Biological activity and molecular docking studies of curcumin-related α,β-unsaturated carbonyl-based synthetic compounds as anticancer agents and mushroom tyrosinase inhibitors

Article abstract of DOI:10.1021/jf501145b

Hyperpigmentation in human skin and enzymatic browning in fruits, which are caused by tyrosinase enzyme, are not desirable. Investigations in the discovery of tyrosinase enzyme inhibitors and search for improved cytotoxic agents continue to be an important line in drug discovery and development. In present work, a new series of 30 compounds bearing α,β-unsaturated carbonyl moiety was designed and synthesized following curcumin as model. All compounds were evaluated for their effects on human cancer cell lines and mushroom tyrosinase enzyme. Moreover, the structure-activity relationships of these compounds are also explained. Molecular modeling studies of these new compounds were carried out to explore interactions with tyrosinase enzyme. Synthetic curcumin-like compounds (2a-b) were identified as potent anticancer agents with 81-82% cytotoxicity. Five of these newly synthesized compounds (1a, 8a-b, 10a-b) emerged to be the potent inhibitors of mushroom tyrosinase, providing further insight into designing compounds useful in fields of food, health, and agriculture.

Full text of DOI:10.1021/jf501145b

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Article
Biological Activity and Molecular Docking Studies of Curcumin
Related #, #- Unsaturated Carbonyl Based Synthetic Compounds
as Anticancer Agents and Mushroom Tyrosinase Inhibitors
Syed Nasir Abbas Bukhari, Ibrahim Jantan, Oya Unsal
Tan, Muhammad Sher, Naeem ul Hassan, and Hua-Li Qin
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2014
Downloaded from http://pubs.acs.org on June 8, 2014
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Biological Activity and Molecular Docking Studies of Curcumin Related α, βꢀ
Unsaturated Carbonyl Based Synthetic Compounds as Anticancer Agents and Mushroom
Tyrosinase Inhibitors
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Syed Nasir Abbas Bukhari1*, Ibrahim Jantan1*, Oya Unsal Tan2, Muhammad Sher3, M.Naeemꢀ
ulꢀHassan3, HuaꢀLi Qin4
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1. Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan
Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia
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2. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara
06100, Turkey
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3. Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan.
4. Department of Pharmaceutical Engineering, School of chemical engineering, Wuhan University
of Technology, 205 Luoshi Road, Wuhan,430070, P.R.China.
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ABSTRACT
Hyperpigmentation in human skin and enzymatic browning in fruits which are caused by
tyrosinase enzyme are not desirable. Investigations in the discovery of tyrosinase enzyme
inhibitors and search for improved cytotoxic agents continue to be an important line in the drug
discovery and development. In present work, a new series of thirty compounds bearing α, βꢀ
unsaturated carbonyl moiety was designed and synthesized following curcumin as model. All
compounds were evaluated for their effects on human cancer cell lines and mushroom tyrosinase
enzyme. Moreover, the structureꢀactivity relationships of these compounds also explained.
Molecular modeling studies of these new compounds were carried out to explore interactions
with tyrosinase enzyme. Synthetic curcuminꢀlike compounds (2aꢀb) were identified as potent
anticancer agents with 81ꢀ82% cytotoxicity. Five of these newly synthesized compounds (1a, 8aꢀ
b, 10aꢀb), emerged to be the potent inhibitors of mushroom tyrosinase providing further insight
into designing compounds useful in fields of food, health and agriculture.
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KEYWORDS: Melanogenesis, Curcuma Longa, Cytotoxicity, Antibrowning, Synthesis
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INTRODUCTION
Throughout history, natural products have offered a rich source of compounds that have
widespread applications in the fields of medicine, food and agriculture1. Structural modification
of natural compounds or synthesis of new compounds, based upon designs following a natural
compound scaffolding have provided us a heaps of important new commercialized drugs within
the medicines, agriculture and food spheres2, 3. An important line in the discovery of modern
anticancer drugs is exploration for enhanced cytotoxic agents principally with safety perspectives
in view4. In line with the safety issues, plant derived natural compounds have always fascinated
scientists worldwide on grounds of being biocompatible, natural compounds are deemed as
potentially safe and effective skin lightening and anticancer agents5.
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Curcumin, flavonoids and numerous dietary chemoꢀpreventive compounds have shown
potential in treating cancer or dermatological disorders6-8. Behind cardiovascular diseases, cancer
is the second principal cause of death in the world. For treating a wide range and types of cancer,
plentiful effort have been started for decades but it is only recently that chemoprevention has
attracted due share of attention. Combinatorial chemistry along with highꢀthroughput screening
technique directed towards molecular targets and cancer cells are wellꢀknown methods for
primary anticancer drug discovery9-11. Curcumin is a plantꢀderived polyphenol obtained from
curcuma longa; Powdered rhizome of this plant, turmeric, has been widely used for color
impartment and flavoring to foods and by tradition for the treatment of various inflammatory
conditions and other diseases12-14. Curcumin (diferuloylmethane) possesses both anticancer and
antityrosinase properties15-17
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Tyrosinase, a ubiquitous dinuclear copper enzyme, catalyzes the hydroxylation of
phenols to catechols and the subsequent oxidation of catechols to quinones. This series of events
take place by activation of dioxygen in the form of a sideꢀonꢀbonded peroxide dicopper (II)
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species, crystallographically18 and spectroscopically identified19, 20. Melanin is a pigmented
heteropolymer synthesized by differentiated melanocytes in specialized cellular organelles
termed melanosomes. The entire process leading to the formation of melanin is called
melanogenesis and is initiated with the first step of tyrosine oxidation to dopaquinone catalyzed
by tyrosinase.
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Melanin plays an important role in protecting human skin, from the harmful effects of
UV radiation, from the sunlight. This is achieved by transfer of mature melanosomes to
neighboring keratinocytes which are arranged in a supranuclear cap, protecting the DNA against
incident ultraviolet light (UV) irradiation21. By controlling skin tone, melanin also determines the
human phenotypic appearance. Although photoprotection is melanin’s main function in human
skin, the accumulation of an abnormal amount of melanin in specific parts of the skin resulting in
more pigmented patches might become an aesthetic problem. In addition, enzymatic browning
caused by tyrosinase system in fruits and fungi is undesirable in, for example, fresh fruits,
beverages, vegetables, and mushrooms. In commercial or domestic processing, handling and
storage of foods, tyrosinase is contemplated to have a critical role. In bruised or cut fruits,
vegetables and other plant foods, tyrosinase causes enzymatic browning which subsequently
leads to a significant decrease in nutritional and market values22. The undesirable phenomenon of
hyperpigmentation in human skin and enzymatic browning in fruits have ushered researchers to
seek new, enhanced and potent tyrosinase inhibitors for use in antibrowning of foods and skin
whitening. Owing to the exclusively important roles in mammalian melanogenesis as well as
enzymatic browning in fruits and fungi, tyrosinase inhibitors have been an important concern
under the research radar for years.
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Some tyrosinase inhibitors have been discovered and reviewed before23-26. Herein, a new
series of thirty compounds bearing α, βꢀcarbonyl moiety was designed and synthesized based on
curcumin template (Figure 1) for potential use in cosmetics, clinical medicines, food industry
and agriculture. Effects of synthesized compounds on human cancer cell lines and mushroom
tyrosinase enzyme were evaluated. Furthermore, structureꢀactivity relationships of synthesized
compounds were also discussed.
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101 MATERIALS AND METHODS
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Materials and General Procedures
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All chemicals and reagents utilized in this research were purchased from SigmaꢀAldrich (St.
Louis, MO, USA), Merck (Darmstadt, Germany) and Acros Organics (Geel, Belgium) of
analytical grade, and were used as supplied, unless otherwise stated. A typical workꢀup included
washing with brine and drying the organic layer with anhydrous magnesium sulfate before
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concentration in vacuo. H and C NMR spectra were recorded with a JEOL ECP spectrometer
operating at 500 MHz, with Me4Si as internal standard and CDCl3 or DMSOꢀd6 as the solvent.
High resolution mass spectra (HRMS) were determined by the Electrospray ionization mass
spectrometry (ESIꢀMS) on MicroTOFꢀQ mass spectrometer (Bruker, Billerica, MA, USA).
Microanalyses data were obtained from the Fison EA 1108 elemental analyzer. Infrared spectra
were recorded using KBr disc on a Perkin Elmer 400 (FTIR) spectrometer. Flash column
chromatography was performed with silica gel 60 (230ꢀ400mesh) (Merck) and thin layer
chromatography (TLC) was carried out on preꢀcoated silica plates (kiesel gel 60 F254, BDH).
Melting points were determined on an electrothermal instrument and are uncorrected.
Compounds were visualized by illumination under ultraviolet (UV) light (254 nm) or by the use
of vanillin stain followed by charring on a hotplate.
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Synthesis of curcumin related α, βꢀunsaturated carbonyl based compounds
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A direct coupling approach was employed in order to synthesize α, βꢀunsaturated carbonyl based
compounds. A general reaction proceeded by base catalyzed ClaisenꢀSchmidt condensation,
carried out by reacting a suitable aromatic aldehyde with different types of ten ketones: at a
molar ratio of 1:2 for synthesizing twenty one compounds (1aꢀ7a; 1bꢀ7b; 1cꢀ7c), and at a molar
ratio of 1:1 for nine compounds (8aꢀ10a; 8bꢀ10b; 8cꢀ10c). A general synthesis method of α, βꢀ
unsaturated carbonyl based compounds is illustrated in Figure 2. Briefly, in a single necked
round bottom flask, selected aromatic aldehyde (20 mmol, 2 equivalant) and ketone (10 mmol, 1
equivalent) were mixed and dissolved in 15 mL of ethanol and stirred at 5°C for a few minutes.
A 40% NaOH solution (in ethanol) was then added into the above solution drop by drop over
several minutes. The mixture obtained was stirred at room temperature (27 °C) for 1ꢀ24 h.
Product formation was indicated by the appearance of precipitate and change in color of the
reaction mixture. TLC was used to monitor the reaction and upon completion, acidified ice was
added to the mixture for quenching the reaction. Column chromatography and/or
recrystallization processes were used to isolate α, βꢀunsaturated carbonyl based compounds.
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2,6-Bis[4-(diethoxymethyl)benzylidene]cyclohexanone (1a)
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Yellow crystals (2.59 g, 54%). mp: 112ꢀ114 ˚C; H NMR (500 MHz, CDCl3) δ: 7.89 (s, 2H),
7.29 (d, J=8Hz, 4H), 6.82 (d, J=8Hz, 4H), 5.89 (s, 2H), 3.42 (q, J=7.5, 8H), 2.32 (t, J=12.0 Hz,
4H), 1.82 (m, 2H), 1.24 (t, J=7.5, 12H); 13C NMR (500 MHz, CDCl3) δ: 186.4, 152.9, 144.4,
136.5, 132.5, 127.3, 126.5, 101.1, 55.0, 28.3, 27.8, 16.2 ; HRMS (ESI) m/z: 479.68 [M+H]+,
Microanalysis calculated for C30H38O5 (478.62), C: 75.28%, H: 8.00%. Found C: 75.42%, H:
8.12%.
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2,6-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclohexanone (1b)
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White powder (2.78 g, 62%). mp: 139ꢀ140 ˚C; δ: 7.94 (s, 2H), 7.55 (d, J=8Hz, 2H), 7.41 (d,
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J=8Hz, 2H), 7.13 (s, 2H), 3.15 (s, 12H), 2.35 (t, J=12.0 Hz, 4H), 1.87 (m, 2H) ; C NMR (500
MHz, CDCl3) δ: 190.7, 149.5, 145.7, 145.6, 140.1, 128.2, 118.6, 117.6, 106.5, 46.8, 29.1, 27.5;
HRMS (ESI) m/z: 451.64 [M+H]+, Microanalysis calculated for C24H26N4O5 (450.49), C:
63.99%, H: 5.82%, N: 12.44%. Found C: 64.12%, H: 5.72%, N: 12.42%.
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3,5-Bis-(4-diethoxymethyl-benzylidene)-tetrahydro-pyran-4-one (2a)
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Pale Yellow crystals (3.06 g, 64%). mp: 106ꢀ108 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.85 (s, 2H),
7.32 (d, J=8Hz, 4H), 6.95 (d, J=8Hz, 4H), 5.52 (s, 2H), 3.62 (q, J=7.5, 8H), 2.69 (s, 4H), 1.18 (t,
J=8, 12H); 13C NMR (500 MHz, CDCl3) δ: 187.2, 152.7, 143.8, 136.7, 131.9, 126.2, 125.2,
101.5, 62.4, 55.2, 16.1; HRMS (ESI) m/z: 481.65 [M+H]+, Microanalysis calculated for
C29H36O6 (480.59), C: 72.48%, H: 7.55%. Found C: 72.62%, H: 7.72%.
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3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]-1-methyl-piperidin-4-one (4b)
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White powder (2.56 g, 55%). mp: 181ꢀ 182 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.76 (s, 2H), 7.45
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(d, J=8Hz, 2H), 7.23 (d, J=8Hz, 2H), 7.13 (s, 2H), 3.21 (s, 12H), 3.03 (s, 4H), 2.19 (s, 3H); C
NMR (500 MHz, CDCl3) δ: 187.5, 148.9, 148.2, 143.2, 141.8, 126.7, 119.6, 118.9, 106.2, 44.5,
46.1, 38.8; HRMS (ESI) m/z: 466.72 [M+H]+, Microanalysis calculated for C24H27N5O5 (465.50),
C: 61.92%, H: 5.85%, N: 15.04%. Found C: 61.99%, H: 5.91%, N: 15.19%.
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1,5-Bis-(4-diethoxymethyl-phenyl)-penta-1,4-dien-3-one (6a)
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Pale brownish solid (1.96 g, 45%). mp: 98ꢀ100 ˚C; H NMR (500 MHz, CDCl3) δ: 7.82 (d,
J=6.5Hz, 4H), 7.71 (d, J=8Hz, 2H) , 7.49 (d, J=6.5Hz, 2H), 7.16 (d, J=7Hz, 4H), 5.25 (s, 2H),
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3.52 (q, J=7Hz, 8H), 1.17 (t, J=7.0, 12H); C NMR (500 MHz, CDCl3) δ: 190.5, 152.2, 148.6,
139.7, 136.4, 135.2, 132.8, 102.2, 55.5, 16.9 ; HRMS (ESI) m/z: 439.62 [M+H]+, Microanalysis
calculated for C27H34O5 (438.56), C: 73.94%, H: 7.81%. Found C: 74.12%, H: 7.89%.
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1,5-Bis-(4-dimethylamino-2-nitro-phenyl)-penta-1,4-dien-3-one (6b)
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Pale yellow solid (2.97 g, 68%). mp: 177ꢀ179 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.70 (d, J=6Hz,
2H), 7.68 (d, J=8Hz, 2H), 7.44 (d, J=6Hz, 2H), 7.25 (d, J=8Hz, 2H), 7.12 (s, 2H), 3.15 (s, 12H);
13C NMR (500 MHz, CDCl3) δ: 187.5, 150.9, 148.6, 144.4, 132.8, 128.1, 117.5, 113.8, 107.9,
46.7; HRMS (ESI) m/z: 409.45 [MꢀH]+, Microanalysis calculated for C21H22N4O5 (410.42), C:
61.45%, H: 5.40%, N: 13.65%. Found C: 61.48%, H: 5.62%, N: 13.77%.
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2,5-Bis-(4-diethoxymethyl-benzylidene)-cyclopentanone (7a)
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Light yellow crystals (3.12 g, 67%). mp: 88ꢀ89 ˚C; H NMR (500 MHz, CDCl3) δ: 7.69 (d,
J=6.5Hz, 4H), 7.42 (s, 2H), 7.21 (d, J=6.5Hz, 4H), 5.45 (s, 2H), 3.62 (q, J=7Hz, 8H), 2.38 (t,
J=7Hz, 4H), 1.22 (t, J=7.0, 12H); 13C NMR (500 MHz, CDCl3) δ: 189.7, 149.4, 145.5, 138.7,
136.4, 133.9, 129.1, 100.9, 56.2, 32.5, 16.1 ; HRMS (ESI) m/z: 465.72 [M+H]+, Microanalysis
calculated for C29H36O5 (464.59), C: 74.97%, H: 7.81%. Found C: 75.18%, H: 7.87%.
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2,5-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclopentanone (7b)
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White powder (2.41 g, 55%). mp: 156ꢀ157 ˚C; H NMR (500 MHz, CDCl3) δ: 7.92 (d, J=6.5Hz,
2H), 7.88 (d, J=6Hz, 2H), 7.75 (d, J=6.5Hz, 2H), 7.44 (s, 2H), 3.10 (s, 12H), 2.45 (t, J=7Hz,
4H) ; 13C NMR (500 MHz, CDCl3) δ: 188.9, 153.2, 149.9, 144.2, 143.8, 125.8, 118.1, 116.4,
108.3, 46.9, 32.6; HRMS (ESI) m/z: 437.72 [M+H]+, Microanalysis calculated for C23H24N4O5
(436.46), C: 63.29%, H: 5.54%, N: 12.84%. Found C: 63.42%, H: 5.48%, N: 12.72%.
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2-(4-Diethoxymethyl-benzylidene)-indan-1-one (8a)
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White powder (2.52 g, 78%). mp: 152ꢀ154 ˚C; H NMR (500 MHz, CDCl3) δ: 7.80 (d, J=8Hz,
2H), 7.69 (s, H), 7.48 (d, J=8Hz, 2H), 7.25 (d, J=7.5Hz, H), 7.20 (d, J=7.5Hz, H), 7.05 (t,
J=7.5Hz, H), 6.94 (t, J=7Hz, H), 5.34 (s, H), 3.49 (q, J=7Hz, 4H), 2.75 (s, 2H), 1.29 (t, J=6Hz,
6H); 13C NMR (500 MHz, CDCl3) δ: 193.2, 144.5, 142.2, 140.1, 139.2, 138.8, 136.5, 130.4,
129.6, 128.1, 125.6, 125.1, 124.2, 102.2, 56.3, 29.9, 16.4 ; HRMS (ESI) m/z: 323.67 [M+H]+,
Microanalysis calculated for C21H22O3 (322.40), C: 78.23%, H: 6.88%. Found C: 78.44%, H:
6.92%.
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2-(4-Dimethylamino-2-nitro-benzylidene)-indan-1-one (8b)
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Yellow semisolid (1.95 g, 63%). mp: 169ꢀ171 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.56 (d, J=8Hz,
H), 7.48 (s, H), 7.32 (d, J=8Hz, H), 7.12 (s, H), 6.98 (d, J=7.5Hz, H), 6.91 (d, J=7.5Hz, H), 6.74
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(t, J=7.5Hz, H), 6.65 (t, J=7Hz, H), 3.22 (s, 6H), 2.81 (s, 2H); C NMR (500 MHz, CDCl3) δ:
192.8, 145.6, 142.6, 141.3, 139.8, 138.2, 137.4, 130.9, 128.7, 128.0, 125.9, 123.8, 119.5, 117.6,
108.6, 46.8, 28.3 ; HRMS (ESI) m/z: 309.55 [M+H]+, Microanalysis calculated for C18H16N2O3
(308.33), C: 70.12%, H: 5.23%, N: 9.09%. Found C: 70.18%, H: 5.59%, N: 9.18%.
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2-(4-Diethoxymethyl-benzylidene)-3,4-dihydro-2H-naphthalen-1-one (9a)
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Light yellow solid (2.67g, 80%). mp: 94ꢀ95 ˚C; H NMR (500 MHz, CDCl3) δ: 7.84 (d, J=8Hz,
2H), 7.59 (s, H), 7.47 (d, J=8.5Hz, 2H), 7.29 (d, J=7.5Hz, H), 7.22 (d, J=7.5Hz, H), 7.12 (t,
J=7.5Hz, H), 7.02 (t, J=7Hz, H), 5.49 (s, H), 3.35 (q, J=7Hz, 4H), 2.29 (t, J=7Hz, 2H), 2.05 (t,
J=7Hz, 2H), 1.17 (t, J=6Hz, 6H); 13C NMR (500 MHz, CDCl3) δ: 188.3, 152.2, 150.1, 148.5,
146.2, 143.9, 137.5, 134.4, 129.2, 127.2, 126.2, 124.8, 117.2, 101.7, 56.2, 29.5, 28.2, 16.0 ;
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HRMS (ESI) m/z: 337.51 [M+H]+, Microanalysis calculated for C22H24O3 (336.42), C: 78.54%,
H: 7.19%. Found C: 78.33%, H: 7.12%.
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2-(4-Dimethylamino-2-nitro-benzylidene)-3,4-dihydro-2H-naphthalen-1-one (9b)
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White powder (2.50 g, 78%). mp: 138ꢀ139 ˚C; H NMR (500 MHz, CDCl3) δ: 7.82 (d, J=6.5Hz,
H), 7.57 (s, H), 7.42 (d, J=6.5Hz, H), 7.35 (s, H), 7.28 (d, J=7Hz, H), 7.20 (d, J=7Hz, H), 7.19 (t,
J=7.5Hz, H), 6.92 (t, J=8Hz, H), 3.14 (s, 6H), 2.47 (t, J=7Hz, 2H), 2.14 (t, J=7Hz, 2H) ; 13C
NMR (500 MHz, CDCl3) δ: 188.7, 152.8, 151.3, 148.9, 146.8, 144.2, 136.8, 134.6, 129.9, 127.6,
126.1, 125.3, 124.2, 107.8, 46.2, 29.9, 27.9; HRMS (ESI) m/z: 323.45 [M+H]+, Microanalysis
calculated for C19H18N2O3 (322.36), C: 70.79%, H: 5.63%, N: 8.69%. Found C: 71.12%, H:
5.52%, N: 8.72%.
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2-[3-(4-Diethoxymethyl-phenyl)-acryloyl]-3,4-dihydro-2H-naphthalen-1-one (10a)
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215
216
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220
Yellow solid (3.16 g, 83%). mp: 121ꢀ122 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.75 (d, J=6Hz, H),
7.52 (d, J=6Hz, H), 7.42 (d, J=8Hz, 2H), 7.39 (d, J=8Hz, 2H), 7.32 (d, J=7Hz, H), 7.29 (d, J=7Hz,
H), 7.17 (t, J=7Hz, H), 6.89 (t, J=7Hz, H), 5.29 (s, H), 3.47 (q, J=7Hz, 4H), 3.29 (t, J=8.5Hz, H),
2.12 (t, J=8Hz, 2H), 1.95 (t, J=7Hz, 2H), 1.24 (t, J=6.5Hz, 6H); 13C NMR (500 MHz, CDCl3) δ:
194.6, 151.1, 148.9, 144.6, 142.3, 139.2, 138.4, 136.2, 135.5, 132.4, 130.6, 129.1, 125.2, 124.2,
103.1, 66.1, 55.7, 30.2, 28.6, 16.7 ; HRMS (ESI) m/z: 379.52 [M+H]+, Microanalysis calculated
for C24H26O4 (378.46), C: 76.17%, H: 6.92%. Found C: 76.21%, H: 6.91%.
221
2-[3-(4-Dimethylamino-2-nitro-phenyl)-acryloyl]-3,4-dihydro-2H-naphthalen-1-one (10b)
1
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Pale yellow powder (2.93 g, 80%). mp: 102ꢀ103 ˚C; H NMR (500 MHz, CDCl3) δ: 7.79 (d,
J=6.5Hz, H), 7.62 (d, J=6.5Hz, H), 7.45 (d, J=7.5Hz, H), 7.32 (s, H), 7.29 (d, J=7.5Hz, H), 7.21
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(d, J=7.5Hz, H), 7.14 (d, J=7.5Hz, H), 7.02 (t, J=7.5Hz, H), 6.94 (t, J=7.5Hz, H), 3.45 (t,
J=8.5Hz, H), 3.10 (s, 6H), 2.22 (t, J=8Hz, 2H), 2.15 (t, J=8Hz, 2H) ; 13C NMR (500 MHz, CDCl3)
δ: 193.8, 152.2, 148.7, 145.2, 142.8, 139.6, 137.1, 136.8, 135.6, 131.9, 130.5, 129.4, 124.8, 123.5,
116.5, 106.2, 65.8, 46.8, 31.3, 28.7 ; HRMS (ESI) m/z: 365.55 [M+H]+, Microanalysis calculated
for C21H20N2O4 (364.39), C: 69.22%, H: 5.53%, N: 7.69%. Found C: 69.34%, H: 5.59%, N:
7.81%.4
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MTT assay
MTT assay was performed for determining the effect of synthetic compounds on normal (MCFꢀ
10A) mammary epithelial cells. MCFꢀ10A cells were maintained in Dulbecco's modified Eagle's
medium/Ham's Fꢀ12 medium (1:1) with 10% foetal calf serum, 2 mM glutamine, insulin (10
ꢁg/mL), hydrocortisone (500 ng/mL), and epidermal growth factor (20 ng/mL). Cells were
passaged every 2 to 3 days using trypsin EDTA (ethylenediaminetetraacetic acid). MCFꢀ10A
cells were plated at a density of 104 cells per millilitre in 96ꢀwell flatꢀbottomed plates. After 24
hours, the medium was carefully aspirated and tested compounds (curcumin analogues) were
added in quadruplicate in 200 ꢁL of medium to give a final concentration of not more than 0.1%
(v/v) dimethylsulfoxide (DMSO). The cells were then permitted to grow for 96 hours at 37°C.
The medium was removed and fresh medium with MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀ
diphenyltetrazolium bromide) (0.4 mg/mL) was added to each well for 3 hours. All medium was
aspirated and the formazan product generated by viable cells was solubilised with 150 ꢁL of
DMSO. Plates were vortexed and the absorbance at 540 nm determined using a microplate
reader. Results were expressed as the percentage inhibition of proliferation compared to controls
containing 0.1% DMSO.
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Anticancer activity
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For determining the anticancer activity a propidium iodide (PI) fluorescence assay was
performed on MCFꢀ7 (breast cancer cell line), PCꢀ3 (prostate cancer cell line), HLꢀ60
(promyelocytic leukemia cell line), Hꢀ460 (lung cancer cell line) and HTꢀ29 (colon cancer cell
line) cell lines27. Propidium iodide (PI), a dye which binds to DNA, provides a rapid and
accurate means for quantitating total nuclear DNA. The principle of this assay is based on the
fact that PI is not able to penetrate an intact cell membrane and the fluorescence signal intensity
of the PI is directly proportional to the amount of DNA in each cell. So the cells which have
damaged membranes or altered permemability profiles are scored as dead cells. For a typical PI
cytotoxicity assay, cells, after trypsinization and counting, were seeded at 3000ꢀ7500 cells/well
in 200 µl media in sterile tissue culture grade 96 well plates and allowed to recover for 24 h in
humidified 5% CO2 incubator at 37 °C. After this, compounds (in 0.1% DMSO) were added onto
triplicate wells with 10 µM concentrations. 0.1% DMSO alone was used as control. Treatment
with test compounds was continued for 48 h. After completion of designated treatment period,
the medium was removed and cells were treated with 25 µl of propidium iodide (50 µg/mL in
water/medium) per well. The above plates with samples were frozen at ꢀ80 °C for 24 h, then
thawed and allowed to equilibrate to room temperature. Plates were read on a fluorometer (Polarꢀ
Star BMG Tech), using 530 nm excitation and 620 nm emission wavelength. Lastly, percent
cytotoxicity of the compounds was calculated by using following formula:
ꢀꢁ − ꢀꢂꢁ
% Cytotoxicity =
× 100
ꢀꢁ
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Where AC= Absorbance reading of control and ATC= Absorbance reading of treated cells. The
results were compared with the standard drug inhibitors flavopiridol (0.5µM).
Tyrosinase assay
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The spectrophotometric assay for tyrosinase was performed according to the reported method
with slight modifications28. Briefly, LꢀDOPA was used as substrate to screen the synthesized
compounds for the diphenolase inhibitory activity of tyrosinase. Positive controls in the assay
were Kojic acid and 4ꢀButylresorcinol. 2% stock solutions of compounds were prepared in
DMSO and diluted using phosphate buffer (pH = 6.8). These synthesized compounds were preꢀ
incubated in 50 mM phosphate buffer (pH = 6.8) at 25 °C for 10 min with thirty units of
mushroom tyrosinase (0.5 mg/mL). LꢀDOPA (0.5 mM) was then added to the reaction mixture.
Reaction was monitored by following formation of LꢀDOPA chrome for 1 min measured by the
change in absorbance at 475 nm. Triplicate measurements were taken for each concentration and
average was used for further calculations. Doseꢀresponse curves were interpolated to obtain the
IC50 values.
Molecular modeling
Molecular modeling studies were performed with MOE (The Molecular Operating Environment)
Version 2011.10, software available from Chemical Computing Group Inc., 1010 Sherbrooke
Street West, Suite 910, Montreal, Canada H3A2R7, http://www.chemcomp.com. The ligands
were built using the builder tool of the MOE program and subjected to energy minimization
(MMFF94x, gradient: 0.05). The Xꢀray crystallographic structure of mushroom tyrosinase (PDB:
2Y9W) 29 was obtained from the Protein Data Bank. The errors of the protein were corrected by
the Structure Preparation process in MOE. After correction, hydrogens were added and partial
charges (Gasteiger methodology) were calculated. Site finder application was used to detect the
possible binding pocket. The default Triangle Matcher placement method was applied for
docking. GBVI/WSA dG scoring function which estimates the free energy of binding of the
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ligand from a given pose was used to rank the final poses. The ligandꢀenzyme complexes with
lowest S score were selected for each ligand.
Statistical analysis
Analysis was carried out using a complete randomized design and treatments were run in
triplicates (n = 3). Statistical analysis was done using analysis of variance (ANOVA) method of
general linear model (GLM) procedure using SAS V8 (Cary, NC, USA) for multiple
comparisons. P < 0.05 was considered to be statistically significant.
297 RESULTS AND DISCUSSION
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Chemistry
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ClaisenꢀSchmidt condensation was used for the synthesis of the desired α, βꢀ
unsaturated carbonyl based compounds30 by reacting different ketones and the appropriate aryl
aldehyde. Twenty one compounds (1aꢀ7a; 1bꢀ7b; 1cꢀ7c) were synthesized in presence of NaOH
at a 1:2 molar ratio in ethanol while some compounds were synthesized in acetic acid by passing
dry HCl gas through the mixture. ClaisenꢀSchmidt condensation was also employed to
synthesize nine compounds (8aꢀ10a; 8bꢀ10b; 8cꢀ10c) similar to chalcone analogues however
employing 1:1 molar ratio of ketone and aldehyde (Figure 2). Both types of catalytic systems,
i.e., NaOH and dry HCl gas mixture, were used to synthesize eight compounds (7a, 8a, 10a, 7b,
8b, 10b, 7c, 8c). Catalytic system employing HCl gas mixture gave high percentage yields
compared to NaOH. Using NaOH as a catalyst resulted in producing mixture of several different
unidentified products necessitating excessive purification using column chromatography.
Spectrophotometric techniques were used to characterize the synthesized compounds. An
elemental analysis of C, H, and N, and melting points are represented. Representative carbon
1
nuclear magnetic resonance spectra were also recorded. NH absorptions were not seen in the H
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NMR spectra of the compounds 4a, 4b and 4c, it has previously been shown by other
researchers30, 31
.
Cytotoxicity towards MCFꢀ10A normal mammary epithelial cells
The rule of three, relating to activityꢀexposureꢀtoxicity, presents the single most difficult
challenge in the design and advancement of drug candidates to the development stage.
Absorption, distribution, metabolism and excretion (ADME) studies are widely used in drug
discovery to optimize this balance of properties necessary to convert lead compounds into drugs
that are both safe and effective for human patients32. Therefore, in vitro cell viability assay was
performed by using the MCFꢀ10A that is a normal cell line and was used as a comparison. MCFꢀ
10A cells were treated with synthetic compounds for 96 hours and cell viability was measured
using the MTT assay. Results are expressed in terms of percentage of toxicity (Table 1). The
compounds were found to be nontoxic and majority of compounds showed more than 95% cell
viability. Therefore, the compounds are biologically safe and can be used as the therapeutic agent
for future drug discovery study.
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Effects on Human Cancer Cell Lines
Anticancer activity of curcumin is one of the most widely explored activities among numerous
others like antiꢀinflammatory activity. In vitro cytotoxicity assays performed in various cell lines
originating from different organs show that curcumin have beneficial effects in, including but
not limited to,; prostate cancer cells33, ovarian cancer cells34, gastric carcinoma cells35, liver
cancer cells, hepatocelꢀ lular carcinoma cells36, cervical cancer cells37, breast cancer cells38,
colon cancer cells39, lung carcinoma cell40, bladder cancer cells41 and pancreatic adenocarcinoma
cell42. This provides strong preliminary data for the justification of clinical studies in humans.
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Synthesized compounds were tested in present investigation for their capacity to inhibit
the growth of five different cancer cell lines (PCꢀ3, MCFꢀ7, HLꢀ60, Hꢀ460 and HTꢀ29).
Compound 2b was found to have highest anticancer activity inhibiting 82% of PCꢀ3 cells; this
was closely followed by compound 2a showing 81% inhibition. These two compounds also show
similarly comparative levels of anticancer activity (75 ꢀ 82%) on the other four cell lines used for
experiments. Compound 2a and 2b were followed by six curcuminꢀlike compounds (2aꢀb, 3aꢀb
and 4aꢀb) which had promising anti cancer activity (59 ꢀ 82%) (Figure 3). This was followed by
twelve compounds (1aꢀc, 2c, 3c, 4c, 5aꢀb, 6aꢀb and 10aꢀb) which show moderate effect (40 ꢀ
58%). Insignificant activity was shown by remaining compounds. For the anticancer activity
experiments positive control used was flavopiridol which show 79% inhibition of Hꢀ460 cells at
0.5µM concentration. Generally, curcumin like compounds show similar inhibitory anticancer
activity on the tested cell lines and it was observed that the average difference in inhibitory
activities was not more than 5% types of tested cancer cell lines. For most active compounds (2a,
2b, 4a and 4b) the median inhibition concentration (IC50) was calculated using GraphPad Prism
software (GraphPad Software, San Diego, CA, USA) and it was observed that compound 2b was
very potent anticancer agent among all other tested compounds with very low IC50 value in the
range of 1.04 to 1.08 µM (Table 2).
Compounds 2a, 2b, 4a and 4b were found to have high potency against different types of
cancer cell lines. It can be depicted from their physicochemical properties (Table 2) that these
compounds have potential to be new lead compounds in developing anticancer agents.
Physicochemical properties of tested compounds were found within optimal range (Molecular
weight <500; clogP <5 and TPSA <140)43, 44
.
Effects on the Diphenolase Activity of Mushroom Tyrosinase
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Tyrosinase inhibitory assay was performed for all synthesized curcuminꢀrelated compounds by
measuring the oxidation of LꢀDOPA as previously reported28. A concentrationꢀdependent
enzyme activity inhibition by the compounds was observed. It was observed that the activity of
remaining enzyme swiftly decreased when the concentration of compound (inhibitor) was
increased. Table 1 summarizes the enzyme inhibition data expressed as IC50. The majority of
synthetic curcumin related compounds (1aꢀc, 2aꢀc, 3aꢀb, 6aꢀc, 7aꢀc, 8aꢀc, 9aꢀc, 10aꢀc) exhibit
<100 ꢁM inhibitory activities against mushroom tyrosinase. With IC50 ranging from 5.34 to
10.11 µM, five compounds (1a, 8aꢀb and 10aꢀb) exhibited promising tyrosinase inhibitiory
activity better than positive control Kojic acid (10.93µM). Two of the synthesized compounds
(8a and 10a), with diethoxymethyl group at position 4, were found to be the strongest inhibitors
among all tested compounds with IC50 values 6.83µM and 5.34µM respectively.
Structureꢀactivity relationship analysis
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It was observed that the presence or absence of groups on the two aromatic rings and the linker
between the two aromatic rings both played key roles in determining the anticancer inhibitory
activity of the various analogs on the tested cancer cells. From their data for SAR of monoꢀ
carbonyl analogues, the research group of G Liang, had shown previously that acetone and
cyclohexanone spacers are much more favorable than the cyclopentanone one45 for enhanced
anticancer activity. Conclusion drawn by Liang’s group is also afforded by present study as all
synthetic compounds containing cyclopentanone ring system (7aꢀ7c) have lesser anticancer
activity when compared to compounds with acetone and cyclohexanone system. In fact, among
these three group of compounds (1, 6, 7), compounds bearing cyclohexanone were found to be
most active.
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Different activity trends were observed among the different series of curcuminꢀrelated
compounds when either linear or cyclic linkers were used between the two aromatic rings of
curcumin related compounds. In general, tetrahydropyranꢀ4ꢀone (2aꢀc) linker containing
compounds exhibited the strongest activities, moderate activity was exhibited by compounds
with NꢀMethylꢀ4ꢀpiperidone (3aꢀc), while three compounds (4aꢀc) with 4ꢀpiperidone showed
good inhibition power however it was lesser compared to compounds bearing NꢀMethylꢀ4ꢀ
piperidone. Bisꢀderivatives, bearing either 1ꢀBenzylꢀ4ꢀpiperidone (5aꢀc), acetone (6aꢀc) or
cyclopentanone (7aꢀc) linker, were less active compared to the rest. Conversely, among the
monoꢀderivatives (8ꢀ10), merely compounds bearing diꢀcarbonyl linker (10aꢀb) were shown to
have moderate growth inhibitory anticancer activity in the tested cell lines.
Presence of a heteroatom linker in groups of compounds (2, 3, 4) resulted in enhanced
anticancer activities compared to the compounds without a heteroatom linker (1, 6, 7). This
finding suggests that flexibility of curcuminꢀlike compounds could improve their anticancer
activities which could be due to the ease of interactions with the DNA of cancer cells. Contrary
to this, compounds with small and rigid linker would be less active due to the difficulty of
interactions with DNA. Association of curcumin flexibility with curcuminꢀDNA interaction
which could be a possible explanation for antitumor activity is a valuable and interesting
finding46, 47
.
Effects of substitution of various groups curcumin related compound activity were
compared. It was observed that introduction of 2ꢀnitroꢀ4ꢀdimethylamine combination on the
aromatic rings improved anticancer activity of curcumin related compounds. In all the three
groups (based on substitution pattern a, b, c), compounds bearing combination of 2ꢀnitro and 4ꢀ
dimethylamine show exceptionally elevated anticancer activity whereas presence of
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diethoxymethyl group at position 4 of rings consequently produces markedly reduced anticancer
activity. Further, presence of pyrrolidine at position 4 of compounds (1ꢀ10c) results in
compounds which are not highly active.
Analysis of the effects of curcumin related compounds on tyrosinase activity produced
structureꢀactivity relationship with new and interesting findings. Based upon different substituted
groups, with two exception (4b and 7b more active than 4a and 7a), it was observed that
compounds having diethoxymethyl group at position 4 of rings (group a) were most active
against the mushroom tyrosinase activity. Like the anticancer activity, substitution of pyrrolidine
at position 4 of compounds (1ꢀ10c) did not produce strongly active compounds for tyrosinase
inhibition. An interesting finding was the presence of diꢀcarbonyl group (10aꢀb) that resulted in
maximum tyrosinase inhibition even better than the positive controls. In general, the monoꢀ
derivatives (8ꢀ10) show better antiꢀtyrosinase activity as compared to bisꢀderivatives (2ꢀ7).
However presence of a heteroatom linker (2, 3, and 4) didn’t result in compounds with a better
antityrosinase activity when compared to those without a heteroatom linker (1, 6, and 7).
Molecular Docking Study
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Tyrosinase enzyme has binuclear copper binding site and tropolone and kojic acid bind the
entrance of this site29, 48. In this study, Site Finder application in MOE was used to find the
possible binding sites in the enzyme. The region consisting of S1 and S2 site was indicated as
active pocket (Figure 4). S1 contains binuclear copper binding site where S2 locates near the
entrance of this site.
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To simplify, docking results were evaluated in groups. First of all, (a) the docking results
of compound 1ꢀ7a (bisꢀderivatives) which have different keton structures were considered.
Compound 1a and 6a gave the same orientation (Figure 5) while the others oriented in a different
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way. It was seen that Val283 and His244 hold 1a and 6a at the entrance of copper binding site by
Hꢀarene interaction of the phenyl rings of both compounds (Figure 6). Furthermore, C=O moiety
of compounds 1a and 6a might form a weak hydrogen bond with backbone NH of His85 (about
4Å). Also, nonpolar part of cyclohexanone of compound 1a made hydrophobic interactions with
Val283 (less than 3Å). Because of the hindrance of nonpolar part of Val283, synthetic
compounds 2aꢀ5a and 7a might be oriented in a different way. (b) The orientations of 8aꢀ10a
(monoꢀderivatives) occurred similar to that of bisꢀderivatives (except 9a) by their interactions
with Val283, His244 and His85 (Figure 6). (c) Compounds carrying pyrrolidine moiety (1ꢀ10c),
were oriented so that C=O groups of the compounds located close to NH of Asn81 instead of
His85. Substitution of pyrrolidine might decrease the flexibilities of these compounds at the
entrance, which might cause a reduction in the activity.
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To conclude, ten series containing thirty novel α, βꢀunsaturated carbonyl based curcumin
related compounds were successfully synthesized using the ClaisenꢀSchmidt condensation
reaction. All synthesized compounds were analyzed for anticancer and antityrosinase activity.
Compounds 2aꢀb were identified as potent anticancer agents with 81ꢀ82% inhibition of cancer
cell lines. Five of these compounds (1a, 8aꢀb, 10aꢀb), emerged to be the potent inhibitors of
Mushroom tyrosinase. Molecular modeling studies were also performed to see the interactions of
synthetic compounds with Xꢀray crystallographic structure of mushroom tyrosinase. It also
provided further insight into designing compounds with structural features that may translate into
higher activity. Our studies have established that these curcuminꢀlike compounds, with
appropriate structural modifications, have the potential to be developed as anticancer agents for
humans and can also be further studied for use in medicines, cosmetic products, food industry as
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well as agricultural purposes. Further studies are required for use and development of these
synthetic compounds in food and agricultural field as antibrowning agents.
ABBREVIATIONS USED
PI,
Propidium
iodide;
DMSO,
Dimethyl
sulfoxide;
LꢀDOPA,
Lꢀ3,
4ꢀ
dihydroxyphenylalanine; MOE, Molecular operating environment; TPSA, Topological polar
surface area.
ACKNOWLEDGEMENTS
Dr. Syed Nasir Abbas Bukhari thanks University Kebangsaan Malaysia for postdoctoral contract.
FUNDING
This research was supported by University Kebangsaan Malaysia (Grants no.: UKMꢀDLPꢀ2013ꢀ
013).
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: (SNAB) snab@pharmacy.ukm.my; snabphd@yahoo.com; snab_hussaini@yahoo.com
(IJ) ibj@pharmacy.ukm.my
CONFLICT OF INTEREST
The authors declare no competing financial interest.
References
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1.
Dias, D. A.; Urban, S.; Roessner, U., A Historical Overview of Natural Products in Drug
Discovery. Metabolites 2012, 2, 303ꢀ336.
2. Guo, Z. R., [Modification of natural products for drug discovery]. Yao xue xue bao =
Acta pharmaceutica Sinica 2012, 47, 144ꢀ57.
3. Valli, M.; Pivatto, M.; Danuello, A.; CastroꢀGamboa, I.; Silva, D. H. S.; Cavalheiro, A.
J.; Araújo, Â. R.; Furlan, M.; Lopes, M. N.; Bolzani, V. d. S., Tropical biodiversity: has it been a
potential source of secondary metabolites useful for medicinal chemistry? Química Nova 2012,
35, 2278ꢀ2287.
4.
Marine drugs 2010, 8, 2702ꢀ20.
5. Prakash, O.; Kumar, A.; Kumar, P.; Ajeet, A., Anticancer Potential of Plants and Natural
Products: A Review. American Journal of Pharmacological Sciences 2013, 1, 104ꢀ115.
6. Brown, A.; Shi, Q.; Moore, T. W.; Yoon, Y.; Prussia, A.; Maddox, C.; Liotta, D. C.;
Bhatnagar, I.; Kim, S. K., Marine antitumor drugs: status, shortfalls and strategies.
Shim, H.; Snyder, J. P., Monocarbonyl Curcumin Analogues: Heterocyclic Pleiotropic Kinase
Inhibitors That Mediate Anticancer Properties. Journal of Medicinal Chemistry 2013, 56, 3456ꢀ
3466.
7.
of clinical trials. Journal of the American Academy of Dermatology 2006, 55, 1048ꢀ1065.
8. Shakibaei, M.; Buhrmann, C.; Kraehe, P.; Shayan, P.; Lueders, C.; Goel, A., Curcumin
Gupta, A. K.; Gover, M. D.; Nouri, K.; Taylor, S., The treatment of melasma: A review
Chemosensitizes 5ꢀFluorouracil Resistant MMRꢀDeficient Human Colon Cancer Cells in High
Density Cultures. PLoS ONE 2014, 9, e85397.
9.
Lu, W.; Ogasawara, M. A.; Huang, P., Models of reactive oxygen species in cancer. Drug
discovery today. Disease models 2007, 4, 67ꢀ73.
22
ACS Paragon Plus Environment

Page 23 of 37
Journal of Agricultural and Food Chemistry
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
10.
Rao, Y. K.; Fang, S.ꢀH.; Tzeng, Y.ꢀM., Differential effects of synthesized 2′ꢀoxygenated
chalcone derivatives: modulation of human cell cycle phase distribution. Bioorganic &
Medicinal Chemistry 2004, 12, 2679ꢀ2686.
11.
Buolamwini, J. K., Novel anticancer drug discovery. Current Opinion in Chemical
Biology 1999, 3, 500ꢀ509.
12.
Shoji, M.; Nakagawa, K.; Watanabe, A.; Tsuduki, T.; Yamada, T.; Kuwahara, S.;
Kimura, F.; Miyazawa, T., Comparison of the effects of curcumin and curcumin glucuronide in
human hepatocellular carcinoma HepG2 cells. Food Chemistry 2014, 151, 126ꢀ132.
13.
Vaughan, R. A.; GarciaꢀSmith, R.; Dorsey, J.; Griffith, J. K.; Bisoffi, M.; Trujillo, K. A.,
Tumor necrosis factor alpha induces Warburgꢀlike metabolism and is reversed by antiꢀ
inflammatory curcumin in breast epithelial cells. International Journal of Cancer 2013, 133,
2504ꢀ2510.
14.
Spice on the Path to Cancer Treatment. Molecules 2011, 16, 4567ꢀ4598.
15. Hosoya, T.; Nakata, A.; Yamasaki, F.; Abas, F.; Shaari, K.; Lajis, N.; Morita, H.,
Basnet, P.; SkalkoꢀBasnet, N., Curcumin: An AntiꢀInflammatory Molecule from a Curry
Curcuminꢀlike diarylpentanoid analogues as melanogenesis inhibitors. J Nat Med 2012, 66, 166ꢀ
176.
16.
Lee, K.ꢀH.; Ab. Aziz, F. H.; Syahida, A.; Abas, F.; Shaari, K.; Israf, D. A.; Lajis, N. H.,
Synthesis and biological evaluation of curcuminꢀlike diarylpentanoid analogues for antiꢀ
inflammatory, antioxidant and antiꢀtyrosinase activities. European Journal of Medicinal
Chemistry 2009, 44, 3195ꢀ3200.
23
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Page 24 of 37
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
17.
Youssef, D.; Nichols, C. E.; Cameron, T. S.; Balzarini, J.; De Clercq, E.; Jha, A., Design,
synthesis, and cytostatic activity of novel cyclic curcumin analogues. Bioorganic & Medicinal
Chemistry Letters 2007, 17, 5624ꢀ5629.
18.
Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M., Crystallographic
evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. The Journal of
biological chemistry 2006, 281, 8981ꢀ90.
19.
Oxygenases. Chemical Reviews 1996, 96, 2563ꢀ2606.
20. Rolff, M.; Schottenheim, J.; Decker, H.; Tuczek, F., CopperꢀO2 reactivity of tyrosinase
Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E., Multicopper Oxidases and
models towards external monophenolic substrates: molecular mechanism and comparison with
the enzyme. Chemical Society Reviews 2011, 40, 4077ꢀ4098.
21.
human melanogenesis. Cell. Mol. Life Sci. 2009, 66, 1493ꢀ1506.
22. Artés, F.; Castañer, M.; Gil, M. I., Revisión: El pardeamiento enzimático en frutas y
Park, H. Y.; Kosmadaki, M.; Yaar, M.; Gilchrest, B. A., Cellular mechanisms regulating
hortalizas mínimamente procesadas Review: Enzymatic browning in minimally processed fruit
and vegetables. Food Science and Technology International 1998, 4, 377ꢀ389.
23.
Jiménez, M.; Chazarra, S.; Escribano, J.; Cabanes, J.; GarcíaꢀCarmona, F., Competitive
Inhibition of Mushroom Tyrosinase by 4ꢀSubstituted Benzaldehydes. Journal of Agricultural
and Food Chemistry 2001, 49, 4060ꢀ4063.
24.
Kubo, I.; KinstꢀHori, I., Flavonols from Saffron Flower:ꢃ Tyrosinase Inhibitory Activity
and Inhibition Mechanism†. Journal of Agricultural and Food Chemistry 1999, 47, 4121ꢀ4125.
24
ACS Paragon Plus Environment

Page 25 of 37
Journal of Agricultural and Food Chemistry
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
25.
Okombi, S.; Rival, D.; Bonnet, S.; Mariotte, A.ꢀM.; Perrier, E.; Boumendjel, A.,
Discovery of Benzylidenebenzofuranꢀ3(2H)ꢀone (Aurones) as Inhibitors of Tyrosinase Derived
from Human Melanocytes. Journal of Medicinal Chemistry 2005, 49, 329ꢀ333.
26.
Parvez, S.; Kang, M.; Chung, H.ꢀS.; Bae, H., Naturally occurring tyrosinase inhibitors:
mechanism and applications in skin health, cosmetics and agriculture industries. Phytotherapy
Research 2007, 21, 805ꢀ816.
27.
Dengler, W. A.; Schulte, J.; Berger, D. P.; Mertelsmann, R.; Fiebig, H. H., Development
of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays. Anti-cancer
drugs 1995, 6, 522ꢀ32.
28.
Bao, K.; Dai, Y.; Zhu, Z.ꢀB.; Tu, F.ꢀJ.; Zhang, W.ꢀG.; Yao, X.ꢀS., Design and synthesis
of biphenyl derivatives as mushroom tyrosinase inhibitors. Bioorganic & Medicinal Chemistry
2010, 18, 6708ꢀ6714.
29.
Ismaya, W. T.; Rozeboom, H. t. J.; Weijn, A.; Mes, J. J.; Fusetti, F.; Wichers, H. J.;
Dijkstra, B. W., Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the
Tetramer Subunits and Interaction with Tropolone. Biochemistry 2011, 50, 5477ꢀ5486.
30.
Dimmock, J. R.; Padmanilayam, M. P.; Puthucode, R. N.; Nazarali, A. J.; Motaganahalli,
N. L.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Kraatz, H.ꢀB.; Prisciak, J. S.; Allen, T. M.; Santos,
C. L.; Balzarini, J.; De Clercq, E.; Manavathu, E. K., A Conformational and Structure−Activity
Relationship Study of Cytotoxic 3,5ꢀBis(arylidene)ꢀ4ꢀpiperidones and Related NꢀAcryloyl
Analogues. Journal of Medicinal Chemistry 2001, 44, 586ꢀ593.
31.
J.
Das, U.; Alcorn, J.; Shrivastav, A.; Sharma, R. K.; De Clercq, E.; Balzarini, J.; Dimmock,
R.,
Design,
synthesis
and
cytotoxic
properties
of
novel
1ꢀ[4ꢀ(2ꢀ
25
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Page 26 of 37
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
alkylaminoethoxy)phenylcarbonyl]ꢀ3,5ꢀbis(arylidene)ꢀ4ꢀpiperidones and related compounds.
European Journal of Medicinal Chemistry 2007, 42, 71ꢀ80.
32.
Nassar, A.ꢀE. F.; Kamel, A. M.; Clarimont, C., Improvingthe decisionꢀmaking process in
structural modification of drug candidates: reducing toxicity. Drug Discovery Today 2004, 9,
1055ꢀ1064.
33.
Hour, T.ꢀC.; Chen, J.; Huang, C.ꢀY.; Guan, J.ꢀY.; Lu, S.ꢀH.; Pu, Y.ꢀS., Curcumin
enhances cytotoxicity of chemotherapeutic agents in prostate cancer cells by inducing
p21WAF1/CIP1 and C/EBPβ expressions and suppressing NFꢀκB activation. The Prostate 2002,
51, 211ꢀ218.
34.
Chan, M. M.; Fong, D.; Soprano, K. J.; Holmes, W. F.; Heverling, H., Inhibition of
growth and sensitization to cisplatinꢀmediated killing of ovarian cancer cells by polyphenolic
chemopreventive agents. Journal of Cellular Physiology 2003, 194, 63ꢀ70.
35.
Koo, J. Y.; Kim, H. J.; Jung, K. O.; Park, K. Y., Curcumin inhibits the growth of AGS
human gastric carcinoma cells in vitro and shows synergism with 5ꢀfluorouracil. Journal of
medicinal food 2004, 7, 117ꢀ21.
36.
Notarbartolo, M.; Poma, P.; Perri, D.; Dusonchet, L.; Cervello, M.; D'Alessandro, N.,
Antitumor effects of curcumin, alone or in combination with cisplatin or doxorubicin, on human
hepatic cancer cells. Analysis of their possible relationship to changes in NFꢀkB activation levels
and in IAP gene expression. Cancer Letters 2005, 224, 53ꢀ65.
37.
Bava, S. V.; Puliappadamba, V. T.; Deepti, A.; Nair, A.; Karunagaran, D.; Anto, R. J.,
Sensitization of taxolꢀinduced apoptosis by curcumin involves downꢀregulation of nuclear factorꢀ
kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization. The
Journal of biological chemistry 2005, 280, 6301ꢀ8.
26
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Page 27 of 37
Journal of Agricultural and Food Chemistry
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
38.
Aggarwal, B. B.; Shishodia, S.; Takada, Y.; Banerjee, S.; Newman, R. A.; BuesoꢀRamos,
C. E.; Price, J. E., Curcumin suppresses the paclitaxelꢀinduced nuclear factorꢀkappaB pathway in
breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clinical
cancer research : an official journal of the American Association for Cancer Research 2005, 11,
7490ꢀ8.
39.
LevꢀAri, S.; Strier, L.; Kazanov, D.; MadarꢀShapiro, L.; DvoryꢀSobol, H.; Pinchuk, I.;
Marian, B.; Lichtenberg, D.; Arber, N., Celecoxib and curcumin synergistically inhibit the
growth of colorectal cancer cells. Clinical cancer research : an official journal of the American
Association for Cancer Research 2005, 11, 6738ꢀ44.
40.
Sen, S.; Sharma, H.; Singh, N., Curcumin enhances Vinorelbine mediated apoptosis in
NSCLC cells by the mitochondrial pathway. Biochemical and Biophysical Research
Communications 2005, 331, 1245ꢀ1252.
41.
Kamat, A. M.; Sethi, G.; Aggarwal, B. B., Curcumin potentiates the apoptotic effects of
chemotherapeutic agents and cytokines through downꢀregulation of nuclear factorꢀkappaB and
nuclear factorꢀkappaBꢀregulated gene products in IFNꢀalphaꢀsensitive and IFNꢀalphaꢀresistant
human bladder cancer cells. Molecular cancer therapeutics 2007, 6, 1022ꢀ30.
42.
LevꢀAri, S.; Vexler, A.; Starr, A.; AshkenazyꢀVoghera, M.; Greif, J.; Aderka, D.; Benꢀ
Yosef, R., Curcumin Augments Gemcitabine Cytotoxic Effect on Pancreatic Adenocarcinoma
Cell Lines. Cancer Investigation 2007, 25, 411ꢀ418.
43.
Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J., Experimental and
computational approaches to estimate solubility and permeability in drug discovery and
development settings. Advanced Drug Delivery Reviews 2001, 46, 3ꢀ26.
27
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Journal of Agricultural and Food Chemistry
Page 28 of 37
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
44.
Ertl, P.; Rohde, B.; Selzer, P., Fast Calculation of Molecular Polar Surface Area as a Sum
of FragmentꢀBased Contributions and Its Application to the Prediction of Drug Transport
Properties. Journal of Medicinal Chemistry 2000, 43, 3714ꢀ3717.
45.
Liang, G.; Shao, L.; Wang, Y.; Zhao, C.; Chu, Y.; Xiao, J.; Zhao, Y.; Li, X.; Yang, S.,
Exploration and synthesis of curcumin analogues with improved structural stability both in vitro
and in vivo as cytotoxic agents. Bioorganic & Medicinal Chemistry 2009, 17, 2623ꢀ2631.
46.
Zsila, F.; Bikadi, Z.; Simonyi, M., Circular dichroism spectroscopic studies reveal pH
dependent binding of curcumin in the minor groove of natural and synthetic nucleic acids.
Organic & Biomolecular Chemistry 2004, 2, 2902ꢀ2910.
47.
Caruso, F.; Rossi, M.; Benson, A.; Opazo, C.; Freedman, D.; Monti, E.; Gariboldi, M. B.;
Shaulky, J.; Marchetti, F.; Pettinari, R.; Pettinari, C., Ruthenium–Arene Complexes of
Curcumin: XꢀRay and Density Functional Theory Structure, Synthesis, and Spectroscopic
Characterization, in Vitro Antitumor Activity, and DNA Docking Studies of (pꢀ
Cymene)Ru(curcuminato)chloro. Journal of Medicinal Chemistry 2011, 55, 1072ꢀ1081.
48.
Sendovski, M.; Kanteev, M.; BenꢀYosef, V. S.; Adir, N.; Fishman, A., First Structures of
an Active Bacterial Tyrosinase Reveal Copper Plasticity. Journal of Molecular Biology 2011,
405, 227ꢀ237.
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622
623
624
625
626
627
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FIGURE CAPTIONS
Figure 1: Chemical modifications in structure of curcumin.
Figure 2: Synthesis of curcumin related α, βꢀunsaturated carbonyl based compounds, Reagents
and conditions: (i) NaOH, EtOH, Room temperature.
Figure 3: Percentage cytotoxicity of selected curcumin like synthetic compounds against various
cancer cell lines.
Figure 4: Binuclear copper binding site (S1) and its entrance (S2) region of mushroom
tyrosinase enzyme.
Figure 5: Superimposition of the best poses of compound 1a, 6a, 8a and 10a (1a, 6a, 8a and 10a
are shown as yellow, blue, pink and green respectively).
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Figure 6: The orientations of compound 1a in tyrosinase active site (1a and residues are shown
as yellow and blue respectively).
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