Syntheses and evaluation of the antioxidant activity of acitretin analogs with amide bond(s) in the polyene spacer

Article abstract of DOI:10.1016/j.ejmech.2009.10.012

Ester analogs of the antipsoriatic drug acitretin were synthesized by coupling either anilines with N-protected indole-3-carboxylic acid, followed by deprotection and coupling with O-monoprotected dicarboxylic acids or Wittig reaction of indole-3-carboxaldehyde, 3-acetyl-1-tosylpyrrole and 4-amino-9-fluorenone with Ph₃P{double bond, long}CHCO₂tBu, followed by N-deprotection, where necessary, and finally coupling with cinnamoyl fluorides. Corresponding free acids were obtained through TFA-mediated carboxyl group deprotection. Although these analogs and acitretin showed very low reducing abilities, analogs 5, 6, 8 and 12 strongly inhibited LOX with IC₅₀ values ranging from 35-65 μM. Acitretin and its analogs 5-7, 10, 11 and 15 inhibited lipid peroxidation more strongly than trolox whereas acitretin and analog 4 were in vivo more potent anti-inflammatory agents on rat paw oedema induced by Carrageenan than indomethacin.

Full text of DOI:10.1016/j.ejmech.2009.10.012

European Journal of Medicinal Chemistry 45 (2010) 298–310
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Syntheses and evaluation of the antioxidant activity of acitretin analogs
with amide bond(s) in the polyene spacer
,
b
b
b,
**
Dimitra Hadjipavlou-Litina a , George E. Magoulas , Marios Krokidis , Dionissios Papaioannou
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b Laboratory of Synthetic Organic Chemistry, Department of Chemistry, School of Natural Sciences, University of Patras, 26504 Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Ester analogs of the antipsoriatic drug acitretin were synthesized by coupling either anilines with N-
protected indole-3-carboxylic acid, followed by deprotection and coupling with O-monoprotected
dicarboxylic acids or Wittig reaction of indole-3-carboxaldehyde, 3-acetyl-1-tosylpyrrole and 4-amino-9-
fluorenone with Ph3P]CHCO2tBu, followed by N-deprotection, where necessary, and finally coupling
with cinnamoyl fluorides. Corresponding free acids were obtained through TFA-mediated carboxyl group
deprotection. Although these analogs and acitretin showed very low reducing abilities, analogs 5, 6, 8
Received 10 July 2009
Received in revised form
6 October 2009
Accepted 7 October 2009
Available online 14 October 2009
and 12 strongly inhibited LOX with IC50 values ranging from 35–65 mM. Acitretin and its analogs 5–7, 10,
Keywords:
Retinoid
11 and 15 inhibited lipid peroxidation more strongly than trolox whereas acitretin and analog 4 were in
vivo more potent anti-inflammatory agents on rat paw oedema induced by Carrageenan than
indomethacin.
Acitretin analog
Antioxidant activity
Anti-inflammatory activity
Lipoxygenase inhibitor
Lipid peroxidation
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
We have recently described the synthesis of easily assembled
ACI analogs in which two double bonds of the tetraene chain have
Acitretin (ACI) is a second generation retinoid which is currently
used orally as a drug against severe psoriasis and other skin
disorders [1a]. Its mechanism of action is not completely under-
stood, and there are few studies focusing on histological and
immunohistochemical differences before and after treatment of
psoriasis with ACI [1b]. Acitretin is known to re-establish a more
normal pattern of cell growth and, in a lesser degree, to have some
effect on immune cell processes [1b]. Plewig and Wagner noted in
vivo significant anti-inflammatory effects with retinoids [2a]. Aci-
tretin was found to inhibit superoxide anion formation [2b]. Its use
is however associated by a number of unwanted side-effects [2c].
The molecule of ACI incorporates an aromatic lipophilic part which
is connected to a carboxylic group (the hydrophilic part of the
retinoid) through a conjugated tetraene chain (Fig. 1). In an effort to
improve the therapeutic index of this compound and its selectivity
towards several biological targets, a variety of ACI analogs have
been synthesized [3].
been replaced by the isosteric amide bond and a third one has been
restricted within the heterocyclic ring of either indole (see e.g.
compounds 1, 2 and 3, Fig. 1) or pyrrole. In addition, the remaining
fourth E double bond has been either retained (1) or isomerized (2)
or reduced (3) whereas the aromatic part of ACI was varied at will
by choosing the appropriate anilines [4]. For the purpose of the
present study, four new analogs with changes in the aromatic part,
namely compounds 4–7, were synthesized. We reasoned that
similar changes may be introduced in the tetraene chain by using
cinnamic acids in the place of anilines as exemplified with ACI
analogs 8 and 9 and their corresponding tert-butyl esters 10 and 11
(Fig. 1). As it will be described below, attempted synthesis of 9
eventually led to the isomeric acid 12. Furthermore, the indole
residue may be substituted by either the pyrrole (analog 13) or the
4-amino-9-fluorenone (analogs 14 and 15) moieties, whereas
structural characteristics of the two types of ACI analogs may be
combined as exemplified with analog 16. Earlier studies have
shown that inclusion of aromatic heterocyclic rings in retinoids is
associated with reduced toxicity [5]. The involvement of
these particular heterocyclic rings in the design of our analogs
stemmed from the anticipation that they could be readily incor-
porated into the spacer by using the commercially available indole-
3-carboxylic acid and -carboxaldehyde or the readily synthesized
* Corresponding author. Tel.: þ302310997627; fax: þ302310997679.
** Corresponding author. Tel.: þ302610962954; fax: þ302610962956.
E-mail addresses: hadjipav@pharm.auth.gr (D. Hadjipavlou-Litina), dapapaio@
chemistry.upatras.gr (D. Papaioannou).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.012

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
299
O
O
9
5
HO
HO
OH
OH
OH
7
1
3
4
2
8
6
MeO
ACI
CA
O
H
N
O
OMe
15
O
N
MeO
MeO
3
5
14
16
4
17
12
1
N
6
X
O
O
O
O
2
13
11
20
1
18
MeO
19
10
7
OMe
OMe
OMe
8
9
9 : X = OH
11 : X = OtBu
H
N
N
MeO
MeO
18
O
OH
O
17
OMe
MeO
19
13
2
12
O
16
14
20
MeO
21
11
10
N
15
2
9
1
HO
4
5
H
N
3
8
N
N
MeO
MeO
O
6
OH
7
12
O
O
O
O
3
OMe
OMe
4
3
O
OMe
O
6
11
13
12
14
9
N
1
OtBu
5
2
10
O
O
17
7
8
H
N
MeO
15
16
OMe
X
13
MeO
4 : X = OtBu
5 : X = OH
6
1
X
7
8
5
O
20
23
18
4
19
21
O
16
N
9
17
2
H
3
Y
10
11
24
15
O
H
N
22
N
R
14
OH
12
13
O
O
14 : X = CO2tBu, Y = H
15 : X = H, Y = CO2tBu
6 : R = H
7 : R = NO2
4
O
O
5
OMe
5
3
OMe
19
O
O
16
15
14
3
2
17
18
4
9
18
O
O
20
12
1
N
6
8
X
2
21
6
13
16
7
N
N
1
11
10
17
H
20
10
11
12
15
24
OMe
7
19
22
MeO
23
14
8
9
OMe
13
8 : X = OH
10 : X = OtBu
16
Fig. 1. Structures of compounds encountered in the present work.
3-acetylpyrrole, which include the hydrolytically stable N–C]C–
CO structural motif. The latter, would allow for the replacement of
either three or two conjugated double bonds of the spacer by the
isosteric structural units NH–CO–C(R)]CH–N(R)–CO (analogs 1–7)
and CO–N(R)–CH]C(R) (analogs 8–11, 13), respectively. Finally, the
implication of tert-butyl esters in the present study was thought of
interest because we had observed, from biological studies of other
types of a,b-unsaturated acids, that their corresponding tert-butyl
esters were more potent inhibitors of lipoxygenase (LOX) than the
free acids [6].
Taking into consideration that ACI and the present ACI analogs
bear structural characteristics of cinnamic acids with established
antioxidant activities, we decided to examine the possibility of ACI
and the currently described analogs to act as inhibitors of LOX
activity and of the peroxidation of biological membranes as well as
to present anti-inflammatory activity. Through these compounds,
we could hopefully determine the effect of the following structural
elements on the biological activity: (a) the variation of the electron
density and/or lipophilicity of the aromatic part, (b) the presence of
one or two amide bonds in various positions of the tetraene chain

300
D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
(spacer), (c) the nature of the heterocyclic ring in the spacer (d) the
presence of an E double bond next to the hydrophilic part of the
molecule and (e) the presence of a free carboxyl group or its cor-
responding lipophilic tert-butyl ester group at the hydrophilic part
of the molecule.
the reaction of fumaric acid and diphenylmethyl bromide in DMF in
the presence of DIPEA at RT. This ester was easily isolated through
routine flash column chromatography (FCC) in 34% yield. On the
other hand, O-tert-butyl fumarate (22) was synthesized through
a Wittig reaction of the commercially available glyoxylic acid
monohydrate with the stabilized phosphorane Ph3P]CHCO2tBu in
DMF at RT [7], followed by aqueous work-up. The thus obtained
ester 22 had the E configuration, as shown by 1H NMR. Coupling of
anilide 20a with acid 22 and of anilides 20b,c with acid 21 using
PyBrOP in the presence of DIPEA and a catalytic amount of
4-dimethylaminopyridine (DMAP) in DCM, at RT for overnight,
afforded the anticipated esters 4 and 23b,c respectively, in 81–86%
yields. Routine deprotection of the afore mentioned esters using
a 25% or 50% solution of TFA in DCM for 1 h gave the projected ACI
analogs 5–7 in 79–90% yields.
2. Results and discussion
2.1. Chemistry
Syntheses of ACI analogs 1, 2 and 3 have been previously
reported [4]. ACI analogs 4–7 were synthesized by coupling initially
the commercially available anilines 17a–c with 1-(tert-butox-
ycarbonyl)indole-3-carboxylic acid (18) [4] using the powerful
coupling
agent
bromotripyrrolidinophosphonium
hexa-
On the other hand, Wittig reaction of Ph3P]CHCO2tBu with the
commercially available indole-3-carboxaldehyde (24) or 4-amino-
9-fluorenone (25) provided adducts 26 and 27, respectively, in
93–95% yields (Scheme 2). It is worth mentioning that compound
27 was obtained as a mixture of isomers (ratio E/Z ¼ 55:45 by 1H
NMR). Crystallization of said mixture from PhMe, gave in low yield
the Z isomer (Z ꢁ 27), as identified by single crystal X-ray crystal-
lography [8]. Similarly, Wittig reaction of Ph3P]CHCO2tBu with
3-acetyl-1-tosylpyrrole (28), readily synthesized from pyrrole [9],
provided the anticipated adduct as a mixture of geometric isomers
(ratio E/Z ¼ 65:35, as shown by 1H NMR). Crystallization from ethyl
acetate/hexane, afforded the desirable pure E isomer. The latter was
fluorophosphate (PyBrOP) and diisopropylethylamine (DIPEA) in
CHCl3 at 40 ꢀC for 4 h. That way, the corresponding N-protected
anilides 19 were obtained in 74–91% yields (Scheme 1). Removal of
the N-protecting group with a 50% solution of trifluoroacetic acid
(TFA) in dichloromethane (DCM) at room temperature (RT) for 1 h
and successive treatment of the thus obtained trifluoroacetate salt
with a 5% NaHCO3 aq. solution, gave the free anilides 20 in 63–70%
overall yield.
Attachment of the fumaryl unit was realized by coupling ani-
lides 20 with the suitably O-monoprotected fumarates 21 or 22
which were prepared through two different approaches. Thus,
O-diphenylmethyl fumarate (21) was synthesized directly through
6
4
7
A
5
8
A
9
N
B
C
NH2
i
H
N
12
OtBu
OtBu
N
1
HO
10
B
C
13
14
+
3
11
16
2
O
O
O
O
17
15
19
18
a: A = C = OCH3, B = H
b: A = B = C = H
c: A = C = H, B = NO2
ii
5
6
10
9
7
8
4
11
12
8
3
A
A
H
N
O
7
11
3
H
N
iii
NH
B
10
15
9
15
4
2
N
13
B
14
19
12
1
6
1
OX
16
2
5
O
O
O
C
13
14
17
C
18
20
4
: X = tBu
23b,c : X = CHPh2
5/6/7 : X = H
vi
O
O
iv
v
HO
HO
HO
OH
OCHPh2
OtBu
O
O
O
21
O
O
HO
O
x H2O
22
Scheme 1. Synthesis of acitretin analogs 4–7. Reagents and conditions: (i) PyBrOP, DIPEA, CHCl3, 40 ꢀC, 4 h, 74–91%; (ii) 50% TFA/DCM, RT, 1 h, then 5% aq. NaHCO3, 63–70%; (iii) 22
(for 20a) or 21 (for 20b,c), PyBrOP, DIPEA, DMAP (cat.), DCM, RT, 12 h, 81–86%; (iv) Ph2CHBr, DIPEA, DMF, RT, 12 h, 34%; (v) Ph3P]CHCO2tBu, DMF, RT, 12 h, 56%; (vi) 50% TFA/DCM
(for 4) or 25% TFA/DCM (for 23b,c), 1 h, RT, 79–90%.

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
301
O
5
3
4
HN
O
1
HN
OtBu
i
2
iv
vi
6
11
10 / 11
8
7
8
[10]
10
9
[11]
vi
24
26
12
6
O
7
8
5
4
OtBu
H
O
1
i
H2N
H2N
H2N
O
9
+
2
3
OtBu
H
10
11
12
15
14
13
25
Z-27
E-27
iv
iv
15
14
8
3
O
5
Ts
4
7
v
ii, iii
O
HN
1
N
OtBu
O
2
13
6
28
29
A
D
A
5
O
3
B
C
B
C
4
viii
[31b]
vii
OH
1
2
F
6
16
9
7
8
D
30
31
a : A = D = H, B,C = OCH2O
b : A = C = D = OCH3, B = H
Scheme 2. Synthesis of acitretin analogs 8–16. Reagents and conditions: (i) Ph3P]CHCO2tBu/DCM, RT, 16 h, 93% (for 26) or Ph3P]CHCO2tBu/DMF, 100 ꢀC, 16 h, 95% (for E/Z ꢁ 27);
(ii) Ph3P]CHCO2tBu/MeCN, 80 ꢀC, 16 h, 64%; (iii) 10 N NaOH/DMSO, RT, 2 h, 98%; (iv) 31a or 31b/DIPEA/CHCl3, 40 ꢀC, 2–3 h, 50–76%; (v) 31a, NaH, THF, RT, 16 h, 65%; (vi) 50% TFA/
DCM, 0 ꢀC, 1 h, 80–90%; (vii) cyanuric fluoride/Py/DCM, ꢁ10 ꢀC, 45 min, 30–50%; (viii) 20a, DIPEA, CHCl3/DMF (85:15), 40 ꢀC, 12 h, 43%.
treated with a 10 N NaOH solution in DMSO in order to remove the
tosyl protecting group. Compound 29 was thus obtained in 63%
overall yield.
positions o- and p- of the aromatic ring and this may be the reason
why the corresponding ester 10, with only one alkoxy group at
position p- of the aromatic ring, does not give this rearrangement.
Finally, analog 16, which is a hybrid of compounds 4 and 11, was
readily obtained by coupling acyl fluoride 31b with anilide 20a in
the presence of DIPEA in CHCl3/DMF (85:15) overnight at 40 ꢀC.
Routine FCC purification of the crude product afforded pure analog
16 in 43% yield (Scheme 2).
The amines 26, E/Z ꢁ 27, pure Z ꢁ 27 and 29, were then
condensed with the cinnamoyl fluorides 31a or 31b, to give the tert-
butyl esters 10, 11, 13, 14/15 and pure 14 in 50–76% yields. These
reactions generally took place in the presence of DIPEA as the base
in CHCl3 at 40 ꢀC for 1.5–3 h, with the exception of the reactants 29
and 31b which required NaH (as the base) in tetrahydrofuran (THF)
at RT for overnight for their efficient condensation. The required
acyl fluorides 31 were readily obtained from the corresponding
commercially available cinnamic acids (30) upon cyanuric fluoride
treatment [10]. Finally, TFA-mediated deprotection of ester 10
provided the corresponding acid 8 in 90% yield. Unexpectedly,
under identical reaction conditions, attempted deprotection of
ester 11, in order to obtain acid 9, led to the rearranged acid 12 in
84% yield. The structure of this compound was deduced by spec-
troscopic data and in particular 1H NMR data. Indeed, two vinylic
protons and the aromatic proton of the indole ring next to N had
been replaced by three protons in the aliphatic region of the
spectrum. A possible mechanism for the formation of this unex-
pected product, through an acid-mediated Michael-type nucleo-
philic attack, is put forward in Fig. 2. The formation of the
resonance form II should be facilitated by the two MeO groups at
2.2. Antioxidant and anti-inflammatory activity
2.2.1. In vitro antioxidant activity studies
In the present investigation, the ACI analogs 1–8 and 10–16 as
well as the parent molecule ACI were studied with regard to their
antioxidant ability as well as their ability to inhibit soybean LOX.
Antioxidants are defined as substances that even at low concen-
tration significantly delay or prevent oxidation of easily oxidizable
substrates. There is an increased interest of using antioxidants for
medical purposes in the recent years. It is well known that free
radicals play an important role in the inflammatory process [11].
Many non-steroidal anti-inflammatory drugs have been reported to
act either as inhibitors of free radical production or as radical
scavengers [12]. Consequently, compounds with antioxidant
properties could be expected to offer protection in rheumatoid

302
D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
arthritis and inflammation and to lead to potentially effective
drugs. Thus, we tested the present ACI analogs with regard to their
antioxidant ability and in comparison to well known antioxidant
agents, such as caffeic acid (CA), nordihydroguairetic acid (NDGA)
and trolox.
similarity to cellular activities such as lipid peroxidation [14]. The
water soluble azo compound AAPH has been extensively used as
a clean and controllable source of thermally produced alkylperoxyl
free radicals [15].
The interaction of the examined compounds with the stable free
radical DPPH is shown in Table 1. This interaction indicates their
radical scavenging ability and electron donating activity in an iron-
free system. Comparing the efficiencies of all the ACI analogs with
those of ACI, only compounds 1 and 8 (both presenting a free
carboxyl function), displayed medium interaction values whereas
compounds 2, 6 and 7 (all with a free carboxyl group) were inactive.
Taking into consideration the mechanism of action of DPPH, analog
1 has both, the electron-rich ring of trimethoxyaniline to offer an
electron to DPPH radical and a free carboxyl group as an efficient
proton source. On the other hand, the comparable interaction value
of the next most potent analog 8 in the series, might be mainly
attributed to the presence of the (E)-3-(1H-indol-3-yl)acrylic acid
substructure.
Several assays should be used in order to assess in vitro
antioxidant activity [13a]. In this way, factors such as solubility or
steric hindrance, which may be of overriding importance in one
environment but not in another, can be varied and the antioxi-
dant ability of a compound in a variety of milieus may be eval-
uated. For these reasons, we have used two different types of
assays to measure in vitro antioxidant activity of ACI analogs: a)
interaction with the stable free radical 1,l-diphenyl-2-picrylhy-
drazyl radical (DPPH), b) interaction with the water soluble azo
compound
2,20-Azobis(2-amidinopropane)
dihydrochloride
(AAPH). Both require a spectrophotometric measurement and
a certain reaction time in order to obtain reproducible results
[13b].
DPPH has been used in a radical scavenging measuring method.
DPPH is a stable free radical in an ethanolic solution. Because of the
unpaired electron of DPPH, it gives a strong absorption maximum
at 517 nm by visible spectroscopy (purple color). In addition, the
unpaired electron of the radical becomes paired in the presence of
All other compounds presented very low, if any, interaction to
DPPH at 100
mM. The low interaction values of ACI and its analogs,
compared to NDGA, should be mainly attributed to the absence of
easily oxidized functionalities like the ones present in NDGA (two
catechol subunits). The absence of hydrogen atoms (phenolic
hydroxyl groups) which could be donated to stabilize the DPPH
radical is obvious within the given results
Comparing the interaction values between the couples of
compounds 4 and 5, 10 and 8, and 11 and 12, it is evident that the
presence of the free carboxyl function is correlated with higher
interaction values. The introduction of an alkyl ester group in the
conjugated side chain did not increase the oxidation potentials,
when compared to the lead compound. The data obtained strongly
suggest that alkyl ester group have no effect on the electron density.
The replacement of the trans with the cis double bond resulted in
very high decrease of the inhibitory value (compound 2, 2%),
a
hydrogen donor (a free radical scavenging antioxidant),
decreasing the absorption. Therefore, DPPH loses color in propor-
tion to the number of electrons captured [13c]. As a result, this
reaction has been widely used as a simple, rapid and convenient
method independent of sample polarity for screening many
samples for radical scavenging activity [13d] as free radical scav-
engers or hydrogen donors [13e]. These advantages made the DPPH
method interesting for testing our analogs.
The use of the AAPH peroxyl radical is recommended as more
appropriate for measuring radical scavenging activity in vitro,
because the activity of the AAPH peroxyl radical shows a greater
OMe
O
OR
N
O
CF3CO2H
MeO
O
OMe
R = H (9) or tBu (11)
O
RO
RO
CF3CO2
N
N
MeO
MeO
MeO
MeO
OH
OH
OMe
OMe
CF3CO2
I
II
O
O
O
RO
CF3CO2H
HO
MeO
MeO
RO
CF3CO2
OH
N
H
N
N
MeO
MeO
CF3CO2H
MeO
MeO
O
OH
OMe
OMe
12
OMe
Fig. 2. Proposed mechanism for the formation of the unexpected product 12.

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
303
MeO
O
OMe
H
N
O
N
OH
N
Me
O
O
O
MeO
O
4 : R = CMe3
5 : R = H
R
Cl
Indomethacin
O
O
N
N
HO
HO
O
H
O
MeO
O
OMe
O
12
8
OMe
Fig. 3. Structures of indomethacin and ACI analogs (redrawn in a similar way to indomethacin) tested for anti-inflammatory activity in the context of the present work.
probably due to unfavourable interaction with the oxidant.
Hybridization of structures 4 and 11 (analog 16) resulted to a very
low interaction value. The interaction remains highly constant after
antioxidant efficiency of several retinoids using the same assay. He
did not find any interaction with DPPH for ACI as well as for all-
trans-retinoic acid. From his studies he concluded that the retinoids
were not efficient antioxidants for hydrophobic radicals. Packer
found that acitretin was quite effective in a different antioxidant
protocol (the phycoerythrin acid/2,20-azobis(2,4-dimethylvaler-
onitrile -AMVN assay) [16].
After treatment with etretinate (the ethyl ester of ACI), a signif-
icant increase in erythrocyte membrane fluidity and in antioxidant
activity as well as a decrease in lipid peroxidation were observed in
erythrocytes from psoriatic patients. The results confirmed the
improvement of the antioxidant defence system and the cell
protection against lipid peroxidation [17]. Taking this under
P
60 min. Lipophilicity, as
assigned for lipophilic contribution of the substituents on the
phenyl ring) at the anilide type analogs, influences the reducing
p values (the sum of the p values,
P
activity e.g 1 >7 (for compound 1
p
¼ ꢁ0.06 whereas for
P
compound 7
p
¼ ꢁ0.28). The role of molar lipophilicity is
contradictory since the two most active analogs 1 and 8 present big
differences in terms of calculated values of lipophilicity e.g. clog P
values 1 ¼1.60 and 8 ¼ 4.63. It is worth to mention that the parent
compound ACI presented zero interaction. The same result has
been found for ACI by Packer [16], who earlier studied the
Table 1
Interaction % with DPPH (DPPH %); % Inhibition of Lipid Peroxidation (AAPH %); In vitro inhibition of soybean lipoxygenase (LOX) (IC50 or %); % Inhibition of Carrageenan-
induced rat paw oedema (ICPE %)
Compds
Clog P [31]
DPPH % @ 100
m
M
AAPH % @ 100
m
M
IC50 mM or % LOX Inh.
ICPE % 0.01 mmol/Kg (ꢂSD)a
63** ꢂ 3.4
20/60 min (ꢂSD)a
(ꢂSD)a
@ 100
m
M (ꢂSD)a
CA
ACI
1
2
3
4
5
6
7
0.82
6.07
1.60
1.60
1.54
3.92
2.16
2.66
2.95
4.63
6.25
5.98
5.5 ꢂ 0.3
17.5 ꢂ 0.8
85 ꢂ 1.6
64 ꢂ 1.2
8 ꢂ 0.5
IC50 ¼ 600 ꢂ 15
mM
na
1% ꢂ 10ꢁ3
26 ꢂ 1.3
2 ꢂ 10ꢁ3
12.5 ꢂ 0.02
5 ꢂ 0.04
10 ꢂ 0.8
1 ꢂ 10ꢁ3
na
21 ꢂ 1.5
13 ꢂ 0.035
8.3 ꢂ 0.06
57% ꢂ 3.2
42% ꢂ 0.8
34 ꢂ 1.2
68 ꢂ 3.2
77 ꢂ 4.1
85 ꢂ 1.9
94 ꢂ 2.2
22 ꢂ 0.7
95 ꢂ 2.1
80 ꢂ 1.6
10% ꢂ 10ꢁ3
IC50 ¼ 100 ꢂ 2.8
m
M
58.6** ꢂ 4.2
46** ꢂ 1.8
IC50 ¼ 35 ꢂ 1.4
mM
IC50 ¼ 52.4 ꢂ 2.5
mM
26% ꢂ 1.3
8
10
11
IC50 ¼ 65 ꢂ 1.8
m
M
34** ꢂ 1.3
IC50 ¼ 82.5 ꢂ 2.7
85% ꢂ 5.6 @ 100
m
m
M
M
85% ꢂ 3.8 @ 50
mM
0% @ 10 mM
12
13
3.90
5.24
12 ꢂ 0.06
8 ꢂ 0.3
57 ꢂ 1.4
46 ꢂ 3.2
100 ꢂ 2.9
49 ꢂ 1.7
IC50 ¼ 45.2 ꢂ 4.4
mM
9* ꢂ 0.7
39% ꢂ 2.9
14 þ 15
na
63% ꢂ 3.3
14
6.15
4.78
na
43% ꢂ 2.5
6 ꢂ 0.02 (60 min)
16
NDGA
Trolox
Indomethacin
5 ꢂ 0.02
42 ꢂ 0.7
63 ꢂ 1.2
69% ꢂ 3.1
81 ꢂ 1.3/83 ꢂ 2.4
47** ꢂ 3.1
The effect on oedema is expressed as percent of weight increase of hind paw (and as percent of inhibition of oedema) in comparison to controls. Each value represents the
mean obtained from 6 to 15 animals in two independent experiments. In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t test); na, no activity under
the reported experimental conditions. SD standard deviation.
a
Values are means ꢂ SD of three or four different determinations. Means within each column differ significantly (p < 0.05).

304
D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
consideration, all the ACI analogs were studied in order to identify
their possible inhibitory activity on lipid peroxidation.
catalytically inactive Fe2þ. Several LOX inhibitors are excellent
ligands for Fe3þ. This inhibition is related to their ability to reduce
the iron species in the active site to the catalytically inactive ferrous
form [23].
In our studies, AAPH was used as a free radical initiator to follow
oxidative changes of linoleic acid to conjugated diene hydroper-
oxide. Compounds 5, 6, 7, 10 and 11 showed substantially higher
inhibition of lipid peroxidation than the reference compound Tro-
lox (63%). Comparing the anti-lipid peroxidation activities of all ACI
analogs with that of ACI, only compounds 7 and 10 showed higher
inhibition (94 and 95%) followed by 11 (80%) and 5 (77%). Also, the
most potent anti-lipid peroxidation sample seemed to be the
mixture of the geometric isomers 14 and 15 containing them in
Perusal of the IC50’s or % inhibition values (Table 1) shows that
the most potent inhibitor, namely compound 5 (IC50 35
found within the subset of the anilide type compounds (1–7), fol-
lowed by compound 12 (IC50 45.2 M) belonging to the other
mM), is
m
subset of compounds (8–15), namely of the cinnamic acid-type. In
these two subsets of compounds, two other potent inhibitors may
be also identified, namely compounds 6 (IC50 52.4
65 M) respectively. Notably, the parent compound ACI showed
essentially negligible interaction with LOX (1%) under our experi-
mental conditions at 100 M.
mM) and 8 (IC50
comparable amounts. The unsubstituted analog
6
presented
m
activity equipotent to ACI. Within the benzodioxolyl-couple, ester
10 (95%) was by far more potent than the corresponding acid 8
(22%). It is interesting to note that the presence of the indole ring
(analog 11) is more favourable than the pyrrole ring (analog 13).
Although high inhibition values (>80%) are observed for
compounds (ACI, 10, 11, 15) with the highest lipophilicities (clog P
5.98–6.15), there are some exceptions, such as compounds 6 and 7
presenting clog P 2.66 and 2.95 and 14 presenting clog P 6.15 (49%).
A clear trend is observed in the series of carboxylic acids 1, 5–7,
wherein increased inhibition of lipid peroxidation is well-corre-
lated to the increase of lipophilicity of said compounds Within the
series of compounds 1–7, compounds bearing a trans double bond
(1, 4–7) are more active than the one bearing the ethylene unit
(reduced double bond, compound 3, 34%) whereas replacement of
the trans with the cis double bond resulted in very high decrease of
the inhibitory value (compound 2, 8%).
The standard inhibitor trolox obviously exerts its inhibitory
effect on lipid peroxidation mainly through the ability of its 6-
hydroxy-5,7,8-trimethylchromane moiety to break the radical
chain. Although no phenol moieties are present in ACI and its
analogs, both ACI and the anilides 1, 4–7 and 15 could break the
radical chain through the initial abstraction of a hydrogen from
either a methyl group of the spacer (ACI) or the amide function
(anilides). The thus created new radicals are efficiently stabilized
through resonance. A nitro group in position meta to the N radical
would provide additional stabilization through its electron-with-
drawing effect and therefore analog 7 should be expected to be, and
actual is, the most effective inhibitor among the analogs of the
anilide type. On the other hand, the tert-butyl esters 10 and 11 will
obviously exert their inhibitory activity on lipid peroxidation
through other mechanism(s), e.g. through interception of the
alkylperoxyl radicals mainly by the indole moiety and possible
formation oxidized derivatives. Of course, ACI might exert its
inhibitory effect, alternatively or in addition to hydrogen abstrac-
tion, through interception of the alkylperoxyl radicals by its
conjugated tetraene chain.
Leukotrienes play an important role as mediators of a variety of
inflammatory and allergic reactions and are derived from the
biotransformation of arachidonic acid catalyzed by Lipoxygenase
(LOX). LOXs play a role in membrane lipid peroxidation by forming
hydroperoxides in the lipid bilayer [18,19]. Inhibitors of LOX have
attracted attention initially as potential agents for the treatment of
inflammatory and allergic diseases but their therapeutic potential
has now been expanded to certain types of cancer and cardiovas-
cular diseases [20,21].
In this context, we decided to further evaluate the synthesized
ACI analogs for their ability to inhibit soybean LOX by the UV
absorbance based enzyme assay [22]. LOXs contain a ‘‘non-heme’’
iron per molecule in the enzyme active site as high spin Fe2þ in the
native state and the high spin Fe3þ in the activated state. Most of
the LOX inhibitors are antioxidants or free radical scavengers [23].
Other studies suggest a relationship between LOX inhibition and
the ability of the inhibitors to reduce Fe3þ at the active site to the
m
A comparison of the inhibition values in the couples of
compounds 4 and 5 and 8 and 10 indicates that the free acid seems
to be stronger inhibitor than the corresponding tert-butyl ester,
whereas the comparison between tert-butyl esters 11 and 13
indicates that the indole ring is probably more important than the
pyrrole ring for the biological activity. Also, isomer 15 seems to be
more potent inhibitor than isomer 14. On the other hand,
comparison of compounds 1, 2 and 3 shows that the double bond
next to the carboxyl group is correlated with higher inhibitory
activity in particular when it has the trans configuration. Further-
more, comparison of acids 1 and 5–7 reveals that the presence of an
electron-withdrawing group, such as the nitro group, in the aniline
ring is correlated with the lowest inhibitory activity, whereas the
highest activity is secured by the presence of two methoxy groups
in the ring. Finally, hybridization of structures 4 and 11 (analog 16)
did not show to offer any advantage over the parent compounds.
Although lipophilicity is referred as an important physicochemical
property for LOX inhibitors [24], herein the most potent compound
5 had an IC50 value of 35
this concept. On the contrary, analogs 8 and 10 had IC50 values of
65 M and 82.5
M and clog P ¼ 4.63 and 6.25, respectively.
Compared to the standard inhibitor CA and taking into
mM and clog P ¼ 2.16 and does not follow
m
m
consideration the mode of action of LOX, the most active analogs of
the present set of compounds do not contain a catechol moiety,
which efficiently scavenges free radicals and might also chelate
Fe3þ and reduce it to the catalytically inactive Fe2þ. Goldreich et al
[25] examined the effect of retinol, all-trans-retinoic acid and
13-cis-retinoic acid on the activity of lipoxygenase-1 and lip-
oxygenase-2 towards linoleic acid. All-trans-retinoic acid and
13-cis-retinoic acid inhibited lipoxygenase-1 activity competitively,
whereas all-trans-retinol inhibited lipoxygenase-1 activity in
a mixed manner. These findings suggest that retinoids may bind to
the active site of the enzyme or simultaneously act as antioxidants.
Thus, it is tempting to propose that our ACI analogs exert their
inhibitory effect mainly through binding to the active site of the
enzyme with different efficiencies and to a lesser degree to their
electron donating abilities. Compounds 5 and 6, the most potent
compounds (with lower lipophilicity values), probably present
different anchorage to the lipophilic region of the enzyme due to
different orientation/position of the molecules. Compared to the
almost inactive ACI, the most active ACI analogs are characterized
by the presence of the indole ring which imposes a conformational
restriction in either the C4 to C6 spacer segment (analogs 8 and 12)
or better the C5 to C7 spacer segment (analogs 5 and 6). Worth
noting is the fact that analog 12 is more potent than analog 8, and
this might be attributed to the additional restriction imposed on
the C5 to C9 spacer segment by the pyrrolidinone ring. It also
evident that the trans configuration of the terminal –CH]CH–CO2H
moiety is preferred (it is also present in the standard inhibitor CA)
over the cis and even more over the flexible –CH2CH2–CO2H moiety
(reduced double bond) and that the first moiety in that region of

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
305
the molecule can be better accommodated than the corresponding
bulkier –CH]CH–CO2tBu moiety.
inhibitory activity of LOX comes from the change of the spacer and
in particular by incorporating the indole ring and by replacing one
or two double bonds with the isosteric amide bond. On the other
hand, improved inhibitory activities on lipid peroxidation are
observed by retaining the afore mentioned changes in spacer and
by replacing either the carboxyl polar group of ACI with the highly
lipophilic tert-butoxycarbonyl group or the electron-rich aromatic
ring of ACI with an electron-deficient phenyl ring, respectively.
Analog 4, presenting in vivo equipotent anti-inflammatory activity
to ACI could be used as a lead compound for the design of long
acting anti-inflammatory agents with LOX inhibitory activity.
2.2.2. Anti-inflammatory activity in vivo
We had observed, from biological studies of other types of a,b-
unsaturated acids, that their corresponding tert-butyl esters were
more potent inhibitors of lipoxygenase (LOX) than the free acids [6].
Several esters of a non-steroidal anti-inflammatory drug have been
synthesized and showed long acting anti-inflammatory activity
[26]. Indomethacin amides and esters are orally active, non-
ulcerogenic, anti-inflammatory agents in an in vivo model of acute
inflammation [27].
The most interesting compounds in this series of ACI analogs,
namely compounds 4, 5, 8 and 12 as well as ACI were selected to be
examined in vivo by using the functional model of Carrageenan-
induced rat paw oedema. Carrageenan-induced oedema is
a non-specific inflammation resulting from a complex of diverse
mediators [28]. As shown in Table 1, ACI and the tert-butyl ester 4
(clog P 3.92) showed superior anti-inflammatory properties than
the commonly used standard indomethacin, whereas free acid 5
(clog P 2.16) induced equipotent inhibition to indomethacin. Lip-
ophilicity seems therefore to be the main cause for the higher
activity of ester 4 compared to acid 5. On the other hand,
compounds 8 and 12 were less potent with the compound 12
presenting the lowest inhibition (9%). This behaviour might be
possibly attributed to the conformational restriction imposed by
the additional five-membered ring in structure 12 compared to the
structure of compound 8 (Fig. 3). Furthermore, it seems that the
decoration of the indole nucleus by the types of substructures
present in compound 12 (and 8) are not as good as the ones in
compounds 4 and 5 for improved or equipotent biological activity
to indomethacin.
4. Experimental section
4.1. General synthetic
Melting points were determined with a Buchi SMP-20 apparatus
and are uncorrected. IR spectra were recorded for KBr pellets on
a Perkin Elmer 16PC FT-IR spectrophotometer. 1H NMR spectra
were obtained at 400.13 MHz and 13C NMR spectra at 100.62 MHz
on a Bruker DPX spectrometer; TMS was used as reference. ESI-MS
were recorded at 30 V, on a Micromass-Platform LC spectrometer
using MeOH as solvent. GC analyses were performed on an Agilent
Technologies 6890 N gas chromatograph fitted with a capillary
column (30.0 m; 250 mm; 0.25 mm nominal) having as stationary
phase HP-5MS 5% Phenyl Methyl Siloxane. Carrier gas flow-rate:
26.4 mL minꢁ1 He; injection port temperature 300 ꢀC; program:
70–300 ꢀC at 14.45 min. Electron Impact mass spectra were recor-
ded at 20 eV on an Agilent Technologies 5975B instrument, tandem
to the above mentioned GC spectrometer. Microanalyses were
performed on a Carlo Erba EA 1108 CHNS elemental analyzer in the
Laboratory of Instrumental Analysis of the University of Patras.
Flash column chromatography was performed on Merck silica gel
60 (230–400 mesh) and TLC on 60 Merck 60F254 films (0.2 mm)
precoated on aluminium foil. Spots were visualized with UV light at
254 nm and charring agents. The eluent systems used were: (A)
PhMe/Hex (1:1), (B) PhMe/AcOEt (95:5), (C) PhMe/AcOEt (9:1), (D)
PhMe/AcOEt (8:2), (E) PhMe/AcOEt (7:3), (F) PhMe/AcOEt (1:1), (G)
AcOEt, (H) CHCl3/MeOH (95:5), (I) CHCl3/MeOH (7:3).
3. Conclusions
ACI analogs, bearing a variety of substituents in the aromatic
part of the molecule, incorporating one or two amide bonds and an
indole or a pyrrole ring in the spacer, and the hydrophilic carboxyl
group or the lipophilic tert-butoxycarbonyl group at the polar end
of the molecule, were readily assembled by combining either
anilines, indole-3-carboxylic acid and 1,4-dicarboxylic acids or
cinnamic acids, indole-3-carboxaldehyde or 3-acetylpyrrole and
the phosphorane Ph3P]CHCO2tBu.
All the chemicals used were of analytical grade and commer-
cially available from Merck. ACI was purchased from CHEMOS
GmbH. ACI analogs 1–3 were synthesized, for the purpose of the
present study, according to literature protocols [4].
These analogs and ACI exhibited in general low reducing abili-
ties. The tert-butyl ester 10 and the carboxylic acid 7 presented
higher inhibition of lipid peroxidation than ACI. Analog 5 showed
the highest inhibitory activity against soybean LOX, in vitro, fol-
lowed by analogs 12, 6 and 8. Finally, ACI and analog 4 showed
higher anti-inflammatory activity, in vivo, than indomethacin.
Our study indicates that LOX or lipid peroxidation inhibitory
activity is not always accompanied by DPPH radical scavenging
activity. Thus, although compounds such as 5, 6, 7, 10 and 11 inhibit
lipid peroxidation potently they present low, if any, DPPH scav-
enging activity. On the other hand, the compounds 5, 6, 8, 10, 11, 12
and 16 are potent LOX inhibitors possessing low to moderate DPPH
radical scavenging activity. This is in accordance with the finding of
Curini et al. [29] who have studied the antioxidant and LOX
inhibitory activity of five natural prenyloxycarboxylic acids and
showed that the most efficient LOX inhibitor (boropinic acid) is not
the most active DPPH radical scavenger. However, a better corre-
lation exists between LOX and lipid peroxidation inhibitory activity.
In general, the comparison of the antioxidant and anti-inflam-
matory effects of the parent molecule ACI and its analogs 1–8, and
10–16 examined in the present work, leads to the conclusion that
the most important contribution to the significant increase of the
4.2. General procedure for the preparation of anilides 19
To a solution of the commercially available anilines 17a–c
(10 mmol) and acid 18 (2.61 g, 10 mmol) [4] in dry CHCl3 (15 mL)
anhydrous DIPEA (6.97 mL, 40 mmol) and PyBrOP (6.99 g,15 mmol)
were added sequentially. The thus obtained solution was set at
40 ꢀC for 4 h. Upon completion, the reaction mixture was diluted
with CHCl3 and washed sequentially once with a 5% aqueous
NaHCO3 solution, twice with H2O and brine. Drying over Na2SO4
and evaporation to dryness left a residue, from which pure amides
19a–c were obtained through FCC.
4.2.1. tert-Butyl 3-(2,4-dimethoxyphenylcarbamoyl)-1H-indole-1-
carboxylate (19a)
Yield: 3.01 g (76%), light red solid, m.p. 132–133 ꢀC, Rf (B): 0.27,
ESI-MS (m/z): 815.16 (2M þ Na), 793.25 (2M þ H), 419.15 (M þ Na),
397.19 (M þ H). IR (KBr, cmꢁ1): 3435, 2971, 1746, 1658. 1H NMR
(CDCl3):
d 8.40 (1H, d, J 8.0 Hz, H-16), 8.22 (1H, dd, J 7.2 and 1.6 Hz,
H-8), 8.21 (1H, d, J 2.8 Hz, H-13), 8.20 (1H, s, H-2), 8.12 (1H, dd, J 6.0
and 1.6 Hz, H-5), 7.40 (1H, td, J 8.0 and 2.8 Hz, H-7), 7.37 (1H, td, J 7.2
and 1.6 Hz, H-6), 6.56 (1H, dd, J 8.0 and 2.8 Hz, H-15), 6.54 (1H, s,

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D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
NH), 3.93 (3H, s, OCH3), 3.83 (3H, s, OCH3),1.71 (9H, s, C(CH3)3) ppm.
13C NMR (CDCl3):
161.6, 156.5 (two C), 149.2, 135.9, 129.4, 127.5,
12/14), 7.16 and 7.11 (2H, two t, J 7.6 Hz, H-5/6), 7.03 (1H, t, J 7.6 Hz,
H-13) ppm. 13C NMR (CDCl3):
166.5, 140.3, 136.7, 129.1, 129.0 (two
d
d
127.2, 125.2, 123.9, 120.9, 120.6, 117.1, 115.5, 103.9, 98.7, 85.0, 55.9,
55.6, 28.1 (three C) ppm. Anal. Calcd. for C22H24N2O5: C, 66.65; H,
6.10; N, 7.07. Found: C, 66.40; H, 6.22; N, 7.38.
C), 126.9, 123.1, 122.6, 121.6, 121.1, 120.2 (two C), 112.4, 111.0 ppm.
Anal. Calcd. for C15H12N2O: C, 76.25; H, 5.12; N, 11.86. Found: C,
76.02; H, 5.40; N, 12.11.
4.2.2. tert-Butyl 3-(phenylcarbamoyl)-1H-indole-1-carboxylate
(19b)
4.3.3. N-(3-Nitrophenyl)-1H-indole-3-carboxamide (20c)
Yield: 0.89 g (63%), yellow solid, m.p. 240–242 ꢀC, Rf (F): 0.36,
ESI-MS (m/z): 320.25 (M þ K), 304.23 (M þ Na), 282.32 (M þ H). IR
(KBr, cmꢁ1): 3373, 3272, 1638, 1372, 856. 1H NMR (DMSO-d6):
Yield: 2.49 g (74%) white foam; Rf (B): 0.32; ESI-MS (m/z): 695.16
(2M þ Na), 673.19 (2M þ H), 375.45 (M þ K), 359.49 (M þ Na),
337.51 (M þ H); IR (neat, cmꢁ1): 3299, 2981, 1741, 1649; 1H NMR
d
11.85 (1H, s, NH), 10.19 (1H, s, NHCO), 8.81 (1H, s, H-11), 8.36 (1H,
(CDCl3):
d
8.21 (1H, d, J 8.0 Hz, H-8), 8.19 (1H, s, H-2), 8.13 (1H, d, J
s, H-1), 8.21 and 8.19 (2H, two d, J 8.0 Hz, H-13/15), 7.90 (1H, d, J
7.6 Hz, H-7), 7.63 (1H, t, J 8.0 Hz, H-14), 7.50 (1H, d, J 7.6 Hz, H-4),
7.22 and 7.18 (2H, two t, J 7.6 Hz, H-5/6) ppm. 13C NMR (CDCl3):
7.6 Hz, H-5), 7.76 (1H, br.s, NH), 7.67 (2H, d, J 8.0 Hz, H-12/16), 7.42
(1H, td, J 7.2 and 1.6 Hz, H-7), 7.40 (2H, t, J 8.0 Hz, H-13/15), 7.38 (1H,
td, J 7.2 and 1.6 Hz, H-6), 7.17 (1H, t, J 8.0 Hz, H-14), 1.18 (9H, s,
d
164.1, 148.5, 141.6, 136.8, 130.4, 129.8, 126.8, 125.9, 122.9, 121.5,
C(CH3)3) ppm; 13C NMR (CDCl3):
d
162.1, 149.1, 137.8, 135.6, 129.1
121.4, 117.4, 114.0, 112.6, 110.4 ppm. Anal. Calcd. for C15H11N3O3: C,
64.05; H, 3.94; N, 14.94. Found: C, 63.88; H, 4.30; N, 15.24.
(two C), 127.8, 127.3, 125.4, 124.4, 123.9, 120.9, 120.1 (two C), 116.4,
115.4, 85.2, 28.1 (three C) ppm. Anal. Calcd. for C20H20N2O3: C,
71.41; H, 5.99; N, 8.33. Found: C, 71.58; H, 5.80; N, 8.62.
4.4. Synthesis of O-monoprotected fumarates 21 and 22
4.2.3. tert-Butyl 3-(3-nitrophenylcarbamoyl)-1H-indole-1-
carboxylate (19c)
4.4.1. (E)-4-(Benzhydryloxy)-4-oxobut-2-enoic acid (21)
To a solution of fumaric acid (90 mmol, 10.44 g) and DIPEA
(180 mmol, 31.4 mL) in DMF (60 mL), was added dropwise a solu-
tion of bromodiphenylmethane (18 mmol, 4.44 g) in DMF (30 mL)
over a period of 2 h. The reaction mixture left to stir at RT overnight,
quenched with ice, and acidified to pH 6 with careful addition of gl.
AcOH. The mixture was extracted twice with AcOEt and the
combined organic layers washed with water and brine, dried over
Na2SO4 and evaporated to dryness. The crude product was sub-
jected to FCC to afford pure ester 21. Yield: 1.73 g (34%), m.p. 141–
142 ꢀC, Rf (G): 0.2, ESI-MS (m/z): 305.17 (M þ Na), 283.65 (M þ H). IR
Yield: 3.47 g (91%), pale yellow oil, Rf (C): 0.37, ESI-MS (m/z):
420.38 (M þ K), 404.43 (M þ Na), 382.12 (M þ H). IR (neat, cmꢁ1):
3254, 2980, 1746, 1657, 1372, 856. 1H NMR (CDCl3):
d 8.49 (1H, t, J
2.0 Hz, H-12), 8.20 (1H, s, H-2), 8.16 (1H, d, J 8.0 Hz, H-8), 8.12 (1H, s,
NH), 8.09 (2H, d, J 6.4 Hz, H-14/16), 7.97 (1H, dd, J 7.2 and 1.2 Hz, H-
5), 7.52 (1H, t, J 8.4 Hz, H-15), 7.40 (1H, td, J 7.2 and 1.2 Hz, H-7), 7.36
(1H, td, J 7.2 and 1.2 Hz, H-6), 1.70 (9H, s, C(CH3)3) ppm. 13C NMR
(CDCl3):
d 162.4, 148.9, 148.6, 139.1, 135.6, 129.9, 128.2, 127.1, 125.8,
125.6, 124.1, 120.9, 118.8, 115.5, 115.4, 114.8, 85.5, 28.1 (three C) ppm.
Anal. Calcd. for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02. Found: C,
63.12; H, 4.88; N, 10.74.
(KBr, cmꢁ1): 3200-2400, 1718, 1692. 1H NMR (DMSO-d6):
d 7.46 (4H,
d, J 7.2 Hz, o-PhH), 7.36 (4H, t, J 7.2 Hz, m-PhH), 7.29 (2H, t, J 7.2 Hz,
p-PhH), 6.90 (1H, s, PhCH), 6.84 (2H, s, CH]CH) ppm. 13C NMR
(DMSO-d6): 165.9, 163.8, 140.3 (two C), 135.6, 132.4, 128.8 (four C),
128.1 (two C), 126.7 (four C), 77.5 ppm. Anal. Calcd. for C17H14O4: C,
72.33; H, 5.00. Found: C, 72.56; H, 4.79.
4.3. General procedure for the preparation of indole derivatives 20
Anilides 19 (5 mmol) were treated with a 50% solution TFA in
DCM (15 mL) at RT for 1 h and then evaporated to dryness. The oily
residues were triturated with Et2O and refrigerated overnight to
give, upon filtration, anilides 20 as the coresponding tri-
fluoroacetate salts. These, were then treated with an excess of a 5%
aqueous NaHCO3 solution and extracted twice with EtOAc. The
organic layers were combined and washed twice with H2O, dried
(Na2SO4) and evaporated to leave a residue. Addition of Et2O and
overnight refrigeration gave pure free indole derivatives 20.
4.4.2. (E)-4-(tert-Butyloxy)-4-oxobut-2-enoic acid (22)
To a solution of glyoxylic acid monohydrate (10 mmol, 0.92 g) in
DMF (15 mL) phosphonate Ph3P]CHCO2tBu (15 mmol, 5.65 g) was
added. The resulting mixture left to stir at RT overnight, diluted
with EtOAc and washed several times with water, dried over
Na2SO4 and evaporated to dryness. The oily residue was triturated
with Et2O and refrigerated. The precipitated Ph3P]O was filtered
off and the filtrate diluted with EtOAc and washed twice with an aq.
solution 0.5 N NaOH. The aqueous phase was carefully acidified to
pH 5 with an aq. solution 5% citric acid and was extracted twice
with EtOAc. The combined organic layers washed twice with water,
dried over Na2SO4 and evaporated to dryness to afford pure ester 22
(0.96 g, 56%) which had identical spectrometric data to those
reported in the literature [7].
4.3.1. N-(2,4-Dimethoxyphenyl)-1H-indole-3-carboxamide (20a)
Yield: 1.04 g (70%), purple solid, m.p. 145–146 ꢀC, Rf (F): 0.29,
ESI-MS (m/z): 615.29 (2M þ Na), 319.27 (M þ Na), 297.23 (M þ H). IR
(KBr, cmꢁ1): 3448, 3167, 1635. 1H NMR (DMSO-d6):
d 11.70 (1H, s,
NH), 8.79 (1H, s, NHCO), 8.24 (1H, d, J 2.8 Hz, H-1), 8.13 (1H, d, J
7.6 Hz, H-7), 7.68 (1H, d, J 8.6 Hz, H-15), 7.47 (1H, d, J 7.6 Hz, H-4),
7.18 and 7.14 (2H, two t, J 7.6 Hz, H-5/6), 6.66 (1H, d, J 2.6 Hz, H-12),
6.54 (1H, dd, J 8.6 and 2.6 Hz, H-14), 3.84 (3H, s, OCH3), 3.78 (3H, s,
OCH3) ppm. 13C NMR (DMSO-d6):
d
162.8, 156.8, 152.1, 136.1, 128.5,
4.5. General procedure for coupling indole derivatives 20 with
fumarates 21 and 22
125.8, 124.8, 121.9, 120.5 (two C), 120.4, 111.9, 110.5, 104.7, 98.7, 55.6,
55.2 ppm. Anal. Calcd. for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45.
Found: C, 69.21; H, 5.13; N, 9.26.
To a suspension of 20a (2 mmol, 0.59 g) and 22 (2.2 mmol,
0.38 g) or 20b,c (2 mmol) and 21 (2.2 mmol, 0.62 g) in DCM
(1.5 mL), were added sequentially DIPEA (6 mmol, 1.05 mL), a cata-
lytic quantity of DMAP and finally PyBrOP (3 mmol, 1.4 g). The
resulting solution was left at RT overnight, diluted with DCM and
washed once with 5% aq. NaHCO3, twice with water, dried over
Na2SO4 and evaporated to dryness. The oily residues were sub-
jected to FCC to afford pure esters 4 and 23b,c.
4.3.2. N-Phenyl-1H-indole-3-carboxamide (20b)
Yield: 0.80 g (68%) pale yellow solid; m.p. 173–175 ꢀC; Rf (F):
0.31; ESI-MS (m/z): 259.40 (M þ Na), 237.11 (M þ H); IR (KBr, cmꢁ1):
3376, 3247, 1643, 1602; 1H NMR (DMSO-d6):
d 11.73 (1H, s, NH), 9.71
(1H, s, NHCO), 8.29 (1H, s, H-1), 8.19 (1H, d, J 7.6 Hz, H-7), 7.77 (2H,
d, J 7.6 Hz, H-11/15), 7.47 (1H, d, J 7.6 Hz, H-4), 7.30 (2H, t, J 7.6 Hz, H-

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
307
4.5.1. tert-Butyl (E)-4-(3-(2,4-dimethoxyphenylcarbamoyl)-1H-
indol-1-yl)-4-oxobut-2-enoate (4)
2), 6.67 (1H, d, J 2.4 Hz, H-16), 6.56 (1H, dd, J 7.6 and 2.4 Hz, H-18),
3.83 (3H, s, OCH3), 3.79 (3H, s, OCH3) ppm. 13C NMR (DMSO-d6):
Yield: 0.73 g (81%), yellow solid, m.p. 118–120 ꢀC, Rf (B): 0.15,
ESI-MS (m/z): 923.55 (2M þ Na), 489.63 (M þ K), 473.62 (M þ Na),
451.66 (M þ H). IR (KBr, cmꢁ1): 3444, 2924, 1714, 1690, 1672. 1H
d 165.6, 163.1, 161.5, 157.6, 153.2, 135.5, 135.1, 132.2, 128.5, 128.2,
126.5, 125.3, 124.6, 121.6, 119.0, 116.3, 115.9, 104.1, 98.8, 55.5,
55.1 ppm. Anal. Calcd. for C21H18N2O6: C, 63.96; H, 4.60; N, 7.10.
Found: C, 64.28; H, 4.41; N, 6.95.
NMR (CDCl3): d 8.56–8.52 (1H, m, H-11), 8.38 (1H, d, J 9.6 Hz, H-19),
8.27 (1H, s, NHCO), 8.16 (1H, s, H-5), 8.08-8.03 (1H, m, H-8), 7.64
(1H, d, J 15.2 Hz, H-3), 7.46 and 7.45 (2H, two t, J 7.6 Hz, H-9/10), 7.06
(1H, d, J 15.2 Hz, H-2), 6.57–6.51 (2H, m, H-16/18), 3.93 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 1.56 (9H, s, C(CH3)3) ppm. 13C NMR
4.6.2. (E)-4-oxo-4-(3-(Phenylcarbamoyl)-1H-indol-1-yl) but-2-
enoic acid (6)
Yield: 0.28 g (85%), yellow solid, m.p. 143–145 ꢀC, Rf (I): 0.32,
ESI-MS (m/z): 691.20 (2M þ Na), 357.30 (M þ Na), 335.11 (M þ H). IR
(KBr, cmꢁ1): 3338, 3138, 1718, 1698, 1644. 1H NMR (DMSO-d6):
(CDCl3): d 163.8, 162.8, 160.9, 156.7, 149.4, 137.9 (two C), 136.3, 131.0,
127.3, 127.1, 126.2, 125.4, 121.0, 120.3, 119.6, 117.3, 103.9, 98.7, 82.6,
55.9, 55.6, 28.0 (three C) ppm. Anal. Calcd. for C25H26N2O6: C, 66.65;
H, 5.82; N, 6.22. Found: C, 66.40; H, 6.01; N, 6.55.
d
10.14 (1H, s, NHCO), 8.97 (1H, s, H-5), 8.44 (1H, d, J 7.0 Hz, H-11),
8.29 (1H, d, J 7.0 Hz, H-8), 7.86 (1H, d, J 15.2 Hz, H-3), 7.76 (2H, d, J
7.6 Hz, H-15/19), 7.52–7.28 (4H, m, H-9/10/16/18), 7.12 (1H, t, J
7.2 Hz, H-17) 7.01 (1H, d, J 15.2 Hz, H-1) ppm. 13C NMR (DMSO-d6):
4.5.2. Benzhydryl (E)-4-oxo-4-(3-(phenylcarbamoyl)-1H-indol-1-
yl)but-2-enoate (23b)
d
166.3, 163.7, 162.1, 139.3, 136.2, 135.8, 132.9, 129.2 (two C), 129.1,
Yield: 0.81 g (81%), white solid, m.p.198–199 ꢀC, Rf (C): 0.39, ESI-
MS (m/z): 523.13 (M þ Na), 501.07 (M þ H). IR (KBr, cmꢁ1): 3326,
129.0, 126.1, 125.4, 124.0, 122.3, 120.7 (two C), 116.9, 116.6 ppm.
Anal. Calcd. for C19H14N2O4: C, 68.26; H, 4.22; N, 8.38. Found: C,
68.01; H, 4.44; N, 8.68.
1720, 1698, 1650, 1598. 1H NMR (DMSO-d6):
d 10.13 (1H, s, NHCO),
8.94 (1H, s, H-5), 8.44 (1H, d, J 7.6 Hz, H-11), 8.27 (1H, d, J 7.6 Hz, H-8),
7.99 (1H, d, J 15.2 Hz, H-3), 7.75 (2H, d, J 8.0 Hz, H-15/19), 7.52 (4H, d, J
7.6 Hz, o-PhH), 7.48-7.29 (11H, m, PhH), 7.25 (1H, d, J 15.2 Hz, H-2),
4.6.3. (E)-4-(3-(3-Nitrophenylcarbamoyl)-1H-indol-1-yl)-4-
oxobut-2-enoic acid (7)
7.01 (1H, s, CHPh2) ppm. 13C NMR (DMSO-d6):
d
164.0, 163.2, 161.8,
Yield: 0.30 g (79%) yellowsolid; m.p. 278–280 ꢀC; Rf (I): 0.26; ESI-
MS (m/z): 418.12 (M þ K), 402.09 (M þ Na), 380.17 (M þ H); IR (KBr,
cmꢁ1): 3420, 3200-2500, 1728, 1702, 1654; 1H NMR (DMSO-d6):
140.2, 139.1, 135.5, 134.2, 134.1, 128.9 (three C), 128.8 (four C), 128.2
(three C), 126.9 (four C), 125.9, 125.2, 123.8, 122.0, 120.4 (three C),
116.8,116.4, 77.9 ppm. Anal. Calcd. for C32H24N2O4: C, 76.78; H, 4.83;
N, 5.60. Found: C, 76.97; H, 4.73; N, 5.39.
d
10.51 (1H, s, NHCO), 8.95 (1H, s, H-5), 8.73 (1H, s, H-15), 8.40(1H, d, J
8.0 Hz, H-17), 8.27 (1H, d, J 8.0 Hz, H-11), 8.14 (1H, d, J 8.0 Hz, H-19),
7.94 (1H, d, J 8.0 Hz, H-8), 7.82 (1H, d, J 15.2 Hz, H-3), 7.65 (1H, t, J
8.0 Hz, H-18), 7.43 and 7.40 (2H, two t, J 8.0 Hz, H-9/10), 6.99 (1H, d, J
4.5.3. Benzhydryl (E)-4-(3-(3-nitrophenylcarbamoyl)-1H-indol-1-
yl)-4-oxo-but-2-enoate (23c)
15.2 Hz, H-2) ppm; 13C NMR (DMSO-d6):
d 166.3, 163.6, 162.5, 148.4,
Yield: 0.94 g (86%), white solid, m.p. 206–207 ꢀC, Rf (B): 0.22, ESI-
MS (m/z): 568.15 (M þ Na), 546.45 (M þ H). IR (KBr, cmꢁ1): 3330,
140.6, 136.3, 135.7, 132.7, 130.6, 129.6, 128.8, 126.2 (two C), 125.5,
122.2, 118.3, 116.6, 116.2, 114.4 ppm. Anal. Calcd. for C19H13N3O6: C,
60.16; H, 3.45; N, 11.08. Found: C, 59.84; H, 3.68; N, 11.38.
1726, 1694, 1656. 1H NMR (DMSO-d6):
d 10.58 (1H, s, NHCO), 8.97
(1H, s, H-5), 8.74 (1H, t, J 1.5 Hz, H-15), 8.43 (1H, dd, J 8.0 and 1.5 Hz,
H-17), 8.28 (2H, dd, J 8.0 and 1.5 Hz, H-19), 8.17 (1H, unresolved dd, J
8.4 Hz, H-8), 7.99 (1H, d, J 15.2 Hz, H-3), 7.96 (1H, unresolved d, J
8.4 Hz, H-8), 7.68 (1H, t, J 8.0 Hz, H-18), 7.53 (4H, d, J 7.8 Hz, o-PhH),
7.47 and 7.46 (2H, twot, J 8.4 Hz, H-9/10), 7.40 (4H, t, J 7.8 Hz, m-PhH),
7.32 (2H, t, 7.6 Hz, p-PhH), 7.26 (1H, d, J 15.2 Hz, H-2), 7.02 (1H, s,
4.7. Procedures for the Wittig reaction between the carbonyl
compounds 24, 25 and 28 and the phosphorane Ph3P]CHCO2tBu
4.7.1. For adduct 26
To a solution of Ph3P]CHCO2tBu (2.2 mmol, 0.83 g) in DCM
(2 mL) was added indole-3-carboxaldehyde (24) (2 mmol, 0.29 g).
The resulting solution left for 16 h at RT and then evaporated to
dryness. The oily residue was subjected to FCC to afford ester 26.
CHPh2) ppm. 13C NMR (DMSO-d6):
d 163.9, 163.2, 161.8, 148.2, 140.3,
140.2 (two C),135.5, 134.2 (two C), 130.4, 129.6,128.8 (four C),128.5,
128.2 (two C), 126.9 (four C), 126.0, 125.9, 125.3, 121.9, 118.1, 116.4,
116.1,114.2, 77.9 ppm. Anal. Calcd. for C32H23N3O6: C, 70.45; H, 4.25;
N, 7.70. Found: C, 70.15; H, 4.58; N, 7.91.
4.7.2. For adduct 27
The procedure has been previously described [9].
4.6. General procedure for TFA-mediated deprotection of esters 4
and 23b,c
4.7.3. For adduct 29
To a solution of Ph3P]CHCO2tBu (2.2 mmol, 0.83 g) in MeCN
(2 mL) was added 1-tosyl-3-acetylpyrrole (28)(2 mmol, 0.53 g). The
resulting solution was set at 80 ꢀC for overnight and then evapo-
rated to dryness. The anticipated ester was obtained, as a mixture of
geometric isomers, through FCC using as an eluant the solvent
system C. Pure E-ester was isolated as a white solid in 64% yield
after crystallization from AcOEt/hexane. The thus obtained ester
(1.2 mmol, 0.43 g) was treated with a 10 N NaOH solution (0.5 mL)
in DMSO (5 mL) at RT for 2 h. The reaction mixture was diluted with
EtOAc and washed twice with water. The organic layer was dried
over Na2SO4 and evaporated to dryness to afford ester 29 in
98% yield.
An ice-cold solution of 50% TFA in DCM (15 mL) was added to
ester 4 (1.5 mmol, 0.68 g) or 25% TFA in DCM (12 mL) to ester 23b or
23c (1 mmol). The resulting solution left at 0 ꢀC for 30 min and then
at RT for additional 30 min. Solvents were removed under reduced
pressure and the residual solids were treated with Et2O and
refrigerated overnight. The precipitates were filtered to afford pure
acids 5–7.
4.6.1. (E)-4-(3-(2,4-Dimethoxyphenylcarbamoyl)-1H-indol-1-yl)-
4-oxobut-2-enoic acid (5)
Yield: 0.53 g (90%), yellow solid, m.p. 273–274 ꢀC, Rf (H): 0.13,
ESI-MS (m/z): 417.39 (M þ Na), 395.45 (M þ H). IR (KBr, cmꢁ1):
3428, 3200-2400, 1702, 1625, 1543. 1H NMR (DMSO-d6):
d
9.41 (1H,
4.7.3.1. tert-Butyl (E)-3-(1H-indol-3-yl)acrylate (26). Yield: 0.45 g
(93%), white solid, m.p. 124–125 ꢀC, Rf (D): 0.33, GC MS (EI):
tR ¼ 11.241 min, m/z (rel.int.), 243 (16, M), 187 (100,
M ꢁ CH2]CMe2), 170 (35, M ꢁ OtBu). IR (KBr, cmꢁ1): 3276, 2982,
s, NHCO), 9.03 (1H, s, H-5), 8.44 (1H, d, J 7.6 Hz, H-11), 8.24 (1H, d, J
7.6 Hz, H-8), 7.86 (1H, d, J 15.2 Hz, H-3), 7.48 (1H, d, J 8.4 Hz, H-19),
7.44 and 7.40 (2H, two t, J 7.6 Hz, H-9/10), 6.98 (1H, d, J 15.2 Hz, H-

308
D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
2926,1678, 1612. 1H NMR (CDCl3):
d
8.77 (1H, br.s, NH), 7.92 (1H, d, J
96.4, 56.5, 56.2, 56.1 ppm. Anal. Calcd. for C12H13FO4: C, 60.00; H,
7.2 Hz, H-7), 7.84 (1H, d, J 16 Hz, H-3), 7.42 (1H, d, J 2.4 Hz, H-5), 7.41
(1H, d, J 7.2 Hz, H-10), 7.27 and 7.24 (2H, two t, J 7.2 Hz, H-8/9), 6.42
(1H, d, J 16 Hz, H-2), 1.57 (9H, s, C(CH3)3) ppm. 13C NMR (CDCl3):
5.45. Found: C, 60.25; H, 5.17.
4.9. Procedures for coupling the cinnamoyl fluorides 31 with the
amines 26, 27 and 29
d
167.8, 137.2, 129.1, 128.5, 128.3, 123.2, 121.3, 120.5, 115.4, 113.6,
111.7, 79.9, 28.4 (three C) ppm. Anal. Calcd. for C15H17NO2: C, 74.05;
H, 7.04; N, 5.76. Found: C, 73.78; H, 7.34; N, 6.03.
4.9.1. For compounds 10, 11, 14/15 and pure 14
To an ice-cold (0 ꢀC) solution of cinnamoyl fluoride 25a or 25b
(1 mmol) and amine 26 or 27 or Z ꢁ 27 (1.05 mmol) in dry CHCl3
(1 mL) DIPEA (2 mmol, 0.35 mL) was added. The thus obtained
solution was set at 40 ꢀC for 2–3 h, diluted with CHCl3 and then
washed twice with water. The organic layer was dried over Na2SO4
and evaporated to dryness. The resulting residues were subjected
to FCC to afford tert-butyl esters 10, 11, 14 and 14/15 (only the data
for pure 14 are provided below).
4.7.3.2. tert-Butyl
(Z)-2-(4-amino-9H-fluoren-9-ylidene)acetate
(Z ꢁ 27). Reddish solid, m.p. 113–114 ꢀC, Rf (B): 0.30, GC-MS (EI):
tR ¼ 13.040, m/z (rel.int.), 293 (9, M), 237 (100, M ꢁ CH2]CMe2),
220 (11, 237 ꢁ OH), 193 (13, 237 ꢁ CO2). IR (KBr, cmꢁ1): 3404, 3338,
1700, 1628. 1H NMR (CDCl3):
d 8.32 (1H, d, J 7.6 Hz, H-14), 7.62 (1H,
d, J 7.6 Hz, H-11), 7.53 (1H, d, J 7.6 Hz, H-5), 7.32 (1H, t, J 6.8 Hz, H-
13), 7.15 (1H, t, J 6.8 Hz, H-12), 7.09 (1H, t, J 7.6 Hz, H-6), 6.71 (1H, d, J
7.6 Hz, H-7), 6.63 (1H, s, H-2), 3.96 (2H, s, NH2), 1.54 (9H, s, (CH3)3)
ppm. 13C NMR (CDCl3):
d
165.9, 146.6, 141.6, 140.4, 138.7, 136.6,
4.9.2. For compound 13
130.3, 128.7, 125.9, 121.5, 121.0, 120.3, 119.9, 115.7, 112.4, 81.0, 28.3
(three C) ppm. Anal. Calcd. for C19H19NO2: C, 77.79; H, 6.53; N, 4.77.
Found: C, 78.00; H, 6.28; N, 4.50.
To an ice-cold suspension of NaH (0.6 mmol, 14.5 mg) in THF
(0.65 mL) was added portionwise amine 29 (0.55 mmol, 0.11 g).
After 30 min, fluoride 31b (0.5 mmol, 0.12 g) was added and the
resulting solution was left at RT for overnight. Evaporation to
dryness and routine FCC purification afforded tert-butyl ester 13.
4.7.3.3. tert-Butyl (E)-3-(1H-pyrrol-3-yl)but-2-enoate (29). Yield:
0.26 g (63% overall yield), white solid, m.p. 93–95 ꢀC, Rf (C): 0.30,
GC-MS (EI): tR ¼ 9.181 min, m/z (rel.int.), 207 (12, M), 151 (93,
M ꢁ CH2]CMe2), 133 (100, 151 ꢁ H2O), 105 (35, 133 ꢁ CO). IR (KBr,
4.9.2.1. tert-Butyl (E)-3-(1-((E)-3-benzo[d][1,3]dioxol-5-yl)acryloyl)-
1H-indol-3-yl)acrylate (10). Yield: 0.30 g (71%), yellow solid, m.p.
141–143 ꢀC, Rf (B): 0.37, ESI-MS (m/z): 857.39 (2M þ Na), 835.35
(2M þ H), 440.21 (M þ Na), 418.18 (M þ H). IR (KBr, cmꢁ1): 2978,
cmꢁ1): 3356, 2974, 1676, 1606. 1H NMR (CDCl3):
d 8.35 (1H, br.s,
NH), 7.04 (1H, q, J 2.6 Hz, H-5), 6.77 (1H, dd, J 4.6 and 2.6 Hz), 6.46
(1H, dd, J 4.6 and 2.6 Hz, H-7), 6.05 (1H, q, J 1.2 Hz, H-2), 2.48 (3H, d,
2904, 1686,1628. 1H NMR (CDCl3):
d 8.53 (1H, d, J 8.2 Hz, H-20), 7.91
J 1.2 Hz, H-8), 1.51 (9H, s, C(CH3)3) ppm. 13C NMR (CDCl3):
d
165.5,
(1H, d, J 15.2 Hz, H-3), 7.87 (1H, d, J 7.2 Hz, H-7), 7.86 (1H, s, H-5),
7.75 (1H, d, J 16 Hz, H-14), 7.43 (1H, t, J 7.2 Hz, H-8), 7.38 (1H, t, J
7.2 Hz, H-9), 7.16 (1H, s, H-16), 7.14 (1H, d, J 8.2 Hz, H-19), 7.03 (1H,
d, J 15.2 Hz, H-2), 6.86 (1H, d, J 7.2 Hz, H-10), 6.53 (1H, d, J 16 Hz,
H-13), 6.04 (2H, s, OCH2O), 1.57 (9H, s, C(CH3)3) ppm. 13C NMR
154.6, 128.1, 119.4, 118.2, 112.8, 106.2, 81.4, 28.2 (three C), 14.3 ppm.
Anal. Calcd. for C12H17NO2: C, 69.54; H, 8.27; N, 6.76. Found: C,
69.86; H, 8.01; N, 6.43.
4.8. General procedure for the preparation of the cinnamoyl
(CDCl3): d 166.6, 164.2, 150.4, 148.6, 147.3, 144.3, 137.0, 134.9, 128.6,
fluorides 31
128.1, 126.7, 125.8, 125.5, 124.4, 120.2, 118.3, 117.1, 114.3, 108.8, 106.7,
101.8, 80.5, 28.3 (three C) ppm. Anal. Calcd. for C23H25NO5: C, 71.93;
H, 5.55; N, 3.36. Found: C, 72.28; H, 5.27; N, 3.12.
To an ice-cold solution (ꢁ10 ꢀC) of cinnamic acids 30 (6 mmol)
and pyridine (42 mmol, 3.38 mL) in DCM (8 mL) was added drop-
wise a solution of cyanuric fluoride (12 mmol, 1.03 mL) in DCM
(4 mL). The resulting solution was left at ꢁ10 ꢀC for 1 h and at RT for
additional 30 min. The reaction mixture was diluted with CHCl3
and washed twice with water. Drying over Na2SO4 and evaporation
to dryness left a residue, from which pure fluorides 31 were
obtained through FCC.
4.9.2.2. tert-Butyl (E)-3-(1-((E)-3-(2,4,5-trimethoxyphenyl)acryloyl)-
1H-indol-3-yl)acrylate (11). Yield: 0.35 g (76%), yellow solid, m.p.
158–159 ꢀC, Rf (B): 0.10, ESI-MS (m/z): 949.67 (2M þ Na), 486.44
(M þ Na), 464.45 (M þ H), 390.26 ([M þ H] ꢁ tBuOH). IR (KBr,
cmꢁ1): 2970, 2944,1702,1676,1636. 1H NMR (CDCl3):
d 8.56 (1H, d, J
7.7 Hz, H-7), 8.21 (1H, d, J 16 Hz, H-3), 7.89 (1H, s, H-5), 7.87 (1H, d, J
7.7 Hz, H-10), 7.77 (1H, d, J 16 Hz, H-14), 7.45 (1H, t, J 7.7 Hz, H-8),
7.37 (1H, t, J 7.7 Hz, H-9), 7.23 (1H, d, J 16 Hz, H-2), 7.08 (1H, s, H-20),
6.54 (1H, s, H-17), 6.52 (1H, d, J 16 Hz, H-13), 3.96 (3H, s, OCH3), 3.95
(3H, s, OCH3), 3.92 (3H, s, OCH3), 1.56 (9H, s, C(CH3)3) ppm. 13C NMR
4.8.1. (E)-3-(Benzo[d][1,3]dioxol-5-yl)acryloyl fluoride (31a)
Yield: 0.58 g (50%), white solid, m.p. 103–104 ꢀC, Rf (A): 0.3, GS-
MS (EI): tR ¼ 8.183, m/z (rel.int.), 194 (100, M), 166 (23, M ꢁ CO), 146
(17, 166 ꢁ HF), 136 (6, 166 ꢁ CH2O). IR (KBr, cmꢁ1): 1819, 1797, 1773,
(CDCl3):
d 166.8, 165.1, 155.1, 153.2, 143.6, 143.2, 137.2, 135.3, 128.2,
1628. 1H NMR (CDCl3):
d
7.73 (1H, d, J 16 Hz, H-3), 7.06 (1H, d, J
127.2, 125.7, 124.3, 120.3, 120.0, 118.0, 117.1, 115.2, 114.8, 113.0, 97.2,
80.5, 56.9, 56.5, 56.3, 28.4 (three C) ppm. Anal. Calcd. for
C27H29NO6: C, 69.96; H, 6.31; N, 3.02. Found: C, 69.70; H, 6.52;
N, 3.38.
8.4 Hz, H-9), 7.05 (1H, s, H-5), 6.85 (1H, d, J 8.4 Hz, H-8), 6.16 (1H,
dd, J 16 and 7.2 Hz, H-2), 6.05 (2H, s, OCH2O) ppm. 13C NMR (CDCl3):
d
157.6 (d, J 335 Hz), 151.3, 151.2 (d, J 11 Hz), 149.0, 128.0, 126.2, 110.1
(d, J 68.2 Hz), 109.1, 107.0, 102.2 ppm. Anal. Calcd. for C10H7FO3: C,
61.86; H, 3.63. Found: C, 61.60; H, 3.90.
4.9.2.3. tert-Butyl (E)-3-(1-((E)-3-(2,4,5-trimethoxyphenyl)acryloyl)-
1H-pyrrol-3-yl)but-2-enoate (13). Yield: 0.14 g (65%), yellow foam,
Rf (C): 0.20, ESI-MS (m/z): 450.30 (M þ Na), 428.31 (M þ H), 372.27
([M þ H] ꢁ CH2]CMe2), 221.36 ([M þ H] ꢁ [pyrrole ꢁ C(Me) ¼
CHCO2tBu]). IR (KBr, cmꢁ1): 2976, 2946, 1704, 1694, 1638. 1H NMR
4.8.2. (E)-3-(2,4,5-Trimethoxyphenyl)acryloyl fluoride (31b)
Yield: 0.43 g (30%), yellow solid, m.p. 93–95 ꢀC, Rf (C): 0.28, GS-
MS: (EI): tR ¼ 9.609, m/z (rel.int.), 240 (100, M), 225 (49, M ꢁ Me),
209 (3, M ꢁ OMe), 197 (20, 225 ꢁ CO), 178 (8, 197 ꢁ F), 163 (5,
178 ꢁ Me). IR (KBr, cmꢁ1): 2944, 2838, 1770, 1604. 1H NMR (CDCl3):
(CDCl3): d 8.21 (1H, d, J 15.4 Hz, H-11), 7.68 (1H, br.s, H-6), 7.42 (1H,
unresolved dd, H-7), 7.10 (1H, d, J 15.4 Hz, H-10), 7.06 (1H, s, H-17),
6.55 (unresolved dd, H-8), 6.52 (1H, s, H-14), 6.08 (1H, s, H-2), 3.95
(3H, s, OCH3), 3.93 (3H, s, OCH3), 3.90 (3H, s, OCH3), 2.48 (3H, s, H-
d
8.10 (1H, d, J 16 Hz, H-3), 6.98 (1H, s, H-9), 6.50 (1H, s, H-6), 6.28
(1H, dd, J 16 and 7.5 Hz, H-2), 3.95 (3H, s, OCH3), 3.90 (3H, s, OCH3),
3.87 (3H, s, OCH3) ppm. 13C NMR (CDCl3):
158.3 (d, J 333.7 Hz),
155.0, 153.7,146.4 (d, J 6.6 Hz),143.4, 113.7,111.3,108.8 (d, J 65.8 Hz),
d
4), 1.51 (9H, s, C(CH3)3) ppm. 13C NMR (CDCl3):
d
166.8, 163.5, 155.0,
153.0, 146.7, 143.7, 143.3, 130.2, 120.4, 118.4, 115.6, 114.8, 112.7, 112.3,

D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
309
110.4, 96.6, 79.8, 56.7, 56.3, 56.1, 28.3 (three C), 16.2 ppm. Anal.
Calcd. for C24H29NO6: C, 67.43; H, 6.84; N, 3.28. Found: C, 67.63; H,
6.57; N, 3.04.
overnight, diluted with DCM and washed once with 5% aq. NaHCO3
and twice with water. The organic layer was dried over Na2SO4 and
evaporated to dryness. Pure analog 16 was obtained through FCC
purification.
4.9.2.4. tert-Butyl
(Z)-2-(4-((E)-3-benzo[d][1,3]dioxol-5-yl)acryla-
Yield: 0.22 g (43%), yellow solid, m.p. 275–276 ꢀC, Rf (E): 0.22,
ESI-MS (m/z): 1055.53 (2M þ Na), 1033.57 (2M þ H), 539.71
(M þ Na), 517.68 (M þ H). IR (KBr, cmꢁ1): 2932, 2834, 1684, 1662. 1H
mido)-9H-fluoren-9-ylidene)acetate (14). Yield: 0.23 g (50%), yellow
solid, m.p. 208–209 ꢀC, Rf (B): 0.15, ESI-MS (m/z): 957.06 (2M þ Na),
935.13 (2M þ H), 490.09 (M þ Na), 468.07 (M þ H), 411.96
([M þ H] ꢁ tBu). IR (KBr, cmꢁ1): 2960, 2922, 1710, 1656, 1630. 1H
NMR (CDCl3, 40 ꢀC):
d 8.61 (1H, dd, J 6.8 and 1.6 Hz, H-11), 8.41 (1H,
d, J 9.2 Hz, H-4), 8.38 (1H, s, NHCO), 8.36 (1H, d, J 9.2 Hz, H-5), 8.28
(1H, d, J 15.2 Hz, H-18), 8.04 (1H, dd, J 6.8 and 1.6 Hz, H-14), 7.50–
7.38 (2H, m, H-12/13), 7.28 (1H, d, J 15.2 Hz, H-17), 7.11 (1H, s, H-9),
6.56 (1H, s, H-24), 6.54 (2H, s, H-2/21), 3.96 (3H, s, OCH3), 3.94 (3H,
NMR (CDCl3, 45 ꢀC):
d 8.72 (1H, br.s, NHCO), 7.71 (1H, d, J 15.6 Hz, H-
18), 7.70–7.60 (2H, m, H-11/14), 7.51 (1H, m, H-13), 7.40–7.20 (3H,
m, H-5/23/24), 7.30 (1H, s, H-20), 7.10–6.94 (2H, m, H-6/12), 6.81
(1H, m, H-7), 6.69 (1H, s, H-2), 6.47 (1H, d, J 15.6 Hz, H-17), 5.99 (2H,
s, OCH3), 3.93 (3H, s, OCH3), 3.92 (3H, s, OCH3), 3.83 (3H, s, OCH3)
s, OCH2O), 1.62 (9H, s, C(CH3)3) ppm. 13C NMR (CDCl3):
d
165.7,
ppm. C NMR (CDCl3, 40 ꢀC):
d
165.3, 161.6, 156.6, 155.0, 153.1,
13
165.6, 149.4 (two C), 148.2, 145.7, 140.8, 139.1 (two C), 135.8, 132.0,
130.8,129.0,128.9,128.2,127.6,127.2, 124.3,121.1,118.6,116.4,108.5,
106.6 (two C), 101.5, 81.3, 28.2 (three C) ppm.
149.4, 143.4, 136.6, 128.7, 126.6, 125.5, 124.7, 124.6, 121.3, 120.9,
119.6, 117.8, 117.3, 114.8, 113.8, 111.8, 104.0, 98.7, 96.7, 56.6, 56.4, 56.1,
56.0, 55.6 ppm. Anal. Calcd. for C29H28N2O7: C, 67.43; H, 5.46; N,
5.42. Found: C, 67.18; H, 5.67; N, 5.75.
4.10. General procedure for carboxyl group deprotection
4.12. General biological assays
tert-Butyl ester 10 or 11 (0.20 mmol) was treated with an ice-
cold solution of 50% TFA in DCM (2 mL) for 1 h and then evaporated
to dryness. Addition of Et2O and overnight refrigeration gave
a precipitate, which upon filtration afforded the acid 8 or 12,
respectively.
DPPH, NDGA and CA were purchased from the Aldrich Chemical
Co. Milwaukee, WI, (USA). Soybean LOX, linoleic acid sodium salt
and indomethacin were obtained from Sigma Chemical, Co. (St.
Louis, MO, USA). Carrageenan type K was commercially available.
For the in vivo experiments, male and female Fischer-344 rats
(180–240 g) were used. For the in vitro tests, a Lambda 20 (Perkin–
Elmer) UV–Vis double beam spectrophotometer was used. Each in
vitro experiment was performed at least in triplicate and the
standard deviation of absorbance was less than 10% of the mean.
4.10.1. (E)-3-(1-((E)-3-benzo[d][1,3]dioxol-5-yl)acryloyl)-1H-indol-
3-yl)acrylic acid (8)
Yield: 0.065 g (90%), white solid, m.p. 240–242 ꢀC, Rf (H): 0.17,
ESI-MS (m/z): 400.03 (M þ K), 384.03 (M þ Na), 362.09 (M þ H). IR
(KBr, cmꢁ1): 3412, 2978, 1698, 1654, 1634. 1H NMR (DMSO-d6):
d
8.89 (1H, s, H-5), 8.52 (1H, d, J 8.0 Hz, H-7), 7.95 (1H, d, J 7.2 Hz, H-
4.13. In vitro assays
20), 7.88 (1H, d, J 15.2 Hz, H-3), 7.79 (1H, d, J 16 Hz, H-14), 7.66 (1H,
s, H-16), 7.60 (1H, d, J 15.2 Hz, H-2), 7.43 and 7.39 (2H, two t,
J 8.0 Hz, H-8/9), 7.36 (1H, d, J 7.2 Hz, H-19), 7.02 (1H, d, J 8.0 Hz, H-
10), 6.63 (1H, d, J 16 Hz, H-13), 6.13 (2H, s, OCH2O) ppm. 13C NMR
4.13.1. Determination of the reducing activity of the stable radical
DPPH [30]
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol
an equal volume of the compounds dissolved in DMSO was added.
The mixture was shaken vigorously and allowed to stand for 20 min
or 60 min. The absorbance at 517 nm was determined spectro-
photometrically and the percentage of activity was calculated. All
tests were undertaken on three replicates and the results were
averaged (Table 1).
(DMSO-d6):
d 168.3, 164.7, 150.4, 148.7, 147.2, 136.8, 135.9, 129.8,
129.3, 128.3, 126.7, 125.9, 124.9, 120.5, 118.8, 117.3, 117.1, 115.5, 109.0,
107.3, 102.3 ppm. Anal. Calcd. for C21H15NO5: C, 69.80; H, 4.18; N,
3.88. Found: C, 69.53; H, 4.38; N, 4.05.
4.10.2. (E)-3-(3-oxo-1-(2,4,5-trimethoxyphenyl)-2,3-dihydro-1H-
pyrrolo[1,2-a]indol-9-yl)acrylic acid (12)
Yield: 0.068 g (84%), white solid, m.p. 235–236 ꢀC, Rf (H): 0.22,
ESI-MS (m/z): 430.57 (M þ Na), 408.60 (M þ H), 390.63
4.13.2. Inhibition of linoleic acid lipid peroxidation [15]
Ten microliters of the 16 mM sodium linoleate solution was
added to the UV cuvette containing 0.93 mL of 0.05 M phosphate
buffer, pH 7.4 prethermostated at 37 ꢀC. The oxidation reaction was
([M þ H] ꢁ H2O),
240.53
([M þ H] ꢁ (MeO)3Ph]),
214.59
(240 ꢁ C2H2), 158 (214 ꢁ 2CO), 141.6 (158 ꢁ OH). IR (KBr, cmꢁ1):
3446, 3200–2400, 1750, 1684, 1618. 1H NMR (DMSO-d6):
d
12.1 (1H,
initiated at 37 ꢀC under air by the addition of 50
m
L of 40 mM AAPH
solution. Oxidation was carried out in the presence of aliquots
(10 L) in the assay without antioxidant. Lipid oxidation was
br.s, CO2H), 8.08–8.02 (1H, m, H-9), 7.88–7.84 (1H, m, H-6), 7.44
(1H, d, J 16 Hz, H-3), 7.42–7.35 (2H, m, H-7/8), 7.05 (1H, s, H-21),
6.69 (1H, s, H-18), 6.20 (1H, d, J 16 Hz, H-2), 4.98 (1H, dd, J 9.2 and
3.2 Hz, H-14), 3.77 (3H, s, OCH3), 3.68 (3H, s, OCH3), 3.62 (1H, dd, J
18 and 9.2 Hz, H-13a), 3.58 (3H, s, OCH3), 2.98 (1H, dd, J 18 and
m
measured in the presence of the same level of DMSO. The rate of
oxidation at 37 ꢀC was monitored by recording the increase in
absorption at 234 nm caused by conjugated diene hydroperoxides.
3.2 Hz, H-13b) ppm. 13C NMR (DMSO-d6):
d 172.2, 168.2, 151.7 (two
C), 149.5, 143.0, 134.9, 132.4, 130.5, 125.2, 124.6, 121.0, 119.4, 117.4,
114.8, 113.6, 108.9, 99.4, 56.9, 56.8, 56.2, 43.2, 34.9 ppm. Anal. Calcd.
for C23H21NO6: C, 67.80; H, 5.20; N, 3.44. Found: C, 68.10; H, 4.98;
N, 3.21.
4.13.3. Soybean LOX inhibition study in vitro [30]
The tested compounds dissolved in DMSO were incubated at RT
with sodium linoleate (0.1 ml) and 0.2 ml of enzyme solution (1/
9 ꢃ 10ꢁ4 w/v in saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleate was recorded at 234 nm and compared with
the appropriate standard inhibitor.
4.11. Preparation of (E)-N-(2,4-dimethoxyphenyl)-1-(3-(2,4,5-
trimethoxyphenyl)acryloyl)-1H-indole-3-carboxamide (16)
4.13.4. In vivo assays: inhibition of the Carrageenan-induced
oedema [30]
Oedemawasinduced intherighthindpawofFisher344rats(150–
200 g) by the intradermal injection of 0.1 ml 2% Carrageenan inwater.
To a solution of anilide 20a (1.05 mmol, 0.31 g) and DIPEA
(2 mmol, 0.35 mL) in CHCl3/DMF (85:15, 1.2 mL) was added fluoride
31b (1 mmol, 0.24 g). The reaction mixture was stirred at 40 ꢀC for

310
D. Hadjipavlou-Litina et al. / European Journal of Medicinal Chemistry 45 (2010) 298–310
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Acknowledgement
We thank the European Social Fund (ESF), Operational Program
for Educational and Vocational Training II (EPEAEK II) and partic-
ularly the Program IRAKLEITOS, for funding the above work. We
also wish to thank Dr C. Hansch and Biobyte Corp. 201 West 4th Str.,
Suite 204, Claremont CA California, 91711, USA for free access to the
C-QSAR program.
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