above mentioned temperature and then left overnight. The next day it
was poured into a mixture of 150 ml of 1M aqueous H SO and crushed ice. The two layers formed
2
4
were vigorously shaken, and the lower organic layer was separated. The aqueous layer was
discarded, while the organic layer was washed successively with ice-cold dilute H SO and water,
2
4
and dried over anhydrous Na SO . After filtration, the solution was stripped down from the solvent
2
4
at ordinary, and then – slightly reduced pressure and the residue was diluted with 150 ml of absolute
EtOH. This was refluxed under the protection of atmospheric moisture until the evolution of CO2
has ceased and treated with 1 g of activated carbon. The resulting mixture was reflux for one more
hour, filtered and evaporated under the diminished pressure, leaving the title product of technical
grade as a straw-colored oil. The latter was redissolved in 200 ml of t-BuOMe, washed successively
with saturated NaHCO solution, water and dried over anhydrous Na SO . The latter was removed
3
2
4
by filtration through a TLC-grade silica-gel pad and evaporated. The thus obtained 3-oxoester was
subjected to vacuum distillation collecting a fraction in a wide range, which was redistilled with a
short Vigreux column, to remove the undesired by products.
An analytically pure substance was prepared by gravity elution silica gel column chromatography
of this products, using Hexan-EtOAc (9:1, v/v) mixture as a mobile phase.

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4
.2.1 Ethyl 4-(2,6-dichlorophenyl)-3-oxobutanoate (2a)
1
H NMR (CDCl , 300MHz) δ (ppm) = 1.25 (t, 3H, J=7.40Hz); 3.47 (s, 2H); 3.79 (s, 2H); 4.16 (q,
3
2H, J=7.42Hz); 7.00 (d, 1H, J=8.23Hz); 7.27 (s, 1H); 7.35 (d, 1H, J=7.78Hz). Yield: 75%.
4.2.2 Ethyl 4-(3,4-dichlorophenyl)-3-oxobutanoate (2b)
1
H NMR (CDCl , 300MHz) δ (ppm) = 1.30 (t, 3H, J=7.13Hz); 3.26 (s, 2H); 3.58 (s, 2H); 4.23 (q,
3
2H, J=7.29Hz); 7.19 (t, 1H, J=8.85Hz); 7.33 (d, 2H, J=7.70Hz). Yield: 69%.
4.2.3 Ethyl 4-(2,6-difluorophenyl)-3-oxobutanoate (2c)
1
H NMR (CDCl , 300MHz) δ (ppm) = 1.27 (t, 3H, J=7.37Hz); 3.54 (s, 2H); 3.92 (s, 2H); 4.19 (q,
3
2H, J=7.51Hz); 6.88 (t, 2H, J=8.24Hz); 7.27 (m, 1H). Yield: 88%.
4.2.4 Ethyl 3-oxo-4-(2,4,6-trimethylphenyl)butanoate (2d)
1
H NMR (CDCl , 300MHz) δ (ppm) = 1.92 (t, 3H, J=6.90Hz); 2.28 (s, 6H); 2.31 (s, 3H); 3.47 (s,
3
2H); 3.51 (s, 2H); 4.21 (q, 2H, J=7.06Hz); 6.90 (s, 2H). Yield: 57%.
4.3 General procedure for the synthesis of Ethyl 3-amino-4-arylcrotonates (3a-d)
A 10 ml glass vial, equipped with a magnetic stirring bar and an aluminium-Teflon crimp top, was
charged with ethyl 4-(2,6-difluorophenyl)-3-oxobutyrate, NH OAc, PhMe, a catalytic amount of
4
o
glacial AcOH and appropriate molecular sieves. The reaction mixture was stirred at 140 C under
o
6
0 W microwave irradiation for 20 minutes, cooled to 50 C and partitioned between t-BuOMe and
saturated NaHCO aqueous solution. The resulting mixture was vigorously stirred, filtered on a
3
Schott funnel to break the resulting emulsion and the upper organic layer was separated. An
aqueous layer was extracted with t-BuOMe and the combined organic phases were washed with
water (until the neutral pH), dried over anhydrous Na SO and filtered. The resulting filtrate was
2
4
stripped down from the solvent under reduced pressure, yielding a crude product, which was
purified by flash chromatography on silica gel column, using Exan-EtOAc (4:1, V/V) mixture as a
mobile phase. The yield of pure ethyl 3-amino-4-(2,6-difluorophenyl) crotonate was 88%.

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4
.3.1 Ethyl 3-amino-4-(2,6-dichlorophenyl)but-2-enoate (3a)
Compound 3a was synthesized from Ethyl 4-(2,6-dichlorophenyl)-3-oxobutanoate 2a (1.0equiv.),
Ammonium acetate (3.0 equiv.) and acetic acid (5 gtt.). Flash chromatography with hexan/EtOAc
(80:20) afforded analytically pure product. Yield: 88%.
1
H NMR (CDCl , 300MHz) δ (ppm) =1.26 (t, 3H, J=7.19Hz); 3.87 (s, 2H); 4.11 (q, 2H,
3
J=7.19Hz); 4.48 (s, 1H); 7.22 (t, 1H, J=8.71Hz); 7.39 (d, 2H, J=7.79Hz).
.3.2 Ethyl 3-amino-4-(3,4-dichlorophenyl)but-2-enoate (3b)
4
Compound 3b was synthesized from Ethyl 4-(3,5-dichlorophenyl)-3-oxobutanoate 2b (1.0equiv.),
Ammonium acetate (3.5 equiv.) and acetic acid (4 gtt.). Flash chromatography with hexan/EtOAc
(80:20) afforded analytically pure product. Yield: 87%.
1
H NMR (CDCl , 300MHz) δ (ppm) =1.15 (t, 3H, J=7.11Hz); 3.40 (s, 2H); 4.05 (q, 2H,
3
J=7.26Hz); 4.58 (s, 1H); 7.03 (d, 1H, J=8.27Hz); 7.24 (s, 1H); 7.31 (d, 1H, J=8.34Hz).
.3.3 Ethyl 3-amino-4-(2,6-difluorophenyl)but-2-enoate (3c)
4
Compound 3c was synthesized from Ethyl 4-(2,6-difluorophenyl)-3-oxobutanoate 2c (1.0equiv.),
Ammonium acetate (3.3 equiv.) and acetic acid (4 gtt.). Flash chromatography with hexan/EtOAc
(80:20) afforded analytically pure product. Yield: 82%.
1
H NMR (CDCl , 300MHz) δ (ppm) =1.24 (t, 3H, J=7.36Hz); 3.51 (s, 2H); 4.09 (q, 2H,
3
J=7.21Hz); 4.60 (s, 1H); 6.91 (t, 2H, J=7.46Hz); 7.33(m, 1H).
.3.4 Ethyl 3-amino-4-mesitylbut-2-enoate (3d)
4
Compound 3d was synthesized from Ethyl 4-mesityl-3-oxobutanoate 2d (1.0equiv.), Ammonium
acetate (3.0 equiv.) and acetic acid (5 gtt.). Flash chromatography with hexan/EtOAc (80:20)
afforded analytically pure product. Yield: 89%.
1
H NMR (CDCl , 300MHz) δ (ppm) =1.37(t, 3H, J=6.68Hz); 2.31 (s, 6H); 2.37 (s, 3H); 3.62(s,
3
2H); 4.11 (q, 2H, J=7.07Hz); 4.47 (s, 1H); 6.89 (s, 1H); 6.91 (s, 1H).

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4
.4 General procedure for the synthesis of 5-Hydroxy-1H-benzo[g]indole-3-carboxylate (4a-d)
To a solution of 1,4-naphthoquinone (1.0 equiv.) in 5mL CH Cl , ZnI (0.1equiv.) was added and
2
2
2
the resulting mixture was heated to boiling temperature. A solution of β-enaminoesters 3a-d,
respectively (1.0 equiv.), in 2 mL of CH Cl was added drop by drop under stirring for 5-10 min.
2
2
After refluxing for additional 30 min, the mixture was cooled to 0-5 °C over night. The precipitated
crystals were filtered off and washed with CH Cl .
2
2
4.4.1 Ethyl 2-(2,6-dichlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4a)
1
Yield: 79%. H NMR (Acetone-d6) δ (ppm) =1.46 (t, 3H, J =7.23Hz ); 3.53 (s, 2H); 4.42 (q, 2H, J
=7.59Hz); 5.02 (s, 1H, OH); 7.37 (t, 1H, J =6.21Hz); 7.39 (d, 2H, J=7.79Hz); 7.42-7.49 (m, 2H);
7
1
1
.77 (s, 1H); 8.01 (dd, 2H, J = 1.5, 8.20Hz); 11.03 (s, 1H, NH). 13C NMR (Acetone-d6) δ (ppm) =
4.8; 17.3; 60.5; 101.7; 104.8; 113.1; 115.9; 126.6; 127.9; 128.1; 128.6; 130.3; 136.7; 139.4; 140.5;
-
44.3; 149.2; 168.0 MS-ESI (m/z): 413.06 [M ]. Anal. Calcd. For C H Cl NO C, 63.78; H, 4.14;
2
2
17
2
3
Cl, 17.12; N, 3.38; O, 11.59 found C, 63.88; H, 4.15; Cl, 17.18; N, 3.65; O, 11.74
4.4.2 Ethyl 2-(3,4-dichlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4b)
1
Yield: 65%. H NMR (Acetone-d6) δ (ppm) =1.44(t, 3H, J =7.19); 4.22 (s, 2H); 4.35 (q, 2H, J
=7.22); 5.15 (s, 1H, OH); 7.25 (d, 1H J =7.24); 7.31(d, 1H, J =7.24); 7.40 (s, 1H); 7.50-7.58 (m,
2H); 7.75 (s, 1H); 7.90 (dd, 2H, J =1.39; 8.15 Hz); 11.39 (s, 1H, NH). 13C NMR (Acetone-d6) δ
(ppm) = 18.2; 35.5; 61.8; 99.4; 105.2; 112.4; 114.8; 127.9; 128.3; 128.7; 130.1; 130.3; 131.0; 132.2;
-
1
32.5; 135.4; 137.4; 138.1; 144.1; 147.0; 168.0 MS-ESI (m/z): 413.06[M ]. Anal. Calcd. For
C H Cl NO C, 63.78; H, 4.14; Cl, 17.12; N, 3.38; O, 11.59 found C, 64.01; H, 4.24; Cl, 17.12; N,
22
17
2
3
3.55; O, 11.68
4.4.3 Ethyl 2-(2,6-difluorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4c)
1
Yield: 68%. H NMR (Acetone-d6) δ (ppm) = 1.39 (t, 3H, J = 7.28); 3.71 (s, 2H); 4.44 (q, 2H, J =
7
.15); 7.01 (t, 2H, J =7.89); 7.34-7.49 (m, 3H); 7.62-7.69 (m, 2H), 7.74 (s, 1H), 7.90 (dd, 2H, J
2.22; 9.03Hz), 11.30 (s, 1H, NH). 13C NMR (Acetone-d6) δ (ppm) = 17.0; 19.5; 72.1; 100.9;
11.4; 112.3; 112.6; 113.1; 113.8; 114.1; 128.4; 129.6; 129.8; 130.7; 130.9; 131.0; 138.2; 144.8;
=
1

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-
1
4
4
49.7; 165.4; 165.9; 170.1 MS-ESI (m/z): 381.12[M ]. Anal. Calcd. For C H F NO C, 69.29; H,
22 17 2 3
.49; F, 9.96; N, 3.67; O, 12.59 found C, 69.32; H, 4.68; F, 10.15; N, 3.68; O, 12.59
.4.4 Ethyl 2-(2,4,6 trimethylbenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4d)
1
Yield: 77%. H NMR (Acetone-d6) δ (ppm) =1.48 (t, 3H, J=7.11); 2.45 (s, 6H); 2.49 (s, 3H); 4.47
(s, 2H); 4.41 (q, 2H, J =7.12); 5.50 (s, 1H, OH); 6.90 (s, 2H); 7.36-7.40 (m, 2H); 7.77 (s, 1H); 8.07-
1
3
8
7
1
3
4
.14 (m, 2H); 10.57(s, 1H, NH). C NMR (Acetone-d6) δ (ppm) = 20.1; 24.2; 25.4; 26.7; 27.0;
0.0; 99.4; 103.0; 111.3; 115.4; 125.6; 125.9; 127.8; 127.9, 128.9; 130.8; 136.8; 137.0; 138.4;
-
44.0; 149.3; 166.1 MS-ESI (m/z): 387.18[M ]. Anal. Calcd. For C H NO C, 77.49; H, 6.50; N,
2
5
25
3
.61; O, 12.39 found C, 77.51; H, 6.45; N, 3.81; O, 12.40.
.5 General procedure for the microwave irradiated synthesis of β-enaminoesters derivatives
6a-n)
(
β-keto esters 5 a-n (1.0 equiv.), Ammonium acetate (3.0 equiv.) and Acetic acid (gtt.) were
dissolved in dry Toluol (6mL) in a 10mL reaction glass vial containing a tiny stirring magnet and
molecular sieves . The vial was sealed tightly with an aluminium-Teflon® crimp top and the
mixture was irradiated for 20 min at a pre-selected temperature of 140°C, with an irradiation power
of 60W. After the reaction, the vial was cooled to 50°C by gas jet cooling. The crude mixture was
portioned between ethyl acetate and saturated solution of Sodium bicarbonate (15mL of each) and
the aqueous layer was extracted with ethyl acetate (3 x 15mL). The combined organic layer were
dried on Sodium sulfate anhydrous, filtered and the solvent was removed under reduce pressure.
Then, final crude compounds were purified by flash chromatography over silica gel.
4.5.1 Ethyl 3-aminobut-2-enoate (6a)
Compound 6a was synthesized from Ethyl 3-(2-fluorophenyl)-3-oxopropanoate 5a(1.0equiv.),
Ammonium acetate (4.0equiv.) and acetic acid (7 gtt.). Flash chromatography with hexan/EtOAc
(80:20 to 70:30) afforded analytically pure product as 89 %.
1
H NMR (CDCl , 300MHz) δ (ppm) =1.24 (t, 3H, J=7.36Hz), 3.51 (s, 3H), 4.09 (q, 2H,
3
J=7.28Hz), 4.60 (s, 1H).