Synthesis and biological activity of derivatives of the marine quinone avarone

Article abstract of DOI:10.1016/j.ejmech.2009.11.033

Nine alkyl(aryl)thio derivatives of the marine sesquiterpene quinone avarone were synthesized by nucleophilic addition of thiols or thiophenol to avarone. In most cases only one regioisomer was obtained. Their cytotoxic activities, brine shrimp lethality

Full text of DOI:10.1016/j.ejmech.2009.11.033

European Journal of Medicinal Chemistry 45 (2010) 923–929
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of derivatives of the marine quinone avarone
a
b
d
d
´
´
ˇ ´ c
´
´
ꢀ
Tatjana Bozic , Irena Novakovic , Miroslav J. Gasic , Zorica Juranic , Tatjana Stanojkovic ,
b
e
a,
*
ꢀ
´
´
ˇ
´
SrCan Tufegdzic , Zoran Kljajic , Dusan Sladic
a Faculty of Science, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia
b
ˇ
Center for Chemistry, ICTM, Njegoseva 12, 11000 Belgrade, Serbia
c Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11001 Belgrade, Serbia
d Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
e Institute of Marine Biology, 85 330 Kotor, P.O. Box 69, Montenegro
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine alkyl(aryl)thio derivatives of the marine sesquiterpene quinone avarone were synthesized by
nucleophilic addition of thiols or thiophenol to avarone. In most cases only one regioisomer was
obtained. Their cytotoxic activities, brine shrimp lethality and antibacterial activity were evaluated, as
well as those of some previously synthesized avarone derivatives. Anti-HIV activity of two derivatives
was tested. Electrochemical properties were determined for all the derivatives in order to obtain more
accurate information on structure–activity relationships. Most derivatives showed cytotoxic activity
Received 15 September 2009
Received in revised form
12 November 2009
Accepted 13 November 2009
Available online 26 November 2009
against tumor cell lines, with IC50 values less than 10
electron-donating substituents. The most active compound was 40-(methylamino)avarone, with IC50
value of 2.4 M to melanoma Fem-X cells, and no cytotoxicity to normal lymphocytes.
Ó 2009 Elsevier Masson SAS. All rights reserved.
mM for some of them, in particular those with
Keywords:
Marine quinone
Avarone
Alkyl(aryl)thio derivatives
Cytotoxic activity
Cyclic voltammetry
m
1. Introduction
There have been reports on activities of derivatives of avarol
[20,24–26], and avarone [8,26,27], but the cytotoxic activity of the
derivatives was not systematically studied. In this paper, as
a continuation of our previous work, we report the synthesis and
characterization of nine new alkyl(aryl)thio derivatives of avarone,
and the results of cytotoxic activity investigation of these
compounds and of four avarone derivatives synthesized previously
[8,28], as well as of avarol and avarone. Alkyl(aryl)thio derivatives
were selected in order to cover the range of redox potentials
between those of alkoxy and akylamino derivatives on one end, and
the chloro derivative on the other. The antibacterial activity and
brine shrimp toxicity, as well as cytotoxicity to peripheral blood
mononuclear cells were also evaluated. Electrochemical parame-
ters of all compounds were determined in order to obtain insight
into possible mechanisms relevant for the activity.
Natural products with a decalin core structure and a quinone/
hydroquinone side chain are often characterized by pronounced
and diverse biological activities [1]. These compounds, which
include the sesquiterpene hydroquinones and quinones from
sponges of the order Dictyoceratida, such as avarol, avarone, ili-
maquinone, nakijiquinone and bolinaquinone, offer promising
opportunities for the development of new drugs and agents to be
applied for elucidation of intracellular events.
The hydroquinone avarol, isolated from the marine sponge
Dysidea avara Schmidt [2,3], and the corresponding quinone avar-
one, have a wide array of pharmacological properties, such as
antitumor [4–6], antibacterial [7,8], antiviral [9], antiinflammatory
[10,11], antipsoriatic [12,13], antioxidant [14,15], and antibiofouling
[16]. On the molecular level the compounds showed DNA damaging
[6,17–19] and enzyme inhibitory [20–23] activities, including
damage to the glycolytic enzyme hexokinase [23]. These activities
have been, at least in part, ascribed to the generation of reactive
oxygen radicals and to modification of biomolecules by nucleo-
philic addition. The biological activities of these compounds were
recently reviewed [1,14].
2. Chemistry
All solvents and reagents used were reagent grade.
Avarol (1) has been isolated from the sponge D. avara collected in
the Bay of Kotor area, Montenegro, as previously described [2].
Avarone (2) was obtained by oxidation of avarol using silver oxide [2].
The alkylthio and arylthio derivatives 3–11 were obtained by
nucleophilic addition of the corresponding thiols to avarone.
* Corresponding author. Tel.: þ381113336679; fax: þ381112636061.
E-mail address: dsladic@chem.bg.ac.rs (D. Sladic´).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.033

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T. Bozi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 923–929
924
Methoxy derivative 12 [28], methylamino derivatives 13 and 14 [8]
and chloro derivative 15 [28] were prepared as previously
described.
Electrochemical behaviour of all the derivatives was studied
using cyclic voltammetry in acetonitrile on a glassy carbon
electrode.
3. Pharmacology
The antiproliferative activity of all the compounds was tested
against three tumor cell lines: human cervix carcinoma HeLa cells,
human malignant melanoma Fem-X cells and human myelogenous
leukemia K-562 cells. Cytotoxicity of some of the derivatives was
tested against human peripheral blood cells. Three derivatives (5, 9
and 11) were chosen by NIH-NCI to be tested against a panel of 60
human tumor cell lines.
Brine shrimp assay [29], i.e. toxicity to the Artemia salina nauplii
was performed, since it is a good indicator of cytotoxicity.
Antibacterial activity of the derivatives was tested against Gram
positive bacteria Staphylococcus aureus ATCC 25923 and Gram
negative bacteria Escherichia coli ATCC 35218.
Two derivatives, namely 5 and 11 were selected by NIH-NCI for
testing the antiviral activity against HIV.
4. Results and discussion
4.1. Chemistry
The optimum reaction conditions for the synthesis of the
alkylthio derivatives 3–9 (Fig. 1) were: equimolar amounts of the
reactants, weakly alkaline medium (mixture of 95% ethanol and
saturated sodium bicarbonate solution (1:1)), nitrogen atmosphere
in order to avoid polymerization, temperature of 60 ꢀC, and reac-
tion time of 30 min. In the reaction with 2-methyl-2-propanethiol
and 1-octanethiol (syntheses of 7 and 8, respectively), making the
mixture more basic by addition of pyridine into the reaction
mixture was necessary, because of the lower reactivity of these
thiol compounds in addition/elimination reaction. In all cases only
40-alkylthio derivatives were obtained, as was shown also in the
reactions with model compounds [30]. Phenylthio derivatives were
obtained under neutral conditions (95% ethanol) in air atmosphere
at 60 ꢀC, the optimum reaction time being 30 min (longer reaction
times resulted in lower yields due to polymer formation), and mole
ratio of quinone–thiophenol 2:1. In contrast to alkylthio derivatives,
a mixture of two products, 30- and 40-(phenylthio)avarone was
obtained, with the latter derivative in higher yield. Other
compounds, the activities of which were investigated, were
30-methoxyavarone [28], 40-chloro-3,4-dihydroavarone [28], 30-
and 40-(methylamino)avarone [8]. It should be noted that in all
cases, the addition/elimination never occurred at position 60, as
discussed previously on the basis of electron density distribution
and steric effects for model compounds [30].
The electrochemical properties of all the synthesized quinones
were analyzed by cyclic voltammetry using a glassy carbon elec-
trode in acetonitrile, in an attempt to correlate oxidative power and
bioactivity (cytotoxicity). All the derivatives underwent reduction,
but their behaviour differed. Most compounds (6,10–15) had vol-
tammograms similar to avarone [31,32], i.e. two reduction waves,
the first one being reversible. Compound 9, 3040-(ethyl-
enedithio)avarone, showed two reduction peaks, both irreversible.
Finally, derivatives 4 and 8, as well as all the branched alkylthio
derivatives (3, 5, 7) showed only one irreversible peak. The differ-
ence in electrochemical behaviour could be ascribed to differences
in interactions with the surface of the electrode. The values of
voltammetric half-peak potentials are given in Table 1.
Fig. 1. Chemical structure of avarone derivatives.
4.2. Pharmacology
The accumulated data on biological activity of quinones rest on
redox processes and/or on Michael-type addition–elimination
reactions. Our previous results obtained with avarol and avarone
supported the mechanism of antitumor action via the reactive
oxygen radicals [6,17,18], but there were also indications of the
relevance of arylation of biomolecules, such as proteins [23,33,34].
Cytotoxic activity in vitro was tested against three tumor cell
lines: human cervical carcinoma, HeLa cells, human melanoma Fem-
X cells and human leukemia K-562 cells. The results are given in
Table 1. Most compounds showed activity to all three cell lines, but
leukemia cells were generally the most susceptible ones, with IC50
values similar to cisplatin for some derivatives, e.g. 12–14. The
exceptions were 40-(methylamino)avarone (13) and 30,40-(ethyl-
enedithio)avarone (9), which were more active to melanoma cells,
although the latter compound showed an overall lowactivity. All the
alkyl(aryl)thio derivatives and 40-chloro-3,4-dihydroavarone (the
3,4-double bond is not essential for the activity [4]) were less (or
equally) active than both avarone and avarol. On the other hand,
methoxy and methylamino derivatives (12–14) were more active
than the parent compounds against most tested cell lines, with IC50
values in the lowmicromolar range. There is a qualitative correlation
between reductionpotentials and cytotoxic activity – the derivatives
with a more negative one-electron reduction potential generally

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925
Table 1
Cytotoxic activity of avarone derivatives and their voltammetric half-peak potentials in acetonitrile.
Compound
Ep/2 (V)a
IC50
(mM)
HeLa
Fem-X
K-562
PBMC
PBMCþPHAb
1
2
3
4
5
6
7
8
–
19.7 ꢃ 2.4
26.8 ꢃ 3.0
67.0 ꢃ 6.1
97.0 ꢃ 1.0
98.0 ꢃ 1.0
25.1 ꢃ 3.1
37.0 ꢃ 2.5
>100
21.7 ꢃ 4.6
23.9 ꢃ 5.2
86.0 ꢃ 3.3
89.5 ꢃ 2.0
89.5 ꢃ 6.3
24.4 ꢃ 3.4
38.0 ꢃ 4.1
>100
75.0 ꢃ 1.3
41.5 ꢃ 4.9
42.5 ꢃ 2.5
9.8 ꢃ 2.0
2.4 ꢃ 0.5
5.2 ꢃ 0.0
32.2 ꢃ 1.0
4.7 ꢃ 0.3
7.3 ꢃ 1.8
4.9 ꢃ 1.3
47.5 ꢃ 1.4
45.5 ꢃ 3.5
39.0 ꢃ 7.0
12.5 ꢃ 0.1
25.7 ꢃ 3.2
>100
91.0 ꢃ 2.7
17.4 ꢃ 2.1
15.2 ꢃ 3.9
2.1 ꢃ 0.0
9.3 ꢃ 1.1
3.6 ꢃ 0.0
11.2 ꢃ 1.0
5.4 ꢃ 0.3
3.0 ꢃ 1.7
4.8 ꢃ 2.4
n.d.d
3.2 ꢃ 0.2
4.5 ꢃ 0.9
n.d.d
ꢂ0.53
ꢂ0.68c
ꢂ0.65c
ꢂ0.75c
ꢂ0.49
ꢂ0.62c
ꢂ0.83c
ꢂ0.61c
ꢂ0.47
ꢂ0.59
ꢂ0.66
ꢂ0.85
ꢂ0.84
ꢂ0.39
n.d.d
n.d.d
n.d.d
n.d.d
n.d.d
n.d.d
n.d.d
21.7 ꢃ 8.4
20.8 ꢃ 0.5
n.d.d
n.d.d
9
10
11
12
13
14
15
Cisplatin
>100
n.d.d
n.d.d
54.8 ꢃ 9.0
45.0 ꢃ 3.7
13.3 ꢃ 1.1
12.6 ꢃ 1.0
11.3 ꢃ 0.2
34.1 ꢃ 1.0
4.0 ꢃ 0.3
n.d.d
n.d.d
9.9 ꢃ 3.0
5.9 ꢃ 0.4
>100
11.2 ꢃ 1.0
5.5 ꢃ 0.2
>100
4.9 ꢃ 0.3
4.2 ꢃ 0.1
n.d.d
n.d.d
33.6 ꢃ 3.0
27.0 ꢃ 6.0
a
vs. saturated calomel electrode.
Phytohemagglutinin.
Irreversible wave.
b
c
d
Not determined.
show a higher activity. This trend supports the concept of the role of
oxygen radicals in the inhibitoryaction of these derivatives on tumor
cells, as previously established for avarol and avarone [6,18]. Among
the alkylthio derivatives the influence of steric effects on the activity
could not be established (e.g. very bulky tert-butylthio derivative 7
was less active than less bulky butylthio derivative 6, but more active
compounds. An alternative explanation could be the presence of an
additional ring in 9, affecting binding to targets, such as DNA. Two
phenylthio regioisomers 10 and 11 had equal activities. However,
unexpectedly, activities of methylamino regioisomers differed, 13
being moreactive tothe melanomacells and 14 tothe leukemia cells.
Selected derivatives were tested for cytotoxicity to lymphocytes
(PBMC – peripheral blood mononuclear cells) – non-stimulated and
stimulated by phytohemagglutinin (PHA) (Table 1). Unfortunately,
most compounds were equally toxic to lymphocytes as to tumor cells,
precluding theirtherapeutical application. Thenotableexceptions are
30-methoxyavarone (12), and 30-(methylamino)avarone (14), which
are more active to the leukemia cells than to the normal cells, and
especially 40-(methylamino)avarone (13), which was not toxic to
either non-stimulated or stimulated PBMC. Having in mind that this
compound is active against melanoma cells at low micromolar
concentration, it is a good candidate for further studies.
than isobutylthio derivative 5). Octylthio derivative
8 was
completely inactive, probably due to excessive lipophilicity and/or
coiling of the hydrocarbon chain around the quinone pharmaco-
phore. The behaviour of 30,40-ethylenedithio derivative 9 is partic-
ularly interesting. It has
a much lower activity than most
alkyl(aryl)thio derivatives, despite the similar reduction potential.
The explanation might lie in the inability of disubstituted derivative
9 to modify proteins by addition, including glycolytic enzyme
hexokinase [23,33,34], in contrast to avarone and the mono-
substituted derivatives. Thus, arylation of cellular nucleophiles is
probably also implicated in the mechanism of action of these
Three compounds (5, 9, 11) were selected by NIH-NCI for in vitro
screening in a panel of 60 human tumor cell lines starting at
a concentration of 1 ꢁ 10ꢂ4 M of the investigated compound [35].
Selected results against the most susceptible cell lines are shown in
Table 2
Selected results on the activity of derivatives 5, 9 and 11 against the NCI/NIH panel of
Table 2. All three compounds have GI50 values below 100 mM with
cell lines.
Cell line
GI50 (mM)
Table 3
5
9
11
Antibacterial activity and brine shrimp test results of avarone derivatives.
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
1.35
4.47
2.29
3.80
11.0
20.0
21.4
14.1
3.89
19.5
2.57
Compound
MIC (
m
g/ml)
LC50 (ppm)
S. aureus
E. coli
A. salina
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
12.5
6.25
n.aa
50
50
25
n.a.a
25
0.18
0.14
0.44
0.20
0.17
0.92
0.89
n.aa
2045
32.33
30.21
0.02
0.34
2.40
0.65
n.t.b
20.4
n.aa
n.aa
25
Non small-cell lung cancer
HOP-92
NCI-H23
4.79
5.50
31.6
13.2
17.0
14.8
n.aa
n.aa
25
Melanoma
LOX IMVI
SK-MEL-2
50
4.07
20.9
11.2
1.70
13.5
20.0
n.aa
n.aa
12.5
12.5
25.0
n.aa
n.aa
50.0
4.0
100
n.aa
n.aa
50
Ovarian cancer
IGROV1
10.0
32.4
18.6
20.4
16.2
16.2
Renal cancer
RXF 393
n.aa
n.aa
25
6.76
Breast cancer
MCF7
MDA-MB-435
T-47D
7.24
15.5
2.63
22.4
17.0
20.4
Doxycycline
2.0
8.13
>100
a
Not active.
Not tested.
b

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T. Bozi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 923–929
Fig. 2. Antiviral activity of derivatives 5(a) and 11(b).
the exception of one cell line with compound 9. Derivative 5 was
generally the most active one, with the GI50 less (or equal) than
10 mM for 11 cell lines (the other 2 compounds against two cell lines
each). Of all types of cells, the highest activity was achieved for
leukemia cell lines for compounds 5 and 11 (four and two cell lines
(out of five tested), respectively, with GI50 less than 10 mM), while 9
showed no apparent specificity for any group of cell lines. Among
the individual cell lines, the greatest activity (log GI50 below ꢂ5.5)
was recorded for MOLT-4 leukemia cell lines by 11, CCRF-CEM and
MOLT-4 leukemia cells and T-47D breast cancer cells by 5, and SK-
MEL-2 melanoma cells by 9. No cell line of colon cancer, CNS cancer
on the activity could not be established. With alkylthio derivatives,
a small increase in lipophilicity of the substituent enhanced the
activity, but a large increase led to a complete loss of activity. A
significant decrease in activity was a consequence of disubstitution
of the quinone ring, probably by hindering additions of cellular
nucleophiles. The brine shrimp test showed good qualitative
correlation with cytotoxicity. None of the derivatives showed
a better antibacterial activity than avarone. The presented results
encourage further studies on avarone derivatives, especially in view
of the high abundance of avarol in the sponge D. avara [2,3].
and prostate cancer had GI50 values less than 10
tested compounds.
mM for any of the
6. Experimental protocols
The results of the brine shrimp test [29] which had shown
previously good agreement with antileukemic activity of avarol and
avarone [36] are shown in Table 3. The test showed again good
qualitative correlation with cytotoxicity against the tested tumor
cell lines – the most active compound was 12, while 9 showed low
activity and 8 was inactive.
Antibacterial activity of the derivatives was tested against path-
ogenic Gram positive bacteria S. aureus ATCC 25923 and Gram
negative bacteria E. coli ATCC 35218 (Table 3). Almost all of the
derivatives were moderatelyactive against S. aureus, and five (5, 8, 9,
12 and 15) of the derivatives showed activityagainst E. coli. However,
avarone was more (or equally) active than any of the derivatives.
The derivatives 5 and 11 were selected by NIH-NCI for testing
the antiviral activity to HIV using a procedure enabling the deter-
mination of the activity of the agents in all the reproductive phases
of the virus, by following the dynamics by which the infected T4
lymphocytes die in presence of the agents [37]. Both derivatives
showed a significant antiviral activity at low micromolar concen-
6.1. Chemistry
NMR spectra were recorded in CDCl3, using tetramethylsilane as
an internal standard, on an Oxford YH NMR spectrometer at
200 MHz for 1H and at 50 MHz for 13C NMR spectra. Mass spectra
were taken on a Finnigan-Mat model 8230 mass spectrometer with
an ion source of 70 eV. UV/Vis spectra were recorded on a Beckman
D-25 spectrophotometer. Polarimetric measurements were per-
formed on a Rudolph research analytical polarimeter, AUTOPOL IV,
in chloroform. Elemental analysis (C, H, S) was performed using
standard micromethods on an ELEMENTAR Vario ELIII C.H.N.S]O
analyser and the results are given in the Appendix.
Cyclic voltammetric measurements were performed in a conical
PAR cell of 25 mL, without cooling, using EG&G PAR173 potentiostat
and a PAR175 programmer, recording the voltammograms by a PAR
RE 0074 X–Y plotter. The working and auxiliary electrodes were of
platinum wire, and before each measurement were cleaned by
keeping them in concentrated sulfuric acid for several hours,
rinsing with water and acetone, and drying. The reference electrode
was the saturated calomel electrode (SCE). The quinones were used
as 1–3 mM solutions in purified acetonitrile. Acetonitrile was
purified by refluxing 2 L of the solvent with 20 g potassium
permanganate and 10 g sodium carbonate, filtration and acidifica-
tion of the filtrate with conc. sulfuric acid, distillation and a final
distillation in dry nitrogen atmosphere using a column of 150 cm
length, filled with Raschig rings. The supporting electrolyte was
tetraethylammonium perchlorate. The measurements were made
in a dry nitrogen atmosphere at 25 ꢀC.
trations (Fig. 2). Derivative 5 afforded 28.5% protection at 2.00 mM,
and derivative 11 12.5% at the same concentration. However, there
was also a high cytotoxicity to uninfected cells, so that the results
were of no significance for further anti-HIV tests.
5. Conclusion
From the presented data on bioactivity of the synthesized
avarone derivatives the following conclusions can be drawn.
Cytotoxic activity against tumor cell lines is similar to that of
avarone, but it could be enhanced by introducing electron-donating
substituents, which lower one-electron reduction potential.
Derivatives of this type, in particular 40-(methylamino)avarone, are
more active to leukemia cells than to normal lymphocytes. The
influence of the steric effects and of the position of the substituents
6.1.1. Synthesis of 40-(isopropylthio)avarone (3)
Avarone (400 mg) was dissolved in 60 mL mixture 95% ethanol-
saturated aqueous solution of NaHCO3 (1:1). A solution of 520
mL
(328 mg) 2-propanethiol in 5 mL 95% ethanol was added, and the

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T. Bozi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 923–929
927
reaction mixture was stirred at 60 ꢀC under nitrogen atmosphere
for 30 min. The dark red solution was neutralized by 5% HCl, and
extracted with toluene. The toluene extract was dried with anhy-
drous Na2SO4, and after evaporation of toluene chromatographed
on a silica gel 60 (Merck) column with toluene as the eluent, and
rechromatographed with the same eluent, affording 40-(iso-
propylthio)avarone (3) as dark yellow oil (111 mg; yield 44%).
derivative was purified by preparative TLC (silica gel G, 1 mm,
toluene–ethyl acetate (9:1)). 40-(Octylthio)avarone (8): dark red oil;
28 mg (yield 20%). [
a
]
¼ 35 (c 0.001, CHCl3); 1H NMR:
d 6.53 (s, H-
D
60), 6.33 (s, H-30), and see Appendix; 13C NMR: 183.9 (C-50),184.0 (C-
20), 152.2 (C-40), 148.3 (C-10), 135.7 (C-60), 125.1 (C-30) and see
Appendix; CIMS (isobutane): 457 [Mþ1]þ; UV/Vis (MeOH): lmax
(log
3) 210 (4.00), 301 (3.59), 435 (3.08). Ep/2 (vs. SCE) ¼ ꢂ0.83 V
[
a
]
¼ 35 (c 0.001, CHCl3); 1H NMR:
d
6.32 (H-60, s), 6.63 (H-30, s)
(1 mM, 50 mV/s; irreversible wave).
D
13
and see Appendix; C NMR: 184.2 (C-20), 184.1 (C-50), 151.3 (C-40),
148.2 (C-10), 135.8 (C-60), 125.5 (C-30) and see Appendix; CIMS
6.1.7. Synthesis of 30,40-(ethylenedithio)avarone (9)
Obtained by the same procedure as 3, but the reaction time was
prolonged to 50 min, and the crude product was chromatographed
on a silica gel column, using petroleum ether (30–50 ꢀC)–benzene
(isobutane): 386 [M]þ; UV/Vis (MeOH): lmax (log
3) 210 (3.86), 300
(3.45), 440 (2.90). Ep/2 (vs. SCE) ¼ ꢂ0.68 V (1 mM, 3.5 mV/s;
irreversible wave).
(3:1) as the eluent. Starting from 214 mg avarone and 57.9
(65 mg) 1,2-ethanedithiol, 30,40-(ethylenedithio)avarone (9) was
obtained as yellow oil (97 mg; yield 70%). [
¼ 14 (c 0.01, CHCl3);
mL
6.1.2. Synthesis of 40-(propylthio)avarone (4)
Obtained by the same procedure as 3, using 433 mg avarone and
a]
D
13
744
m
L (469 mg) 1-propanethiol. 40-(Propylthio)avarone (4): orange
1H NMR: 6.47 (H-60, s) and see Appendix; C NMR: 180.6 (C-20),
d
oil; 139 mg (yield 55%). [
a]
¼ 10 (c 0.001, CHCl3); 1H NMR:
d
6.56
180.3 (C-50), 147.2 (C-10), 138.2 (C-30), 137.2 (C-40), 135.8 (C-60) and
D
13
(H-60, s), 6.63 (H-30, s) and see Appendix; C NMR: 185.0 (C-50),
184.9 (C-20), 152.1 (C-40), 148.3 (C-10), 135.6 (C-60), 125.2 (C-30) and
see Appendix; CIMS (isobutane): 387 [Mþ1]þ; UV/Vis (MeOH):
see Appendix; CIMS (isobutane): 403 [Mþ1]þ; UV/Vis (MeOH):
lmax (log 3) 210 (4.95), 298 (3.67), 536 (3.08). Ep/2 (vs.
SCE) ¼ ꢂ0.61 V and ꢂ0.86 V (3 mM, 200 mV/s; irreversible waves).
lmax (log 3) 209 (3.59), 288 (3.20), 438 (3.62). Ep/2 (vs.
SCE) ¼ ꢂ0.65 V (1 mM, 50 mV/s; irreversible wave).
6.1.8. Synthesis of 30-(phenylthio)avarone (10) and 40-
(phenylthio)avarone (11)
6.1.3. Synthesis of 40-(isobutylthio)avarone (5)
Avarone (313 mg) and thiophenol (51 mL, 55 mg) were dissolved
Obtained by the same procedure as 3, using 194 mg avarone and
mL (56 mg) 2-methyl-1-propanethiol. After the first column
in 50 mL ethanol, and the reaction mixture was stirred at 60 ꢀC for
30 min. The dark red product was chromatographed on a silica gel
60 (Merck) column with toluene as the eluent, affording first
30-(phenylthio)avarone (10) as dark yellow crystals (29 mg; yield
71
chromatography, the first fraction was purified by preparative TLC
(silica gel G, 1 mm, eluent petroleum ether (40–70 ꢀC)–diethyl
ether (97:3)), affording 40-(isobutylthio)avarone (5) as yellow oil
14%). M.p. 100–102 ꢀC; [
a
]
D
¼ 5 (c 0.01, CHCl3); 1H NMR:
d 6.44 (H-
(81 mg; yield 65%). [
a
]
¼ 9 (c 0.01, CHCl3); 1H NMR:
d
6.37 (H-60, s),
60, d, 4J ¼ 2.2 Hz), 5.86 (H-40, d, 4J ¼ 2.2 Hz) and see Appendix; 13C
NMR: 184.9 (C-20), 184.5 (C-50), 153.4 (C-30), 148.4 (C-10), 135þ.7
(C-60), 130.4 (C-40) and see Appendix; CIMS (isobutane): 420 [M] ;
D
6.56 (H-30, s) and see Appendix; 13C NMR: 184.3 (C-20), 184.0 (C-50),
150.3 (C-40), 147.5 (C-10), 136.0 (C-60), 126.3 (C-30) and see
Appendix; CIMS (isobutane): 400 [M]þ; UV/Vis (MeOH): lmax
UV/Vis (MeOH): lmax (log 3) 253 (3.28), 290 (3.08), 443 (3.76). Ep/2
(log
3
) 244 (3.74), 303 (3.69), 435 (3.11). Ep/2 (vs. SCE) ¼ ꢂ0.72 V
(vs. SCE) ¼ ꢂ0.47 V (1 mM, 50 mV/s; reversible wave). The second
(1 mM, 50 mV/s; irreversible wave).
product was 40-(phenylthio)avarone (11), obtained as light yellow
crystals (111 mg, yield 53%). M.p. 134–136 ꢀC; [
a
]
D
¼ 21 (c 0.1,
6.1.4. Synthesis of 40-(butylthio)avarone (6)
Obtained by the same procedure as 3, using 448 mg avarone and
172 mL (144 mg) 1-butanethiol. Rechromatography was not neces-
sary. 40-(Butylthio)avarone (6): light yellow oil; 258 mg (yield 90%).
CHCl3); 1H NMR: 6.50 (H-60, s), 5.84 (H-30, s) and see Appendix;
d
13C NMR: 184.5 (C-20), 184.2 (C-50), 154.9 (C-40), 146.6 (C-10), 136þ.3
(C-60), 130.5 (C-30) and see Appendix; CIMS (isobutane): 420 [M] ;
UV/Vis (MeOH): lmax (log 3) 250 (3.78), 298 (3.69), 440 (3.18). Ep/2
(vs. SCE) ¼ ꢂ0.49 V (1 mM, 50 mV/s; reversible wave).
[
a]
¼ 74 (c 0.01, CHCl3); 1H NMR:
d
6.37 (H-60, s), 6.56 (H-30, s) and
D
see Appendix; 13C NMR: 183.9 (C-50), 183.8 (C-20), 152.1 (C-40), 148.3
(C-10), 135.6 (C-60), 125.1 (C-30) and see Appendix; CIMS (isobu-
6.1.9. Synthesis of 30-methoxyavarone (12)
tane): 400 [M]þ; UV/Vis (MeOH): lmax (log
3) 248 (3.73), 305 (3.72),
Obtained as previously described [28]. Ep/2 (vs. SCE) ¼ ꢂ0.66 V
435 (3.2). Ep/2 (vs. SCE) ¼ ꢂ0.49 V and ꢂ0.73 V (1 mM, 100 mV/s;
(1 mM, 50 mV/s; reversible wave).
the first wave was reversible).
6.1.10. Synthesis of 40-(methylamino)avarone (13) and 30-
(methylamino)avarone (14)
6.1.5. Synthesis of 40-(tert-butylthio)avarone (7)
Obtained by the same procedure as 3, using 316 mg avarone and
Obtained as previously described [8]. Ep/2 (vs. SCE) ¼ ꢂ0.85 V for
704
mL (444 mg) 2-methyl-2-propanethiol, but with addition of
13 and ꢂ0.84 V for 14 (1 mM, 50 mV/s; reversible wave).
1 mL pyridine into the reaction mixture. After the rechromatog-
raphy, the derivative was purified by preparative TLC (silica gel G,
1 mm, toluene–ethyl acetate (9:1)). 40-(tert-Butylthio)avarone (7):
6.1.11. Synthesis of 40-chloro-3,4-dihydroavarone (15)
Obtained as previously described [28]. Ep/2 (vs. SCE) ¼ ꢂ0.39 V
(1 mM, 50 mV/s; reversible wave).
yellow oil; 106 mg (yield 53%). [
a
]
¼ 31 (c 0.001, CHCl3); 1H NMR:
D
d
6.47 (H-60, s), 6.76 (H-30, s) and see Appendix; 13C NMR: 184.6
(C-50), 184.4 (C-20), 149.9 (C-40), 148.9 (C-10), 136.2 (C-60), 128.4 (C-
6.2. Pharmacology
30) and see Appendix; CIMS (isobutane): 401 [Mþ1]þ; UV/Vis
(MeOH): lmax (log
3) 208 (3.89), 300 (3.53), 434 (3.01). Ep/2 (vs.
6.2.1. Antiproliferative activity
SCE) ¼ ꢂ0.62 V (1 mM, 3.5 mV/s; irreversible wave).
Stock solutions of the compounds were prepared in dimethyl
sulfoxide (DMSO) at a concentration of 10 mM and afterwards
diluted with a nutrient medium (RPMI 1640), supplemented with
6.1.6. Synthesis of 40-(octylthio)avarone (8)
Obtained by the same procedure as 3, using 303 mg avarone and
L-glutamine (3 mM), streptomycin (100 mg/ml) and penicillin
(100 IU/ml), 10% heat inactivated (56 ꢀC) fetal bovine serum (FBS)
and 25 mM Hepes, adjusted to pH 7.2 by bicarbonate solution and
168
mL (142 mg) 1-octanethiol, but with addition of 1 mL pyridine
into the reaction mixture. After the rechromatography, the

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T. Bozi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 923–929
928
applied to target cells to various final concentration ranging from
0 to 100 M. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) was dissolved (5 mg/ml) in phosphate buffer
saline, pH 7.2, and filtered through Millipore filter, 0.22 m, before
6.2.4. Antiviral activity
Antiviral activity to HIV was tested at the NIH-NCI according to
the standard procedure [37].
m
m
use. RPMI 1640 cells culture medium, FBS and MTT were Sigma
Chemicals products.
Acknowledgment
HeLa cells and Fem-X cells were cultured as monolayers in the
nutrient medium. K-562 cells were grown as a suspension culture
in the same nutrient medium. The cells were grown at 37 ꢀC in 5%
CO2 and humidified air atmosphere. Peripheral blood mononuclear
cells (PBMC) were separated from whole heparinized blood of
healthy volunteers by LymphoprepÔ gradient centrifugation.
Interface cells, washed three times with Haemaccel (aqueous
This work was supported by the Ministry of Science of the
Republic of Serbia (Grant 142026).
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.11.033
solution supplemented with 145 mM Naþ, 5.1 mM Kþ, 6.2 mM Ca2þ
,
145 mM Clꢂ and 35 g/l gelatin polymers, pH 7.4) were counted and
resuspended in the nutrient medium.
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