Psoralen derivatives as inhibitors of NF-κB/DNA interaction: the critical role of the furan ring

Article abstract of DOI:10.1007/s11030-015-9586-2

Abstract: Simplified analogues of previously reported NF-κB/DNA interaction inhibitors, lacking the furan moiety, were synthesized and evaluated by performing experiments based on electrophoretic mobility shift assay (EMSA). The synthetic modifications led to simpler coumarin derivatives with lower activity allowing to better understand the minimal structural requirement for the binding to NF-κB. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-015-9586-2

Mol Divers
DOI 10.1007/s11030-015-9586-2
SHORT COMMUNICATION
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Psoralen derivatives as inhibitors of NF- B/DNA interaction:
the critical role of the furan ring
Giovanni Marzaro1 · Ilaria Lampronti2 · Monica Borgatti2 · Paolo Manzini1 ·
Roberto Gambari2,3 · Adriana Chilin1
Received: 2 September 2014 / Accepted: 21 March 2015
© Springer International Publishing Switzerland 2015
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Abstract Simplified analogues of previously reported NF-
Keywords Coumarin derivatives · Psoralen · NF- B ·
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B/DNA interaction inhibitors, lacking the furan moiety,
Transcription · 3D-QSAR
were synthesized and evaluated by performing experiments
based on electrophoretic mobility shift assay (EMSA). The
synthetic modifications led to simpler coumarin derivatives
with lower activity allowing to better understand the minimal
Introduction
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structural requirement for the binding to NF- B.
Nuclear factor kappa-B (NF- B) is a key transcription
Graphical abstract
factor involved in several biological processes, including
inflammation [1,2], oxidative stress [3], cancer [4–6] and
regulation of microRNA expression [7–11]. Therefore, the
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identification of novel NF- B inhibitors is of great rele-
vance in translational research aiming at the development
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of novel therapeutic approaches. NF- B is known as the
key transcription factor regulating the expression of pro-
inflammatory genes, such as those encoding the production
α
of the pro-inflammatory cytokines including TNF- , IL-1 β,
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IL-6, iNOS and COX-2 and IL-8 [12]. This role of NF- B
was highlighted by several research groups on the basis of
studies regarding the inflammatory responses to bacterial
infection, as in the case of clinical complications in cys-
tic fibrosis patients infected with Pseudomonas aeruginosa
[1,2,12–14]. In this case, we and other groups have reported
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that inhibition of NF- B/DNA interactions with decoy mole-
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cules targeting the NF- B transcription factor [1,15,16],
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-015-9586-2) contains supplementary
material, which is available to authorized users.
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antisense oligonucleotides targeting NF- B mRNA [17,18]
or low molecular weight drugs selectively binding NF- B
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domains involved in NF- B/DNA recognition [12,19] lead
to efficient inhibition of the expression of IL-8 and IL-6
Adriana Chilin
adriana.chilin@unipd.it
B
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genes. In addition, NF- B is deeply involved in cancer, reg-
1
ulating a large set of anti-apoptotic genes [4–6], such as the
Department of Pharmaceutical and Pharmacological Sciences,
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NF- B dependent TRAF1 and TRAF2 [20]. Furthermore,
University of Padova, Via Marzolo 5, 35131 Padova, Italy
2
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NF- B has been demonstrated to control the expression of
Department of Life Sciences and Biotechnology, University
of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy
disease-associated microRNAs, such as miR-146a [7], miR-
26a [8], miR-125b [9], and miR-23a [10]. Therefore, the
3
Biotechnology Center, University of Ferrara, Via Fossato di
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Mortara 64, 44121 Ferrara, Italy
identification of new molecules directly targeting NF- B
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Table 1 Structures of simplified THBP analogues
R"
n
3
7
1
O
R'
O
O
O
Fig. 1 Structure of reference compound THBP
R
Compd
1
n
1
R
Rꢀ
Rꢀꢀ
H
N
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and/or inhibiting NF- B functions (the most important of
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which are NF- B/DNA interactions at the level of promoter
sites of NF- B regulated genes) is very important for anti-
inflammatory treatments [12], anti-cancer approaches [11],
as well as miRNA-therapeutic strategy useful for treatment
of several diseases caused by alteration of microRNA activity
(the so called miRNA-therapeutics) [21,22]. The importance
of inhibiting NF- B activity is also supported by recent
patent applications in the field of novel therapeutic strate-
gies [23–25] and clinical trials [26].
Recently, we have found that a tetrahydrobenzopsoralen
derivative bearing a suitable amide function (THBP; Fig. 1)
is a good inhibitor of NF- B/DNA interaction. Moreover,
COOH
H
H
CH3
κ
H
N
COOH
COOH
2
3
4
2
1
2
CH3
CH3
CH3
H
N
κ
CH3
CH3
H
N
COOH
COOH
H
N
κ
5
6
7
1
2
2
H
CH2CH2OCH3
CH2CH2OCH3
CH2CH2OCH3
combining docking and 3D-QSAR experiments, we have
hypothesized a plausible binding mode between THBP and
H
N
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NF- B [27].
COOH
COOH
H
As previously reported, the anilide function resulted
essential for the activity, as well as the terminal carboxylic
function (devoted to establish an H-bond with a Lys residue
H
N
CH3
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of NF- B) [27]. Conversely, on the furan side, the role of
the furan ring was not yet determined: alkylfuran derivatives
were active as well as tetrahydrobenzofuran derivatives, even
if the latter ones were more active than the former ones. In
our research efforts towards the assessment of the minimal
7 position of coumarin) able to act as H-bond acceptor with
the Asp276(B) amide. The chain length in the spacer portion
was modulated with one to two methylene spacers, because
the introduction of an additional methylene function remark-
ably decreased the activity, as previously observed [27]. The
m-aminobenzoic acid and p-aminobenzoic acid were chosen
as the amide substituent on the 3-side chain, since these func-
tional groups demonstrated the best activity in the previously
reported series of psoralen derivatives [27].
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structurerequired for inhibitionof NF- B/DNA interactions,
we planned to simplify the polycyclic skeleton of the “lead
compound”, eliminating the tetrahydrobenzofuran moiety,
thus obtaining coumarin acetanilides and propionanilides
(Table 1).
Simple coumarins have been indeed shown to exhibit bio-
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logical activities on NF- B driven pathways involved in
human pathologies. For instance, hydroxycoumarin deriva-
tives have been reported as potent apoptosis inducers and pro-
posed as possible anti-tumour and anti-inflammatory agents
[28–32]. Furthermore, potent anti-inflammatory effects were
also exhibited by coumarin-containing plant extracts or
natural-occurring coumarins [33–38]. From a synthetic point
of view, coumarins are easier to access and they tolerate a
higher degree of structural diversity than psoralens. Hence,
if coumarin acetanilides and propionanilides would maintain
Results and discussion
Compound 1–7 were synthesized starting from known
alkyl (7-hydroxy-2-oxo-2H-benzopyran-3-yl)acetates or
propionates (8–11) (Scheme 1) [39,40]. The 7-hydroxy
group was etherified with methyl iodide or 2-methoxyethyl
chloride in alkaline medium at reflux in acetone and the 7-O-
alkylethers 12–18 were submitted to basic hydrolysis of the
ester function at the 3-side chain using MAOS (Microwave
Assisted Organic Synthesis) techniques to reduce reaction
times and improve yields [41]. Acid derivatives 19–25
were activated as acyl chlorides and then reacted with the
appropriate anilines (e.g., m-amino or p-aminobenzoic acid)
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NF- B/DNA interaction inhibition properties, they would
allow the preparation of larger libraries of compounds with
respect to psoralen analogues. In our simplified coumarin
class the benzofuran ring of the parent THBP was replaced
with a methoxy or a methoxyethoxy group, in order to
maintain a functional group (i.e., the ether oxygen at the
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Scheme 1 Reagents and
conditions: a methyl iodide or
2-methoxyethyl chloride,
K2CO3, reflux, 2–5 h; b 1M
NaOH, propan-2-ol, MW,
120 ◦C, 5 min; c (i) SOCl2, r.t.,
3 h; (ii) m-aminobenzoic acid or
p-aminobenzoic acid, THF,
TEA/H2O, r.t
OAlk
a
OAlk
n
1
2
1
2
1
2
2
Alk
Me
Et
R
H
H
R'
Me
n
n
12
13
14
15
16
17
18
O
R'
O
Me
HO
O
O
O
O
O
b
Me Me
Me
R
R
8-11
12-18
Et
Me
H
Me
Me
Et
CH2CH2OMe
CH2CH2OMe
n
1
2
Alk
Me
Et
R
H
H
H
8
9
10
11
Et
Me CH2CH2OMe
1
2
Me Me
Et
Me
R"
OH
n
R
H
R'
Me
n
n
c
19
20
21
22
23
24
25
1
2
1
2
1
2
2
R'
O
R'
O
O
O
O
H
Me
O
O
O
Me
Me
H
Me
R
R
1-7
R'
19-25
Me
n
R
R"
CH2CH2OMe
CH2CH2OMe
H
H
N
1
2
3
4
5
6
7
1
2
1
2
1
2
1
H
H
Me
Me
Me
Me
Me CH2CH2OMe
COOH
COOH
H
N
Me
Me
H
CH2CH2OMe
CH2CH2OMe
H
N
H
COOH
Me CH2CH2OMe
Table 2 Predicted and actual
pIC50 values for title compounds
Compd
Predicted
pIC50
Predicted
pIC50
Measured
Measured pICc50
(1st
(2nd
IC50(μM)c
model)a
model)b
1
4.15
4.21
3.98
3.82
4.51
4.40
4.24
4.80d
4.15
4.21
3.98
3.82
3.15
3.02
2.95
4.80d
300
3.52
3.52
3.22
3.30
<2
2
300
3
600
4
500
5
>10000
5000
>10000
10
6
2.30
<2
7
THBP
5.00
a Values obtained considering the structures of 1–7 as flexible during the superimposition on THBP
b Values obtained considering the QM-fully minimized structures of 1–7 during the superimposition on
THBP
c Values obtained through EMSA experiments
d Values reported in ref 28
to give the desired anilides 1–7, according to previously
reported methods [27]. Special attention should be paid to
this step because decarboxylation of coumarin acids to the
corresponding 3-methyl derivatives can easily occur at tem-
perature higher than room temperature [27].
THBP were used in QSAR modeling. The first protocol con-
sidered compounds 1–7 as flexible during superimposition,
whereas in the second one only the QM-fully minimized
structures of 1–7 were used. The different results obtained for
compounds 1 (bearing the 7-methoxy function) and 5 (bear-
ing the 7-methoxyethoxy group) are reported in Fig. 2 as an
example.
The ability of the designed compounds to inhibit the NF-
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B/DNA complex formation was preliminarily predicted
using the 3D-QSAR model previously reported [27] (see pre-
dicted pIC50 values in Table 2).
Due to the increased flexibility of the novel coumarin
compounds, two different alignment protocols on reference
While for the methoxyderivatives 1-4 no differences were
found between the two alignment protocols, for the more
flexible 5–7 the methoxyethyl functions showed large dif-
ferences in the spatial placement. This is also reflected in
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Fig. 2 Result of the superimposition between THBP (green carbon
stick) and compounds 1 (panels A and B) and 5 (panels C and D) with
Discovery Studio 3.1 suite. During superimposition, compounds 1 and
5 were considered as flexible, i.e. all the single bond but the amide were
left free to rotate (panel A for 1 and panel C for 5 respectively) or were
considered in the QM-fully minimized structures (panel B for 1 and
panel D for 5 respectively)
the predicted pIC50 values (see Table 2). Generally, the 3D-
QSAR model predicted all the methoxyethyl derivative as
more potent than the methoxy ones when a flexible alignment
protocol was used. In particular, compound 5 resulted as the
most promising one in the series. On the other hand, starting
from the minimized structures, the methoxyethyl derivatives
were predicted almost ineffective.
In order to obtain preliminary indications on the biological
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activity of the synthesized compounds on NF- B function,
electrophoretic mobility shift assays (EMSA) studies were
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performed. Purified p50 NF- B protein was employed and
the results obtained (see Fig. 3 for EMSA gels and Table 2
for IC50 and the relative pIC50 values) demonstrated that the
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methoxy derivatives 1–4 were able to inhibit NF- B/DNA
interactions, with compounds 1 and 2 being the most active
(i.e., the two derivatives more closely related to the parent
THBP). Their potency was lower than the other psoralen-
Fig. 3 Effects of compounds 1–7 on the molecular interactions
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based NF- B inhibitors studied by our group [27,42], while
32
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between NF- B p50 and P-labelled target NF- B double stranded
6 showed very weak inhibitory activity. As depicted in Fig. 3,
compounds 1–4 and 6 completely abrogate the formation
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oligonucleotide. Compounds were first incubated with NF- B and
32
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then the -P-labelled target NF- B oligonucleotide was added.
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of the NF- B/DNA complex at the highest tested con-
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NF- B/DNA complexes were analyzed by polyacrylamide gel elec-
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trophoresis. Arrows indicate NF- B/DNA complexes; asterisks indi-
centration (10 mM; see the disappearance of the retarded
radioactive bands indicated by the arrows). Compounds 5
and 7 displayed no inhibitory activity in these experimental
conditions.
The EMSA data, therefore, agreed with the activities trend
predicted by the 3D-QSAR model based on the alignment of
the QM-fully minimized structures (see predicted pIC50 val-
ues with model 2 in Table 2; r2 = 0.943). However, the
model over-estimated the pIC50 of the novel compounds.
Clearly, the presence of a larger conjugated system (the pso-
ralen moiety) was strictly required for the effective inhibition
cate the free 32-P-labelled target NF- B probe. a = 10 mM, b = 1 mM,
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c = 0.1 mM, d = 0.01 mM
the methoxy derivative adopts the same pose in the respect
to reference compound, the methoxyethyl derivative did not
fit in with a comparable pose (Fig. 4).
We believe that the flexibility of the methoxyethyl chain
could impair the needed binding to NF- B/DNA binding
region. Indeed, superimposing the conformation of com-
pound 5 (generated with the flexible protocol) on THBP
docked in NF- B, a clash with Gln274(B) was observed
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κ
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of NF- B/DNA interaction.
(Fig. 5).
In order to further rationalize the biological data, docking
experiments were carried out on compounds 1 and 5. While
Hence, it could be argued that the 7-O-methoxyethyl chain
had to be folded as in Fig. 2 in order to occupy the cor-
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of the parent NF- B/DNA interaction inhibitor THBP could
have been advantageous from a synthetic point of view,
allowing the synthesis of large libraries of novel compounds.
Unfortunately, the elimination of the furan ring yielded com-
pounds with lower potency with respect to THBP, thus
demonstrating the critical role of the furan ring in the bind-
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ing to NF- B. While the methoxy derivatives showed a
reduced inhibitory potency, more hindered ethers resulted
almost inactive. The negative role of methyl group in the
benzene ring was confirmed, as it caused a reduction in activ-
ity, while the length of the spacer portions in the 3 position
did not significantly influenced the activity. We can conclude
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that the furan ring is essential for inhibition of NF- B/DNA
interactions. These results should be taken in consideration in
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designing novel NF- B inhibitors based on coumarin scaf-
fold. At least at this stage of our SAR studies, the minimum
Fig. 4 Best poses obtained for compound 1 (yellow carbon stick) and
for compound 5 (light gray carbon stick). The pose of reference com-
pound (green carbon stick) is also reported for comparison purpose.
Lys272(A), Lys275(B) and Asp276(B) are depicted as stick and were
included in the picture to highlight the binding site
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requirementforcoumarincompoundstointeractwithNF- B
are: (i) an anilide moiety bearing a carboxylic acid function
at meta or para position; (ii) a spacer between the coumarin
core and the amide function of 1 or 2 carbon atoms length;
(iii) a conjugated system constituted by the psoralen core.
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The identified novel inhibitors of NF- B/DNA interaction
deserves in our opinion to be considered for other studies for
determining their activities as pro-apoptotic, anti-tumour and
anti-inflammatory agents in vitro and in vivo as described in
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the case of other NF- B inhibitors [28–38].
Experimental methods
Chemistry
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Fig. 5 Clash between 5 and Gln274(B) in the binding site of NF- B.
The minimized structure of 5 was superimposed to the docked pose
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-595-010M
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of reference compound and then visualized in contest with NF- B.
Lys272(A), Lys275(B) and Asp276(B) are depicted as stick and were
included in the picture to highlight the binding site; Gln274(B) and 5
are depicted as sphere. The clash between the protein and the compound
is indicated by the red arrow
1
melting point apparatus and are uncorrected. The H and
13C-NMR spectra were recorded on a Bruker 300-AMX
spectrometer with TMS as an internal standard. NMR sig-
nal abbreviations: s = singlet, d = doublet, dd = doublet of
doublets, t = triplet, q = quartet, m = multiplet, br s = broad
singlet. Coupling constants are given in Hz and the relative
peaks area were in agreement with all assignment. Elemental
analysis were performed on a Perkin-Elmer 2400 analyzer.
Mass spectra were performed on an Applied Biosystem
Mariner System 5220 with direct injection of the sample.
Microwave assisted reactions were performed on a CEM
rect binding zone, but this conformation suffered a penalty
energy of about 43 Kcal/mol, as measured through QM cal-
culations. This observation further suggests the importance
of considering a plausible bio-active conformation in 3D-
QSAR modeling. In fact, as observed by other authors [43],
the energy penalty between the fully minimized structure and
the bio-active conformation for a compound should be lower
than 5 or 9 Kcal/mol, depending on the number of rotatable
bonds.
ꢀ
Discover monomode reactor with the temperature moni-
Conclusions
toredbyabuilt-ininfraredsensorandtheautomaticcontrolof
the power; all the reactions were performed in closed devices
with pressure control. Starting methyl (7-hydroxy-2-oxo-
2H-benzopyran-3-yl)acetate (8), ethyl (7-hydroxy-2-oxo-
Since coumarin ethers are more readily accessible than pso-
ralen analogues, the simplification of the polycyclic skeleton
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2H-benzopyran-3-yl)propionate (9), methyl (7-hydroxy-8-
methyl-2-oxo-2H-benzopyran-3-yl)acetate (10), and ethyl
(7-hydroxy-8-methyl-2-oxo-2H-benzopyran-3-yl)propionate 2H, CH2CH2COOCH2CH3), 2.43 (s, 3H, 4-CH3), 2.30 (s,
CH2CH2COOCH2CH3), 3.91 (s, 3H, OCH3), 2.97 (t, J =
7.6 Hz, 2H, CH2CH2COOCH2CH3), 2.60 (t, J = 7.6 Hz,
(11) were prepared according to literature methods [39,40,
44].
3H, 8-CH3), 1.25 (t, J = 7.1 Hz, 3H, CH2CH2COOCH2CH3).
Anal. calcd. for C17H20O5: C, 67.09; H, 6.62; found: C,
67.10; H, 6.61.
General procedure for 7-O-methylethers 12–15
General procedure for 7-O-methoxyethylethers 16–18
A mixture of 8 or 9 or 10 or 11 (3.0 mmol), methyl iodide
(3.3 mmol), and K2CO3 (6.0 mmol) in acetone (10 mL) was
refluxed for 2 h. After cooling the solid was filtered off and
the reaction mixture was evaporated under reduced pressure
to give 12–15.
A mixture of 8 or 9 or 10 or 11 (3.0 mmol), 2-methoxyethyl
chloride (3.3 mmol), and K2CO3 (6.0 mmol) in acetone
(10 mL) was refluxed for 5 h. After cooling the solid was
filtered off and the reaction mixture was evaporated under
reduced pressure to give 16–18.
Methyl 2-(7-methoxy-4-methyl-2-oxo-2H-benzopyran-3-
yl)acetate (12)
Methyl 2-[7-(2ꢀ-methoxyethoxy)-4-methyl-2-oxo-2H-
benzopyran-3-yl]acetate (16)
Yield 94 %; pale yellow solid; mp: 121 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.54 (d, J = 8.8 Hz, 1H, 5-H), 6.87
(dd, J = 8.8, 2.5 Hz, 1H, 6-H), 6.82 (d, J = 2.5 Hz,
1H, 8-H), 3.87 (s, 3H, CH2COOCH3), 3.71 (s, 5H, OCH3
and CH2COOCH3), 2.39 (s, 3H, 4-CH3). Anal. calcd. for
C14H14O5: C, 64.12; H, 5.38; found: C, 64.13; H, 5.37.
Yield 44 %; pale yellow solid; mp: 103 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.54 (d, J = 8.8 Hz, 1H, 5-H),
6.91 (dd, J = 8.8, 2.5 Hz, 1H, 6-H), 6.84 (d, J
=
2.5 Hz, 1H, 8-H), 4.20–4.15 (m, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.80–3.76 (m, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.71 (s, 5H, CH2COOCH3 and CH2CO
OCH3), 3.46 (s, 3H, OCH3), 2.39 (s, 3H, 4-CH3). Anal.
calcd. for C16H18O6: C, 62.74; H, 5.92; found: C, 62.76; H,
5.93.
Ethyl 3-(7-methoxy-4-methyl-2-oxo-2H-benzopyran-3-
yl)propionate (13)
Yield90%;paleyellowsolid;mp:77 ◦C;1H NMR(300MHz,
CDCl3): δ 7.51 (d, J = 8.7 Hz, 1H, 5-H), 6.86 (dd, J = 8.7,
2.4 Hz, 1H, 6-H), 6.80 (d, J = 2.4 Hz, 1H, 8-H), 4.12
(q, J = 7.1 Hz, 2H, CH2CH2COOCH2CH3), 3.86 (s, 3H,
OCH3), 2.96 (t, J = 7.8 Hz, 2H, CH2CH2COOCH2CH3),
2.60 (t, J = 7.8 Hz, 2H, CH2CH2COOCH2CH3), 2.44 (s,
3H, 4-CH3), 1.25 (t, J = 7.1 Hz, 3H, CH2CH2COOCH2CH3).
Anal. calcd. for C16H18O5: C, 66.19; H, 6.25; found: C,
66.21; H, 6.26.
Ethyl 3-[7-(2ꢀ-methoxy)ethoxy-4-methyl-2-oxo-2H-
benzopyran-3-yl)propionate (17)
Yield 57 %; pale yellow solid; mp: 110 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.51 (d, J = 8.8 Hz, 1H, 5-H), 6.89
(dd, J = 8.8, 2.5 Hz, 1H, 6-H), 6.82 (d, J = 2.5 Hz,
1H, 8-H), 4.17 (t, J = 4.5 Hz, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 4.12 (q, J = 7.0 Hz, 2H, COOCH2CH3),
3.78 (t, J = 4.5 Hz, 2H, OCH2CH2OCH3 or OCH2CH2
OCH3), 3.46 (s, 3H, OCH3), 2.96 (t, J = 7.6 Hz, 2H,
CH2CH2COO), 2.60 (t, J = 7.6 Hz, 2H, CH2CH2COO),
2.43(s, 3H, 4-CH3), 1.23(t, J = 7.0 Hz, 3H, COOCH2CH3).
Anal. calcd. for C18H22O6: C, 64.66; H, 6.63; found: C,
64.66; H, 6.65.
Methyl 2-(4,8-dimethyl-7-methoxy-2-oxo-2H-benzopyran-
3-yl)acetate (14)
Yield 94 %; pale yellow solid; mp: 156 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.46 (d, J = 9.1 Hz, 1H, 5-H), 6.84
(d, J = 9.1 Hz, 1H, 6-H), 3.92 (s, 3H, OCH3), 3.73 (s, 2H,
CH2COOCH3), 3.71 (s, 3H, CH2COOCH3), 2.38 (s, 3H, 4-
CH3), 2.30 (s, 3H, 8-CH3). Anal. calcd. for C15H16O5: C,
65.21; H, 5.84; found: C, 65.21; H, 5.87.
Ethyl 3-[4,8-dimethyl-7-(2ꢀ-methoxy)ethoxy-4-methyl-2-
oxo-2H-benzopyran-3-yl)propionate (18)
Yield 39 %; pale yellow solid; mp: 121 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.41 (d, J = 8.6 Hz, 1H, 5-H), 6.83
(dd, J = 8.6, 2.5 Hz, 1H, 6-H), 4.20 (t, J = 4.5 Hz,
2H, OCH2CH2OCH3 or OCH2CH2OCH3), 4.08 (q, J =
7.2 Hz, 2H, COOCH2CH3), 3.80 (t, J = 4.5 Hz, 2H,
OCH2CH2OCH3 or OCH2CH2OCH3), 3.48 (s, 3H, OCH3),
2.97 (t, J = 7.5 Hz, 2H, CH2CH2COO), 2.60 (t, J = 7.5 Hz,
Ethyl 3-(4,8-dimethyl-7-methoxy-2-oxo-2H-benzopyran-3-
yl)propionate (15)
Yield 95 %; pale yellow solid; mp: 127 ◦C; 1H NMR
(300 MHz, CDCl3): δ 7.44 (d, J = 8.8 Hz, 1H, 5-H), 6.83
(d, J = 8.8 Hz, 1H, 6-H), 4.12 (q, J = 7.1 Hz, 2H,
123

Mol Divers
2H, CH2CH2COO), 2.43 (s, 3H, 4-CH3), 2.33 (s, 3H, 8-
CH3), 1.23 (t, J = 7.2 Hz, 3H, COOCH2CH3). Anal. calcd.
for C19H24O6: C, 65.50; H, 6.94; found: C, 65.48; H, 6.96.
Anal. calcd. for C15H16O5: C, 65.21; H, 5.84; found: C,
65.22; H, 5.85.
2-[7-(2ꢀ-Methoxyethoxy)-4-methyl-2-oxo-2H-benzopyran-
3-yl]acetic acid (23)
General procedure
for 7-alkoxy-2-oxo-2H-benzopyran-3-yl)acetic or propionic
acids 19–25
Yield68%;off-whitesolid;mp:155 ◦C;1H NMR(300MHz,
CDCl3): δ 7.56 (d, J = 8.8 Hz, 1H, 5-H), 6.94 (dd, J =
8.8, 2.4 Hz, 1H, 6-H), 6.86 (d, J = 2.4 Hz, 1H, 8-H),
4.19 (t, J = 4.4 Hz, 2H, OCH2CH2OCH3), 3.81–3.76
(m, 5H, OCH2CH2OCH3 and OCH2CH2OCH3), 3.47 (s,
2H, CH2COOH), 2.43 (s, 3H, 4-CH3). Anal. calcd. for
C15H16O6: C, 61.64; H, 5.52; found: C, 61.65; H, 5.50.
A mixture of 7-O-alkylethers (2.0 mmol) in propan-2-ol
(2.0 mL) and 2 M NaOH (2.0 mL) was microwave irradi-
ated at 120 ◦C (power set point 200 W; ramp time 2 min;
hold time 5 min). After cooling the reaction mixture was
poured into 1 M HCl (10 mL) and the resulting precipitate
was filtered off to give 19–25.
3-[7-(2ꢀ-Methoxyethoxy)-4-methyl-2-oxo-2H-benzopyran-
2-(7-Methoxy-4-methyl-2-oxo-2H-benzopyran-3-yl)acetic
acid (19)
3-yl]propionic acid (24)
Yield 71 %; off-white solid; mp: 204 ◦C; 1HNMR (300 MHz,
CDCl3): δ 7.56 (d, J = 8.8 Hz, 1H, 5-H), 6.89 (dd, J = 8.8,
2.5 Hz, 1H, 6-H), 6.84 (d, J = 2.5 Hz, 1H, 8-H), 3.88 (s, 3H,
OCH3), 3.76 (s, 2H, CH2COOH), 2.43 (s, 3H, 4-CH3). Anal.
calcd. for C13H12O5: C, 62.90; H, 4.87; found: C, 62.92; H,
4.88.
Yield55%;off-whitesolid;mp:150 ◦C;1H NMR(300MHz,
CDCl3): δ 7.52 (d, J = 8.7 Hz, 1H, 5-H), 6.91 (dd, J = 8.7,
2.5 Hz, 1H, 6-H), 6.83 (d, J = 2.5 Hz, 1H, 8-H), 4.17 (t, J =
4.4 Hz, 2H, OCH2CH2OCH3 or OCH2CH2OCH3), 3.78 (t,
J = 4.4 Hz, 2H, OCH2CH2OCH3 or OCH2CH2OCH3),
3.46(s, 3H, OCH3), 2.97(t, J = 7.3 Hz, 2H, CH2CH2COOH),
2.67 (t, J = 7.3 Hz, 2H, CH2CH2COOH), 2.44 (s, 3H, 4-
CH3). Anal. calcd. for C16H18O6: C, 62.74; H, 5.92; found:
C, 62.77; H, 5.91.
3-(7-Methoxy-4-methyl-2-oxo-2H-benzopyran-3-
yl)propionic acid (20)
Yield74%;off-whitesolid;mp:184 ◦C;1H NMR(300MHz,
CDCl3): δ 7.52 (d, J = 8.7 Hz, 1H, 5-H), 6.87 (dd, J =
8.7, 2.5 Hz, 1H, 6-H), 6.81 (d, J = 2.5 Hz, 1H, 8-H), 3.87
(s, 3H, OCH3), 2.98 (t, J = 7.5 Hz, 2H, CH2CH2COOH),
2.68 (t, J = 7.5 Hz, 2H, CH2CH2COOH), 2.44 (s, 3H, 4-
CH3). Anal. calcd. for C14H14O5: C, 64.12; H, 5.38; found:
C, 64.15; H, 5.38.
3-[4,8-Dimethyl-7-(2ꢀ-methoxyethoxy)-2-oxo-2H-
benzopyran-3-yl]propionic acid (25)
Yield88%;off-whitesolid;mp:194 ◦C;1H NMR(300MHz,
CDCl3): δ 7.40 (d, J = 8.9 Hz, 1H, 5-H), 6.81 (d, J =
8.9 Hz, 1H, 6-H), 4.16 (t, J = 4.7 Hz, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.77(t, J = 4.7 Hz, 2H, OCH2CH2OCH3
or OCH2CH2OCH3), 3.44 (s, 3H, OCH3), 2.91 (t, J =
7.9 Hz, 2H, CH2CH2COOH), 2.53 (t, J = 7.9 Hz, 2H,
CH2CH2COOH), 2.39 (s, 3H, 4-CH3), 2.27 (s, 3H, 8-CH3).
Anal. calcd. for C16H18O6: C, 63.74; H, 6.29; found: C,
63.76; H, 6.32.
2-(4,8-Dimethyl-7-methoxy-2-oxo-2H-benzopyran-3-
yl)acetic acid (21)
Yield76%;off-whitesolid;mp:176 ◦C;1H NMR(300MHz,
CDCl3): δ 7.49 (d, J
=
8.8 Hz, 1H, 5-H), 6.88 (d,
J = 8.8 Hz, 1H, 6-H), 3.93 (s, 3H, OCH3), 3.77 (s, 2H,
CH2COOH), 2.45 (s, 3H, 4-CH3), 2.31 (s, 3H, 8-CH3). Anal.
calcd. for C14H14O5: C, 64.12; H, 5.38; found: C, 64.14; H,
5.39.
General procedure for amides 1–7
A solution of acids 18–25 (1.0 mmol) in thionyl chloride
(25 mL) was stirred at room temperature until a clear solu-
tion was formed. The solvent was evaporated under nitrogen
stream at room temperature and the residue was dissolved in
dry tetrahydrofuran (10 mL). The solution was added drop-
wise to a mixture of 3-aminobenzoic acid or 4-aminobenzoic
acid (2.0 mmol) in triethylamine (0.3mL, 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=3. The resulting precipitate was filtered off and crystal-
lized from propan-2-ol to give 1–7.
3-(4,8-Dimethyl-7-methoxy-2-oxo-2H-benzopyran-3-
yl)propionic acid (22)
Yield77%;off-whitesolid;mp:131 ◦C;1H NMR(300MHz,
CDCl3): δ 7.44 (d, J = 8.8 Hz, 1H, 5-H), 6.84 (d, J =
8.8 Hz, 1H, 6-H), 3.91 (s, 3H, OCH3), 2.98 (t, J
=
7.2 Hz, 2H, CH2CH2COOH), 2.66 (t, J = 7.2 Hz, 2H,
CH2CH2COOH), 2.44 (s, 3H, 4-CH3), 2.30 (s, 3H, 8-CH3).
123

Mol Divers
4-[2ꢀ-(7ꢀꢀ-Methoxy-4ꢀꢀ-methyl-2ꢀꢀ-oxo-2H-benzopyran-3ꢀꢀ-
yl)acetylamino]benzoic acid (1)
3-[3ꢀ-(4ꢀꢀ,8ꢀꢀ-Dimethyl-7ꢀꢀ-methoxy-2ꢀꢀ-oxo-2H-benzopyran-
3ꢀꢀ-yl)propionylamino]benzoic acid (4)
From 22 and 3-aminobenzoic acid: yield 42 %; pale yel-
From 19 and 4-aminobenzoic acid: yield 37 %; pale yel-
low solid; mp: > 300 ◦C; 1H NMR (300 MHz, DMSO-d6):
1
low solid; mp: 296 ◦C; H NMR (300 MHz, DMSO-d6):
δ 10.11 (br s, 1H, CONH), 8.18 (s, 1H, 2-H), 7.78 (d,
J = 7.6 Hz, 2H, 6-H), 7.63 (d, J = 8.4 Hz, 1H, 5ꢀꢀ-H),
7.59 (d, J = 7.6 Hz, 1H, 4-H), 7.39 (t,J = 7.6 Hz, 1H, 5-
H), 7.03 (d, J = 8.4 Hz, 1H, 6ꢀꢀ-H), 3.89 (s, 3H, OCH3),
2.89 (t, J = 7.1 Hz, 2H, 2ꢀ-H or 3ꢀ-H), 2.50 (t, J = 7.1 Hz,
2H, 2ꢀ-H or 3ꢀ-H), 2.40 (s, 3H, 4ꢀꢀ-CH3), 2.20 (s, 3H, 8ꢀꢀ-
CH3). 13C NMR (75 MHz, DMSO-d6): δ 171.09, 167.63,
161.31, 159.48, 150.93, 148.25, 139.86, 131.68, 129.39,
124.34, 124.00, 123.63, 121.28, 120.29, 114.17, 112.24,
107.70, 56.51, 35.56, 23.78, 12.20, 8.39. Anal. calcd. for
C22H21NO6: C, 66.83; H, 5.35; N, 3.54; found: C, 66.82; H,
5.38; N, 3.56. HRMS (ESI-TOF) for C22H20NO6 [M−H]−:
calcd.: 394.1291, found: 394.1309.
10.45 (br s, 1H, CONH), 7.88 (d, J = 7.9 Hz, 2H, 2-H
δ
and 6-H), 7.80–7.66 (m, 3H, 5ꢀꢀ-H, 6ꢀꢀ-H and 8ꢀꢀ-H), 6.99
(d, J = 7.9 Hz, 2H, 3-H and 5-H), 3.87 (s, 3H, OCH3),
3.76 (s, 2H, 2ꢀ-H), 2.42 (s, 3H, 4ꢀꢀ-CH3). 13C NMR (75
MHz, DMSO-d6): δ 168.35, 166.86, 161.77, 161.06, 153.36,
149.69, 143.17, 130.35, 126.62, 124.97, 118.24, 116.77,
113.43, 112.16, 100.45, 55.83, 34.90, 15.25. Anal. calcd. for
C20H17NO6: C, 65.39; H, 4.66; N, 3.81; found: C, 65.41; H,
4.68; N, 3.80. HRMS (ESI-TOF) for C20H16NO6 [M−H]−:
calcd.: 366.0978, found: 366.1016.
4-[3ꢀ-(7ꢀꢀ-Methoxy-4ꢀꢀ-methyl-2ꢀꢀ-oxo-2H-benzopyran-3ꢀꢀ-
yl)propionylamino]benzoic acid (2)
4-{2ꢀ-[7ꢀꢀ-(2-Methoxyethoxy)-4ꢀꢀ-methyl-2ꢀꢀ-oxo-2H-
benzopyran-3ꢀꢀ-yl]acetylamino}benzoic acid (5)
From 20 and 4-aminobenzoic acid: yield 18 %; pale yel-
low solid; mp: > 300 ◦C; 1H NMR (300 MHz, DMSO-d6):
delta 10.33 (br s, 1H, CONH), 7.86 (d, J = 8.4 Hz,
2H, 2-H and 6-H), 7.72–7.63 (m, 3H, 5ꢀꢀ-H, 6ꢀꢀ-H and 8ꢀꢀ-
H), 6.98–6.91 (m, 2H, 3-H and 5-H), 3.85 (s, 3H, OCH3),
2.88 (t, J = 7.1 Hz, 2H, 2ꢀ-H or 3ꢀ-H), 2.56 (t, J =
7.1 Hz, 2H, 2ꢀ-H or 3ꢀ-H), 2.41 (s, 3H, 4ꢀꢀ-CH3). 13C NMR
(75 MHz, DMSO-d6): δ 170.81, 166.84, 161.46, 160.74,
153.17, 147.65, 143.13, 130.29, 126.42, 124.86, 120.93,
118.23, 113.47, 111.96, 100.33, 55.76, 34.99, 23.12, 14.67.
Anal. calcd. for C21H19NO6: C, 66.13; H, 5.02; N, 3.67;
found: C, 66.14; H, 5.00; N, 3.70. HRMS (ESI-TOF) for
C21H18NO6 [M − H]−: calcd.: 380.1134, found: 380.1185.
From 23 and 4-aminobenzoic acid: yield 73 %; pale yel-
low solid; mp: > 300 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ 10.39 (br s, 1H, CONH), 7.85 (d, J = 8.6 Hz, 2H, 2-H and
6-H), 7.76 (d, J = 9.1 Hz, 1H, 5ꢀꢀ-H), 7.65 (d, J = 8.6 Hz,
2H, 3-H and 5-H), 7.02–6.97 (m, 2H, 6ꢀꢀ-H and 8ꢀꢀ-H),
4.24–4.20 (m, 2H, OCH2CH2OCH3 or OCH2CH2OCH3),
3.76 (s, 3H, OCH3), 3.71–3.67 (m, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.33 (s, 2H, 2ꢀ-H), 2.41 (s, 3H, 4ꢀꢀ-CH3).
13C NMR (75 MHz, DMSO-d6): δ 168.50, 166.83, 161.03,
160.94, 153.27, 149.65, 143.14, 130.33, 126.61, 124.92,
118.21, 116.78, 113.45, 112.46, 100.94, 70.06, 67.57, 58.11,
34.89, 15.22. Anal. calcd. for C22H21NO7: C, 64.23; H, 5.14;
N, 3.40; found: C, 64.24; H, 5.17; N, 3.40. HRMS (ESI-
TOF) for C22H20NO7 [M − H]−: calcd.: 410.1240, found:
410.1268.
3-[2ꢀ-(4ꢀꢀ,8ꢀꢀ-Dimethyl-7ꢀꢀ-methoxy-2ꢀꢀ-oxo-2H-benzopyran-
3ꢀꢀ-yl)acetylamino]benzoic acid (3)
4-{3ꢀ-[7ꢀꢀ-(2-Methoxyethoxy)-4ꢀꢀ-methyl-2ꢀꢀ-oxo-2H-
benzopyran-3ꢀꢀ-yl]propionylamino}-benzoic acid (6)
From 21 and 3-aminobenzoic acid: yield 93 %; pale yel-
low solid; mp: > 300 ◦C; 1H NMR (300 MHz, DMSO-d6):
δ 10.30 (br s, 1H, CONH), 8.21 (s, 1H, 2-H), 7.79 (d, J
= 7.7 Hz, 1H, 6-H), 7.70 (d, J = 8.7 Hz, 1H, 5ꢀꢀ-H), 7.60
(d, J = 7.7 Hz, 1H, 4-H), 7.40 (t, J= 7.6 Hz, 1H, 5-H),
7.08 (d, J = 8.7 Hz, 1H, 6ꢀꢀ-H), 3.92 (s, 3H, OCH3), 3.75
(s, 2H, 2ꢀ-H), 2.42 (s, 3H, 4ꢀꢀ-CH3), 2.21 (s, 3H, 8ꢀꢀ-CH3).
13C NMR (75 MHz, DMSO-d6): δ 168.81, 167.62, 161.63,
159.73, 151.06, 150.24, 139.88, 131.71, 129.44, 124.41,
124.15, 123.62, 120.34, 117.11, 114.13, 112.36, 107.79,
56.53, 35.33, 15.75, 8.38. Anal. calcd. for C21H19NO6:
C, 66.13; H, 5.02; N, 3.67; found: C, 66.15; H, 5.03; N,
3.69. HRMS (ESI-TOF) for C21H18NO6 [M − H]−: calcd.:
380.1134, found: 380.1197.
From 24 and 4-aminobenzoic acid: yield 62 %; pale yellow
solid; mp: 248 ◦C; 1H NMR (300 MHz, DMSO-d6): δ 10.22
(br s, 1H, CONH), 7.85 (d, J = 8.7 Hz, 2H, 2-H and 6-
H), 7.71–7.62 (m, 3H, 5ꢀꢀ-H, 6ꢀꢀ-H and 8ꢀꢀ-H), 6.98–6.92 (m,
2H, 3-H and 5-H), 4.21–4.17 (m, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.70–3.65 (m, 2H, OCH2CH2OCH3 or
OCH2CH2OCH3), 3.33 (s, 3H, OCH3), 2.88 (t, J = 7.4,
2H, 2ꢀ-H or 3ꢀ-H), 2.56 (t, J = 7.4, 2H, 2ꢀ-H or 3ꢀ-H), 2.40 (s,
3H, 4ꢀꢀ-CH3). 13C NMR (75 MHz, DMSO-d6): δ 170.81,
166.83, 160.74, 160.65, 153.11, 147.63, 143.13, 130.28,
126.45, 124.86, 120.97, 118.22, 113.52, 112.28, 100.84,
10.06, 67.52, 58.10, 35.00, 23.11, 14.65. Anal. calcd. for
123

Mol Divers
C23H23NO7: C, 64.93; H, 5.45; N, 3.29; found: C, 64.94; H,
5.47; N, 3.30. HRMS (ESI-TOF) for C23H22NO7 [M−H]−:
calcd.: 424.1396, found: 424.1415.
Computational methodologies
All the computational studies were carried out on a 4 CPU
(Intel Core2 Quad CPU Q9550, 2.83 GHz) ACPI ×64 Linux
workstation with Ubuntu 12.04 64 bit operating system. The
4-{3ꢀ-[4ꢀꢀ,8ꢀꢀ-Dimethyl-7ꢀꢀ-(2-methoxyethoxy)-2ꢀꢀ-oxo-2H-
benzopyran-3ꢀꢀ-yl]propionylamino}-benzoic acid (7)
κ
NF- B/DNA complex was downloaded from Protein Data
Bank (PDB code 1NFK) and modified with Chimera 5.3.1
Software [47]. The binding pose of the reference compound
(THBP) was derived from our previously published results
[27]. All the QM studies have been performed with Firefly
8.0 software [48] (which is partially based on the GAMESS-
US source code) at the B3LYP level with 6-31+G(d,p) basis
set [49]. All the input files for Firefly have been prepared with
the graphical interface of Avogadro 1.1.0 software [50]. The
molecular structure have been prepared with MarvinSketch
5.5 software [51]. The lowest energy conformations and the
degree of protonation at pH 7.4 were determined with Open-
Babel 2.2.3 software [52], using the MMFF94s force field.
Thedockingstudies were performedwithAutoDock4.2soft-
ware with the AutoDock Tools 1.5.4 graphical interface [53].
For compound 1 and 5, the Gasteiger charges were com-
puted and the nonpolar hydrogen atom were merged with
ADT, thus preparing the appropriate PDBQT files for the
molecular docking studies. The superimpositions of mole-
cules have been done using the Discovery Studio 3.1 suite
[54]. All the 3D-QSAR studies have been performed using
Open3DQSAR 2.02 software [55].
From 25 and 4-aminobenzoic acid: yield 18 %; pale yel-
1
low solid; mp: 292 ◦C; H NMR (300 MHz, DMSO-d6):
δ 10.14 (br s, 1H, CONH), 7.81 (d, J = 8.4 Hz, 2H, 2-
H and 6-H), 7.63–7.56 (m, 3H, 3-H, 5-H and 5ꢀꢀ-H), 7.04
(d, J = 9.0 Hz, 1H, 6ꢀꢀ-H), 4.22 (t, J = 4.4 Hz, 2H,
OCH2CH2OCH3 or OCH2CH2OCH3), 3.73–3.68 (m, 2H,
OCH2CH2OCH3 or OCH2CH2OCH3), 3.33 (s, 3H, OCH3),
2.88–2.85 (m, 2H, OCH2CH2OCH3 or OCH2CH2OCH3),
2.59–2.54 (m, 2H, 2ꢀ-H or 3ꢀ-H), 2.59–2.54 (m, 2H, 2ꢀ-H or
3ꢀ-H), 2.40 (s, 3H, 4ꢀꢀ-CH3), 2.20 (s, 3H, 8ꢀꢀ-CH3). 13C NMR
(75 MHz, DMSO-d6): δ 170.84, 166.82, 160.77, 158.26,
147.76, 143.12, 130.28, 130.18, 124.86, 123.46, 120.77,
128.22, 113.72, 112.10, 108.31, 70.30, 67.99, 58.30, 35.05,
23.15, 14.68, 7.96. Anal. calcd. for C24H25NO7: C, 65.59; H,
5.73; N, 3.19; found: C, 65.61; H, 5.75; N, 3.21. HRMS (ESI-
TOF) for C24H24NO7 [M − H]−: calcd.: 438.1553, found:
438.1575.
Biology
Electrophoretic mobility shift assay (EMSA)
EMSA was performed as previously described [45]. Briefly,
3D-QSAR modeling
double-stranded synthetic oligodeoxynucleotides mimick-
The alignment of the compounds were performed with the
Discovery Studio 3.1 suite, using the “field fit” algorithm
(contributions: 80 % steric field; 20 % electronic). Two types
of alignment were performed: in the first one, the structure
of the novel derivatives were superimposed on the previosly
reported binding pose of THBP [27], considering all the
bonds as rotatable (increments allowed: 10◦) except for the
amide ones. The second alignment was obtained superimpos-
ing the QM-fully minimized structure of the novel derivatives
on the previously reported binding pose of THBP, consid-
ering all the bonds as unrotatable. In both the case, the
structure of THBP was considered as rigid. The two aligned
datasets were used for inputted in our previously reported
3D-QSAR models, thus outputting the predicted pIC50 val-
ues (see Table 2).
ꢀ
κ
κ
ing the NF- B binding (NF- B, sense: 5 -CGC TGG
GGA CTT TCC ACG G-3ꢀ) were employed. Oligodeoxynu-
cleotides were labeled with
γ32
P-ATP using 10 Units of
T4-polynucleotide-kinase (MBI Fermentas) in 500 mM
Tris-HCl, pH 7.6, 100 mM MgCl2, 50 mM DTT, 1 mM sper-
γ32
midine, 1mMEDTAinthepresenceof50 mCi
P-ATP)in
a volume of 20 μL for 45 min at 37 ◦C. Reaction was brought
to 150 mM NaCl and 150 ng complementary oligodeoxynu-
cleotide 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 else-
where in a total volume of 20 μL containing buffer TF plus
κ
5 % glycerol, 1 mM dithiothreitol, 10 ng of human NF- B
κ
p50 protein with or without 10 ng of NF- B p65 protein
(Promega) and different concentrations of compounds [46].
After a incubation of 20 min at room temperature, 0.25 ng
of 32P-labeled oligonucleotides 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 (0.25× TBE buffer: 22 mM Tris borate,
0.4 mM EDTA) on 6 % polyacrylamide gels. Gels were dried
and subjected to standard autoradiographic procedures.
Molecular docking
κ
The structure of NF- B in complex with the DNA has been
importedinChimera, andthewatermoleculesandthenucleic
acid were removed. The so handled protein was imported
in AutoDock Tools. The hydrogen atoms were added and
the Gasteiger charges were computed. After the addition
123

Mol Divers
of the AutoDock atom types, the nonpolar hydrogen atoms
were merged and the protein was saved in the appropriate
PDBQT file format required by AutoGrid and AutoDock
modules. The grid box for docking simulations (centered at:
x = −11.11, y = 18.23, z = 15.33) was characterized by
0.375 Å spacing. For both compounds 1 and 5, 10 runs with a
maximum of 2,500,000 energy evaluations were carried out,
using Lamarkian genetic algorithm searching engine. Cluster
analysis was performed on docked results, with a root-mean-
square tolerance of 2.0 Å.
Quantum Mechanical calculations. The fully minimized
structure of compounds 1–7 were determined with the Firefly
QC package (which is partially based on the GAMESS-US
source code), setting the parameters as follows: $basis gbasis
=N31 ngasuss = 6 ndfunc = 1; npfunc = 1; dffsp = .true.; $pcm
solvnt =water; $contrl scftyp = rohf; runtyp = energy; dfttyp
= b3lyp; icharge = −1; mult = 1. The QM based energy of
compound 5 after the flexible alignment on THFC has been
computed using the same parameters as above, with the only
exception of runtyp, which was set to energy.
9. Tan G, Niu J, Shi Y, Ouyang H, Wu ZH (2012) NF-kappaB-
dependent microRNA-125b up-regulation promotes cell survival
by targeting p38alpha upon ultraviolet radiation. J Biol Chem
287:33036–33047. doi:10.1074/jbc.M112.383273
10. Rathore MG, Saumet A, Rossi JF, de Bettignies C, Tempe D, Lecel-
lier CH, Villalba M (2012) The NF-kappaB member p65 controls
glutamine metabolism through miR-23a. Int J Biochem Cell Biol
44:1448–1456. doi:10.1016/j.biocel.2012.05.011
11. Galardi S, Mercatelli N, Farace MG, Ciafre SA (2011) NF-κB and
c-Jun induce the expression of the oncogenic miR-221 and miR-
222 in prostate carcinoma and glioblastoma cells. Nucleic Acids
Res 39:3892–3902. doi:10.1093/nar/gkr006
12. Gambari R, Borgatti M, Lampronti I, Fabbri E, Brognara E, Bianchi
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