Structure-based design, synthesis and preliminary anti-inflammatory activity of bolinaquinone analogues

Article abstract of DOI:10.1016/j.ejmech.2010.11.028

As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone (BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin E₂ (PGE₂). This optimization process led to the identification of three strictly correlated compounds with comparable and higher inhibitory potency than BLQ on PGE₂ production. To evaluate the affinity of BLQ and its analogues for hsPLA₂, surface plasmon resonance (SPR) experiments were performed.

Full text of DOI:10.1016/j.ejmech.2010.11.028

European Journal of Medicinal Chemistry 46 (2011) 488e496
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based design, synthesis and preliminary anti-inflammatory activity
of bolinaquinone analogues
,
a
a
a
Carmen Petronzi a, Rosanna Filosa a , Antonella Peduto , Maria Chiara Monti , Luigi Margarucci ,
Antonio Massa b, Simona Francesca Ercolino a, Valentina Bizzarro a, Luca Parente a, Raffaele Riccio a,
Paolo de Caprariis a
*
a Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento di Chimica, Università di Salerno, Via Ponte Don Melillo, Stecca 7, 84084 Fisciano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone
(BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new
hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-
like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin
E2 (PGE2). This optimization process led to the identification of three strictly correlated compounds with
comparable and higher inhibitory potency than BLQ on PGE2 production. To evaluate the affinity of BLQ
and its analogues for hsPLA2, surface plasmon resonance (SPR) experiments were performed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 6 April 2010
Received in revised form
15 November 2010
Accepted 17 November 2010
Available online 24 November 2010
Keywords:
Bolinaquinone analogues
hsPLA2
anti-Inflammatory drugs
1. Introduction
A2 include several unrelated protein families with common enzy-
matic activity but with different substrate specificities, cofactor
The search for biologically active natural products from marine
sources continues to be an important scientific field that offers
promising opportunities for the development of new compounds
endowed with pharmacological properties.
Natural products with a quinoide system and a decalin-type or
related aromatic side chain are characterized by pronounced and
several biological properties (Chart 1).
For instance, the marine sesquiterpene quinones such as boli-
naquinone (1) (BLQ) [1], nakijiquinone AeD [2] (2aed) and naki-
jiquinone G-H [3] (2eef), ilimaquinone [4] (3), avarol [5] (4),
smenospongine (5), smenospongidine (6), smenospongiarine [6]
(7) display antimicrobial, antiviral, anti-inflammatory and cyto-
toxic activities [7].
Among this class, the marine metabolite bolinaquinone
expresses its anti-inflammatory activity by inhibition of secretory
phospholipase A2 (sPLA2) [1].
requirements, subcellular localization and cellular functions.
Based on biophysical and biochemical properties more than 15
different forms of PLA2 enzymes have been identified [8].
For their cellular location PLA2 enzymes are classified as cyto-
solic PLA2 (cPLA2), intracellular PLA2 (iPLA2) and secretory PLA2
(sPLA2) [9].
Among the calcium dependent sPLA2, type IIA (sPLA2-IIA) is an
isoform first isolated and purified from rheumatoid synovial fluid.
Increased plasma levels of this enzyme were found in diseases that
involve systemic inflammation such as sepsis, rheumatoid arthritis,
and cardiovascular disease [10,11].
Therefore, compounds that can selectively block secretory PLA2
activity and the assessment of their molecular mechanism of
enzyme inactivation are of paramount importance in the field of
anti-inflammatory drugs.
Among terpenoidic hydroquinone class, avarol was able to
inhibit human recombinant synovial phospholipase A2 activity
Phospholipases A2 (PLA2) are a class of lipolytic enzymes that
catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids
liberating free fatty acids and lysophospholipids [8]. Phospholipases
with an IC50 ¼ 158
mM, while quinone derivative avarone failed to
show inhibitory activity [12]. BLQ, sharing a hydroxyl-p-quinone
moiety connected to a trans-decalin terpene unit in a rearranged
drimane skeleton is one of the most active metabolites with
a selective profile against secretory PLA2’s [1].
* Corresponding author.
E-mail address: rfilosa@unisa.it (R. Filosa).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.028

C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
489
R
Indeed, on the basis of these premises, we decided to rely on
some well-reasoned structural changes of the basic molecule 1, in
the attempt to improve its pharmacological behaviour (Scheme 1).
To generate the chelation of calcium ion responsible of the
competitive inhibition process of hsPLA2, we generated focused
collection of hydroxyl-benzoquinone analogues. Therefore, the
quinone-type building block was not changed entirely but simpli-
fied by elimination of methoxy group.
In order to explore the non-covalent interactions with the large
hydrophobic surface of the active site, thedrimane moiety of BLQ
was replaced with different hydrophobic structures such as
n-pentyl, benzyl, naphthyl and biphenyl units, also they were
shifted from position 3 to 5.
OH
HN
COOH
O
H
O
O
O
OCH3
H
OH
1: Bolinaquinone
2a: R= H
Nakijiquinone A
2b: R= iPr
Nakijiquinone B
2c: R= CH2OH
Nakijiquinone C
2d: R= CH(CH3)OH
Nakijiquinone D
R
OCH3
O
HN
To evaluate the impact of the single linker atom replacement on
the overall profile of these molecules, we have substituted the
carbon-linked of quinoide derivative with an NH or O bridge.
Optimization of the aromatic region was performed by intro-
duction of different sterical hindrance structures as phenyl, naph-
thyl, biphenyl, thianthrenyl and dibenzofuranyl moiety directly
attached to the quinoide core.
The purpose of the present study is to investigate the anti-
inflammatory activity of a series of 2-hydroxy-1,4-benzoquinones,
as well as to define the structureeactivity relationships (SAR) of this
new class. Moreover, preliminary investigations on the compounds
synthesized in their potency against hsPLA2 are evaluated.
This report describes the synthesis of new compounds that ach-
ieved good potency in the biochemical assay and cell-based system.
O
O
O
H
H
OH
OH
3: Ilimaquinone
2e: R=2-(4-imidazolyl)-ethyl
Nakijiquinone G
2f: R= (CH2)4-NH-C(=NH)-NH2
Nakijiquinone H
NHR
HO
H
O
OH
O
H
OH
2. Chemistry
Synthesis of compounds 8aed (Scheme 2) was accomplished in
three steps.
4: Avarol
5: R= H
Smenospongine
6: R= CH2CH2Ph
Smenospongidine
7: R= iPentyl
Commercially available 2,4-dimethoxyphenyl boronic acid (10)
was subjected to Suzuki coupling with several benzylic halides
(11bed), to yield derivatives 12bed. After deprotection,
compounds 13aed were oxidized with Fremy’s salt yielding
quinones 8aed. In the case of derivative 8a we oxidized commer-
cially available n-hexylresorcinol.
Smenospongiarine
Chart 1. Formulas of natural sesquiterpene quinones and hydroquinones.
Compound 8e was synthesized starting from 2,4,5-trimethoxy-
benzaldehyde (14) that was converted, via oxidation [18], to the
phenol derivative 15 which reacted with 2-naphthyl boronic acid
providing diaryl ether derivative 16 (Scheme 3).
Treatment of 16 with ammonium cerium nitrate in acetonitrile
yielded desired compound.
For derivative 8f, 2,4-dimethoxy aniline was submitted to Ull-
mann-type coupling with 2-bromonaphthalene to provide N-(2,4-
dimethoxyphenyl)-naphthyl-2-amine (18). The resulting amine
was deprotected and finally oxidized with Fremy’s salt to give
quinone 8f (Scheme 4).
BLQ shows the ability to inhibit strongly hsPLA2, bee venom PLA2
(bvPLA2), with IC50 values of 0.2 and 0.1 mM, respectively; remark-
ably, this product also exerts no effects on cytosolic PLA2 (cPLA2) [1].
Moreover, bolinaquinone is able to reduce the inflammatory
response of adjuvant arthritis by inhibiting PGE2, NO, and TNF
production in paw homogenates without affecting PGE2 levels in
the stomach [13].
In a recent work [14] we have proposed, for the inactivation
mechanism of hsPLA2 [15] by BLQ, a key process in the inhibition
consisting in the occupation of the active site’s hydrophobic pocket
by the sesquiterpene unit of the inhibitor.
The molecular basis of the human group IIA secretory phospo-
lipase A2 inactivation by BLQ is completely due to a non-covalent
event involving the 2-hydroxy-1,4-benzoquinone responsible for
the chelation with Ca2þ ions in the region of binding [16,17], ori-
enting the inhibitor within the binding site.
Mainly, the oxygen atoms of hydroxyl-quinone system are of
primary importance for the interaction, namely, both the C-1
carbonyl group and the deprotonated C-2 hydroxyl residue coor-
dinate the calcium ion in a bidentate fashion.
For the second set of analogues 9aeg, a different procedure was
followed (Scheme 5).
Commercially available 1-iodo-2,4-dimethoxybenzene (20)
was subjected to Suzuki coupling, under microwave irradiation,
with several boronic acids (21aeg), yielding the corresponding
compounds, which were O-demethylated to provide resorcinol
derivatives (23aeg) that were finally oxidized with Fremy’s salt
yielding quinones.
3. Biological results
Following our extensive investigation on the PLA2 inactivation
mechanism by BLQ we have developed a series of simplified
derivatives related to the natural product.
3.1. Effects of BLQ and its analogues on LPS-induced PGE2
production by LPS-stimulated RAW 264.7 cells
The general design of novel inhibitors against inflammation-
related diseases was guided by the modular composition of this
metabolite.
To assess the effect of BLQ and compounds 8aef and 9aeg on
LPS-induced PGE2 production, RAW 264.7 macrophages cells were

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C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
OH
2
H
O
1
6
3
O
NH
4
O
5
OCH3
BLQ
8a
8b
8c
8d
8e
8f
OH
2
O
3
NO2
1
6
4
OH
Cl
O
5
R1
9a
9b
9c
9d
9e
S
S
O
9f
9g
Scheme 1. Modular composition of the bolinaquinone library.
OH
B
R
O
R
R
O
iii
i
ii
HO
R
Br
MeO
OMe
MeO
OMe
OH
HO
OH
12 b-d
10
11 b-d
13 a-d
8a-d
R = Ph
R = Ph
R = n-Pentyl
R = Ph
R = p-Biphenyl
R = 2-Napht
R = p-Biphenyl
R = 2-Napht
R = p-Biphenyl
R = 2-Napht
Scheme 2. Reagents and conditions: (i) K3PO4, Pd(OAc)2, PPh3, toluene, 110 ꢀC, 16 h; (ii) BBr3, CH2Cl2, ꢁ15 ꢀC to rt, 18 h; (iii) NO(KSO3)2, Na2CO3 THFeH2O, rt, 10 h.
O
MeO
MeO
MeO
MeO
O
O
O
O
MeO
MeO
OH
H
ii
i
iii
OH
OMe
OMe
OMe
14
15
16
8e
Scheme 3. Reagents and conditions: (i) H2O2, H2SO4, MeOH, rt, 2.5 h; (ii) 2-naphthyl boronic acid, Cu(OAc)2, pyridine, CH2Cl2, rt, 48 h; (iii) CAN, CH3CN, rt, 1 h.
treated with various concentrations of analytes for 30 min and PGE2
was induced by co-treatment with LPS (1 g/mL) for another 24 h.
Neither LPS nor samples were added to the control group.
Indomethacin (Indom.) was used as reference. Effects of BLQ and its
analogues on LPS-induced PGE2 production by LPS-stimulated RAW
264.7 cells are summarized in Table 1.
The introduction of an aryl group has been shown to signifi-
cantly affect the potency of such derivatives. As shown in Table 1
the benzyl ring was well tolerated, while replacement with the
biphenyl moiety was found to be deleterious (IC50 values 26.0 for
8b vs 56.7 for 8c). Likewise, the n-hexyl analogue 8a showed
a significant reduction in potency.
m
Analysis of the structureeactivity relationships revealed the
influence of the substituent at position 5 in the 2-hydroxy-1,4-
benzoquinone ring.
In contrast, 5-naphthyl derivatives 8d, 8e and 8f displayed an
IC50 values in the micromolar range which is similar to the value
obtained with BLQ. Besides compound 8d exerted an inhibitory
H
N
H
N
H
O
NH2
N
ii
i
iii
HO
OH
MeO
OMe
O
OH
MeO
OMe
17
18
19
8f
Scheme 4. Reagents and conditions: (i) 2-Bromonaphthalene, Pd(OAc)2, PPh3, t-BuONa, toluene, 110 ꢀC, 40 h; (ii) BBr3, CH2Cl2, ꢁ15 ꢀC to rt, 19 h; (iii) NO(KSO3)2, Na2CO3, THFeH2O,
rt, 2.5 h.

C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
491
R
OH
I
R
OMe
R
O
O
OH
iii
ii
i
R
B
MeO
OMe
HO
MeO
OH
OH
9a-g
20
21a-g
22a-g
23a-g
R = Ph(pCl)
R = Ph(pCl)
R = Ph(pCl)
R = Ph(mNO2)
R = Ph(pOH)
R = 2-Napht
R = p-Biphenyl
R = Ph(mNO2)
R = Ph(pOH)
R = 2-Napht
R = p-Biphenyl
R = Ph(mNO2)
R = Ph(pOH)
R = 2-Napht
R = p-Biphenyl
R = 2-Dibenzofuranyl
R = 2-Thianthrenyl
R = 2-Dibenzofuranyl
R = 2-Thianthrenyl
R = 2-Dibenzofuranyl
R = 2-Thianthrenyl
Scheme 5. Reagents and conditions: (i) K2CO3, PdCl2(dppf), Ethanol, MW, 25 min; (ii) BBr3, CH2Cl2, ꢁ15 ꢀC to rt, 18 h; (iii) NO(KSO3) 2, EtOAceH2O, Na2HPO4, 0 ꢀC to rt, 20 h.
potency higher than the lead compound 1 with an observed IC50 of
4.0 M.
Remarkably 8d inhibitor, as shown in Fig. 1, showed good ability
As expected, the increase of response units (RU) in the associ-
ation phase and the slope of the dissociation phase of the complex
are clearly dependent on the analyte concentration.
m
in reducing LPS-induced PGE2 release with different degrees of
potency. In particular, compound 8d showed a concentration
The analysis of the ascendant region of the sensograms allowed
to extrapolate the ka values, whereas the time required to
completely dissociate the complex and the slope of the curves in
their descending region directly depends on kd of the complex.
On the basis of these experiments, the dissociation constants
KDSPR of the ligand-hsPLA2 complexes were calculated using a 1:1
Langmuir algorithm and summarized in Table 1.
Even if the use of Biacore 2000 was not completely suitable in
the study of small moleculeseprotein interaction, the obtained data
were enough accurate to get preliminary SAR analysis.
dependent inhibition of PGE2 release (ꢁ66% at 10
mM and ꢁ90% at
100 M, panel b).
m
Furthermore a few compounds were synthesized with varia-
tions in the aromatic system directly attached to quinone ring. The
analogues lacking the methyl spacer were inactive (IC50 values from
41.0 to 65.9 for compounds 9aeg) suggesting that the linker
between the hydrophobic pocket and quinone ring is essential for
the activity.
Next, to determine the affinityofBLQ anditsanaloguesfor hsPLA2,
surface plasmon resonance (SPR) experiments were performed.
The analysis with SPR elucidated that for hsPLA2’s interaction
the aromatic groups instead of n-hexyl chain must be present (1 vs
8a). The hsPLA2 interaction would appear to require a bicycle ring in
3.2. Surface plasmon resonance experiments
SPR is an optical technique suitable for characterizing macro-
molecular interactions [19,20] based on the evanescent wave
phenomenon to measure changes in refractive index very close to
a sensor surface. The binding between a compound in solution and
its ligand immobilized on the sensor surface results in a change in
the refractive index.
The interaction is monitored in real time and the amount of
bound ligand and rates of association and dissociation can be
measured. First, hsPLA2 was immobilized on a CM-5 sensor chip
and, then, tested compounds were singly injected over the enzyme-
modified sensor chip at different concentrations from 50 nM to
1000 nM, in presence of Ca2þ ion (CaCl2, 500
mM).
In Fig. 2, the sensograms obtained from the binding of hsPLA2-IIA
to 8d at four different concentrations inpresence of CaCl2 are shown.
Table 1
Evaluation of the affinity for hsPLA2 and inhibition of PGE2 production from LPS-
treated RAW 264.7 cells by BLQ and the synthetic quinones.
IC50(m
M)b
a
KDSPR
Sd (KDSPR
)
BLQ
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
9f
4.88 ꢂ 10ꢁ7
4.28 ꢂ 10ꢁ5
4.58 ꢂ 10ꢁ7
4.55 ꢂ 10ꢁ7
3.88 ꢂ 10ꢁ7
5.01 ꢂ 10ꢁ7
9.59 ꢂ 10ꢁ7
7.01 ꢂ 10ꢁ7
7.49 ꢂ 10ꢁ7
3.33 ꢂ 10ꢁ7
5.03 ꢂ 10ꢁ5
1.03 ꢂ 10ꢁ6
2.87 ꢂ 10ꢁ7
4.67 ꢂ 10ꢁ6
2.93 ꢂ 10ꢁ7
3.93 ꢂ 10ꢁ5
2.73 ꢂ 10ꢁ7
3.27 ꢂ 10ꢁ7
3.14 ꢂ 10ꢁ7
3.49 ꢂ 10ꢁ7
8.27 ꢂ 10ꢁ7
4.05 ꢂ 10ꢁ7
4.49 ꢂ 10ꢁ7
2.49 ꢂ 10ꢁ7
3.63 ꢂ 10ꢁ5
9.11 ꢂ 10ꢁ7
2.40 ꢂ 10ꢁ7
2.01 ꢂ 10ꢁ6
5.2 ꢃ 0.5
87.1 ꢃ 10.5
26.0 ꢃ 3.6
56.7 ꢃ 3.4
4.0 ꢃ 0.9
13.0 ꢃ 0.2
18.0 ꢃ 0.6
41.0 ꢃ 3.4
40.5 ꢃ 5.6
52.1 ꢃ 2.8
65.9 ꢃ 9.3
61.7 ꢃ 7.2
55.1 ꢃ 3.3
60.5 ꢃ 9.3
Fig. 1. Effect of BLQ and analogue 8d on PGE2 release by LPS-stimulated mouse
macrophage-like cells. Compounds were added to the cells 30 min before LPS (1 mg/mL).
9g
a
Experimental dissociation constants of BLQ and ligands.
IC50
Supernatants were collected after 24 h for PGE2 assay. Results are mean ꢃ s.e.m. of 3
experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs LPS (Student’s t
test).
b
(mM) on PGE2 release by LPS-stimulated mouse macrophage-like cells.
Results are mean ꢃ s.e.m. of 3 experiments performed in triplicate.

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C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
assessed for purity by analytical high performance liquid chroma-
tography (HPLC), using an Agilent 1100 series, UV detector moni-
toring at 254 nm, HP Chem Station software, and a Waters C18
RP-column (5
m
m, 300 mm ꢂ 3.9 mm). All target compounds were
found to be >95% purity. MS spectrometry analysis ESI-MS was
carried out on a Finnigan LCQ Deca ion trap instrument. Microanal-
yses were carried out on Carlo Erba 1106 elemental analyzer.
5.2. General procedure for the synthesis of 1-substituted-2,4-
dimethoxybenzene derivatives 12bed
Fig. 2. Sensograms obtained from the binding of hsPLA2 to 8d in presence of CaCl2 at
four different concentrations (50 nM grey curve; 300 nM green curve, 700 nM blue
curve, 1000 nM red curve). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article).
2,4-Dimethoxyphenyl boronic acid (0.205 g, 1.5 equiv.) in dry
toluene (4 mL) was reacted at room temperature for 10 min with
benzyl halide (1.0 equiv.) in the presence of K3PO4 (2.0 equiv.), PPh3
(0.02 equiv.) and Pd(OAc)2 (0.01 equiv.) under argon atmosphere.
Bromine derivative (1.0 equiv.) was added and the reaction was
heated at 100 ꢀC for 16 h. A solution of NaOH (1 M) was added and the
crude product was extracted with diethyl ether. Final compounds
were purified by column chromatography over silica gel.
the hydrophobic pocket and to tolerate various building blocks
including large and hydrophobic aryl ring like dibenzofuran instead
of no-planar tricyclic derivative as thianthrene (9f vs 9g). The
presence of a condensate bicycle group should be favored and the
introduction of a methylene bridge increases binding affinity
probably because the compounds should fit better with the enzyme
binding domain (8ced vs 9dee).
5.2.1. 1-Benzyl-2,4-dimethoxybenzene (12b)
Elution with hexane/EtOAc (90:10) afforded 12b (97%) as pale
oil.
1H NMR (CDCl3, 300 MHz)
d
3.92 (s, 6H), 4.03 (s, 2H), 6.55 (dd,
J ¼ 1.1 Hz,1H), 6.62 (s, 1H), 7.11 (d, J ¼ 7.0 Hz,1H), 7.32e7.44 (m, 5H).
13C NMR (CDCl3, 300 MHz)
35.9, 55.9, 100.9, 107.1, 120.3, 126.3,
4. Results and discussion
d
Prostaglandins and leukotrienes are the products of arach-
idonate cascade and mediator systems exerting numerous vascular
and inflammatory effects.
Group II secretory PLA2 can act as a signalling agent contributing
to the inflammatory response. Group IIA hsPLA2 has been reported
to release arachidonic acid in some systems and may provide the
substrate for both cyclooxygenase (COX) and 5-lipoxigenase (5-LO)
products formation in mast cells.
The purpose of the present study was to investigate the
modulatory effect of the natural compound bolinaquinone and its
analogues on PGE2 production on mouse macrophage cell line RAW
264.7.
Our in vitro test indicated that almost all compounds inhibited
PGE2 production with different potency.
In particular, this paper addresses the influence of substituents
on the 2-hydroxyl-1,4-benzoquinone series in the structure-based
design of anti-inflammatory compounds.
The data we gained on the binding with hsPLA2, except for
derivatives 9aec and 9f was in good agreement with cellular
analysis. In conclusion, we have described the discovery and opti-
mization of a novel series of hydroxyquinones with potent anti-
inflammatory activity. Further explorations to clarify potential and
selectivity modulation on PGE2 production are ongoing in our group.
128.3, 129.3, 130.3, 141.6, 158.9, 159.2.
5.2.2. 1-[(1,10-Biphenyl)-4-yl]-2,4-dimethoxybenzene (12c)
Elution with light petroleum/EtOAc (90:10) afforded 12c (91%)
as yellow oil.
1H NMR (CDCl3, 300 MHz)
d 3.92 (s, 6H), 4.13 (s, 2H), 6.42 (d,
J ¼ 8.1 Hz,1H), 6.56 (s,1H), 7.05 (s,1H), 7.31(s, 2H), 7.40 (d, J ¼ 7.1 Hz,
1H), 7.47 (t, J ¼ 7.7 Hz, 2H), 7.55 (m, 2H), 7.62 (q, J ¼ 8.1 Hz, 2H).
13C NMR (CDCl3, 300 MHz)
d 35.0, 56.2, 102.0, 106.1, 120.3, 127.7,
128.0, 129.5, 130.3, 131.0, 134.0, 136.5, 140.5, 159.0.
5.2.3. 1-[(2-Naphthyl)methyl]-2,4-dimethoxybenzene (12d)
Elution with hexane afforded 12d (70%) as light-yellow oil.
1H NMR (CDCl3, 300 MHz)
d 3.85 (s, 6H), 4.16 (s, 2H), 6.42 (dd,
J ¼ 4.0 Hz, 1H), 6.55 (s, 1H), 7.08 (s, 1H), 7.37e7.47 (m, 3H), 7.63 (s,
1H), 7.86 (t, J ¼ 8.6 Hz, 3H).
13C NMR (CDCl3, 300 MHz)
d 36.7, 56.5, 101.0, 107.7, 122.3, 125.1,
126.0, 127.0, 128.0, 131.3, 133.5, 134.7, 137.2, 160.0.
5.3. General procedure for the synthesis of 1-substituted-2,4-
benzenediol derivatives (13bed, 23aeg) and 1-(2-Naphthylamino)-
2,4-benzenediol (19)
5. Experimental
The 2,4-dimethoxybenzene aryl derivatives (1.0 equiv.) in dry
dichloromethane (12 mL), cooled to ꢁ15 ꢀC, were reacted in the
presence of boron tribromide (8.8 equiv.). The mixture was stirred
for 18 h then deionised water was added, followed by dichloro-
methane. The combined organic layer was dried over Na2SO4,
filtered, and purified to obtain desired compounds.
5.1. General
Microwaves experiments were performed in a CEM Discover
monomode reactor (CEM Corp., Matthews, NC). All reactions were
conducted in a speciallyadapted cylindrical Pyrex vessel. All reagents
were analytical grade and purchased from SigmaeAldrich (Milano-
Italy). Flash chromatography was performed on Carlo Erba silica gel
60 (230 O 400 mesh; Carlo Erba, Milan, Italy). TLC was carried out
using plates coated with silica gel 60F 254 nm purchased from Merck
(Darmstadt, Germany). Melting points were determined in open
capillary tubes on a Electrothermal 9100 apparatus and are uncor-
rected. 1H and 13C NMR spectra were registered on a Bruker AC 300.
Chemical shifts are reported in ppm. All target compounds were
5.3.1. 1-Benzyl-2,4-benzenediol (13b)
Elution with hexane/EtOAc (70:30) afforded 13b (95%) as white
powder.
1H NMR (CDCl3, 300 MHz)
d 4.10 (s, 2H), 4.75 (br, 2H), 6.35
(s, 1H), 6.38 (d, 1H, J ¼ 7.1 Hz), 6.93 (d, 1H, J ¼ 8.1 Hz), 7.22e7.30 (m,
3H), 7.31 (d, 2H, J ¼ 7.4 Hz).
13C NMR (CDCl3, 300 MHz)
d 35.3, 104.1, 109.0, 122.2, 126.3,
128.5, 131.0, 141.7, 156.8, 157.7. Mp: 110 ꢀC.

C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
493
5.3.2. 1-[(1,10-Biphenyl)-4-yl]-2,4-benzenediol (13c)
Elution with CHCl3/MeOH (95:5) afforded 13c (80%) as pale
powder.
1H NMR (CDCl3, 300 MHz)
1H NMR (CDCl3, 300 MHz)
d
6.48 (s, 1H), 6.50 (m, 1H), 7.20 (d,
J ¼ 7.4 Hz, 1H), 7.38 (d, J ¼ 7.2 Hz, 1H), 7.49 (t, J ¼ 8.3 Hz, 2H), 7.46
(m, 2H), 7.68 (t, J ¼ 8.3 Hz, 4H).
d
3.93 (s, 2H), 4.85 (br, 2H), 6.46
13C NMR (CDCl3, 300 MHz)
d 104.1, 109.0, 119.8, 127.7, 127.9,
(s, 1H), 6.53 (s, 1H), 7.02 (d, 1H, J ¼ 8.1 Hz), 7.33 (s, 1H), 7.45 (d, 1H,
128.4, 129.3, 130.7, 135.4, 136.5, 157.1, 158.8. Mp: 400 ꢀC dec.
J ¼ 7.8 Hz), 7.53 (t, J ¼ 7.6 Hz, 2H), 7.61e7.73 (m, 4H).
13C NMR (CDCl3, 300 MHz)
d
35.6,104.1,109.0, 122.2,127.7,128.8,
5.3.10. 1-(2-Dibenzofuranyl)-2,4-benzendiol (23f)
Elution with hexane/EtOAc (90:10 to 60:40) afforded 23f (83%)
as white solid.
129.3, 131.1, 134.0, 136.5, 140.5, 156.8. Mp: 117.6 ꢀC.
5.3.3. 1-[(2-Naphthyl)methyl]-2,4-benzenediol (13d)
1H NMR (CD3OD, 300 MHz)
d 6.85 (s, 1H), 6.87 (s, 1H), 7.48 (t,
Elutionwith CHCl3/MeOH (95:5) afforded 13d (70%) as pale solid.
J ¼ 6.5 Hz,1H), 7.68 (t, J ¼ 7.2 Hz, 1H), 7.79 (d, J ¼ 8.5 Hz, 1H), 7.96 (q,
1H NMR (CD3OD, 300 MHz)
d
4.08 (s, 2H), 6.35 (d, 1H, J ¼ 8.1 Hz),
J ¼ 7.2 Hz, 2H), 8.13 (d, J ¼ 7.2 Hz, 1H), 9.0 (s, 1H).
6.42 (s, 1H), 6.93 (d, 1H, J ¼ 8.1 Hz), 7.42 (m, 3H), 7.62 (s, 1H),
7.83e7.92 (m, 3H).
13C NMR (CD3OD, 300 MHz)
d 104.1, 109.0, 111.6, 112.3, 113.0,
119.8, 121.0, 121.5, 123.3, 124.7, 130.7, 132.9, 137.8, 145.8, 156.8, 157.1,
13C NMR (CD3OD, 300 MHz)
d 36.0, 105.0, 110.0, 122.5, 125.1,
158.8. Mp: 400 ꢀC dec.
126.0, 127.6, 128.0, 131.3, 133.7, 135.2, 157.0, 158.0. Mp: 128 ꢀC.
5.3.11. 1-(2-Thianthrenyl)-2,4-benzenediol (23g)
5.3.4. 1-(2-Naphthylamino)-2,4-benzenediol (19)
Elution with light petroleum/EtOAc (50:50), afforded 19 as
a dark brown solid (85%).
Elution with hexane/EtOAc (95:5) afforded 23g (77%) as pale oil.
1H NMR (CDCl3, 300 MHz)
d 6.56 (s, 1H), 6.75 (s, 1H), 7.09 (d,
J ¼ 8.5 Hz, 1H), 7.22e7.30 (m, 2H), 7.43 (d, J ¼ 8.1 Hz, 1H), 7.51e7.57
1H NMR (CDCl3, 300 MHz)
d
4.92 (br, 1H), 5.15 (br, 1H), 6.13 (s,
(m, 3H).
1H), 6.47 (m, 1H), 6.63 (s, 1H), 6.92 (s, 1H), 7.06 (d, 1H, J ¼ 8.5 Hz),
7.15 (d, 1H, J ¼ 8.9 Hz), 7.42 (m, 2H), 7.6 1 (d, 1H, J ¼ 7.6 Hz), 7.92 (d,
2H, J ¼ 9.2 Hz).
13C NMR (CDCl3, 300 MHz)
d 104.1, 109.0, 119.8,127.0, 127.9, 130.5,
130.7, 131.8, 132.3, 135.6, 136.6, 137.7, 138.2, 157.1, 158.8. Mp: 310.2 ꢀC.
13CNMR (CDCl3, 300 MHz)
d
103, 108, 110, 119,122, 124, 127, 129,
5.4. General procedures for the synthesis of 2-hydroxy-2,5-
cyclohexadiene-1,4-dione-5-substituted derivatives (8aed, 8f)
134, 142, 149. MS (ESI) m/z 253.29. Mp: 238.3 ꢀC.
5.3.5. 1-(4-Chlorophenyl)-2,4-benzenediol (23a)
Elution with hexane/EtOAc (90:10 to 80:20) afforded 23a (76%)
as pale solid.
To a solution of the appropriate dihydroxyl derivatives
(1.0 equiv.) in tetrahydrofuran (6 mL) at room temperature, a solu-
tion of Fremy’s salt (2.5 equiv.) in 0.7 mL of 15% Na2CO3, (1.6 equiv.)
was added. The solution was stirred vigorously until the reaction
was completed (10 h for 8aed, 19 h for 8f). Desired compounds
were obtained after purification by flash chromatography.
1H NMR (CD3OD, 300 MHz)
d
6.37 (m, 2H), 7.10 (d, J ¼ 8.5 Hz,
1H), 7.35 (d, J ¼ 9.4 Hz, 2H), 7.52 (d, J ¼ 8.1 Hz, 2H).
13C NMR (CD3OD, 300 MHz)
d 104.1, 109.0, 119.8, 128.3, 129.4,
130.7, 133.2, 134.6, 157.1, 158.8. Mp: 68.9 ꢀC.
5.4.1. 5-Hexyl-2-hydroxy-2,5-cyclohexadiene-1,4-dione (8a)
Elutionwith hexane/EtOAc (50:50) afforded 8a(60%) as yellow oil.
5.3.6. 1-(3-Nitrophenyl)-2,4-benzenediol (23b)
Elution with hexane/EtOAc (80:20) afforded 23b (86%) as yellow
1H NMR (CDCl3, 300 MHz)
d
1.05 (m, 3H), 1.23 (m, 5H), 1.31 (m,
3H), 2.5 (t, 2H, J ¼ 8.1 Hz), 6.15 (s, 1H), 6.37 (s, 1H), 8.60 (br, 1H).
13C NMR (CDCl3, 300 MHz)
40.5, 110.1, 125.8, 128.7, 129.1, 133.0,
oil.
1H NMR (CDCl3, 300 MHz)
d
6.50 (s, 1H), 6.60 (d, J ¼ 8.1 Hz, 1H),
d
7.23 (d, J ¼ 8.3 Hz, 1H), 7.65 (t, J ¼ 7.6 Hz, 1H), 7.88 (d, J ¼ 7.4 Hz, 1H),
8.23 (d, J ¼ 8.3 Hz, 1H), 8.41 (s, 1H).
135.3, 137.5, 169.0, 181.3, 187.2. MS (ESI) m/z: 213.01. Anal. Calcd for
C13H10O3: C 72.89, H 4.71, O 22.41. Found: C 72.40, H 4.11, O 21.99.
13C NMR (CDCl3, 300 MHz)
d 104.1, 109.0, 119.8, 120.0, 122.5,
130.2, 131.2, 134.0, 137.4, 148.9, 157.1, 158.8. Mp: 138.2 ꢀC.
5.4.2. 5-Benzyl-2-hydroxy-2,5-cyclohexadiene-1,4-dione (8b)
Elutionwithhexane/EtOAc(50:50) afforded 8b(60%)asyellowoil.
5.3.7. 1-(4-Hydroxyphenyl)-2,4-benzenediol (23c)
Elution with light petroleum/EtOAc (85:15 to 80:20) afforded
23c (80%) as white solid.
1H NMR (CDCl3, 300 MHz)
d
3.83 (s, 2H), 6.12 (s, 1H), 6.37 (s, 1H),
7.20 (d, 2H, J ¼ 8.2 Hz), 7.39 (m, 3H), 8.60 (br, 1H).
13C NMR (CDCl3, 300 MHz)
40.5, 110.1, 125.8, 128.7, 129.1, 133.0,
d
1H NMR (DMSO-d6, 300 MHz)
d
6.26 (dd, 1H, J ¼ 8.1 Hz), 6.36 (s,
135.3, 137.5, 169.0, 181.3, 187.2. MS (ESI) m/z: 213.01. Anal. Calcd for
C13H10O3: C 72.89, H 4.71, O 22.41. Found: C 72.40, H 4.11, O 21.99.
1H), 6.71 (d, 2H, J ¼ 8.4 Hz), 6.94 (d, 1H, J ¼ 7.8 Hz), 7.24 (d, 2H,
J ¼ 8.2 Hz), 9.16 (s, 3H).
13C NMR (DMSO-d6, 300 MHz)
d
104.1, 109.0, 116.4, 119.8, 128.1,
5.4.3. 5-[(1,10-Biphenyl)-4-yl]-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (8c)
129.3, 130.7, 158.1, 159.8. Mp: 118.6 ꢀC.
Elution with EtOAc afforded 8c (55%) as brown oil.
5.3.8. 1-(2-Naphthyl)-2,4-benzenediol (23d)
Elution with hexane/EtOAc (70:30) afforded 23d (70%) as pale-
yellow solid
1H NMR (CDCl3, 300 MHz)
d 3.84 (s, 2H), 6.15 (s, 1H), 6.44 (s, 1H),
7.26 (s, 1H), 7.46 (d, 2H, J ¼ 7.1 Hz) 7.56 (t, 2H, J ¼ 7.7 Hz), 7.60 (t, 4H,
J ¼ 5.5 Hz).
1H NMR (CD3OD, 300 MHz)
d
6.45 (s, 1H), 6.48 (m, 1H), 6.98 (d,
13C NMR (CDCl3, 300 MHz)
d 40.5,111.0,127.6,128.0, 129.3, 130.0,
J ¼ 7.1 Hz, 1H), 7.30e7.53 (m, 4H), 7.67 (d, J ¼ 8.1 Hz, 1H), 7.82e7.90
133.0, 135.3, 136.7, 169.0, 180.3, 188.5. MS (ESI) m/z: 289.09. Anal.
Calcd. for C19H14O3: C 78.61, H 4.86, O 16.53. Found: C 78.43, H 4.65,
O 16.02.
(m, 2H).
13C NMR (CD3OD, 300 MHz)
d 104.1, 109.0, 119.8, 124.9, 126.2,
128.1, 129.2, 130.7, 133.1, 134.2, 157.1, 158.8. Mp: 125.7 ꢀC.
5.4.4. 5-[(2-Naphthyl)methyl]-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (8d)
5.3.9. 1-[4-(1,10-Biphenyl)]-2,4-benzenediol (23e)
Elution with hexane/EtOAc (90:10 to 70:30) afforded 23e (82%)
as white solid.
Elution with hexane/EtOAc (50:50) afforded 8d (50%) as orange
oil.

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C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
1H NMR (CDCl3, 300 MHz)
d
3.93 (s, 2H), 6.14 (s, 1H), 6.31 (s, 1H),
1H NMR (CDCl3, 300 MHz)
d 3.71 (s, 3 H), 3.84 (s, 3H), 5.81 (br,
7.26 (s, 1H), 7.57 (d, 2H, J ¼ 6.9 Hz), 7.64 (s, 1H), 7.82e7.84 (m, 3H),
1H), 6.22 (d, 1H, J ¼ 8.9 Hz), 6.53 (s, 1H), 7.25 (d, 1H, J ¼ 8.9 Hz), 7.33
(t, 1H, J ¼ 7.5 Hz), 7.45 (m, 3H), 7.67 (d, 1H, J ¼ 7.6 Hz), 7.91 (d, 2H,
J ¼ 9.2 Hz).
8.64 (br, 1H).
13C NMR (CDCl3, 300 MHz)
d 41.0, 110.1, 125.0, 126.0, 127.0, 127.6,
128.0, 131.8, 133.0, 135.2, 135.7, 169.0, 180.3, 188.5. MS (ESI) m/z:
263.0. Anal. Calcd for C17H12O3: C 77.26, H 4.58, O 18.16. Found: C
76.90, H 4.20, O 18.07.
13C NMR (CDCl3, 300 MHz)
d 58.2, 102.8, 108.0, 110.3, 119.1, 120.2,
124.5, 126.6, 128.1, 134.0, 144.1, 150.3. MS (ESI) m/z: 280.46.
5.8. General procedures for the synthesis of compounds (22aeg)
5.4.5. 5-(2-Naphthylamino)-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (8f)
1-Iodo-2,4-dimethoxybenzene 20 (0.200 g, 1.0 equiv.) in ethanol
(4 mL) was reacted with arylboronic acid (1.2 equiv.), K2CO3
(2.0 equiv.), PdCl2(dppf) (5% mol). MW irradiation of 60 W, for
25 min, was used, the temperature being ramped from room
temperature to 110 ꢀC. To the resulting mixture water and ethyl
acetate (20 mL of each) were added. Final compounds were purified
by flash chromatography over silica gel.
Elution with EtOAc/hexane (90:10) afforded 8f as a violet solid
(50%).
1H NMR (CD3OD, 300 MHz)
d 2.2 (s, 1H), 5.5 (s, 1H), 7.00 (m, 2H),
7.20 (t, 1H, J ¼ 7.6 Hz), 7.32 (t, 1H, J ¼ 8.1 Hz), 7.52 (d, 1H, J ¼ 8.8 Hz),
7.66e7.80 (m, 3H).
13C NMR (CD3OD, 300 MHz)
d 103.2, 108.5, 110.0, 116.1, 122.3,
124.0, 126.4, 128.3, 132.6, 135.0, 148.0, 180.5, 181.2. MS (ESI) m/z:
280.4. Anal. Calcd for C16H11 NO3: C 72.45, H 4.18, N 5.28, O 18.09.
Found: C 73.05, H 3.99, N 5.67, O 17.97. Mp: 389 ꢀC.
5.8.1. 1-(4-Chlorophenyl)-2,4-dimethoxybenzene (22a)
Elution with hexane/EtOAc (95:5) afforded 22a (70%) as white
solid.
1H NMR (CDCl3, 300 MHz)
d
3.80 (s, 6H), 6.64 (m, 2H), 7.25 (d,
5.5. 2-(2,4,5-Trimethoxyphenoxy)naphthalene (16)
J ¼ 8.0 Hz, 1H), 7.39 (d, J ¼ 7.2 Hz, 2H), 7.47 (d, J ¼ 8.5 Hz, 2H).
13C NMR (CDCl3, 300 MHz)
d 56.2, 55.9, 100.9, 107.1, 117.9, 129.3,
129.4, 129.9, 133.2, 134.6, 158.8, 160.6. Mp: 48.2 ꢀC.
2,4,5-Trimethoxyphenol 15 (0.280 g, 1 equiv.) in dry dichloro-
methane (10 mL) was reacted with 2-naphthalenboronic acid
(2.2 equiv.), Cu(OAc)2 (1.5 equiv.) and pyridine (2.5 equiv.) at room
temperature for 48 h. The reaction mixture was diluted with water
and extracted with dichloromethane. Final compound was purified
by column chromatography over silica gel on silica gel with
a mixture of hexane/EtOAc (50:50) as eluent to give 16 (0.450 g,
97%) as a light-yellow oil.
5.8.2. 1-(3-Nitrophenyl)-2,4-dimethoxybenzene (22b)
Elution with hexane/EtOAc (95:5) afforded 22b (80%) as yellow
solid.
1H NMR (CDCl3, 300 MHz)
d 3.93 (s, 6H), 6.64 (m, 2H), 7.25 (d,
J ¼ 8.1 Hz, 1H), 7.55 (t, J ¼ 7.8 Hz, 1H), 7.85 (d, J ¼ 7.0 Hz, 1H), 8.15 (d,
J ¼ 8.3 Hz, 1H), 8.42 (s, 1H).
1H NMR (CDCl3, 300 MHz)
d 3.84 (s, 6H), 3.93 (s, 3H), 6.76 (s, 2H),
13C NMR (CDCl3, 300 MHz)
d 56.2, 55.9, 100.9, 107.1, 117.9, 120.0,
122.5, 129.9, 130.2, 134.0, 137.4, 148.9, 158.8, 160.6. Mp: 85.9 ꢀC.
7.15 (s, 1H), 7.31 (s, 1H), 7.43 (d, 2H, J ¼ 8.1 Hz), 7.56 (d, 1H,
J ¼ 6.4 Hz), 7.75 (d, 1H, J ¼ 8.5 Hz), 7.90 (d, 2H, J ¼ 8.1 Hz).
13C NMR (CDCl3, 300 MHz)
d 60.0, 100.2, 105.3, 110.7, 123.6,
5.8.3. 20,40-Dimethoxy-[1,10-biphenyl]-4-ol (22c)
Elution with CH2Cl2/MeOH (95:5 to 50:50) afforded 22c (74%) as
white solid.
126.5, 127.2, 129.1, 135.6, 140.3, 143.0, 145.2, 153.6.
1H NMR (CDCl3, 300 MHz)
d 3.85 (s, 3H), 3.93 (s, 3H), 5.02 (br,
5.6. 5-(2-Naphthyloxy)2-hydroxy-2,5-cyclohexadiene-
1,4-dione (8e)
1H), 6.64 (m, 2H), 6.90 (d, 2H, J ¼ 6.8 Hz), 7.10 (d,1H, J ¼ 8.1 Hz), 7.40
(d, 2H, J ¼ 8.5 Hz).
13C NMR (CDCl3, 300 MHz)
d 56.2, 55.9, 100.9, 107.1, 116.4, 117.9,
A water solution of ammonium cerium(IV) nitrate (CAN)
(2.5 equiv.) was added rapidly to 2-(2,4,5-trimethoxyphenoxy)
naphthalene (16) (0.199 g, 1 equiv.) in acetonitrile (16 mL) at room
temperature. After 1 h the solvent was evaporated. The mixture
was diluted with water, extracted with ethyl acetate. Column
chromatography, with a mixture of hexane/EtOAc (40:60) as eluent,
provided final compound 8e (0.085 g; 53%) as yellow solid.
129.3, 129.9, 157.4, 158.8, 160.6. Mp: 48.2 ꢀC.
5.8.4. 1-(2-Naphthyl)-2,4-dimethoxybenzene (22d)
Elution with hexane/EtOAc (95:5 to 90:10) afforded 22d (70%) as
pale-yellow solid.
1H NMR (CDCl3, 300 MHz)
d 3.55 (s, 3H), 3.82 (s, 3H), 6.68 (m,
1H NMR (CDCl3, 300 MHz)
d 5.10 (br,1H), 5.79 (s,1H), 6.10 (s,1H),
2H), 7.25 (d, 1H, J ¼ 8.1 Hz), 7.40e7.60 (m, 4H), 7.70 (d, 2H,
J ¼ 8.5 Hz), 8.00 (t, 1H, J ¼ 9.2 Hz).
7.00 (d, 1H, J ¼ 8.3 Hz), 7.30 (s, 1H), 7.53 (m, 2H), 7.60 (d, 1H,
13C NMR (CDCl3, 300 MHz)
d 56.2, 55.9, 100.9, 107.1, 117.9, 125.1,
J ¼ 7.6 Hz), 7.70 (d, 1H, J ¼ 6.9 Hz), 8.00 (d, 1H, J ¼ 7.6 Hz).
13C NMR (CDCl3, 300 MHz)
d 106.1, 110.5, 126.2, 127.5, 128.3,
126.3, 126.8, 127.2, 128.3, 129.9, 133.1, 134.2, 136.7, 158.8, 160.6. Mp:
118.9 ꢀC.
129.0, 131.1, 132.7, 135.6, 150.5, 169.6, 183.0, 185.4. MS (ESI) m/z:
288.41. Anal. Calcd for C16H10O4: C 72.18, H 3.79, O 24.04. Found: C
73.07, H 4.01, O 24.78. Mp: 104.5 ꢀC.
5.8.5. 1-[4-(1,10-Biphenyl)]-2,4-dimethoxybenzene (22e)
Elution with hexane/EtOAc (95:5 to 90:10) afforded 22e (75%) as
white solid.
5.7. N-(2,4-Dimethoxyphenyl)naphthyl-2-amine (18)
1H NMR (CDCl3, 300 MHz)
d
3.81 (s, 3H), 3.94 (s, 3H), 6.68 (m,
2H), 7.20e7.40 (m, 3H), 7.55 (t, 3H, J ¼ 6.2 Hz), 7.70e7.80 (m, 4H).
2-4-Dimethoxyaniline 17 (0.377 g, 5 equiv.) in dry toluene
was reacted with 2-bromonaphthalene (1.0 equiv.), Pd(OAc)2
(0.04 equiv.), PPh3 (0.16 equiv.), sodium tert-butoxide (1.2 equiv.).
The mixture was heated at 100 ꢀC for 40 h and after neutralized
with a solution of NH4Cl. The crude product was extracted with
diethyl ether and the final compound was purified by column
chromatography over silica gel, using light petroleum/EtOAc
(90:10) as eluent, to give 18 as a dark orange oil (0.137 g, 77%).
13C NMR (CDCl3, 300 MHz)
d 55.9, 56.2, 100.9, 107.1, 117.9, 127.7,
127.9, 128.4, 129.3, 129.9, 135.4, 136.5, 158.8, 160.6. Mp: 115.3 ꢀC.
5.8.6. 1-(2-Dibenzofuranyl)-2,4-dimethoxybenzene (22f)
Elution with hexane/EtOAc (95:5) afforded 22f (80%) as white
solid.
1H NMR (CDCl3, 300 MHz)
d
3.83 (s, 3H), 3.91 (s, 3H), 6.68e6.69
(m, 2H), 7.29e7.36 (m, 6H), 7.96e7.99 (m, 2H).

C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
495
13C NMR (CDCl3, 300 MHz)
113.0,117.9,119.4,121.0,122.5,123.3,124.7,129.9,134.0,136.4,145.8,
156.3, 158.8. Mp: 116.8 ꢀC.
d
56.2, 55.9, 100.9, 107.1, 111.6, 112.1,
5.9.5. 5-[4-(1,10-Biphenyl)]-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (9e)
Elution with hexane/EtOAc (50:50) afforded 9e (48%) as orange
oil.
5.8.7. 1-(2-Thianthrenyl)-2,4-dimethoxybenzene (22g)
Elution with hexane/EtOAc (95:5) afforded 22g (77%) as yellow
solid.
1H NMR (CDCl3, 300 MHz)
(m, 3H), 7.61e7.68 (m, 6H), 8.74 (br, 1H).
13C NMR (CDCl3, 300 MHz)
110.1, 126.9, 127.7, 127.9, 127.8,
d 6.25 (s, 1H), 7.00 (s, 1H), 7.20e7.32
d
1H NMR (CDCl3, 300 MHz)
d
3.83 (s, 3H), 3.93 (s, 3H), 6.63 (m,
129.3, 131.5, 132.7, 135.7, 136.5, 145.7, 169.0, 181.3, 187.0. MS (ESI) m/
z: 275.09. Anal. Calcd for C18H12O3: C 78.25, H 4.38, O 17.37. Found:
C 78.10, H 4.08, O 17.15.
2H), 7.25e7.27 (m, 4H), 7.29 (d, 2H, J ¼ 7.8 Hz), 7.51 (d, 2H,
J ¼ 8.11 Hz).
13C NMR (CDCl3, 300 MHz)
d 55.9, 56.2, 100.9, 107.1, 117.9, 127.0,
127.9, 129.9, 130.5, 131.8, 132.3, 135.6, 136.6, 137.7, 138.2, 158.8,
5.9.6. 5-(2-Dibenzofuranyl)-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (9f)
160.6. Mp: 140.4 ꢀC.
Elution with hexane/EtOAc (60:40) afforded 9f (56%) as brown
solid.
1H NMR (DMSO-d6, 300 MHz)
d
6.11 (s,1H), 6.99 (s,1H), 7.30e7.36
(m, 3H), 7.46 (d, J ¼ 7.4 Hz, 1H), 7.8e8.0 (m, 3H), 11.0 (br, 1H).
13C NMR (DMSO-d6, 300 MHz)
110.1, 111.5, 111.6, 113.0, 119.1,
5.9. General procedure for the synthesis of 2-hydroxy-2,5-
cyclohexadiene-1,4-dione 5-substituted derivatives (9aeg)
d
121.0, 121.7, 123.3, 124.7, 129.6, 132.7, 134.0, 145.7, 145.8, 156.3, 169.0,
181.3, 187.0. MS (ESI) m/z: 290.0. Anal. Calcd for C18H10O4: C 74.48, H
3.47, O 22.05. Found: C 74.13, H 3.40, O 21.98. Mp: 400 ꢀC dec.
Dihydroxyl derivatives 23aeg (0.064 g,1.0 equiv.) in ethyl acetate
(15 mL) cooled to 0 ꢀC were reacted, under argon with Fremy’s salt
(2.5 equiv.) and Na2HPO4(H2O)2 (1.6 equiv.) at room temperature for
20 h. The aqueous layer was back-extracted with ethyl acetate. Final
compounds were purified by flash chromatography.
5.9.7. 5-(2-Thianthrenyl)-2-hydroxy-2,5-cyclohexadiene-
1,4-dione (9g)
Elution with CHCl3/MeOH (90:10) afforded 9g (44%) as yellow
5.9.1. 5-(4-Chlorophenyl)-2-hydroxy-2,5-cyclohexadiene-
1,4-dione (9a)
oil.
1H NMR (CD3OD, 300 MHz)
d
6.42 (d, 1H, J ¼ 7.6 Hz), 6.45 (s, 1H),
Elution with hexane/EtOAc (80:20) afforded 9a (55%) as purple
6.99 (d, 1H, J ¼ 8.1 Hz), 7.58e7.61 (m, 2H), 7.62e7.67 (m, 2H), 7.82 (t,
2H, J ¼ 7.4 Hz), 8.52 (br, 1H).
solid.
1H NMR (CD3OD, 300 MHz)
d 5.66 (s, 1H), 6.50 (s, 1H), 7.28 (d,
13C NMR (CD3OD, 300 MHz)
d 111.1, 125.5, 126.6, 127.9, 130.7,
J ¼ 8.1 Hz, 2H), 7.38 (d, J ¼ 7.2 Hz, 2H), 8.51 (s, 1H). 13C NMR (CD3OD,
131.8, 132.7, 136.9, 137.6, 138.2, 145.7, 169.0, 182.3, 188.0. MS (ESI) m/
z: 338.99. Anal. Calcd for C18H10O3S2: C 63.89, H 2.98, O 14.18, S
18.95. Found: C 63.70, H 2.50, O 14.04, S 18.46.
300 MHz)
d 110.1, 127.8, 128.8, 130.7, 132.7, 133.5, 145.7, 169.0, 181.3,
187.0.MS(ESI)m/z:235.01. Anal. CalcdforC12H7ClO3:C61.43,H3.01,Cl
15.11, O 20.46. Found: C 61.08, H 2.90, Cl 15.01, O 20.10. Mp: 400 ꢀC dec.
5.9.2. 5-(3-Nitrophenyl)-2-hydroxy-2,5-cyclohexadiene-
1,4-dione (9b)
6. Biological protocols
Elution with EtOAc afforded 9b (60%) as purple solid. 1H NMR
6.1. Cell culture
(CDCl3, 300 MHz)
7.83 (d, J ¼ 8.1 Hz, 1H), 8.36 (s, 1H).
13C NMR (CDCl3, 300 MHz)
110.1, 120.3, 121.3, 129.6, 132.5,
133.5, 132.7, 145.7, 148.3, 169.0, 181.3, 187.0. MS (ESI) m/z: 246.05.
Anal. Calcd for C12H7NO5: C 58.78, H 2.88, N 5.71, O 32.63. Found: C
58.10, H 2.44, N 5.10, O 32.08. Mp: 400 ꢀC dec.
d
6.30 (s, 1H), 6.90 (s, 1H), 7.64 (t, J ¼ 8.4 Hz, 2H),
The mouse macrophage-like cell line RAW 264.7, was purchased
from American Type Culture Collection (ATCC, LGC Promochem,
Sesto San Giovanni, Milan, Italy). RAW 264.7 macrophages were
grown in DMEM supplemented with 10% heat-inactivated FBS,
d
4 mM L-Glutamine, 1000 units/mL penicillin and 10000 mg/mL
streptomycin (all from Cambrex Bioscience, Verviers, Belgium) at
37 ꢀC in an atmosphere of 95% O2 and 5% CO2. RAW 264.7 cells were
plated at a density of 5 ꢂ 105cells/well in 6-well culture plates. Cells
were allowed to adhere overnight, rinsed twice and cultured for
16 h in serum-free DMEM. After pre-incubation for 16 h, the test
compounds were added 30 min before co-treatment with LPS
5.9.3. 5-(4-Hydroxyphenyl)-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (9c)
Elution with EtOAc/MeOH (80:20) afforded 9c (50%) as brown
solid.
1H NMR (CD3OD, 300 MHz)
d
6.62 (s, 1H), 6.70 (s, 1H), 6.94 (d,
J ¼ 9.3 Hz, 2H), 7.53 (d, J ¼ 8.7 Hz, 2H), 8.05 (br, 1H).
13C NMR (CD3OD, 300 MHz)
110.1, 115.8, 125.2, 127.8, 132.7,
(1
DMSO on the day of the experiment and diluted with serum-free
DMEM at appropriate concentrations (1e100 M). The final
mg/mL) for another 24 h. Test compounds were dissolved in
d
157.7, 169.0, 181.3, 187.0. MS (ESI) m/z: 214.2. Anal. Calcd for
C12H8O4: C 66.67, H 3.73, O 29.60. Found: C 66.01, H 3.60, O 29.15.
Mp: 400 ꢀC dec.
m
concentration of DMSO was adjusted to 0.1% (v/v). Control groups
also received the same amount of DMSO.
5.9.4. 5-(2-Naphthyl)-2-hydroxy-2,5-cyclohexadiene-
6.2. PGE2 enzyme immunoassay
1,4-dione (9d)
Elution with hexane/EtOAc (50:50) afforded 9d (53%) as light-
RAW 264.7 supernatants were analyzed for PGE2 using
a commercially available kit (Cayman Chem, Ann Arbor, MI, USA).
Due to the rapid metabolism to an inactive metabolite of PGE2 in
vivo, concentrations detected in samples are very low: a more
accurate index of PGE2 biosynthesis and excretion is obtained
by converting PGE2 to a single, stable derivative (13,14-dihydro-
15-keto-prostaglandin A2) easily quantifiable by an enzyme
immunoassay (EIA) procedure. Briefly, the assay is based on the
yellow oil.
1H NMR (CD3OD, 300 MHz)
d
6.74 (s, 1H), 7.37 (s, 1H), 7.42 (m,
1H), 7.55e7.62 (m, 2H), 7.74 (d, 1H, J ¼ 8.1 Hz), 7.93 (t, 3H, J ¼ 7.8 Hz).
13C NMR (CD3OD, 300 MHz)
110.1, 123.5, 125.1, 126.0, 126.4,
d
127.7, 128.1, 128.2, 132.7, 133.2, 133.6, 134.8, 145.7, 169.0, 181.3, 187.0.
MS (ESI) m/z: 249.2. Anal. Calcd for C16H10O3: C 76.79, H 4.03 O
19.18. Found: C 76.70, H 3.99, O 19.07.

496
C. Petronzi et al. / European Journal of Medicinal Chemistry 46 (2011) 488e496
competition between free PGE2 and a PGE2 acetylcholinesterase
conjugate (PGE2 tracer) for a limited amount of PGE2-specific
monoclonal antibody; the amount of PGE2 tracer binding to
monoclonal antibody will be inversely proportional to the
concentration of PGE2 in the well. The results are shown as
mean ꢃ SEM of three separate experiments made in triplicate.
Rate constants for association (ka) dissociation (kd) and the
dissociation constant (KD) were obtained by globally fitting data
from all the injection of different concentration of each compound,
using the BIAevaluation software, using the simple 1:1 Langmuir
binding model.
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