Design, synthesis and biological evaluation of novel trimethylangelicin analogues targeting nuclear factor kB (NF-kB)

Article abstract of DOI:10.1016/j.ejmech.2018.03.080

A series of trimethylangelicin (TMA) derivatives were designed and synthesized to overcome the unwanted effects of TMA, promising agent for treatment of inflammation-related diseases and other pathologies, such as cystic fibrosis. The new generation TMA analogues bore hindered substituents at the 4 position in order to minimize or avoid the photoreactions with DNA. Among them, the 4-isopropyl-6-ethyl derivative 23 exhibited TMA-like inhibitory activity on NF-κB/DNA interactions but it proved unable to photoreact with pyrimidine bases of DNA, nor to induce any other DNA damage. The isopropyl analogue 23 was proven to lack mutagenicity when assayed through Ames test and exhibited no anti-proliferative activity on cystic fibrosis IB3-1 cells, displaying at the same time inhibition of the TNF-α induced release of the NF-κB regulated PDGF-B chain, IL-10, IL-15, IL-17 and IFN-γ. Therefore compound 23 deserves further assay to determine its anti-inflammatory properties, since it lacks photoreaction properties and mutagenicity-related side effects.

Full text of DOI:10.1016/j.ejmech.2018.03.080

Accepted Manuscript
Design, synthesis and biological evaluation of novel trimethylangelicin analogues
targeting nuclear factor kB (NF-kB)
Giovanni Marzaro, Ilaria Lampronti, Elisabetta D'Aversa, Gianni Sacchetti, Giorgia
Miolo, Christian Vaccarin, Giulio Cabrini, Maria Cristina Dechecchi, Roberto Gambari,
Adriana Chilin
PII:
S0223-5234(18)30323-4
10.1016/j.ejmech.2018.03.080
EJMECH 10345
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 January 2018
Revised Date: 12 March 2018
Accepted Date: 29 March 2018
Please cite this article as: G. Marzaro, I. Lampronti, E. D'Aversa, G. Sacchetti, G. Miolo, C. Vaccarin,
G. Cabrini, M.C. Dechecchi, R. Gambari, A. Chilin, Design, synthesis and biological evaluation of
novel trimethylangelicin analogues targeting nuclear factor kB (NF-kB), European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.080.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel
Trimethylangelicin Analogues Targeting
Nuclear Factor kB (NF-kB)
Giovanni Marzaroa,1, Ilaria Lamprontib,1, Elisabetta D’Aversab, Gianni Sacchettib, Giorgia Mioloa,
Christian Vaccarina, Giulio Cabrinic, Maria Cristina Dechecchic, Roberto Gambarib, Adriana
Chilin*,a
aDepartment of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, I-
35131 Padova, Italy
bDepartment of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, I-44121
Ferrara, Italy
cLaboratory of Molecular Pathology, Department of Pathology and Diagnostics, University Hospital of
Verona, I-37126 Verona, Italy
1First two authors contributed equally to this work and should be considered co-first authors
*Corresponding author. E-mail address: adriana.chilin@unipd.it
Keywords: furocoumarin, angelicin, synthesis, NF-κB
Abstract
A series of trimethylangelicin (TMA) derivatives were designed and synthesized to overcome the
unwanted effects of TMA, promising agent for treatment of inflammation-related diseases and other
pathologies, such as cystic fibrosis. The new generation TMA analogues bore hindered substituents
at the 4 position in order to minimize or avoid the photoreactions with DNA. Among them, the 4-
isopropyl-6-ethyl derivative 23 exhibited TMA-like inhibitory activity on NF-κB/DNA interactions
but it proved unable to photoreact with pyrimidine bases of DNA, nor to induce any other DNA
damage. The isopropyl analogue 23 was proven to lack mutagenicity when assayed through Ames
test and exhibited no anti-proliferative activity on cystic fibrosis IB3-1 cells, displaying at the same
time inhibition of the TNF-α induced release of the NF-κB regulated PDGF-B chain, IL-10, IL-15,
IL-17 and IFN-γ. Therefore compound 23 deserves further assay to determine its anti-inflammatory
properties, since it lacks photoreaction properties and mutagenicity-related side effects.
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INTRODUCTION
Nuclear Factor κB (nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB) is a key
transcription factor involved in several biological processes (such as inflammation and apoptosis)
[1,2]. NF-κB is activated by several intra-cellular as well as extra-cellular stimuli [3]. Examples are
bacterial or viral products [4], cytokines [5], oxidant-free radicals [6], ultraviolet irradiation [7].
After a complex, but well characterized process of activation [8] NF-κB translocates to the nucleus,
where induces the transcription of target genes [3,9]. Inappropriate NF-κB activation/regulation
causes inflammatory diseases [10], supporting the concept that NF-κB should be considered a target
for therapeutic interventions [11]. In particular, NF-κB is a key transcription factor regulating a set
of pro-inflammatory genes involved in the inflammatory status of patients affected by cystic
fibrosis (CF) [12-14], a life-limiting autosomal recessive disorder caused by mutations in the gene
for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride-conducting
transmembrane channel that regulates anion transport and mucociliary clearance in the airways
[15,16]. In these last years small molecule and gene-based therapies have been developed targeting
the CFTR defect, in order to modify the disease and to improve the quality of life for CF patients
[17,18].
With respect to the issues of modulating CFTR function and controlling inflammatory status of CF,
TMA (4,6,4’-trimethylangelicin) (Fig. 1) is a small-molecule exhibiting both corrector and
potentiator activities of CFTR [19]. TMA displays also anti-inflammatory properties by targeting
NF-κB and reducing IL-8 expression [20], therefore demonstrating to be a promising agent for
treatment of cystic fibrosis. Unfortunately, TMA was also shown to have potential drawbacks, such
as phototoxicity and mutagenicity [21,22].
Figure 1. Structure of 4,6,4’-trimethylangelicin (TMA).
Recently we found that other TMA analogues/isosters shared the same anti-inflammatory properties
[23]: indeed, they inhibited NF-κB/DNA interaction and they reduced NF-κB-mediated IL-8 gene
expression in human bronchial epithelial CF IB3-1 cells, demonstrating again their potential interest
for a possible development in the anti-inflammatory therapy. Due to our interest in this field [24-
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26], we went on modifying the TMA scaffold in order to identify new compounds with comparable
biological properties in the respect of TMA but with reduced or abolished side effects.
RESULT AND DISCUSSION
Chemistry
Bearing in mind that the photoreaction of TMA with DNA is a photocycloaddition between a
double bond of furocoumarin nucleus (lactone or furan double bond) and thymine (or cytosine)
double bond [27], in order to minimize or avoid the covalent photoreactions with DNA we designed
new analogues with substituents at the 4 position much more hindered than the classic methyl
group. In the 6 position of furocoumarin backbone a methyl or an ethyl group were inserted, while
the furan ring was not modified in the respect of the parent TMA, as the previous studies indicated
that changes in this moiety were detrimental for activity [23].
The new TMA analogues were synthesized according to Scheme 1, adopting a well known
synthetic strategy [28,29]. The starting 7-hydroxybenzopyran-2-ones (3-9) were synthesized
condensing 4-methylresorcinol (1) or 4-ethylresorcinol (2) with the appropriate acetoacetic ester
derivatives. Compounds 3-9 were functionalized with chloroacetone obtaining the O-α-ketoethers
10-16. The intermediates ethers were finally cyclized in anhydrous alkaline medium affording the
desired angelicins 17-23.
Scheme 1. Synthesis of TMA analogues. Reaction conditions: a. Ethyl acetoacetate (for 3) or ethyl propionylacetate
(for 4,5) or ethyl 3-oxohexanoate (for 6,7) or ethyl isobutyrylacetate (for 8,9), H2SO4, 1 h, 40-92%; b. Chloroacetone,
K2CO3, reflux, 6 h, 74-96%; c. KOH, EtOH abs., reflux, 1 h, 17-47%. See Table 1 for R specification.
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Table 1. R substituent for TMA analogues.
Compounds
1
2
R
Me
Et
R’
-
-
Et
Me
Et
Me
Et
Me
Et
Me
Et
Et
Pr
Pr
iPr
iPr
3, 10, 17
4, 11, 18
5, 12, 19
6, 13, 20
7, 14, 21
8, 15, 22
9, 16, 23
Biology
The angelicin derivatives were then tested for their inhibitory activity on NF-κB/DNA interactions.
Moreover, since NF-κB is implicated in cell proliferation and survival [1-3], the effects of TMA
analogues were tested also on the proliferation of cystic fibrosis IB3-1 cells [30]. The effects of
TMA derivatives on NF-κB/DNA interactions were verified by electrophoretic mobility shift assay
32
(EMSA), using purified NF-κB p50 and P-labeled target NF-κB double-stranded oligonucleotide
mimicking the NF-κB consensus sequence.
We first demonstrated that, as elsewhere published, TMA was able to inhibit the interactions
32
between NF-κB p50 and a P-labeled target NF-κB double-stranded oligonucleotide (Figure S1 in
Supplementary Data). Second, the TMA analogues 17-23 were comparatively assayed to verify
their ability to inhibit NF-κB/DNA interactions.
Figure 2. Representative EMSA experiment results depicting the effects of TMA analogues (17-23) at 12.5-100 µM
concentrations on the molecular interactions between NF-κB p50 and 32P-labeled target NF-κB double-stranded
oligonucleotide. Arrows indicate NF-κB/DNA complexes and the free 32P-labeled target NF-κB probe.
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As shown in the representative example reported in Figure 2, all the TMA analogues exhibited
inhibitory activity on NF-κB/DNA interactions, but compounds 18 and 23 were found to be the
most effective, clearly showing a strong inhibitory effects when added at 12.5 µM.
A summary of EMSA data is reported in Table 2, together with the effects of TMA analogues on
IB3-1 cell growth. For the analysis of the effects on cell proliferation, monolayers of 60% confluent
IB3-1 cells (derived from a CF patient with a ∆F-508/W1282X mutant genotype) were seeded in
12-well plate, treated with different compound concentrations and the cell number/mL determined
after 48 h of culture, obtaining the IC50 values reported in Table 2.
The results show that compounds 18 and 23 efficiently inhibit NF-κB/DNA interactions displaying
low IC50 values (8.0 µM and 7.4 µM, respectively). However, while compound 18 exhibited some
anti-proliferative activity on IB3-1 cells (IC50 = 50 µM), compound 23 was unable to inhibit IB3-1
cell growth even when added at 200 µM, thus proving to be an interesting NF-κB inhibitor without
cytotoxic effects.
Table 2. Effects of the TMA analogues on inhibition of NF-κB/DNA complexes and proliferation
of cystic fibrosis IB3-1 cells.
Compounds Inhibition of NF-κB/DNA complex (IC50)
IB3-1 (IC50)
>200 µM
50 µM
24.0 µM
8.0 µM
17
18
19
20
21
22
23
17.8 µM
21.9 µM
19.8 µM
18.9 µM
7.4 µM
>200 µM
200 µM
162.5 µM
>200 µM
>200 µM
Inhibition of NF-κB/DNA interaction and cell growth are expressed as IC50 (concentration leading to 50% inhibition of
NF-κB/DNA interaction and IB3-1 cell growth).
In this respect, we further demonstrated that 7.4 µM concentration of compound 23 was unable to
exert cytotoxic effects on cystic fibrosis IB3-1 cells (Table 2) as well as in other in vitro cultured
cell lines, such as K562 leukemia and U251 glioma cell lines (unpublished results). In conclusion
on the basis of the effects on NF-kB/DNA interactions and on cell proliferation of IB3-1 cells
(Table 2), compound 23 resulted the most interesting one. Therefore, we investigated its
photochemical behavior in order to verify if the photosensitizing properties were abolished
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introducing a bulky substituent in the 4 position. First of all the ability of compound 23 to form an
intercalative molecular complex with DNA in the ground state has been investigated by linear flow
dichroism (LD). Differently from TMA, that was efficiently complexed inside the macromolecule,
compound 23 was unable to intercalate inside the double helix, even at the highest sensitivity (data
reported in Supporting Information).
To exclude any further interaction with DNA, compound 23 was also evaluated for its ability to
undergo covalent photocycloaddition with bases of DNA in the absence and in the presence of
UVA light, taking TMA as reference compound. The photobinding properties of the compounds
were tested in the presence of salmon testes DNA with and without UVA light irradiation.
The irradiated solutions were opportunely treated and analyzed to detect the formation of
furocoumarin-pyrimidine bases photocycloadducts, which were then isolated (TLC) and analyzed
(NMR). Differently from TMA, able to form cycloadducts with thymine characterized by a strong
fluorescence (data not shown), with compound 23 we did not detect any significant band on the
TLC plate and no band had the capacity to photoreverse under 254 nm light to the initial compound
and thymine, typical of photocycloadducts between furocoumarins and DNA [31].
The presence of induced DNA damage in pBR322 plasmid DNA was studied by agarose gel
electrophoresis. Open circular DNA, OC, is formed when single strand breaks are present at one
strand. Linearization of the plasmid DNA (L) indicates that double strand breaks are induced by the
damaging compounds. These forms of DNA have different macromolecular structures and can be
distinguished as they behave differently in agarose gel electrophoresis.
Figure 3. pBR322 DNA in the presence of TMA and 23 (100 µM) in the dark (0 J/cm2) and irradiated with increasing
doses of UVA light. Dark (DNA SC supercoiled), UVA irradiated DNA (at 7.5 J/cm2, DNA UVA) and linear DNA
(DNA L) samples were taken as controls. OC = open circular, L = linear, SC = supercoiled.
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In Figure 3, as regard TMA samples, the increasing amount of single strand breaks (see the
increasing darkening of the OC bands by increasing the radiant exposures) indicates a larger
number of TMA induced lesions able to cut the DNA at one strand. At the same time, the SC DNA
bands become lighter and the photo-addition products formed slow down SC DNA migration as
irradiation augmented. None of these damages was visible for compound 23, even when the
experiments were performed under UVA light. Thus, compound 23 was unable to intercalate inside
the double helix, nor to photoreact with pyrimidine bases of DNA, nor to induce any other DNA
damage, providing evidence that this treatment is not able to exert any action of DNA, even when
as an isolate substrate.
In order to verify the potential mutagenicity of compound 23, it was assayed for genotoxicity
evaluation through Ames test employing the histidine-requiring Salmonella typhimurium mutants
TA 97A, TA98, TA100 and TA1535 strains. The histidine-requiring Salmonella typhimurium
mutant strains described are particularly appropriate to preliminary check genotoxic potential of
new chemical compounds because both of them have a plasmid that induce an increasing of error-
prone repair of DNA damage (pKM101), and mutations that induce an increased permeability to
larger molecules through a defective lipopolysaccharide layer (rfa mutations) and avoid the
possibility to repair excision DNA damage (uvrB mutations) [32,33].
The genetic properties of the Salmonella typhimurium mutants TA 97A, TA98, TA100 and TA1535
strains allow to check frameshift mutation and base-pair substitution through the increasing number
of revertant colonies in presence of mutagenic chemical compounds, with and without metabolic
activation induced by S9 mix. The number of spontaneous revertants is relatively constant, but it
dramatically increases - generally in a dose-dependent way - when a mutagen is added to the plate.
In general, the cut-off to discriminate between a mutagenic and non-mutagenic result is considered
a 2-fold revertants increase over the response of the negative control (spontaneous revertants). For
the above reported considerations, the Ames test is generally considered an appropriate preliminary
screening tool to determine the mutagenic potential of new chemicals for different industrial
applications, from agro-chemicals, pharmaceuticals and medical devices to health products (for e.g.
personal cares, cosmetics, nutraceuticals, etc.). For the high significant predictive capacity, Ames
test is required by the regulatory agencies for registration or acceptance of new compounds [56].
The results obtained for TMA and compound 23 using the Salmonella typhimurium mutants TA98
and TA100 are reported in Table 3.
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Table 3. Mutagenicity of TMA and lack of mutagenicity of compound 23 on Salmonella
typhimrium TA98 and TA100 strains.
TMA (4,6,4’-trimethylangelicin)
TA98 –S9
revertant colonies SD
(average)
TA98 +S9
revertant colonies SD
(average)
Conc (µl/plate)
t/c*
t/c
DMSO
C+
1
5
10
24.67
576.50
25.50
15.50
28.50
24.00
30.00
3.51
119.50
7.78
3.54
13.44
5.66
1.00
23.37
1.03
0.63
1.16
0.97
1.22
27.33
502.50
20.50
1.53
20.51
4.95
26.87
84.85
1.00
18.38
0.75
1.90
4.50
8.25
6.86
52.00
123.00
225.50
187.50
50
100
45.96
37.48
1.41
TA100 –S9
TA100 +S9
Conc (µl/plate)
revertant colonies
(average)
213.67
3871.00
212.00
220.50
225.50
232.50
221.00
SD
t/c
revertant colonies SD
(average)
t/c
DMSO
C+
1
5
10
30.35
272.94
28.08
23.33
6.36
1.00
18.12
0.99
1.03
1.06
1.09
1.03
210.33
2852.00
232.00
210.50
218.50
243.00
529.50
23.97
1.00
13.56
1.10
1.00
1.04
1.16
2.52
50.91
10.51
9.19
10.61
11.31
120.92
50
100
19.09
8.46
Compound 23
Conc (µl/plate)
TA98 –S9
TA98 +S9
revertant colonies SD
(average)
revertant colonies
SD
t/c
t/c
(average)
68.0
1054.7
77.3
82.0
90.7
DMSO
C+
1
5
10
7.2
1.0
15.5
1.1
1.2
1.3
1.2
1.2
112.7
331.0
94.0
71.3
82.0
74.7
71.3
23.4
40.6
12.0
22.7
5.3
1.0
2.9
0.8
0.6
0.7
0.7
0.6
74.4
16.8
14.0
9.2
15.5
6.9
50
100
83.3
84.0
1.2
1.2
TA100 –S9
TA100 +S9
Conc (µl/plate)
revertant colonies
SD
t/c
revertant colonies SD
(average)
t/c
(average)
101.3
478.4
76.0
99.3
83.0
DMSO
C+
1
5
10
7.8
26.9
2.6
1.0
4.7
0.8
1.0
0.8
0.9
0.9
118.3
285.0
135.0
126.7
120.0
138.3
180.0
12.5
5.4
27.8
12.6
8.7
1.0
2.4
1.1
1.1
1.0
1.2
1.5
1.2
11.5
10.1
6.1
50
100
88.7
87.3
2.9
17.3
In bold are values indicating mutagenicity (> 2.00). * t/c = treated/control.
While TMA displayed mutagenic activity (in bold are values indicating mutagenicity, i.e. the
treated/control values > 2.00), compound 23 did not display genotoxic activity at all the
concentrations tested with all the Salmonella strains. These data were confirmed using the
Salmonella typhimurium mutants TA 97A and TA1535 strains (Table in Supplementary Data).
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In order to verify whether compound 23 displays inhibitory effects on pro-inflammatory genes, IB3-
1 were treated with 10 ng/ml TNF-α for 24 hours in the absence or in the presence of a 24-hours
pre-treatment step with 1 µM compound 23 [23]. The secretome was then analysed using the Bio-
plex approach using a 27-plex kit, analysing the pg/ml of 27 proteins present in the supernatant,
including cytokines, chemokines and growth factors [23]. The 27-plex list was matched with the list
of NF-κB regulated genes published by Pahl [3] (updated in https://www.bu.edu/nf-kb/gene-
resources/target-genes/), including the recently validated IL-13 [34], IL-15 [35] and IL-17 [36]. The
results Venn’s diagram (shown in the upper part of Figure 4) demonstrates that 23 proteins were in
common. From this list of 23 NF-κB regulated genes TNF-α was not analyzed, since TNF-α was
exogenously added to induce IB3-1 pro-inflammatory genes. Further, 4 proteins were not
considered (IL-13, GM-CSF, MIP-1α, MIP-1β) for the low level of released (< 5 pg/ml; data not
shown). The results concerning the remaining 18 proteins are reported in the lower part of Figure 4
and demonstrated that treatment with compound 23 caused inhibition of release of 5 proteins
(PDGF-B chain, IL-10, IL-15, Il-17 and IFN-γ) higher that 20%. The highest level of inhibition by
compound 23 was found for IL-10 and IFN-γ.
Figure 4. A. Venn’s diagram comparing the list of NF-κB regulated genes published by Pahl [3] (implemented with IL-
13, IL-15 and IL-17) [34-36] with the 27-plex proteins. B. Changes of release caused by treatment of TNF-α induced
IB3-1 cells with 1 µM compound 23. The following proteins were not considered: TNF-α (since TNF-α was used for
IB3-1 induction) and IL-13, GM-CSF, MIP-1α, MIP-1β (since they release was lower that 5 pg/ml; data not shown).
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CONCLUSIONS
In conclusion we have synthesized and validated a TMA derivative, compound 23, which exhibits
TMA-like inhibitory activity on NF-κB/DNA interactions without displaying TMA unwanted
effects. First of all, compound 23 was unable to intercalate inside the double helix, nor to photoreact
with pyrimidine bases of DNA, nor to induce any other DNA damage. In addition compound 23
was proven to lack mutagenicity when assayed through Ames test employing the histidine-requiring
Salmonella typhimurium mutants TA 97A, TA98, TA100 and TA1535 strains. On the other hand,
compound 23 exhibited inhibitory effects on some NF-κB regulated genes (PDGF-B chain, IL-10,
IL-15, Il-17 and IFN-γ) induced in cystic fibrosis IB3-1 cells by TNF-α treatment. Therefore, in
consideration of the role of NF-κB in regulating inflammation in cystic fibrosis [12-14,37,38],
compound 23 can be proposed for further characterization as a possible anti-inflammatory agent to
be used in combination with other anti-inflammatory molecules used at sub-optimal concentrations.
EXPERIMENTAL SECTION
Chemistry
General Information. All commercial chemicals and solvents used were analytical grade and were
used without further purification. Microwave assisted reactions were performed on a CEM Discover
monomode reactor in closed devices with the temperature monitored by a built-in infrared sensor
and the automatic control of the power. Melting points (uncorrected) were determined using a
Gallenkamp MFB-595-010M capillary melting point apparatus. Analytical thin layer
chromatography (tlc) was performed on pre-coated silica gel plates (Merck 60-F-254, 0.25 mm),
which were developed on a mixture of CHCl3/MeOH (9/1). The NMR spectra were recorded on a
Bruker 300-AMX spectrometer with TMS as internal standard. Coupling constants are given in Hz.
HRMS spectra were acquired using a XEVO G2-S Qtof (Waters) mass spectrometer with direct
injection of the sample and collecting data in positive ion mode. Purity of compounds was
determined by elemental analysis. Elemental analysis were performed on a Perkin-Elmer 2400
analyser and all values were within ± 0.4 % of the theoretical values.
Synthesis of alkyl-7-hydroxycoumarins 3-9.
General procedure. To a solution of 1 or 2 (10.0 mmol) in the appropriate acetoacetic ester (10.0
mmol), H2SO4 (7 mL) was added dropwise and the mixture was stirred at room temperature for 1 h.
The solution was poured into an ice/water mixture (100 g) and the obtained precipitate was filtered
and washed with water to give the corresponding alkyl-7-hydroxycoumarin.
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1
6-Ethyl-7-hydroxy-4-methylbenzopyran-2-one (3). From 2: yield 92%: mp 197 °C. H-NMR
(DMSO-d6): 10.52 (broad s, 1H, 7-OH), 7.44 (s, 1H, 5-H), 6.72 (s, 1H, 8-H), 6.09 (q, J = 1.1 Hz,
1H, 3-H), 2.59 (q, J = 7.5 Hz, 2H, -CH2-CH3), 2.37 (d, J = 1.1 Hz, 3H, 4-Me), 1.15 (t, J = 7.5 Hz,
3H, -CH2-CH3).
1
4-Ethyl-7-hydroxy-6-methylbenzopyran-2-one (4). From 1: yield 73%: mp 151 °C. H-NMR
(DMSO-d6): 10.52 (broad s, 1H, 7-OH), 7.52 (s, 1H, 5-H), 6.72 (s, 1H, 8-H), 6.05 (t, J = 1.2 Hz,
1H, 3-H), 2.76 (qd, J = 7.4, 1.2 Hz, 2H, -CH2-CH3), 2.18 (s, 3H, 6-Me), 1.21 (t, J = 7.4 Hz, 3H, -
CH2-CH3).
1
4,6-Diethyl-7-hydroxybenzopyran-2-one (5). From 2: yield 83%: mp 108 °C. H-NMR (DMSO-
d6): 10.51 (broad s, 1H, 7-OH), 7.48 (s, 1H, 5-H), 6.73 (s, 1H, 8-H), 6.05 (t, J = 1.1 Hz, 1H, 3-H),
2.78 (qd, J = 7.4, 1.1 Hz, 2H, 4-CH2-CH3), 2.59 (q, J = 7.5 Hz, 2H, 6-CH2-CH3), 1.21 (t, J = 7.4 Hz,
3H, 4-CH2-CH3), 1.15 (t, J = 7.5 Hz, 3H, 6-CH2-CH3).
1
7-Hydroxy-6-methyl-4-propylbenzopyran-2-one (6). From 1: yield 90%: mp 166 °C. H-NMR
(DMSO-d6): 10.52 (broad s, 1H, 7-OH), 7.53 (s, 1H, 5-H), 6.73 (s, 1H, 8-H), 6.05 (t, J = 1.1 Hz,
1H, 3-H), 2.71 (td, J = 7.5, 1.1 Hz, 2H, -CH2-CH2-CH3), 2.18 (s, 3H, 6-Me), 1.63 (sest, J = 7.5 Hz,
2H, -CH2-CH2-CH3), 0.97 (t, J = 7.5 Hz, 3H, -CH2-CH2-CH3).
1
6-Ethyl-7-hydroxy-4-propyl-benzopyran-2-one (7). From 2: yield 90%: mp 127 °C. H-NMR
(DMSO-d6): 10.52 (broad s, 1H, 7-OH), 7.48 (s, 1H, 5-H), 6.73 (s, 1H, 8-H), 6.04 (t, J = 1.1 Hz,
1H, 3-H), 2.72 (td, J = 7.6, 1.2 Hz, 2H, -CH2-CH2-CH3), 2.59 (q, J = 7.5 Hz, 2H, -CH2-CH3), 1.63
(sest, J = 7.6 Hz, 2H, -CH2-CH2-CH3), 1.15 (t, J = 7.5 Hz, 3H, -CH2-CH3), 0.97 (t, J = 7.6 Hz, 3H, -
CH2-CH2-CH3).
1
7-Hydroxy-4-isopropyl-6-methylbenzopyran-2-one (8). From 1: yield 40%: mp 147 °C. H-NMR
(CDCl3): 7.41 (s, 1H, 5-H), 6.96 (s, 1H, 8-H), 6.17 (d, J = 1.1 Hz, 1H, 3-H), 3.27 (sept/d, J = 7.2 H,
1.1 Hz, 1H, -CH-(CH3)2), 2.31 (s, 3H, 6-Me), 1.31 (d, J = 7.2 Hz, 6H, -CH-(CH3)2).
1
6-Ethyl-7-hydroxy-4-isopropylbenzopyran-2-one (9). From 2: yield 52%: mp 168 °C. H-NMR
(CDCl3): 7.41 (s, 1H, 5-H), 7.05 (s, 1H, 8-H), 6.17 (d, J = 1.1 Hz, 1H, 3-H), 3.30 (sept/d, J = 7.2 H,
1.1 Hz, 1H, -CH-(CH3)2), 2.71 (q, J = 7.5 Hz, 2H, -CH2-CH3), 1.51 (d, J = 7.2 Hz, 6H, -CH-
(CH3)2), 1.26 (t, J = 7.5 Hz, 3H, -CH2-CH3).
Synthesis of alkyl-7-O-(2’-oxopropyl)ethers 10-16.
General procedure. A mixture of alkyl-7-hydroxybenzopyran-2-one (5.0 mmol), chloroacetone
(0.5 mL, 5.9 mmol) and anhydrous K2CO3 (2.1 g, 15.0 mmol) in acetone (75 mL) was refluxed 6 h.
After cooling, the solid was filtered off and washed with fresh acetone. The solvent was evaporated
11

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from the combined filtrate and washings to give the corresponding alkyl-7-(2’-oxopropyloxy)-2H-
1-benzopyran-2-one.
1
6-Ethyl-4-methyl-7-(2’-oxopropyloxy)benzopyran-2-one (10). From 3: yield 87%: mp 142 °C. H-
NMR (DMSO-d6): 7.52 (s, 1H, 5-H), 6.91 (s, 1H, 8-H), 6.19 (q, J = 1.4 Hz, 1H, 3-H), 4.99 (s, 2H, -
OCH2), 2.68 (q, J = 7.6 Hz, 2H, -CH2-CH3), 2.40 (d, J = 1.4 Hz, 3H, 4-Me), 2.19 (s, 3H, -COCH3),
1.20 (t, J = 7.6 Hz, 3H, -CH2-CH3).
1
4-Ethyl-6-methyl-7-(2’-oxopropyloxy)benzopyran-2-one (11). From 4: yield 76%: mp 127 °C. H-
NMR (CDCl3): 7.39 (s, 1H, 5-H), 6.59 (s, 1H, 8-H), 6.15 (t, J = 1.1 Hz, 1H, 3-H), 4.62 (s, 2H, -
OCH2), 2.77 (qd, J = 7.4, 1.1 Hz, 2H, -CH2-CH3), 2.34 (s, 3H, 6-Me), 2.32 (s, 3H, -COCH3), 1.21
(t, J = 7.4 Hz, 3H, -CH2-CH3).
1
4,6-Diethyl-7-(2’-oxopropyloxy)benzopyran-2-one (12). From 5: yield 74%: mp 89 °C. H-NMR
(DMSO-d6): 7.56 (s, 1H, 5-H), 6.92 (s, 1H, 8-H), 6.13 (t, J = 1.0 Hz, 1H, 3-H), 4.99 (s, 2H, -
OCH2), 2.81 (qd, J = 7.4, 1.0 Hz, 2H, 4-CH2-CH3), 2.68 (q, J = 7.5 Hz, 2H, 6-CH2-CH3), 2.19 (s,
3H, -COCH3), 1.23 (t, J = 7.4 Hz, 3H, 4-CH2-CH3), 1.19 (t, J = 7.5 Hz, 3H, 6-CH2-CH3).
6-Methyl-7-(2’-oxopropyloxy)-4-propylbenzopyran-2-one (13). From 6: yield 74%: mp 109 °C.
1H-NMR (DMSO-d6): 7.61 (s, 1H, 5-H), 6.91 (s, 1H, 8-H), 6.13 (t, J = 1.1 Hz, 1H, 3-H), 4.99 (s,
2H, -OCH2), 2.74 (td, J = 7.6, 1.1 Hz, 2H, -CH2-CH2-CH3), 2.26 (s, 3H, -COCH3), 2.19 (s, 3H, 6-
Me), 1.64 (sest, J = 7.6 Hz, 2H, -CH2-CH2-CH3), 0.98 (t, J = 7.6 Hz, 3H, -CH2-CH2-CH3).
1
6-Ethyl-7-(2’-oxopropyloxy)-4-propylbenzopyran-2-one (14). From 7: yield 96%: mp 118 °C. H-
NMR (DMSO-d6): 7.57 (s, 1H, 5-H), 6.92 (s, 1H, 8-H), 6.14 (t, J = 1.1 Hz, 1H, 3-H), 4.99 (s, 2H, -
OCH2), 2.74 (td, J = 7.6, 1.2 Hz, 2H, -CH2-CH2-CH3), 2.68 (q, J = 7.3 Hz, 2H, -CH2-CH3), 2.19 (s,
3H, -COCH3), 1.65 (sest, J = 7.6 Hz, 2H, -CH2-CH2-CH3), 1.19 (t, J = 7.3 Hz, 3H, -CH2-CH3), 0.98
(t, J = 7.6 Hz, 3H, -CH2-CH2-CH3).
4-Isopropyl-6-methyl-7-(2’-oxopropyloxy)benzopyran-2-one (15). From 8: yield 73%: mp 118 °C.
1H-NMR (CDCl3): 7.44 (s, 1H, 5-H), 6.60 (s, 1H, 8-H), 6.15 (d, J = 1.1 Hz, 1H, 3-H), 4.62 (s, 2H, -
OCH2), 3.26 (sept/d, J = 7.4, 1.1 Hz, 1H, -CH-(CH3)2), 2.35 (s, 3H, -COCH3), 2.33 (s, 3H, 6-Me),
1.31 (d, J = 7.4 Hz, 6H, -CH-(CH3)2).
6-Ethyl-4-isopropyl-7-(2’-oxopropyloxy)benzopyran-2-one (16). From 9: yield 92%: mp 80 °C.
1H-NMR (CDCl3): 7.44 (s, 1H, 5-H), 6.61 (s, 1H, 8-H), 6.18 (d, J = 1.1 Hz, 1H, 3-H), 4.62 (s, 2H, -
OCH2), 3.28 (sept/d, J = 7.1, 1.1 Hz, 1H, -CH-(CH3)2), 2.77 (q, J = 7.6 Hz, 2H, -CH2-CH3), 2.32 (s,
3H, -COCH3), 1.32 (d, J = 7.1 Hz, 6H, -CH-(CH3)2), 1.27 (t, J = 7.6 Hz, 3H, -CH2-CH3).
12

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Synthesis of alkyl-2H-furo[2,3-h]-1-benzopyran-2-ones 17-23.
General procedure. To an ethanolic solution (50 mL) of alkyl-7-(2’-oxopropyloxy)-2H-1-
benzopyran-2-one (1.0 mmol), a 4% ethanolic potassium hydroxide solution (15 mL) was added
and the mixture was refluxed in the dark for 1 h. The solution was cooled, diluted with water (50
mL) and acidified with diluted HCl. The ethanol was removed under reduced pressure and the
solution extracted with CHCl3 (3 x 50 mL). The organic phase was evaporated under reduced
pressure and the residue was purified by column chromatography to give the corresponding
furobenzopyranone.
4,9-Dimethyl-6-ethyl-2H-furo[2,3-h]-1-benzopyran-2-one (17). From 10: yield 47%: mp 148 °C.
1H-NMR (DMSO-d6): 7.87 (q, J = 1.1 Hz, 1H, 8-H), 7.47 (s, 1H, 5-H), 6.32 (q, J = 1.3 Hz, 1H, 3-
H), 2.89 (q, J = 7.6 Hz, 2H, -CH2-CH3), 2.48 (d, J = 1.1 Hz, 3H, 9-Me), 2.41 (d, J = 1.3 Hz, 3H, 4-
Me), 1.29 (t, J = 7.6 Hz, 3H, -CH2-CH3). HRMS (ESI-TOF) for C15H15O3 [M + H]+: calcd.:
243.1021, found: 243.1059. Anal. calcd. for C15H14O3: C, 74.36; H 5.82; found: C, 74.10; H, 5.95.
6,9-Dimethyl-4-ethyl-2H-furo[2,3-h]-1-benzopyran-2-one (18). From 11: yield 30%: mp 96 °C.
1H-NMR (DMSO-d6): 7.88 (q, J = 1.3 Hz, 1H, 8-H), 7.55 (s, 1H, 5-H), 6.27 (t, J = 1.0 Hz, 1H, 3-
H), 2.89 (qd, J = 7.6, 1.0 Hz, 2H, -CH2-CH3), 2.50 (s, 3H, 6-Me), 2.43 (d, J = 1.3 Hz, 3H, 4-Me),
1.27 (t, J = 7.6 Hz, 3H, -CH2-CH3). HRMS (ESI-TOF) for C15H15O3 [M + H]+: calcd.: 243.1021,
found: 243.1123. Anal. calcd. for C15H14O3: C, 74.36; H 5.82; found: C, 74.61; H, 5.92.
4,6-Diethyl-9-methyl-2H-furo[2,3-h]-1-benzopyran-2-one (19). From 12: yield 40%: mp 115 °C.
1H-NMR (DMSO-d6): 7.87 (q, J = 1.1 Hz, 1H, 8-H), 7.52 (s, 1H, 5-H), 6.27 (t, J = 1.1 Hz, 1H, 3-
H), 2.95-2.85 (m, 4H, 4 and 6-CH2-CH3), 2.41 (d, J = 1.1 Hz, 3H, 9-Me), 1.29 (t, J = 7.5 Hz, 3H, 4
or 6-CH2-CH3), 1.19 (t, J = 7.5 Hz, 3H, 4 or 6-CH2-CH3). HRMS (ESI-TOF) for C16H17O3 [M +
H]+: calcd.: 257.1178, found: 257.1246. Anal. calcd. for C16H16O3: C, 74.98; H 6.29; found: C,
75.10; H, 6.51.
6,9-Dimethyl-4-propyl-2H-furo[2,3-h]-1-benzopyran-2-one (20). From 13: yield 17%: mp 146 °C.
1H-NMR (DMSO-d6): 7.87 (q, J = 1.3 Hz, 1H, 8-H), 7.54 (s, 1H, 5-H), 6.26 (s, 1H, 3-H), 2.82 (t, J
= 7.6, 1.0 Hz, 2H, -CH2-CH2-CH3), 2.50 (s, 3H, 6-Me), 2.42 (d, J = 1.3 Hz, 3H, 9-Me), 1.68 (sest, J
= 7.6 Hz, 2H, -CH2-CH2-CH3), 1.00 (t, J = 7.6 Hz, 3H, -CH2-CH3). HRMS (ESI-TOF) for C16H17O3
[M + H]+: calcd.: 257.1178, found: 257.1246. Anal. calcd. for C16H16O3: C, 74.98; H 6.29; found:
C, 75.06; H, 6.45.
6-Ethyl-9-methyl-4-propyl-2H-furo[2,3-h]-1-benzopyran-2-one (21). From 14: yield 32%: mp 138
°C. 1H-NMR (DMSO-d6): 7.88 (q, J = 1.2 Hz, 1H, 8-H), 7.53 (s, 1H, 5-H), 6.27 (t, J = 1.1 Hz, 1H,
3-H), 2.90 (q, J = 7.5 Hz, 2H, 6-CH2-CH3), 2.84 (td, J = 7.5, 1.2 Hz, 2H, 4-CH2-CH2-CH3), 2.42 (d,
J = 1.2 Hz, 3H, 9-Me), 1.68 (sest, J = 7.5 Hz, 2H, 4-CH2-CH2-CH3), 1.29 (t, J = 7.5 Hz, 3H, 4-CH2-
13

ACCEPTED MANUSCRIPT
CH2-CH3 or 6-CH2-CH3),1.00 (t, J = 7.6 Hz, 3H, 4-CH2-CH2-CH3 or 6-CH2-CH3). HRMS (ESI-
TOF) for C17H19O3 [M + H]+: calcd.: 271.1334, found: 271.1390. Anal. calcd. for C17H18O3: C,
75.53; H 6.71; found: C, 75.37; H, 6.45.
6,9-Dimethyl-4-isopropyl-2H-furo[2,3-h]-1-benzopyran-2-one (22). From 15: yield 35%: mp 124
1
°C. H-NMR (CDCl3): 7.42 (q, J = 1.2 Hz, 1H, 8-H), 7.33 (s, 1H, 5-H), 6.28 (s, 1H, 3-H), 3.36
(sept, J = 6.9 Hz, 1H, -CH-(CH3)2), 2.54 (s, 3H, 6-Me), 2.53 (d, J = 1.2 Hz, 3H, 9-Me), 1.35 (d, J =
6.9 Hz, 6H, -CH-(CH3)2). HRMS (ESI-TOF) for C16H17O3 [M + H]+: calcd.: 257.1178, found:
257.1214. Anal. calcd. for C16H16O3: C, 74.98; H 6.29; found: C, 74.72; H, 6.10.
6-Ethyl-4-isopropyl-9-methyl-2H-furo[2,3-h]-1-benzopyran-2-one (23). From 16: yield 35%: mp
101 °C. 1H-NMR (Acetone-d6): 7.64 (s, 1H, 8-H), 7.52 (s, 1H, 5-H), 6.18 (s, 1H, 3-H), 3.46 (sept, J
= 6.4 Hz, 1H, 4-CH-(CH3)2), 2.91 (q, J = 7.3 Hz, 2H, 6-CH2-CH3), 2.44 (s, 3H, 9-Me), 1.37-1.29
13
(m, 9H, 6-CH2-CH3 e -CH-(CH3)2). C-NMR (Acetone-d6): δ 164.80 (CO), 162.00 (C4), 157.75
(C6a), 149.89 (C9b), 144.17 (C8), 126.21 (C6), 120.70 (C5), 118.58 (C9a or C9), 117.79 (C9a or
C9), 114.90 (C4a), 109.98 (C3), 30.60 ((CH3)2C), 24.21 (CH3CH2), 23.15 ((CH3)2C), 15.54
(CH3CH2), 10.71 (9-Me). HRMS (ESI-TOF) for C17H19O3 [M + H]+: calcd.: 271.1334, found:
271.1424. Anal. calcd. for C17H18O3: C, 75.53; H 6.71; found: C, 75.34; H, 6.56.
Biology
EMSA (Electrophoretic Mobility Shift Assays). Electrophoretic mobility shift assays were
32
performed using double stranded P-labeled oligonucleotides as target DNA [23]. Binding
reactions were set up in binding buffer (10% glycerol, 0.05% NP-40, 10 mM Tris–HCl pH 7.5, 50
mM NaCl, 0.5 mM DTT, 10 mM MgCl ), in the presence of 0.1 µl/20 µl of NF-κB-p50 (50 gsu)
2
(PROMEGA, Madison, WI, USA) and 0.25 ng of labeled oligonucleotide, in a total volume of 20
µl. After 30 min binding at room temperature samples were electrophoresed at constant voltage
(200 V for 30 min) through a low ionic strength (0.25 TBE buffer) (1 TBE/40.089 M Tris–borate,
0.002 M EDTA) on 6% polyacrylamide gels until the tracking dye (bromophenol blue) reached the
bottom of a 16 cm slab. Gels were dried and exposed for autoradiography with intensifying screens
at 80°C. Images were scanned and analyzed with the Image Lab ChemiDoc (Biorad). In these
experiments, DNA/protein complexes migrate through the gel with slower efficiency. In studies on
the inhibitors of protein/DNA interactions, addition of the reagents was as follows: (i) nuclear
factors; (ii) active principles; (iii) binding buffer; (iv) labeled oligonucleotides mimicking the
binding sites for TF to be modulated. The nucleotide sequence of double-stranded target DNA
utilized in these experiments was 5’-CGC TGG GGA CTT TCC ACG G-3’ (sense strand, NF-κB)
14

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[50]. The synthetic oligonucleotides utilized in this study were purchased from Sigma Genosys
(Sigma Genosys, Cambs, UK).
Cell cultures and cell proliferation assay. IB3-1 cells [30], derived from a CF patient with a
DF508/W1282X mutant genotype and immortalized with adeno12/SV40, were grown in LHC-8
supplemented with 5% FBS in the absence of gentamycin, at 37°C/5% CO2 [23]. The effects of
compounds (TMA derivatives) on the IB3-1 cell proliferation were analyzed as elsewhere described
(RS1). In order to determine IC50 values the following concentrations were used 12.5, 25, 50 µM,
100, 200 and 800 µM. Cells were counted with a Z2 Coulter Counter (Coulter Electronics, Hialeah,
FL, USA). The cell number/ml was determined as IC50 after 2 days of culture, when untreated cells
are in log phase of cell growth. For induction of pro-inflammatory genes, monolayers of 60%
confluent IB3-1 cells were seeded in 6- or 12-well plates in LHC-8 medium and after 24 hours
compound 23 was added, 5 hours before stimulation with 80 ng/ml TNF-α (ORF Genetics,
Kopavogur, Iceland), and incubated for a further 24 hours.
Bioplex analysis. Cytokines, released from cells into tissue culture supernatants, were measured by
Bioplex cytokine assay (Bio-Rad Laboratories, Hercules, CA) as suggested by the manufacturer
(Luminex technology) [39]. The Bioplex cytokine assay is designed for the multiplexed quantitative
measurement of multiple cytokines in a single well using as little as 50 µl of sample. In our
experiments, the premixed multiplex beads (27-plex) of the Bioplex human cytokines were used. 50
µl of cytokine standards or samples (supernatants recovered from treated cells and diluted to 2
µg/µl) were incubated with 50 µl of anti-cytokine conjugated beads in 96-well filter plates for 30
min at room temperature with shaking. Plates were washed by vacuum filtration three times with
100 µl of Bioplex wash buffer, 25 µl of diluted detection antibody were added, and plates were
incubated for 30 min at room temperature with shaking. After three filter washes, 50 µl of
streptavidin–phycoerythrin were added, and the plates were incubated for 10 min at room
temperature with shaking. Finally, plates were washed by vacuum filtration three times, beads were
suspended in Bioplex assay buffer, and samples were analyzed on a Bio-Rad 96-well plate reader
using the Bioplex Suspension Array System and Bioplex Manager software (Bio-Rad Laboratories,
Hercules, CA).
Interaction with salmon testes DNA
Linear flow dichroism. Linear flow dichroism spectra were measured as described in
Supplementary Data.
Irradiation procedure. UVA irradiation was carried out by using Philips HPW 125 lamps, mainly
emitting at 365 nm. Blue The total energy hitting the sample was monitored by means of a
15

ACCEPTED MANUSCRIPT
radiometer (Variocontrol, Waldmann, Villingen-Schwenningen, Germany), equipped with a
Variocontrol UV Sensor (Waldmann). The radiant power emitted by the UVA lamp was about 8
mW/cm2. The samples were maintained at room temperature during irradiation.
Isolation of photoadducts. Ethanol concentrated solutions of the compounds (2 mg/mL) were
added dropwise to salmon testes DNA (Sigma-Aldrich) in 10 mM NaCl and 1 mM EDTA solution
(1.5 × 10−3 M) to achieve a DNA/compound ratio of about 40.The mixture was irradiated with 15
J/cm2 UVA light in a glass dish. After irradiation, the DNA was precipitated with 1 M NaCl and 2
volumes of cold ethanol; the precipitated DNA was collected, washed with 80% ethanol, dried and
then dissolved in water. The final solutions were hydrolyzed with 0.5 M HCl, heated at 100°C for 1
h, neutralized with NaOH and extracted three times with chloroform. Then, the organic layers were
collected, dried under high vacuum and dissolved in ethanol. The hydrolyzed mixtures were
separated by thin-layer chromatography (TLC; F254 plates, 0.25 mm, Merck, Darmstadt, Germany)
eluting with ethyl acetate/ethanol (9:1). Bands of interest, visualized with a Mineral Light Lamp
VL-4LC VILBERT-LOURMAT, were scraped off and the expected photoproducts extracted with
1
chloroform and ethanol and then analyzed by NMR. H-NMR were performed in deuterated
acetone, deuterated DMSO or CDCl3 using a Bruker AMX300 spectrometer (300 MHz).
pBR322 DNA strand breaks. Each pBR322 DNA sample (150 ng) (Life Technologies, Monza,
Italy) dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) was irradiated with
increasing BL and UVA radiation exposures (0, 2.5, 5.0, 7.5, J/cm2) in the presence of 300 µM of
the compounds. The samples were loaded on 1% agarose gel, after addition of 1 µL of gel loading
buffer (0.25% Bromophenol blue, BPB; 0.25% xylene cyanol; and 40%, w/v, sucrose) to each of
them. The electrophoretic run was carried out in TAE buffer (40 mM Tris-acetate, 1 mM EDTA,
pH 8.0) at 25 V for 6 h, using the GNA-100 electrophoretic apparatus (Pharmacia, Uppsala,
Sweden). After staining with ethidium bromide (1 µg/mL in TAE buffer) for 20 min, the DNA
bands were visualized with a UV transilluminator and detected with Bio-Rad GelDoc EZ (Bio-Rad
Laboratories, Inc, Hercules, CA, USA). Samples of DNA alone in the dark and after irradiation
were used as controls.
Mutagenicity assay (Ames test). Mutagenicity assay was performed for TMA and compound 23,
following the plate incorporation method with the histidine-requiring Salmonella typhimurium
mutant TA97A, TA98, TA100 and TA1535 strains purchased by Molecular Toxicology Inc.
(Boone, NC, USA; moltox.com) [32,33]. All strains (100 µl per plate of fresh overnight cultures)
were checked with and without the addition of 0.5 ml of a 5% S9 exogenous metabolic activator (S9
mix). The lyophilized post-mitochondrial supernatant S9 mix (Aroclor 1254-induced, Sprague–
Dawley male rat liver in 0.154 M KCl solution), commonly used for the activation of pro-mutagens
16

ACCEPTED MANUSCRIPT
to mutagenic metabolites (Molecular Toxicology, Inc., Boone, NC, USA) was stored at - 80 °C
before use. The concentration tested for all the samples were 1, 5, 10, 50, 100 µg/plate (see
following table for molar concentrations). A fully-grown culture of the appropriate tester strain (0.1
ml) was added to 2 ml molten top agar (0.6% agar, 0.5% NaCl, 0.5 mM L-histidine/biotin solution)
at 46 °C, together with 0.1 ml of each sample solution at different concentrations, and 0.5 ml S9
mix for assays with metabolic activation. The ingredients were thoroughly mixed and poured onto
minimal glucose agar plates (1.5% agar in 2% Vogel–Bonner medium E with 5% glucose solution).
DMSO was used as a negative control (100 µl/plate). Positive controls were prepared as follows: 2-
aminoanthracene (2 µg/plate) and 2-nitrofluorene (2 µg/plate) for TA 97A, TA98 and TA1535 with
and without metabolic activator (S9 mix) respectively; 2-aminoanthracene (2 µg/plate) and sodium
azide (2 µg/plate) for TA100, with and without metabolic activator (S9 mix) respectively. The
plates were incubated at 37 °C for 72 h and then the his+ revertants were checked and counted
using a Colony Counter 560 Suntex (Antibioticos, Italy). A sample was considered mutagenic when
the observed number of colonies was at least twofold over the spontaneous level of revertants
[32,33]. All determinations were made in triplicate. Relative standard deviations were computed
using the statistical software STATISTICA 6.0 (StatSoft Italia srl).
Author contributions
A.C., G.Marzaro and C.V. synthesized the compounds. G.Miolo performed the photoactivity
measurements. I.L., E.D., G.S., G.C. and MC. D. performed the other biological assays. A.C. and
R.G. conceived the idea and wrote the main manuscript text. All authors approved the final version
of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
The project was supported by FFC (Fondazione Ricerca Fibrosi Cistica) Projects FFC#1/2016
(Adriana Chilin) and FFC#3/2016 (Roberto Gambari).
Supplementary Data
Detailed analytical data for compound 23; HRMS for compounds 17-22; EMSA experiment on
TMA; LD measurement; mutagenicity of compound 23 on Salmonella typhimrium TA97A and
1535 strains.
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Highlights
•
•
TMA (trimethylangelicin) is a promising agent for treatment of inflammation-related diseases,
unfortunately showing unwanted side effects (photoreactivity and mutagenicity).
New generation TMA analogues with hindered substituent at the 4 position were designed and
synthesized.
•
•
All the title compounds inhibited NF-κB/DNA interactions.
A lead compound was selected for the good inhibitory activity on NF-κB/DNA interactions, for its
inhibitory effects on some NF-κB regulated genes and for the absence of TMA side effects.