Synthesis and biological evaluation of new N-alkylcarbazole derivatives as STAT3 inhibitors: Preliminary study

Article abstract of DOI:10.1016/j.ejmech.2012.11.004

The signalling pathway of Janus tyrosine Kinases-Signal Transducers and Activators of Transcription (JAK-STAT) is activated by a number of cytokines, hormones (GH, erythropoietin and prolactin), and growth factors. JAK-STAT signalling is involved in regul

Full text of DOI:10.1016/j.ejmech.2012.11.004

European Journal of Medicinal Chemistry 60 (2013) 112e119
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new N-alkylcarbazole derivatives as STAT3
inhibitors: Preliminary study
,
a
b
e
a
Carmela Saturnino a , Chiara Palladino , Mariagrazia Napoli , Maria Stefania Sinicropi , Antonio Botta ,
*
Marina Sala a, Alessandra Carcereri de Prati c, Ettore Novellino d, Hisanori Suzuki c
a Department of Pharmaceutical Science, University of Salerno, Via Ponte Don Melillo 8, 84024 Fisciano (SA), Italy
b Department of Chemistry, University of Salerno, Via Ponte Don Melillo 8, 84024 Fisciano (SA), Italy
c Department of Life and Reproduction Science, University of Verona, Strada Le Grazie 8, 37134, Verona, Italy
d Department of Pharmaceutical and Toxicologic Chemistry, University of Napoli ‘Federico II’, Via Domenico Montesano 49, 80131 Napoli, Italy
e Department of Pharmaceutical Science, University of Calabria, Arcavarcate di Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The signalling pathway of Janus tyrosine Kinases-Signal Transducers and Activators of Transcription (JAK-
STAT) is activated by a number of cytokines, hormones (GH, erythropoietin and prolactin), and growth
factors. JAK-STAT signalling is involved in regulation of cell proliferation, differentiation and apoptosis.
These activities are due to different members of JAK-STAT family consisting of: JAK1, JAK2, JAK3, Tyk2 and
STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6. Recent studies suggest a key role for STAT family
proteins, in particular for STAT3, in selectively inducing and maintaining a pro-carcinogenic inflamma-
tory microenvironment, that promote tumour cells transformation. Moreover, a striking correlation
between cancer development/progression and STAT3 persistent activation exists, probably due to STAT3
promoting of the pro-oncogenic inflammatory pathways, like NF-kB, IL-6 and JAK family kinases. Recent
study demonstrated that carbazoles can inhibit STAT3 mediated transcription. From these evidences,
STAT3 represents a therapeutic target, so we have synthesized a new set of N-alkylcarbazole derivatives
substituted in positions 2, 4 and 6, to evaluate their activity on STAT3. Some of these compounds showed
an interesting activity as STAT3 selective inhibitors; in particular, compounds 9a 9b and 9c revealed to
inhibit the STAT3 activation for the 50%, 90% and 95%, respectively.
Received 17 October 2011
Received in revised form
2 October 2012
Accepted 7 November 2012
Available online 4 December 2012
Keywords:
Carbazoles
Antitumoral
Cytotoxicity
Inhibitors
STAT3
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
It is well established that the binding of a cytokine or growth factor
to its receptor activates the receptor-associated tyrosine kinases,
Signal transducers and activators of transcription (STATs) are
latent cytoplasmic transcription factors that once activated trans-
duce signals from the cell membrane to the nucleus. To date, seven
STATs have been identified in mammals (STATs 1, 2, 3, 4, 5A, 5B, 6).
Among them, STAT3 is the most intimately linked to tumorigenesis.
In fact, STAT3 is constitutively activated and overexpressed in
various tumour types such as breast carcinoma [1,2], prostate
cancer [3], melanoma [4], multiple myeloma [5], and leukemia [6].
In normal cells, where STAT3 is present in latent form, its acti-
vation can be induced by different cytokines, such as interleukins
(IL2, IL5, IL6, IL7, IL9, IL10, IL11, IL15), by EGF (epidermal growth
factor), PDGF (platelet-derived growth factor), GH (growth
hormone), leptin, CT-1 (cardiotrophin-1), G-CSF (granulocyte
colony-stimulating factor), and CSF-1 (colony-stimulating factor-1).
known as JAK1e3 and Tyk2. First, one or more members of the JAK
family (JAK 1, 2, 3 and Tyk 2) of tyrosine kinases associated with
a transmembrane receptor is phosphorylated and activated after
ligandereceptor interaction. Activated JAKs recruit and phosphor-
ylate one or more of the STAT proteins on tyrosine residues. Tyro-
sine phosphorylated STATs dimerize, translocate to the nucleus and
bind specific promoter elements to regulate gene expression [7a]. A
lot of papers state that STAT3 is implicated in promoting cell
survival and it has been found to be persistently activated in many
cancers. Therefore STAT3 is thought to confer protection against
apoptosis in many of these transformed cells. Recent studies [7b]
demonstrate that regulation of anti-apoptotic pathways by STAT3
is largely due to transcriptional control of genes that encode anti-
apoptotic proteins (Bcl-xL, Bcl-2) and proliferation regulatory
proteins (Cyclin D1, survivin).
Recent studies demonstrated the carbazole inhibition of: SVR
(murine endothelial cells) cells proliferation; proinflammatory
* Corresponding author. Tel.: þ39 089969769; fax: þ39 089969602.
E-mail address: saturnino@unisa.it (C. Saturnino).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.004

C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
113
cytokine synthesis in particular of IL-15; rac activation (vascular
2. Results
endothelial growth factor) VEGF-stimulated and STAT3-mediated
transcription [8]. Moreover, in other studies carbazole derivatives
showed a cytotoxic activity against a countless number of cancer
cells: murine leukemia L1210 cell line [9a], SKeNeSH human
neuroblastoma (NB) cells [9b], human solid cancer cells (PC3,
DU145, and PA 1) [9c], human leukemic topoisomerase II sensitive
(HL60N) and resistant (HL60 MX2) cell lines [9d].
From the aforementioned manuscripts, it seems that the
carbazole moiety can be regarded as a privileged structure in the
search of new antiproliferative chemotypes and several modifi-
cations of the cabazole scaffold led to different activity profiles
against different tumor cell lines. Nevertheless, analysis of these
studies reveals that the N9 position was never targeted and
functionalized. Therefore, in the present paper a small series of
new carbazoles modified by N-alkylation with C5, C6, C7 alkyl
chains was obtained. Such a structural modification was also
attempted to modulate the lipophilic properties of the carbazole
ligands. Moreover, the alkyl chains were functionalized with
dimethyl 5-hydroxyisophthalate or methyl salicylate as substitu-
ents. The characterization and biological evaluation of new N-
alkylcarbazole derivatives as potential STAT3 inhibitors is also
reported.
2.1. Chemistry
The synthesis of N-alkylcarbazole derivatives was accomplished
using two different carbazole scaffolds. The compounds 3aec, 4aec
and 5, 6 were achieved from the 2 or 4 hydroxycarbazoles 1,
commercially available, in two steps of reaction as shown in
Scheme 1. The first step was the N-alkylation of the hydrox-
ycarbazoles with 1,5-dibromopentane; 1,6-dibromohexane or 1,7-
dibromoheptane, to obtain the intermediates 2aec. These N-alky-
lated derivatives, in the second step were reacted to dimethyl 5-
hydroxyisophthalate or methyl salicylate to give the final prod-
ucts. The compounds 8aec and 9aec were obtained from the same
reactions, as shown in Scheme 2, from the 6-methoxy-1,4-dimethyl-
9H-carbazole 7. The compound 7 was synthesized from 5-methoxy-
1H-indole, hexane-2,5-dione and 4-methylbenzenesulfonic acid
dissolved in ethanol as shown in Scheme 2.
All synthesized compounds were characterized by NMR analysis
and the elemental analysis carried out on them, are in agreement
with the proposed formulations.
The compounds 8aec and 9aec were also characterized by mass
spectrometry. The mass spectra of compounds 8aec and 9aec
Scheme 1. Synthetic route for the preparation of 3a, 3b, 3c, 4a, 4b, 4c, 5, 6 compounds.

114
C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
Scheme 2. Synthetic route for the preparation of 8a, 8b, 8c, 9a, 9b, 9c compounds.
show the molecular ion and the fragmentation of the aryl-esther
group and sometimes of the OCH3 group.
DNA-binding activity at 50
compounds were ineffective also at higher doses (100
m
M
(Fig. 1a) whereas all other
M)
m
(Table 1). In the Supplementary materials we also reported time-
and dose-dependent effectiveness of 9aec. Moreover, the
compounds 9a, 9b and 9c were unable to inhibit IFNy induced
2.2. Pharmacology
In order to analyse the effect of a new set of N-alkylcarbazole
derivatives on STAT3 signalling pathway, EMSA and Western blot
analysis were performed in THP-1 cells treated with IL-6 (20 ng/
ml) for 15 min. IL-6 increased predominantly the STAT3 DNA-
binding activity as indicated by EMSA/supershift experiments
with anti-STAT3 antibody (data not shown) in line with previous
report [10]. Among different compounds examined only 9a, 9b
and 9c were able to inhibit, with different effectiveness, STAT3
STAT1 nor TNF-
a
þ LPS-induced NF-kB activation (Fig. 2a, b). One
of the critical steps leading to the activation of STATs is their
phosphorylation on specific tyrosine residues and successive
translocation into the nucleus. In line with above described data,
Western Blot analysis showed that the compounds 9a, 9b and 9c
decreased, in different grade, IL-6-induced tyrosine705 phos-
phorylation of cytosolic STAT3 without affecting the total amount
of STAT3 protein (Fig. 1c).

C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
115
Fig. 2. Effect of compounds 9aec on STAT1 and NF-kB activation in THP-1 cell line.
THP-1 cells (1.5 ꢁ 106/mL) were pretreated with 50
m
M of the compounds 9a, 9b or 9c
for 30 min and then stimulated with INFg (a) or TNFa plus LPS (b) for 30 min. Nuclear
extracts were then prepared and DNA-binding analysis was performed. EMSA shows
that compounds 9b, 9a and 9c are ineffective on DNA-binding activity of STAT1 nor NF-
kB activated with INFg or TNFa plus LPS respectively. The gels are representative of five
independent experiments.
Fig. 1. Effect of compounds 9aec on STAT3 activation in THP-1 cell line. (a) THP-1 cells
(1.5 ꢁ 106/mL) were pretreated with 50
mM of the compounds 9a, 9b or 9c for 30 min
and then stimulated with IL-6 (20 ng/mL) for 15 min. Nuclear extracts were then
prepared and DNA-binding analysis was performed. EMSA shows that compounds 9b,
9a and 9c decrease DNA-binding activity of STAT3 activated by 20 ng/ml IL6. The gels
are representative of five independent experiments. (b) Densitometric analysis of
EMSA were performed as described in experimental protocols (Section 4.6) and are
expressed as mean ꢂ S.D. of five experiments. (c) THP-1 cells (1.5 ꢁ 106/mL) were
subsequent translocation to the nucleus for binding to specific DNA
sequences in the promoter of target genes [13,14] involved in
apoptosis, cell survival, angiogenesis and metastasis. In the present
study, we demonstrated that some of the tested compounds sup-
pressed, with different effectiveness, the STAT3 phosphorylation
and its nuclear translocation. In particular, compound 9a, 9b and 9c
revealed to inhibit the STAT3 activation for the 50%, 90% and 95%,
respectively. These compounds represent a new class of carbazole
leads endowed with antiproliferative activities. In fact, the addition
of an N-alkyl chain provided an additional interaction point to the
target protein as small modifications of its terminal phenyl ring
cause dramatic changes in the activity profile (compare 8a, 8b and
8c with 9a, 9b and 9c). Also among the active ligand the length of
the alkyl linker seems to be critical for the activity (compare 9a
with 9b and 9c). If some considerations of the structure-activity
relationships (SARs) can be drawn, at present a deeper under-
standing of the chemical features responsible for the inhibition of
the STAT3 activation would require a more systematic SAR study
starting from the present preliminary work.
pretreated with 50 mM of the compounds 9a, 9b or 9c for 30 min and then stimulated
with IL-6 (20 ng/mL) for 15 min. Whole-cell extracts were prepared and p-STAT3 was
detected by Western blot analysis. The same blot was stripped and reprobed with
STAT3 antibody to verify equal protein loading.
3. Discussions and conclusions
Research within the past four years has provided convincing
evidence for an oncogenic role of STAT3, a nuclear transcription
factor belonging to the seven member STAT gene family of tran-
scription factors, in different human cancers so that STAT3 is
emerging as a critical transcription activator biomarker in antigenic
therapy of tumour [11]. Therefore, inhibiting STAT3 pathway may
be an effective approach to suppress tumour growth. The IL-6-
induced activation of STAT3 is mediated by JAKs through a cyto-
plasmic domain of gp 130 of the IL-6 receptor [12]. The STAT3
phosphorylation at Tyr705 causes its homodimerization and
4. Experimental section
All reagents and solvents were purchased from SigmaeAldrich
s.r.l. (Milan, Italy), the 1,5-dibromopentane, 1,6-dibromohexane
and 1,7-dibromoheptane were purified on Al2O3. The reactions
were monitored by thin-layer chromatography (TLC), using silica
gel aluminum plates Whatman K6F and Allumina (Merck), with
fluorescence indicators and opportune solvents. A >95% purity
could be inferred from the 1H-NMR spectra. Melting points were
taken on a Gallenkamp melting point apparatus and were uncor-
rected. The organic extracts were dried over sodium sulphate dry
(Merck). The elemental analyses for C, H, N, were performed
according to standard microanalytical procedures. Molecular
weights were determined by ESI mass spectrometry. ESI-MS anal-
ysis in positive and negative ion mode, were made using a Finnigan
LCQ ion trap instrument, manufactured by Thermo Finnigan (San
Jose, CA, USA), equipped with the Excalibur software for processing
the data acquired. The sample was dissolved in a mixture of
acetonitrile and methanol (50/50) and injected directly into the
electrospray source, using a syringe pump, which maintains
Table 1
Effect of examined compounds on STAT3 activation in THP-1 cell line.a
Compound
Dose (
m
M)
% of STAT 3
inhibition
3a
3b
3c
4a
4b
4c
5
100
100
100
100
100
100
100
100
100
100
100
50
1 ꢂ 0.5
1 ꢂ 0.5
5 ꢂ 1.1
20 ꢂ 2.2
5 ꢂ 1.2
6 ꢂ 1.3
1 ꢂ 0.3
3 ꢂ 0.4
3 ꢂ 0.3
1 ꢂ 0.3
6 ꢂ 1.2
50 ꢂ 5.2
95 ꢂ 3.2
90 ꢂ 6.5
6
8a
8b
8c
9a
9b
9c
50
50
a
THP-1 cells (1.5 ꢁ 106/mL) were pretreated with 50
mM or 100 mM of all the
constant flow at 5 ml/min. The temperature of the capillary was set
at 220 ꢀC.
synthesized compounds for 30 min and then stimulated with IL-6 (20 ng/mL) for
15 min. Nuclear extracts were subjected to EMSA analysis. Densitometric analysis of
the gels were performed as described in experimental protocols (Section 4.6) and
the inhibition of STAT3 DNA-binding is reported as mean percentage ꢂ SD respect to
IL-6 induction alone.
1H NMR spectra were recorded at 298 K on a Bruker Avance
300 MHz spectrometer, using CDCl3 and referred to internal tet-
ramethylsilane. All chemicals used for pharmacological assays were

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C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
of the highest analytical grade, purchased from Sigma Chemical
Company, Milan, Italy, unless otherwise specified. RPMI 1640
medium, Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine (FBS) serum were obtained from Lonza, Verviers, BE.
4.3.2. Dimethyl 5-(6-(2-hydroxy-9H-carbazol-9-yl)hexyloxy)
isophthalate (3b)
(
d ppm, CDCl3 300 MHz): 4.17 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.05 [(OHeC12H7N)eCH2e
(CH2)4eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.93 [(OHeC12H7N)e
CH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.88 [(OHe
C12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 6H, s]; 1.27e2.00
[(OHeC12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 8H, m];
6.70e8.30 [(OHeC12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2,
10H,m].
Mass spectrum: 475.53 [(OHeC12H7N)eCH2e(CH2)4eCH2Oe
C6H3e(OCOCH3)2].
Elemental analysis of 3b: calcd. for C28H29NO6 (%): C 70.72, H
6.15, N 2.95. Found (%): C 70.54, H 6.23, N 2.86.
4.1. Synthesis of 6-methoxy-1,4-dimethyl-9H-carbazole (7)
5-methoxy-1H-indole (6.8 mmol) was dissolved in ethanol
(10 ml) and stirred at room temperature. To this solution were
added hexane-2,5-dione (6.8 mmol) and 4-methylbenzenesulfonic
acid (6.04 mmol). The mixture was stirred at reflux for 6 h. After the
reaction, the solvent was evaporated, poured into water and
extracted with chloroform (3 ꢁ 30 ml). The organic layer was dried
over anhydrous MgSO4, filtered and the solvent removed by
evaporation. The resulting residue was purified on a silica gel
column chromatography in petroleum ether/ethyl acetate (8/2) to
give as white crystals (90% yield) (Scheme 2) [15e18].
4.3.3. Dimethyl 5-(7-(2-hydroxy-9H-carbazol-9-yl)heptyloxy)
isophthalate (3c)
(d ppm, CDCl3 300 MHz): 4.20 [(OHeC12H7N)eCH2e(CH2)5e
4.2. Synthesis of N-alkyl carbazoles (2aec; 7aec)
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.11 [(OHeC12H7N)eCH2e(CH2)5e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 3.94 [(OHeC12H7N)eCH2e(CH2)5e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 3.91 [(OHeC12H7N)eCH2e(CH2)5e
CH2OeC6H3e(OCOCH3)2, 6H, s]; 1.12e1.94 [(OHeC12H7N)eCH2e
(CH2)5eCH2OeC6H3e(OCOCH3)2, 10H, m]; 6.80e8.30 [(OHe
TBEA (benzyltriethylamonium bromide) (2.73 mmol), was dis-
solved in an aqueous solution of NaOH 50% p/p (15 ml) and toluene
(20 ml) and stirred for 15 min. After was added a solution of
a
suitable hydroxycarbazole or 6-methoxy-1,4-dimethyl-9H-
C12H7N)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 10H, m].
carbazole (5.45 mmol) in toluene (20 ml). To this mixture was
added 1,5-dibromopentane or 1,6-dibromohexane or 1,7-
dibromoheptane (16.3 mmol) and was stirred at reflux for 48 h.
After the reaction, the solvent was evaporated, poured into water
and extracted with chloroform (5 ꢁ 50 ml). The organic layer was
dried over anhydrous MgSO4, filtered and the solvent removed by
evaporation. The resulting residue was purified on a silica gel
column chromatography in petroleum ether/ethyl acetate (9/1) to
give as white/yellow solid (60% yield) (7aec). The resulting residue
was extracted with hexane (3 ꢁ 20 ml), ethyl acetate, and diethyl
ether. Final product was purified on a silica gel column chroma-
tography in chloroform to give as yellow/orange solid (60% yield)
(2aec) (Schemes 1 and 2) [16e18].
Mass spectrum: 489.56 [(OHeC12H7N)eCH2e(CH2)5eCH2Oe
C6H3e(OCOCH3)2].
Elemental analysis of 3c: Calcd. for C29H31NO6 (%): C 71.15, H
6.38, N 2.86. Found (%): C 71.02, H 6.49, N 2.92.
4.3.4. Dimethyl 5-(5-(4-hydroxy-9H-carbazol-9-yl)pentyloxy)
isophthalate (4a)
(d ppm, CDCl3 250 MHz): 4.22 [(OHeC12H7N)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.16[(OHeC12H7N)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 3.96 [(OHeC12H7N)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 3.90 [(OHeC12H7N)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 6H, s]; 1.23e2.11 [(OHeC12H7N)eCH2e
(CH2)3eCH2OeC6H3e(OCOCH3)2, 6H, m]; 6.56e8.41 [(OHe
C12H7N)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 10H, m].
4.3. General method for the synthesis of compounds 3aec, 4aec,
9aec
Mass spectrum: 461.51 [(OHeC12H7N)eCH2e(CH2)3eCH2Oe
C6H3e(OCOCH3)2].
Elemental analysis of 4a: calcd. for C27H27NO6 (%): C 70.27, H
5.90, N 3.03. Found (%): C 70.15, H 5.98, N 3.14.
Dimethyl 5-hydroxyisophthalate (l.35 mmol) and Cs2CO3
(2.7 mmol) were dissolved in DMF (10 ml). A solution of N-bro-
moalkyl-substituted carbazole (1.35 mmol) in DMF was added at
room temperature. The mixture was stirred for 20 h. After the
reaction, the solvent was evaporated, poured into water and
extracted with chloroform (5 ꢁ 50 ml). The organic layer was dried
over anhydrous MgSO4, filtered and the solvent removed by
evaporation. Final product was an orange/brown solid (80% yield)
(Schemes 1 and 2).
4.3.5. Dimethyl 5-(6-(4-hydroxy-9H-carbazol-9-yl)hexyloxy)
isophthalate (4b)
(
d ppm, CDCl3 300 MHz): 4.10 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.32 [(OHeC12H7N)eCH2e
(CH2)4eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.99 [(OHeC12H7N)e
CH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.88 [(OHe
C12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 6H, s]; 1.21e2.12
[(OHeC12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 8H, m];
6.57e8.50 [(OHeC12H7N)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2,
10H, m].
4.3.1. Dimethyl 5-(5-(2-hydroxy-9H-carbazol-9-yl)pentyloxy)
isophthalate (3a)
(
d
ppm, CDCl3 300 MHz): 4.20 [(OHeC12H7N)eCH2e(CH2)3e
Mass spectrum: 475.53 [(OHeC12H7N)eCH2e(CH2)4eCH2Oe
C6H3e(OCOCH3)2].
Elemental analysis of 4b: calcd. for C28H29NO6 (%): C 70.72, H
6.15, N 2.95. Found (%): C 70.59, H 6.27, N 2.84.
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.10 [(OHeC12H7N)eCH2e
(CH2)3eCH2OeC6H3e(OCOCH3)2, 2H, t]; 4.03 [(OHeC12H7N)e
CH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.94 [(OHe
C12H7N)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 6H, s]; 1.27e2.02
[(OHeC12H7N)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 6H, m];
6.80e8.22 [(OHeC12H7N)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2,
10H,m].
4.3.6. Dimethyl 5-(7-(4-hydroxy-9H-carbazol-9-yl)heptyloxy)
isophthalate (4c)
(
d ppm, CDCl3 300 MHz): 4.23 [(OHeC12H7N)eCH2e(CH2)5e
Mass spectrum: 461.51 [(OHeC12H7N)eCH2e(CH2)3eCH2Oe
C6H3e(OCOCH3)2].
Elemental analysis of 3a: Calcd. for C27H27NO6 (%): C 70.27, H
5.90, N 3.03. Found (%): C 70.14, H 5.96, N 3.12.
CH2OeC6H3e(OCOCH3)2, 1H, s]; 4.25 [(OHeC12H7N)eCH2e
(CH2)5eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.98 [(OHeC12H7N)e
CH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.93 [(OHe
C12H7N)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 6H, s]; 1.20e2.10

C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
117
[(OHeC12H7N)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 10H, m];
6.50e8.40 [(OHeC12H7N)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2,
10H, m]].
Mass spectrum: 489.56 [(OHeC12H7N)eCH2e(CH2)5eCH2Oe
C6H3e(OCOCH3)2].
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2]; 20.4[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2].
Mass spectrum: 517.61 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)4eCH2OeC6H3e(OCOCH3)2].
Elemental analysis of 9b: calcd. for C31H35NO6 (%): C 71.93, H
6.82, N 2.71. Found (%): C 71.82, H 6.96, N 2.59.
Elemental analysis of 4c: calcd. for C29H31NO6 (%): C 71.15, H
6.38, N 2.86. Found (%): C 71.02, H 6.49, N 2.97.
4.3.9. Dimethyl 5-(7-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
4.3.7. Dimethyl 5-(5-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
heptyloxy)isophthalate (9c)
pentyloxy)isophthalate (9a)
1H NMR (
d ppm CDCl3 300 MHz): 3.95 [(CH3OeC6H3eC6H3e
1H NMR(
d
ppm CDCl3 250 MHz): 4.06 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 3H, s]; 4.48
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e
(OCOCH3)2, 2H, t]; 4.00 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)5eCH2OeC6H3e(OCOCH3)2, 2H, t]; 3.93 [(CH3OeC6H3e
C6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 6H, s];
2.77 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e
(OCOCH3)2, 3H, s]; 2.84 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)5eCH2OeC6H3e(OCOCH3)2, 3H, s]; 1.23e1.87 [(CH3OeC6H3e
C6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2, 10H, m];
6.80e8.40 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2Oe
C6H3e(OCOCH3)2, 8H, m].
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 3H, s]; 4.56 [(C
H3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOC
H3)2, 2H, t]; 4.10 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 4.00 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2, 6H, s]; 2.79
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e
(OCOCH3)2, 3H, s]; 2.74 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)3eCH2OeC6H3e(OCOCH3)2, 3H, s]; 1.21e2.00 [(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2,
6H, m]; 6.91e8.50 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3e
CH2OeC6H3e(OCOCH3)2, 8H, m].
13C NMR (
d ppm CDCl3 300 MHz): 166.5 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2] 159, 153.5,
139.5, 136.2, 131.4, 128.9, 124.28, 123.6, 122.1, 120.4, 117.5, 113.4,
109.2, 106.6 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2Oe
13C NMR(
d
ppm CDCl3 300 MHz): 166.5 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2] 159, 153.5,
139.5, 136.2, 131.4, 128.9, 124.28, 123.6, 122.1, 120.4, 117.5, 113.4,
109.2, 106.6 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2Oe
C6H3e(OCOCH3)2];
(CH2)5eCH2OeC6H3e(OCOCH3)2];
68.2[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
56.3[(CH3OeC6H3eC6H3e
C6H3e(OCOCH3)2];
(CH2)3eCH2OeC6H3e(OCOCH3)2];
68.2[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
56.3[(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2]; 52.4[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2];
44.8[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2-(CH2)5eCH2OeC6H3e
(OCOCH3)2]; 30.7, 29.2, 29.1, 27.2, 26.1[(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2]; 20.3[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e(OCOCH3)2];
20.4[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3e
(OCOCH3)2].
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2]; 52.4[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2];
44.8[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e
(OCOCH3)2]; 30.7, 30.5, 29.1[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)3eCH2OeC6H3e(OCOCH3)2];
20.3[(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2]; 20.4[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3e(OCOCH3)2].
Mass spectrum: 503.59 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)3eCH2OeC6H3e(OCOCH3)2].
Mass spectrum: 531.64 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)5eCH2OeC6H3e(OCOCH3)2].
Elemental analysis of 9a: Calcd. for C30H33NO6 (%): C 71.55, H
6.61, N 2.78. Found (%): C 71.44, H 6.81, N 2.91.
Elemental analysis of 9c: calcd. for C32H37NO6 (%): C 72.29, H
7.01, N 2.63. Found (%): C 72.14, H 7.18, N 2.75.
4.3.8. Dimethyl 5-(6-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
hexyloxy)isophthalate (9b)
4.4. General method for the synthesis of compounds 5, 6, 8aec
1H NMR (
d ppm CDCl3 250 MHz): 3.95 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 3H, s]; 4.52
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCO
CH3)2, 2H, t]; 4.05 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4e
CH2OeC6H3e(OCOCH3)2, 2H, t]; 3.94 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2,6H, s]; 2.79
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCO
CH3)2, 3H, s]; 2.83 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4e
CH2OeC6H3e(OCOCH3)2, 3H, s]; 1.21e1.90 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2, 8H, m]; 6.80e
8.30 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e
(OCOCH3)2, 8H, m].
Methyl salicylate (l.35 mmol) and Cs2CO3 (2.7 mmol) were
dissolved in DMF (10 ml). A solution of N-bromoalkyl-substituted
carbazole (1.35 mmol) in DMF was added at room temperature. The
mixture was stirred for 20 h. After the reaction, the solvent was
evaporated, poured into water and extracted with chloroform
(5 ꢁ 50 ml). The organic layer was dried over anhydrous MgSO4,
filtered and the solvent removed by evaporation. Final product was
an orange/brown solid (80% yield) (Schemes 1 and 2).
4.4.1. Methyl 2-(6-(2-hydroxy-6-methoxy-9H-carbazol-9-yl)
hexyloxy)benzoate (5)
13C NMR (
d
ppm CDCl3 300 MHz): 166.5 [(CH3OeC6H3eC6H3e
(d ppm, CDCl3 300 MHz): 4.25 [(OHeC12H7N)eCH2e(CH2)4e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2] 159, 153.5,
139.5, 136.2, 131.4, 128.9, 124.28, 123.6, 122.1, 120.4, 117.5, 113.4,
109.2, 106.6 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2Oe
CH2OeC6H3eOCOCH3, 1H, s]; 4.18 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 2H, t]; 4.09 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 2H, t]; 3.89 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 3H, s]; 1.21e1.97 [(OHeC12H7N)eCH2e
(CH2)4eCH2OeC6H3eOCOCH3, 8H, m]; 6.70e8.10 [(OHeC12H7N)e
CH2e(CH2)4eCH2OeC6H3eOCOCH3, 11H, m].
Mass spectrum: 417.5 [(OHeC12H7N)eCH2e(CH2)4eCH2Oe
C6H3eOCOCH3].
Elemental analysis of 5: calcd. for C26H27NO4 (%): C 74.80, H 6.52,
N 3.35. Found (%): C 74.59, H 6.64, N 3.44.
C6H3e(OCOCH3)2];
(CH2)4eCH2OeC6H3e(OCOCH3)2];
68.2[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
56.3[(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2]; 52.4[(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e(OCOCH3)2];
44.8[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3e
(OCOCH3)2]; 30.8, 29.2, 26.9, 26.1[(CH3OeC6H3eC6H3e(CH3)2eN)e
CH2e(CH2)4eCH2OeC6H3e(OCOCH3)2]; 20.3[(CH3OeC6H3eC6H3e

118
C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
4.4.2. Methyl 2-(6-(5-hydroxy-3-methoxy-9H-carbazol-9-yl)
Elemental analysis of 8c: calcd. for C30H35NO4 (%): C 76.08, H
hexyloxy)benzoate (6)
7.45, N 2.96. Found (%): C 75.96, H 7.52, N 3.02.
(d ppm, CDCl3 300 MHz): 4.32 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 1H, s]; 4.09 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 2H, t]; 3.99 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 2H, t]; 3.90 [(OHeC12H7N)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3,3H, s]; 1.26e2.19 [(OHeC12H7N)eCH2e
(CH2)4eCH2OeC6H3eOCOCH3, 8H, m]; 6.54e8.50 [(OHeC12H7N)e
CH2e(CH2)4eCH2OeC6H3eOCOCH3, 11H, m].
4.5. Cell culture
Human monocytic leukemia THP-1 cells (American Type Culture
Collection, Manassas, VA) were cultured in RPMI 1640 supple-
mented with 10% FBS, 100 UI/ml penicillin, 100
mg/ml streptomycin
and 40
m
g/ml gentamycin in a 5% CO2 atmosphere at 37 ꢀC.
Mass spectrum: 417.5 [(OHeC12H7N)eCH2e(CH2)4eCH2Oe
C6H3eOCOCH3].
4.6. Electrophoretic mobility shift assay e EMSA
Elemental analysis of 6: calcd. for C26H27NO4 (%): C 74.80, H 6.52,
N 3.35. Found (%): C 74.69, H 6.70, N 3.46.
Nuclear extracts of THP-1 cells were prepared according to
Osborn et al. [19] in presence of 10 g/ml leupeptin, 5 g/ml anti-
pain, 5 g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride.
m
m
4.4.3. Methyl 2-(5-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
m
pentyloxy) benzoate (8a)
Eight micrograms of nuclear extract were incubated with 2e
5 ꢁ 104 cpm of 32P-labeled double-stranded oligonucleotides, the
consensus STAT1/3 DNA binding site (sis-inducible factor-binding
recognition element, SIE/m67) from the c-fos promoter (50-gtcga-
(d ppm CDCl3 300 MHz): 3.89 [(CH3OeC6H3eC6H3e(CH3)2eN)e
CH2e(CH2)3eCH2OeC6H3eOCOCH3, 3H, s]; 4.52 [(CH3OeC6H3e
C6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3eOCOCH3, 2H, t]; 4.00
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3eOCOC
H3, 2H, t]; 3.86 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3e
CH2OeC6H3eOCOCH3, 3H, s]; 2.80 [(CH3OeC6H3eC6H3e(CH3)2e
N)eCH2e(CH2)3eCH2OeC6H3eOCOCH3, 3H, s]; 2.83 [(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3eOCOCH3, 3H,
CATTTCCCGTAAATCg-30), in a 15
m
l reaction mixture containing
20 mM Hepes, pH 7.9, 50 mM KCl, 0.5 mM dithiothreitol, 0.1 mM
EDTA, 2 g of poly(dI-dC), 1 g of salmon sperm DNA, and 10%
m
m
glycerol. Products were fractionated on a non-denaturing 5%
polyacrylamide gel. The gels were dried and autoradiographed and
the intensity of hybridization was quantified using the public
domain NIH Image 1.61 program (developed at the U.S. National
Institutes of Health and available on http://rsb.info.nih.gov/nih-
image/). Supershift assay was performed by incubating the
s];
1.10e2.00
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)3e
CH2OeC6H3eOCOCH3, 6H, m]; 6.60e8.40 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)3eCH2OeC6H3eOCOCH3, 9H, m].
Mass spectrum: 445.55 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)3eCH2OeC6H3eOCOCH3].
nuclear extracts in a binding buffer for 1 h at 4 ꢀC with 1
ml of
Elemental analysis of 8a: calcd. for C28H31NO4 (%): C 75.48, H
7.01, N 3.14. Found (%): C 75.35, H 7.12, N 3.26.
antibody before addition of labelled oligonucleotide. Polyclonal
antibodies against STAT3 and STAT1 were purchased from Santa
Cruz Biotecnology (Santa Cruz, CA).
4.4.4. Methyl 2-(6-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
hexyloxy)benzoate (8b)
4.7. Western blot analysis
(d ppm CDCl3 250 MHz): 3.99 [(CH3OeC6H3eC6H3e(CH3)2eN)e
CH2e(CH2)4eCH2OeC6H3eOCOCH3, 3H, s]; 4.53 [(CH3OeC6H3e
C6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3eOCOCH3, 2H, t]; 4.04
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3eOCOC
H3, 2H, t]; 3.94 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 3H, s]; 2.82 [(CH3OeC6H3eC6H3e(CH3)2e
N)eCH2e(CH2)4eCH2OeC6H3eOCOCH3, 3H, s]; 2.84 [(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3eOCOCH3, 3H,
Cells were homogenized at 4 ꢀC in 20 mM HEPES, pH 7.4, con-
taining 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet-P40
(NP-40), 20% glycerol, protease cocktail inhibitors (GE Healthcare,
Amersham Place, UK) and phosphatase cocktail inhibitors. Aliquots
of the cell lysate (40 mg total protein/lane) were loaded on 7.5% SDS-
polyacrylamide gels. Electrophoresis was performed at 100 V with
a running buffer containing 0.25 M Tris HCl, pH 8.3, 1.92 M glycine,
and 1% SDS. The resolved protein were electroblotted onto a PVDF
membrane (Immobilon P, Millipore, Bedford MA) and incubated
overnight at 4 ꢀC with rabbit anti-phospho-Tyr705Stat3 (Cell
Signaling Technology, Beverly, MA). After washing, membranes
were developed using anti-rabbit IgG peroxidase-conjugated anti-
body (Cell Signaling Technology) and chemiluminescent detection
system (Immun-StarÔ WesternCÔ Kit, Bio-Rad, Hercules, CA).
Blotted proteins were detected and quantified using the ChemiDoc
XRS Imaging System (Bio Rad). After stripping, membranes were
re-hybridized with rabbit anti-STAT3 antibodies (Santa Cruz
Biotechnology).
s];
1.30e1.90
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)4e
CH2OeC6H3eOCOCH3, 8H, m]; 6.60e8.00 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)4eCH2OeC6H3eOCOCH3, 9H, m].
Mass spectrum: 459.58 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)4eCH2OeC6H3eOCOCH3].
Elemental analysis of 8b: calcd. for C29H33NO4 (%): C 75.79, H
7.24, N 3.05. Found (%): C 75.62, H 7.38, N 3.14.
4.4.5. Methyl 2-(7-(6-methoxy-1,4-dimethyl-9H-carbazol-9-yl)
heptyloxy)benzoate (8c)
(d ppm CDCl3 250 MHz): 3.95 [(CH3OeC6H3eC6H3e(CH3)2eN)e
CH2e(CH2)5eCH2OeC6H3eOCOCH3, 3H, s]; 4.50 [(CH3OeC6H3e
C6H3e(CH3)5eN)eCH2e(CH2)3eCH2OeC6H3eOCOCH3, 2H, t]; 3.95
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3eOCOC
H3, 2H, t]; 3.85 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5e
CH2OeC6H3eOCOCH3, 3H, s]; 2.87 [(CH3OeC6H3eC6H3e(CH3)2e
N)eCH2e(CH2)5eCH2OeC6H3eOCOCH3, 3H, s]; 2.95 [(CH3Oe
C6H3eC6H3e(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3eOCOCH3, 3H,
Acknowledgments
We wish to thank Italian Minister of University and Research for
financial support of this work. The authors are grateful to Prof. P.
Longo for useful discussions and Dr. I. Immediata for helpful tech-
nical support.
s];
1.20e1.90
[(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e(CH2)5e
CH2OeC6H3eOCOCH3, 10H, m]; 6.76e8.12 [(CH3OeC6H3eC6H3e
(CH3)2eN)eCH2e(CH2)5eCH2OeC6H3eOCOCH3, 9H, m].
Mass spectrum: 473.60 [(CH3OeC6H3eC6H3e(CH3)2eN)eCH2e
(CH2)5eCH2OeC6H3eOCOCH3].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.004.

C. Saturnino et al. / European Journal of Medicinal Chemistry 60 (2013) 112e119
119
(c) A.G. Rufine, N. Lionel, T. Vincent, B. Jenny, C. Philip, A. Fabrice, M. Pascale,
Bioorg. Med. Chem. 18 (2010) 6865e6873;
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