Synthesis and cytotoxicity evaluation of novel asymmetrical mono-carbonyl analogs of curcumin (AMACs) against vero, HeLa, and MCF7 cell lines

Article abstract of DOI:10.3390/scipharm86020025

A series of novel asymmetrical mono-carbonyl analogs of curcumin (AMACs) were synthesized and evaluated for cytotoxic activity using BSLT and MTT assay against Vero, HeLa, and MCF7 cell lines. The structures of the synthesized compounds were confirmed by FTIR,¹H-NMR,¹³C-NMR, and mass spectral data. The results of the cytotoxicity evaluation showed that the synthesized compounds exhibited moderate to very high toxic activity in BSLT (LC₅₀ value 29.80–1704.23 μM); most of the compound exhibited cytotoxic activity against HeLa cell lines, which is comparable to the activity of cisplatin (IC₅₀ value 40.65–95.55 μM), and most of the compound tested against MCF7 cell lines exhibited moderate to very high cytotoxic activity (IC₅₀ value 7.86–35.88 μM). However, the selectivity index (SI) of the compounds was low (<1–1.96). Among the synthesized compounds, compound 1b was the most cytotoxic and selective against MCF7 cell lines. It could be considered for further development to obtain the more active and selective chemotherapeutic agents against breast cancer.

Full text of DOI:10.3390/scipharm86020025

Scientia
Pharmaceutica
Article
Synthesis and Cytotoxicity Evaluation of Novel
Asymmetrical Mono-Carbonyl Analogs of Curcumin
(AMACs) against Vero, HeLa, and MCF7 Cell Lines
ID
ID
Pekik Wiji Prasetyaningrum, Anton Bahtiar and Hayun Hayun *
Faculty of Pharmacy, Universitas Indonesia, Depok 16424, West Java, Indonesia; pekikwiji@gmail.com (P.W.P.);
anton.bahtiar@gmail.com (A.B.)
* Correspondence: hayun.ms@ui.ac.id; Tel.: +62-21-727-0031
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 May 2018; Accepted: 5 June 2018; Published: 7 June 2018
Abstract: A series of novel asymmetrical mono-carbonyl analogs of curcumin (AMACs) were
synthesized and evaluated for cytotoxic activity using BSLT and MTT assay against Vero, HeLa,
and MCF7 cell lines. The structures of the synthesized compounds were confirmed by FTIR,
1H-NMR, 13C-NMR, and mass spectral data. The results of the cytotoxicity evaluation showed
that the synthesized compounds exhibited moderate to very high toxic activity in BSLT (LC50 value
29.80–1704.23
comparable to the activity of cisplatin (IC50 value 40.65–95.55
against MCF7 cell lines exhibited moderate to very high cytotoxic activity (IC50 value 7.86–35.88
µ
M); most of the compound exhibited cytotoxic activity against HeLa cell lines, which is
M), and most of the compound tested
M).
µ
µ
However, the selectivity index (SI) of the compounds was low (<1–1.96). Among the synthesized
compounds, compound 1b was the most cytotoxic and selective against MCF7 cell lines. It could be
considered for further development to obtain the more active and selective chemotherapeutic agents
against breast cancer.
Keywords: asymmetrical mono-carbonyl analogs of curcumin; AMACs; synthesis; cytotoxicity; Vero;
HeLa; MCF7; cell lines
1. Introduction
Cancer is one of the main causes of death worldwide, especially in developing countries.
Breast and cervix uterine cancer have the highest cancer incidence in Indonesian female populations
with 48,998 and 20,928 cases, respectively. The cancer mortality pofile reported in 2014 showed that
the mortality caused by the two cancers were 21.4% and 10.3% [1]. For many years, chemotherapeutic
agents have been developed and used to treat cancer. Unfortunately, there is no drug shows good
selectivity for cancer cells. Many chemotherapeutic drugs produce serious chronic and delayed
toxicities that may be irreversible, particularly in the heart, lungs, and kidneys [2]. Curcumin
(diferuloylmethane) demonstrated various biological activities such as growth suppression in a
wide variety of tumor cells, as well as chemopreventive effects on certain types of cancers with
low toxicity [
of its low chemical stability, low solubility, poor absorption, and rapid metabolism, resulting in
low bioavailability and weak in vivo biological activity [ ]. Many curcumin analogs have been
3]. Nevertheless, it has not yet been accepted as a therapeutic compound because
4–7
synthesized and investigated, such as mono-carbonyl analogs of curcumin (MACs), to improve its
bioactivity, stability, and bioavailability.
The mono-carbonyl analogs of curcumin (MACs) exhibited a potency of 10–30 times for cell
lines and cellular proteins compared to curcumin [
8–11]. Some MACs compounds with acetone and
cyclohexanone as a linker between the two phenyl rings inhibited the growth of leukemia, colon, renal,
Sci. Pharm. 2018, 86, 25; doi:10.3390/scipharm86020025
www.mdpi.com/journal/scipharm

Sci. Pharm. 2018, 86, 25
2 of 13
melanoma, ovarian, central nervous system (CNS), and prostate cancer cells better than cisplatin [12].
The MACs pharmacokinetic profile is much more stable than curcumin, resulting in higher tumor
regression [
with different constituents on the two phenyl rings have been developed and reported to show
antioxidant, anti-inflammatory, antimicrobial [13 17], and antitumor properties [18]. However, reports
8,12]. Nowadays, some of the asymmetrical mono-carbonyl analogs of curcumin (AMACs)
–
on studies of AMACs compounds as anti-cancer agents are still limited. To further explore AMACs as
anticancer compounds, we report the synthesis and in vitro cytotoxicity evaluation of novel AMACs
(1a–1e and 2a–2e, Scheme 1) against Vero, HeLa, and MCF7 Cell lines.
2. Materials and Methods
2.1. Chemistry
2.1.1. General Procedures
All solvents, chemicals, and reagents were obtained commercially and used without purification.
Purity tests of the products were performed using thin layer chromatographic (TLC) method on silica
gel 60 F254 plates (Merck, Darmstadt, Germany). Melting points were determined in the capillary tube
using melting point apparatus (Stuart Scientific, Bibby Sterilin, Staffordshire, UK) and are uncorrected.
Infrared (IR) spectra were recorded on an FTIR 8400S spectrophotometer (Shimadzu, Kyoto, Japan).
1H-NMR and 13C-NMR spectra were recorded on Nuclear Magnetic Resonance (NMR) spectrometer
(Agilent, Santa Clara, CA, USA) at 500 MHz for 1H and 125 MHz for 13C using tetramethylsilane
(TMS) as internal standard, and High-resolution mass spectra (HRMS) were measured with a Waters
LCT Premier XE (ESI-TOF) (Waters Corp., Milford, MA, USA) system in negative mode.
2.1.2. Synthesis of (2E)-2-(phenylmethylidene)cyclohexan-1-one and Analogs
The syntheses were performed according to the synthesis method used for 2-benzylidene acetone
by replacing acetone with cyclohexanone [19]. A mixture of aromatic aldehyde (0.32 mol) and
cyclohexanone (0.88 mol) was added to a solution of NaOH (10%) dropwise while stirring for 2 h.
The mixture was neutralized with dilute HCl to pH 7, the organic layer was separated, and the water
layer was extracted with 16 mL of toluene. The toluene layer was mixed with the organic layer, washed
with 16 mL of water, dried with anhydrous sodium sulfate, and evaporated using rotary vacuum
evaporator to give the crude product. The crude product was used as the starting material for the next
step without further purification.
2.1.3. Synthesis of Asymmetrical Mono-Carbonyl Analogs of Curcumin (AMACs) (1a–1e)
The synthesis of the compounds was performed by aldol condensation of
(2E)-2-(phenylmethylidene)cyclohexan-1-one or its analogs and vanillin under acidic condition.
The mixture of (2E)-2-(phenylmethylidene)cyclohexan-1-one or its analogs (0.005 mol) and vanillin
(0.01 mol) in ethanol (10 mL) was heated under reflux condition until dissolved and a drop of diluted
HCl/ethanol (1 drop: 1 mL) was added and stirred for 30 mins. The progress of the reaction was
monitored using TLC method. Upon completion, the solvent was evaporated, and the solid material
obtained was triturated with a cold mixture of glacial acetic acid/water (1:1) and filtered using
the Buchner funnel. The solid product obtained was washed with a cold mixture of glacial acetic
acid/water (1:1), dried, and purified by column chromatography with a mixture of the appropriate
ratio of n-hexane and ethyl acetate.
(2E,6E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-(phenylmethylidene)cyclohexan-1-one (1a). The
compound was a bright yellow powder, in a 50.0% yield, m.p.: 149–151 ◦C and Rf = 0.8 (ethyl acetate:
n-hexane = 1:2). FTIR (KBr)
1647 (C=O), 1587, 1531, 1448 (C=C), 1174 (C-O). 1H-NMR (500 MHz, CDCl3),
υ
max cm−1: 3211 (OH), 2999 (CH aromatic), 2837 (CH aliphatic),
δ/ppm: 1.80 (m, 2H,

Sci. Pharm. 2018, 86, 25
3 of 13
C-CH2-Ccyclohexanone); 2.93 (m, 4H, =C-CH2-Ccyclohexanone); 3.91 (s, 3H, OCH3); 5.89 (s, 1H, OH); 6.95
(d, 1H, J = 8.5, HAr); 7.00 (s, 1H, HAr; 7.09 (d, 1H, J = 8.3 Hz, HAr); 7.33 (t, 1H, J = 7.4, HAr); 7.39 (t,
2H, J = 7.3 Hz, HAr); 7.46 (d, 2H, J = 7.2 Hz, HAr); 7.75 and 7.79 (s, 1H Ar-CH=C and 1H, C=CH-Ar).
13C-NMR (100 MHz, CDCl3)
28.7 (1C, =C- H2-Ccyclohexanone), 56.0 (O-
137.5 (4C, -C=C-) 146.4 (2C, CAr-O), 190.3 (1C, C=O) [20]. HRESIMS (m/z) found 319.1346 ([M
δ
/ppm: 23.1 (1C, C-
CH2-Ccyclohexanone), 28.5 (1C, =C-CH2-Ccyclohexanone),
C
CH3), 113.4, 114.5, 124.6, 128.6, 130.4, 134.2 (10C, CAr), 136.1,
−
H]−)
calculated masses for C21H19O3: 319.1334.
(2E,6E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-[(4-methoxyphenyl)methylidene] cyclohexan-1-one
◦
(
1b). The compound was a yellow powder, in a 2.7% yield, m.p.: 133–136 C and Rf = 0.55 (ethyl
acetate: n-hexane = 1:2). FTIR (KBr)
υ
max cm−1: 3431 (OH), 3003 (CH aromatic), 2935 (CH aliphatic),
1734 (C=O), 1656, 1593 and 1512 (C=C), 1161 (C-O). 1H NMR (500 MHz, CDCl3),
δ
/ppm: 1.80 (m, 2H,
C-CH2-Ccyclohexanone); 2.92 (m, 4H, =C-CH2-Ccyclohexanone); 3.84 (s, 3H, CH3-O); 3,91 (s, 3H, CH3-O);
5.86 (s, 1H, OH); 6.92 (d, 1H, J = 8.5, HAr); 6.99 (s, 1H, HAr); 7.08 (d, 1H, J = 8.3 Hz, HAr); 7.46 (d,
2H, J = 8.3, Hz, HAr); 7.73 and 7.76 (s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz,
CDCl3)
δ/ppm: 23.1 (1C, C-CH2-Ccyclohexanone), 28.5 (2C, =C-CH2-Ccyclohexanone), 56.0 (1C, OCH3),
55.4 (1C, OCH3), 113.3, 114.4, 124.5, 128.5, 132.3 (9C, CAr), 134.4, 137.0 (4C, -C=C-), 146.4,146.5,160.0
(3C, CAr-O) 190.1 (1C, C=O) [20]. HRESIMS (m/z) found 349.1432 ([M
C22H21O4 : 349.1440.
−
H]−) calculated masses for
(2E,6E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-[(4-fluorophenyl)methylidene] cyclohexan-1-one (1c).
The compound was a light yellow powder, in a 47.3% yield, m.p.: 129–131 ◦C and Rf = 0.52 (ethyl
acetate : n-hexane = 1:2). FTIR (KBr)
1734 (C=O), 1656, 1604 and 1514 (C=C), 1220 (C-F), 1155 (C-O). 1H-NMR (500 MHz, CDCl3),
υ
max cm−1: 3313 (OH), 3003 (CH aromatic), 2939 (CH aliphatic),
δ/ppm:
1.82 (m, 2H, C-CH2-Ccyclohexanone); 2.89 (t, 2H, J = 7.1 Hz, =C-CH2-Ccyclohexanone); 2.93 (t, 2H, J = 6.1 Hz,
=C-CH2-Ccyclohexanone); 3.93 (s, 3H, CH3-O); 5.90 (s, 1H, -OH); 6.96 (d, 1H, J = 9.3 Hz, HAr); 7.00 (s, 1H,
HAr); 7.11 (d, 3H, J = 8.2 Hz, HAr); 7.44 (dd, 2H, J = 5.5 Hz, HAr); 7.75 (s, 2H, Ar-CH=C). 13C NMR
(100 MHz, CDCl3)
δ/ppm: 23.1 (1C, C-CH2-Ccyclohexanone), 28.7 (2C, =C-CH2-Ccyclohexanone), 56.0 (1C,
OCH3), 113.4, 114.5, 115.5, 115.6, 124.6, 128.5, 132.3, 134.1 (9C, CAr), 135.5, 136.0, 137.8, 146.4 (4C, C=C),
163.7 (CAr-F), 146.7, 160.7 (2C, CAr-O), 190.1 (1C, C=O) [20]. HRESIMS (m/z) found 337.1270 ([M
−
H]−) calculated masses for C21H18FO3 : 337.1240.
(2E,6E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-[(4-chlorophenyl)methylidene] cyclohexan-1-one (1d).
◦
The compound was a light yellow powder, in a 6.1% yield, m.p.: 150–154 C and Rf = 0.75 (ethyl
acetate:n-hexane = 1:2). FTIR (KBr)
1734 (C=O), 1658, 1604 and 1514 (C=C), 1163 (C-O), 833 (C-Cl). H-NMR (500 MHz, CDCl3),
υ
max cm−1: 3296 (OH), 3003 (CH aromatic), 2939 (CH aliphatic),
1
δ/ppm:
1.80 (m, 2H, C-CH2-Ccyclohexanone); 2.87 (t, 2H, J = 7.3 Hz, =C-CH2-Ccyclohexanone); 2.93 (t, 2H, J = 7.3 Hz,
=C-CH2-Ccyclohexanone); 3.92 (s, 3H, CH3-O); 5.86 (s, 1H, OH); 6.96 (d, 1H, J = 8 2 Hz, HAr); 6.99 (s,
1H, HAr); 7.08 (d, 1H, J = 8.2 Hz, HAr); 7.37 (d, 2H, J = 8.8 Hz, HAr); 7,39 (d, 2H, J = 9.3 Hz, HAr),
7.72, 7.74 (s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CDCl3)
C-CH2-Ccyclohexanone), 28.5, 28.6 (2C, =C-C
δ
/ppm: 23.0 (1C,
H2-Ccyclohexanone), 56.1 (1C, OCH3), 113.4, 114.5, 124.7, 128.5
(C15), 129.2, 131.6., 134.1 (9C, CAr), 134.6, 135.2, 136.8, 137.8 (4C, -C=C-), 134.0. (CAr-Cl), 146.6, 146.7
(2C, CAr-O), 196.1 (1C, C=O) [20]. HRESIMS (m/z) found 353.0947 ([M
C21H18ClO3 : 353.0945.
−
H]−) calculated masses for
(2E,6E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]-6-[(4-methylphenyl)methylidene] cyclohexan-1-one (1e).
The compound was a yellow powder, in a 13.2% yield, m.p.: 130–131 ◦C and Rf = 0.75 (ethyl acetate :
n-hexane = 1:2). FTIR (KBr)
(C=O), 1653, 1593 and 1462 (C=C), 1161 (C-O). H-NMR (500 MHz, CDCl3),
υ
max cm−1: 3323 (OH), 3007 (CH aromatic), 2939 (CH aliphatic), 1734
1
δ/ppm: 1.79 (m, 2H,
C-CH2-Ccyclohexanone); 2.38 (s, 3H, CH3-Ar), 2.91 (t, 2H, J = 5.0 Hz, =C-CH2-Ccyclohexanone); 2.93 (t, 2H,

Sci. Pharm. 2018, 86, 25
4 of 13
J = 5.0 Hz, =C-CH2-Ccyclohexanone), 3.92 (s, 3H, CH3-O); 5.86 (s, 1H, -OH); 6.96 (t, 3H, J = 8.2 Hz, HAr);
6.99 (s, 1H, HAr); 7.08 (d, 1H, J = 8.3 Hz, C15=CH-C18)); 7.22 (d, 2H, J = 9.0 Hz, HAr); 7.37 (d, 2H, J = 9.0
Hz, HAr), 7,74 and 7.77 (s, 1H, s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CDCl3)
δ
/ppm: 21.5 (1C, CH3-Ar), 23.1 (1C, C-CH2-Ccyclohexanone), 28.6, 28.7 (2C, =C-CH2-Ccyclohexanone),
56.0 (CH3-O), 113.4, 114.5, 124.6, 128.6, 129.2, 130.5, 133.3, 134.3 (10C, CAr), 134.6, 135.2, 136.8, 137−.8
(4C, -C=C-), 146.4, 146.5 (2C, CAr-O), 190.3 (1C, C=O) [20]. HRESIMS (m/z) found 333.1492 ([M
calculated masses for C22H21O3: 333.1491.
−
H] )
2.1.4. Synthesis of Diethylamine Mannich Base of AMACs (2a–2e)
The syntheses were performed according to the method used for the synthesis of di-Mannich
bases of curcumin and the synthesis of 2-[(2,6-dimethylmorpholin-4-yl)methyl]-4-[(E)-2-{3[(E)-2-{3-
[(2,6-dimethylmorpholin-4-yl)methyl]-4-hydroxy-5-methoxyphenyl}ethenyl]-1H-pyrazol-5yl}etheyl]-6-
methoxyphenol reported previously [21,22]. Compound 1a–1e (2 mmol) separately were dissolved
in ethanol, cooled in an ice bath, and diethylamine (5–7 mmol) and formaldehyde solution 37%
(5–7 mmol) were added slowly. The mixture was stirred for 30 min at room temperature and then
refluxed for 7–11 h. The progress of the reaction was monitored by TLC. After the reaction was
completed, the solvent was evaporated to obtain the solid residue. The residue was dissolved
in methanol (40 mL) and evaporated to a residue. The residue was then dissolved in methanol
(50 mL), warmed, and poured slowly with constant stirring into about 400 mL of cold distilled water.
The solvent was decanted, and the precipitate obtained was filtered off, washed with cold distilled
water, dried at room temperature, and then purified by column chromatography.
(2E,6E)-2-({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl}methylidene)-6-(phenylmethy◦lidene)cyclohexan-
1-one (2a). The compound was a caramel-like solid, in a 65.5% yield, m.p.: 79–80 C and Rf = 0.51
(ethyl acetate: ethanol = 1:1). FTIR (KBr)
1599 (C=C), 1269 (C-N), 1157 (C-O). H-NMR (500 MHz, CD3OD),
υ
max cm−1: 3053 (CH aromatic), 2972 (C-H), 1660 (C=O),
1
δ
/ppm: 1.18 (t, 6H, J = 7.2 Hz,
CH3-CH2-), 1.77 (m, 2H, C-CH2-Ccyclohexanone); 2.77 (q, 4H, J = 7.0 Hz, CH3-CH2-N), 2.89 and 2.94 (t,
4H, J = 7.0 Hz, =C-CH2-Ccyclohexanone), 3.85 (s, 3H, CH3-O), 3.95 (s, 2H, Ar-CH2-N), 6.92 (s, 1H, HAr),
7.04 (s, 1H, HAr); 7.33 (t, 1H, J = 8,7 Hz, HAr); 7.40 (t, 2H, J = 7.4 Hz HAr), 7.45 (d, 2H, J = 10.9 Hz,
HAr), 7.66 and 7.68 (s, 1H, s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CD3OD)
δ
/ppm: 10.9 (2C,
C
H3-CH2) 24.1 (1C, C-
C
H2-Ccyclohexanone), 29.3 and 29.9 (2C, C-CH2-Ccyclohexanone),
47.5 (1C, CH3- H2-N-), 56.9 (1C, Ar-
C
C
H2-N), 56.4 (1C, C
H3-O), 114.9, 122.2, 126.6, 126.4. 129.5,
129.7, 131.3, 133.9 (10C, CAr), 137.2, 137.3, 137.9, 139.7 (4C, -C=C-), 149,7 and 153.0 (2C, CAr-O), 191.8
−
(1C, C=O) [20] HRESIMS (m/z) found 404.2285 ([M
−
H] ) calculated masses for C26H30NO3: 404.2226.
(2E,6E)-2-({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl}methylidene)-6-[(4-methoxyphenyl)methylidene]
cyclohexan-1-one (2b). The compound was an orange sticky powder, in a 46.8% yield, m.p.: 98–99
◦C and Rf = 0.51 (ethyl acetate: ethanol = 1:1). FTIR (KBr) max cm−1: 3059 (CH aromatic), 2970
υ
(CH aliphatic), 1734 (C=O), 1556 (C=C), 1595 and 1510 (C=C aromatic) 1271 (C-N), 1155 (C-O). 1H
NMR (500 MHz, CD3OD), /ppm: 1.17 (t, 6H, J = 7.1, CH3-CH2-), 1.78 (m, 2H, C-CH2-Ccyclohexanone);
δ
2.77 (q, 4H, J = 7.1 Hz, CH3-CH2-N), 2.88 and 2.92 (t, 2H, J = 5.4 Hz, =C-CH2-Ccyclohexanone and t, 2H,
J = 5.4 Hz, =C-CH2-Ccyclohexanone), 3.81 (s, 3H, CH3-O-), 3.85 (s, 3H, CH3-O-), 3.91 (s, 2H, Ar-CH2-N),
6.89 (s, 1H, HAr), 6.96 (d, 2H, J = 8.2, HAr), 7.01 (s, 1H, HAr); 7.44 (d, 2H, J = 8.8 Hz, HAr), 7.64 and 7.65
(s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C NMR (100 MHz, CD3OD)
(1C, C- H2-Ccyclohexanone), 29.4 and 29.6 (2C, C-
H3-O), 55.8 (1C, H3-O), 57.0 (1C, Ar- H2-N), 114.8, 122.8, 126.7, 126.2, 126.7, 133.4 (9C, CAr), 129.7,
δ
/ppm: 11.0 (2C,
CH3-CH2) 24.1
C
C
H2-Ccyclohexanone), 47.4 (2C, CH3- H2-N-), 56.3 (1C,
C
C
C
C
135.6, 137.7, 139.3 (4C, -C=C-), 149.6, 152.8 and 161.7 (3C, CAr-O), 191.9 (1C, C=O) [20]. HRESIMS
(m/z) found 434.2101 ([M − H]−) calculated masses for C27H32NO4 : 434.2332.

Sci. Pharm. 2018, 86, 25
5 of 13
(2E,6E)-2-({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl}methylidene)-6-[(4-fluorophenyl)methylidene]
cyclohexan-1-one (2c). The compound was an orange powder, in a 33.01% yield, m.p.: 79–81 ◦C and
Rf = 0.48 (ethyl acetate: ethanol = 1:1). FTIR (KBr)
1734 (C=O), 1656 (C=C), 1595 and 1492 (C=C aromatic) 1271 (C-N),1224 (C-F), 1157 (C-O). 1H-NMR
(500 MHz, CD3OD), /ppm: 1.18 (t, 6H, J = 7.2 Hz, CH3-CH2-), 1.77 (m, 2H, C-CH2-Ccyclohexanone);
υ
max cm−1: 3041 (CH aromatic), 2937 (CH aliphatic),
δ
2.77 (q, 4H, J = 7.2 Hz, CH3-CH2-N), 2.87 and 2.94 (t, 2H, J = 5.5 Hz, =C-CH2-Ccyclohexanone and t,
2H, J = 5.2 Hz, =C-CH2-Ccyclohexanone); 3.85 (s, 3H, CH3-O), 3.93 (s, 2H, Ar-CH2-N), 6.92 (s, 1H, HAr),
7.03 (s, 1H, HAr); 7.14 (d-d, 2H, J = 8.8 Hz, HAr), 7,49 (d-d, 2H, J = 5.5 Hz, HAr), 7.65 and 7.66 (s, 1H,
Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CD3OD)
(1C, C- H2-Ccyclohexanone), 29.4 and 29.6 (2C, C-
H3-O), 57.0 (1C, Ar- H2-N), 114.9, 116.3, 116.5, 122.3, 126.4, 133.5, 133.9 (9C, CAr), 136.1, 137.7, 139.3,
δ/ppm: 10.9 (2C,
CH3-CH2) 24.0
C
C
H2-Ccyclohexanone), 47.4 (2C, CH3- H2-N-), 56.3 (1C,
C
C
C
139.8 (4C, -C=C-), 153.2 and 163.0 (2C, CAr-O), 165.0 (1C, CAr-F), 191.6 (1C, C=O) [20]. HRESIMS (m/z)
found 422.2178 ([M − H]−) calculated masses for C26H29FNO3 : 422.2132.
(2E,6E)-2-({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl}methylidene)-6-[(4-chlorophenyl)methylidene]
cyclohexan-1-one (2d). The compound was an orange powder, in a 76.93% yield, m.p.: 95–97 ◦C and
Rf = 0.45 (ethyl acetate: ethanol = 1:1). FTIR (KBr)
1734 (C=O), 1656 (C=C), 1597 and 1491 (C=C aromatic) 1271 (C-N), 1157 (C-O), 839 (C-Cl). H-NMR
υ
max cm−1: 3032 (CH aromatic), 2972 (CH aliphatic),
1
(500 MHz, CD3OD), δ/ppm: 1.18 (t, 6H, J = 7.2 Hz, CH3-CH2-), 1.78 (m, 2H, C-CH2-Ccyclohexanone);
2.79 (q, 4H, J = 7.2 Hz, CH3-CH2-N), 2.87 and 2.95 (t, 2H, J = 5.1 Hz, =C-CH2-Ccyclohexanone and t,
2H, J = 7.3 Hz, =C-CH2-Ccyclohexanone); 3.85 (s, 3H, CH3-O), 3.94 (s, 2H, Ar-CH2-N), 6.93 (s, 1H, HAr),
7.04 (s, 1H, HAr); 7.42 (d, 2H, J = 8.4 Hz, HAr), 7.44 (d, 2H, J = 8.7 Hz, HAr), 7.63 and 7.66 (s, 1H,
Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CD3OD)
(1C, C- H2-Ccyclohexanone), 29.2 and 29.6 (2C, C-
H3-O), 56.9 (1C, Ar- H2-N), 114.9, 122.5, 126.5, 129.6, 133.7, 132.8 (9C, CAr), 136.1, 137.7, 138.5, 139.8
δ/ppm: 11.0 (2C,
C
H3-CH2), 24.1
C
C
H2-Ccyclohexanone), 47.4 (2C, CH3-
CH2-N), 56.3 (1C,
C
C
(4C, -C=C-), 153.2 and 149.7 (2C, CAr-O), 139.9 (1C, CAr-Cl), 191.6 (1C, C=O) [20]. HRESIMS (m/z)
found 438.1881 ([M − H]−) calculated masses for C26H29ClNO3 : 438.1837.
(2E,6E)-2-({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl}methylidene)-6-[(4-methylphenyl)methylidene]
◦
cyclohexan-1-one (2e). The compound was an orange sticky powder, in a 76.9% yield, m.p.: 86–89 C and
Rf = 0.48 (ethyl acetate: ethanol = 1:1). FTIR (KBr)
1734 (C=O), 1664 (C=C), 1599 and 1498 (C=C aromatic) 1269 (C-N), 1157 (C-O). 1H-NMR (500 MHz,
CD3OD), /ppm: 1.18 (t, 6H, J = 7.2 Hz, CH3-CH2-), 1.77 (m, 2H, C-CH2-Ccyclohexanone), 2.35 (s, 3H,
υ
max cm−1: 3032 (CH aromatic), 2974 (CH aliphatic),
δ
CH3-Ar), 2.80 (q, 4H, J = 7.2 Hz, CH3-CH2-N), 2.90 and 2.94 (t, 2H, J = 7.3 Hz, =C-CH2-Ccyclohexanone
and t, 2H, J = 5.4 Hz, =C-CH2-Ccyclohexanone); 3.85 (s, 3H, CH3-O), 3.94 (s, 2H, Ar-CH2-N), 6.92 (s,
1H, HAr), 7.04 (s, 1H, HAr); 7.23 (d, 2H, J = 7.8 Hz, HAr), 7.37 (d, 2H, J = 7,9 Hz, HAr), 7.65 and 7.67
(s, 1H, Ar-CH=C and s, 1H, C=CH-Ar). 13C-NMR (100 MHz, CD3OD)
δ
/ppm: 10.9 (2C,
H2-Ccyclohexanone), 47.5 (2C,
H2-N), 114.9, 122.2, 126.3, 126.7, 130.2, 131.5, 134.1 (10C,
CH3-CH2),
21.4 (1C,
C
H3-Ar), 24.1 (1C, C-
C
H2-Ccyclohexanone), 29.3 and 29.6 (2C, C-
C
CH3- H2-N), 54 (1C,
C
C
H3-O), 56.9 (1C, Ar-
C
CAr), 134.3, 137.0, 137.6, 139.5 (4C, -C=C-), 149.7 and 152.9 (2C, CAr-O), 191.6 (1C, C=O) [20]. HRESIMS
(m/z) found 419.1925 ([M − H]−) calculated masses for C27H32NO3: 419.2384.
2.2. Cytotoxicity Test
2.2.1. Brine Shrimp Lethality Test
The assay was carried out according to the principle and protocol previously described by
Meyer [23], with slight modification. Artemia salina L. eggs were inserted into a box containing
seawater; the box was placed under UV lamp, and after 48 h the eggs hatched into larvae and ready
for the test. The compounds (1a
–1e, 2a–2e) were diluted in 10 mL seawater containing 10 larvae
(1% DMSO (v/v)) until concentrations 20, 200, 500, and 1000 ppm were reached. After 24 h, the live

Sci. Pharm. 2018, 86, 25
6 of 13
and dead shrimp were counted. The mortality rate (%) was obtained by comparing the number of
total dead larvae and the total number of larvae. The experiment was conducted in triplicate. The
concentrations (dose)-response (% mortality) data were transformed into a straight line using a logit
transformation, and the concentration required to kill 50% of the population (LC50 values) was derived
from the best fit line obtained by linear regression analysis.
2.2.2. MTT Proliferation Assay
The cytotoxic activity of the synthesized compounds was evaluated against human cervix
carcinoma (HeLa, ATCC CCL-2) cell lines, human estrogen-dependent breast carcinoma (MCF7,
ATCC HTB-22) cell lines, and kidney of an African green monkey (Vero, ATCC CCL-81) as a normal
cell lines using the methyl thiazolyl tetrazolium (MTT) method conducted according to the MTT assay
protocol published by the American Type Culture Collection (ATCC) [24]. Cisplatin was used as a
reference drug. Curcumin was also evaluated as a comparative compound on the tests against Vero
and MCF7 cell lines, and doxorubicin was evaluated as an additional comparator on the test against
MCF7 cell lines. The assay detects the reduction of yellow tetrazolium (MTT) by metabolically active
cells to be purple formazan measured using spectrophotometry [24].
The cells lines were seeded into 96-well plates at a density of 5000 cells per well, replenished
with growth media consisting of Dulbecco’s Modified Eagle’s medium (D-MEM) for Vero or Roswell
Park Memorial Institute (RPMI) 1640 medium for MCF7 and HeLa, 5% Fetal Bovine Serum (FBS),
◦
100 U/mL penicillin, and 100
µ
g/mL streptomycin. The cells were incubated at 37 C in 5% CO2
for 24 h. Then, a series concentrations of the tested compounds (1.5, 3.1, 6.2, 12.5, 25.0, 50.0, 100.0,
and 200.0 g/mL) were added to each well of the plate and incubated for 48 h. After that, 10 L fresh
µ
µ
solution of MTT reagent was added to each well, and the plate was incubated in a CO2 incubator
◦
at 37 C for 4 h. After the purple precipitate was obtained, the cells were dissolved in ethanol
and their optical density was recorded at 595 nm. The experiment was performed in triplicate.
Percent proliferation inhibition was calculated using the following formula:
ꢀ
ꢁ
(At − Ab)
(Ac − Ab)
Viability cells inhibition (%) = 100 −
× 100%
At = absorption of test compound, Ab = absorption of blank, Ac = absorption of control.
The concentration of the synthesized compounds required to inhibit 50% of the growth of the cell
lines (IC50 values) was calculated by analyzing the relationship between concentrations and percent
(%) inhibitions using GraphPad Prism 7 version 7.00 for Windows, GraphPad Software, La Jolla, CA,
USA, www.graphpad.com [25].
3. Results and Discussion
3.1. Chemistry
The novel asymmetrical mono-carbonyl analogs of curcumin (AMACs) (1a
–1e) and its
diethylamine Mannich base derivatives (2a 2e) compounds were synthesized to further explore as
–
anticancer compounds. The synthetic route of the compounds is shown in Scheme 1. The intermediate
compounds, (2E)-2-(phenylmethylidene)cyclohexan-1-one and its analogs, were synthesized by the
Claisen-Schmidt reaction between benzaldehyde or its analogs with cyclohexanone in the presence
of aqueous alkali according to the preparation method of 4-phenylbut-3-en-2-one [19]. The aldol
condensation of the intermediate compounds was obtained with vanilin with the addition of
diluted HCl/ethanol under reflux conditions for 30 min gave AMACs (1a
reaction of 1a 1e with diethylamine and formaldehyde under reflux condition in ethanol for 7–11 h
(TLC monitoring) afforded the diethylamine Mannich bases of AMACs compounds (2a–2e).
The FTIR spectra of compounds 1a
1e showed absorption bands at 3200–3500 cm−1 due to the
presence of the OH group. The bands at about 1100 cm−1 correspond to C-O-C ether, whereas the
–1e). Finally, the Mannich
–
–

Sci. Pharm. 2018, 86, 25
7 of 13
α
,
β
-unsaturated carbonyl groups of the AMACs were observed as strong bands at about 1600 cm−1
In the 1H-NMR spectra, protons of OH phenolic and OCH3 group appear as a singlet at
5.86–5.90 ppm
(1H) and as a singlet at 3.91–3.93 ppm (3H), respectively. The two protons of ethenyl chain of the
compounds appeared as two singlets at 7.72–7.79 ppm (2H), indicating an asymmetrical compound.
The FTIR spectra of compounds 2a 2e showed the disappearance of OH phenolic. The bands at
1151–1271 cm−1 correspond to C-O-C and C-N, whereas the
-unsaturated carbonyl groups of
the compounds were observed as strong bands at 1,734 cm−1. In the 1H-NMR spectra, protons of
the OCH3 group appeared as a singlet at 3.85 ppm (3H). The two protons of the ethenyl chain of
.
δ
δ
–
α,β
δ
the compounds were observed as two singlets in the range of 7.63 to 7.71 ppm (1H, respectively).
The protons of diethylamine groups were observed at 1.16–1.18 as a triplet (6H) and 2.75–2.80 ppm as
a quintet (4H), and the protons of methylene adjacent N to the phenyl ring were observed as a singlet
(2H) at 3.90–3.95 ppm. The proton signal of OH phenolic disappeared because of exchangeable with
deuterium from CD3OD used as a solvent in the experiment [20]. Furthermore, the structures of the
compounds were supported by 13C-NMR and HR-MS, which showed complete agreement with the
expected molecular structures.
Scheme 1. Synthesis of the title compounds (1a–1e and 2a–2e). Conditions: (i) r.t., 2 h.; (ii) ethanol,
reflux, diluted HCl/ethanol, 30 min.; and (iii) ethanol, reflux, 7–11 h.
3.2. Cytotoxic Activity
The cytotoxic activity of the compounds was evaluated firstly using a brine shrimp lethality test
(BSLT) method as a preliminary test. All the synthesized compounds exhibited toxic activity with LC50
value in the range of 29.80–1704.23
had moderate toxicity (LC50 value > 100–1000
activity (LC50 > 30–100 g/mL), and 1e and 2d had very high toxic activity (LC50 < 30 µg/mL) [26
µ
M (13.06–714.49
µ
g/mL) (Table 1). Compounds 1b
1c 1d 2b and 2c had high toxic
27].
, 2a, and 2e
µg/mL), compound 1a
,
,
,
µ
,
The BSLT is a rapid, inexpensive, and simple method used to predict the toxicity level of the compounds.
The method is not specific to antitumor activity. However, a positive correlation was found between
BSLT toxicity and cytotoxicity toward some cell lines [27]. Therefore, in the present study, all the
synthesized compounds were followed up to be evaluated for their potential as an anti-cancer agent.

Sci. Pharm. 2018, 86, 25
8 of 13
The cytotoxic activity of the compounds was evaluated against HeLa and Vero cell lines and
for certain selected compounds against MCF7 cell lines. The IC50 values and selectivity index (SI)
that obtained from the MTT assay are presented in Table 1 and Figure 1. Most of the synthesized
compounds (1b
–1e and 2a
–2e) exhibited cytotoxic activity against HeLa cell lines with IC50 value in
the range of 40.65–95.55
µM. In our experiment, cisplatin exhibited an IC50 value of 67.59
µM, but an
earlier study [28] reported a much lower value (12.3 µM). The reason for this difference could be due
to differences in the conditions of the assay [26]. Based on the experimental data, the cytotoxicity of
the compound was comparable with that of cisplatin. Unfortunately, all the synthesized compounds
exhibited higher cytotoxic activity on Vero cell lines compared to HeLa cell lines (IC50 value 3.94–16.15
µM). As a result, the SI of the synthesized compounds was less than 1, indicating that the synthesized
compounds were more toxic to a normal cell than to cervix carcinoma cells. Their SI values were
lower than that of cisplatin, which had an SI value of 1.26. The results also showed that most of the
diethylamine Mannich base derivatives of AMACs (2a
against Hela cell lines than that of the parent compounds (1a
study that showed that the introduction of Mannich bases enhancing the biological activity of the
compounds [29 33]. However, the increasing cytotoxicity was not selective because the effect was also
–
2e) exhibited slightly higher cytotoxic activity
–
1e). This result is in line with a previous
–
observed at Vero cell lines.
The IC50 values of compounds 2a
–
2d to Vero cell lines were in the range of 3.94–7.28
g/mL
35], the compounds were considered highly toxic to the normal cell. Therefore,
the cytotoxicity of 2a 2d against MCF7 cell lines was not evaluated. The results of the MTT assay
against MCF7 cell lines showed that compound 1a exhibited noncytotoxic activity (IC50 > 100 M),
whereas compounds 1b 1e and 2e exhibited cytotoxic activity with IC50 values in the range of
7.86–35.88 M. Based on the data obtained, the cytotoxic activity of the synthesized compound
µM
(1.73–3.17
µg/mL). Based on the cytotoxicity criteria of a pure compound (IC50 < 4 µ
or <10 µM) [34
,
–
µ
–
µ
is more selective to MCF7 cells rather than to HeLa cell lines. Among the synthesized compounds
evaluated, compound 1b was the highest cytotoxic and selective compound against MCF7 cell lines
with an IC50 value of 7.86
higher cytotoxic activity (IC50 value 7.86
10.47 and 12.85 M, respectively). However, the cytotoxicity was very low when compared with
doxorubicin, which exhibits an IC50 value <2.94 M (Table 1, Figure 1). Moreover, the selectivity index
µM (2.75
µg/mL) and an SI value of 1.96. The compound exhibited slightly
µM) than that of curcumin and cisplatin (IC50 values of
µ
µ
(SI) of the compounds to Vero and MCF7 was lower than that of curcumin and cisplatin with SI values
of 1.96, 3.00, and 6.61, respectively (Table 1). The greater the SI value, the safer the compound.
The cytotoxicity of compound 1b containing a 4-OCH3 group at the phenyl ring A (Table 1)
was higher than 1a, 1d, 1c, and 1e containing 4-H, 4-Cl, 4-F, and 4-CH3, respectively. The results
were in line with earlier reported findings that substituent on 4-position of the phenyl ring of the
AMACs or MACs significantly influenced the cytotoxicity of the compounds. In addition, a weak
electron-donating substitution in 4-position was reported to be the most favorable to the cytotoxic
activity of a compound [12,18]. Our data indicate that the electron-withdrawing substitution at
4-position reduced the cytotoxic activity. The effect differs from the electron-withdrawing substitution
at 2-position which enhances the cytotoxic activity [12].
As per a standard used earlier, pure compounds are further considered for evaluation as
chemotherapeutic agents in preclinical studies using an animal model, should have a potency of
10 µM (4 µg/mL) or less in cell culture studies and an SI value less than 2 [26,35]. Compound 1b could
be considered as a new lead compound for further development to obtain more active and selective
chemotherapeutic agents against breast cancer.

Sci. Pharm. 2018, 86, 25
9 of 13
Table 1. Cytotoxicity of the synthesized compounds against brine shrimp and Vero, Hela, and MCF7
cell lines.
IC50 (µM) (1)
SI (2)
Hela MCF7
Substuents
LC50 (µM)
Compd
No
R1
R2
BSLT
Vero
Hela
MCF7
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
2a
2b
2c
H
OCH3
F
Cl
CH3
H
OCH3
F
Cl
CH3
-
-
H
H
H
H
H
X
X
X
X
X
-
196.63
322.63
177.36
204.09
78.71
921.08
80.21
88.37
29.80
1704.23
nt
13.98 ± 0.04
15.43 ± 0.34
13.39 ± 0.39
14.06 ± 0.18
16.15 ± 0.18
4.14 ± 0.21
7.29 ± 0.12
4.23 ± 0.32
3.94 ± 0.07
15.02 ± 0.14
31.41 ± 0.41
84.66 ± 2.09
nt
>100
>100
7.86 ± 1.05
10.94 ± 0.79
35.88 ± 4.57
10.39 ± 0.36
nt
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
-
<1
1.96
1.28
<1
1.55
-
-
-
-
1.03
3.00
6.61
-
95.55 ± 7.19
49.15 ± 1.17
55.60 ± 1.49
61.19 ± 2.86
46.61 ± 1.54
69.29 ± 3.17
41.10 ± 0.16
40.65 ± 0.98
76.61 ± 4.27
nt
nt
nt
nt
2d
2e
10
14.55 ± 1.96
10.47 ± 1.10
12.85 ± 1.35
< 2.94
11 Curcumin
12 Cisplatin
13 Doxorubicin
-
-
nt
nt
67.59 ± 2.04
1.26
-
-
nt
(1)
(2)
Values are the mean
±
SD (n = 3); nt = not tested. SI = Selectivity Index = IC50 value normal cell/IC50 value
cancer cell. X = CH2-N(CH2-CH3)2.
Figure 1. Cytotoxicity of the synthesized compounds against Vero, HeLa, and MCF7 Cell lines.
nt = not tested.
Figure 2 depict the morphological analysis of untreated MCF7 cells (a) and Vero cell (b) versus
treated MCF7 and Vero cell with respect to compound 1b (7.88
(e and f), and cisplatin (12.59 M) (g and h). The figures compare the cytotoxicity of the compounds at
the same concentration against human breast cancer cells MCF7 and normal cell Vero.
µM) (c and d), curcumin (8.48 µM)
µ

Sci. Pharm. 2018, 86, 25
10 of 13
Figure 2. Morphological assessment of MCF7 cells (left) and Vero cells (right) using MTT assay.
) untreated cells; ( ) cells treated with compound 1b at 7.88 M; ( ) cells treated with curcumin
at 8.48 µM; and (g,h) cells treated with cisplatin at 12.59 µM.
(
a,
b
c,d
µ
e,f
4. Conclusions
A series of asymmetrical mono-carbonyl analogs of curcumin (AMACs) were successfully
synthesized. All the synthesized compounds exhibited moderate to very high toxicity based on
BSLT, most of them exhibited comparable cytotoxic activity with cisplatin against HeLa cell lines,
and the selected compound exhibited moderate to very high cytotoxic activity against MCF7 cell lines.
However, all compounds had low SI (<1–1.96). Among the synthesized compounds, compound 1b
showed the highest cytotoxic and selective activity against MCF7 cell lines. This compound could be
considered for further development to obtain the more active and selective chemotherapeutic agents
against breast cancer.

Sci. Pharm. 2018, 86, 25
11 of 13
Author Contributions: H.H. and A.B. conceived and designed the experiments; P.W.P. performed the experiments;
H.H. and P.W.P. analyzed the data; and H.H. and P.W.P. wrote the paper.
Acknowledgments: We thank the Directorate of Research and Community Services, Directorate of Higher
Education of the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia for the
financial support of this research (PDUPT Research Grant, 2017). We are also thankful to Chemistry Study
Program, Faculty of Natural Sciences, Bandung Institute of Technology (ITB), Bandung, Indonesia for recording
NMR spectral data; to Central Laboratory of Universitas Padjadjaran, Bandung, Indonesia for recording HR-MS
data; to the Department of Chemistry, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia and Institute
for Research and Community Service, Primate Research Center, Bogor Agricultural University, Bogor, Indonesia,
for MTT viability assay; and to Center for Chemistry Research, the Indonesian Institute of Sciences, Serpong,
Tangerang, Indonesia for BSLT assay.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
The World Health Organization. Cancer Country Profile, 2014. Available online: http://www.who.int/
cancer/country-profiles/idn_en.pdf?ua (accessed on 11 December 2017).
2.
Roche, V.F. Cancer and chemotherapy. In Foye’s Principles of Medicinal Chemistry, 11th ed.; Lemke, T.L.,
Williams, D.A., Roche, V.F., Zito, S.W., Eds.; Lippincott Wiliams and Wilkins: Baltimore, MD, USA, 2016;
pp. 1199–1266.
3.
4.
Basnet, P.; Skalko-Basne, N. Curcumin: An Anti-Inflammatory Molecule from a Curry Spice on the Path to
Cancer Treatment. Molecules 2011, 16, 4567–4598. [CrossRef] [PubMed]
Anand, P.; Thomas, S.G.; Kunnumakkara, A.B.; Sundaram, C.; Harikumar, K.B.; Sung, B.; Tharakan, S.T.;
Misra, K.; Priyadarsini, I.K.; Rajasekharan, K.N.; et al. Biological activities of curcumin and its analogues
(Congeners) made by man and Mother Nature. Biochem. Pharmacol. 2008, 76, 1590–1611. [CrossRef]
[PubMed]
5.
6.
Anand, P.; Kunnumakkara, A.B.; Newman, R.A.; Aggarwal, B.B. Bioavalibility of curcumin: Problems and
promises. Mol. Pharm. 2007, 4, 807–818. [CrossRef]
Sharma, R.A.; Euden, S.A.; Platton, S.L.; Cooke, D.N.; Shafayat, A.; Hewitt, H.R.; Marczylo, T.H.; Morgan, B.;
Hemingway, D.; Plummer, S.M.; et al. Phase I clinical trial of oral curcumin: Biomarkers of systemic activity
and compliance. Clin. Cancer Res. 2004, 10, 6847–6854. [CrossRef] [PubMed]
7.
Garcea, G.; Berry, D.P.; Jones, D.J.; Singh, R.; Dennison, A.R.; Farmer, P.B.; Sharma, R.A.; Steward, W.P.;
Gescher, A.J. Consumption of the putative chemopreventive agent curcumin by cancer patients: Assessment
of curcumin levels in the colorectum and their pharmacodynamic consequences. Cancer Epidemiol.
Biomark. Prev. 2005, 14, 120–125.
8.
9.
Shetty, D.; Kim, Y.J.; Shim, H.; Snyder, J.P. Eliminating the Heart from the Curcumin Molecule: Monocarbonyl
Curcumin Mimics (MACs). Molecules 2015, 20, 249–292. [CrossRef] [PubMed]
Ohori, H.; Yamakoshi, H.; Tomizawa, M.; Shibuya, M.; Kakudo, Y.; Takahashi, A.; Takahashi, S.; Kato, S.;
Suzuki, T.; Ishioka, C.; et al. Synthesis and biological analysis of new curcumin analogues bearing an
enhanced potential for the medicinal treatment of cancer. Mol. Cancer Ther. 2006, 5, 2563–2571. [CrossRef]
[PubMed]
10. Yin, S.; Zheng, X.; Yao, X.; Wang, Y.; Liao, D. Synthesis and Anticancer Activity of Mono-Carbonyl Analogues
of Curcumin. J. Cancer Ther. 2013, 4, 113–123. [CrossRef]
11. Adams, B.K.; Ferstl, E.M.; Davis, M.C.; Herold, M.; Kurtkaya, S.; Camalier, R.F.; Hollingshead, M.G.;
Kaur, G.; Sausville, E.A.; Rickles, F.R.; et al. Synthesis and biological evaluation of novel curcumin analogs
as anti-cancer and anti-angiogenesis agents. Bioorg. Med. Chem. 2004, 12, 3871–3883. [CrossRef] [PubMed]
12. 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.
Bioorg. Med. Chem. 2009, 17, 2623–2631. [CrossRef] [PubMed]
13. Zhao, C.; Liu, Z.; Liang, G. Promising curcumin-based drug design: Monocarbonyl analogues of curcumin
(MACs). Curr. Pharm. Res. 2013, 19, 2114–2135. [CrossRef]

Sci. Pharm. 2018, 86, 25
12 of 13
14. Zhang, Y.; Zhao, L.; Wu, J.; Jiang, X.; Dong, L.; Xu, F.; Liang, G. Synthesis and evaluation of a series of novel
asymmetrical curcumin analogs for the treatment of inflammation. Molecules 2014, 19, 7287–7307. [CrossRef]
[PubMed]
15. Zhang, Y.; Jiang, X.; Peng, K.; Chen, C.; Fi, L.; Wang, Z.; Feng, J.; Liu, Z.; Zhang, H.; Liang, G.; et al. Discovery
and evaluation of novel anti-inflammatory derivatives of natural bioactive curcumin. Drug Des. Dev. Ther.
2014, 8, 2161–2171. [CrossRef]
16. Aluwi, M.F.M.F.; Rullah, K.; Yamin, B.M.; Leong, S.W.; Bahari, M.N.A.; Lim, S.J.; Lam, K.W. Synthesis of
unsymmetrical monocarbonyl curcumin analogues with potent inhibition on prostaglandin E2 production
in LPS-induced murine and human macrophages cell lines. Bioorg. Med. Chem. Lett. 2016, 26, 2531–2538.
[CrossRef] [PubMed]
17. Bandgar, B.P.; Kinkar, S.N.; Chavan, H.V.; Jalde, S.S.; Shaikh, R.U.; Gacche, R.N. Synthesis and biological
evaluation of asymmetric indole curcumin analogs as potential anti-inflammatory and antioxidant agents.
J. Enzym. Inhib. Med. Chem. 2014, 29, 7–11. [CrossRef] [PubMed]
18. Li, Q.; Chen, J.; Luo, S.; Xu, J.; Huang, Q.; Liu, T. Synthesis and assessment of the antioxidant and antitumor
properties of asymmetric curcumin analogues. Eur. J. Med. Chem. 2015, 93, 461–469. [CrossRef] [PubMed]
19. Furniss, B.S.; Hannaford, A.J.; Smith, P.W.G.; Tatchell, A.R. Vogel’s Textbook of Practical Organic Chemistry,
5th ed.; Longman Group Ltd.: London, UK, 1989; pp. 1032–1033.
20. Silverstein, R.M.; Webster, F.X.; Kiemle, D.J. Spectrometric Identification of Organic Compounds, 7th ed.;
John Wiley & Sons, Inc.: New York, NY, USA, 2005.
21. Geschickter, C.F.; Meadow, J.R. Curcumin Derivatives. U.S. Patent 3,479,345, 18 November 1969.
22. Untung, J.; Iskandarsyah, I.; Hayun, H. 2-[(2,6-Dimethylmorpholin-4-yl)methyl]-4-[(E)-2-{3-[(E)-2-{3-[(2,6-
dimethylmorpholin-4-yl)methyl]-4-hydroxy-5-methoxyphenyl}ethenyl]-1H-pyrazol-5-yl}ethenyl]-6-
methoxyphenol. Molbank 2017, 3, M949. [CrossRef]
23. Meyer, B.N.; Ferrigni, N.R.; Putnam, J.E.; Jacobson, L.B.; Nichols, D.E.; McLaughlin, J.L. Brine shrim.p.:
A convenient general bioassay for active plant constituents. Planta Med. 1982, 45, 31–34. [CrossRef] [PubMed]
24. MTT Cell Proliferation Assay.
Available online:
https://www.atcc.org/~/media/
DA5285A1F52C414E864C966FD78C9A79.ashx (accessed on 5 October 2017).
25. GraphPad Software, Inc. Available online: www.graphpad.com (accessed on 13 December 2017).
26. Badisa, R.B.; Darling-Reed, S.F.; Joseph, P.; Cooperwood, J.S.; Latinwo, L.M.; Goodman, C.B. Selective
Cytotoxic Activities of Two Novel synthetic Drugs on Hyman Breast Carcinoma MCF-7 Cells. Anticancer Res.
2009, 29, 2993–2996. [PubMed]
27. Chisalberti, E.L. Detection and Isolation of Bioactive Natural Products. In Bioactive Natural Products, Detection,
Isolation, and Structur Determination, 2nd ed.; Colegate, S.M., Molyneux, R.J., Eds.; Taylor and Francis Groups,
LLC: Boca Raton, FL, USA, 2008; p. 18.
28. Aydin, S.; Becit, M.; Basaran, A.; Basaran, N. Effects of curcumin on cisplatin cytotoxicity in HeLa cells.
Toxicol. Lett. 2016, 258, S259–S260. [CrossRef]
29. Bala, S.; Sharma, N.; Kajal, A.; Kamboj, S.; Saini, V. Mannich Bases: An Important Pharmacophore in Present
Scenario. Int. J. Med. Chem. 2014, 2014, 191072. [CrossRef] [PubMed]
0
30. Liu, C.; Dong, L.; Wang, S.; Wang, Q. Synthesis and antiproliferative activity of pterostilbene and 3 -methoxy
pterostilbene Mannich base derivatives against Hela cells. Mol. Divers. 2015, 19, 737–743. [CrossRef]
[PubMed]
31. Yerdelen, K.O.; Gul, H.I.; Sakagami, H.; Umemura, N. Synthesis and biological evaluation of
1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one and its aminomethyl derivatives. J. Enzym. Inhib.
Med. Chem. 2015, 30, 383–388. [CrossRef] [PubMed]
32. Reddy, M.V.B.; Su, C.-R.; Chiou, W.-F.; Liu, Y.-N.; Chen, R.Y.; Bastow, K.F.; Lee, K.-H.; Wu, T.-S. Design,
synthesis, and biological evaluation of Mannich bases of heterocyclic chalcone analogs as cytotoxic agents.
Bioorg. Med. Chem. 2008, 16, 7358–7370. [CrossRef] [PubMed]
33. Roman, G. Mannich bases in medicinal chemistry and drug design. Eur. J. Med. Chem. 2015, 89, 743–816.
[CrossRef] [PubMed]

Sci. Pharm. 2018, 86, 25
13 of 13
34. Schmitz, F.J.; Bourden, B.F.; Toth, S.I. Antitumor and Cytotoxic Compounds from Marine Organisms.
In Marine Biotechnology, Pharmaceutical and Bioactive Natural Products; Attaway, D.H., Zaborsky, O.R., Eds.;
Plenum Press: New York, NY, USA, 1993; Volume 1, p. 198.
35. Burger, A.M.; Fiebig, H.H. Preclinical Screening for New Anticancer Agents. In Handbook of Anticancer
Pharmacokinetics and Pharmacodynamics, Cancer Drug Discovery and Development; Figg, W.D., McLeod, H.L.,
Eds.; Humana Press Inc.: Totowa, NJ, USA, 2004; pp. 36–37.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).