Long Chain Alkyl Esters of Hydroxycinnamic Acids as Promising Anticancer Agents: Selective Induction of Apoptosis in Cancer Cells

Article abstract of DOI:10.1021/acs.jafc.7b01388

Cancer is the major cause of morbidity and mortality worldwide. Hydroxycinnamic acids (HCAs) are naturally occurring compounds and their alkyl esters may possess enhanced biological activities. We evaluated C4, C14, C16, and C18 alkyl esters of p-coumaric, ferulic, sinapic, and caffeic acids (19 compounds) for their cytotoxic activity against four human cancer cells and also examined their effect on cell cycle alteration and apoptosis induction. The tetradecyl (1c) and hexadecyl (1d) esters of p-coumaric acid and tetradecyl ester of caffeic acid (4c), but not the parental HCAs, were selectively effective against MOLT-4 (human lymphoblastic leukemia) cells with IC₅₀ values of 0.123 ± 0.012, 0.301 ± 0.069 and 1.0 ± 0.1 μM, respectively. Compounds 1c, 1d, and 4c significantly increased apoptotic cells in sub-G1 phase and activated the caspase-3 enzyme in MOLT-4 cells. Compound 1c was 15.4 and 23.6 times more potent than doxorubicin and cisplatin, respectively, against the drug resistant MES-SA-DX5 uterine sarcoma cells. These p-coumarate esters were several times less effective against NIH/3T3 fibroblast cells. Docking studies showed that 1c may cause cytotoxicity by interaction with carbonic anhydrase IX. In conclusion, long chain alkyl esters of p-coumaric acid are promising scaffolds for selective apoptosis induction in cancer cells.

Full text of DOI:10.1021/acs.jafc.7b01388

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Article
Long Chain Alkyl Esters of Hydroxycinnamic Acids as Promising
Anticancer Agents: Selective Induction of Apoptosis in Cancer Cells
José C J M D S Menezes, Najmeh Edraki, Shrivallabh Kamat, Mahsima Khoshneviszadeh,
Zahra Kayani, Hossein Hadavand Mirzaei, Ramin Miri, Nasrollah Erfani, Maryam Nejati, José
A. S. Cavaleiro, Tiago B. Silva, Luciano Saso, Fernanda M. Borges, and Omidreza Firuzi
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01388 • Publication Date (Web): 18 Jul 2017
Downloaded from http://pubs.acs.org on July 18, 2017
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Long Chain Alkyl Esters of Hydroxycinnamic Acids as Promising Anticancer
Agents: Selective Induction of Apoptosis in Cancer Cells
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Short titleꢀ Hydroxycinnamate alkyl esters induce apoptosis in cancer cells
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José C. J. M. D. S. Menezes1,2†, Najmeh Edraki3, Shrivallabh P. Kamat2, Mahsima
Khoshneviszadeh3, Zahra Kayani3, Hossein Hadavand Mirzaei3, Ramin Miri3, Nasrollah
Erfani4, Maryam Nejati3, José A. S. Cavaleiro1, Tiago Silva5, Luciano Saso6, Fernanda
Borges5*, Omidreza Firuzi3*
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1 Department of Chemistry & QOPNA, University of Aveiro, 3810ꢀ193 Aveiro, Portugal.
2 Department of Chemistry, Goa University, Taleigao 403 206 Goa, India.
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Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, 71345ꢀ1149 Iran.
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Institute for Cancer Research (ICR), School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran.
5 CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
4169ꢀ007 Porto, Portugal.
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Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University of
Rome, Italy.
†Present addressꢀ Section of Functional Morphology, Faculty of Pharmaceutical Sciences,
Nagasaki International University, Huis Ten Bosch Cho 2825ꢀ7 Sasebo, Nagasaki Japan 859ꢀ
3298
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*Corresponding authors:
Omidreza Firuzi, MD, PhD
Medicinal and Natural Products Chemistry Research Center
Shiraz University of Medical Sciences
Shiraz, Iran
Phone: (+98)ꢀ711ꢀ230ꢀ3872
Fax: (+98)ꢀ711ꢀ233ꢀ2225
Email: firuzio@sums.ac.ir
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Fernanda Borges, PhD
CIQUP/Department of Chemistry and Biochemistry
Faculty of Sciences, University of Porto
4169ꢀ007 Porto, Portugal
Eꢀmail: fborges@fc.up.pt
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Abstract
Cancer is the major cause of morbidity and mortality worldwide. Hydroxycinnamic acids
(HCAs) are naturallyꢀoccurring compounds and their alkyl esters may possess enhanced
biological activities. We evaluated C4, C14, C16 and C18 alkyl esters of pꢀcoumaric, ferulic,
sinapic and caffeic acids (19 compounds) for their cytotoxic activity against four human cancer
cells and also examined their effect on cell cycle alteration and apoptosis induction. The
tetradecyl (1c) and hexadecyl (1d) esters of pꢀcoumaric acid and tetradecyl ester of caffeic acid
(4c), but not the parental HCAs, were selectively effective against MOLTꢀ4 (human
lymphoblastic leukemia) cells with IC50 values of 0.123 ± 0.012, 0.301 ± 0.069 and 1.0 ± 0.1
µM, respectively. Compounds 1c, 1d, and 4c significantly increased apoptotic cells in subꢀG1
phase and activated the caspaseꢀ3 enzyme in MOLTꢀ4 cells. Compound 1c was 15.4 and 23.6
times more potent than doxorubicin and cisplatin, respectively, against the drug resistant MESꢀ
SAꢀDX5 uterine sarcoma cells. These pꢀcoumarate esters were several times less effective
against NIH/3T3 fibroblast cells. Docking studies showed that 1c may cause cytotoxicity by
interaction with carbonic anhydrase IX. In conclusion, long chain alkyl esters of pꢀcoumaric acid
are promising scaffolds for selective apoptosis induction in cancer cells.
Keywords: Cancer cell, cytotoxic activity, hydroxycinnamic acids, apoptosis, caspaseꢀ3
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Abbreviations
Hydroxycinnamic acids (HCAs)
pꢀCoumaric acid (p-CA)
Ferulic acid (FA)
Sinapic acid (SA)
Caffeic acid (CA)
Carbonic anhydrase IX (CAꢀIX)
3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT)
Fetal bovine serum (FBS)
Phosphate buffered saline (PBS)
Dimethyl sulfoxide (DMSO)
Propidium Iodide (PI)
Poly(vinylidenedifluoride) (PVDF)
5ꢀ(1ꢀ(4ꢀmethylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)thiopheneꢀ2ꢀsulfonamide (MPTTS)
Lamarckian Genetic Algorithm (LGA)
RootꢀMeanꢀSquare Deviation (RMSD)
Hypoxia inducible factorꢀ1α (HIFꢀ1α)
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1. Introduction
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Cancer is the major cause of morbidity and mortality worldwide. Different types of
cancer have caused 8.2 million deaths in 2012 and this number is escalating to 13.1 million in
20301. Chemotherapy is the main treatment modality available to cancer patients. However, the
resistance of cancer cells to chemotherapeutic agents and their side effects limits the use of these
agents and therefore have constantly encouraged the discovery of novel antitumoral agents with
better safety and efficacy profiles2ꢀ3.
Hydroxycinnamic acids (HCA), or phenylpropanoids, including pꢀcoumaric acid (1a),
ferulic acid (2a), sinapic acid (3a) and caffeic acid (4a) (Table 1), are phenolic compounds
naturally found in fruits and vegetables4ꢀ6. Hexadecyl (1d) and octadecyl (1e) esters of p-CA
have been found in sweet potato7. Similarly, long chain alkyl esters of FA were identified in
roots of Ipomoea batatas8, while octadecyl caffeate (4e) and coumarate (1e) are both present in
the root surface of sweet potato9. HCAs and their ester, amide, and glycoside derivatives have
also shown various biological activities5, 10ꢀ13. In particular, anticancer effect of these natural
phenolic compounds has been studied by several investigators14ꢀ18
.
One of the limiting factors for the pharmacological use of HCAs is their hydrophilicity,
which hinders their efficiency to interact with cell membranes and subsequently lowers their
efficacy11ꢀ12. Esterification can significantly increase the partition coefficient and lipophilicity
and this may improve the compound’s ability in crossing the membranes and lead to its higher
pharmacological effectiveness. Previous reports from our and others’ laboratories have also dealt
with the effect of esterification on biological activities of HCAs. In this regard, different alkyl or
aryl esters of HCAs have demonstrated enhanced in vitro antioxidant, neuroprotective and antiꢀ
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cancer potential compared to their parental compounds12ꢀ13, 16ꢀ23. The anticancer effects of FA
and CA have been shown in several reports17, 21, 24ꢀ25; however, less attention has been focused on
pꢀCA and its esters.
In our previous study, long chain alkyl esters of HCAs were synthesized and their
antioxidant activity was assessed13. Following our ongoing program of the study of biological
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activities of HCAs
and in order to obtain new insights on their mechanism of action as
anticancer agents, herein we evaluated the cytotoxic potential of longꢀchain alkyl esters of HCAs
(1bꢀ1e, 2bꢀ2d, 3bꢀ3e and 4bꢀ4e) against four different human cancer and one nonꢀcancer cell
lines. Their cytotoxic effects were investigated on cell cycle progression and apoptosis using
flow cytometry and Western blotting, respectively. A molecular docking study was also
performed for evaluating the anticancer potential of these HCAs as inhibitors of carbonic
anhydrase IX (CAꢀIX).
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2. Materials and Methods
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2.1. Chemicals
MTT (3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) was purchased
from SigmaꢀAldrich (Saint Louis, MO, USA) and penicillin/streptomycin were purchased from
Invitrogen (San Diego, CA, USA). Fetal bovine serum (FBS), phosphate buffered saline (PBS)
and trypsin were purchased from Biosera (Ringmer, UK). Roswell Park Memorial Institute
(RPMI) 1640 medium was from PAA (Austria). Doxorubicin was purchased from EBEWE
Pharma (Unterach, Austria) and cisplatin from Mylan (Athens, Greece). Dimethyl sulfoxide
(DMSO) and isopropanol were from Merck (Darmstadt, Germany).
The other chemical reagents and solvents utilized for synthesis were purchased from
SigmaꢀAldrich Química S.A. and Merck (Sintra, Portugal). Thin layer chromatography (TLC)
was performed on silica gel 60 F254 precoated on aluminum plates acquired from Merck
(Darmstadt, Germany) and spots were visualized under a UV lamp at 254 nm.
1H, 13C NMR, and DEPT135 data were recorded at room temperature on a Brüker AMX
300 spectrometer at 400 and 100 MHz, respectively. Mass spectra were obtained on a VG
AutoSpec and microTOF (focus) mass spectrometer. A Biotage Initiator Microwave Synthesizer
was used for synthesis. The purity of the final products (>97% purity) was verified by highꢀ
performance liquid chromatography (HPLC) equipped with a UV detector. Chromatograms were
obtained in an HPLC/DAD system, a Jasco instrument (pumps model 880ꢀPU and solvent
mixing model 880ꢀ30, Tokyo, Japan), equipped with a commercially prepacked Nucleosil RPꢀ18
analytical column (250 mm x 4.6 mm, 5 ꢁm, MachereyꢀNagel, Duren, Germany), and UV
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detection (Jasco model 875ꢀUV) at the maximum wavelength of 254 nm. The mobile phase
consisted of a methanol/water or acetonitrile/water (gradient mode, room temperature) at a flow
rate of 1 mL/min. The chromatographic data was processed in a Compaq computer, fitted with
CSW 1.7 software (DataApex, Czech Republic).
2.2. Synthesis
2.2.1. Synthesis of butyl cinnamates (1b, 2b, 3b, and 4b)
The butyl cinnamates were obtained by the following general procedure: The appropriate
cinnamic acid (0.5 g), nꢀbutyl alcohol (15 mmol) and 2 drops of concentrated sulphuric acid
were mixed in a glass vial (5 mL) and sealed. The temperature of the reaction in the MW reactor
was maintained at 20 ºC higher than the boiling point of butanol for 5 min. After the reaction the
mixture was cooled to room temperature, and the crude product was extracted with ethyl acetate.
The organic phase was washed with water, dried and the solvent evaporated. The residue was
purified using silica gel flash chromatography and dichloromethane or ethyl acetate as eluent.
2.2.2. Synthesis of tetradecyl, hexadecyl, and octadecyl HCAsꢀ
The long chain alkyl cinnamates were obtained by a twoꢀstep procedure: Step 1:
Meldrum’s acid and the appropriate alcohol in equimolar quantities were refluxed in dry toluene
(5 mL) for 4 h. After the reaction mixture had cooled the crude product was extracted with
diethyl ether. The ether phases were combined dried and then evaporated. These half esters of
malonic acid were used in the next reaction without further purification. Step 2: The respective
half ester of malonic acid and the appropriate benzaldehyde derivative in equimolar quantities
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were added to cyclohexane (3ꢀ5 mL). Anhydrous pyridine (1 equiv.) and aniline (1.6 equiv.)
were then, added to the mixture and it was refluxed for 6ꢀ10 h. After neutralization (pHꢀ7), the
crude products were extracted with diethyl ether. The organic phases were combined, dried and
the solvent was evaporated. The residues were purified by flash chromatography using petroleum
ether/ ethyl acetate mixtures. The short and long chain alkyl HCAs (1bꢀ1e, 2bꢀ2d, 3bꢀ3e and 4bꢀ
4e) structural assignments were in agreement with the previously reported data12ꢀ13, 27ꢀ29
.
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(E)ꢀButylꢀ3ꢀ(4ꢀhydroxyphenyl)propenoate 1b; H NMR (DMSOꢀd6): δ 0.85 (t, 3H, J =
6.8 Hz, CH3), 1.19 (m, 2H, CH2), 1.47 (m, 2H, CH2), 3.50 (t, 2H, J = 6.6 Hz, CH2), 6.31 (d, 1H,
J = 16.0 Hz, Hꢀα), 6.65 (d, 2H, J = 8.7 Hz, Hꢀ3, 5), 7.50 (m, 3H, Hꢀ2, 6, β), 9.43 (s, 1H, OH);
13C NMR (DMSOꢀd6) δ 14.4 (CH3), 18.9 (CH2), 31.7 (CH2), 64.2 (CH2), 114.7 (Cꢀα), 116.2 (Cꢀ
3, 5), 125.6, 130.7 (Cꢀ2, 6), 145.0 (Cꢀβ), 160.3 (Cꢀ4), 167.1 (CO).
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(E)ꢀTetradecylꢀ3ꢀ(4ꢀhydroxyphenyl)propenoate 1c13, 28; H NMR (CDCl3): δ 0.88 (t, 3H,
J = 6.6 Hz, Hꢀ14'), 1.26 (bs, 22H, Hꢀ3'ꢀ13'), 1.69 (m, 2H, Hꢀ2'), 4.19 (t, 2H, J = 6.6 Hz, Hꢀ1'),
5.81 (bs, 1H, OH), 6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.85 (d, 2H, J = 8.7 Hz, Hꢀ3, 5), 7.43 (d, 2H, J
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= 8.4 Hz, Hꢀ2, 6), 7.63 (d, 1H, J = 15.9 Hz, Hꢀβ). C NMR (CDCl3): δ 14.1 (Cꢀ14'), 22.7 (Cꢀ
13'), 26.0 (Cꢀ3'), 28.7 (Cꢀ2'), 29.27 (Cꢀ4'), 29.33 (Cꢀ11'), 29.6 (Cꢀ5'ꢀ10'), 31.9 (Cꢀ12'), 64.8 (Cꢀ
1'), 115.5 (Cꢀα), 115.9 (Cꢀ3, 5), 127.1 (Cꢀ1), 129.9 (Cꢀ2, 6), 144.5 (Cꢀβ), 157.9 (Cꢀ4), 167.9
(CO); HRESIMS: m/z 383.2554 (M + Na)+; Calcd for C23H36O3Na+: 383.2557.
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(E)ꢀHexadecylꢀ3ꢀ(4ꢀhydroxyphenyl)propenoate 1d28; H NMR (CDCl3): δ 0.88 (t, 3H, J
= 6.6 Hz, Hꢀ16'), 1.1ꢀ1.8 (m, 28H, Hꢀ2'ꢀ15'), 4.19 (t, 2H, J = 6.6 Hz, Hꢀ1'), 5.70 (bs, 1H, OH),
6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.85 (d, 2H, J = 8.6 Hz, Hꢀ3, 5), 7.42 (d, 2H, J = 8.5 Hz, Hꢀ2, 6),
7.62 (d, 1H, J = 15.9 Hz, Hꢀβ).
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(E)-Octadecylꢀ3ꢀ(4ꢀhydroxyphenyl)propenoate 1e28; H NMR (CDC13): 0.88 (t, 3H, J =
6.7 Hz, Hꢀ18'), 1.25ꢀ1.71 (m, 32H, Hꢀ2'ꢀ16'), 4.18 (t, 2H, J = 6.7 Hz, Hꢀ1'), 5.16 (s, 1H, OH),
6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.84 (d, 2H, J = 8.7 Hz, Hꢀ3, 5), 7.44 (d, 2H, J = 8.6 Hz, Hꢀ2, 6),
7.62 (d, 1H, J = 15.9 Hz, Hꢀβ).
(E)ꢀButylꢀ3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)propenoate 2b12; H NMR (CDCl3): δ 0.97 (t,
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3H, J = 7.4 Hz, CH3), 1.39ꢀ1.49 (m, 2H, CH2), 1.65ꢀ1.72 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.20
(t, 2H, J = 6.7 Hz, CH2), 5.97 (s, 1H, OH), 6.29 (d, 1H, J = 15.9 Hz, Hꢀα), 6.92 (d, 1H, J = 8.1
Hz, Hꢀ5), 7.03 (d, 1H, J = 1.8 Hz, Hꢀ2), 7.07 (dd, 1H, J = 8.1, 1.8 Hz, Hꢀ6), 7.61 (d, 1H, J = 15.9
Hz, Hꢀβ); 13C NMR (DMSOꢀd6): δ 14.7 (CH3), 20.1 (CH2), 31.7 (CH2), 56.8 (OCH3), 65.2
(CH2), 110.2 (Cꢀ5), 115.6 (Cꢀ2), 116.5 (Cꢀα), 123.9 (Cꢀ6), 127.9 (Cꢀ1), 145.6 (Cꢀβ), 147.7 (Cꢀ4),
158.1 (Cꢀ3), 168.3 (CO).
(E)ꢀTetradecylꢀ3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)propenoate 2c13; H NMR (CDCl3): δ
1
0.87 (t, 3H, J = 6.6 Hz, Hꢀ14'), 1.25 (bs, 22H, Hꢀ3'ꢀ13'), 1.68 (m, 2H, Hꢀ2'), 3.93 (s, 3H, OCH3),
4.18 (t, 2H, J = 6.7 Hz, Hꢀ1'), 5.87 (bs, 1H, OH), 6.29 (d, 1H, J = 15.9 Hz, Hꢀα), 6.91 (d, 1H, J =
8.1 Hz, Hꢀ5), 7.03 (s, 1H, Hꢀ2), 7.07 (dd, 1H, J = 8.1, 1.8 Hz, Hꢀ6), 7.60 (d, 1H, J = 15.9 Hz, Hꢀ
β).
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(E)ꢀOctadecylꢀ3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)propenoate 2e13; H NMR (CDCl3): δ
0.87 (t, 3H, J = 6.6 Hz, Hꢀ18'), 1.25 (bs, 30H, Hꢀ3'ꢀ17'), 1.68 (m, 2H, Hꢀ2'), 3.93 (s, 3H, OCH3),
4.19 (t, 2H, J = 6.8 Hz, Hꢀ1'), 5.86 (bs, 1H, OH), 6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.90 (d, 1H, J =
8.1 Hz, Hꢀ5), 7.04 (s, 1H, Hꢀ2), 7.07 (dd, 1H, J = 8.1, 1.8 Hz, Hꢀ6), 7.60 (d, 1H, J = 15.9 Hz, Hꢀ
β).
(E)ꢀButylꢀ3ꢀ(3,5ꢀdimethoxyꢀ4ꢀhydroxyphenyl)propenoate 3b27; HNMR (DMSOꢀd6): δ
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0.91 (t, 3H, J = 7.3 Hz, CH3), 1.33ꢀ1.42 (m, 2H, CH2), 1.56ꢀ1.64 (m, 2H, CH2), 3.80 (s, 6H, 2 ×
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OCH3), 4.12 (t, 2H, J = 6.4 Hz, CH2), 6.53 (d, 1H, J = 15.9 Hz, Hꢀα), 7.03 (s, 2H, Hꢀ2, 6), 7.55
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(d, 1H, J = 15.9 Hz, Hꢀβ), 8.94 (s, 1H, OH); C NMR (DMSOꢀd6): δ 13.6 (CH3), 18.8 (CH2),
30.4 (CH2), 56.1 (2 × OCH3), 63.4 (CH2), 106.2 (Cꢀ2, 6), 115.0 (Cꢀα), 124.4 (Cꢀ1), 138.3 (Cꢀ4),
145.3 (Cꢀβ), 148.0 (Cꢀ3, 5), 166.7 (CO); EIꢀMS, 280 (88,M•+).
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(E)ꢀTetradecylꢀ3ꢀ(3,5ꢀdimethoxyꢀ4ꢀhydroxyphenyl)propenoate 3c13; H NMR (CDCl3): δ
0.87 (t, 3H, J = 6.6 Hz, Hꢀ14'), 1.25 (bs, 22H, Hꢀ3'ꢀ13'), 1.69 (m, 2H, Hꢀ2'), 3.91 (s, 6H, 2 ×
OCH3), 4.18 (t, 2H, J = 6.6 Hz, Hꢀ1'), 5.81 (s, 1H, OH), 6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.77 (s,
2H, Hꢀ2, 6), 7.58 (d, 1H, J = 15.9 Hz, Hꢀβ); 13C NMR (CDCl3): δ 14.1 (Cꢀ14'), 22.7 (Cꢀ13'), 26.0
(Cꢀ3'), 28.8 (Cꢀ2'), 29.29 (Cꢀ4'), 29.33 (Cꢀ11'), 29.6 (Cꢀ5'ꢀ10'), 31.9 (Cꢀ12'), 56.3 (2 × OCH3),
64.6 (Cꢀ1'), 105.1 (Cꢀ2, 6), 116.1 (Cꢀα), 126.0 (Cꢀ1), 137.1 (Cꢀ4), 144.8 (Cꢀβ), 147.2 (Cꢀ3, 5),
167.21 (CO); HRESIMS: m/z 443.2762 (M + Na)+; Calcd for C25H40O5Na+: 443.2768.
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(E)ꢀHexadecylꢀ3ꢀ(3,5ꢀdimethoxyꢀ4ꢀhydroxyphenyl)propenoate 3d13; 1H NMR (CDCl3): δ
0.87 (t, 3H, J = 6.6 Hz, Hꢀ16'), 1.25 (bs, 26H, Hꢀ3'ꢀ15'), 1.69 (m, 2H, Hꢀ2'), 3.91 (s, 6H, OCH3 ×
2), 4.18 (t, 2H, J = 6.6 Hz, Hꢀ1'), 5.80 (s, 1H, OH), 6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.77 (s, 2H,
Hꢀ2, 6), 7.58 (d, 1H, J = 15.9 Hz, Hꢀβ); 13C NMR (CDCl3): δ 14.1 (Cꢀ16'), 22.7 (Cꢀ15'), 26.0 (Cꢀ
3'), 28.8 (Cꢀ2'), 29.3 (Cꢀ4'), 29.33 (Cꢀ13'), 29.7 (Cꢀ5'ꢀ12'), 31.9 (Cꢀ14'), 56.3 (OCH3 × 2), 64.6
(Cꢀ1'), 105.1 (Cꢀ2, 6), 116.1 (Cꢀα), 126.0 (Cꢀ1), 137.1 (Cꢀ4), 144.78 (Cꢀβ), 147.2 (Cꢀ3, 5), 167.2
(CO). HRESIMS: m/z 471.3078 (M + Na)+; Calcd for C27H44O5Na+: 471.3081.
1
(E)ꢀOctadecylꢀ3ꢀ(3,5ꢀdimethoxyꢀ4ꢀhydroxyphenyl)propenoate 3e13, 28; H NMR (CDCl3): δ
0.87 (t, 3H, J = 6.6 Hz, Hꢀ18'), 1.25 (bs, 30H, Hꢀ3'ꢀ17'), 1.70 (m, 2H, Hꢀ2'), 3.92 (s, 6H, 2 ×
OCH3), 4.19 (t, 2H, J = 6.6 Hz, Hꢀ1'), 5.78 (s, 1H, OH), 6.30 (d, 1H, J = 15.9 Hz, Hꢀα), 6.77 (s,
2H, Hꢀ2, 6), 7.59 (d, 1H, J = 15.9 Hz, Hꢀβ); 13C NMR (CDCl3): δ 14.1 (Cꢀ18'), 22.7 (Cꢀ17'), 26.0
(Cꢀ3'), 28.8 (Cꢀ2'), 29.3 (Cꢀ4'), 29.33 (Cꢀ15'), 29.7 (Cꢀ5'ꢀ14'), 31.9 (Cꢀ16'), 56.3 (2 × OCH3), 64.6
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(Cꢀ1'), 105.1 (Cꢀ2, 6), 116.1 (Cꢀα), 126.0 (Cꢀ1), 137.1 (Cꢀ4), 144.8 (Cꢀβ), 147.2 (Cꢀ3, 5), 167.24
(CO); HRESIMS: m/z 499.3398 (M + Na)+; Calcd for C29H48O5Na+: 499.3394.
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(E)ꢀButylꢀ3ꢀ(3,4ꢀdihydroxyphenyl)propenoate 4b12; H NMR (DMSOꢀd6): δ 0.90 (t, 3H,
J = 7.5 Hz, CH3), 1.32ꢀ1.42 (m, 2H, CH2), 1.56ꢀ1.65 (m, 2H, CH2), 4.11 (t, 2H, J = 6.6 Hz, CH2),
6.27 (d, 1H, J = 15.9 Hz, Hꢀα), 6.76 (d, 1H, J = 8.1 Hz, Hꢀ5), 7.00 (dd, 1H, J = 8.1, 2.0 Hz, Hꢀ6),
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7.05 (d, 1H, J = 2.0 Hz, Hꢀ2), 7.46 (1H, d, J = 15.9 Hz, Hꢀβ), 9.60 (2H, s, 3, 4ꢀOH); C NMR
(DMSOꢀd6): δ 23.3 (CH3), 28.4 (CH2), 40.0 (CH2), 73.2 (CH2), 123.8 (Cꢀ5), 124.3 (Cꢀ2), 125.5
(Cꢀα), 131.2 (Cꢀ6), 135.2 (Cꢀ1), 154.7 (Cꢀβ), 155.3 (CꢀOH), 158.1 (CꢀOH), 176.5 (CO).
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(E)ꢀTetradecylꢀ3ꢀ(3,4ꢀdihydroxyphenyl)propenoate 4c13, 29; H NMR (CDCl3): δ 0.88 (t,
3H, J = 6.6 Hz, Hꢀ14'), 1.26 (bs, 22H, Hꢀ3'ꢀ13'), 1.67 (m, 2H, Hꢀ2'), 4.18 (t, 2H, J = 6.7 Hz, Hꢀ
1'), 5.65 (s, 1H, OH), 5.66 (s, 1H, OH), 6.27 (d, 1H, J = 15.9 Hz, Hꢀα), 6.87 (d, 1H, J = 8.1 Hz,
Hꢀ5), 7.02 (dd, 1H, J = 8.1, 1.9 Hz, Hꢀ6), 7.09 (d, 1H, J = 1.8 Hz, Hꢀ2), 7.57 (d, 1H, J = 15.9 Hz,
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Hꢀβ); C NMR (CDCl3): δ 14.0 (Cꢀ14'), 22.6 (Cꢀ13'), 25.9 (Cꢀ3'), 28.4 (Cꢀ2'), 29.2 (Cꢀ4', 11'),
29.6 (Cꢀ5'ꢀ10'), 31.8 (Cꢀ12'), 64.7 (Cꢀ1'), 114.3 (Cꢀα), 115.4 (Cꢀ5), 115.9 (Cꢀ2), 122.3 (Cꢀ6),
127.7 (Cꢀ1), 143.6 (Cꢀ3), 144.4 (Cꢀβ), 146.0 (Cꢀ4), 167.5 (CO); HRESIMS: m/z 399.2508 (M +
Na)+; Calcd for C23H36O4Na+: 399.2506.
(E)ꢀHexadecylꢀ3ꢀ(3,4ꢀdihydroxyphenyl)propenoate 4d13, 29; 1H NMR (CDCl3): δ 0.87 (t, 3H,
J = 6.6 Hz, Hꢀ16'), 1.25 (bs, 26H, Hꢀ3'ꢀ15'), 1.67 (m, 2H, Hꢀ2'), 4.18 (t, 2H, J = 6.7 Hz, Hꢀ1'),
5.69 (bs, 2H, 2 × OH), 6.27 (d, 1H, J = 15.9 Hz, Hꢀα), 6.87 (d, 1H, J = 8.1 Hz, Hꢀ5), 7.02 (dd,
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1H, J = 8.1, 1.8 Hz, Hꢀ6), 7.09 (d, 1H, J = 1.8 Hz, Hꢀ2), 7.57 (d, 1H, J = 15.9 Hz, Hꢀβ); C
NMR (DMSOꢀd6): δ 13.4 (Cꢀ16'), 21.6 (Cꢀ15'), 24.9 (Cꢀ3'), 27.7 (Cꢀ2'), 28.1 (Cꢀ4', 13'), 28.5 (Cꢀ
5'ꢀ12'), 30.8 (Cꢀ14'), 63.2 (Cꢀ1'), 113.5 (Cꢀα), 114.3 (Cꢀ5), 115.2 (Cꢀ2), 120.8 (Cꢀ6), 125.0 (Cꢀ1),
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144.5 (Cꢀ3), 145.1 (Cꢀβ), 147.9 (Cꢀ4), 166.12 (CO); HRESIMS: m/z 427.2815 (M + Na)+; Calcd
for C25H40O4Na+: 427.2819.
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(E)ꢀOctadecylꢀ3ꢀ(3,4ꢀdihydroxyphenyl)propenoate 4e13, 29; H NMR (DMSOꢀd6): δ 0.84
(t, 3H, J = 6.6 Hz, Hꢀ18'), 1.22 (bs, 30H, Hꢀ3'ꢀ17'), 1.61 (m, 2H, Hꢀ2'), 4.09 (t, 2H, J = 6.5 Hz,
Hꢀ1'), 6.25 (d, 1H, J = 15.9 Hz, Hꢀα), 6.75 (d, 1H, J = 8.1 Hz, Hꢀ5), 7.0 (dd, 1H, J = 8.1, 1.8 Hz,
Hꢀ6), 7.04 (d, 1H, J = 1.8 Hz, Hꢀ2), 7.45 (d, 1H, J = 15.9 Hz, Hꢀβ), 9.17 (s, 1H, OH), 9.61 (s,
1H, OH); 13C NMR (DMSOꢀd6): δ 13.4 (Cꢀ18'), 21.6 (Cꢀ17'), 24.9 (Cꢀ3'), 27.8 (Cꢀ2'), 28.2 (Cꢀ4',
15'), 28.5 (Cꢀ5'ꢀ14'), 30.8 (Cꢀ16'), 63.2 (Cꢀ1'), 113.5 (Cꢀα), 114.2 (Cꢀ5), 115.2 (Cꢀ2), 120.8 (Cꢀ
6), 125.0 (Cꢀ1), 144.5 (Cꢀβ), 145.1 (Cꢀ3), 147.9 (Cꢀ4), 166.1 (CO); HRESIMS: m/z 455.3135 (M
+ Na)+; Calcd for C27H44O4Na+: 455.3132.
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HLꢀ60 (human promyelocytic leukemia), MCFꢀ7 (human breast adenocarcinoma),
MOLTꢀ4 (human acute lymphoblastic leukemia) and NIH/3T3 (mouse embryonic fibroblast)
cells were obtained from the National Cell Bank of Iran at Pasteur Institute, Tehran, Iran. MESꢀ
SA/DX5 (human sarcoma cells multiꢀresistant) cells were purchased from SigmaꢀAldrich. All
cells were grown in monolayer cultures and maintained at 37 °C in humidified air containing 5%
CO2 except for HLꢀ60 and MOLTꢀ4 cells that were grown in suspension. Cells were maintained
in RPMI 1640 containing 10% FBS, and 100 units/mL of penicillinꢀG and 100 µg/mL of
streptomycin. Only for HLꢀ60 cells, RPMI 1640 containing FBS at the concentration of 20% was
used.
2.4. Cytotoxicity assay
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30ꢀ31
The cytotoxic activities of the compounds were evaluated by MTT reduction assay
.
One hundred ꢁL of cell suspension having a density of 50,000 cells/mL were seeded into each
well of a 96ꢀwell microplate and incubated overnight at 37 °C. Three to four concentrations of
compounds, ranging from 50 nM to 100 µM, determined by the potency of the compound, were
added to the wells in triplicate and the plates were incubated for another 72 h at 37 °C. All
compounds were dissolved in DMSO, except for compounds 2e, 3e and 4e, which were
solubilized in isopropanol. The final concentrations of DMSO or isopropanol in the wells did not
exceed 0.25%. This concentration of the solvents did not affect the viability of the cell lines
tested in this study. At the end of the incubation, the growth medium was replaced with 0.5
mg/mL of MTT dissolved in fresh RPMI 1640 without phenol red. After an additional 4 h of
incubation at 37 °C, the formazan crystals were dissolved in 200 µL DMSO and the optical
absorbance was measured at 570 nm (applying a background correction at 655 nm) using a Bioꢀ
Rad microplate reader (Model 680). The cell viability expressed as percent inhibition for each
concentration of the compound was calculated with reference to the control and the IC50 values
were calculated with the CurveExpert software version 1.34 for Windows. Each experiment was
repeated 3ꢀ4 times.
2.5. Cell cycle analysis
The analysis of cells in different phases of the cell cycle and the subꢀG1 phase were
monitored using propidium iodide (PI)ꢀbased assessment of cell cycle by flow cytometry.
MOLTꢀ4 cells were seeded in 12ꢀwell plates (1×106 cells /well) and treated with different
concentrations of 1c (30, 100 and 300 nM), 4c (300, 1000 and 3000 nM) and 1d (100, 300 and
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1000 nM) for 24 and 48 h. At the end of the incubation, the cells in each well were collected and
washed with PBS. Then, they were fixed with 70% ethanol overnight at ꢀ20 °C. After 24 h, the
fixed cells were washed with PBS and subsequently stained with DNA staining solution (PI 20
ꢁg/mL and RNase 200 ꢁg/mL) at room temperature for 30 min in the dark. Twenty thousand
cells of each sample were analyzed using a FACS Calibur flow cytometer (BD Biosciences,
USA) and the percentage of the cells in subꢀG1, G0/G1, S, and G2/M phases were calculated
using CellQuest (BD, USA) software.
2.6. Preparation of cell lysates for immunoblotting
MOLTꢀ4 cells were seeded at a density of 250,000 cells/mL in 12ꢀwell plates and were
incubated at 37 °C for 24 h. The alkyl ester derivatives of HCAs were introduced into the cell
cultures and they were further incubated for 24 h. The cells were harvested and lysed for 30 min
in iceꢀcold RIPA lysis buffer (TrisꢀHCl 250 mM, NaCl 750 mM, NPꢀ40 5%, EDTA 5 mM,
sodium deoxycholate 2.5 %, SDS 0.5%) with frequent vortex mixing. PMSF and a protease
inhibitor cocktail (Roche) were added to the extraction buffer in order to inhibit the protease
enzymes. The lysates were centrifuged at 15,000 g for 20 min at 4 °C. Supernatants were isolated
in fresh tubes and stored at ꢀ80 °C before use. Protein contents of the cell extracts were measured
by a BCA protein assay kit (QuantiꢀPro ®BCA, Sigma–Aldrich, St. Louis, USA) using bovine
serum albumin as the protein standard.
2.7. Immunoblotting of caspase-3
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The activation of the caspaseꢀ3 enzyme was detected by immunoblotting. Twenty ꢁg of
total proteins were loaded on 12% SDSꢀpolyacrylamide gels and electrophoresis was performed
at a constant voltage. The proteins were then electrotransferred onto poly(vinylidene difluoride)
(PVDF) membranes and nonꢀspecific binding sites were blocked with 5% fatꢀfree milk dissolved
in Trisꢀbuffered saline containing Tween 20 (TBSꢀT, 150 mM NaCl and 50 mM Tris-HCl pH
7.4, 0.1% Tween 20) at room temperature for 40 min. Proteins were then detected by specific
primary antibodies, rabbit monoclonal antiꢀcaspaseꢀ3 (dilution 1:400, clone 8G10, Cell
o
Signaling, USA) and antiꢀactin (dilution 1:2000, SigmaꢀAldrich, USA) overnight at 4 C.
Membranes were washed three times in TBST buffer and then incubated with secondary
antibody (goat antiꢀrabbit horseradish peroxidase conjugated IgG, dilution 1:1000, Santa Cruz
Biotechnology) for 1 h at room temperature. After 3 further washes in TBST buffer,
immunoreactive bands were visualized using enhanced chemiluminescence detection substrate
(Thermo Fisher Scientific, Waltham, MA). Images were acquired with an image acquiring
system (G: BOX Chemi, Syngene, Cambridge, U.K.) with GeneSnap software. The density of
the protein bands was quantified by GeneTools software (Syngene), followed by normalization
to the βꢀactin as a loading control. All experiments were replicated at least three times.
2.8. Target prediction for anticancer activity of HCAs
The method that we applied for exploring the plausible molecular targets responsible for
the antiꢀcancer potential of HCAs, consisted of the following steps32:
1. Extraction of molecular targets of interest for HCAs from Drug Bank, ChemMapper 33,
DINIES34, SuperPred35 and BindingDB36,
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2. Gathering targets based on highest structural and physiochemical similarities of their
ligands with HCA derivatives.
3. Selecting molecular targets involved in the mechanism of antiꢀcancer action based on
CancerResource33, 37
.
4. Text mining with the purpose of investigation of the exact molecular target (among
selected targets in steps 2 and 3) involved in the anticancer activity of the test compounds.
2.9. Molecular docking
A molecular docking study of alkyl HCAs was conducted in order to further explore the
mechanisms of cytotoxicity as inhibitors of CAꢀIX and evaluation of their structural requirement
for the binding interaction with this potential target. The Xꢀray crystal data of threeꢀdimensional
structure of human CAꢀIX in complex with 5ꢀ(1ꢀ(4ꢀmethylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀ
yl)thiopheneꢀ2ꢀsulfonamide (MPTTS, PDB ID: 5FL6) was acquired from the Protein Data Bank
(http://www.rcsb.org). The crystal structure of the protein was further refined by removing the
coꢀcrystallized ligand and water molecules and the protein was converted to pdbqt format using
Autodock Tools (1.5.4). The active site was derived from the Xꢀray crystal structure data. The
3D coordinates of studied ligands were generated by using Chem3D Ultra 8 and energetically
minimized by the conjugate gradient algorithm (gradient convergence value of 0.1 kcalmolꢀ1 was
used) using the HyperChem software. The Gastiger charges (empirical atomic partial charges)
were calculated by HyperChem software and the torsional degrees of freedom were assigned on
the PDB files. The pdbqt files were generated for each ligand separately using ADT 1.5.4. The
grid maps of the active site for enzyme were calculated by AutoꢀGrid with three dimensions of
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60 × 60 × 60 (x, y, z), grid spacing of 0.375 Ǻ and the cubic grids were centered on the binding
site of MPTTS as the cognate ligand.
Docking computations were performed using the Lamarckian genetic algorithm (LGA).
For this purpose, maximum numbers of evaluations were set to 2,500,000, the numbers of GA
runs were 50 and the maximum numbers of generations were set as 27,000 and all other options
were set as default. Resulting conformers differing by less than 1.0 Å in positional rootꢀmeanꢀ
square deviation (RMSD) were clustered together and represented by the result with the topꢀ
ranked free energy of binding interaction.
2.10. Statistical analysis
The data were expressed as the mean ± S.D. or S.E.M. and were analyzed by oneꢀway
ANOVA using the SPSS software version 14.0 for Windows with LSD post hoc test, when
applicable.
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3. Results
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3.1. Cytotoxicity of alkyl hydroxycinnamates against cancer cells
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Cytotoxic activities of hydroxycinnamic acids (HCAs) including 1a, 2a, 3a, 4a and their
medium and long chain alkyl ester derivatives (butyl "b", tetradecyl "c", hexadecyl "d" and
octadecyl "e" esters, Table 1) were evaluated against 4 cancer cell lines (HLꢀ60, MCFꢀ7, MOLTꢀ
4 and MESꢀSA/DX5) and on the nonꢀcancer NIH/3T3 cells using MTT reduction assay. The cell
viability assay results were compared with cisplatin and doxorubicin, which were used as
standard anticancer agents (Table 2).
3.2. Effect of alkyl hydroxycinnamates on cancer cell cycle
In order to explore the plausible mechanism of cytotoxicity of the most potent HCA
esters, the cellꢀcycle distribution of MOLTꢀ4 cells incubated with 1c, 1d and 4c were evaluated
using propidium iodide (PI)ꢀbased assessment of cell cycle by flow cytometry after 24 and 48 h.
Figures 1 and 2 illustrate the distribution of the cells in the G0/G1, S and G2/M phases of cell
cycle. The results indicated that the number of apoptotic cells in subꢀG1 phase was significantly
increased after treatment with different concentrations of test compounds after 24 and 48 h. The
cell cycle profile of MOLTꢀ4 cells treated with increasing concentrations of 1c at 30, 100 and
300 nM after 24 h indicated that the percentage of cells in subꢀG1 phase showed a doseꢀ
dependent increase compared to control cells. Similarly, exposure of cells to 1c for 48 h resulted
in a concentrationꢀdependent increase in the percentage of apoptotic cells. Similar patterns of
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doseꢀ and timeꢀdependent responses were observed in a subꢀG1 fraction of cells treated with 1d
and 4c.
3.3. Effect of alkyl hydroxycinnamates on the activation of caspase-3
Three of the most effective alkyl hydroxycinnamates were incubated with MOLTꢀ4
cancer cells for 24 h and the total protein was extracted and analyzed by immunoblotting for
determination of caspaseꢀ3 activation, which was verified by the cleavage of caspaseꢀ3 and the
appearance of a smaller 17 KD fragment. As shown in Figure 3, exposure of MOLTꢀ4 cells to 1c
(0.5 µm), 4c (2.5 µm) and 1d (2.5 µm) resulted in the cleavage of caspaseꢀ3 and the appearance
of the cleaved caspaseꢀ3 peptide (17 KD), accompanied by reduction of proꢀcaspaseꢀ3 (35 KD)
protein.
3.4. Carbonic anhydrase IX as the potential molecular target for alkyl
hydroxycinnamates
All linear alkyl ester derivatives of HCAs were modeled with Hyperchem 7. Then targets
were collected from target prediction databases, through a process schematically shown in Figure
4. We used a textꢀmining method and examined experimental applications to cancer studies
based on the existing literature. As the final finding, carbonic anhydrase isoforms were identified
as putative targets for HCA derivatives.
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3.5. Molecular modeling study: interaction of HCAs in complex with carbonic anhydrase
IX
The crystal structure of CAꢀIX (PDB code 5FL6) in complex with MPTTS was used for
molecular docking study. For validation of the docking procedure, RMSD (root mean square) of
experimental conformer was calculated by reꢀdocking of MPTTS in CAꢀIX’s active sites. The
topꢀranked conformer of MPTTS into the binding site of CAꢀIX superimposed well over the Xꢀ
ray coordinates of experimental one with the RMSD value of 1.03Ǻ (Figure 5).
Docking results and comparative binding interactions of 1a and 1c, into CAꢀIX complex,
are depicted in Figure 6B and 6A, respectively. Based on the docking results, the corresponding
binding free energies of 1a and 1c into CAꢀIX were ꢀ5.35 and ꢀ5.80 Kcal/mol, respectively.
Compound 1a showed 3 hydrogen binding interactions with Asn66, His68 and Thr200 of CAꢀ
IX. Esterification of the acidic moiety of compound 1c resulted in favorable hydrogen bonding
interactions with Asn66, His68, Gln71 and Thr200 of CAꢀIX catalytic site, which was observed
in all CAꢀIX inhibitors. Moreover, 1c was also positioned in a close interaction (less than 3Å)
with Leu199 and Thr201, which are conserved residues in the catalytic site of all carbonic
anhydrases.
Comparing the binding interaction of 1c with its methoxylated counterparts 2c (∆G= ꢀ
5.21 Kcal/mol) and 3c (∆G= ꢀ4.77 Kcal/mol), it could be concluded that introduction of methoxy
substitution into the phenyl ring might distort the binding orientation of ester pendant into CAꢀIX
catalytic site and hinder the hydrogen bonding interaction with key residues such as Asn66,
His68, and Gln71. Compound 2c takes part in hydrogen bonding interactions with Thr200 and
Gln92, while 3c only interacted with Thr200. Dislocation of tetradecyl ester side chain was
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demonstrated in the case of 2c and 3c (Figure 6C and 6D). Moreover, addition of hydroxyl
moieties into the phenyl ring of HCAs, also had negative effects on the binding orientation of the
compound in the CAꢀIX active site; 4c (containing two hydroxyl groups at R2 and R3 position),
is devoid of any Hꢀbond interactions with Asn66, His68, and Gln71, and is only involved in Hꢀ
bond interaction with Thr200, Thr201, and Gln92 (Figure 6E).
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Discussion
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Cytotoxic activities of hydroxycinnamic acids (HCAs) and their medium and long chain
alkyl ester derivatives were evaluated against 4 human and one nonꢀcancer cell line using MTT
reduction assay. The effects of the most potent compounds were examined on cell cycle and
induction of apoptosis.
The MTT assay results showed that among different tested HCA derivatives, tetradecyl
(1c) and hexadecyl (1d) esters of pꢀCA were the most potent compounds against MOLTꢀ4 cells
with IC50 values of 0.123 and 0.317 µM, respectively. Interestingly, these derivatives showed
IC50 values (0.317 and 0.414 µM, respectively) against multidrug resistant cell line MESꢀ
SA/DX5 that were much lower than those of doxorubicin (1.9 µM) and cisplatin (2.9 µM),
standard chemotherapeutic agents (Table 2). Doxorubicin and cisplatin are commonly used
chemotherapeutic agents for treatment of several solid tumors and leukemias and have been
utilized by us and other authors as standard cytotoxic agents. All pꢀCA derivatives were inactive
against HLꢀ60 and MCFꢀ7 cells, which shows that these compounds may specifically affect
certain cancer cell lines and not others. On the basis of the obtained cytotoxic data, the following
structureꢀactivity relationships could be deduced for the synthesized compounds of this study:
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Influence of aromatic substitution pattern on cytotoxic activity
In general, pꢀCA ester derivatives (1bꢀe) showed higher cytotoxic effects against MOLTꢀ
4 and MESꢀSA/DX5 cells, followed by caffeate (4bꢀe) and ferulate esters (2bꢀe), while sinapate
esters (3bꢀe) showed the lowest anticancer effects against these cells.
The number of hydroxyl functions and the nature of mꢀsubstituents on the aromatic ring
seems to influence the cytotoxic activity of the test compounds. In this case, the presence of a
single 4ꢀOH function was the most favorable substitution pattern (as in pꢀCA derivatives),
followed by the presence of a 3,4ꢀdihydroxy (catechol) moiety (as in CA derivatives).
The results obtained in HLꢀ60, MCFꢀ7 cells do not allow a straightforward evaluation of
the influence of the aromatic substitution pattern due to lower activity levels. However, CA (4a)
and its ester derivatives (4bꢀe), bearing a catechol moiety, were the only group of compounds
that showed a moderate activity against HLꢀ60 cells.
Effect of carboxylic acid esterification on cytotoxicity
Esterification of the carboxylic acid function generally improved the cytotoxic activity of
all HCA derivatives against MOLTꢀ4 and MESꢀSA/DX5 cells. For instance, pꢀCA (1a) was
inactive in these cellular models (IC50 > 100 µM), while the ester derivatives 1bꢀe showed a
significant improvement in cytotoxic effect against these cell lines (Table 2).
Length of alkyl chain and physicochemical properties
With the exception of butyl caffeate (4b) in HLꢀ60 cells, tetradecyl esters were the most
active derivatives in all cancer cellular models. The existence of an optimal chain length points
towards specific structural and physicochemical requirements that allow effective transport
and/or diffusion across the cell membrane and interaction with intracellular targets. Among
them, lipophilicity (as determined by the partition coefficient LogP, Table 1), stands out as an
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important physicochemical descriptor of bioactivity. As shown in Table 1, the naturallyꢀ
occurring carboxylic acids (1a, 2a, 3a and 4a) are the most hydrophilic compounds in this series
(1.36 < Log P < 1.87), which may hinder their cellular uptake and intracellular accumulation,
resulting in the poor cytotoxic activity observed. Accordingly, esterification of the carboxylic
acid moiety improved the derivatives’ lipophilicity, as could be observed in increased LogP
values. However, chain lengths longer than the optimal size may increase the interaction with the
phospholipids in the membrane and decrease the solubility in the intracellular compartments,
which may justify the observed decrease in cytotoxicity in the current cellular models. This
observation was in accordance with previous reports regarding the antioxidant activity and
membrane affinity of FA ester derivatives38ꢀ39. Similar observation of a biphasic correlation
29
between lipophilicity and anticancer activity of caffeate esters has also been reported . In the
present case, the tetradecyl ester moiety may provide optimal membrane anchoring, orienting the
HCA moiety towards its intracellular targets and thus providing maximum activity.
Other authors have screened the effect of different alkyl esters ranging from C3 to C22 of
CA and FA against different cancer cell lines. They identified C12 and C16 caffeate esters, and
also C8 and C12 ferulate esters as the optimum alkyl chain length against MCFꢀ7, HCTꢀ116
(colon adenocarcinoma), CCIꢀH460 (lung adenocarcinoma), SFꢀ26 (CNS tumor) and AGS
(gastric adenocarcinoma) cells with an IC50 range of 2.5 to 18.7 µg/ml. The C14 ester was not
included in this study and the authors did not find any preferential effect against a certain type of
cancer 21.
It has been previously shown that hexyl caffeate and hexyl ferulate exhibit cytotoxicity
against certain types of cancer including MCFꢀ7 cells18. Uwai and colleagues have shown that
C11ꢀC14 caffeate esters have the highest cytotoxicity against Raw264.7 macrophage cells, with
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C11 ester being the most potent in inhibition of NO in these cells29. The biphasic correlation
between ClogP, the index of lipophilicity, and cytotoxicity indicated that the cytotoxicity of
caffeate esters was directly correlated with lipophilicity, having an optimum range29.
The effect of these compounds against cancer cells was also compared with nonꢀcancer
cells. Derivatives 1c and 1d, with remarkable submicromolar potency against MOLTꢀ4 and
MESꢀSA/DX5 cells, were almost devoid of cytotoxicity against nonꢀmalignant NIH/3T3 cells.
Moreover, meta substitution with hydroxyl group could affect the selectivity of compounds
against cancer cells; Although the presence of a 3,4ꢀdihydroxy (catechol) group in the aromatic
ring broadened the cytotoxic activity spectrum to HLꢀ60 cells (4bꢀe, Table 2), it also induced
toxic effects against nonꢀcancer cells, which raises selectivity issues.
We also examined the effect of the most active compounds on cell cycle; At 300 nM the
percentage of subꢀG1 cells exposed to 1c for 24 and 48 h (27.0 and 54.7%, respectively) were
higher than the percentages related to compounds 4c (4.9 and 6.5%, respectively) and 1d (17.7
and 34.7%, respectively). Therefore based on these findings, induction of apoptosis could be
considered as a conceivable mechanism of anticancer activity of these HCA esters.
On the other hand, caspaseꢀ3 assessment results were in accordance with cell cycle
analysis that evidenced the induction of apoptosis by compounds 1c, 1d, and 4c. Caspaseꢀ3 is an
executioner caspase and its activation was one of the signs of apoptosis induction in cells25, 40ꢀ42
.
It has been shown that FA was capable of apoptosis induction in prostate cancer cells PCꢀ
3 and LNCaP24. However, very high doses (300ꢀ500 µM) were tested in this study, which cannot
be compared to our findings on FA and its esters. In a recent study, methylꢀcaffeate showed
induction of apoptosis in MCFꢀ7 cells, involving the caspaseꢀ3 and poly (ADPꢀribose)
polymeraseꢀ1 (PARP1) activation25.
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Various studies have shown that inhibition of matrix metalloproteinase (MMP) and
vascular endothelial growth factor (VEGF)15, 43ꢀ44; inhibition of NFꢀkB, Bclꢀ2; activation of Bax,
Bak and JNK19, 45; cell cycle arrest by inhibition of cyclin D1, E, B1 and induction of p16, p21,
p1746ꢀ48 are the common mechanisms involved in the antiꢀproliferative and apoptosis inducing
effects of HCA derivatives.
In our target prediction study, carbonic anhydrase isoforms were identified as putative
targets for HCA derivatives, which was also corroborated by previous literature reports49.
Carbonic anhydrases are abundant zinc metalloenzymes that catalyze the reversible
interconversion of CO2 and HCO3ꢀ 50. Several different carbonic anhydrase isoforms have been
identified, among which CAꢀIX, a membraneꢀassociated glycoprotein, may have an important
role in cancer 51 and was overexpressed as a result of hypoxia in many cancer cells 52ꢀ53 through
54
the activation of hypoxia inducible factorꢀ1α (HIFꢀ1α) . CAꢀIX was considered to be a good
candidate for cancer targeted therapy, as its expression was very limited in normal tissues, while
it was highly expressed in various cancers55. Different studies have shown that the cytotoxic
activity of phenolic acid esters including CA, FA, pꢀCA as well as other phenolic compounds is
possibly due to the inhibition of carbonic anhydrase49, 56ꢀ57
.
From the findings of the docking study, it might be concluded that esterification improves
the binding pose of HCAs into the CAꢀIX binding site, however, this improvement does not
seem to be sufficient to explain the dramatic effect of esterification on the cytotoxicity as it was
observed in the case of 1c compared to 1a against MOLTꢀ4 and MESꢀSA/DX5 cells. The
superior cytotoxic potential of tetradecyl and other ester derivatives over the parental compounds
could also be largely attributed to the improved physicochemical properties by esterification,
increased lipophilicity and cell membrane permeability of HCAs. Alternatively, it could be
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assumed that these compounds have additional mechanisms of action, through which, the
considerable increase of activity caused by esterification could be explained.
Regarding the pharmacokinetic properties of HCAs, it has been shown that free pꢀCA has
high bioavailability after oral intake but data for its conjugated forms is limited58. It was further
observed that pꢀCA as a metabolite from its ester conjugates was detected at lower
concentrations in plasma indicating its different metabolic pathway compared to the free pꢀCA58.
The free forms of HCA are easily absorbed in the upper gastrointestinal tract59. For example,
detection of 24% of pꢀCA 24h after its administration has shown less metabolism and faster
uptake of this free HCA compared to cinnamic acid60. An extensive review of the
pharmacokinetics and biological activities of HCA discusses the high rate of absorption of free
pꢀCA and FA compared to CA from concluded from findings of various studies6.
In an in vivo study, p-CA and gallic acid were orally administered to rats and their blood
concentrations and pharmacokinetic profiles were measured61. It was found that pꢀCA was
quickly absorbed from the gastrointestinal tract and 10 minutes after oral administration, the
portal vein serum concentration of intact pꢀCA was 165.7 µM. Interestingly, this study reported
that the bioavailability of pꢀCA was 70 times higher than gallic acid61. On the other hand,
another study in Beagle dogs has shown that maximum plasma concentration of FA is 0.977
µM62.
Since, long chain alkyl esters of HCAs are way more lipophilic compared to the parental
compounds, it would be reasonable to presume that these compounds are absorbed into the
lymphatic circulation, due to their lipophilic nature63. On the other hand, since the lymphatic
system transports lipophilic compounds, it can be suggested that incorporation of the HCAs into
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nanostructured lipid carriers may further increase their accumulation in the lymphatic system
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.
In conclusion, the findings of this study demonstrate that long chain alkyl ester
derivatives of pꢀCA (compounds 1c and 1d) show remarkable submicromolar potency against
certain cancer cells and are selective towards these cells over nonꢀmalignant cells. Therefore,
they represent promising scaffolds as anticancer agents that selectively induce apoptosis in
cancer cells.
Author contributions
J.C.J.M.D.S.M: Performed experiments, wrote the paper; N.E.: Designed the study, wrote
the paper; S.P.K.: Performed experiments; M.K: Performed experiments; Z.K.: Performed
experiments; H.H.M.: Performed experiments; R.M.: Designed the study, wrote the paper; N.E.:
Designed the study, wrote the paper; M.N.: Performed experiments; J.A.S.C.: Performed
experiments; T.S.: Performed experiments, wrote the paper; L.S.: Designed the study, wrote the
paper; F.B. and O.F.: Designed the study, supervised the project, wrote the paper.
Conflict of interest statement
The authors hereby declare that they have no conflict of interest.
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Acknowledgements
The financial supports from the Vice Chancellor for Research, Shiraz University of
Medical Sciences (Grant numbers 5360 and 94ꢀ01ꢀ12ꢀ11136); University of Aveiro and Goa
University for laboratory facilities; Fundação para a Ciência e a Tecnologia (FCT, Portugal),
European Union, QREN, FEDER and COMPETE for funding the QOPNA and CIQUP research
units (projects PEstꢀC/QUI/UI0062/2013 and UID/QUI/00081/2015, POCIꢀ01ꢀ0145ꢀFEDERꢀ
006980) and research grants for JCJMDS Menezes from QOPNA are gratefully acknowledged.
613
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