Development of a novel furocoumarin derivative inhibiting NF-κB dependent biological functions: Design, synthesis and biological effects

Article abstract of DOI:10.1016/j.ejmech.2011.07.032

Nuclear Factor kappaB (NF-κB) plays a very important role in the control of gene expression and is deeply involved in several human pathologies. Accordingly, molecules targeting NF-κB dependent biological functions are considered of great interest. Virtua

Full text of DOI:10.1016/j.ejmech.2011.07.032

European Journal of Medicinal Chemistry 46 (2011) 4870e4877
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of a novel furocoumarin derivative inhibiting NF-
biological functions: Design, synthesis and biological effects
kB dependent
Monica Borgatti a, Adriana Chilin b, Laura Piccagli a, Ilaria Lampronti a, Nicoletta Bianchi a, Irene Mancini a,
**
,
a,
*
Giovanni Marzaro b, Francesco dall’Acqua b, Adriano Guiotto b , Roberto Gambari
a BioPharmaNet, Department of Biochemistry and Molecular Biology, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Via Fossato di Mortara 74,
I-44100 Ferrara, Italy
b Department of Pharmaceutical Sciences, University of Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclear Factor kappaB (NF-
involved in several human pathologies. Accordingly, molecules targeting NF-
k
B) plays a very important role in the control of gene expression and is deeply
B dependent biological
functions are considered of great interest. Virtual screening of furocoumarin libraries against NF- B p50
allowed to rank compounds in respect to their expected ability to bind NF- B and the identified
compound might be considered for the development of analogs to be tested for biological activity on
inhibition of NF- B/DNA complex formation. The data reported in the present paper suggest that,
following this approach, the best ranked compounds identified by virtual screening (a) strongly bind in
silico to NF- B double
B and (b) efficiently inhibit the molecular interactions between 32P-labeled NF-
stranded DNA and p50 or p50/p65 complex. These data allowed to develop a novel lead of great interest
for inhibiting NF- B dependent biological functions. This novel molecule (compound 2), bearing a methyl
group in the 9 position of the psoralen nucleus, exhibits high efficiency in inhibiting NF- B/DNA inter-
actions. In addition, we found that compound 2 is a potent inhibitor of IL-8 gene expression in TNF-
treated IB3-1 cystic fibrosis cells. Taken together, our data indicate that compound 2 might find an
Received 25 March 2011
Received in revised form
13 July 2011
Accepted 18 July 2011
Available online 23 July 2011
k
k
k
k
Keywords:
k
k
Furocoumarin
Virtual screening
Docking
Cystic fibrosis
Interleukin-8
k
k
TNF-
a
a
important place in the set of molecules of interest for the development of pharmaceutical strategies
against the inflammatory phenotype of cystic fibrosis.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
with Pseudomonas aeruginosa [16e20]. Accordingly, targeting NF-
k
B appears to be a relevant therapeutic strategy, as recently
reviewed [21e23]. Unfortunately, targeting NF- B is not a simple
task. First of all, several proteins belong to the NF- B family,
including RelA (also known as p65), RelB, cRel/Rel, p50 and p52,
originating homo- and hetero-dimers, the most common of them
being p50/p65 and p52/RelB. Moreover, the metabolic regulation of
NF-kB biological functions involves several control levels, one of the
most important being the interaction with inhibitory proteins
Nuclear factor kappaB (NF-
biological processes, including cell cycle regulation [1e3], expres-
sion of specific genes [4,5], regulation of cell differentiation [6,7]
and apoptosis [8e11]. On the other hand, alteration of NF-kB
activity is associated with several human pathologies, including
osteoporosis [12], rheumatoid arthritis [13] and cancer [14,15]. In
k
B) plays a critical role in several
k
k
addition, NF-kB is one among the master transcription factors
responsible for inflammation in cystic fibrosis (CF) cells infected
belonging to the I
these, I plays a major role as recently reviewed by Ferreiro and
Komives [24], generating a complex with the NF- B homo- or
hetero-dimers; this molecular interaction prevents NF- to
translocate to the nucleus and exert its regulatory functions on
transcription of target genes [24e26]. Activation of NF- B is oper-
ated by the I B kinase (IKK) complex, which is composed by the two
catalytic subunits IKK and IKK and a regulatory subunit, the NF-
kappaB essential modulator (NEMO, also known as IKK ) [27].
Upon different stimuli, IKK phosphorilates the N-terminal signal
response domain of NF- B-bound causing subsequent
kB (inhibitor of NF-kB) family [24,25]. Among
kBa
k
Abbreviations: VS, virtual screening; PSO, psoralen; ANG, angelicin; TMP, 4,5’,8-
trimethylpsoralen; TMA, trimethylangelicin; IL-8, interleukin-8; EMSA, electro-
kB
phoretic mobility shift assay; CF, cystic fibrosis; TNF-
a
, tumor necrosis factor
a; NF-
k
k
B, nuclear factor kappaB; I , inhibitory protein
k
B
a
a of nuclear factor kappaB; PCR,
polymeraseechain reaction; RT-qPCR, reverse transcription quantitative PCR; FBS,
fetal bovine serum.
* Corresponding author. Tel.: þ39 0532 974443; fax: þ39 0532 974500.
** Corresponding author. Tel.: þ39 049 8275351; fax: þ39 049 827 5366.
E-mail addresses: adriano.guiotto@unipd.it (A. Guiotto), gam@unife.it
(R. Gambari).
k
a
b
g
k
IkBa,
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.032

M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
4871
polyubiquitinylation and proteasome-directed degradation, thus
leading NF- B to be translocated to the nucleus. At the nuclear
level, NF- B binds to the DNA target elements present in NF-
regulated genes, as well as to co-activators of gene transcription
[28]. In any case, the availability of NF- B inhibitors is considered
k
k
kB
k
a major objective of therapeutic intervention.
In this respect, the concerted actions of researchers involved in
bioinformatics, medicinal chemistry, cellular and molecular biology
appear to be crucial for the development of novel drug candidates
for important human pathologies [29e31]. In this context we have
recently reported the possible use of a structured based virtual
Fig. 1. Structures of synthesized compounds.
related with the observation that the introduction of a methyl
group increases the inhibitory activity of furocoumarins (for
screening (VS) procedure to identify possible NF-kB binders
[32e34], demonstrating that this in silico screening approach is
suitable for the identification of low-molecular-weight compounds
instance psoralen and angelicin) on NF-
kB/DNA interactions. As
reported in Figs. 2 and 4, 50,8-trimethylpsoralen (TMP) and trime-
that inhibit NF-kB/DNA interactions and NF-kB dependent functions
thylangelicin (TMA) inhibit the interactions between p50 NF-kB
and target 32P-labeled DNA with higher efficiency in comparison to
non-methylated psoralen and angelicin.
[32e34]. VS against NF-kB p50 using docking simulations was
applied starting from a three-dimensional (3D) database containing
more than 4.6 million commercially available structures. Docking
Methyl (7-hydroxy-2-oxo-2H-3-benzopyranyl)acetates 3e4 [46]
were condensed with 1-chloropinacolone using a new MAOS
(microwave-assisted organic synthesis) protocol to give the ethers
5e6 (Scheme 1). In this way, starting products were irradiated at
130 ꢀC for 20 min in triethylamine and water to give the desired
ethers with higher yield and reduced reaction times, if compared to
previously reported methods [46]. Subsequent cyclization of the
ketoalkyloxy side chain in alkaline medium afforded psoralenacetic
acids 7e8, which were finally reacted with DL-phenylalanine
through activation of the carboxyl group via acyl chloride to give
the amides 1a and 2. Special attention should be paid to this step
because decarboxylation of psoralen acids can easily occur at
temperature higher than room temperature. Finally, since a chiral
center is present in the amide moiety of compound 1a, the L-
enantiomer 1b was also synthesized starting from L-phenylalanine,
simulations to p50 NF-
known inhibitors of NF-
with docking results, the highest-scored compound displayed a high
level of inhibitory activity in EMSA experiments (inhibition of NF- B/
DNA interactions) and on biological functions dependent on NF-
kB were performed with a test set of six
kB-DNA interactions [32,33]. In agreement
k
kB
activity (inhibition of IL-8 gene expression in IB3-1 CF cells). In
a more recent study, we constructed a focus library of differently
substituted furocoumarins and analogs for an in silico screening
against NF-
belonging to the furocoumarin family [34]. We identified several
furocoumarin derivatives expected to be active on NF- B dependent
functions: four of the five best ranked compounds (the commercially
available ones) displayed interesting activities (inhibition of NF- B/
kB with the aim of finding more potent NF-kB inhibitors
k
k
DNA interactions and IL-8 gene expression [34]), demonstrating the
success of our VS approach.
in order to evaluate the influence of the stereochemistry on NF-kB-
The rationale of this report is based on the evidence that VS, in
combination with molecular biology approaches, allows the iden-
tification of lead compounds that can be further modified to
generate bioactive molecules useful in experimental therapy of
human pathologies [29,34]. Following a recently published study
[29], in the present work we have synthesized the most active
compound identified through VS and a structurally-related deriv-
DNA interactions.
In order to obtain a first indication on biological activity, elec-
trophoretic mobility shift assay (EMSA) was performed. This
approach allows to rank even large set of newly synthesized
compounds in respect to the effects on molecular interactions
between NF-kB and target DNA sequences [32e34].
ative. Their activity on NF-
kB/DNA interactions was determined by
2.2. Biological activity of the compounds 1aeb and 2: EMSA studies
EMSA (Electrophoretic Mobility Shift Assay) and TNF-
a
induced
expression of interleukin-8 (IL-8) in cystic fibrosis (CF) cells was
evaluated by quantitative reverse transcription and polymer-
aseechain reaction (RT-PCR). This cellular system is very attractive,
since it is well known that the hallmark in CF airway pathology is
a characteristic elevated concentration of pro-inflammatory cyto-
kines and chemokines, the most important of which is IL-8
[35e37]. On the other hand, it is firmly established that down-
To determine biological activity of compounds 1a, 1b and 2 in
EMSA studies purified p50 NF-
results obtained, shown in Fig. 3A, demonstrate that all the tested
compounds are active in inhibiting NF- B/DNA interactions, being
kB protein was first employed. The
k
compound 2 the most efficient. Accordingly, further analysis was
performed on this compound (Fig. 3B) demonstrating an IC50 of about
30
a
m
M and an efficiency in inhibiting the interaction of NF-
kB-DNA to
stream activation of nuclear transcription factors, including NF-
[17e19], is required for a cascade of pro-inflammatory cytokines
and chemokines, first of all the NF- B dependent IL-8, which is well
known to play a role of master gene in PMN recruitment in CF lung
[20,38,39]. Accordingly, candidate drugs interfering with NF- B/
kB
reconstituted p50/p65 heterodimer (Fig. 3C). Interestingly,
compound 2 is far more active than psoralen, TMP, angelicin and TMA
(Table 1) in inhibiting NF- B/DNA interactions. This set of experi-
ments suggests that compound 2 might be a good candidate for
developing molecules inhibiting the expression of NF- B regulated
genes. Interestingly, compound 2 shows docking activity to NF-
(Fig. 4). Tyr57, Thr143, Lys144 and Lys145 are the amino acids
k
k
k
k
DNA interactions and inhibiting IL-8 expression in CF cells are of
great importance [38,40e45].
kB
apparently involved in the interactions between NF-kB and
2. Results and discussion
compound 2.
On the basis of these observations, compound 2 was further
characterized for its possible activity on the expression of
interleukin-8 (IL-8) gene. It is firmly established that IL-8 gene
2.1. Chemistry
Compound 1, identified by VS as the best ranked ligand to p50-
p50 dimer [34], was synthesized along with its closely related
analog 2, bearing a methyl group in the 9 position of the psoralen
nucleus (Fig. 1). The rationale for synthesizing compound 2 was
expression is regulated by NF-
cules inhibiting NF- B/DNA interactions might exhibit inhibitory
activities on NF- B regulated genes [35e37], we were interested to
determine the activity of compound 2 on IL-8 gene expression.
kB [20e23]; therefore, since mole-
k
k

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M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
Fig. 2. Structures of psoralen, TMP, angelicin and TMA and their effects on the molecular interactions between NF-
nucleotide. Compounds were first incubated with NF- B oligonucleotide was added. NF-
B and then the 32P-labeled target NF-
gel electrophoresis. Arrows indicate NF- B probe.
B/DNA complexes; asterisks indicate the free 32P-labeled target NF-
k
B p50 and 32P-labeled target NF-
B/DNA complexes were analyzed by polyacrylamide
kB double stranded oligo-
k
k
k
k
k
2.3. Biological activity of compound 2: inhibition of IL-8 mRNA
accumulation in TNF- treated IB3-1 CF cells
genes, including the gene coding for the pro-inflammatory protein
IL-8 are activated following infection with P. aeruginosa, or treat-
a
ment with TNF-a of IL-1b [20,38,39]. This feature is very important
In order to determine the biological activity of compound 2 on
IL-8 gene expression we therefore employed IB3-1 CF cells treated
in the pathophysiology of CF, since several clinical complications
are caused by exacerbation of this inflammatory response. Cells
were incubated overnight in the presence of compounds 2 and then
with 80 ng/ml of TNF-
a
in the presence of 10, 20 and 50
mM
compound 2. In the IB3-1 CF cellular system NF-
k
B dependent
treated with TNF-a (80 ng/ml). After 1 day of incubation, cellular
Fig. 3. Effects of compounds 1a, 1b, 2 and TMA on the molecular interactions between NF-
oligonucleotide. Compounds were first incubated with NF-
B and then the 32P-labeled target NF-
acrylamide gel electrophoresis. Arrows indicate NF-
B/DNA complexes; asterisks indicate the free 32P-labeled target NF-
k
B p50 (A and B) and NF-
B oligonucleotide was added. NF-
B probe.
k
B p50/p65 (C) and 32P-labeled target NF-
B/DNA complexes were analyzed by poly-
kB double stranded
k
k
k
k
k

M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
4873
Table 1
Inhibitory activity on NF-kB/DNA interactions of
compounds 2 in respect to other furocoumarins.
a
Compound
IC50
2
0.03 ꢁ 0.002
3 ꢁ 0.2
Psoralen
TMP
Angelicin
TMA
0.5 ꢁ 0.1
2.5 ꢁ 0.4
1 ꢁ 0.2
a
IC50 is the inhibitory concentration leading to 50%
B/DNA interactions.
inhibition of NF-
k
Moreover our data allow to develop a novel lead of great
interest for inhibiting NF- B dependent biological functions. This
k
novel molecule (compound 2), bearing a methyl group in the 9
position of the psoralen nucleus, exhibits higher efficiency in
Fig. 4. Stereo view of the complex formed by NF-kB p50 homodimer and the docked
compound 2 (bottom side of the panel). The amino acids Tyr57, Thr143, Lys144 and
Lys145, involved in the interactions with compound 2, are highlighted.
RNA was isolated for RT-qPCR analysis and the cellular superna-
tants isolated for determination of the release of IL-8.
The results obtained are reported in Fig. 5, which demonstrates
that compounds 2 inhibits TNF-a induced IL-8 mRNA accumulation
(Fig. 5A) and IL-8 protein release (Fig. 5B) exhibiting fair dos-
eeresponse effects. While statistically-significant differences were
not observed when compound 2 was used at 10
mM concentration,
the highest effects were observed at 50 M, being 30
m
mM compound
2 still active (Fig. 5, panels A and B). Interestingly, compound 2
inhibits IL-8 gene expression without major effects on IB3-1 cell
growth (compare panels A and B of Fig. 5 to panel C). These results
demonstrate that treatment of IB3-1 CF cells with compound 2
significantly reduces the TNF-a-dependent transcription of the pro-
inflammatory mediator IL-8 and the relative release of IL-8.
3. Conclusions
The data reported in the present paper suggest that the best
ranked compound(s) identified through VS approach (compounds
1aeb) efficiently inhibit the molecular interactions between 32P-
labeled NF-kB double stranded DNA and p50 or p50/p65 complex.
These data demonstrate that docking simulations are suitable for
predictive studies on binding affinity of small compounds to
a difficult target as NF-kB homodimer and heterodimer.
Fig. 5. Effects of compound 2 on IL-8 gene expression in TNF-
were exposed for 24 h to the indicated concentrations of compound 2 in the presence
of TNF- as described in methods. After this incubation time, RNAs (A) and cellular
a treated cells. IB3-1 cells
a
supernatants (B) were isolated. Quantitative RT-PCR was performed and IL-8 mRNA
content was quantified in respect to control uninduced IB3-1 cells (A). The pg/ml of IL-
8 were quantified by Bio-plex analysis [52,53] and the results obtained are shown in
panel B. Panel C shows the effects of compound 2 on cell proliferation. (ꢂ) ¼ untreated
Scheme 1. Reagents and conditions: (a) 1-chloropinacolone, TEA/H2O, MW, 130 ꢀC,
20 min; (b) 1 M NaOH, propan-2-ol, reflux, 4 h; (c) (i) SOCl2, r.t., 3 h; (ii) DL-Phe or L-
Phe, TEA/H2O, r.t.
IB3-1 cells; (þ) ¼ TNF- treated IB3-1 cells (n ¼ 3). The results represent the average ꢁ
a
SD. p < 0.001, highly significant; p < 0.005, significant; n.s., not significant.

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M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
inhibiting NF-
k
B/DNA interactions and strongly binds in silico to
constants are given in Hz and the relative peaks area were in
agreement with all assignment. Elemental analysis were performed
on a PerkineElmer 2400 analyzer. Mass spectra were performed on
a Applied Biosystem Mariner System 5220 with direct injection of
the sample. Microwave-assisted reactions were performed on
a CEM DiscoverÒ monomode reactor with the temperature moni-
tored by a built-in infrared sensor and the automatic control of the
power; all the reactions were performed in closed devices with
pressure control. Compounds 3 and 4 were prepared according to
literature methods [46]. Purity for all the tested compounds was
determined by elemental analyses and was found equal or more
than 95%.
NF-
k
B. Very good agreement does exist between docking studies
B) and
(showing high binding efficiency of compound 2 to NF-
k
EMSA experiments (showing higher inhibitory activity). These
data are consistent with the observation that psoralens-like
molecules bearing methyl groups (TMP and TMA) are more
active in inhibiting NF-
reference compounds psoralen and angelicin. About potency in
inhibiting NF- B/DNA interactions, compound 2 is the most
kB/DNA interactions in respect to the
k
active furocoumarin derivative recently studied by our research
group.
The results presented in this study identify compound 2 as
a lead compound for generating libraries of structurally-related
molecules, whose synthesis and evaluation will allow to establish
structure-activity relationships (SAR) for this class of compounds.
Works are in progress about design and synthesis of novel
structurally-related furocoumarins bearing modifications in the
points of molecular diversity (such as the substituents of furan ring
or benzene ring, the length of the alkyl chains in 3 position of the
pyrone ring, the aminoacid condensed on the carboxylic function of
the 3-side chain) in order to define the role of each functional
group.
4.1.1. General procedures for methyl [7-(30,30-dimethyl-20-
oxobutoxy)-2-oxo-2H-benzopyran-3-yl]acetates 5e6
A mixture of 3 or 4 (10.0 mmol) and 1-chloropinacolone
(17.0 mmol) in triethylamine (3.6 mL, 26.0 mmol) and water
(3.6 mL) was microwave irradiated at 130 ꢀC (power set point
200 W; ramp time 2 min; hold time 20 min). After cooling the
reaction mixture was poured into 1 M HCl (300 mL) and the
resulting precipitate was filtered off to give 5e6.
With respect to possible biomedical applications, our data
suggest that compound 2 might be of interest for experimental
4.1.2. Methyl [4-methyl-7-(30,30-dimethyl-20-oxobutoxy)-2-oxo-
2H-benzopyran-3-yl]-acetate (5)
therapy of CF. In fact, compound 2 inhibits NF-
and IL-8 gene expression in TNF- treated IB3-1 CF cells. Inter-
estingly, and relevant to possible biomedical applications of our
data, NF- B inhibitors affecting IL-8 gene expression are employed
k
B/DNA interactions
Yield 98%; mp: 158 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.55 (d,
a
J ¼ 8.9 Hz, 1H, 5eH), 6.91 (dd, J ¼ 8.9, 2.6 Hz, 1H, 6eH), 6.68 (d,
J ¼ 2.6 Hz, 1H, 8eH), 4.96 (s, 2H, OCH2COC(CH3)3), 3.71 (s, 5H,
CH2COOCH3 and CH2COOCH3), 2.39 (s, 3H, 4-CH3), 1.27 (s, 9H,
C(CH3)3). Anal. calcd. for C19H22O6: C, 65.88; H, 6.40; found: C,
65.85; H, 6.47. HRMS (ESI-TOF) for C19H23O6 [M þ H]þ: calcd.:
347.1495, found: 347.1558.
k
in clinical trials for CF. For instance Saadane et al. [40] demon-
strated that treatment of with parthenolide and stimulation with
IL-1
these cytokines and prevents NF-
and I B Kinase complex activity. Therefore parthenolide inhibits
B kinase, resulting in stabilization of cytoplasmic I , which in
turn leads to inhibition of NF- B translocation and attenuation of
subsequent inflammatory responses. I B kinase may be a good
b
and/or TNF leads to inhibition of IL-8 secretion induced by
k
B activation, I degradation,
kBa
k
4.1.3. Methyl [4,8-dimethyl-7-(30,30-dimethyl-20-oxobutoxy)-2-oxo-
I
k
k
B
a
2H-benzopyran-3-yl]-acetate (6)
k
Yield 94%; mp: 144 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.40 (d,
k
J ¼ 8.9 Hz, 1H, 5eH), 6.59 (d, J ¼ 8.9 Hz, 1H, 6eH), 5.00 (s, 2H,
OCH2COC(CH3)3), 3.73 (s, 2H, CH2COOCH3), 3.70 (s, 3H,
CH2COOCH3), 2.39 (s, 3H, 4-CH3), 2.36 (s, 3H, 8-CH3), 1.27 (s, 9H,
C(CH3)3). Anal. calcd. for C20H24O6: C, 66.65; H, 6.71; found: C,
66.69; H, 6.72. HRMS (ESI-TOF) for C20H25O6 [M þ H]þ: calcd.:
361.1651, found: 361.1698.
target, and parthenolide might be promising treatments for the
excessive inflammation in CF. In addition, Cigana et al. [41]
demonstrated azithromycin as powerful inhibitor of NF-kB and
AP-1 DNA binding, leading to anti-inflammatory effects in CF cells,
indicating inhibition of transcription of pro-inflammatory genes as
possible mechanism, thus providing a rationale for the possible
use of specific TF inhibitors for therapy. Finally, Tchilibon et al. [42]
in a systematic search for candidate drugs that might be used
therapeutically to suppress IL-8 secretion in CF, identified a potent
and efficacious series of amphiphilic pyridinium salts. The mech-
anism of action of these compounds appears to involve inhibition
of signaling of the NF-
promoter.
Taken together, our data indicate that compound 2 might find an
important place in the set of molecules of interest for the devel-
opment of pharmaceutical strategies against the inflammatory
phenotype of CF.
4.1.4. General procedures for (30-tert-butyl-70-oxo-7H-furo[3,2-g]
[1]benzopyran-60-yl)acetic acids 7e8
A mixture of 5 or 6 (10.0 mmol) in propan-2-ol (40 mL) and 1 M
NaOH (60 mL) was refluxed for 4 h. After cooling the mixture was
acidified with 5 M HCl, the resulting precipitate was filtered off and
crystallized from ethyl acetate to give 7e8.
kB and AP-1 transcription factors to the IL-8
4.1.5. (30-tert-butyl-50-methyl-70-oxo-7H-furo[3,2-g] [1]
benzopyran-60-yl)acetic acid (7)
Yield 94%; mp: 264 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.94 (s, 1H,
20eH), 7.44 (s, 1H, 40-H or 90eH), 7.40 (s, 1H, 40eH or 90eH), 3.83 (s,
2H, OCH2COOH), 2.57 (s, 3H, 50eCH3), 1.46 (s, 9H, C(CH3)3). Anal.
calcd. for C18H18O5: C, 68.78; H, 5.77; found: C, 68.73; H, 5.80. HRMS
(ESI-TOF) for C18H17O5 [MꢂH]ꢂ: calcd.: 313.1076, found: 313.1129.
4. Experimental section
4.1. Chemistry
4.1.6. (30-tert-butyl-50,90-dimethyl-70-oxo-7H-furo[3,2-g] [1]
benzopyran-60-yl)acetic acid (8)
All commercial chemicals and solvents used were analytical
grade and were used without further purification. Analytical thin
layer chromatography (TLC) was performed on pre-coated silica gel
plates (Merck 60-F-254, 0.25 mm). Melting points were determined
on a Gallenkamp MFB-595-010 M melting point apparatus and are
uncorrected. The 1H NMR spectra were recorded on a Bruker 300-
AMX spectrometer with TMS as an internal standard. Coupling
Yield 83%, mp: 222 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.79 (s, 1H,
20eH), 7.41 (s, 1H, 40eH), 3.83 (s, 2H, OCH2COOH), 2.59 (s, 3H,
20eCH3 or 50-CH3), 2.54 (s, 3H, 20-CH3 or 50ꢂCH3), 1.45 (s, 9H,
C(CH3)3). Anal. calcd. for C19H20O5: C, 69.50; H, 6.14; found: C,
69.54; H, 6.18. HRMS (ESI-TOF) for C19H19O5 [MeH]ꢂ: calcd.:
327.1232, found: 327.1297.

M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
4875
4.1.7. General procedures for 2-[20-(3”-tert-butyl-7”-oxo-7H-furo
[3,2-g] [1]benzopyran-6”-yl)-acety-lamino]-3-phenylpropionic
acids 1aeb and 2
prepared using Maestro graphical interface, as described in detail
elsewhere [32e34]. The Standard-Precision (SP) method as
implemented in Glide, was employed as first for p50 NF-kB. The
A solution of acids 7e8 (1.0 mmol) in thionyl chloride (25 mL)
was stirred at room temperature until a clear solution was formed.
The solvent was evaporated under nitrogen stream and the residue
was dissolved in tetrahydrofuran (10 mL). The solution was added
dropwise to a mixture of DL-phenylalanine or L-phenylalanine
(0.3 g, 2.0 mmol) in triethylamine (0.3 mL, 2.1 mmol) and water
(5 mL). After reaction completion, the mixture was diluted with
water (40 mL) and acidified with 1 M HCl to pH ¼ 3. The resulting
precipitate was filtered off and crystallized from propan-2-ol to
give 1aeb and 2.
maximum number of poses per ligand to pass to the grid-
refinement calculation was set to 10,000. Only one good pose for
each molecule was retained. Subsequently, all possible redundant
structures were eliminated from the database using the table
project implemented in Glide. Based on the G-score function, the
highest-ranking 1000 docked ligands were selected and collected
in a new multifile for further docking runs. After a minimization
process in the OPLS_2001 force field by premin script, the picked
out compounds were docked again into DNA recognition site, using
the more CPU time-intensive and accurate Extra-Precision (XP)
method.
Finally, the first ten molecules with the highest-ranking poses
(XP G-score value) against the most protein target were individu-
ated as promising. The molecules were docked into the protein
targets applying the accurate XP method as described above but in
this case the total number of docked retained poses for each
compound was set to 100. The subsequent selection of the putative
bioactive conformation within a cluster of poses for each ligand,
were based on both G-score and E-model values.
4.1.8. DL-2-[20-(3”-tert-butyl-5”-methyl-7”-oxo-7H-furo[3,2-g] [1]
benzopyran-6”-yl)-acetyl-amino]-3-phenylpropionic acid (1a)
Yield 45%; mp: 244 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.91 (s, 1H,
2”eH), 7.45 (s, 1H, 4”ꢂH or 9”eH), 7.43 (s, 1H, 4”ꢂH or 9”eH),
7.16e6.75 (m, 5H, H Ar Phe), 4.80e4.71 (m, 1 H, 2eH), 3.66 (d,
J ¼ 14.1 Hz, 1H, 20eH), 3.55 (d, J ¼ 14.1 Hz, 1H, 20eH), 3.24 (dd,
J ¼ 14.1, J ¼ 4.9 Hz, 1H, 3eH), 3.03 (dd, J ¼ 14.1, J ¼ 8.0 Hz, 1H, 3eH),
2.55 (s, 3 H, 5”ꢂCH3), 1.47 (s, 9 H, C(CH3)3); 13C NMR (75 MHz,
acetone-d6):
d 172.8, 168.7, 160.9, 156.2, 149.5, 149.4, 141.8, 137.6,
129.7, 129.0, 128.0, 126.2, 123.4, 118.2, 117.2, 116.0, 99.14, 53.5, 36.6,
33.6, 30.6, 29.6, 15.4. Anal. calcd. for C27H27NO6: C, 70.27; H, 5.90; N,
3.03; found: C, 70.25; H, 5.93; N, 3.04. HRMS (ESI-TOF) for
C27H26NO6 [MeH]ꢂ: calcd.: 460.1760, found: 460.5102.
4.3. Electrophoretic mobility shift assay (EMSA)
Electrophoretic Mobility Shift Assay (EMSA) was perfomed as
previously described [48e50]. Briefly, double stranded synthetic
oligodeoxynucleotides mimicking the NF-
50-CGC TGG GGA CTT TCC ACG G-30) have been employed. Oligo-
deoxynucleotides were labeled with
32P-ATP using 10 Units of T4-
kB binding (NF-kB, sense:
4.1.9. L-2-[20-(3”-tert-butyl-5”-methyl-7”-oxo-7H-furo[3,2-g] [1]
benzopyran-6”-yl)-acetyl-amino]-3-phenylpropionic acid (1b)
g
Yield 27%; mp: 158 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.90 (s, 1H,
polynucleotide-kinase (MBI Fermentas) in 500 mM TriseHCl, pH
2”eH), 7.42 (s, 1H, 4”-H or 9”eH), 7.41 (s, 1H, 4”-H or 9”eH),
7.14e6.95 (m, 5H, H Ar Phe), 4.80e4.71 (m, 1 H, 2eH), 3.65 (d,
J ¼ 14.1 Hz, 1H, 20eH), 3.54 (d, J ¼ 14.1 Hz, 1H, 20eH), 3.23 (dd,
J ¼ 14.1, J ¼ 4.9 Hz, 1H, 3eH), 3.02 (dd, J ¼ 14.1, J ¼ 8.0 Hz, 1H, 3eH),
2.53 (s, 3 H, 5”eCH3), 1.47 (s, 9 H, C(CH3)3); 13C NMR (75 MHz,
7.6, 100 mM MgCl2, 50 mM DTT, 1 mM spermidine, (1 mM EDTA in
the presence of 50 mCi g ml for 45 min at
32P-ATP) in a volume of 20
37 ꢀC. Reaction was brought to 150 mM NaCl and 150 ng comple-
mentary oligodeoxynucleotide was added. Reaction temperature
was increased to 100 ꢀC for 5 min and left diminishing to room
temperature overnight. Binding reactions were set up as described
acetone-d6):
d 172.8, 168.6, 160.9, 156.2, 149.5, 149.4, 141.8, 137.7,
129.7, 129.0, 128.0, 126.1, 123.4, 118.2, 117.2, 116.0, 99.15, 53.6, 36.6,
33.7, 30.6, 29.6, 15.4. Anal. calcd. for C27H27NO6: C, 70.27; H, 5.90; N,
3.03; found: C, 70.28; H, 5.88; N, 3.03. HRMS (ESI-TOF) for
C27H26NO6 [MeH]ꢂ: calcd.: 460.1760, found: 460.5214.
elsewhere [49] in a total volume of 20
5% glycerol, 1 mM dithiothreitol, 10 ng of human NF-
with or without 10 ng of NF- B p65 protein (Promega) and
m
l containing buffer TF plus
k
B p50 protein
k
different concentrations of compounds. After a incubation of
20 min at room temperature, 0.25 ng of 32P-labeled oligonucleo-
tides were added to the samples for further 20 min at room
temperature and then they were electrophoresed at constant
voltage (200 V) under low ionic strength conditions (0.25ꢃ TBE
buffer: 22 mM Tris borate, 0.4 mM EDTA) on 6% polyacrylamide
gels. Gels were dried and subjected to standard autoradiographic
procedures [49].
4.1.10. DL-2-[20-(3”-tert-butyl-5”,9”-dimethyl-7”-oxo-7H-furo[3,2-
g] [1]benzopyran-6”-yl)-acety-lamino]-3-phenylpropionic acid (2)
Yield 45%; mp: 120 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.77 (s, 1H,
2”eH), 7.43 (s, 1H, 4”eH), 7.11e6.85 (m, 5H, H Ar Phe), 4.77e4.70
(m, 1 H, 2eH), 3.66 (d, J ¼ 14.1 Hz, 1H, 20eH), 3.56 (d, J ¼ 14.1 Hz, 1H,
20eH), 3.24 (dd, J ¼ 14.1, J ¼ 5.5 Hz, 1H, 3eH), 3.02 (dd, J ¼ 14.1,
J ¼ 8.5 Hz, 1H, 3eH), 2.61 (s, 3 H, 5”-CH3 or 9”eCH3), 2.54 (s, 3 H,
5”eCH3 or 9”eCH3), 1.46 (s, 9 H, C(CH3)3); 13C NMR (75 MHz,
4.4. Cell cultures
acetone-d6):
d 172.8, 168.8, 160.9, 155.2, 149.8, 147.1, 141.5, 137.6,
130.1, 129.0, 128.0, 126.2, 122.4, 117.8, 115.9, 114.3, 108.1, 53.5, 36.5,
33.6, 30.6, 29.7. Anal. calcd. for C28H29NO6: C, 70.72; H, 6.15; N, 2.95;
found: C, 70.77; H, 6.16; N, 2.93. HRMS (ESI-TOF) for C28H28NO6
[MeH]ꢂ: calcd.: 474.1917, found: 474.1879.
IB3-1 cells (LGC Promochem), derived from a CF patient with
DF508/W1282X mutant genotype and immortalized with
adeno12/SV40 [51], were grown in LHC-8 basal medium (Invi-
trogen, Carlsbad, CA), supplemented with 5% FBS in the absence of
gentamycin, at 37 ꢀC/5% CO2.
a
4.2. Docking of compounds into DNA binding of NF-kB
All the compound library were docked into putative binding site
of the NF- B targets employing Glide software [47]. The 3D struc-
tures of the NF- BeDNA complexes were retrieved from the Protein
Data Bank (PDB codes: 1NFK and 1LE9) [33,34]. Starting from the
co-crystallized complexes, the NF- B p50ep50 homodimer (from
4.5. Proliferation assay
k
k
IB3-1 cells were seeded at a density of 100,000 cells in 24 wells
plates in LHC-8 medium in presence of 5% FBS. After adhesion, cells
were starved in serum free LHC-8, drug was added at serial dilu-
tions (as indicate in figure) and incubated for further 3 days. After
this period cells were washed with PBS and detached with trypsin/
k
1NFK), p50 monomer chain A (p50a from 1NFK), p50 monomer
chain B (p50b from 1NFK; p50b from 1LE9) for docking were

4876
M. Borgatti et al. / European Journal of Medicinal Chemistry 46 (2011) 4870e4877
EDTA. Cells were resuspended in DMEM medium and counted with
a Sysmex XE-2100 Cytometer (Dasit, Milan, Italy).
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Fibrosis Research Foundation (grant # 15/2004 to RG). RG is granted
by Fondazione CARIPARO and by MUR COFIN-2007.

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