Improved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative

Article abstract of DOI:10.1016/j.bmc.2010.06.089

Several lines of evidence indicate that histone acetyltransferases (HATs) are novel drug targets for treatment of diseases like, for example, cancer and inflammation. The natural product anacardic acid is a starting point for development of small molecule inhibitors of the histone acetyltransferase (HAT) p300/CBP associated factor (PCAF). In order to optimize the inhibitory potency, a binding model for PCAF inhibition by anacardic acid was proposed and new anacardic acid derivatives were designed. Ten new derivatives were synthesized using a novel synthetic route. One compound showed a twofold improved inhibitory potency for the PCAF HAT activity and a twofold improved inhibition of histone acetylation in HEP G2 cells.

Full text of DOI:10.1016/j.bmc.2010.06.089

Bioorganic & Medicinal Chemistry 18 (2010) 5826–5834
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improved inhibition of the histone acetyltransferase PCAF by an anacardic
acid derivative
Massimo Ghizzoni a, André Boltjes a, Chris de Graaf b, Hidde J. Haisma a, Frank J. Dekker a,
*
a Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, Anthonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
b Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Department of Pharmacochemistry, Faculty of Exact Sciences, VU University Amsterdam,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Several lines of evidence indicate that histone acetyltransferases (HATs) are novel drug targets for treat-
ment of diseases like, for example, cancer and inflammation. The natural product anacardic acid is a start-
ing point for development of small molecule inhibitors of the histone acetyltransferase (HAT) p300/CBP
associated factor (PCAF). In order to optimize the inhibitory potency, a binding model for PCAF inhibition
by anacardic acid was proposed and new anacardic acid derivatives were designed. Ten new derivatives
were synthesized using a novel synthetic route. One compound showed a twofold improved inhibitory
potency for the PCAF HAT activity and a twofold improved inhibition of histone acetylation in HEP G2
cells.
Received 3 June 2010
Revised 23 June 2010
Accepted 29 June 2010
Available online 3 July 2010
Keywords:
Anacardic acid
Histone acetyltransferase
p300/CBP associated factor (PCAF)
Salicylate derivatives
Enzyme inhibition
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Nuclear factor-
an important role in inflammation. When activated, NF-
cates to the nucleus, where it binds to the B recognition site of
j
B (NF-
j
B) mediated signal transduction plays
jB translo-
For several diseases, treatment with the currently available
drugs is not satisfactory, which demonstrates the need for concep-
tually new drugs for new therapeutic targets. The epigenetic regu-
lation of gene transcription is an area of research in which new
therapeutic targets are being discovered. Gene transcription is reg-
ulated by, among others, histone acetyltransferases (HATs) (Fig. 1).1
Deregulation of histone acetylation has been linked to several
diseases, including cancer and inflammation, which suggests that
HATs are potential anti-inflammatory targets.2–4
Ultimately, we aim to explore the role of histone acetyltransfer-
ases in inflammation by development of small molecule inhibitors.
Inflammation involves a variety of inflammatory mediators whose
transcription is enhanced during the disease process by global his-
tone acetylation as well as gene specific histone acetylation. Sev-
eral studies showed a disturbed balance between HAT and HDAC
activity in inflammatory diseases. Asthma patients show increased
HAT activity in bronchial biopsies, whereas the activity and expres-
sion of HDACs are decreased.5 The same was observed in lung tis-
sue for chronic obstructive pulmonary disease (COPD) patients
compared to healthy subjects. Tissue of patients with COPD
showed reduced HDAC activity and increased histone H4 acetyla-
tion in the interleukin-8 promotor.6
j
target genes and to cofactors with intrinsic HAT activity such as
p300/CBP and PCAF.7 This leads to global acetylation of lysine
residues in histone H4 and opening of the chromatin structure,
enabling transcription of proinflammatory genes, including IL-8.8
This mechanism is supported by the observation that Legionella
pneumophila-induced IL-8 release from alveolar epithelial cells is
decreased by HAT inhibition and enhanced by HDAC inhibition.
These findings suggest that restoring the HAT/HDAC balance by
small molecule inhibitors of histone acetylation provides opportu-
nities for treatment of inflammation.6,9–11
Figure 1. Acetylation of histone lysine residues by histone acetyltransferases
* Corresponding author. Tel.: +31 50 3638030; fax: +31 50 3637953.
E-mail address: f.j.dekker@rug.nl (F.J. Dekker).
provides potential anti-inflammatory drug targets. Anacardic acid is
permeable small molecule inhibitor of the histone acetyltransferase PCAF.
a cell-
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.089

M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
5827
Figure 2. (a) Binding mode of the natural substrate CoA bound to the HAT PCAF active site (PDB code 1CM0). Only the pantothenic acid and the pyrophosphate of CoA are
shown. The substrate carbon atoms are shown in purple. Docked binding configurations of anacardic acid (b) (carbons atoms in green), compound 4e (c) (carbon atoms in
pink), and compound 6d (d) (carbon atoms in orange). Helix a4 and beta strand b4 are represented by yellow ribbons. Important binding residues are shown as ball-and-
sticks with grey carbon atoms. Oxygen, nitrogen sulfur, phosphor, and hydrogen atoms are colored red, blue, yellow, orange, and cyan, respectively. Protein–ligand as well as
w6-ligand and protein-w6 H-bonds described in the text are shown as black or grey dotted lines.
Only a few small molecule inhibitors of HATs are currently avail-
able. Potent and selective bisubstrate inhibitors for the HATs p300
and PCAF have been described, however the lack of cell permeabil-
ity represents a disadvantage.12 Another well known class of inhib-
itors is represented by isothiazolones, which are cell-permeable
and potent; however their reactivity limits their specificity.13–16
Furthermore, the natural products curcumin,17 plumbagin,18 gar-
cinol19 and anacardic acid20 (AA, Fig. 1) have been reported as
inhibitors of different classes of HATs. Interestingly, it has been
inhibitory potency for the recombinant HAT PCAF enzyme and
for histone acetylation in HEP G2 cells was studied.
2. Results and discussion
2.1. Molecular docking studies
The crystal structure of PCAF in complex with Coenzyme A (CoA)
has been published previously.23 Using this structure, docking stud-
ies were performed in order to propose a possible binding mode for
anacardic acid. We hypothesized that the salicylate group of anacar-
dic acid mimics the pyrophosphate group of CoA, which forms an
extensive H-bond network with the backbone amide nitrogens of
V582, G584, G586, and T587, with the side chain hydroxyl of
T587, and with the water molecule w6 (Fig. 2a). This active site
water molecule is further stabilized by a H-bond network with
the backbone of E580, K583, and Y585 (Fig. 2a). Docking simulations
including this essential water molecule indeed yielded a (highest
ranked) pose of anacardic acid similar to the CoA binding mode
(Fig. 2b), in which the carboxylic group accepts H-bonds from the
backbone of K583, G584, G586, and w6, the hydroxyl group donates
a H-bond to the T587 side chain in the pyrophosphate binding pock-
et, and the long alkyl chain of anacardic acid protrudes deep into the
hydrophobic pantothenic acid binding pocket between beta strand
demonstrated that anacardic acid suppresses the NF-jB pathway,
which shows the potential of this compound to suppress inflamma-
tion.21 Several benzamide derivatives of anacardic acid have been
studied for inhibition of p300, however they were either equally po-
tent p300 HAT inhibitors compared to anacardic acid or they acti-
vated the p300 HAT.20,22
In this study we describe hypothesis-driven optimization of the
HAT inhibitory potency of anacardic acid. We proposed a binding
model for anacardic acid in the PCAF active site using molecular
modeling techniques. Based on this model, a series of new anacar-
dic acid derivatives was designed. A convenient synthetic route
was developed to synthesize these derivatives. We replaced the
6-alkyl chain of anacardic acid by different substituents in order
to improve the potency and the drug likeness of these inhibitors.
A series of 10 anacardic acid derivates was synthesized and their

5828
M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
(a)
(b)
OH
OH
O
O
O
O
O
O
O
O
O
O
O
a
b
c
c
R
R
OTf
(c)
(d)
(e)
3 a-e
4 a-e
anacardic acid (AA) 4a
1
2 a-e
R
b
OH
OH
O
O
O
O
O
O
R
R
6d
5d
Scheme 1. Synthesis of anacardic acid derivatives: Reagents and conditions: (a) CuI, PdCl2(PPh3)2, Et2NH, HC„CR, CH3CN, 70 °C; (b) H2, Pd/C, MeOH, 40 °C; (c) KOH 5 N, THF,
55 °C.
4 (b4) and alpha helix 4 (a4) (Fig. 2b). The anacardic acid binding
mode is further stabilized by aromatic edge-to-face stacking be-
tween the salicylate ring and Y616. This molecular docking pose
was used as starting point for the design of new molecules and to
rationalize structure–activity relationships. First of all, the role of
the hydrophobic chain in the binding to the pantothenic acid pocket
O
O
O
O
O
O
a
O
b
O
O
BnO
BnO
OTf
HO
HO
R
R
BnO
7
9
8
between
a4 and b4 was investigated. Secondly, it was hypothesized
R
that incorporation of a second aromatic ring could further stabilize
ligand binding via additional aromatic stacking interactions with
Y616. Thirdly, the acidity of the carboxyl group was modulated by
introduction of substituents on the salicylate ring. In silico predicted
binding modes of novel potent anacardic acid-based PCAF ligands
(Fig. 2c and d) will be discussed in Section 2.3.
OBn OBn
OBn OBn
OH
OH
O
a
b
O
O
BnO
OTf
11 a-b
10
R
12 a-b
2.2. Chemistry
(a)
(b)
A convenient synthetic method was developed in order to syn-
thesize a diverse series of compounds with a salicylate functional-
ity. Several syntheses of anacardic acid have been described so far;
however these routes require harsh conditions that are not com-
patible with many functional groups.24–26 Therefore, a new syn-
thetic route was developed, in which a Sonogashira coupling is
the key step (Scheme 1).
Scheme 2. Synthesis of olivetolic acid derivatives: Reagents and conditions: (a) CuI,
PdCl2(PPh3)2, Et2NH, HC„CR, CH3CN, 70 °C; (b) H2, Pd/C, MeOH, 40 °C.
Triflate 1 was synthesized according to previously published
procedures,27 starting from commercially available 2,6-dihydroxy-
benzoic acid. Different terminal alkynes were coupled in presence
of CuI, diethylamine, and PdCl2(PPh3)2 to give the correspondent
alkynyls 2. The Sonogashira reaction, performed in acetonitrile at
70 °C on oil bath, required 2 h to give the product with high yields
(80–90%). The same reaction was also performed in acetonitrile at
100 °C with microwave irradiation. In this case, the product was
obtained in comparable yields after just 30 min. Hydrogenation,
using palladium on activated carbon as catalyst, was used to re-
duce the akynyl triple bond to a single bond. The best yields (60–
80%) were obtained if hydrogenation was performed for 16 h, with
3 atm H2-pressure in methanol at 40 °C. In case of compound 2d,
hydrogenation under the described conditions gave the correspon-
dent cis-alkene 5d (J = 12.1 Hz). To obtain the alkane 3d, it was nec-
essary to perform hydrogenation for 40 h. The last step in this
synthetic route was the cleavage of the acetonide protecting group.
This was achieved by saponification with KOH 5 N at 55 °C with
yields between 50% and 80%.
alkane 9. Cleavage of the acetonide 9 to yield 12 poses a challenge.
Saponification by hydroxide (OHꢀ) can only be performed under
forcing conditions due to deprotonation of the 4-hydroxy group
and concomitant electron-donation to the carboxylate. Acidic
cleavage of the acetonide will result in decarboxylation.30 We
designed another route to avoid these problems (Scheme 2). Build-
ing block 1031 was employed for Sonogashira coupling to terminal
alkenes. The resulting alkynyls gave the desired product upon
hydrogenation.
Anacardic acid derivatives with an alkoxy function in the
2-position were synthesized starting from 13 (Scheme 3). Com-
pound 13 was converted to 14 using a Williamson ether synthesis
with benzyl bromine (yield 83%). In contrast, reaction of 13 with
tetradecylchloride did not give the desired product. Compound
16 was obtained by Mitsunobu coupling between 13 and tetradec-
anol (yield 29%). Saponification of 14 and 16 provided the corre-
sponding free carboxylic acids 15 and 17 with yields of 49% and
66%, respectively. Compound 18 was synthesized from anacardic
acid using a catalytic amount of H2SO4 in methanol.
Structures of 4-hydroxy-6-alkyl-salicylates 12a and 12b
(Scheme 2) are closely related to another natural compound
known as olivetolic acid, an intermediate in the biosynthesis of
cannabinoids.28 A synthetic route towards these compounds has
been described by Dushin and Danishefsky.29 This route implicates
a Sonogashira coupling on 7, followed by hydrogenation to give the
2.3. Enzyme inhibition assay
The synthesized compounds were tested for inhibition of the
HAT PCAF using an indirect ELISA assay in order to study their bind-
ing properties and structure–activity relationship. The ELISA was

M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
5829
was synthesized to study the importance of a free carboxylic acid
for the interaction with PCAF, however the water solubility of 18
is to low for testing at 1 mM concentration in our assay.
O
O
O
O
O
OH
OH
O
O
O
O
c
a
The role of the hydrophobic interactions for the ligand potency
was investigated by modifications of the hydrophobic alkyl chain.
Compounds 4c and 4d showed inhibition comparable to anacardic
acid, indicating that introduction of an aromatic ring in the chain
did not affect the inhibitory properties. On the other hand, com-
pounds 4b and 12b were less potent than the correspondent AA
4a and 12a, indicating that one-third reduction of the chain length
causes loss of potency. Also compound 15, with a benzyl group in-
stead of the long alkylic chain, showed less than 20% inhibition at
1 mM. These data suggest that hydrophobic interactions at the end
of the CoA binding pocket are important for binding. This observa-
tion is in line with the proposed binding mode of anacardic acid
(Fig. 2b), which is stabilized by hydrophobic interactions deep in
Bn
Bn
OH
13
15
14
b
OH
OH
O
O
O
O
O
c
O
13
13
17
16
OH
OH
OH
O
d
O
O
the pantothenic acid pocket between a4 and b4 (Fig. 1a).
Interestingly, incorporation of a 3-(2-methoxyphenoxy)propyl
moiety 4e provided equal inhibition to AA 4a. In the highest ranked
docking pose of 4e (Fig. 2c), the 2-methoxyphenoxy ring stacks be-
tween the aromatic rings of Y616 and F617, whereas the 2-meth-
oxy group can be accommodated in a small additional pocket
between L589, V576 and Y616. Compound 4e provides a good
platform for further structural variation in order to optimize the
inhibitory potency of this compound class.
14
14
18
(AA) 4a
Scheme 3. Reagents and conditions: (a) Benzylbromide, K2CO3, DMF; (b) tetradec-
anol, PPh3, DIAD, THF; (c) KOH 5 N, THF, 55 °C; (d) H2SO4, MeOH, reflux.
Compound 6d is the only compound that proved to be more
potent than anacardic acid in our assay. We measured for this com-
pound an IC50 of 662 64 lM (see Supplementary data). Remark-
ably, compounds 4c and 4d, also including a second benzyl group,
but lacking the cis-double bond, did not show increased potency
compared to anacardic acid 4a. In the highest ranked docking pose
of compound 6d, the salicylate moiety is hydrogen bonded to the
pyrophosphate binding pocket as described for anacardic acid 4a
(Fig. 2d). In addition, the geometry of the cis-double bond forces
the phenoxy ring in an optimal conformation for face-to-edge aro-
matic stacking with Y616 and orientates the hydrophobic tail of the
molecule deep into the hydrophobic binding cleft between a4 and
b4 (Fig. 2d). This conformational restriction could explain the in-
creased potency of 6d compared to 4d. Compound 6d has improved
druglike properties compared to anacardic acid, because it has a
more favorable lopP and the long aliphatic chain has been replaced
by a more compact substituent. The salicylates are fully deproto-
nated at pH 7.4, which gives a c log P value of 5.1 for anacardic acid
and 2.0 for 6d (Chemical properties, Chemdraw). In conclusion,
compounds 6d and 4e provide a starting point for the design of
new PCAF inhibitors.
Figure 3. Inhibition of the HAT PCAF by different compounds at 1 mM. Data are
expressed as percentage of PCAF activity (n = 3, P <0.05 compared to AA 4a).
*
based on detection of acetylated lysine residues on a histone H4
peptide using an anti-acetyl-lysine antibody. Human recombinant
PCAF was used as source of HAT activity. Anacardic acid (AA) 4a
showed around 40% inhibition at 1 mM (Fig. 3), which would give
an estimated IC50 value between 1 and 2 mM. The potency mea-
sured in our assay is 100-fold lower than the estimation by Balsubr-
amanyam et al.20 but in line with the IC50 reported by Wu et al.32 The
differences might be explained by the fact that Wu et al.32 used com-
parable Ac-CoA concentrations, whereas Balsubramanyam et al.20
used lower Ac-CoA concentrations in the enzyme inhibition assay.
Furthermore, differences in assay buffer composition or differences
in the enzyme source might cause differences in IC50 values.
Several modification of the salicylate moiety were investigated
to slightly modulate the acidity of the carboxyl group. Compounds
12a and 12b showed comparable potency as observed for com-
pounds AA 4a and 4b, respectively. These data show that the intro-
duction of a hydroxyl group in the 4-position of the salicylate does
not influence the inhibitory properties. Introduction of an alkoxy
group in the 6-position of the salicylate ring 17 did not lead to
significant changes in potency. The methyl ester of anacardic acid
2.4. Cell-based study
Since compound 6d was more potent than anacardic acid 4a, we
decided to test the effects of 6d on histone acetylation in a cell-
based study. Histone H4 acetylation levels in HEP G2 cells were
measured using an anti-acetyl histone H4 antibody. The HDACs
inhibitor suberoylanilide hydroxamic acid (SAHA) was used to in-
crease the basal histone acetylation. Cells were treated for 24 h with
5 lM SAHA and co-treated with different concentration of 6d and
anacardic acid 4a. The cells were treated with sub-toxic concentra-
tions of the inhibitors (see Supplementary data). Figure 4a shows
that SAHA increased histone H4 acetylation levels significantly.
Compound 6d decreased the acetylation induced by SAHA in HEP
G2 cells at 30 and 60 lM. The decrease is stronger than observed
for a comparable concentration of anacardic acid (AA) 4a. Quantifi-
cation of the western blot results (Fig. 4b) showed a significant in-
creased inhibition for compound 6d compared to AA 4a (P <0.01).
These data demonstrate that compound 6d is a useful tool to inhibit
histone acetylation in cell-based studies. Differences between the

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M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
4. Experimental sections
4.1. Chemistry
Chemicals were purchased from Sigma-Aldrich and Acros
Organics, and used without further purification. Solvents were
dried before use according to known procedures and all the reac-
tions were carried-out under a nitrogen atmosphere. Analytical
thin-layer chromatography (TLC) was performed on aluminium
sheets of Silica Gel 60 F254. Ultraviolet light, KMnO4 solution or
ninhydrin solution were used to visualize bands. All microwave
irradiation reactions were carried out in a Biotage Initiator™
Microwave Synthesizer. Column chromatography was performed
with MP Ecochrom Silica Gel 32–63, 60 Å. 1H and 13C NMR spectra
were recorded on a Varian Gemini-200. 13C spectra were recorded
using the attached proton test (APT) pulse sequence. Chemical shift
values are reported as part per million (d) relative to residual sol-
vent peaks (CDCl3, 1H d = 7.26, 13C d = 77.16 or CD3OD, 1H d =
3.31, 13C d = 49.00). The coupling constants (J) are reported in Hertz
(Hz). Electrospray ionization mass spectra (ESI-MS) were recorded
on an Applied Biosystems/SCIEX API3000-triple quadrupole mass
spectrometer. High-resolution mass spectra (HR-MS) were re-
corded using a flow injection method on a LTQ-Orbitrap XL mass
spectrometer (Thermo Electron, Bremen, Germany) with a resolu-
tion of 60,000 at m/z 400. Protonated testosterone (lock mass m/
z = 289.2162) was used for internal recalibration in real time. Melt-
ing points were determined on an Electrothermal digital melting
point apparatus and are uncorrected.
4.1.1. Synthetic procedure 1
Figure 4. Cell-based studies on histone H4 acetylation levels in HEP G2 cells. (a)
Western blot of histone extracts of HEP G2 cells after 24 h treatment with different
concentrations of the indicated compounds. Coomassie brilliant blue staining was
used to determine equal amount of loading. (b) Quantification of Histone H4
acetylation levels: values are shown in comparison to histone acetylation levels of
non-treated cells (100%) and expressed as mean SEM of three independent
Freshly distilled diethylamine (3.0 mmol, 0.30 mL) and the
alkyne (2.2 mmol) were subsequently added to a solution of the
triflate (2.0 mmol), CuI (0.20 mmol, 38 mg), and PdCl2(PPh3)2
(0.01 mmol, 70 mg) in degassed anhydrous acetonitrile (6.0 mL).
The mixture was either stirred for 2 h at 70 °C on oil bath or sub-
jected to microwave irradiation for 30 min at 100 °C (70 W). Water
(10 mL) was added and the mixture was extracted with EtOAc
(3 ꢁ 30 mL). The combined organic phases were washed with brine
(1 ꢁ 60 mL), dried over Na2SO4 and filtered. The solvent was evap-
orated under reduced pressure and the residue was purified by col-
umn chromatography.
*
**
experiments. P <0.05, P <0.01 compared to SAHA stimulated samples. #P <0.01
compared to SAHA—AA 4a (60 M) treated samples.
l
effective concentrations between the inhibition of the recombinant
enzyme and the cell-based studies might originate from factors
such as differences in the assay buffer composition, differences in
the Ac-CoA concentration or differences in the enzyme source. Fur-
thermore, the fact that higher concentrations of AA 4a and 6d were
required to inhibit the recombinant enzyme PCAF compared to the
cell-based studies could indicate that these compounds target dif-
ferent histone acetyltransferases in the cell-based studies.
4.1.2. Synthetic procedure 2
A suspension of the starting material (1.5 mmol) and Pd/C (10%)
(0.15 mmol) in methanol (30 mL) was shaken overnight at 40 °C,
with 3 atm H2-pressure in a Parr apparatus. The mixture was filtered
through Celite, and the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography.
3. Conclusion
In this study we describe the development of a salicylate deriv-
ative 6d with improved inhibition of histone acetylation compared
to the natural product anacardic acid. A binding configuration of
anacardic acid in the HAT PCAF active site was proposed using
molecular modeling. In this binding model, the salicylate is
bounded in the pyrophosphate binding pocket and the alkyl chain
is located in the CoA substrate binding cleft. Based on this model,
new anacardic acid derivatives were designed in order to explore
the role of the alkyl chain and the importance of the salicylate moi-
ety. A convenient synthetic route towards anacardic acid was
developed and 10 derivatives were synthesized. Testing of the
inhibitory potency for the PCAF HAT activity showed that one
derivative had a twofold improved inhibitory potency for the
HAT PCAF recombinant enzyme. Moreover, this derivative showed
also a twofold improved potency for inhibition of histone H4 acet-
ylation in cell-based studies on HEP G2 cells. This is the first report
on hypothesis-driven optimization of the HAT PCAF inhibition.
4.1.3. Synthetic procedure 3
The starting material (1.0 mmol) was dissolved in THF (2.0 mL).
KOH 5 N (10 mmol, 2.0 mL) was added and the solution was stirred
overnight at 55 °C. The reaction mixture was diluted with EtOAc
(10 mL) and acidified with 1 N HCl (20 mL). The mixture was
extracted with EtOAc (3 ꢁ 30 mL). The organic phases were
collected, washed with brine (1 ꢁ 50 mL), dried over Na2SO4 and
filtered. The solvent was evaporated and the product was purified
by column chromatography or crystallization.
4.1.4. 2,2-Dimethyl-5-(pentadec-1-ynyl)-4H-benzo[d][1,3]-
dioxin-4-one (2a)
The product was obtained using Section 4.1.1 starting from 1
(0.65 g, 2.0 mmol) and 1-pentadecyne (0.46 g, 2.2 mmol), using
oil bath heating. The crude product was purified using column
chromatography with EtOAc/Hex 1:6 (v/v) as eluent to yield 2a

M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
5831
(0.65 g, 85%) as a brown solid; Rf = 0.60 (EtOAc/Hex 1:2); 1H NMR
(200 MHz, CDCl3): d = 0.87 (3H, t, J = 6.5 Hz), 1.20–1.65 (22H, m),
1.70 (6H, s), 2.50 (2H, t, J = 7.1 Hz), 6.85 (1H, d, J = 8.5 Hz), 7.18
(1H, d, J = 7.6 Hz), 7.38 ppm (1H, t, J = 7.9 Hz); 13C NMR (50 MHz,
CDCl3): d = 14.3, 20.2, 22.9, 25.9, 28.7, 29.2, 29.4, 29.6, 29.7, 29.9,
32.1, 78.9, 98.8, 105.7, 114.2, 116.6, 126.5, 129.1, 134.9, 156.6,
161.6 ppm; MS (ESI): m/z 385.5 [M+H]+.
NMR (50 MHz, CDCl3): d = 25.8, 56.0, 57.9, 85.1, 91.2, 105.8,
111.8, 115.0, 117.8, 121.0, 122.2, 124.6, 129.3, 135.0, 147.2,
149.8, 156.6, 158.8 ppm; MS (ESI) m/z: 356.2 [M+NH4]+.
4.1.9. 2,2-Dimethyl-5-pentadecyl-4H-benzo[d][1,3]dioxin-4-one
(3a)
The product was obtained using Section 4.1.2 starting from 2a
and it was purified by column chromatography using EtOAc/Hex
1:6 (v/v) as eluent. The product was obtained as colorless solid
(0.31 g, 60%); Rf = 0.61 (EtOAc/Hex 1:2); 1H NMR (200 MHz, CDCl3):
d = 0.87 (3H, t, J = 6.5 Hz), 1.20–1.60 (26H, m), 1.69 (6H, s), 3.08
(2H, t, J = 7,6 Hz), 6.79 (1H, d, J = 8.2 Hz), 6.92 (1H, d, J = 7.6 Hz),
7.38 ppm (1H, t, J = 7.9 Hz). 13C NMR (50 MHz, CDCl3): d = 14.3,
22.8, 25.8, 29.5, 29.6, 29.8, 31.3, 32.1, 34.5, 105.1, 112.2, 115.2,
125.2, 135.2, 148.7, 157.2, 160.3 ppm; MS (ESI): m/z 389.5 [M+H]+.
4.1.5. 2,2-Dimethyl-5-(dec-1-ynyl)-4H-benzo[d][1,3]dioxin-4-
one (2b)
The product was obtained using Section 4.1.1 starting from 1
(0.65 g, 2.0 mmol) and 1-decyne (0.30 mg, 2.2 mmol) using oil bath
heating. It was purified by column chromatography using EtOAc/
Hex 1:5 (v/v) as eluent. The product was obtained as a brown solid
(0.52 g, 86%); Rf = 0.58 (EtOAc/Hex 1:1); 1H NMR (200 MHz, CDCl3):
d = 0.86 (3H, t, J = 6.5 Hz), 1.27–1.65 (12H, m), 1.68 (6H, s), 2.48
(2H, t, J = 6.9 Hz), 6.84 (1H, d, J = 8.2 Hz), 7.17 (1H, d, J = 7.6 Hz),
7.38 ppm (1H, t, J = 8.1 Hz); 13C NMR (50 MHz, CDCl3): d = 14.2,
20.1, 22.8, 25.8, 28.6, 29.1, 29.2, 29.3, 32.0, 78.8, 98.7, 105.5,
114.2, 116.5, 126.5, 129.0, 134.8, 156.6, 159.1 ppm; MS (ESI): m/z
315.3 [M+H]+.
4.1.10. 2,2-Dimethyl-5-decyl-4H-benzo[d][1,3]dioxin-4-one (3b)
The product was obtained using Section 4.1.2 starting from 2b
and it was purified by column chromatography using EtOAc/Hex
1:5 as eluent. The product was obtained as a white solid (0.29 g,
65%); Rf = 0.60 (EtOAc/Hex 1:1); 1H NMR (200 MHz, CDCl3):
d = 0.87 (3H, t, J = 6.8 Hz), 1.25–1.66 (16H, m), 1.69 (6H, s), 3.08
(2H, t, J = 7.6 Hz), 6.78 (1H, d, J = 8.2 Hz), 6.92 (1H, d, J = 7.6 Hz),
7.38 ppm (1H, t, J = 7.9 Hz); 13C NMR (50 MHz, CDCl3): d = 14.3,
22.8, 25.8, 28.7, 29.5, 29.6, 29.8, 31.3, 32.1, 33.4, 34.5, 105.1,
112.2, 115.2, 125.2, 135.2, 148.7, 157.2, 160.4 ppm; MS (ESI): m/z
319.3 [M+H]+.
4.1.6. 5-((4-Heptylphenyl)ethynyl)-2,2-dimethyl-4H-benzo[d]
[1,3]dioxin-4-one (2c)
The product was obtained using Section 4.1.1 starting from 1
(0.65 g, 2.0 mmol) and 1-ethynyl-4-heptylbenzene (0.44 g,
2.2 mmol) using oil bath heating. The residue was purified by col-
umn chromatography using EtOAc/Hex 1:14 (v/v) as eluent. The
product was obtained as a brown solid (0.66 g, 88%); Rf = 0.62
(EtOAc/Hex 1:5); 1H NMR (200 MHz, CDCl3): d = 0.88 (3H, t,
J = 6.6 Hz), 1.26–1.73 (8H, m), 1.59 (2H, m), 1.73 (6H, m), 2.62
(2H, t, J = 7.65 Hz), 6.89–6.93 (1H, m), 7.17 (2H, d, J = 8.2 Hz),
7.28–7.33 (1H, m), 7.42–7.46 (1H, m), 7.50–7.57 ppm (2H, d,
J = 8.2 Hz); 13C NMR (50 MHz, CDCl3): d = 14.2, 22.8, 25.9, 29.2,
29.3, 31,3, 31.9, 36.1, 87.2, 96.8, 105.7, 114.0, 117.0, 120.2, 125.8,
128.5, 128.6, 132.1, 134.9, 144.2, 156.8, 159.0 ppm; MS (ESI): m/z
377.3 [M+H]+.
4.1.11. 5-(4-Heptylphenethyl)-2,2-dimethyl-4H-
benzo[d][1,3]dioxin-4-one (3c)
The product was obtained using Section 4.1.2 starting from 2c
and it was purified by column chromatography using EtOAc/Hex
1:12 as eluent. The product was obtained as dark yellow solid
(0.55 g, 97%); Rf = 0.41 (EtOAc/Hex 1:5); 1H NMR (200 MHz, CDCl3):
d = 0.88 (3H, t, J = 6.5 Hz), 1.30 (8H, m), 1.60 (2H, m), 1.69 (6H, m),
2.56 (2H, t, J = 7.6), 2.86 (2H, t, J = 8.0), 3.38 (2H, t, J = 8.0), 6.79–
6.90 (2H, m), 7.06–7.19 (4H, m), 7.34–7.42 ppm (1H, m); 13C
NMR (50 MHz, CDCl3): d = 14.2, 22.8, 25.8, 29.3, 29.4, 31.7, 31.9,
35.7, 36.8, 37.2, 105.1, 105.2, 112.3, 115.5, 125.5, 125.9, 127.2,
128.4, 128.6, 128.9, 135.2, 139.0, 140.6, 147.4, 157.2, 160.4 ppm;
MS (ESI): m/z 381.3 [M+H]+.
4.1.7. 2,2-Dimethyl-5-((4-(pentyloxy)phenyl)ethynyl)-4H-
benzo[d][1,3] dioxin-4-one (2d)
The product was obtained using Section 4.1.1 starting from 1
(0.65 g, 2.0 mmol) and 1-eth-1-ynyl-4-(pentyloxy)benzene
(0.41 g, 2.2 mmol) using either oil bath heating or microwave irra-
diation. The residue was purified by column chromatography using
EtOAc/Hex 1:6 (v/v) as eluent. The product was obtained as brown
oil. The yield was 80% (0.58 g) after oil bath heating and 84%
(0.61 g) after microwave irradiation; Rf = 0.57 (EtOAc/Hex 1:2);
1H NMR (200 MHz, CDCl3): d = 0.72 (3H, t, J = 6.9 Hz), 1.21–1.17
(4H, m), 1.50–1.52 (8H, m), 3.74 (2H, t, J = 6.6 Hz), 6.62–6.68 (3H,
m), 7.03–7.08 (1H, m), 7.20 (1H, d, J = 7.9 Hz), 7.31–7.36 ppm
(2H, m); 13C NMR (50 MHz, CDCl3): d = 14.1, 22.5, 25.8, 28.2,
29.0, 68.1, 86.7, 96.9, 105.7, 113.8, 114.6, 114.9, 116.7, 126.0,
128.2, 133.7, 134.9, 135.3, 156.7, 159.1, 159.8 ppm; MS (ESI): m/z
365.2 [M+H]+.
4.1.12. 2,2-Dimethyl-5-(4-(pentyloxy)phenethyl)-4H-benzo[d]
[1,3]dioxin-4-one (3d)
A suspension of the starting material 2d (0.55 g, 1.5 mmol) and
Pd/C 10% (16 mg, 0.15 mmol) in methanol (30 mL) was stirred un-
der 3 atm H2-pressure at 40 °C for 36 h in a PARR apparatus. The
mixture was filtered through Celite and the filtrate was concen-
trated under reduced pressure. The residue was purified by column
chromatography using EtOAc/Hex 1:14 (v/v) as eluent. The product
was obtained as yellow oil (0.51 g, 93%); Rf = 0.38 (EtOAc/Hex
1:10); 1H NMR (200 MHz, CDCl3): d = 0.88 (3H, t, J = 3.5 Hz),
1.38–1.44 (4H, m), 1.70 (6H, s), 1.73–1.79 (2H, m), 2.84 (2H, t,
J = 8.1 Hz), 3.36 (2H, t, J = 7.9 Hz), 3.93 (2H, t, J = 6.8 Hz), 6.80–
6.84 (m, 2H), 6.86 (1H, d, J = 7.7 Hz), 7.06 (1H, d, J = 8.5 Hz),
7.14–7.16 (m, 2H), 7.35–7.39 ppm (m, 1H); 13C NMR (50 MHz,
CDCl3): d = 14.1, 22.5, 25.7, 28.3, 29.1, 34.9, 36.7, 36.9, 68.0,
105.1, 112.2, 114.4, 115.4, 125.5, 129.3, 129.6, 133.7, 135.2,
147.2, 157.2 157.4, 160.0 ppm; MS (ESI): m/z 369.4 [M+H]+.
4.1.8. 5-(3-(2-Methoxyphenoxy)prop-1-yn-1-yl)-2,2-dimethyl-
4H-benzo [d][1,3]dioxin-4-one (2e)
The product was obtained using Section 4.1.1 starting from 1
(0.65 g, 2.0 mmol) and 1-methoxy-2-(prop-2-yn-1-yloxy)benzene
(0.36 g, 2.2 mmol) using either oil bath heating or microwave irra-
diation. The residue was purified by column chromatography using
EtOAc/Hex 1:5 (v/v) as eluent. The product was obtained as yellow
oil in 88% yield (0.60 g) after bath heating and 86% yield (0.58 g)
after microwave irradiation; Rf = 0.43 (EtOAc/Hex 1:2); 1H NMR
(200 MHz, CDCl3): d = 1.79 (6H, s), 3.97 (3H, s), 5.16 (2H, s), 6.98–
7.07 (4H, m), 7.29–7.35 (2H, m), 7.46–7.54 ppm (1H, m); 13C
4.1.13. 5-(3-(2-Methoxyphenoxy)propyl)-2,2-dimethyl-4H-
benzo[d][1,3] dioxin-4-one (3e)
The product was obtained using Section 4.1.2 starting from 2e
and it was purified by column chromatography using EtOAc/Hex
1:8 (v/v) as eluent. The product was obtained as yellow oil
(0.46 g, 90%); Rf = 0.49 (EtOAc/Hex 1:2); 1H NMR (200 MHz, CDCl3):

5832
M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
d = 1.70 (3H, m), 2.13–2.24 (2H, m), 3.28 (2H, t, J = 7.6 Hz), 3.87
(3H, s), 4.09 (2H, t, J = 6.6 Hz), 6.82 (1H, d, J = 7.0 Hz), 6.90 (4H,
m), 6.98 (1H, d, J = 7.6 Hz), 7.36–7.44 ppm (1H, t, J = 7.9 Hz); 13C
NMR (50 MHz, CDCl3): d = 26.4, 31.1, 31.5, 56.7, 69.1, 105.8,
112.7, 114.1, 116.2, 121.7, 126.1, 126.1, 135.9, 147.7, 149.3,
150.3, 158.0, 161.0 ppm; MS (ESI) m/z 360.3 [M+H]+.
(4H, m), 7.12–7.29 ppm (1H, m); 13C NMR (50 MHz, CD3OD):
d = 32.4, 21.7, 56.6, 69.7, 106.4, 113.6, 115.1, 116.1, 122.2, 122.4,
123.2, 134.3, 145.6, 149.9, 151.0, 162.5, 174.1; HRMS m/z [MꢀH]+
calcd for C17H17O5 301.1081, found 301.1074.
4.1.19. (Z)-2,2-Dimethyl-5-(4-(pentyloxy)styryl)-4H-benzo[d]
[1,3]dioxin-4-one (5d)
4.1.14. 2-Pentadecyl-6-hydroxybenzoic acid (anacardic acid, 4a)
The product was obtained using Section 4.1.3 starting from 3a
and it was purified by crystallization from EtOAc/Hex 1:6 (v/v) at
4 °C. The product was obtained as a white solid (0.30 g, 85%);
Rf = 0.31 (100% EtOAc); mp: 90–91 °C; 1H NMR (200 MHz, CDCl3):
d = 0.88 (3H, t, J = 6.5 Hz), 1.20–1.50 (24H, m), 1.55–1.60 (2H, m),
2.97 (2H, t, J = 7.6 Hz), 6.77 (1H, d, J = 7.3 Hz), 6.87 (1H, d,
J = 8.2 Hz), 7.36 ppm (1H, t, J = 7.9 Hz); 13C NMR (50 MHz, CDCl3):
d = 14.4, 22.9, 29.6, 29.7, 29.9, 30.1, 32.2, 32.3, 36.7, 110.6, 116.1,
123.0, 135.7, 148.1, 163.9, 176.4 ppm; HRMS: m/z [MꢀH]ꢀ, calcd
for C22H35O3 347.2586, found 347.2576.
The product was obtained using the Section 4.1.2 starting from
2d and purified by column chromatography using EtOAc/Hex 1:14
(v/v) as eluent. The product was obtained as yellow oil (0.44 g,
80%); Rf = 0.51 (EtOAc/Hex 1:2); 1H NMR (200 MHz, CDCl3):
d = 1.10–1.19 (3H, m), 1.54–1.67 (4H, m), 1.92–2.07 (8H, m),
4.09–4.18 (2H, m), 6.90–6.93 (2H, m), 7.01–7.10 (2H, m), 7.16–
7.23 (3H, m), 7.23–7.25 (1H, m), 7.35–7.39 ppm (1H, m); 13C
NMR (50 MHz, CDCl3): d = 14.8, 23.2, 26.5, 28.9, 29.7, 68.6, 106.2,
112.8, 114.8, 115.1, 116.1, 116.6, 126.1, 128.3, 129.7, 131.1,
131.2, 134.4, 135.7, 143.0, 159.0, 161.0 ppm; MS (ESI): m/z 367.3
[M+H]+.
4.1.15. 2-Decyl-6-hydroxybenzoic acid (4b)
4.1.20. (Z)-2-Hydroxy-6-(4-(pentyloxy)styryl)benzoic acid (6d)
The product was obtained using Section 4.1.3 starting from 5d
and it was purified by column chromatography using EtOAc/Hex
1:5 (v/v) as eluent. The product was obtained as yellow solid
(0.18 mg, 55%); Rf = 0.67 (100% EtOAc); mp: 94–95 °C; 1H NMR
(400 MHz, CDCl3): d = 0.91 (3H, t, J = 7.0 Hz), 1.33–1.42 (4H, m),
1.71–1.78(2H, m), 3.89 (2H, t, J = 6.4), 6.58 (1H, d, J = 12.1), 6.68
(2H, d, J = 8.4), 6.77 (1H, d, J = 8.3), 6.83 (1H, d, J = 12.1), 6.93 (1H,
d, J =8.2), 7.00 (2H, d, J = 8.4), 7.27–7.32 ppm (1H, m); 13C NMR
(50 MHz, CDCl3): d = 14.1, 22.6, 28.3, 29.0, 68.0, 110.3, 114.2,
114.6, 116.9, 122.9, 128.7, 129.0, 129.3, 130.6, 135.8, 142.6,
158.4, 163.6, 175.5 ppm; HRMS: m/z [MꢀH]+ calcd for C20H21O4
325.1445, found 325.1443.
The product was obtained using Section 4.1.3 starting from 3b
and it was purified by column chromatography using EtOAc/Hex
1:6 (v/v) as eluent. The product was obtained as pale yellow solid
(0.14 g, 51%); Rf = 0.53 (100% EtOAc); mp: 81–82 °C; 1H NMR
(200 MHz, CDCl3): d = 0.87 (3H, t, J = 6.5 Hz), 1.20–1.45 (14H, m),
1.50–1.60 (2H, m), 2.98 (2H, t, J = 7,6 Hz), 6.78 (1H, d, J = 7.6 Hz),
6.87 (1H, d, J = 7.6 Hz), 7.36 ppm (1H, t, J = 7.9 Hz); 13C NMR
(50 MHz, CDCl3): d = 14.3, 22.8, 29.4, 29.5, 29.6, 29.8, 30.0, 32.1,
32.2, 36.6, 110.5, 116.0, 122.9, 135.6, 148.0, 163.8, 176.3 ppm;
HRMS: m/z [MꢀH]ꢀ calcd for C17H25O3 277.1804, found 277.1807.
4.1.16. 2-(4-Heptylphenethyl)-6-hydroxybenzoic acid (4c)
The product was obtained using Section 4.1.3 starting from 3c
and it was purified by column chromatography using EtOAc/Hex
1:2 (v/v) as eluent. The product was obtained as white powder
(0.23 g, 67%); Rf = 0.4 (100% EtOAc); mp: 110–111 °C; 1H NMR
(200 MHz, CDCl3): d = 1.03 (3H, t, J = 5.5 Hz), 1.43–1.46 (8H, m),
1.71–1.78 (2H, m), 2.72 (2H, t, J = 7.6 Hz), 3.00–3.08 (2H, m),
3.40–3.48 (2H, m), 6.86 (1H, d, J = 7.5 Hz), 7.06 (1H, d, J = 7.5 Hz),
7.20 (4H, m), 7.39–7.56 ppm (1H, m); 13C NMR (50 MHz, CDCl3):
d = 14.2, 22.8, 29.3, 29.5, 31.7, 32.0, 35.7, 38.0, 38.7, 110.6, 116.4,
123.2, 127.0, 128.4, 128.5, 129.0, 135.8, 139.0, 140.8, 146.7,
163.8, 176.1 ppm; HRMS: m/z [MꢀH]ꢀ calcd for C22H27O3
339.1965, found 339.1969.
4.1.21. Benzyl 2,4-bis(benzyloxy)-6-(((trifluoromethyl)sulfonyl)-
oxy) benzoate (10)
Triflic anhydride (35 mmol, 5.9 mL) was added dropwise to a
solution of benzyl 2,4-bis(benzyloxy)-6-hydroxybenzoate (30 mmol,
13 g) and pyridine (0.11 mol, 8.1 mL) in dry dichloromethane
(30 mL) at 0 °C. The resulting mixture was stirred for 1.5 h at 0 °C.
The excess of triflic anhydride was quenched by dropwise addition
of water (5 mL). The mixture was slowly poured into a saturated
solution of NaHCO3 (50 mL), the two layers were separated in a sep-
aration funnel, and the organic layer was collected. The water layer
was washed with dichloromethane (2 ꢁ 50 mL). The combined
organic layers were washed with water (1 ꢁ 100 mL), brine
(1 ꢁ 100 mL), and dried over Na2SO4 and concentrated under re-
duced pressure. The crude product was purified by column chroma-
tography using EtOAc/Hex 1:10 (v/v) as eluent to yield the product
as a white solid (9.6 g, 56%); Rf = 0.46 (EtOAc/Hex 1:4); 1H NMR
(400 MHz, CDCl3): d = 5.04 (s, 2H), 5.05 (s, 2H) 5.33 (s, 2H), 6.54
(s, 1H), 6.59 (s, 1H), 7.34 ppm (m, 15H); 13C NMR (50 MHz, CDCl3):
d = 67.9, 71.1, 71.3, 100.7, 100.8, 110.9, 127.5, 127.8, 128.5, 128.7,
128.8, 128.9, 129.1, 135.5, 135.7, 148.3, 158.8, 161.6, 163.3 ppm;
MS (ESI): m/z 590.2 [M+NH4]+.
4.1.17. 2-Hydroxy-6-(4-(pentyloxy)phenethyl)benzoic acid (4d)
The product was obtained using Section 4.1.3 starting from 3d
and it was purified by column chromatography using EtOAc/Hex
1:5 (v/v) as eluent. The product was obtained as yellow solid
(0.17 g, 51%); Rf = 0.43 (100% EtOAc); mp: 98–99 °C; 1H NMR
(200 MHz, CD3OD): d = 0.94 (3H, t, J = 7.0 Hz), 1.38–1.45 (m, 4H),
1.72–1.75 (m, 2H), 2.70–2.85 (2H, m), 3.10–3.20 (2H, m), 3.92 (t,
J = 6.5 Hz), 6.66–6.80 (m, 4H), 7.08 (2H, d, J = 8.5 Hz), 7.23–
7.27 ppm (m, 1H); 13C NMR (50 MHz, CD3OD): d = 13.4, 22.5,
28.4, 29.2, 37.7, 38.7, 68.0, 106.4, 114.3, 115.1, 122.2, 129.2,
133.2, 134.4, 144.9, 157.8, 161.7, 171.7 ppm; HRMS m/z [MꢀH]+
calcd for C20H23O4 327.1601, found 327.1614.
4.1.22. Benzyl 2,4-bis(benzyloxy)-6-(pentadec-1-yn-1-yl)-
benzoate (11a)
The product was obtained using Section 4.1.1 starting from 10
(1.2 g, 2.0 mmol) and 1-pentadecyne (0.46 mg, 2.2 mmol) using
either oil bath heating or microwave irradiation. It was purified
by column chromatography using EtOAc/Hex 1:25 (v/v) as eluent.
The product was obtained as colorless oil in 83% yield (1.1 g) for oil
bath heating and 88% (1.1 g) for microwave irradiation; Rf = 0.53
(EtOAc/Hex 1:7 v/v); 1H NMR (400 MHz, CDCl3): d = 0.88 (3H, t,
J = 6.3 Hz,), 1.20–1.60 (22H, m), 2.28 (2H, t, J = 6.6 Hz,), 5.00 (1H,
s), 5.02 (1H, s), 5.33 (1H, s), 6.50 (1H, d, J = 2.4 Hz), 6.63 (1H, d,
4.1.18. 2-Hydroxy-6-(3-(2-methoxyphenoxy)propyl)benzoic
acid (4e)
The product was obtained using Section 4.1.3 starting from 3e
and it was purified by column chromatography using EtOAc/Hex
1:4 (v/v) as eluent. The product was obtained as white powder
(0.22 g, 73%); Rf = 0.42 (EtOAc); mp: 147–148 °C; 1H NMR
(200 MHz, CD3OD): d = 2.03–2.11 (2H, m), 3.09 (2H, t, J = 7.6 Hz),
3.83 (3H, s), 3.98 (2H, t, J = 6.5 Hz), 6.75–6.79 (2H, m), 6.89–6.94

M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
5833
J = 2.4 Hz), 7.24–7.39 ppm (15H, m); 13C NMR (50 MHz, CDCl3):
d = 14.2, 19.5, 22.7, 28.6, 29.0, 29.2, 29.4, 29.6, 29.7, 31.9, 66.9,
70.2, 70.5, 77.8, 94.6, 101.1, 109.6, 119.8, 124.1, 127.1, 127.6,
127.9, 128.0, 128.2, 128.4, 128.5, 128.7, 136.0, 136.2, 136.3,
156.8, 160.2, 166.9 ppm; MS (ESI): m/z 631.4 [M+H]+.
1:2 (v/v) as eluent. The product was obtained as white solid
(0.12 g, 49%); Rf = 0.39 (100% EtOAc); mp: 122–123 °C; 1H NMR
(200 MHz, CD3OD): d = 5.27 (2H, s), 6.59 (1H, d, J = 8.2), 6.67 (1H,
d, J = 8.2), 7.35 (5H, m), 7.48–7.53 ppm (1H, m). 13C NMR
(50 MHz, CD3OD): d = 73.0, 105.2, 112.0, 112.2, 121.1, 129.8,
130.1, 136.4, 137.8, 160.5, 164.5, 171.5 ppm; HRMS: m/z [MꢀH]ꢀ
calcd for C14H11O4 243.0662, found 243.0666.
4.1.23. Benzyl 2,4-bis(benzyloxy)-6-(dec-1-yn-1-yl)benzoate
(11b)
The product was obtained using Section 4.1.1 starting from 10
(1.2 g, 2.0 mmol) and 1-decyne (0.30 g, 2.2 mmol) using micro-
wave irradiation. It was purified by column chromatography using
EtOAc/Hex 1:20 (v/v) as eluent. The product was obtained as
brown oil (0.89 g, 79%); Rf = 0.66 (EtOAc/Hex 1:4); 1H NMR
(400 MHz, CDCl3): d = 0.88 (t, J = 6.6, 3H), 1.27–1.55 (m, 12H),
2.28 (t, J = 6.9, 2H), 4.99 (s, 2H), 5.02 (s, 2H), 5.33 (s, 2H), 6.50
(1H, d, J = 4.3 Hz), 6.62 (1H, d, J = 4.3 Hz), 7.27–7.40 ppm (m,
15H); 13C NMR (50 MHz, CDCl3): d = 14.1, 19.4, 22.6, 28.5, 28.9,
29.1, 29.2, 31.8, 66.9, 70.2, 70.5, 94.6, 101.1, 109.6, 119.8, 124.1,
127.0, 127.5, 127.8, 127.9, 128.2, 128.3, 128.5, 128.6, 135.9,
136.2, 136.3, 156.8, 160.2, 166.9 ppm; MS (ESI): m/z 561.4 [M+H]+.
4.1.28. 2,2-Dimethyl-5-(tetradecyloxy)-4H-benzo[d][1,3]dioxin-
4-one (16)
5-Hydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one
(13)
(0.99 g, 5.1 mmol) and 1-tetradecanol (1.6 g, 7.5 mmol) were dis-
solved in dry THF (15 mL), and the reaction mixture was cooled
down to 0 °C. PPh3 (2.0 g, 7.5 mmol) and DIAD (1.5 mL, 7.5 mmol)
were added, and the mixture was first stirred for 1 h at 0 °C and
then overnight at room temperature. The reaction mixture was
concentrated under reduced pressure, and the compound was
purified by column chromatography using EtOAc/Hex 1:12 as elu-
ent. The product was obtained as white solid (0.54 g, 27%);
Rf = 0.33 (EtOAc/Hex 1:10); 1H NMR (200 MHz, CDCl3): d = 0.84
(3H, m), 1.09–1.33 (22H, m), 1.51 (2H, m), 1.70 (6H, s), 4.03 (2H,
t, J = 6.8 Hz), 6.50 (1H, d, J = 7.4 Hz), 6.57 (1H, d, J = 8.5 Hz), 7.32–
7.39 ppm (1H, m); 13C NMR (50 MHz, CDCl3): d = 14.09, 22.8,
25.7, 25.9, 26.8, 27.5, 29.1, 29.5, 29.6, 29.7, 32.2, 69.5, 103.6,
105.2, 106.5, 109., 136.3, 157.9, 158.0, 161.2 ppm; MS (ESI): m/z
391.3 [M+H]+.
4.1.24. 2,4-Dihydroxy-6-pentadecylbenzoic acid (12a)
The product was obtained using Section 4.1.2 starting from 10a
and purified by column chromatography using EtOAc/Hex 1:3 (v/v)
as eluent. The product was obtained as a white solid (0.49 g,
89.5%); Rf = 0.19 (100% EtOAc); mp: 124–125 °C; 1H NMR
(400 MHz, CD3OD): d = 0.88 (3H, t, J = 6.8 Hz), 1.22–1.31 (24H,
m), 1.51–1.56 (2H, m), 2.86 (2H, t, J = 7.7 Hz), 6.14 (1H, d,
J = 2.2 Hz), 6.18 ppm (1H, d, J = 2.2 Hz); 13C NMR (50 MHz, CD3OD):
d = 13.1, 22.4, 29.1, 29.2, 29.4, 29.6, 31.7, 31.8, 36.2, 100.3, 103.5,
110.4, 148.7, 162.2, 165.5, 173.4 ppm; MS (ESI): m/z 363.4;
[MꢀH]ꢀ; HRMS: m/z [MꢀH]ꢀ calcd for C22H35O4 363.2540, found
363.2542.
4.1.29. 2-Hydroxy-6-(tetradecyloxy)benzoic acid (17)
The product was obtained using Section 4.1.3 starting from 16
and it was purified by column chromatography using EtOAc/ Hex
1:5 (v/v) as eluent. The product was obtained as pale pink solid
(0.23 g, 66%); Rf = 0.63 (100% EtOAc); mp: 93–94 °C; 1H NMR
(200 MHz, CDCl3): d = 0.87 (3H, t, J = 6.8 Hz), 1.21–1.26 (22H, m),
1.86–1.90 (2H, m), 4.21 (2H, t, J = 6.2), 6.46 (1H, d, J = 8.2), 6.69
(1H, d, J = 8.5), 7.33–7.41 ppm (1H, m). 13C NMR (50 MHz, CDCl3):
d = 14.2, 22.8, 25.9, 26.0, 26.7, 27.5, 28.9, 29.2 29.5, 29.6, 29.7,
32.1, 70.9, 101.8, 102.3, 112.2, 134.2, 135.6, 158.2, 171.0 ppm;
HRMS: m/z [MꢀH]ꢀ calcd for C21H33O4 349.2384, found 349.2383.
4.1.25. 2-Decyl-4,6-dihydroxybenzoic acid (12b)
The product was obtained using Section 4.1.2 starting from 10b
and it was purified by column chromatography using EtOAc/Hex
1:3 (v/v) as eluent. The product was obtained as a pale orange solid
(0.34 g, 76.5%); Rf = 0.48 (100% EtOAc); mp: 107–108 °C; 1H NMR
(400 MHz, CD3OD): d = 0.90 (3H, t, J = 6.8 Hz), 1.29–1.33 (m,
14H), 1.54–1.57 (2H, m), 2.86–2.90 (2H, t, J = 7.9 Hz), 6.14 (1H, d,
J = 2.6 Hz), 6.19 ppm (1H, d, J = 2.6 Hz); 13C NMR (50 MHz, CD3OD):
d = 14.4, 23.7, 30.5, 30.7, 30.8, 30.9, 33.1, 33.2, 37.6, 101.7, 111.4,
111.7, 150.2, 163.5, 166.8, 173.5 ppm; HRMS: m/z [MꢀH]ꢀ calcd
for C17H25O4 293.1758, found 293.1761.
4.1.30. Methyl 2-hydroxy-6-pentadecylbenzoate (18)
The starting material 4a (0.17 g, 0.5 mmol) was dissolved in
5 mL methanol. Concentrated H2SO4 (0.5 mL) was slowly added
and the solution was stirred for 4 h at reflux. The mixture was then
cooled down to room temperature, diluted with 20 mL dichloro-
methane and extracted with NaOH 1 M (3 ꢁ 20 mL). The organic
layer was washed with brine (1 ꢁ 15 mL), dried over Na2SO4 and
filtered. The solvent was evaporated to give the pure product as
a pail yellow solid (0.12 g, 65%); Rf = 0.73 (EtOAc/Hex 1:6); mp:
77–78 °C; 1H NMR (200 MHz, CD3OD): d = 0.85–0.88 (3H, m),
1.26–1.53 (26H, m), 2.88 (2H, t, J = 7.6), 3.96 (3H, s), 6.72 (1H, d,
J = 6.8), 6.82 (1H, d, J = 7.8), 7.25–7.29 ppm (1H, m); 13C NMR
(50 MHz, CD3OD): d = 14.3, 22.8, 25.1, 25.7, 29.5, 29.7, 29.8, 30.0,
32.1, 32.3, 34.1, 36.8, 52.2, 112.0, 115.7, 122.6, 134.3, 146.3,
162.7, 172.1 ppm; HRMS: m/z [MꢀH]ꢀ calcd for C23H37O3
361.2746, found 361.2748.
4.1.26. 5-(Benzyloxy)-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-
one (14)
The starting material 13 (2.2 g, 11 mmol) was dissolved in DMF
(20 mL), and cooled to 0 °C. K2CO3 (3.1 g, 22 mmol) was added and
benzylbromide (1.5 mL, 12 mmol) was added dropwise, and the
mixture was stirred for 1 h at 0 °C and then overnight at room tem-
perature. The reaction mixture was extracted with EtOAc
(3 ꢁ 20 mL), and the combined organic layers were washed with
brine (1 ꢁ 40 mL), dried with MgSO4, and filtered. The solvent
was evaporated to give the pure compound as a yellow solid
(2.6 g, 84%); Rf = 0.23 (EtOAc/Hex 1:5); 1H NMR (200 MHz, CDCl3):
d = 1.71 (6H, s), 5.25 (2H, s), 6.53 (1H, d, J = 8.2 Hz), 6.63 (1H, d,
J = 8.5 Hz), 7.26–7.43 (5H, m), 7.53–7.57 ppm (1H, m); 13C NMR
(50 MHz, CDCl3): d = 25.8, 70.8, 104.2, 105.1, 105.4, 107.3, 109.6,
126.8, 127.9, 128.7, 136.4, 158.0, 158.2, 160.5 ppm; MS (ESI): m/z
285.2 [M+H]+.
4.2. Cell culture and histone extraction
All cell culture reagents were purchased from Invitrogen. The
human cancer cell line HEP G2 (liver) was cultured in Dulbecco’s
modified Eagle Medium (DMEM) containing 10% heat-inactivated
fetal serum bovine (FBS), 50 IU/mL penicillin, and 50 mg/mL strep-
tomycin at 37 °C in a humidified atmosphere containing 5% CO2.
For the experiments, cells were seeded in T-175 flasks, allowed
to grow until about 70% confluency and FBS-starved for 16 h. Cells
were then treated at sub-toxic (see Supplementary data) concen-
4.1.27. 2-(Benzyloxy)-6-hydroxybenzoic acid (15)
The product was obtained using Section 4.1.3 starting from 14
and it was purified by column chromatography using EtOAc/Hex

5834
M. Ghizzoni et al. / Bioorg. Med. Chem. 18 (2010) 5826–5834
tration of inhibitor for 24 h. The histone extraction was performed
as previously described in literature with minor modifications.33
Protein concentrations of samples were determined using the
Bradford Assay using BSA as a standard for calibration.
the co-crystallized CoA. One active site water molecule (pdb ID6)
was kept for the docking. Fifteen poses were generated for each
compound. The docking results were analyzed using MOE 2008.10
(www.chemcomp.com) and Molegro Virtual Docker (www.mole-
gro.com). Pictures were generated using PyMOL (www.pymol.org).
4.3. Immunoblot protocol
A. Supplementary data
Approximately 5 lg of extracted histones were loaded on a
12.5% polyacrylamide gel, resolved by SDS–PAGE electrophoresis
and electroblotted to PVDF membranes. The membranes were
incubated with rabbit anti-acetyl-Histone H4 antibody (Millipore,
06-598) followed by a swine anti-rabbit HRP conjugated antibody
(DakoCytomation, P0217). Bands were visualized using AEC detec-
tion kit (Sigma). Bands were scanned using a BioRad GS-710 den-
sitometer and quantified using ImageJ quantification software.
Equal amount of loading was determined by Coomassie blue stain-
ing. The experiments were performed in triplicate.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.089. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
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An indirect ELISA was used to test the inhibition of the HAT
PCAF activity. A solution 0.1% of bovine serum albumin (BSA) in
100 mM HEPES 7.4 was used as buffer unless differently specified.
C96 Maxisorp 96-well plate (Nunc) was coated overnight at 4 °C
with 50
Na2CO3 pH 9.4. After washing with buffer, the wells were blocked
with 200 L of a solution 3% BSA in buffer for 1 h at room temper-
ature (rt). The plate was washed with buffer, and then coated with
50 L per well of a solution 300 nM biotinylated histone H4 pep-
tide (Millipore, 12-372) for 1 h at rt. After washing, 40 L reaction
buffer (0.1% BSA + 0.16% Triton X-100 in 100 mM HEPES pH 7.4)
was added to the wells, followed by 20 L recombinant PCAF
240 nM in reaction buffer, and 10 L of the selected inhibitor solu-
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incubation, 10 L solution 40 M Ac-CoA in reaction buffer was
lL per well of a solution 19 lg/mL streptavidin in 0.2 M
l
l
l
l
l
l
l
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buffer and incubated with 100 lL per well of chromogen solution
(0.1 mg/ml 3,30,5,50-tetramethyl benzidine + 0.003% H2O2 in 0.1 M
acetate buffer pH 5.5). The reaction was terminated by addition
of 100 lL H2SO4 1.0 M and the optical extinction read at 450 nm.
The positive controls were the values of wells with no inhibitor,
which were set as 100%. The negative controls were the values of
wells with no Ac-CoA and were set as 0%. Stock solutions of inhib-
itors were prepared in dimethyl sulfoxide (DMSO) and diluted in
reaction buffer. The final concentration of DMSO was tested not
to have any effect on the assay. Each compound was tested in trip-
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4.5. Molecular docking
The Crystal structure of PCAF chain B was downloaded form Pro-
tein Data Bank (code 1CM0). All molecules were drawn using
Chemaxon MarvinSketch (www.chemaxon.com) and prepared
(structure recognition and protonation) using SPORES (www.tcd.u-
ni-konstanz.de/research/spores.php). Molecular docking simula-
tions were performed using PLANTS v1.6.34,35 The docking site
center was determined by considering all residues within 5 Å from