New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

Article abstract of DOI:10.1016/j.bmc.2010.07.026

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several h

Full text of DOI:10.1016/j.bmc.2010.07.026

Bioorganic & Medicinal Chemistry 18 (2010) 6275–6281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New antitumoral agents I: In vitro anticancer activity and in vivo acute
toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-
3-one and derivatives
b
a
José Agustín Quincoces Suarez a, , Daniela Gonçales Rando , Reginaldo Pereira Santos ,
*
Carolina Passarelli Gonçalves a, Elizabeth Ferreira b, João Ernesto de Carvalho c, , Luciana Kohn ,
Durvanei Augusto Maria d, Fernanda Faião-Flores d, Dirk Michalik e, Maria Cristina Marcucci f,
Christian Vogel g
c
*
a Laboratory of Organic Synthesis, Universidade Bandeirante de São Paulo (UNIBAN), São Paulo, Brazil
b LAPEN, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo (USP), São Paulo, Brazil
c Division of Pharmacology and Toxicology, Multidisciplinary Center for Chemical, Biological and Agricultural, State University of Campinas, UNICAMP, Campinas, Brazil
d Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil
e Leibniz-Institut für Katalyse, Rostock, Germany
f Laboratory of Natural Products, Universidade Bandeirante de São Paulo (UNIBAN), São Paulo, Brazil
g Institut für Chemie, University Rostock, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2010
Revised 8 July 2010
Accepted 12 July 2010
Available online 16 July 2010
This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pen-
tadien-3-one 1 and its derivatives 2–5. This set of synthetic compounds exhibited high antitumoral activ-
ities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460,
melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing
the resistance phenotype for adriamycin NCI-ADR/RES, prostate PC-3, and leukemia K-562. Compounds
were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as
well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29
was observed for all tested compounds and suggests further studies with models of colon cancer. Com-
Dedicated to Professor Dr. Klaus Peseke on
his 70th birthday.
pounds 1, 2, and 4 showed significant cytotoxic activity with IC50 values 62.3 lM for all human cancer
Keywords:
cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate.
Ó 2010 Elsevier Ltd. All rights reserved.
Synthesis
Diarylpentadienone derivatives
Cytotoxicity
LD50 toxicity
Antineoplastic agents
1. Introduction
thy since it can be expressed in terms of prophylactic and growth
inhibition effects.7
Compounds chemically known as 1,5-diaryl-1,4-pentadien-3-ones
have been widely explored as promising pharmacological agents,
exhibiting potent anti-oxidative,1 anti-inflammatory,1,2 anti-HIV,3,4
and insecticide5 activities.
These are structural analogs of the natural product curcumin
(1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)
which is found as a major pigment in the Indian spice turmeric
Curcumalonga, Zingiberaceae.6 They share not only similar chemical
structures but also similar biological properties as antioxidant and
anticancer promising agents. The antitumoral activity is notewor-
Organic synthesis provides a cheaper and more efficient
approach to obtain large amounts of ‘natural inspired’ chemicals
to therapeutics than by extracting few milligrams from the nature.
In 1927, Glaser and Tramer8 reported the first synthesis of 1,5-
bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one with 60%
of yield by starting from the vanillin and acetone in the presence
of concentrated hydrochloric acid as catalyst.
Further Ramanan and Rao9 synthesized this product in 1989 by
starting from 4-O-methoxymethylvanillin and acetone in alkaline
medium, obtaining a yield of 42% after purification by thin-layer
chromatography.
In 1997, Sardjiman et al.10 developed a new variant synthetic
approach using equimolar quantities of vanillin and acetone in
the presence of concentrated hydrochloric acid, reporting a yield
of raw product of 89%. For this reason the melting point indicated
* Corresponding authors. Tel.: +55 11 2967 9148; fax: +55 11 2967 9064 (J.A.Q.S.);
tel.: +55 19 2139 2850; fax: +55 19 2139 2852 (J.E.d.C.).
E-mail addresses: quinco99@hotmail.com (J.A. Quincoces Suarez), carval-
ho@cpqba.unicamp.br (J.E. de Carvalho).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.026

6276
J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
in this procedure was 58 °C less than the one reported by Glaser
and Tramer.8
Compound 2, 1,5-bis(4-acetoxy-3-methoxyphenyl)-1,4-penta-
dien-3-one, was obtained as described earlier by our group.2
The prenylated derivative 3 was synthesized from 1 and prenyl
bromide in the presence of potassium carbonate in dry N,N-
dimethylformamide. The reaction mixture was stirred for 8 h at
40 °C and was completed after 8 h.
Veratraldehyde, acetone and concentrated hydrochloric acid re-
acted at 40 °C and under ultrasonic irradiation (40 kHz) to obtain
compound 4 with good yield.
Artico et al.3 also obtained this substance 1 year later, but with a
low yield of 18%. But they reported a melting temperature range
(114–116 °C) lower than the one reported by Glaser and Tramer,
what suggests that this compound was not obtained pure despite
of purification by column chromatography.
Only in 1998, compound 1,5-bis(4-hydroxy-3-methoxyphenyl)-
1,4-pentadien-3-one 1 was isolated and characterized in the rhizomes
of Curcuma domestica by Masuda et al.1 The authors also reported the
anti-oxidative and anti-inflammatory activity of this pentadienone.
The method for the preparation of 1,5-bis(4-hydroxy-3-
methoxyphenyl)-1,4-pentadien-3-one11 1, by Claisen condensa-
tion, starting from vanillin or vanillin derivatives, respectively,
and acetone in acid medium under ultrasonic irradiation condi-
tions with good yield and purity, was patented in 2002.
Ohori et al.12 development in 2006 a very important work about
curcumin analogs. These authors included compound 4 in this
study and reported their potent antitumoral activity, but they did
not cite our published patent in this paper.
Finally, compound 5 was obtained starting from 1 and malono-
nitrile in the presence of ammonium acetate and acetic acid under
Knoevenagel reaction conditions.
3. Results and discussion
The chemical structure of the 1,5-diaryl-1,4-pentadien-3-ones
and derivatives was determined by spectroscopic methods and ele-
mental analysis, thus excluding any other alternative structures
(Section 5).
Table 1 exhibits the cytotoxic effects of compounds 1–5 toward
nine different human cancer cell lines.
Liang et al.13 synthesized in 2009 compound 1 and found a sig-
nificative cytotoxic activity toward a diverse panel of cultured hu-
man tumor cell lines, but they did not cite our published patent in
their paper.
Two series of curcumin analogs, a total of 24 compounds, were
synthesized and evaluated as new antitumoral agents by Fuchs
et al.14 in 2009. They included in this list compounds 1 and 4 too
and found that compound 1 was 5–8 times more potent than cur-
cumin. Our published patent was not present in the list of cited
references.
Compounds 1, 2, and 4 exhibited a better potency than the ana-
logs 3 and 5, since their IC50 values were less than 2.29 lM, which
can be considered to show very promising activities. Indeed, com-
pound 1 was found to be the most potent one with selectivity for
melanoma and breast cancer cells lines. Compound 2 had a low
selectivity for renal cell line and compound 4 did not show any
selectivity at all.
Compounds 3 and 5 presented higher selectivity for some can-
cer cells lines. This was observed for compound 3, to leukemia and
colon cell lines, and for compound 5, to ovarian expressing the
resistance phenotype for adriamycin NCI-ADR/RES, melanoma
and colon.
More than 85% of the modern therapeutic drugs, including the
anticancer agents, are obtained from synthetic or semi-synthetic
approaches.
Cancer or neoplasia represents the collective name for more
than 100 diseases characterized by the uncontrolled growth of
abnormal cells that may affect almost any tissue of the body. It af-
fects more than 11 million people every year and it is responsible
for 7 million deaths per year, which may be translated, from a sta-
tistical point of view, as 12.5% of world deaths.15
These differences in potency found for compounds 3 and 5,
compared to the other analogs, have inspired us to design preli-
minary steric studies using computer-aided tools.
These methodologies could arise some hypothesis about the
reasons behind the different biological behaviors presented by
those compounds.
Unfortunately, despite of all knowledge and advances in the
cancer research, it is still urgent the need for new and effective
agents capable of bringing this disease under control, mainly in
consequence of drug toxicity and development of resistance to
the actual chemotherapeutic agents.
It is preconized that effective novel anticancer drugs require not
only disease specificity, but also minimal toxic side-effects. Drug
capability for precise cancer therapy without consequence to the
function of normal cells has yet to be developed.
This paper reports the in vitro cytotoxic effects produced by five
diarylpentadienone derivatives, which were synthesized under
ultrasonic reaction conditions, against a panel of relevant human
tumor cell lines as well as against peritoneal normal macrophages
to determine the selectivity of compounds. It also reports the
in vitro antitumoral assays against the murine tumoral cell lines
melanoma B16F10 and lymphocytic leukemia L1210. Finally, intra-
peritoneal LD50 values for the most promising analogs were also
obtained in mice.
Figure 1 shows the electrostatic potential maps calculated onto
surfaces obtained for all five compounds.
Prenyl group at the 4-position of both aromatic rings creates
important increase in the overall volume of the compound 3 and
could be related to the low cytotoxic activity obtained for this ana-
log. This result could also be related to lipophilic factors since the
prenyl side chain is known as a high apolar group.
Minor differences can be observed in the electrostatic potential
map of this compound when compared to the maps obtained to the
other analogs. Actually, no relationship between activity and the
electrostatic potential can be established to the series. This does
not imply that this parameter is not important to the cytotoxic
activity of the compounds and a larger series could shed light on
this point.
Compound 5 presents major differences of shape and volume
characteristics. The cyano groups, which substitute the carbonyl
group of the other structures, lead one of the aromatic ring to an
‘out-of-plane’ conformation as well as induce a different distribu-
tion of the electrostatic potential of the molecule. These differ-
ences, however, do not seem to be as important as the steric
hindrance at 4-position of the aromatic rings since compound 5
presented better activity than compound 3.
It is noteworthy, however, that these results are preliminary ones
and more specific structure activity studies have to be accomplished
in order to understand the efficiency of this series of compounds as
promising antitumoral agents.
2. Chemistry
The synthesis of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pen-
tadien-3-one, 1, can be accomplished by Claisen condensation
starting from vanillin and acetone in a molar ratio of 2:1 at 5 °C,
in the presence of concentrated hydrochloric acid under ultrasonic
irradiation conditions (40 kHz) for 1 h11 (Scheme 1).

J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
6277
Scheme 1. Synthesis of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives. Reagents and condition: (a) acetic acid anhydride/sodium acetate, 1 h,
reflux; (b) prenyl bromide/K2CO3/DMF; (c) malononitrile/NaAc/HAc/Knoevenagel,))) = ultrasonic irradiation.
The line average indicated in Table 1 shows that HT-29 colon
Table 1
cells were the most sensible tumor line to the tested compounds,
followed by the leukemia (K-562), ovarian expressing multidrug
resistance phenotype (NCI-ADR/RES) and kidney (786-O).
Antitumoral activities of diarylpentadienones, by means of IC50 values (
different human tumor cell lines
lM), toward
X
Considering that compounds 1 and 2 were the most potent ones
of the series, they were also assayed against murine tumoral cell
lines (melanoma B16F10 and lymphocytic leukemia L1210) as well
as against peritoneal normal macrophages (Table 2).
MeO
RO
OMe
OR
Table 2 shows that compound 1 was more potent than com-
pound 2 for murine cancer cells lines but also exhibited higher
cytotoxic profile against normal cells (macrophages) since the
IC50 values for leukemia and macrophages were almost the same.
This toxicity for macrophages may be related with an anti-inflam-
matory effect already observed and reported for compound 1 by
Masuda et al.1 and for compound 2 by Paulino et al.2
Compound
R
X
1
2
3
4
5
H
O
O
O
O
COMe
CH2CH@C(Me)2
Me
H
C(CN)2
Park et al.16 showed a reduction in Cox-2 expression after expo-
sure of T24 bladder tumor cells to curcumin. More recently, Chad-
alapaka et al.17 reported inhibition of 253JB-V and KU7 bladder
cancer cell growth using 10–25 mM/L of curcumin, showing a de-
Human cell lines
Compounds
1
2
3
4
5
UACC-62
MCF-7
NCI-ADR
786-O
NCI-460
K-562
PC-03
0.09
0.12
0.83
1.99
1.53
1.84
1.25
2.2
4.31
1.09
3.12
0.66
1.7
1.41
0.95
1.39
1.48
30.6
18.2
7.22
7.87
34.1
4.8
60.5
30.3
2.12
2.00
2.37
3.36
1.86
1.83
2.32
2.32
2.43
3.34
5.24
2.97
7.14
6.89
7.59
4.25
115
crease in the expression of NF-jB-dependent genes, Cyclin D1, sur-
vivin and Bcl-2. In agreement with these literature data, our results
show that the strain of colon cancer (HT-29) is the most sensitive
to the compounds studied and those with high expression of
Cox-2.
The acute toxicity assay (LD50) is also an important item in the
development of new drugs, mainly against neoplasias, and also
important for the subsequent studies. For this reason, this assay
OVCAR-03
HT-29
2.3
4.04
3.37

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J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
Figure 1. Electrostatic potential mapped onto the surfaces calculated to the five analogs. Negative electrostatic potential regions are represented in red color (high electronic
density) while positive electrostatic potential areas are shown in dark blue color (low electronic density).
Table 2
treatment period. No mortality occurred immediately or during
the 14-day observation period; body weight dose was considered
as the ‘Limit test’ as recommend by the acute toxicity testing pro-
Antiproliferative effects of compounds 1 and 2 on B16F10 melanoma cells, L1210
leukemia cells, and mice macrophages
Compounds
Cell line (
lM)
cedures. Administration of further higher doses was considered
physiologically unsound and, is not generally recommended. The
treated animals did not show any significant alteration in water
or food consumption or in body weight during the experimental
period. Furthermore, the animals showed no symptoms of diar-
rhea, piloerection, convulsion, and sleepiness.
B16F10
L1210
Macrophages
1
2
21.47 2.7
220.2 10.3
56.1 3.8
245.6 12.4
51.81 1.92
274.7 11.5
According to Loomis,18 compounds with LD50 values ranging
from 5 to 15 g/kg are considered to be practically non-toxic.
was also applied to compounds 1 and 2 which demonstrated the
most promising antitumoral activities.
4. Conclusions
The animals treated with compound 2 (intraperitoneal) pre-
sented the first toxic symptoms with the dose of 2.5 g/kg of animal
body weight. The LD50 value was determined by linear regression
and was found to be 3.68 g/kg after 14 days of observation.
Compound 1 exhibited a much lower acute toxicity (8.54 g/kg)
which is almost two fold lower compared to the acetylated deriv-
ative. This could be related to the higher liposolubility of com-
pound 2 but further studies could clarify these results.
In the present work, we report the synthesis of new 1,5-diaryl-
1,4-pentadien-3-one derivatives with good yield and purity under
ultrasonic irradiation conditions. Compound 1 showed significant
cytotoxic activity with IC50 values of 1.35 lM or lower for all hu-
man cancer cell lines. Lethal doses determination after intraperito-
neal acute administration of this compound revealed very low
toxicity rate (LD50 = 8.54 g/kg). For this reason, compound 1 could
be applicable as a promising new antineoplastic agent with the
The animal treated with these compounds did not develop any
clinical signs of toxicity either immediately or during the post-

J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
6279
desirable profile of high efficiency and selectivity. Even compound
2, by its antitumoral and toxicity characteristics, could be consid-
ered, at least, as a good lead to further drug design studies. With
less potency, the other compounds also had anticancer activity,
especially against colon cell lines. Apparently, colon cancer may
be a target for diarylpentadienone derivatives.
1639, 1593 (C@C); 1250 (C–O) cmꢀ1
.
1H NMR (300 MHz, CDCl3):
3
d = 7.69 (d, 2H, H-1/H-5, J1,2=4,5 = 15.8 Hz); 7.14 (d, 2H, 2 H-20,
4
3
4
J2 ,6 = 1.8 Hz); 7.12 (dd, 2H, 2 H-60, J5 ,6 = 8.1 Hz, J2 ,6 = 1.8 Hz);
0
0
0
0
0
0
3
6.92 (d, 2H, H-2/H-4, J1,2=4,5 = 15.8 Hz); 6.87 (d, 2H, 2 H-50,
3
0
0
J5 ,6 = 8.1 Hz); 5.51 (m, 2H, 2 @CHprenyl); 4.60 (m, 4H, 2 CH2prenyl);
3.90 (s, 6H, 2 MeO); 1.77 (s, 6H, 2 CH3prenyl); 1.72 (s, 6H, 2
CH3prenyl). 13C NMR (75.5 MHz, CDCl3): d = 188.6 (C-3); 150.5 (C-
40); 149.4 (C-30); 143.0 (C-1/C-5); 138.1 (@C(CH3)2); 127.5 (C-10);
123.4, 122.6 (C-2/C-4, C-60); 119.3 (C-50); 112.5 (C-20); 109.9
(@CHprenyl); 65.4 (CH2prenyl); 56.0 (MeO); 25.5, 18.0 (CH3prenyl).
Anal. Calcd for C29H34O5: C, 75.32; H, 7.36. Found: C, 75.27; H,
7.41; EI-MS m/e: 462 (M+).
5. Experimental
5.1. Chemical synthesis
TLC was carried out on silica gel 60 GF254 (Merck) with detec-
tion by UV light (k = 254 nm) and/or by charring with iodine. IR
spectra were recorded with a Nicolet 205 FT-IR spectrometer. 1H
NMR (300.13 MHz) and 13C NMR (75.5 MHz) spectra were re-
corded on Bruker instrument ARX 300 with deuterochloroform or
DMSO-d6 as solvent. The mass spectra were recorded on an AMD
402/3 spectrometer (AMD Intectra GmbH). Elemental analyses
were performed on a Leco CHNS-932 instrument.
5.1.4. 1,5-Bis(3,4-dimethoxyphenyl)-1,4-pentadien-3-one (4)
Veratraldehyde (7.8 mmol, 1.3 g) and acetone (3.9 mmol,
0.29 ml) were contacted at a molar ratio of 2:1 at 40 °C, under
ultrasonic irradiation (40 kHz) in the presence of concentrated
hydrochloric acid for 2 h. The mixture was poured onto water/ice
and the crude product was extracted with ethyl acetate. The com-
bined organic extracts were washed with sodium hydrogen car-
bonate solution, then with water, dried with anhydrous sodium
sulfate and evaporated under reduced pressure to furnish, after
recrystallization from methanol, yellow powder of 4 (2.4 g, 87%
yield); mp 115–120 °C; IR (KBr): 3057 (@CH); 2999, 2956, 2935
(CH3); 2835(–CH); 1645 (C@O); 1593, 1512 (C@C); 1261 (C–O)
5.1.1. 1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-
one (1)
Vanillin (0.02 mol; 3.04 g) and acetone (0.01 mol; 0.73 ml) were
contacted at a molar ratio of 2:1, at 40 °C, under ultrasonic irradi-
ation (40 kHz) in the presence of concentrated hydrochloric acid
for 2 h. Then, the mixture was poured onto water/ice. The crude
product was dissolved in a sodium hydroxide solution, and the fil-
trate treated with hydrochloric acid. The formed product was fil-
tered, and washed with distilled water until a neutral pH value
was achieved. Yield obtained: 92% of the crude product (yellow-
green powder).
cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 7.67 (d, 2H, H-1/H-5,
3
4
J1,2=4,5 = 16.0 Hz); 7.38 (d, 2H, 2 H-20, J2 ,6 = 1.5 Hz); 7.30 (dd,
0
0
3
4
2H, 2 H-60, J5 ,6 = 8.0 Hz, J2 ,6 = 1.5 Hz); 7.20 (d, 2H, H-2/H-4,
0
0
0
0
3
3
J1,2=4,5 = 16.0 Hz); 7.00 (d, 2H, 2 H-50, J5 ,6 = 8.0 Hz); 3.81 (s, 6H,
0
0
2 MeO); 3.79 (s, 6H, 2 MeO). Anal. Calcd for C21H22O5: C, 71.19;
H, 6.22. Found: C, 71.15; H, 6.18.
For further purification, the crude product was recrystallized
from acetonitrile/water. Purity = 100% (determined by HPLC), yel-
low powder, mp 155 °C, TLC Rf 0.82 in hexane/EtOAc 1:2; IR
(KBr): 3415 (OH); 3070 (@CH); 2959 (CH3); 2848 (–CH); 1588
5.1.5. 1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-
ylidenmalononitrile (5)
1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (1)
(3.26 g, 0.01 mol), malononitrile (0.66 g, 0.01 mol), ammonium
acetate (3.5 g, 0.045 mol), acetic acid (9.5 ml), and toluene
(50 ml) are added following Cope’s variant, heating under reflux
for 8 h. Then the solution was concentrated under reduced pres-
sure to a syrup. The residue was poured in water and extracted
with ethyl acetate. The combined organic layers were washed with
sodium hydrogen carbonate solution, then with water, dried with
anhydrous sodium sulfate and evaporated until dryness. Further
purification of the product was not necessary; orange powder of
5 (2.84 g, 76% yield); mp 216 °C; IR (Nujol): 3352, 3198 (OH),
(C@O); 1513 (C@C) cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 9.68
3
(s, 2H, 2 OH); 7.65 (d, 2H, H-1/H-5, J1,2=4,5 = 16.0 Hz); 7.37 (d,
4
3
2H, 2 H-20, J2 ,6 = 1.9 Hz); 7.20 (dd, 2H, 2 H-60, J5 ,6 = 8.0 Hz,
0
0
0
0
4
3
0
0
J2 ,6 = 1.9 Hz); 7.16 (d, 2H, H-2/H-4, J1,2=4,5 = 16.0 Hz); 6.85 (d,
3
0
0
2H,
2 2
H-50, J5 ,6 = 8.0 Hz); 3.85 (s, 6H, MeO). 13C NMR
(75.5 MHz, DMSO-d6): d = 188.2 (C-3); 149.6 (C-40); 148.2 (C-30);
142.9 (C-1/C-5); 126.5 (C-10); 123.5 (C-60); 123.2 (C-2/C-4); 115.9
(C-50); 111.6 (C-20); 55.9 (MeO). Anal. Calcd for C19H18O5: C,
69.94; H, 5.52. Found: C, 69.91; H, 5.48; EI-MS m/e: 326 (M+).
2246, 2208 (CN); 1602, 1515 (C@C); 1273 (C–O) cmꢀ1
.
1H NMR
5.1.2. 1,5-Bis(4-acetoxy-3-methoxyphenyl)-1,4-pentadien-3-
one (2)
(300 MHz, CDCl3): d = 9.40 (br, 2H, 2 OH); 7.56 (d, 2H, H-1/H-5,
3
4
J1,2=4,5 = 15.8 Hz); 7.15 (d, 2H, 2 H-20, J2 ,6 = 1.6 Hz); 7.12 (dd,
Compound 2 was obtained as described earlier by our group.2
0
0
3
4
2H, 2 H-60, J5 ,6 = 8.1 Hz, J2 ,6 = 1.6 Hz); 6.99 (d, 2H, 2 H-50,
0
0
0
0
3
3
0
0
J5 ,6 = 8.1 Hz); 6.73 (d, 2H, H-2/H-4, J1,2=4,5 = 15.8 Hz); 3.76 (s,
5.1.3. 1,5-Bis[3-methoxy-4-(3-methyl-but-2-enyloxy)phenyl]-
1,4-pentadien-3-one (3)
6H, 2 MeO). Anal. Calcd for C22H18N2O4: C, 70.59; H, 4.82; N,
7.49. Found: C, 70.53; H, 4.79; N, 7.51; EI-MS m/e: 374 (M+), 359
(M+ꢀCH3), 357 (M+ꢀOH), 348 (M+ꢀCN), 343 (M+ꢀOCH3).
To a stirred solution of 1,5-bis(4-hydroxy-3-methoxyphenyl)-
1,4-pentadien-3-one (1) (0.652 g, 2 mmol) in dry DMF (10 ml),
anhydrous potassium carbonate (0.828 g, 6 mmol) was added
and the solution was stirred for 30 min at 40 °C under argon. Pre-
nyl bromide (0.596 g, 4 mmol) was added drop-wise and the
resulting mixture was heated and stirred at the same temperature
for 8 h under argon. Then the mixture was poured into cold water
(50 ml) and extracted with chloroform. The combined organic lay-
ers were washed with diluted sodium hydrogen sulfate solution,
then with water, dried with Na2SO4 and evaporated under reduced
pressure. The residue was purified by column chromatography on
silica gel 60 (63–200 mesh, Merck) with hexane/ethyl acetate 2:1.
Yield 0.49 g (53%); colorless oil, TLC Rf 0.48 in hexane/EtOAc 2:1; IR
(KBr): 3035 (@CH); 2962, 2929 (CH3); 2885 (–CH); 1664 (C@O);
5.2. Cytotoxic effects screening
5.2.1. Cytotoxic effects on human tumor cell lines
Since it is known that different cell lines display different sensi-
tivities toward a cytotoxic compound, the use of more than one cell
line is therefore considered to be necessary in the detection of
cytotoxic compounds. Bearing this in mind, cell lines of different
histological origin were used in the present study.
Human tumor cell lines UACC-62 (melanoma), MCF-7 (breast),
NCI-460 (lung, non-small cells), OVCAR-03 (ovarian), PC-03 (pros-
tate), HT-29 (colon), 786-O (renal), and NCI-ADR/RES (ovarian

6280
J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
expressing phenotype multiple drugs resistance) were kindly pro-
vided by National Cancer Institute (NCI).
Stock cultures were grown in a medium containing 5 ml of
RPMI-1640 (Gibco BRL, Life Technologies) and supplemented with
ities of the animals were opened under sterile conditions under a
laminar flow and exposed. The acute toxicity tests were conducted
some years ago and followed the OECD (Organization for Economic
Co-operation and Development) protocol (OECD425).20,21
5% of fetal bovine serum. Gentamicin (50
experimental cultures. Cells in 96-well plates (100
were exposed to four concentrations of samples in dimethyl sulf-
oxide at 37 °C, 5% of CO2 in air for 48 h.
Then, a 50% of trichloroacetic acid solution was added and after
incubation for 30 min at 4 °C, washing and drying, the cell prolifer-
ation was determined by spectrophotometric quantification
(540 nm) of the cellular protein content using sulphorhodamine
B assay.
Sulphorhodamine B (SRB) is an aminoxantine with a bright pink
color that has two sulfonic groups that bind to protein’s amino
acids basic terminals of cells fixed with trichloroacetic acid. There-
fore, this non-clonogenic methodology permits a high sensitive
protein dosage with a straight relationship to cell culture.19
l
g/ml) was added to the
Two milliliters of the saline solution, containing 5000 U antico-
agulant heparin (Liquemine-Roche), were injected into the cavity,
followed by a massage and collection of the peritoneal wash irriga-
tion liquid, which was centrifuged at 2000 rpm for 10 min at 4 °C.
The suspension was resuspended with RPMI-1640 culture med-
ium supplemented with 10% bovine fetal serum, with the count cell
number adjusted to 106/ml in a Neubauer chamber. Aliquots of the
macrophages were cultivated on plates with 96 flat bottom wells,
incubated with and without compounds 1 and 2 diluted in Miglyol
810Ò. After 24 h, the plates were submitted to the cytotoxicand anti-
proliferative effect assessment by the MTT colorimetric method.
ll cells/well)
5.2.4. Culture of leukemia murine L1210
L1210 lymphocyte murine leukemia cells were obtained from
American Type Culture Collection (ATCC—clone CCL 219). These
cells were grown in Dulbecco’s modified Eagle’s medium contained
5.2.2. Cytotoxic and antiproliferative effects on B16F10
melanoma cells, L1210 leukemia cells, and mice macrophages
Analyses of cytotoxic and antiproliferative effects against
B16F10 and L1210 tumor cell lines and mice macrophages were
performed with compounds 1 and 2. Solutions with different con-
centrations were obtained by diluting the substance with Miglyol
810Ò.
The colorimetric methodology for the respiratory route of mito-
chondria MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazo-
lium bromide) was used, as described by Mosmann.20 The MTT
assay is a colorimetric assay that relies on the enzymatic reduction
antibiotics, L-glutamine (70 mM), supplemented with 10% fetal bo-
vine serum. Culture cell growth was measured by cell count using a
Neubauer chamber. After treatment with drug, the cell viability
was quantified by using an MTT [3-(4,5-dimethyl-2-thiazyl)-2,5-
diphenyl-2H-tetrazolium bromide] standard spectrophotometric
assay. After this treatment, the content of the plates was centri-
fuged for 2 min at 1800 rpm, at 4 °C. The supernatant was removed
and 100 ll of dimethyl sulfoxide were added to dissolve formazan
crystals. The absorbances were obtained by an ELISA reader (Titer-
Tek Multiskan) at 540 nm wavelength and the inhibitory potencies
of
a
yellow tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-
of the compounds tested were expressed as IC50
.
diphenyltetrazolium bromide (MTT), which forms a purple forma-
zan crystal in metabolically active cells. The formazan can then be
solubilized producing a concentration dependent colorimetric sig-
nal at 540–570 nm proportional to the cell number and activity.
This results in a sensitive assay that is readily adaptable to a 96-
well microplate format to analyze a large numbers of samples
using a microplate absorbance reader.
After growth and in vitro confluence of normal and tumoral
cells, different concentrations of compounds 1 and 2 diluted in Mi-
glyol 810Ò and the diluent-control (Miglyol 810Ò) were added on
the adhering cells, incubated for 24 h. After the referred period,
5.3. Medium lethal dose assays (LD50
)
Toxicological tests were performed using 20 male albino Swiss
mice, weighting approximately 25 g each, divided equally in trea-
ted and control groups.
Animals were maintained in the test rooms at least for 7 days
for adaptation. They were then submitted to fast for 12 h before
administering the test substances by intraperitoneal administra-
tion after weight determination. The tested doses were of 1, 2.5,
and 5 g/kg of animal body weight, respectively. They were ob-
served for a minimum of 14 days for signals of toxicity and deter-
mination of number of deaths. The LD50 values were assessed by
Litchfield and Wilcoxon’s method.22
10 ll of MTT (5 mg/ml) were added and incubated for 3 h at 5%
CO2 and 37 °C. After this period, the content of the culture plates
was centrifuged for 2 min at 1800 rpm, at 4 °C.
The supernatant was removed and 100 ll of dimethyl sulfoxide
were added to dissolve formazan crystals previously constituted
and precipitated. The absorbances were obtained by an ELISA read-
er (TiterTek Multiskan) at 540 nm wavelength.
After growth and in vitro confluence of normal and tumoral
cells, different concentrations of compounds 1 and 2 diluted in Mi-
glyol 810Ò and the diluent-control (Miglyol 810Ò) have been added
on the adhering cells, incubated for 24 h. After the referred period,
10 ll of MTT (5 mg/ml) were added and incubated for 3 h at 5% CO2
and 37 °C. After this period, the content of the culture plates was
centrifuged for 2 min at 1800 rpm, at 4 °C. The supernatant was re-
5.4. Computer-aided studies
The three-dimensional structures of each of the five pentadie-
nones were constructed employing the HyperChem 7 software.23
The crystallized structure of 1,5-bis(4-methylphenyl)-1,4-pentadi-
en-3-one was retrieved from the Acta Crystallographica Section E:
Structure Reports Online (entry code to cif file: bq2087, resolution
at 1.04 Å)24 and was applied as geometry references in the building
up of all compounds.
The energy minimization was carried out employing the Hyper-
Chem 8 MM+ force field without any restriction, followed by calcu-
lating the partial atomic charges using RM1 semiempirical method,
also implemented in HyperChem software.
Finally, geometry optimization and electrostatic partial atomic
charges (Chelpg) were computed using the ab initio method HF/
6-31G (Gaussian G03).25 The electrostatic potential (EP) was
mapped on a surface constructed to each of the five analogs. Neg-
ative electrostatic potential regions are represented in red color
(high electronic density) while positive electrostatic potential
areas are shown in dark blue color (low electronic density).
moved and 100 ll of dimethyl sulfoxide were added to dissolve
formazan crystals previously constituted and precipitated. The
absorbances were obtained by an ELISA reader (TiterTek Multis-
kan) at 540 nm wavelength.
Commercial chemotherapics as paclitaxel and etoposide were
considered as standard drugs.
5.2.3. Cytotoxic effect on peritoneal normal macrophages
Peritoneal macrophages were obtained from normal Balb-C
mice by the following procedure: after anesthesia, abdominal cav-

J. A. Quincoces Suarez et al. / Bioorg. Med. Chem. 18 (2010) 6275–6281
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Acknowledgments
The authors are grateful to Universidade Bandeirante de São Pau-
lo, UNIBAN, to the State of São Paulo Research Foundation, FAPESP
(2004/11351-0 and 2006/00116-5) and the National Council for Sci-
entific and Technological Development CNPq for financial support.
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