Preliminary antiproliferative evaluation of natural, synthetic benzaldehydes and benzyl alcohols

Article abstract of DOI:10.4067/S0717-97072013000300003

Vanillin, o-vanillin, natural and synthetic benzaldehydes and benzyl alcohols were assessed for antiproliferative effects using different human cell lines. Benzyl alcohols were synthesized from benzaldehydes reduced with NaBH4 in methanol solution. A new method for deprotection of ether compounds with TiCl4 solution was achieved with better performance, than previously reported. Twenty four compounds were tested. The in vitro growth inhibition assay was based on sulphorhodamine dye to quantify cell viability. Catechol 9 derived from piperonal as well as compounds 4 and 12 showed higher cytotoxicity on breast and prostate cancer cell lines (MDA-MB-231 and PC-3 respectively). o-Vanillin 5 has the highest cytotoxicity for all cell lines. IC50 values of 35.40 ± 4.2 ?M Breast MDA-MB231; 47.10 ± 3.8 ?M Prostate PC-3; 72.50 + 5.4 ?M Prostate DU-145; 85.10 + 6.5 ?M and Colon HT-29, were obtained without toxicity towards dermal human fibroblast (DHF cells).

Full text of DOI:10.4067/S0717-97072013000300003

J. Chil. Chem. Soc., 58, Nº 3 (2013)
PRELIMINARY ANTIPROLIFERATIVE EVALUATION OF NATURAL, SYNTHETIC
BENZALDEHYDES AND BENZYL ALCOHOLS
ALEJANDRO MADRID 1,*, LUIS ESPINOZA 1, KAREN CATALÁN 1, CESAR GONZALEZ 1, IVÁN MONTENEGRO
1, MARCO MELLADO 1,ENRIQUE WERNER2, MAURICIO CUELLAR 3 AND JOAN VILLENA 4,*
1Departamento de Química, Universidad Técnica Federico Santa María, Av. España N° 1680, Valparaíso, Chile.
2Departamento de Ciencias Básicas, Campus Fernando May Universidad del Bío-Bio, Avda.Andres Bello s/n casilla 447, Chillán, Chile.
3Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña N° 1093, Valparaíso, Chile.
4Centro de Investigaciones Biomédicas (CIB), Escuela de Medicina, Universidad de Valparaíso, Av. Hontaneda N° 2664, Valparaíso, Chile.
(Received: January 7, 2013 - Accepted: March 8, 2013)
ABSTRACT
Vanillin, o-vanillin, natural and synthetic benzaldehydes and benzyl alcohols were assessed for antiproliferative effects using different human cell lines.
Benzyl alcohols were synthesized from benzaldehydes reduced with NaBH4 in methanol solution. A new method for deprotection of ether compounds with TiCl4
solution was achieved with better performance, than previously reported. Twenty four compounds were tested. The in vitro growth inhibition assay was based
on sulphorhodamine dye to quantify cell viability. Catechol 9 derived from piperonal as well as compounds 4 and 12 showed higher cytotoxicity on breast and
prostate cancer cell lines (MDA-MB-231 and PC-3 respectively). o-Vanillin 5 has the highest cytotoxicity for all cell lines. IC50 values of 35.40 ± 4.2 μM Breast
MDA-MB231; 47.10 ± 3.8 μM Prostate PC-3; 72.50 + 5.4 μM Prostate DU-145; 85.10 + 6.5 μM and Colon HT-29, were obtained without toxicity towards dermal
human fibroblast (DHF cells).
Key words: Antiproliferative; Benzaldehydes; Benzyl Alcohols; o-vainillin.
Chemicals
INTRODUCTION
The natural and synthetic benzaldehydes were purchased from Sigma-
Aldrich (St. Louis, MO, USA). All other chemicals (reagents and solvents)
were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis,
MO, USA).
Methoxybenzaldehydes effect over cancer cells has been reported, vanillin
(4-hydroxy-3 methoxybenzaldehyde) exhibits a potent anti-proliferative effect
on a broad spectrum of cancer cell lines. In 1986, Ohta et al. first tested the
anti-mutagenic effect of vanillin on bacteria and found that vanillin could
reduce 4-nitroquinoline-1-oxide1. Another compound from this family is
anisaldehyde, which exhibits a concentration dependent cytotoxicity against
murine B16 melanoma cells2. Another in vitro study has demonstrated
inhibitor effect on hemoglobin S polymerization, of isovanillin, o-vainillin,
m-hydroxybenzaldehide and the p-hydroxybenzaldehyde3.
It was reported by King et al., that vanillin is effective on the repair
of mutations in colon cancer cells line HCT-116, this essay suggests colon
cancer cells to be suitable for studying vanillin anti-mutagenic effect and
its cytotoxicity relationship4. On the other hand, in 2002 da Silva et al. have
reported that vanillin inhibits spontaneous mutation in bacteria5. Studies in
Drosophila showed that vanillin inhibited mitomycin C-induced mutations
and dramatically increases recombinations6, demonstrating that vanillin
is a modifying factor that blocks the mutagenic pathway, and consequently
directs the mitomycin-induced lesions into a recombinational repair. Other
experimental tests suggested that post-treatment with vanillin protects against
point mutations and chromosome aberrations induced by chemical and physical
agents.7
General procedure for the reduction of benzaldehydes
All benzaldehydes were reduced using the method developed by Brown8
was slightly modified in this synthesis. A solution of piperonal 7 (300.0 mg, 2.0
mmol) in methanol (50 mL), sodium borohydride (176.0 mg, 4.65 mmol) was
added in small portions and carefully. The reaction mixture was stirred at -10
ºC for 2 h. After work-up as in the reduction of alcohols, the resulting residue
was recrystallized from hexane to give a tan solid identified as compound 22
(188.8 mg, 68.4 %); m.p.: 52.3-54.5 ºC. IR (cm−1): 3349 (O-H); 2918 (C-H);
1610 (C=C); 1432 (-CH ); 815 (-C-H). 1H- NMR: 6.86 (s, 1H, H-3); 6.79 (m,
2H, H-5 and H-6); 5.952(s, 2H, OCH2O); 4.57 (s, 2H, CH2OH); 1.77 (s, 1H,
OH). 13C-NMR: 148.5 (C-2); 147.5 (C-1); 133.7 (C-4); 118.6 (C-5); 109.8 (C-
6); 108.4 (C-3); 101.1 (OCH2O); 63.8 (CH2OH).
General procedure for acetylation and methylation of benzaldehydes
and benzyl alcohols.
Acetylation and methylation of benzaldehydes and benzyl alcohols of this
work were used typical protocols in synthesis organic9.
The purpose of the present work is to examine the effect of a series of 24
compounds related with benzaldehydes and benzyl alcohols. These compounds
were tested on one human tumor breast cancer cell line (MDA-MB-231), one
human colorectal cancer cell line (HT-29), two human prostate cancer cell lines
(PC-3, DU-145), and one dermal human fibroblast cell line (DHF).
Procedure for the cleavage with TiCl4 solution
The new method of synthesis consisted of a TiCl4 solution (0.9 mL, 8.20
mmol) cooled to −20 °C, was slowly added to a solution of piperonal 7 (300 mg,
2 mmol) in CH2Cl (20 mL) at −10 °C under an atmosphere of N2 with gentle
stirring. The reacti2on was continued for 4 h at −20 °C. After this, the mixture
was taken up in water and then extracted with ethyl acetate (3 × 50 mL). The
watery layer was discarded and the organic layer was washed to neutrality with
a saturated solution of NaHCO3. The organic layer was dried over MgSO ,
filtered and evaporated. Then it was absorbed on silica, chromatographe4d
by CC eluting with mixtures of petroleum ether/EtOAc increasing polarity
(19.0:1.0→13.0:7.0) to obtain a white solid identified as compound 9 (188.8
mg, 68.4 %); m.p.: 149.8–150.8 °C. IR (cm−1): 3329 (O-H); 2916 (C-H);
(C=O); 1616 (C=C); 1430 (-CH2); 809 (-C-H). 1H- NMR: 9.81 (s, 1H, CHO);
7.49 (b.s., 1H, OH); 7.44 (b.s., 1H, OH); 7.41 (dd, 1H, J = 8.1, 1.2 Hz, H-5);
7.28 (s, 1H, H-3); 6.73 (d, 1H, J = 7.9 Hz, H-6). 13C-NMR: 190.8 (CHO); 151.9
(C-1); 145.6 (C-2); 129.2 (C-4); 121.5 (C-5); 115.5 (C-6); 114.6 (C-3).
EXPERIMENTAL SECTION
General
Unless otherwise stated, all chemical reagents purchased (Merck or
Aldrich) were of the highest commercially available purity and were used
without previous purification. IR spectra were recorded as thin films in a FT-IR
Nicolet 6700 spectrometer and frequencies are reported in cm-1. Low resolution
mass spectra were recorded on an Agilent 5973 spectrometer at 70eV ionising
voltage in a DB-5 m, 30 m x 0.25 mm x 0.25 um column, and dates are given
as m/z (% rel. int.). 1H, 13C, 13C DEPT-135, sel. gs1D 1H NOESY, gs2D HSQC
and gs2D HMBC spectra were recorded in CDCl3 solutions and are referenced
to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.0 ppm for 1H and 13C,
respectively, on a Bruker Avance 400 Digital NMR spectrometer, operating at
400.1 MHz for 1H and 100.6 MHz for 13C.
Cell Lines
The experimental cell cultures were obtained from American Type
Culture Collection (Rockville, MD, USA). All cancer cell lines were grown
e-mail: alejandro.madrid@usm.cl
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
in DMEM-F12 medium containing 10% FCS, 100 U/mL penicillin, 100 μg/
mL streptomycin and 1 mM glutamine. Cells are seeded into 96 well microtiter
plates in 100 μL at plating density of 3 × 103 cells/well. After 24 h incubation at
37 °C (under a 5% humidified carbon dioxide to allow cell attachment) the cells
were treated with different concentrations of drugs (aldehydes and derivatives)
and incubated for 72 h under the same conditions. Stock solution of compounds
was prepared in ethanol and the final concentration of this solvent was kept
constant at 0.1%. Control cultures received only 0.1% ethanol.
two broad signals at δ = 7.49 (b.s., 1H) and δ = 7.44 (b.s., 1H) are now observed
in 1H-NMR spectrum.
Biological Results
These natural compounds used in this study were chosen because they are
predominant flavor components of some plants used in the food industry, for
this reason, foods fortified with these substances (natural or synthetic
derivatives) have a potential role preventing diseases.
The in vitro cytotoxicity evaluation of natural and synthetic compounds
1-24 (see Figure 1 and 2) natural and synthesized indicated that cell viability
expressed as % vs. control vehicle (ethanol 0.1%) was dose-dependent (μM).
Doxorubicin was used as positive control in this study (IC50 value < 10 μM).
Among IC values for compounds 4, 5, 10 and 12 were the most potent, are
summarize5d0 in Table 1 known as the micromolar concentration that produces
50% cell growth inhibition after 72 hours of drug exposure.
In vitro Growth Inhibition Assay
The sulforhodamine B assay according to the method of Skehan et al.10
was used with some modifications11. Briefly, the cells were set up as 3 ×
103 cells per well of a 96-well, flat-bottomed 200 μL microplate. Cells were
incubated at 37 °C in a 5% humidified CO2 plus 95% air mixture and treated
with the compounds at different concentrations for 72 hours. At the end of
drug exposure, cells were fixed with 50% trichloroacetic acid at 4 °C (TCA
final concentration 10%). After washing with destilled water, cells were stained
with 0.1% sulforhodamine B (Sigma-Aldrich, St. Louis, MO, USA), dissolved
in 1% acetic acid (50 μL/well) for 30 min, and subsequently washed with 1%
acetic acid to remove unbound stain. Protein-bound stain was solubilized with
100 μL of 10 mM unbuffered Tris base. The cell density was determined using
a fluorescence plate reader (wavelength 540 nm). Values shown are the mean
+ SD three independent experiments in triplícate. Untreated cells were used as
a negative control while, cells treated with doxorubicin were used as a positive
control.
RESULTS AND DISCUSSION
Chemistry
All benzaldehydes were reduced using NaBH4 in methanol solution
obtaining8 the corresponding benzyl alcohols with almost 100 percent yield,
the high reduction performance was achieved by the only in those compounds
which showed no free hydroxyl as case of piperonal (Scheme 1), whereas in
those molecules with a free hydroxyl as vanillin yield decreased to a half, and
for molecules bearing two or more free hydroxyl groups yield decreased in
about 20%. Based on the low reduction yields, other methods such as LiAlH4
or zinc in acid media were tested to increase efficiency, for aldehydes with two
or more hydroxyl groups, but with both methods the yields remained low; to
solve this problem, molecules were subjected to reactions of methylation and
acetylation, to block the free hydroxyl, and thus increase the benzyl alcohol
yield close to 100%, (Scheme 1). All the compounds were characterized
by NMR, IR and MS spectral data and their structures were confirmed by
comparison with spectral data in literature.
Figure 1. Natural and synthetic Benzaldehydes.
Figure 2. Natural and synthetic Benzyl Alcohols.
We are showing only the biologically active compounds with IC50 lower
than 100 μM, while cytotoxicity exhibited by benzyl alcohols in tested cancer
cell lines were higher than 100 μM. In this preliminary study, the differences
in biological activity attributable to the position of the substituents on the
aromatic ring are observed for in the different molecules tested.
The cytotoxic effects of vanillin are well known12. However, o-vanillin
was the most active compound in all the cell lines, especially in breast
cancer cells MDA-MB231. The structures difference is in the position of the
hydroxyl group, therefore o-vanillin may generate an intramolecular hydrogen
bond, decreasing in polarity and increasing its ability to pass through cell
membranes13.
On the other hand, the cytotoxic activity of these molecules could be
explained by the formation of Schiff bases derived from benzylic aldehydes.
Amino groups play an important role in the tertiary structure of cellular
enzymes such as tyrosinase, involving the amino group hydrogen bonding
which is essential to maintain the tertiary structure of the enzymes14. The low
conformational stabilities of native proteins make them easily susceptible to
denaturation by altering the balance of weak non-covalent bonds that maintain
the native conformation. Native proteins form a sort of intramolecular
micelle in which the non-polar Schiff base portion is largely out of contact
with the water-based environment and the aromatic ring provides stability
Scheme 1. General scheme of synthesis of Benzaldehydes derivatives
Conditions and reagents: a. NaBH , MeOH, -10 ºC, 4 h, 22; 95.1%; 21;
92.3%. b. i) TiCl4/CH2Cl2; N2, -20 °C, 44h iii) H2O, r.t, 20 h; 9; 68.4%.c. Ac2O,
DMAP, CH2Cl , 2 h, r.t.; 25; 98.1%. d. i) NaBH4, MeOH, 4 h -10 ºC.; ii) MeOH,
Na2CO3; 2 h r.t2.; 19; 67.6%. e. (CH3)2SO4/K2CO3, acetone, r.t.; 10; 60.2%.
Cleavage of the methylenedioxy ring of piperonal to obtain catechol
9 (68.4% yield) was achieved using TiCl solution. The absence of the
methylenedioxy singlet 6.07 ppm confirmed 4the presence of catechol, instead
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
to the system15. This would be enhanced in our active compounds due to the
presence of electron donor groups (OH and OCH ). Compound 9 has two
hydroxyl substituents in position p and m and comp3 ound 12 possesses three
methoxy substituents: one in p position and two in o position, which should
stabilize the Schiff base and increase cytotoxicity. Thus, the higher activity
of compounds 4 and 5 in comparision to the compounds 9 and 12 is directly
related to the existence of intramolecular hydrogen bonding and to proton
transfer in the equilibrium16. This tautomeric equilibrium has been confirmed,
at room temperature by NMR spectroscopic studies17,18. Compound 4 can form
an intramolecular hydrogen bond which would decrease the effect attributed
to the tautomerism, therefore o-vanillin (compound 5) is the molecule with the
highest non-specific cytotoxicity.
the tested compounds10. Unlike Ho’s assays, we used a concentration range
between 0 and 100 µM for all the tested compounds. We obtained an IC50 for
vanillin >100 µM on cancer cells HT-29 and an IC50 = 85.10 ± 6.5 µM for
o-vanillin in the same cell line. Therefore the cytotoxic effect reported by Ho
can be explained because the range of concentrations used is some thousand
times higher than the concentration we used for this cell line test.
We are reporting the toxicity of o-vanillin on prostate cancer cell lines
PC-3 and DU-145, calculated IC are 47.10 + 3.8 and 72.50 + 5.4 µM
respectively. However, the highest 5t0oxicity was observed against breast cancer
cell line MDA-MB231, presenting an IC50 of 35.40 ± 4.2 µM, lower than the
other tested compounds (for example compound 4 has an IC of 59.90 ± 3.9
µM). Compounds 9 and 12 have lower cytotoxic effect in b5r0east cancer cell
line, being their IC were 82.70 ± 6.7 and 78.71 ± 8.3 µM respectively, lower
than those presente5d0 by compounds 4 and 5 (See Table 1).
Cytotoxicity assays performed by Ho et al. on colon cancer cells HT-
29 were made in a high concentrations range of 0-10mM (to 6.58 mM) for
Table 1. Cytotoxic activity (IC50; µM) of natural and synthetic Benzaldehydes and Benzyl Alcohol derivatives against various human cancer cell lines.
Compounds
Breast MDA-MB231
59.90 + 3.9
Prostate PC-3
84.15 + 8.5
47.10 + 3.8
77.45 + 8.97
71.93 + 9.2
8.44 + 0.05
Prostate DU-145
Colon HT-29
DHF
>100
4
-
-
5
35.40 + 4.2
72.50 + 5.4
85.10 + 6.5
>100
9
12
82.70 + 6.7
-
-
42.40 + 6.2
82.40 + 9.2
13.3 ± 1.80
78.71 + 8.3
-
-
Doxorubicin
0.46 + 0.06
2.50 ± 0.04
2.15 + 0.02
12. Ho K. L., Yazan L., Ismail N., Ismail M. Cancer Epidemiol. 33, 155,
(2009).
4. CONCLUSIONS
13. Ashwood V., Field M., Horwell D., Julien-Larose C., Lewthwaite R.,
McCleary S., Pritchard M., Raphy J., Singh L. J. Med. Chem. 44, 2276,
(2001).
In chemical synthetic terms, the development of a new method for
deprotection of ether compounds with TiCl4 solution was achieved with better
performance than that previously reported by our group19.
14. Hassan M. Pure Appl. Chem. 79, 2277, (2007).
15. Kubo I. and Kinst-Hori I. J. Agric. Food Chem. 46, 1268, (1998).
16. Blonski C., De moissac D., Périé J. and Sygusch J. Biochem. J. 323, 71,
(1997).
17. Sharif S., Powell D. R., Schagen D., Steiner T., Toney M. D., Fogle E. and
Limbach H. H. Acta Cryst. B62, 480, (2006).
We can conclude that benzyl alcohols showed no significant cytotoxicity
in the cancer cell lines tested. Aldehydes 4 and 5 showed higher cytotoxic
activity than 9 and 12 compounds against the selected cancer cell lines. These
results indicate that compound 5 exerts this cytotoxic and selective effect on
all cancer cells lines over a wide concentration range without effects in non-
tumoral cell line (DHF).
18. Ambroziaka K., Rozwadowskia Z., Dziembowskaa T., Biegb B. J. Mol.
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19. Madrid A., Espinoza L., Montenegro I., Villena J. and Carrasco H.
Molecules 16, 4632, (2011).
In order to continue with this preliminary work, we will study mechanisms
of cell death such as caspase activity and cytochrome c release, triggered by
these four compounds 4, 5, 9 and 12.
ACKNOWLEDGMENTS
The authors thank DGIP of Universidad Técnica Federico Santa María for
financing (Project DGIP Nº 13.11.36, PAC 2010-2012 for A. M. and PIIC 2012
for I.M.), supporting this research.
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