Optimization of benzoquinone and hydroquinone derivatives as potent inhibitors of human 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2016.10.046

Aiming to assess the biological activities of synthetic 1,4-benzoquinones, we previously synthesized different libraries of benzoquinones with lipophilic and bulky alkyl- or aryl-substituents that inhibited 5-lipoxygenase (5-LO). The high potency of 4,5-dimethoxy-3-alkyl-1,2-benzoquinones on 5-LO led to the idea to further modify the structures and thus to improve the inhibitory potential in?vitro and in?vivo as well as to investigate SARs. Systematic structural optimization through accurate structure-based design resulted in compound 30 (3-tridecyl-4,5-dimethoxybenzene-1,2-diol), an ubiquinol derivative that exhibited the strongest anti-inflammatory effect, with a 10-fold improved 5-LO inhibitory activity (IC₅₀?=?28?nM) in activated neutrophils. Moreover, 30 significantly reduced inflammatory reactions in the carrageenan-induced mouse paw oedema and in zymosan-induced peritonitis in mice. Compound 30 (1?mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30?min after zymosan, outperforming zileuton at a dose of 10?mg/kg. The binding patterns of the quinone- and hydroquinone-based 5-LO inhibitors were analyzed by molecular docking. Together, we elucidated the optimal alkyl chain pattern of quinones and corresponding hydroquinones and reveal a series of highly potent 5-LO inhibitors with effectiveness in?vivo that might be useful as anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2016.10.046

Accepted Manuscript
Optimization of benzoquinone and hydroquinone derivatives as potent inhibitors of
human 5-lipoxygenase
Antonella Peduto, Maria Scuotto, Verena Krauth, Fiorentina Roviezzo, Antonietta
Rossi, Veronika Temml, Veronica Esposito, Hermann Stuppner, Daniela Schuster,
Bruno D'Agostino, Chiara Schiraldi, Mario de Rosa, Oliver Werz, Rosanna Filosa
PII:
S0223-5234(16)30908-4
10.1016/j.ejmech.2016.10.046
EJMECH 9011
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2016
Revised Date: 19 October 2016
Accepted Date: 20 October 2016
Please cite this article as: A. Peduto, M. Scuotto, V. Krauth, F. Roviezzo, A. Rossi, V. Temml, V.
Esposito, H. Stuppner, D. Schuster, B. D'Agostino, C. Schiraldi, M. de Rosa, O. Werz, R. Filosa,
Optimization of benzoquinone and hydroquinone derivatives as potent inhibitors of human 5-
lipoxygenase, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.046.
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Graphical Abstract
Optimization of Benzoquinone and Hydroquinone
Derivatives as Potent Inhibitors
Leave this area blank for abstract info.
of Human 5-Lipoxygenase
Antonella Peduto, Maria Scuotto, Verena Krauth, Fiorentina Roviezzo,
Antonietta Rossi, Veronika Temml, Veronica Esposito, Hermann Stuppner, Daniela Schuster,
Bruno D’Agostino, Chiara Schiraldi,Mario De Rosa, Oliver Werz, Rosanna Filosa

ACCEPTED MANUSCRIPT
Optimization of Benzoquinone and
Hydroquinone
Derivatives as Potent Inhibitors
of Human 5-Lipoxygenase
Antonella Pedutoa,f#, Maria Scuotto a #, Verena Krauthb#, Fiorentina Roviezzoc, Antonietta
Rossic, Veronika Temmld, Veronica Espositoc, Hermann Stuppnerd, Daniela Schustere,
Bruno D’Agostinoa, Chiara Schiraldi a, Mario de Rosa a, Oliver Werz*b, Rosanna Filosa*a
aDepartment of Experimental Medicine, Second University of Naples, Via Costantinopoli, 16 - 80138 Naples,
Italy;
bChair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14,
D-07743 Jena, Germany
eDepartment of Pharmacy, University of Naples Federico II, Via D. Montesano 49, Naples, Italy
d,eInstitute of Pharmacy/Pharmacognosyd or Pharmaceutical Chemistrye, University of Innsbruck, Center for
f
Molecular Biosciences Innsbruck, Innrain 80/82, AT-6020 Innsbruck, Austria. Dermofarma, Via Cortenocera,
82030 S. Salvatore Telesino, BN, Italy
#these authors contributed equally
*Corresponding authors at: Department of Experimental Medicine, Second University of Naples, Via
Costantinopoli, 16 - 80138 Naples, Italy; Phone: 0815667663rosanna.filosa@unina2.it (R. Filosa) and Chair of
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743
Jena, Germany: Phone: +49-3641-949801, E-mail: oliver.werz@uni-jena.de (O.werz).
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Abstract
Aiming to assess the biological activities of synthetic 1,4-benzoquinones, we previously
synthesized different libraries of benzoquinones with lipophilic and bulky alkyl- or aryl-
substituents that inhibited 5-lipoxygenase (5-LO). The high potency of 4,5-dimethoxy-3-
alkyl-1,2-benzoquinones on 5-LO led to the idea to further modify the structures and thus to
improve the inhibitory potential in vitro and in vivo as well as to investigate SARs. Systematic
structural optimization through accurate structure-based design resulted in compound 30 (3-
tridecyl-4,5-dimethoxybenzene-1,2-diol), an ubiquinol derivative that exhibited the strongest
anti-inflammatory effect, with a 10-fold improved 5-LO inhibitory activity (IC50 = 28 nM) in
activated neutrophils. Moreover, 30 significantly reduced inflammatory reactions in the
carrageenan-induced mouse paw oedema and in zymosan-induced peritonitis in mice.
Compound 30 (1 mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30 min after
zymosan, outperforming zileuton at a dose of 10 mg/kg. The binding patterns of the quinone-
and hydroquinone-based 5-LO inhibitors were analyzed by molecular docking. Together, we
elucidated the optimal alkyl chain pattern of quinones and corresponding hydroquinones and
reveal a series of highly potent 5-LO inhibitors with effectiveness in vivo that might be useful
as anti-inflammatory drugs.
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1.
Introduction
Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LO)
pathway and are linked to diverse inflammatory disorders. Intervention with LTs represents a
pertinent pharmacological approach against inflammatory diseases, and anti-LT therapy has
been validated in clinical trials of asthma and allergic rhinitis, with potential in other
respiratory and allergic disorders [1], as well as in cardiovascular diseases such as
atherosclerosis, myocardial infarction, stroke and abdominal aortic aneurysm [2]. In addition,
such inhibitors may be tools for the elucidation of the involvement of 5-LO and LTs in
diverse biological processes.
Over the last two decades, several studies have shown that quinone derivatives possess a
number of biological and pharmacological applications, and hydroquinones are of
considerable scientific interest because of their versatile biological activities, where also the
corresponding (hydro) quinone may display multiple actions [3]. Our groups have been
interested for a long time in the synthesis and the biological evaluation of anti-cancer and
anti-inflammatory agents including quinone-based compounds [4-15]. Within the context of
our investigations towards the synthesis of quinone derivatives with prospects for therapeutic
use, we recently studied the natural compound embelin and RF-Id, a synthetic derivative of
bolinaquinone (Figure 1) [7-8].
The high potency against 5-LO and the promising in vivo efficacy of the corresponding
synthesized compounds stimulated us to further modify the structures and thus, to improve the
inhibitory potential as well as to investigate SARs of this class of compounds. Starting from
embelin, the alkyl chain length in position 3 (n-undecyl residue) was varied by introducing
saturated linear n-alkyl residues or isoprenoid side chains. Next, one or two hydroxyl groups
were methylated in 2- and 5-position, respectively, and finally, the 2,5-dimethoxy-1,4-
benzoquinone core was replaced by a 4,5-methoxy-1,2-benzoquinones backbone (ortho-
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quinone structures) leading to compounds decorated with the same linear and prenylated
chains as for the other groups [10-11].
We have shown that the increase of the n-alkyl side-chain length determines the 5-LO
inhibitory activity in neutrophils stimulated with the Ca2+-mobilizing agent A23187. Thus,
ortho-quinones with simple n-undecyl (25) or n-dodecyl (26) and n-tridecyl (27) residues
were most potent in intact human neutrophils with IC50 values in the submicromolar range
(approx. 50-100 nM) [10-11]. Starting from this basis and considering that modifications on
the quinone scaffold did not affect the polarity of the linear chain as reported in the literature
so far, we were interested, whether the presence of a polar group (i.e. hydroxyl) in the n-alkyl
chain would affect the potency against 5-LO. Thus, we took into account the marked anti-
inflammatory properties of Idebenone (2,3-dimethoxy-5-methyl-6(10-hydroxydecyl)-1,4-
benzoquinone) [16-18], a synthetic analogue of coenzyme Q10 (CoQ10), with potent
antioxidant activity, able to inhibit the enzymatic metabolism of arachidonic acid by
cyclooxygenase and lipoxygenases, with potential anti-inflammatory activity in the central
nervous system [18]. Notably, introduction of a polar group into the lipophilic n-alkyl chain
may also improve water solubility and thus bioavailability. We therefore envisioned the
preparation of quinone derivatives bearing a long (i.e., > C8) hydrophilic chain in order to
evaluate the potential of such modification for interference with 5-LO activity. Furthermore,
we aimed to obtain broader insights into the relationship between the molecular structure and
the biological activity against 5-LO of the most active quinone derivatives (25, 26, 27) and
their reduced hydroquinone forms (28, 29, 30). Finally we decided to synthesize different
hydroquinones with the same alkyl chain (33, 34), to evaluate if different positions of the
hydroxyl group would allow for better interference with 5-LO's active site.
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Results and discussion
2.1 Chemistry
Hydroxylated ortho-quinones 10-12 and para-quinones 13-15 were synthesized starting from
1,2,4,5-tetramethoxybenzene, which was subjected to an ortho-metalation reaction in the
presence of n-BuLi and tetramethylethylenediamine (TMEDA). The lithium derivative was
reacted with different alkyl bromides 4-6 giving intermediates 7-9. Silylation of bromo-
alcohols 1-3 was performed with tert-butyl dimethyl silyl chloride furnishing desired
compounds 4-6 with good yields. Cerium ammonium nitrate (CAN)-mediated oxidative
cleavage provided a mixture of 4,5-dimethoxy-1,2-benzoquinones (10-12) and 2,5-
dimethoxy-1,4-benzoquinones (13-15) following a synthetic procedure previously described
by us [10] (Scheme 1). Removal of the protecting group from tetramethoxy intermediates 7,
8, and 9 by acidic cleavage furnished the desired compounds 16, 17, and 18 in good yields
(Scheme 1). The synthesis of compounds 21 and 23 started from 10‐tert‐butyl dimethyl
silanyloxy decanal (19) which was reacted with 1,2,4,5-tetramethoxybenzene following the
same experimental conditions as described above, furnishing compound 20 with 28% yield.
Subsequent CAN-mediated oxidative cleavage at -10°C for 10 min allowed removing the
TBDMS group and oxidizing to obtain para-quinone derivative 21. Under these conditions,
the ortho-quinone analog rapidly decomposed. Acetylation of 20 with acetic anhydride and
further treatment with CAN provided compound 23 with 20% yield (Scheme 2). 4,5-
Dimethoxybenzene-1,2-diols (28, 29 and 30) were obtained from the corresponding ortho-
quinones after treatment with NaBH4 in ethanol (Scheme 3), with the same reaction
conditions we designed and synthesized compounds 33 and 34 to test the influence of
hydroxyl group.
2.2. Evaluation of structure-activity relationships and molecular docking studies
In order to assess the effects of the synthesized compounds on 5-LO product synthesis, a cell-
free assay using isolated human recombinant 5-LO and a cell-based assay using human
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neutrophils were applied. The cell-free assay identifies compounds that directly interfere with
5-LO catalytic activity, whereas the cell-based test system considers cellular regulatory
aspects of 5-LO product synthesis as well. As such, the cell-based assay offers several
possible points of attack of a given compound (e.g., inhibition of 5-LO-activating protein
(FLAP) or coactosine-like protein (CLP), interference with lipid hydroperoxides, protein
kinases, Ca+2 mobilization, and 5-LO translocation) [4]. The reference 5-LO inhibitor N-[1-
(1-benzothien-2-yl)ethyl]-N-hydroxyurea (zileuton) was used to control the 5-LO activity
assays.
Results for inhibition of 5-LO in cell-free assays and in intact cells by the test compounds, as
well as more detailed concentration-response curves are presented in Tables 1 to 3 and in
Figures 2 and 3. Of note, the ortho-quinone 27 and its reduced form 30, equipped with a C13
n-alkyl chain lacking hydroxyl groups, were revealed as most potent direct 5-LO inhibitors
with IC50 values in cell-free assays of 10 and 60 nM, respectively. Interestingly, variation of
the substrate concentration in cell-free assays revealed no competition of 27 or 30 with AA,
instead inhibition of 5-LO by the test compounds was even enhanced at higher AA
concentrations (≥ 20 µM) especially for the potent ortho-quinone 27 (Figure 4), suggesting a
non- or uncompetitive rather than a competitive mode of action. From this result, we
concluded that the compounds do not bind to the AA binding site but interfere at a different
point of attack with 5-LO activity.
To locate the alternative binding site for the quinones in 5-LO, we performed a pocket finder
calculation implemented in MOE [19]. The pocket finder identified two alternative cavities
that could act as binding sites for the quinones: a wide cavity on the opposite side of the
substrate channel around and a canyon-like site situated between the membrane binding C2
domain and the catalytic domain (Figure 5). The latter is positioned near a binding site
suggested for pirinixic acid derivatives [20] and ideally shaped to accommodate the long
lipophilic n-alkyl chains with polar groups at the ω position to form hydrogen bond
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interactions at the end. In the docking, the most common interaction partners are Arg101 and
Gln141 on one side of the channel and Tyr383, Tyr81, Trp102, and Glu622 on the other
(Figure 6A). Typically, two or more of these residues form hydrogen bonds with the oxygen
atoms from the substituted quinone ring, as shown in Figure 6 for compounds 15, 23, and 27.
The additional hydroxyl group at the ω-end of the n-alkyl chain in para-quinones (15 and 23)
forms hydrogen bonds with either Arg101 or Gln141 in the docking simulation, but
apparently does not improve the 5-LO inhibitory activity in cell-free assays (Table 2). As
shown in Table 1, also introduction of the hydroxyl group in the ω-position of the n-alkyl
chain of ortho-quinones resulted in a decrease of potency (for example, 26 vs 12 and 25 vs
11). Of interest, however, is compound 23 that in addition to the ω-hydroxyl contains an
acetyl group at C-1 of the n-alkyl chain which confers potent inhibition of 5-LO in cell-free
assays (Table 2). It is interesting to note that the 5-LO inhibitory activity of the compounds in
the cell-based test system was not diminished by hydroxyl substitution, in particular, efficient
5-LO inhibitory activity was evident for compound 12 (IC50 = 120 nM).
The ortho-quinones were sometimes considerably more active than the para-quinone
derivatives (i.e., 10 vs 13, 11 vs 14). In the docking simulation the ortho-quinones formed
interactions with Tyr383, which were not predicted for the para-quinones, due to a slightly
different tilt of the ring. This confirms our previous finding that more favorable interactions of
the ortho-quinone versus the para-quinone scaffold in the binding of 5-LO may exist [10]. On
the other hand, additional interactions of the ortho-quinones may contribute to inhibition of 5-
LO product synthesis, for example interaction with other targets (i.e. FLAP, CLP, p38MAPK,
ERK or CaMKII).
The 5-LO inhibitory potencies of the compounds are in line with computational predictions,
as shown in Figure 6. The docking simulation suggests that the substituted ring of the
quinones is usually positioned at the polar solvent accessible region of the binding pocket,
forming hydrogen bonds with Tyr383, Tyr81, Trp102, and Glu622 (Figure 6B). The
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lipophilic n-alkyl chain fills the hydrophobic grove that leads to the other opening. Arg101
and Gln141 serve as anchor points for the hydroxyl group present in some of the derivatives
(Figure 6C). The carboxylate group added in compound 23 does not form additional
hydrogen bonds, however both the hydroxyl group at the alkyl chain and the ring seem to be
ideally positioned for interaction with Gln141, Tyr81, and Glu622 (Figure 6D).
With the accepted general concept that quinones are reduced in situ in the intracellular
environment, where high concentration of GSH create a reducing milieu, the formed active
hydroquinones are able to scavenge radicals and therefore act also as antioxidants [21-22]. In
the case of the hydroquinones 28, 29, and 30, they showed a comparable activity against
isolated 5-LO versus the corresponding quinones (Figures 2, 3); under cell-based conditions,
30 was highly active with an IC50 of 28 nM. Because both the quinone core and the n-alkyl
chain play an important role in 5-LO inhibition, we were interested in derivatives of 30 in
order to explore the influence of the hydroquinone nucleus. Compounds 33 and 34, with the
optimal length of the n-alkyl chain (i.e. 13 carbons) but altered methylation pattern of the
hydroxyl groups, demonstrated a clear decrease of activity, especially in cell-based assays
(IC50 = 1600 nM for 33 and 310 nM for 34, versus 30 with IC50 = 28 nM; Table 3 and Figure
3). Molecular docking studies support a similar binding mode for 30, 33 and 34, which
explore equivalent spaces of the binding cavity (Fig. 7).
2.3 Anti-inflammatory effectiveness of derivatives 27, 30, in carrageenan-induced paw edema
The ortho-quinone 27 and its reduced form 30 were the most potent inhibitors of 5-LO in cell-
based systems within this series. Thus, we investigated their effectiveness in two different in
vivo models of acute inflammation. First, we evaluated their effect on carrageenan-induced
paw oedema, which represents a well-established model of acute inflammation [23].
Intraplantar injection of carrageenan led to an increase in oedema formation, expressed as
increase in hind paw volume, with a maximum reached at 4 hrs post-carrageenan application,
a second peak at 48 hrs, and remaining elevated up to 72 hrs (Figure 8). Compound 27 (1
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mg/kg, i.p.) significantly reduced paw oedema during the early (4 hrs) and late (48 hrs) time
points. Furthermore, in agreement with the results from intact cells, the corresponding
hydroquinone 30 was efficient in the in vivo study, in particular at the later phase. Thus,
treatment of mice with 1 mg/kg 27 or 30 reduced paw volume at 4 hrs post carrageenan
equally well (by 75% and 77.4%, respectively), while 30 was more efficient in repressing
oedema formation at 48 and 72 hrs versus 27.
2.4 Anti-inflammatory effectiveness of derivatives 27 and 30 in zymosan-induced peritonitis
We investigated the effectiveness of 27 and 30 in another model of acute inflammation,
namely zymosan-induced mouse peritonitis, which is strongly related to the formation of LTs.
Compounds 27 and 30 (1 mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30 min
after zymosan, outperforming zileuton at a dose of 10 mg/kg, i.p. (Table 4). After 4 hrs post
zymosan 27 and 30 (1 mg/kg, i.p.) prevented cell infiltration about equally well to zileuton at
10 mg/kg, i.p (Table 4).
3. Conclusions
In this report we present the synthesis, structure determination, and the in vitro and in vivo
evaluation of a series of new quinone analogues aiming to find novel lead structures for the
development of 5-LO inhibitors. Our findings support that also the hydroquinone scaffold can
interact with high-affinity with 5-LO in a specific manner exhibiting high effectiveness in
inflammatory models in vivo. We suggest that these compounds might represent privileged
structures for the selective modulation of LT biosynthesis as well as for obtaining more potent
and target-focused anti-inflammatory drugs.
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4. Experimental section
4.1 Chemistry
All reagents were analytical grade and purchased from Sigma–Aldrich (Milano, Italy). Flash
chromatography was performed on Carlo Erba silica gel 60 (230–400 mesh; CarloErba,
Milan, Italy). TLC was carried out using plates coated with silica gel 60F 254 nm purchased
1
from Merck (Darmstadt, Germany). H and 13C NMR spectra were registered on a Brucker
AC 300. Chemical shifts are reported in ppm. The abbreviations used are follows: s, singlet;
d, doublet; dd double doublet; bs, broad signal. MS spectrometry analysis ESI-MS was
carried out on a Finnigan LCQ Deca ion trap instrument. Microanalyses were carried out on a
Carlo Erba 1106 elemental analyzer. Melting points were performed by Stuart melting point
SMP30 and are uncorrected.
The purity (95% or higher) of all final products that were evaluated for bioactivity was
assessed by HPLC. The analytical HPLC analyses were carried out on Beckman Coulter 125
S, equipped with two high pressure binary gradient delivery systems, a System Gold 166
variable-wavelength UV e vis detector and a Rheodyne 7725i injector (Rheodyne, Inc.,
Cotati, CA, USA) with a 20-mL stainless steel loop.
For the analytical tests, compounds were prepared dissolving in methanol (0.5 mg/mL). Each
solution (20 mL) was injected in a Jupiter Phenomenex RP 18 (4.5 _ 250 mm) analytical
column. The mobile phase was a combination mixture of H2O + 0.1% TFA (solvent A) and
CH3CN + 0.1% TFA (solvent B). The elution was made in gradient from 5% of B to 71% in
35 min, the flow rate was 1.0 mL/min.
4.2 General procedure for synthesis of compounds 4, 5 and 6
To a solution of compounds1, 2 or 3 (4.0 mmol) in CH2Cl2 (12 mL), stirred at 0 °C, imidazole
(1.5 eq.), DMAP (0.1 eq.) and TBDMSCl (1.2 eq.) were added. The mixture was stirred over
night at room temperature, washed with water and brine. Organic layer was dried over MgSO4
and the solvent was evaporated under reduced pressure. Crude mixture was purified through
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flash chromatography eluting with hexane/ethyl acetate 8:2, affording desired product as
colorless oil.
4.2.1 (8-bromooctyloxy)(tert-butyl)dimethylsilane (4)
Yield: 76%. 1H NMR (CDCl3, 300 MHz): δ: 0.07 (s, 6H); 0.92 (s, 9H); 1.3 (s, 6H); 1.41-1.60
(m, 4H); 1.87 (m, 2H); 3.43 (t, 2H, J=6Hz); 3.62 (t, 2H, J=6Hz).
4.2.2 (10-bromodecyloxy)(tert-butyl)dimethylsilane (5)
1
Yield: 85%. H NMR (CDCl3, 300 MHz): δ: 0.06 (s, 6H); 0.89 (s, 9H); 1.3 (s, 10H); 1.41-
1.60 (m, 4H); 1.85 (m, 2H); 3.43 (t, 2H, J=6Hz); 3.62 (t, 2H, J=6Hz).
4.2.3 (11-bromoundecyloxy)(tert-butyl)dimethylsilane (6)
1
Yield: 57%. H NMR (CDCl3, 300 MHz): δ: 0.07 (s, 6H); 0.92 (s, 9H); 1.3 (s, 12H); 1.41-
1.60 (m, 4H); 1.87 (m, 2H); 3.43 (t, 2H, J=6Hz); 3.62 (t, 2H, J=6Hz).
4.3 General procedure for synthesis of compounds 7, 8 and 9
To a solution of tetramethoxybenzene (350 mg, 1.7 mmol, 1 eq.) [23]. and TMEDA (1.1 eq.)
in anhydrous THF (7.5 mL) was added dropwise a solution of n-BuLi (1.2 eq.) at -20°C. The
reaction was stirred for 30 min and then was warmed to -10 °C and stirred at this temperature
for 30 min. The alkyl halide 4-6 (1.05 eq) was added dropwise, and the reaction was stirred
overnight at room temperature. Saturated NH4Cl was added to the reaction, and the aqueous
phase was extracted with diethyl ether. The organic phases were combined and dried over
MgSO4. Removal of the organic solvent under reduced pressure afforded crude mixture that
was purified through flash chromatography eluting with hexane/ethyl acetate 95:5.
4.3.1 (8-(2,3,5,6-tetramethoxyphenyl)octyloxy)(tert-butyl)dimethylsilane (7)
Yield: 59%. 1H NMR (CDCl3, 300 MHz): δ: 0.06 (s, 6H); 0.89 (s, 9H); 1.3 (s, 6H); 1.41-1.60
(m, 4H); 1.65 (m, 2H); 2.61 (t, 2H, J=8Hz); 3.61 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.87 (s, 6H);
6.43 (s, 1H).
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4.3.2 (10-(2,3,5,6-tetramethoxyphenyl)decyloxy)(tert-butyl)dimethylsilane (8)
1
Yield: 37%. H NMR (CDCl3, 300 MHz): δ: 0.06 (s, 6H); 0.89 (s, 9H); 1.3 (s, 10H); 1.41-
1.60 (m, 4H); 1.65 (m, 2H); 2.63 (t, 2H, J=8Hz); 3.62 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.87 (s,
6H); 6.43 (s, 1H).
4.3.3 (11-(2,3,5,6-tetramethoxyphenyl)undecyloxy)(tert-butyl)dimethylsilane (9)
1
Yield: 63%. H NMR (CDCl3, 300 MHz): δ: 0.06 (s, 6H); 0.90 (s, 9H); 1.3 (s, 12H); 1.41-
1.60 (m, 4H); 1.63 (m, 2H); 2.61 (t, 2H, J=8Hz); 3.62 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.87 (s,
6H); 6.43 (s, 1H).
4.4 General procedure for synthesis of compounds 10-15
To a solution of compounds 7-9 (0.5 mmol) in CH3CN (6 mL), was added a solution of CAN
(2.5 mmol) in CH3CN-H2O (11 mL, 7:3) dropwise at -10 °C (salt-ice bath). The reaction was
stirred for 10 minutes at -10 °C and diluted with water and extracted with ethyl acetate. The
organic layer was washed two times with distilled water, dried over Na2SO4, and concentrated
in vacuo. The residue was purified over silica gel using hexane:EtOAc (50:50) as eluent to
give desired compounds.
4.4.1 3-(8-hydroxyoctyl)-4,5-dimethoxycyclohexa-3,5-diene-1,2-dione (10)
1
Yield: 36%. H NMR (CDCl3, 300 MHz): δ: 1.26 (s, 10H); 1.54-1.58 (m, 2H); 2.41 (t, 2H,
J=8Hz); 3.64 (t, 2H, J=6Hz); 3.92 (s, 6H); 5.70 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 23.4,
25.7, 29.3, 32.7, 56.8, 62.0, 63.0, 101.3, 102.2, 130.5, 159.0, 166.4, 178.7, 180.7. ESI(m/z):
297.3 [M+ + 1].
4.4.2 3-(10-hydroxydecyl)-4,5-dimethoxycyclohexa-3,5-diene-1,2-dione (11)
1
Yield: 45%. H NMR (CDCl3, 300 MHz): δ: 1.28 (s, 14H); 1.54-1.58 (m, 2H); 2.41 (t, 2H,
J=8Hz); 3.64 (t, 2H, J=6Hz); 3.91 (s, 6H); 5.69 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 22.2,
25.8, 27.9, 32.9, 56.2, 61.6, 69.7, 103.0, 105.4, 128.1, 160.0, 160.8, 181.1. ESI(m/z): 325.4
[M+ + 1].
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4.4.3 3-(11-hydroxyundecyl)-4,5-dimethoxycyclohexa-3,5-diene-1,2-dione (12)
1
Yield: 37%. H NMR (CDCl3, 300 MHz): δ: 1.26 (s, 16H); 1.54-1.58 (m, 2H); 2.41 (t, 2H,
J=8Hz); 3.64 (t, 2H, J=6Hz); 3.91 (s, 6H); 5.69 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 22.6,
25.7, 28.0, 29.4, 32.7, 50.8, 56.7, 63.1, 102.2, 119.3, 151.7, 161.1, 181.7, 182.9. ESI(m/z):
361.3 [M+ + 23].
4.4.4 3-(8-hydroxyoctyl)-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (13)
1
Yield: 36%. H NMR (CDCl3, 300 MHz): δ: 1.30 (s, 10H); 1.54-1.58 (m, 2H); 2.44 (t, 2H,
J=8Hz); 3.66 (t, 2H, J=6Hz); 3.80 (s, 3H); 4.04 (s, 3H); 5.73 (s, 1H). 13C NMR (CDCl3, 300
MHz): δ 23.0, 25.7, 28.6, 29.4, 32.7, 56.4, 61.4, 63.0, 105.4, 130.6, 155.9, 158.7, 182.5,
183.6. ESI(m/z): 319.6 [M+ + 23].
4.4.5 3-(10-hydroxydecyl)-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (14)
1
Yield: 28%. H NMR (CDCl3, 300 MHz): δ: 1.28 (s, 14H); 1.54-1.58 (m, 2H); 2.44 (t, 2H,
J=8Hz); 3.64 (t, 2H, J=6Hz); 3.82 (s, 3H); 4.05 (s, 3H); 5.74 (s, 1H). 13C NMR (CDCl3, 300
MHz): δ 22.9, 25.7, 28.6, 29.4, 33.2, 56.4, 61.4, 63.0, 104.9, 130.6, 155.9, 158.7, 182.5,
183.6. ESI(m/z): 325.4 [M+ + 1].
4.4.6 3-(11-hydroxyundecyl)-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (15)
1
Yield: 26%. H NMR (CDCl3, 300 MHz): δ: 1.29 (s, 16H); 1.54-1.58 (m, 2H); 2.44 (t, 2H,
J=8Hz); 3.66 (t, 2H, J=6Hz); 3.82 (s, 3H); 4.07 (s, 3H); 5.75 (s, 1H). 13C NMR (CDCl3, 300
MHz): δ 23.1, 25.7, 28.6, 29.4, 32.8, 56.4, 61.4, 63.0, 105.4, 130.7, 155.9, 158.7, 182.4,
183.6. ESI(m/z): 339.5 [M+ + 1].
4.5 General procedure for synthesis of compounds 16-18
A solution of appropriate tert-butyl-dimethylsilane 7-9 (0.3 mmol) in a mixture of THF
(5mL), H2O (1.4 mL) and HCOOH (4.2 mL) was stirred at room temperature for 30 min.
Saturated NaHCO3 was added and the aqueous phase was extracted with ethyl acetate. The
organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The
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residue was purified over silica gel using hexane:EtOAc (80:20-50:50) as eluent to give
desired compounds colorless oils.
4.5.1 8-(2,3,5,6-tetramethoxyphenyl)octan-1-ol (16)
1
Yield: 51%. H NMR (CDCl3, 300 MHz): δ: 1.3 (s, 8H); 1.41-1.60 (m, 4H); 2.61 (t, 2H,
J=8Hz); 3.62 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.85 (s, 6H); 6.42 (s, 1H). 13C NMR (CDCl3, 75
MHz): δ:24.6, 25.7 (2C), 29.3 (2C), 29.4, 30.7, 32.7, 32.8 (2C), 56.2, 60.9, 63.0, 96.5, 131.0,
140.9 (2C), 148.8 (2C). ESI(m/z): 327.8 [M+ + 1].
4.5.2 10-(2,3,5,6-tetramethoxyphenyl) decan-1-ol (17)
1
Yield: 56%. H NMR (CDCl3, 300 MHz): δ: 1.3 (s, 12H); 1.41-1.60 (m, 4H); 2.61 (t, 2H,
J=8Hz); 3.62 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.85 (s, 6H); 6.42 (s, 1H). 13C NMR (CDCl3, 75
MHz): δ:24.6, 25.7 (2C), 29.3 (4C), 29.4, 30.7, 32.7, 32.8 (2C), 56.2, 60.9, 63.0, 96.5, 131.0,
140.9 (2C), 148.8 (2C) ESI(m/z): 377.5 [M+ + 23].
4.5.3 11-(2,3,5,6-tetramethoxyphenyl)undecan-1-ol (18)
1
Yield: 53%. H NMR (CDCl3, 300 MHz): δ: 1.3 (s, 14H); 1.41-1.60 (m, 4H); 2.61 (t, 2H,
J=8Hz); 3.62 (t, 2H, J=6Hz); 3.79 (s, 6H); 3.85 (s, 6H); 6.42 (s, 1H). 13C NMR (CDCl3, 75
MHz): δ:24.6, 25.7 (3C), 29.3 (4C), 29.4, 30.7, 32.7, 32.8 (2C), 56.2, 60.9, 63.0, 96.5, 131.0,
140.9 (2C), 148.8 (2C) ESI(m/z): 391.4 [M+ + 23].
4.5.4 (10-hydroxy-10-(2,3,5,6-tetramethoxyphenyl)decyloxy)(tert-butyl)dimethylsilane (20)
To a solution of tetramethoxybenzene (210mg, 1.03 mmol, 1 eq.). [24] and TMEDA (1.1 eq.)
in anhydrous THF (7.5 mL) was added dropwise a solution of n-BuLi (1.05 eq.) at -50 °C.
The reaction was stirred for 30 min and then was warmed to -10 °C and stirred at this
temperature for 30 min. A solution of 10‐tert‐butyl dimethyl silanyloxy decanal (19) [25]
(1.05 eq) in 5 mL of dry THF was added dropwise, and the reaction was stirred overnight at
room temperature. Saturated NH4Cl was added to the reaction, and the aqueous phase was
extracted with diethyl ether. The organic phases were combined and dried over MgSO4.
Removal of the organic solvent under reduced pressure afforded crude mixture that was
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purified through flash chromatography eluting with hexane/ethyl acetate 90:10. Desired
compound was obtained as a colorless oil (Yield: 28%). 1H NMR (CDCl3, 300 MHz): δ: 0.06
(s, 6H); 0.89 (s, 9H); 1.3 (s, 10H); 1.41-1.60 (m, 4H); 1.65 (m, 2H); 3.62 (t, 2H, J=6Hz); 3.79
(s, 6H); 3.87 (s, 6H); 4.91 (m, 1H); 6.43 (s, 1H).
4.5.5 3-(1,10-dihydroxydecyl)-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (21)
To a solution of compounds 20 (60 mg, 0.125 mmol) in CH3CN (1.4 mL), was added a
solution of CAN (2.5 mmol) in CH3CN-H2O (2.6 mL, 7:3) dropwise at -10 °C (salt-ice bath).
The reaction was stirred for 10 minutes at -10 °C and diluted with water and extracted with
ethyl acetate. The organic layer was washed two times with distilled water, dried over
Na2SO4, and concentrated in vacuo. The residue was purified over silica gel using petroleum
ether:EtOAc (15:85) as eluent to give desired compound as orange oil (Yield: 21%). 1H NMR
(CDCl3, 300 MHz): δ: 1.3 (s, 10H); 1.41-1.60 (m, 4H); 1.65 (m, 2H); 3.62 (t, 2H, J=6Hz);
3.79 (s, 3H); 4.12 (s, 3H); 4.78 (m, 1H); 5.76 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 23.2,
25.7, 29.0, 30.8, 32.8, 58.3, 62.2, 105.5, 131.6, 155.9, 158.7, 182.1, 183.0. ESI(m/z): 341.5
[M+ + 1].
4.5.6 10-acetoxy-10-(2,3,5,6-tetramethoxyphenyl)decyloxy)(tert-butyl)dimethylsilane (22)
Acetic anhydride (0.119 mL, 1.24 mmol, 4.0 eq.) TEA (0.087 mL, 0.622 mmol, 2.0 eq.) and
DMAP (8mg, 0.065 mmol, 0.21 eq.) were added to a stirred solution of 20 ( 148mg, 0.311
mmol) in anhydrous diethyl ether (2.0 mL) under nitrogen atmosphere. The reaction mixture
was stirred for 2 h at room temperature. Saturated NaHCO3 was added and the aqueous phase
was extracted with diethyl ether. The organic phases were combined and dried over MgSO4.
Removal of the organic solvent under reduced pressure afforded crude mixture that was
purified through flash chromatography eluting with hexane/ethyl acetate 80:20. Desired
compound was obtained as a colorless oil (Yield: 71%). 1H NMR (CDCl3, 300 MHz): δ: 0.06
(s, 6H); 0.89 (s, 9H); 1.3 (s, 10H); 1.41-1.60 (m, 4H); 1.65 (m, 2H); 2.09 (s, 3H); 3.62 (t, 2H,
J=6Hz); 3.85 (s, 6H); 3.87 (s, 6H); 6.27 (m, 1H); 6.50 (s, 1H).
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4.5.7 3-(1-acetoxy-10-hydroxydecyl)-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (23)
To a solution of compounds 22 (114 mg, 0.218 mmol, 1eq.) in CH3CN (2 mL), was added a
solution of CAN (2.5 mmol) in CH3CN-H2O (3.7 mL, 7:3) dropwise at -10 °C (salt-ice bath).
The reaction was stirred for 10 minutes at -10 °C and diluted with water and extracted with
ethyl acetate. The organic layer was washed two times with distilled water, dried over
Na2SO4, and concentrated in vacuo. The residue was purified over silica gel using petroleum
1
ether:EtOAc (15:85) as eluent to give desired compound a yellow oil (Yield: 20%). H NMR
(CDCl3, 300 MHz): δ: 1.3 (s, 10H); 1.41-1.60 (m, 4H); 1.65 (m, 2H); 2.09 (s, 3H); 3.62 (t,
2H, J=6Hz); 3.81 (s, 3H); 4.11 (s, 3H); 5.77 (s, 1H); 5.82 (m, 1H). 13C NMR (CDCl3, 300
MHz): δ 19.9, 23.2, 25.7, 29.0, 30.8, 32.8, 58.3, 63.5, 105.5, 131.6, 155.9, 158.7, 170.8,
182.1, 183.0. ESI(m/z): 383.6 [M++1].
4.6 General procedure for synthesis of compounds 28, 29, 30
To a suspension of starting (0.092 mmol) in ethanol (3.0 mL), cooled at 0°C, NaBH4 was
slowly added (2.5 eq.). The mixture was reacted for 1 h at the same temperature. The excess
of NaBH4 was neutralized with HCl 1M, the mixture was diluted with water and extracted
three times with diethyl ether. The organic phases were combined and dried over MgSO4.
Removal of the organic solvent under reduced pressure afforded crude mixture that was
purified through flash chromatography eluting with hexane/ethyl acetate 70:30.
4.6.1 3-undecyl-4,5-dimethoxybenzene-1,2-diol (28)
1
Yield: 83%. H NMR (CDCl3). δ 0.90 (t, 3H, J = 8 Hz); 1.37 (s, 16H); 1.60 (m, 2H); 2.68 (t,
2H, J= 8 Hz); 3.81 (s, 6H); 4.58 (OH); 5.36 (OH), 6.42 (s, 1H). 13C NMR (CDCl3, 75 MHz):
δ: 14.1, 23.1, 24.8, 28.1, 28.3, 29.4, 29.5, 29.7, 30.0, 32.1 (2C), 56.1, 60.1, 98.7, 124.3, 135.2,
139.5, 140.2, 147.8. ESI(m/z): 347.1 [M++23].
4.6.2 3-dodecyl-4,5-dimethoxybenzene-1,2-diol (29)
1
Yield: 63%. H NMR (CDCl3). δ 0.90 (t, 3H, J = 8 Hz); 1.37 (s, 18H); 1.52 (m, 2H); 2.62 (t,
2H, J= 8 Hz); 3.78 (s, 6H); 4.83 (OH); 5.62 (OH), 6.42 (s, 1H). 13C NMR (CDCl3, 75 MHz):
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δ: 14.1, 23.1, 24.8, 28.1, 28.3, 29.4, 29.5, 29.7 (2C), 30.0, 32.1 (2C), 56.1, 60.1, 98.7, 124.3,
135.2, 139.5, 140.2, 147.8. ESI(m/z): 361.3 [M++23].
4.6.3 3-tridecyl-4,5-dimethoxybenzene-1,2-diol (30)
1
Yield: 40%. H NMR (CDCl3, 300 MHz): δ 0.90 (t, 3H, J = 8 Hz); 1.37 (s, 20H); 1.52 (m,
2H); 2.62 (t, 2H, J= 8 Hz); 3.78 (s, 6H); 4.73 (OH); 5.80 (OH), 6.42 (s, 1H). 13C NMR
(CDCl3, 75 MHz): δ: 14.1, 23.1, 24.8, 28.1, 28.3 (2C),, 29.4 (2C), 29.5, 29.7, 30.0, 32.1 (2C),
56.1, 60.1, 98.7, 124.3, 135.2, 139.5, 140.2, 147.8. ESI(m/z): 353.4 [M++1].
4.6.4 5-methoxy-3-tridecylbenzene-1,2,4-triol (33)
Yield: 80%. 1H NMR (CDCl3, 300 MHz): δ 0.90 (t, 3H); 1.28 (s, 20H); 1.52 (m, 2H); 2.67 (t,
2H Hz); 3.8 (s, 3H); 5.34 (OH), 6.42 (s, 1H).
13C NMR (CDCl3, 300 MHz): δ: 14.1, 22.7, 23.9, 28.1, 29.5, 32.0, 56.6, 102.2, 117.0, 136.0,
137.7, 139.9, 161.1. ESI(m/z): 339.6 [M++1].
4.6.5 2,5-dimethoxy-3-tridecylbenzene-1,4-diol (34)
Yield: 70%. 1H NMR (CDCl3, 300 MHz): δ 0.92 (t, 3H); 1.31 (s, 20H); 1.56 (m, 2H); 2.62 (t,
2H); 3.72 (s, 3H); 3.79 (s, 3H); 6.37 (s, 1H).
13C NMR (CDCl3, 300 MHz): δ 14.1, 22.7, 24.5, 29.6, 31.9, 56.2, 61.8, 96.7, 122.4, 137.1,
138.9, 141.5, 143.0 ESI(m/z): 375.2 [M++23].
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4.7 Biological evaluation and assay systems
4.7.1. Materials
Zileuton was purchased from Sequoia Research Products (Oxford, UK), zymosan from Sigma
(Milan, Italy) and LTC4 enzyme immunoassay (EIA) from Cayman Chemical,
(Inalco, Milan, Italy).
4.7.2. Cells and isolation
Neutrophils were freshly isolated from leukocyte concentrates obtained from the Institute of
Transfusion Medicine, University Hospital Jena as described [26]. Briefly, human peripheral
blood was collected in heparinized tubes (16 I.E. heparin/ml blood) by venipuncture from
fasted (12 h) adult healthy volunteers, with consent, and leukocyte concentrates were prepared
by centrifugation (4000 x g, 20 min, 20 °C). The subjects had no apparent inflammatory
conditions and had not taken anti-inflammatory drugs for at least ten days prior to blood
collection. Neutrophils were immediately isolated by dextran sedimentation and
centrifugation on Nycoprep cushions (PAA Laboratories, Linz, Austria) and hypotonic lysis
of erythrocytes was performed as described [26]. Neutrophils were finally resuspended in
PBS pH 7.4 containing 1 mg/ml glucose and 1 mM CaCl2 (PGC buffer) (purity > 96-97%).
4.7.3 Animals
Male CD-1 mice (35–40 g; Charles River) (30 rats, 50 mice) were housed at the Department
of Pharmacy (Naples, Italy) in a controlled environment (23 °C, humidity range of 40–70%
and 12 h light/dark cycles) and provided with standard rodent chow and water. Animals were
allowed to acclimatize for 4 days before the experiments and were subjected to a 12 h light–
12 h dark schedule. Experiments were conducted during the light phase. Animal care was in
compliance with Italian regulations on protection of animals used for experimental and other
scientific purpose (Ministerial Decree 116/92) as well as with the European Economic
Community regulations (Official Journal of the European Community L 358/1 12/18/1986).
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4.7.4 Mouse paw oedema
Mice were lightly anaesthetized by inhalation of enflurane and depth of anesthesia was
assessed by checking both abdominal and pedal withdrawal reflex. They were then given a
subplantar injection of 50 ꢀL of carrageenan 1% (w v−1). Paw volume was measured using a
hydroplethismometer specially modified for small volumes (Ugo Basile, Milan, Italy)
immediately before subplantar injection (basal value) and 2, 4, 6, 24, 48 and 72 h thereafter.
Mice were divided into seven groups (n = 6) and received an i.p. injection of compounds or
vehicle (DMSO), 30 min before the carrageenan injection.
4.7.5 Zymosan-Induced Peritonitis in Mice
For zymosan-induced peritonitis in mice, 27, 30 or zileuton at the indicated dose or vehicle
(0.5 mL of 0.9% saline solution containing 2% DMSO) was given i.p. 30 min before zymosan
i.p. injection (0.5 mL of suspension of 2 mg/mL in 0.9% w/v saline). Mice were killed by
inhalation of CO2 at 30 min and 4 h after zymosan injection, for LTC4 evaluation and cell
infiltration, respectively [27] Exudates were collected by peritoneal lavage with 2 mL of cold
PBS, the cells in the exudates were counted and LTC4 was measured by EIA.
4.7.6 5-LO activity assays
For analysis of 5-LO products in intact cells, neutrophils (5 × 106) were resuspended in 1 ml
PGC buffer, preincubated for 15 min at 37 °C with test compounds or vehicle (0.1% DMSO)
and Ca2+-ionophore A23187 (2.5 µM) plus 20 µM arachidonic acid was added. After 10 min
at 37 °C the reaction was stopped on ice by addition of 1 ml of methanol. 30 µl 1 N HCl and
500 µl PBS, and 200 ng prostaglandin (PG)B1 were added and the samples were subjected to
solid phase extraction on C18-columns (100 mg, UCT, Bristol, PA, USA). 5-LO products
(LTB4 and its trans-isomers, and 5-H(P)ETE) were analyzed by RP-HPLC and quantities
calculated on the basis of the internal standard PGB1. Cys-LTs C4, D4 and E4 were not
detected (amounts were below detection limit), and oxidation products of LTB4 were not
determined.
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For analysis of 5-LO activity in cell-free assays, E. coli BL21 cells were transformed with
pT3-5-LO plasmid (provided by Dr. Olof Radmark, Karolinska Institute, Stockholm, Sweden),
recombinant 5-LO protein was expressed and purified on an ATP-agarose column as
described previously [28]. Aliquots of semi-purified 5-LO (0.5 µg) were diluted with ice-cold
PBS containing 1 mM EDTA, and 1 mM ATP was added (final volume was 1 ml). Samples
were pre-incubated with the test compounds or vehicle (0.1% DMSO) as indicated. After 15
min at 4 °C, samples were pre-warmed for 30 sec at 37 °C, and 2 mM CaCl2 plus the
indicated concentrations of arachidonic acid were added to initiate 5-LO product formation.
After 10 min at 37 °C, the reaction was stopped by addition of 1 ml ice-cold methanol, and the
formed metabolites were analyzed by RP-HPLC as described [29] 5-LO products include the
all-trans isomers of LTB4 and 5-H(P)ETE.
4.8 Docking protocol
The crystal structure 3o8y was prepared by correcting four virtual mutations (Glu13Trp,
His14Phe, Gly75Trp, and Ser76Leu) to represent the wild type enzyme rather than the
crystallized stable 5-LO [30]It was then energetically minimized in Discovery Studio (version
3.5, Biovia Inc. www.biovia.com), and 52 hydrogen atoms were added for docking. The
docking experiments were performed using GOLD version 5.2 [31] with GoldPLP as scoring
function. The binding site was located using the Pocketfinder tool within MOE[32] For the
docking, the binding site was defined around Ile167 in a 10 Å radius. All other settings were
kept default. Protein-ligand interactions were visualized using LigandScout 3.1[33]
4.9 Statistical analysis
Data obtained are expressed as mean ± S.E. of single determinations performed in three or
four independent experiments at different days. IC50 values were graphically calculated from
averaged measurements at 4-5 different concentrations of the compounds using SigmaPlot
12.0 (Systat Software Inc., San Jose, USA). Statistical evaluation of the data was performed
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by one-way ANOVA followed by a Bonferroni or TukeyeKramer post-hoc test for multiple
comparisons respectively. A p value <0.05 (*) was considered significant.
Acknowledgements
This work was supported by the grant PON Nutrafast no. 01_01226 sponsored by the
Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) and PON 03 PE_00069_03
Sviluppo e sperimentazione di molecule ad azione nutraceutica e cosmeceutica.
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24

ACCEPTED MANUSCRIPT
O
HO
OH
O
Id
O
O
HO
MeO
MeO
10
10
OH
OH
O
O
Embelin
Idebenone
Fig.1
25

ACCEPTED MANUSCRIPT
A
B
100
75
50
25
0
100
75
50
25
0
28
25
28
25
0.01 0.1
1
10
0.01 0.1
(µM)
1
(µM)
29
26
100
75
50
25
0
100
29
26
75
50
25
0
0.01 0.1
(µM)
1
0.01 0.1
(µM)
1
150
100
50
100
75
50
25
0
0
0.01
0.1
1
0.01 0.1
1
idebenone (µM)
idebenone (µM)
Fig. 2
26

ACCEPTED MANUSCRIPT
A
B
125
100
75
50
25
0
27
30
33
34
27
30
33
34
100
75
50
25
0
0.01 0.1
1
10
(µM)
(µM)
Fig. 3
27

ACCEPTED MANUSCRIPT
cmpd. 27
2 µM
cmpd. 30
2 µM
5 µM
5 µM
100
75
50
25
0
10 µM
20 µM
40 µM
80 µM
100
75
50
25
0
10 µM
20 µM
40 µM
80 µM
0.003
0.03
0.3
0.003
0.03
0.3
(µM)
(µM)
Fig. 4
28