Synthesis and biological activities of thio-avarol derivatives

Article abstract of DOI:10.1021/np800318m

Eleven new thio-avarol derivatives (3-13) were synthesized. Their antimicrobial, brine shrimp lethality, and free-radical scavenging activities and acetylcholinesterase inhibition, together with 12 already reported semisynthetic thio-avarol derivatives (14-25), were evaluated. Structure-activity relationships among these thio derivatives were determined.

Full text of DOI:10.1021/np800318m

1850
J. Nat. Prod. 2008, 71, 1850–1853
Synthesis and Biological Activities of Thio-avarol Derivatives#
Boris Pejin, Carmine Iodice, Giuseppina Tommonaro, and Salvatore De Rosa*
Istituto di Chimica Biomolecole CNR, Via Campi Flegrei, 34, I-80078 Pozzuoli (Napoli), Italy
ReceiVed May 29, 2008
Eleven new thio-avarol derivatives (3-13) were synthesized. Their antimicrobial, brine shrimp lethality, and free-
radical scavenging activities and acetylcholinesterase inhibition, together with 12 already reported semisynthetic thio-
avarol derivatives (14-25), were evaluated. Structure-activity relationships among these thio derivatives were determined.
Avarol is a marine sesquiterpenoid hydroquinone, previously
isolated from the marine sponge Dysidea aVara Schmidt (Dictyocera-
tida),1,2 with interesting pharmacological properties3 including anti-
inflammatory,4 antitumor,5 antioxidant,6 antiplatelet,7 anti-HIV,8
and antipsoriatic9,10 effects. Previous studies demonstrated the
antioxidant properties of avarol, which inhibits superoxide genera-
tion and microsomal lipid peroxidation.4,6 The biological activities
of this compound have been correlated with its redox chemistry
and its ability to effect radical production, while the terpenoid
moiety plays a marginal role in biological processes.11 These
interesting properties and the previous findings that avarone, the
quinone of avarol, reacts toward protein sulfhydryl groups12 and
that 3′-(salicylthio) avarol (11) could be a promising antipsoriatic
agent13 prompted us to prepare further sulfhydryl derivatives of
avarol and to extend the evaluation to other biological properties.
In this paper we report the synthesis of 11 new thio-avarol
derivatives (3-13), the antimicrobial, brine shrimp lethality, and
free-radical scavenging activities, and acetylcholinesterase inhibition
of these new derivatives together with 12 already described thio
derivatives (14-25).14 This represents a wide series of thio-avarol
derivatives, with different polarities. Brine shrimp lethality15 was
used as an indicator of cytotoxicity. This assay was demonstrated
to be in excellent agreement with L5 178y (mouse lymphoma cells)
and L12 10 (leukemia cells) assays, using avarol (1) and avarone
(2).16 The free-radical scavenging assay is a simple test evaluating
the potential antioxidant activity of compounds, using 2,2-diphenyl-
1-picrylhydrazyl (DPPH).17,18 Acetylcholinesterase (AChE) inhibi-
tion was detected by a TLC bioautographic assay.19,20 AChE is
the enzyme involved in the metabolic hydrolysis of acetylcholine
at cholinergic synapses in the central and peripheral nervous system.
The abnormal activity of this enzyme is one factor responsible for
Alzheimer’s disease, the most common cause of senile dementia
in later life. AChE inhibitors are still the best drugs currently
available for the management of this disease.21
Results and Discussion
Avarol (1) was isolated from the sponge Dysidea aVara,1
collected in the Bay of Naples, Italy. Avarone (2) was obtained by
Ag2O oxidation of avarol, in ethanol, as previously reported.1 Thio
derivatives (3-25) (Figure 1) were generally obtained by slowly
adding the corresponding thio compound dissolved in ethanol to a
solution of avarone in ethanol.13 Using 3-mercaptobenzoic acid only
one derivative was obtained with substitution at 3′ (3) of the
benzoquinone ring, as well as with thiosalicylic acid, previously
reported (14).22 On the one hand, for the 2-mercaptobenzyl alcohol,
thiolactic acid, and 2-mercaptobenzothiazole two isomers were
obtained with substitution at 3′ (4, 6, and 8) and 4′ (5, 7, and 9),
as well as with thiophenol (15 and 16, with substitution at 3′ and
Figure 1. Chemical structures of avarol derivatives.
4′, respectively), thioglycol (20 and 21, with substitution at 3′ and
4′, respectively), thioglycerol (22 and 23, with substitution at 3′
and 4′, respectively), and thioglycolic acid (24 and 25, with
substitution at 3′ and 4′, respectively), previously reported.14 On
the other hand, for p-thiocresol three isomers were obtained with
substitution at 3′ (17), 4′ (18), and at both 3′ and 4′ (19), as
previously reported.14 Only for the thiocholesterol were four isomers
obtained; in fact, in addition to 3′- (11), 4′- (12), and 3′,4′-
# Dedicated to the memory of Prof. Rodolfo Nicolaus.
* Corresponding author. Tel: +39 081 8675029. Fax: +39 081 8041770.
E-mail: sderosa@icmib.na.cnr.it.
10.1021/np800318m CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/13/2008

Synthesis of Thio-aVarol DeriVatiVes
Journal of Natural Products, 2008, Vol. 71, No. 11 1851
Table 1. Biological Activities of Avarol (1), Avarone (2), and Thio-avarol Derivatives (3-25)
antimicrobial MIC (µg/mL)a
compound
B. s.
M. l.
S. c.
A. salina LC50 (95% C.L.)b
DPPH IC50 (µM)c
AChE (µg)d
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
25
10
10
N.A.
N.A.
10
10
1.0
10
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
100
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.18 (0.32/0.10)
0.14 (0.32/0.07)
14.73 (67.54/4.52)
>50
10.64 (18.55/5.06)
27.25 (57.41/14.80)
>50
N.A.
N.A.
N.A.
N.A.
18.0
32.1
31.8
25.3
90.0
34.6
35.3
29.5
31.2
N.A.
31.4
71.4
41.5
34.0
10.7
N.A.
63.7
N.A.
N.A.
N.A.
N.A.
62.5
71.4
40.5
52.3
10
1
1
10
>10
1
1
10
>10
>10
>10
>10
>10
1
10
N.A.
10
N.A.
N.A.
N.A.
N.A.
10
N.A.
N.A.
N.A.
N.A.
0.01
N.A.
N.A.
N.A.
N.A.
N.A.
10
10
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.01
10
100
N.A.
100
N.A.
10
100
10
10
10
0.01
1.0
N.A.
N.A.
15.11 (45.37/6.04)
0.97 (1.54/0.11)
33.10 (54.53/20.44)
0.23 (0.95/0.10)
24.87 (43.23/14.77)
N.A.
N.A.
N.A.
1.50 (3.71/0.21)
0.23 (1.13/0.12)
8.50 (14.85/4.45)
12.83 (23.70/7.09)
N.A.
100
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
10
22
23
24
25
10
1
1
100
10
gentamicin
nystatin
trolox
galanthamine
100
25.8
0.01
a B. s. )Bacillus subtilis; M. l. ) Micrococcus luteus; S. c. ) Saccharomyces cereVisiae. N.A. ) MIC > 100 µg/mL. b LC50 values are expressed in
ppm; 95% C.L.) 95% confidence limits. N.A. ) LC50 > 100 ppm. c Concentration that promotes 50% of DPPH reduction. N.A. ) IC50 > 100 µM.
d Amounts given, in µg, are the minimum inhibitory quantities applied on the TLC plates. N.A. ) >50 µg.
thiocholesterol avarol (13), 4′-thiocholesterol avarone (10) was also
isolated. The position of the substituent was determined by the
analysis of 1H NMR spectra. Signals of protons in the benzoquinone
ring are doublets in 3′-substituted compounds and singlets in 4′-
substituted compounds, while 3′,4′-disubstituted compounds show
only one proton signal as a singlet.
The AChE inhibition tests showed a moderate activity (1 µg)
for all avarol derivatives with a carboxylic acid group in the
molecule (3, 6, 7, 24, and 25). In comparison, the alkaloid
galanthamine used clinically for the treatment of Alzheimer’s
disease21 inhibited the enzyme at 0.01 µg. Because most inhibitors
of AChE are alkaloids that often possess several side effects,23 it
is important to search for new AChE inhibitors not belonging to
this structural class.
The antimicrobial activity evaluation was carried out using a
liquid culture of three bacterial strains (Escherichia coli, Bacillus
subtilis, and Micrococcus luteus) and yeast (Saccharomyces cer-
eVisiae), by a serial dilution, and the results are reported in Table
1. None of the tested compounds showed effects against the Gram-
negative bacterium E. coli. Only compound 8 showed good
antifungal activity, comparable with that of nystatin, against the
yeast S. cereVisiae. Further, compound 8 showed high antibacterial
activity, more than gentamicin (MIC 10 µg/mL), against the Gram-
positive bacterium M. luteus (MIC 0.01 µg/mL). Compounds 14
and 25 showed high antibacterial activity, more active than
gentamicin (MIC 1.0 µg/mL), against the Gram-positive bacterium
B. subtillis (MIC 0.01 µg/mL) and were moderately active against
M. luteus. Compounds 3, 6, 7, 15, 20, 22, 23, and 24 were
moderately active (MIC 10 µg/mL) against B. subtillis.
Results obtained in the brine shrimp test (Table 1) showed that
all avarol derivatives are less active than avarol (1) and avarone
(2). Only compounds 15, 17, and 23 showed interesting cytotoxic
activity, with LC50 ) 0.97, 0.23, and 0.23 ppm, respectively.
Generally, the introduction of a substituent on the hydroquinone
ring of the avarol skeleton reduces the antioxidant activity, evaluated
by the free-radical scavenging assay using 2,2-diphenyl-1-picryl-
hydrazyl (DPPH)17,18 as a TLC spray reagent. The results are
reported in Table 1. Avarol derivatives with substitution at 3′ (3,
4, 6, 8, 11, 15, 17, 22, and 24) are more active than those with
substitution at 4′ (5, 7, 9, 12, 16, 18, 17, 23, and 25). Avarol (1)
showed the most potent antioxidant activity, with IC50 ) 18 µM,
while 6, 9, and 10 exhibited moderate potencies with IC50 ) 34,
95, and 98 µM, respectively.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP 370 polarimeter, using a 10 cm microcell. UV spectra
were obtained on a Varian DMS 90 spectrophotometer. NMR spectra,
recorded at the NMR Service of Istituto di Chimica Biomolecolare del
CNR (Pozzuoli, Italy) on a Bruker Avance-400 operating at 400 MHz,
using an inverse probe fitted with a gradient along the Z-axis, in CDCl3,
using the residual CDCl3 resonance at 7.26 ppm as internal references.
Only chemical shifts of hydroquinone are reported because all other
signals belonging to the sesquiterpenoid portion were earlier reported 1,2
and thio residues are generally well-known.24 LRMS and HRMS were
recorded on a JEOL JMS D-300 and an AEI MS-50, respectively.
Column chromatography was carried out on Merck silica gel 60.
Materials. Avarol (1) was isolated from the sponge Dysidea aVara,1
which was collected in the Bay of Naples, Italy. A voucher specimen
is maintained in the collection of the ICB-CNR. Avarone (2) was
prepared from avarol by oxidation with Ag2O as previously described.1
Avarol-3′-thiosalicylate (14), avarol-3′-thiophenol (15), avarol-4′-
thiophenol (16), avarol-3′-thiocresol (17), avarol-4′-thiocresol (18),
avarol-3′-4′-dithiocresol (19), avarol-3′-thioglycol (20), avarol-4′-
thioglycol (21), avarol-3′-thioglycerol (22), avarol-4′-thioglycerol (23),
avarol-3′-thioglycolate (24), and avarol-4′-thioglycolate (25) were
obtained as previously reported.13 3-Mercaptobenzoic acid, 2-mercap-
tobenzyl alcohol, thiolactic acid, 2-mercaptobenzothiazole, and thio-
cholesterol were obtained from Sigma-Aldrich (Milano, Italy). Ace-
tylcholinesterase, 1-naphthyl acetate, and the rest of the reagents used
in the biological tests were obtained from Sigma Chemicals (St. Louis,
MO). Fast Blue B salt was from Fluka (Milano, Italy).

1852 Journal of Natural Products, 2008, Vol. 71, No. 11
Pejin et al.
Synthesis of Avarol-3′-thiobenzoate (3). 3-Mercaptobenzoic acid
(100 mg) was dissolved in EtOH (5 mL), added to a solution of avarone
(2) (100 mg) in EtOH (10 mL), and stirred for 2 h at room temperature.
After evaporation of EtOH, the residue was chromatographed on a Si
gel column and eluted with CHCl3-MeOH (95:5) to give avarol-3′-
thiobenzoate (3) (77 mg; yield 52%): amorphous solid; [R]D -0.3 (c
0.25, CHCl3); UV (MeOH) λmax (log ε) 224 (5.03), 314 (4.38); 1H NMR
(CDCl3) δ 6.90 (H-6′, d, J ) 2.7 Hz), 6.76 (H-4′, d, J ) 2.7 Hz) and
see Supporting Information; EIMS m/z 468 [M + 2]+ (0.4), 466 [M]+
(10), 276 (18), 258 (22), 191 (28), 189 (30), 135 (30), 107 (35), 95
(100); HREIMS m/z 466.2183 (calcd for C28H34O4S, 466.2178).
Synthesis of 3′-(Benzylthio)avarol (4) and 4′-(Benzylthio)avarol
(5). 2-Mercaptobenzyl alcohol (100 mg) was dissolved in EtOH (5 mL),
added to a solution of avarone (100 mg) in EtOH (10 mL), and stirred
for 2 h at 60 °C. After evaporation of EtOH the residue was
chromatographed on a Si gel column and eluted with petroleum
ether-Et2O-HOAc (7:3:0.1). The more polar component was 3′-
(benzylthio)avarol (4) (28 mg; yield 19%): amorphous solid; [R]D 0.8
ether-Et2O (9:1) to give, in order of polarity, avarone-4′-thiocholesterol
(10) as the less polar component (42 mg; yield 18%): amorphous solid;
[R]D -6.1 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 230 (4.49), 306
(4.12); 1H NMR (CDCl3) δ 6.52 (H-6′, s), 6.37 (H-4′, s) and see
Supporting Information; EIMS m/z 714 [M + 2]+ (0.4), 712 [M]+ (8),
522 (15), 508 (10), 191 (15), 189 (13), 135 (20), 107 (18), 95 (100);
HREIMS m/z 712.5257 (calcd for C48H72O2S, 712.5253), avarol-3′,4′-
thiocholesterol (13) (14 mg; yield 4%): amorphous solid; [R]D -0.5 (c
0.01, CHCl3); UV (MeOH) λmax (log ε) 232 (4.55), 307 (4.33); 1H NMR
(CDCl3) δ 6.80 (H-6′, s) and see Supporting Information; EIMS m/z
1116 [M + 2]+ (0.4), 1114 [M]+ (5), 924 (18), 910 (12), 191 (18), 189
(17), 135 (25), 107 (25), 95 (100); HREIMS m/z 1114.8575 (calcd for
C75H118O2S2, 1114.8573), avarol-4′-thiocholesterol (12) (19 mg; yield
8%): amorphous solid; [R]D -1.1 (c 0.01, CHCl3); UV (MeOH) λmax
(log ε) 234 (4.31), 308 (4.00); 1H NMR (CDCl3) δ 6.82 (H-6′, s), 6.74
(H-4′, s) and see Supporting Information; EIMS m/z 716 [M + 2]+
(0.3), 714 [M]+ (6), 524 (10), 510 (8), 191 (18), 189 (12), 135 (20),
107 (25), 95 (100); HREIMS m/z 714.5412 (calcd for C48H74O2S,
714.5409), and avarol-3′-thiocholesterol (11) as the most polar com-
ponent (48 mg; yield 21%): amorphous solid; [R]D -7.7 (c 0.4, CHCl3);
1
(c 0.25, CHCl3); UV (MeOH) λmax (log ε) 238 (4.21), 315 (3.94); H
NMR (CDCl3) δ 6.77 (H-6′, d, J ) 2.9 Hz), 6.67 (H-4′, d, J ) 2.9 Hz)
and see Supporting Information; EIMS m/z 454 [M + 2]+ (0.5), 452
[M]+ (12), 262 (22), 191 (30), 135 (22), 107 (30), 95 (100); HREIMS
m/z 452.2380 (calcd for C28H36O3S, 452.2385). The less polar com-
ponent was 4′-(benzylthio)avarol (5) (5 mg; yield 4%): amorphous solid;
[R]D 2.4 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 258 (4.16), 332
(3.95); 1H NMR (CDCl3) δ 6.96 (H-6′, s), 6.78 (H-4′, s) and see
Supporting Information; EIMS m/z 454 [M + 2]+ (0.4), 452 [M]+ (10),
262 (18), 248 (12), 191 (22), 189 (18), 135 (30), 107 (35), 95 (100);
HREIMS m/z 452.2389 (calcd for C28H36O3S, 452.2385).
1
UV (MeOH) λmax (log ε) 234 (4.54), 308 (4.12); H NMR (CDCl3) δ
6.81 (H-6′, d, J ) 2.7 Hz), 6.61 (H-4′, d, J ) 2.7 Hz) and see Supporting
Information; EIMS m/z 716 [M + 2]+ (0.4), 714 [M]+ (8), 524 (8),
510 (8), 191 (15), 189 (15), 135 (23), 107 (20), 95 (100); HREIMS
m/z 714.5405 (calcd for C48H74O2S, 714.5409).
Biological Assays. Antimicrobial activity was carried out using liquid
culture of three bacterial strain, E. coli (DSM 498), B. subtilis subsp.
Spizizenii (DSM 347), and M. luteus (DSM 348) grown in nutrient
broth (Oxoid) at 37 °C, and the yeast S. cereVisiae (DSM 70449) grown
in medium M186 (peptone 5 g/L, glucose 10 g/L, yeast extract 3 g/L,
malt extract 3 g/L) at 37 °C. The MIC was determined by a serial
dilution, in duplicate, starting from 100 µg/mL to 0.01 µg/mL. The
bacterial and yeast growth was observed after 48 h of incubation.
Cytotoxic activity was evaluated by the brine shrimp (Artemia salina)
test in triplicate. The compounds were dissolved in DMSO (at least 2
mg/200 µL DMSO) to reach final concentrations of 100, 10, and 1
ppm, in 5 mL of artificial seawater using 10 freshly hatched larvae of
A. salina.15 Briefly, for each dose tested, surviving shrimps were
counted after 24 h, and the data statistically analyzed by the Finney
program,25 which affords LD50 values with 95% confidence intervals.
Free-radical scavenging activity was performed in MeOH, at different
concentrations (5, 10, 20, 50, and 100 µM). Solutions of each
compounds were prepared and adjusted to 2 mL total volume with 0.7
mL of DPPH-MeOH solution (6 mg/50 mL; 0.1 mM final concentra-
tion). The absorbance at 517 nm was determined after 30 min, and the
percent free-radical inhibition was calculated and plotted to obtain the
IC50 value. Trolox, a synthetic antioxidant compound, was used as
positive control standard. The IC50 value denotes the concentration of
compound required to scavenge 50% DPPH free radical.
Acethylcolinesterase inhibition was performed dissolving the samples
in MeOH at a concentration of 1 mg/mL. From this main solution was
performed a serial dilution in order to obtain lower concentration of
samples (0.1; 0.01; 0.001 mg/mL), and 10 µL of each solution was
applied to TLC plates to test 10, 1, 0.1, and 0.01 µg of samples to
detect the minimum concentration that inhibited AChE. Galanthamine
was used as positive control. The assay was carried out as described
by Marston et al.20 Briefly, a stock solution of acetylcholinesterase
(1000 U in 150 mL of Tris-hydrochloric acid buffer pH 7.8) was
obtained, which was stabilized adding bovine serum albumin (150 mg).
A 10 µL aliquot of each solution of the samples was applied to the
TLC plates, dried to remove the solvent, and then sprayed with enzyme
stock solution. For incubation of the enzyme, the plate was kept at 37
°C for 20 min in a humid atmosphere. For the detection of the enzyme,
solutions of 1-naphthyl acetate (250 mg in 100 mL of EtOH) and of
Fast Blue B salt (400 mg in 160 mL of distilled H2O) were mixed and
sprayed onto the plate. Acethylcolinesterase inhibition activity was
detected by a white spot on a purple background after 1-2 min.
Synthesis of Avarol-3′-thiolactate (6) and Avarol-4′-thiolactate
(7). Thiolactic acid (100 µL) dissolved in EtOH (5 mL) was added to
a solution of avarone (100 mg) in EtOH (10 mL) and stirred for 3 h at
60 °C. After evaporation of EtOH the residue was chromatographed
on a Si gel column and eluted with petroleum ether-Et2O-HOAc (1:
1:0.1). Further purification was performed by HPLC (Kromasil C18),
using CH3CN-H2O (9:1) as a mobile phase, to give avarol-3′-thiolactate
(6) (11 mg; yield 8%): amorphous solid; [R]D -4.2 (c 0.1, CHCl3);
1
UV (MeOH) λmax (log ε) 213 (4.31), 315 (3.67); H NMR (CDCl3) δ
6.85 (H-6′, d, J ) 2.7 Hz), 6.67 (H-4′, d, J ) 2.7 Hz) and see Supporting
Information; EIMS m/z 420 [M + 2]+ (0.3), 418 [M]+ (8), 228 (16),
214 (8), 191 (15), 189 (13), 135 (30), 107 (35), 95 (100); HREIMS
m/z 418.2183 (calcd for C24H34O4S, 418.2178), and avarol-4′-thiolactate
(7) (23 mg; yield 18%): amorphous solid; [R]D 11.8 (c 0.2, CHCl3);
1
UV (MeOH) λmax (log ε) 279 (4.43), 319 (4.23); H NMR (CDCl3) δ
6.85 (H-6′, s), 6.76 (H-4′, s) and see Supporting Information; EIMS
m/z 420 [M + 2]+ (0.5), 418 [M]+ (10), 228 (14), 214 (10), 191 (20),
189 (10), 135 (30), 107 (30), 95 (100); HREIMS m/z 418.2184 (calcd
for C24H34O4S, 418.2178).
Synthesis of Avarol-3′-thiobenzothiazole (8) and Avarol-4′-
thiobenzothiazole (9). 2-Mercaptobenzothiazole (100 mg) was dis-
solved in EtOH (5 mL), added to a solution of avarone (100 mg) in
EtOH (10 mL), and stirred for 7 h at 60 °C. After evaporation of EtOH
the residue was chromatographed on a Si gel column and eluted with
petroleum ether-Et2O (3:2). Further purification was performed by
HPLC, using CH3CN-H2O (9:1) as a mobile phase, to give avarol-
3′-thiobenzothiazole (8) (18 mg; yield 12%): amorphous solid; [R]D
-4.6 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 220 (4.90), 277 (4.49),
319 (4.29); 1H NMR (CDCl3) δ 7.00 (H-6′, d, J ) 3.0 Hz), 6.84 (H-4′,
d, J ) 3.0 Hz) and see Supporting Information; EIMS m/z 481 [M +
2]+ (1.3), 479 [M]+ (15), 289 (14), 275 (10), 191 (18), 189 (12), 135
(30), 107 (25), 95 (100); HREIMS m/z 479.1955 (calcd for
C28H33NO2S2, 479.1952), and avarol-4′-thiobenzothiazole (9) (15 mg;
yield 10%): amorphous solid; [R]D 15.1 (c 0.1, CHCl3); UV (MeOH)
1
λmax (log ε) 218 (4.91), 274 (4.45), 320 (4.38); H NMR (CDCl3) δ
6.98 (H-6′, s), 6.92 (H-4′, s) and see Supporting Information; EIMS
m/z 481 [M + 2]+ (0.9), 479 [M]+ (10), 289 (18), 275 (8), 191 (15),
189 (15), 135 (25), 107 (20), 95 (100); HREIMS m/z 479.1950 (calcd
for C28H33NO2S2, 479.1952).
Synthesis of Avarone-4′-thiocholesterol (10), Avarol-3′-thiocho-
lesterol (11), Avarol-4′-thiocholesterol (12), and Avarol-3′,4′-thio-
cholesterol (13). Thiocholesterol (100 mg) was dissolved in EtOH (5
mL), added to a solution of avarone (100 mg) in EtOH (10 mL), and
stirred for 3 h at 60 °C. After evaporation of EtOH, the residue was
chromatographed on a Si gel column and eluted with petroleum
Acknowledgment. This research was supported by CNR-Rome. One
of the authors (B.P.) acknowledges an Italian Minister of Foreign Affairs
fellowship. The assistance of Mr. V. Mirra is gratefully acknowledged.
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.

Synthesis of Thio-aVarol DeriVatiVes
Journal of Natural Products, 2008, Vol. 71, No. 11 1853
(12) Belisario, M. A.; Pecce, R.; Maturo, M.; De Rosa, S. Toxicology 1994,
86, 89–108.
(13) Amigo´, M.; Paya´, M.; De Rosa, S.; Terencio, M. C. Br. J. Pharmacol.
2007, 152, 353–365.
(14) Amigo´, M.; Terencio, M. C.; Paya´, M.; Iodice, C.; De Rosa, S. Bioorg.
Med. Chem. Lett. 2007, 17, 2561–2565.
References and Notes
(1) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 15, 3401–
3404.
(2) De Rosa, S.; Minale, L.; Riccio, R.; Sodano, G. J. Chem. Soc., Perkin
Trans. 1 1976, 1408, 1414.
(3) De Rosa, S. Mediterranean Marine Organisms as Source of New
Potential Drugs. In Natural Products in the New Millennium: Prospects
and Industrial Application; Rauter, A. P., Palma, F. B., Justino, J.,
Arau´jo, M. E., dos Santos, S. P., Eds.; Kluwer Academic Publishers:
Dordrecht, 2002; pp 441-461.
(4) Ferra´ndiz, M. L.; Sanz, M. J.; Bustos, G.; Payá, M.; Alcaraz, M. J.;
De Rosa, S. Eur. J. Pharmacol. 1994, 253, 75–82.
(5) Shen, Y. C.; Lu, C. H.; Chakraborty, R.; Kuo, Y. H. Nat. Prod. Res.
2003, 17, 83–89.
(15) Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols,
D. E.; McLaughlin, J. L. Planta Med. 1982, 45, 31–34.
(16) Crispino, A.; De Giulio, A.; De Rosa, S.; Strazzullo, G. J. Nat. Prod.
1989, 52, 646–648.
(17) Takao, T.; Kitatani, F.; Watanabe, N.; Yagi, A.; Sakata, K. Biosci.
Biotechnol. Biochem. 1994, 51, 1780–1783.
(18) Cuendet, M.; Hostettmann, K.; Potterat, O.; Dyatmiko, W. HelV. Chim.
Acta 1997, 80, 1144–1152.
(19) Rhee, I. K.; van de Meent, M.; Ingkaninaa, K.; Verpoorte, R.
J. Chromatogr. A 2001, 915, 217–223.
(6) Belisario, M. A.; Maturo, M.; Pecce, R.; De Rosa, S.; Villani, G. R.
(20) Marston, A.; Kissling, J.; Hostettmann, K. Phytochem. Anal. 2002,
13, 51–54.
(21) Scott, L. J.; Goa, K. L. Drugs 2000, 60, 1095–1122.
(22) Amigo´, M.; Terencio, M. C.; Mitova, M.; Iodice, C.; Paya´, M.; De
Rosa, S. J. Nat. Prod. 2004, 67, 1459–1463.
(23) U.S. Food and Drug Administration Alert [5/2005].
(24) Pouchert, C. J.; Behnke, J. The Aldrich Library of 13C and 1H FT
NMR Spectra; Aldrich Chemical Company, Inc., 1993; Vols. 1-3.
(25) Finney D. J. Probit Analysis, 3rd ed.; Cambridge University Press:
Cambridge, U.K, 1971.
Toxicology 1992, 72, 221–233.
(7) Belisario, M. A.; Maturo, M.; Avagnale, G.; De Rosa, S.; Scopacasa,
F.; De Caterina, M. Pharmacol. Toxicol. 1996, 79, 300–304.
(8) Sarin, P. S.; Sun, D.; Thornton, A.; Mu¨ller, W. E. G. J. Natl. Cancer
Inst. 1987, 78, 663–666.
(9) Sipkema, D.; Osinga, R.; Schatton, W.; Mendola, D.; Tramper, J.;
Wijffels, R. H. Biotechnol. Bioeng. 2005, 90, 201–222.
(10) Amigo´, M.; Paya´, M.; Braza-Bo¨ıls, A.; De Rosa, S.; Terencio, M. C.
Life Sci. 2008, 82, 256–264.
(11) De Rosa, S.; De Giulio, A.; Strazzullo, G. Trends Org. Chem. 1991,
2, 127–141.
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