Cytotoxicity of some synthetic bis(arylidene) derivatives of cyclic ketones towards cisplatin-resistant human ovarian carcinoma cells

Article abstract of DOI:10.1007/s00044-020-02532-5

Symmetrical α,α?-bis(arylidene)ketones were prepared by acid-catalyzed aldol condensations between aliphatic ketones (e.g., cyclopentanone, 4-alkylcyclohexanones, tetrahydropyran-4-one, and tetrahydrothiopyran-4-one) and two equivalents of an aromatic hydroxyaldehyde (e.g., 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, vanillin, isovanillin, and 3-fluoro-4-hydroxybenzaldehyde). Most of the compounds were cytotoxic towards the cisplatin-resistant human ovarian cancer cell line A2780-CP70 as well as the non-resistant line A2780.

Full text of DOI:10.1007/s00044-020-02532-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2020) 29:935–941
https://doi.org/10.1007/s00044-020-02532-5
ORIGINAL RESEARCH
Cytotoxicity of some synthetic bis(arylidene) derivatives of cyclic
ketones towards cisplatin-resistant human ovarian carcinoma cells
Hinal Patel1 Begum Mothia1 Jaison Patel1 Olatunde Fasanya1 Kartheek Sooda2 Farideh Javid2 Peter B. Wyatt1
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Received: 16 July 2019 / Accepted: 13 March 2020 / Published online: 17 April 2020
© The Author(s) 2020
Abstract
Symmetrical α,αʹ-bis(arylidene)ketones were prepared by acid-catalyzed aldol condensations between aliphatic ketones (e.g.,
cyclopentanone, 4-alkylcyclohexanones, tetrahydropyran-4-one, and tetrahydrothiopyran-4-one) and two equivalents of an
aromatic hydroxyaldehyde (e.g., 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, vanillin, isovanillin, and 3-fluoro-4-
hydroxybenzaldehyde). Most of the compounds were cytotoxic towards the cisplatin-resistant human ovarian cancer cell line
A2780-CP70 as well as the non-resistant line A2780.
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Keywords Ovarian cancer Dienone Cytotoxicity Curcumin Carboplatin Cisplatin
Introduction
prepared a group of symmetrically substituted bis(aryli-
dene)ketones containing new examples along with some
previously known compounds. We have then examined the
ability of these substances to inhibit the growth of both
A2780 human ovarian carcinoma cells and the related
cisplatin-resistant cells, A2780-CP70.
Ovarian cancer is estimated to kill more than 180,000
women each year worldwide (World Health Organization
2019). A particular challenge is the frequent development of
resistance towards cisplatin, one of the important che-
motherapeutic agents (Galluzzi et al. 2014). Curcumin (1),
which is a major constituent of the spice turmeric, inhibits
the proliferation of cisplatin-resistant ovarian cancer cells
by multiple mechanisms (Weir et al. 2007). Although cur-
cumin has low toxicity, its low potency, rapid metabolism,
and low oral availability limit its clinical use and have
prompted the evaluation of analogues, particularly α,α′-bis
(arylidene)ketones (Adams et al. 2004, Selvendiran et al.
2007). Such compounds are easily prepared through che-
mical synthesis (Scheme 1) and considerable structural
diversity can be obtained by combining sets of readily
available starting materials. In our present study we have
Material and methods
‘Petrol’ refers to the fraction of petroleum spirit with bp
40–60 °C. Flash chromatography was performed on BDH
silica gel (33–70 μm). All new compounds were >95% pure
as assessed by TLC and high field NMR. Melting points
were determined using a Reichert hot stage microscope and
are uncorrected. NMR spectra were recorded on Jeol EX270
and Bruker AMX400 spectrometers. Mass spectra were
provided by the EPSRC NMSF at Swansea University. Low
resolution, EI and CI measurements were performed on a
Quattro II triple quadrupole instrument. High resolution
measurements were made using a Finnigan MAT 900 XCT
high resolution spectrometer. DEPT and/or HSQC experi-
* Farideh Javid
fajavid@hud.ac.uk
ments were used to assist the assignment of all reported 13
NMR spectra.
C
* Peter B. Wyatt
p.b.wyatt@qmul.ac.uk
Aldol condensations to prepare the bis(arylidene)ketones
were performed by analogy with existing literature (Sard-
jiman et al. 1997, Youssef et al. 2004) using the conditions
and product isolation procedures exemplified for the pre-
paration of ketone 10 below. The syntheses of the following
aldol products have been reported previously: 2 (Leow et al.
1
Department of Chemistry, School of Biological and Chemical
Sciences, Queen Mary University of London, Joseph Priestley
Building, Mile End Road, London E1 4NS, UK
2
Department of Pharmacy, School of Applied Sciences, University
of Huddersfield, Huddersfield HD1 3DH, UK

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Medicinal Chemistry Research (2020) 29:935–941
Scheme 1 General scheme for
formation of bis(arylidene)
ketone analogs of curcumin (1)
by aldol condensation
2014), 3 (Sardjiman et al. 1997), 5 (Leow et al. 2014), 6
(Sardjiman et al. 1997), 8 (Inayama et al. 1976), 14 (Bairam
et al. 2017), 16 (Costi et al. 2004, Artico et al. 1998), 19
(Tan et al. 2014).
2,6-bis(3-fluoro-4-hydroxybenzylidene)-4-
methylcyclohexanone (9)
3-Fluoro-4-hydroxybenzaldehyde (0.9 g, 6.42 mmol) and
4-methylcyclohexanone (0.39 ml, 3.21 mmol) gave title
compound 9 as a yellow powder (0.36 g, 31%); mp
163–165 °C (from EtOH); vmax (KBr)/cm–1 3134 (O–H),
1652 (C=O), 1589 (C=C); δH (400 MHz, DMSO-d6) 1.05
(3H, d, J 7 Hz, CH3), 1.78–1.82 (1H, m, H-4), 2.50–2.53
(2H, m, overlapping with DMSO-d5, H-3ax, 5ax), 2.94 (2H,
dd, J 16, 3 Hz, H-3eq, 5eq), 7.03 (2H, t, J 9 Hz, H-5′, 5′′),
7.23 (2H, dd, J 2, 9 Hz, 6′, 6′′), 7.37 (2H, dd, J 13, 2 Hz, H-
2′, 2′′), 7.51 (2H, s, CH=), 10.4 (2H, br s, OH); δC
(101 MHz, DMSO-d6), 21.3 (Me), 28.6 (C-4), 35.6 (C-3, 5),
117.7 (d, J 3 Hz, CH), 118.0 (d, J 18 Hz, CH), 127.0 (d, J
6 Hz, Cquat), 127.7 (d, J 3 Hz, CH), 133.6 (Cquat), 135.1 (d, J
2 Hz, =CH), 145.9 (d, J 12 Hz, C–O), 150.6 (d, J 241 Hz,
CF), 188.2 (C=O); δF (376 MHz, DMSO-d6) –132.3;
HRMS (ESI) m/z found 357.1294; calculated for
C21H19F2O3 (M + H+) 357.1297.
2,5-bis(3-fluoro-4-hydroxybenzylidene)
cyclopentanone (4)
3-Fluoro-4-hydroxybenzaldehyde (0.90 g, 6.42 mmol) and
cyclopentanone (0.29 ml, 3.21 mmol) gave the title com-
pound 4 as a green powder (0.19 g, 18%); mp 289 °C
(decomp, from EtOH); vmax (KBr)/cm–1 3373 (O–H), 1653
(C=O), 1578 (C=C); δH (400 MHz, DMSO-d6), 3.09 (4H,
s, H-3,4), 7.12 (2H, t, J 9 Hz, H-5′, 5′′), 7.40 (2H, s, CH=),
7.44 (2H, d, J 9 Hz, H-6ʹ, 6), 7.55 (2H, d, J 13 Hz, H-2′, 2′′),
10.6 (2H, br s, OH); δC (101 MHz, DMSO-d6) 25.7 (C-3, 4),
118.0 (d, J 3 Hz, CH), 118.1 (d, J 12 Hz, CH), 127.2 (d, J
6 Hz, Cquat), 128.0 (d, J 3 Hz, CH), 131.5 (d, J 2 Hz, CH),
135.8 (=Cquat), 146.5 (d, J 13 Hz, C–O), 150.8 (d, J 242 Hz,
CF), 194.8 (C=O); δF (376 MHz, DMSO-d6), –132.0;
HRMS (ESI) m/z found 329.0990; calculated for
C19H15F2O3 (M + H+) 329.0984.
2,6-bis(4-hydroxybenzylidene)-4-
ethylcyclohexanone (10)
2,6-bis(3-fluoro-4-hydroxybenzylidene)
cyclohexanone (7)
4-Hydroxybenzaldehyde (1.22 g, 10 mmol) and 4-
ethylcyclohexanone (0.63 g, 5 mmol) were stirred toge-
ther in methanol (5 mL) at 30 °C until a clear solution
was obtained. Concentrated hydrochloric acid (1 mL)
was added and the mixture was stirred at 30 °C for 1 day.
The mixture was then treated with cold AcOH-water
(20 mL; 1:1), and filtered. The precipitate was purified by
recrystallization from methanol in the presence of char-
coal to give 2,6-bis(4-hydroxybenzylidene)-4-ethylcy-
clohexanone (10) (0.83 g, 50%), as yellow crystals; mp
229–231 °C (from MeOH); vmax (Nujol)/cm–1 3363
(OH), 1627 (C=O), 1573 (C=C); δH (400 MHz, DMSO-
d6), 0.85 (3H, t, J 7 Hz, CH3CH2), 1.37 (2H, quintet, J
7 Hz, CH2CH3), 1.51–1.59 (1H, m, H-4), 2.51 (2H, t, J
13 Hz, H-3ax, 5ax), 3.00 (2H, d, J 13 Hz, H-3eq, 5eq), 6.87
(4H, d, J 9 Hz, H-3′, 5′, 3′′, 5′′), 7.41 (4H, d, J 9 Hz, H-
3-Fluoro-4-hydroxybenzaldehyde (0.90 g, 6.42 mmol) and
cyclohexanone (0.33 mL, 3.21 mmol), and methanol (3 mL)
gave the title compound 7 as a green powder (0.05 g, 5%), mp
236–240 °C (from EtOH); vmax (KBr)/cm–1 3366 (O–H), 1653
(C=O), 1590 (C=C); δH (400 MHz, DMSO-d6), 1.73 (2H,
quintet, J 6 Hz, H-4), 2.86 (4H, t, J 6 Hz, H-3, 5), 7.03 (2H, t, J
9 Hz, H-5′, 5′′), 7.24 (2H, d, J 8 Hz, 6′, 6′′), 7.37 (2H, dd, J 13,
2 Hz, H-2′, 2′′), 7.52 (2H, s, CH=), 10.4 (2H, br s, OH); δC
(101 MHz, DMSO-d6) 22.3 (C-4), 27.7 (C-3, 5), 117.7 (d, J
3 Hz, CH), 118.0 (d, J 18 Hz, CH), 127.0 (d, J 6 Hz, Cquat
)
127.7 (d, 3 Hz, CH), 134.5 (=Cquat), 134.8 (CH=), 145.9 (d, J
12 Hz, C–O), 150.6 (d, J 241 Hz, CF), 188.4 (C=O). δF
(376 MHz, DMSO-d6) –132.3; HRMS (ESI) m/z found
343.1141; calculated for C20H17F2O3 (M + H+) 343.1140.

Medicinal Chemistry Research (2020) 29:935–941
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2′, 6′, 2′′, 6′′), 7.57 (2H, s, =CH), 9.94 (2H, s, OH); δC
(101 MHz, DMSO-d6), 11.3 (CH3CH2), 27.8 (CH3CH2),
33.4 (C-3, 5), 34.9 (C-4), 115.5 (CH), 126.4 (Cquat),
132.31 (CH), 132.35 (CH), 136.1 (CH), 158.2 (C–O),
188.4 (C=O); HRMS (ESI) m/z found 335.1641;
C22H23O3 (M + H+) requires 335.1642.
heating at 30 °C for 15 h and 60 °C for 6 h followed by
recrystallization (EtOH) gave the title compound 13 as a
yellow-green powder (0.16 g, 20%), mp 183–185 °C; vmax
(KBr)/cm–1 3153 (O–H), 1653 (C=O), 1589 (C=C); δH
(400 MHz, DMSO-d6), 0.86 (3H, t, J 7 Hz, CH3CH2), 1.39
(2H, quintet, J 7 Hz, CH3CH2), 1.57–1.62 (1H, m, H-4),
2.45–2.60 (2H, m, overlapping with DMSO-d5, H-3ax,
5ax), 2.98 (2H, dd, J 16, 3 Hz, H-3eq, 5eq), 7.03 (2H, t, J
9 Hz, H-5′, 5′′), 7.21 (2H, dd, J 9, 2 Hz, H-6′, 6′′), 7.38
(2H, dd, J 13, 2 Hz, H-2′, 2′′), 7.53 (2H, s, CH=C); δC
(101 MHz, DMSO-d6), 11.3 (CH3CH2), 27.7 (CH3CH2),
33.1 (C-3,5), 34.8 (C-4), 117.7 (d, J 3 Hz, C-5′, 5′′), 118.0
(d, J 18 Hz, C-2′, 2′′), 127.0 (C-1′, 1′′), 127.7 (C-6′, 6′′),
133.6 (=Cquat), 135.1 (=CH), 145.9 (d, J 12 Hz, C-4′, 4′′),
150.6 (d, J 241 Hz, C–F), 188.5 (C=O); δF (376 MHz,
DMSO-d6), –132.3; HRMS (ESI) m/z found 371.1451;
calculated for C22H21F2O3 (M + H+) 371.1453.
4-ethyl-2,6-bis(3-hydroxy-4-methoxybenzylidene)
cyclohexanone (11)
3-Hydroxy-4-methoxybenzaldehyde (1.52 g, 10 mmol) and
4-ethylcyclohexanone (5 mmol) gave the title compound 11
as yellow crystals (0.16 g, 8%), mp 182–184 °C (from
EtOH); vmax (KBr)/cm–1 3423 (O–H), 1647 (C=O), 1571
(C=C), δH (400 MHz, DMSO-d6), 0.87 (3H, t, J 7 Hz, Me),
1.35–1.42 (2H, m, CH2Me), 1.51–1.63 (1H, m, H-4), 2.52
(2H, t, J 16 Hz, H-3ax, 5ax), 3.01 (2H, d, J 15 Hz, H-3eq, 5eq),
3.82 (6H, s, OMe), 6.96–7.03 (6H, m, Ar-H), 7.51 (2H,
s, =CH), 9.21 (1H, br s, OH); δC (101 MHz, DMSO-d6),
11.3 (CH3CH2), 27.9 (CH3CH2), 33.4 (C-3, 5), 34.9 (C-4),
55.6 (OMe), 112.0 (CH), 117.1 (CH), 122.9 (CH), 128.3
(Cquat), 133.1 (Cquat), 136.1 (=CH), 146.3 (C–O), 148.5
(C–O), 188.6 (C=O); HRMS (ESI) m/z found 395.1851;
calculated for C24H27O5 (M + H+) 395.1853.
4-tert-butyl-2,6-bis(4-hydroxy-3-
methoxybenzylidene)cyclohexanone (15)
Vanillin (1.52 g, 10 mmol) and 4-tert-butylcyclohexanone
(0.77 g, 5 mmol) gave the title compound 15 (0.61 g,
30%) as yellow crystals, mp 194–196 °C (from EtOH);
vmax (KBr)/cm–1 3337 (O–H), 1647 (C=O), 1589 (C=C),
δH (400 MHz, DMSO-d6) 0.94 (9H, s, Me3C), 1.32–1.42
(1H, m, H-4), 2.42–2.52 (2H, m, overlapping with
DMSO-d5 signal, H-3ax,5ax), 3.08 (2H, d, J 14 Hz, H-3eq,
5eq), 3.81 (6H, s, OMe), 6.88 (2H, d, J 8 Hz, H-5′, 5′′),
7.04 (2H, dd, J 8, 2 Hz, H-6′, 6′′), 7.12 (2H, d, J 2 Hz, H-
2′, 2′′), 7.56 (2H, d, J 2 Hz, =CH), 9.52 (2H, br s, OH); δC
(101 MHz, DMSO-d6), 27.1 (Me3C), 29.1 (C-3, 5), 32.1
(Me3C), 43.8 (C-4), 55.6 (MeO), 114.7 (C-2′, 2′′), 115.6
(C-5′, 5′′), 124.2 (C-6′, 6′′), 126.9 (Cquat), 133.3 (Cquat),
136.1 (=CH), 147.4 (C–O), 147.8 (C–O), 188.5 (C=O);
HRMS (ESI) m/z found 423.2163; calculated for
C26H31O5 (M + H+) 423.2166.
4-ethyl-2,6-bis(4-hydroxy-3-methoxybenzylidene)
cyclohexanone (12)
Vanillin (1.52 g, 10 mmol) and 4-ethylcyclohexanone
(0.63 g, 5 mmol) reacted to give a crude product which
was dissolved in chloroform, then applied to a plug of silica
gel. Elution with MeCN, pooling and evaporation of
appropriate fractions, then recrystallization from EtOH gave
the title compound 12 (0.20 g, 10%) as a dark yellow
powder mp 183–184 °C; vmax (KBr)/cm–1 3337 (O–H),
1647 (C=O), 1577 (C=C), δH (400 MHz, DMSO-d6), 0.86
(3H, t, J 7 Hz, Me), 1.37–1.40 (2H, m, CH2), 1.51–1.64
(1H, m, H-4), 2.50–2.57 (2H, m, overlapping with DMSO-
d5, H-3ax, 5ax), 3.03 (2H, d, J 16 Hz, H-3eq, 5eq), 3.82 (6H, s,
OMe), 6.86 (2H, d, J 8 Hz, H-5′, 5ʺ), 7.03 (2H, dd, J 8,
1.5 Hz, H-6′, 6ʺ), 7.11 (2H, d, J 1.5 Hz, H-2′, 2ʺ), 7.58 (2H,
s, CH=), 9.51 (2H, s, OH); δC (101 MHz, DMSO-d6), 11.3
(CH3CH2), 27.9 (CH3CH2), 33.4 (C-3, 5), 35.0 (C-4), 55.6
(OMe), 114.9 (C-2′), 115.6 (C-5′), 124.1 (C-6′), 126.9
(Cquat), 132.6 (Cquat), 136.4 (=CH), 147.4 (C–O), 147.8
(C–O), 188.5 (C=O); HRMS (ESI) m/z found 395.1855;
calculated for C24H27O5 (M + H+) 395.1853.
2,5-bis(3-hydroxy-4-methoxybenzylidene)
tetrahydro-4H-pyran-4-one (17)
3-Hydroxy-4-methoxybenzaldehyde (1.52 g, 10 mmol),
tetrahydro-4H-pyran-4-one (0.51 g, 5.09 mmol), MeOH
(5 mL) concentrated HCl (1 mL) were heated together at
30 °C for 15 h. 50% Aqueous AcOH (20 mL) was added
and the crude product was filtered off, then recrystallized
from EtOH to give the title compound 17 as yellow crystals
(0.36 g, 20%), mp 272–273 °C; vmax (KBr)/cm–1 3294
(O–H), 1663 (C=O), 1597 (C=C); δH (400 MHz, DMSO-
d6) 3.83 (6H, s, OMe), 4.88 (4H, s, OCH2), 6.87–6.91 (4H,
m), 7.01 (2H, d, J 8 Hz), 7.53 (2H, s, =CH), 9.28 (2H, br s,
OH); δC (101 MHz, DMSO-d6) 55.6 (OMe), 67.8 (OCH2),
112.1 (CH), 117.3 (CH), 123.3 (CH), 127.1 (Cquat), 131.5
2,6-bis(3-fluoro-4-hydroxybenzylidene)-4-
ethylcyclohexanone (13)
4-Ethylcyclohexanone (0.295 mL, 2.14 mmol) and 3-
fluoro-4-hydroxybenzaldehyde (0.90 g, 6.42 mmol) after

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(Cquat), 135.0 (=CH), 146.5 (C–O), 149.2 (C–O), 184.4
(C=O); HRMS (ESI) m/z found 369.1332; calculated for
C21H21O6 (M + H+) 399.1333.
Results and discussion
The aldol condensation reaction between aromatic alde-
hydes and aliphatic ketones in appropriate stoichiometric
proportions gives α,α′-bis(arylidene)ketones (Scheme 1).
Use of hydroxybenzaldehyde starting materials readily
allows the construction of symmetrical products that
resemble curcumin in the sense that two phenol or cate-
chol rings are connected by a linking group containing an
unsaturated ketone. Some substances of this general type
are known to have activity against various cancer cell
lines. These include HeLa human cervix cancer (Costi
et al. 2004), leukemia (Tan et al. 2014), and HEKT293T
human embryonic kidney cancer (Leow et al. 2014).
There is literature precedent for conducting aldol con-
densation reactions under either acidic or basic conditions.
However, base would deprotonate phenolic hydroxyl
groups that are present in the aldehyde substrates, probably
necessitating the protection of the hydroxyl groups (Artico
et al. 1998). We therefore decided to perform the reactions
directly on the hydroxybenzaldehydes in the presence of
hydrochloric acid (general approach of Sardjiman et al.
1997). Examples were selected to investigate the effect of
including methoxyl or fluorine atom substituents in addition
to the phenolic hydroxyl groups.
2,5-bis(3-fluoro-4-hydroxybenzylidene)
tetrahydropyran-4-one (18)
Tetrahydropyran-4-one (0.198 mL, 2.14 mmol), 3-
fluoro-4-hydroxybenzaldehyde (0.60 g, 4.28 mmol) and
methanol (2.14 mL), after heating at 60 °C for 2 d, gave a
crude product that was subjected to flash chromato-
graphy (CH2Cl2-EtOAc, 9:1) followed by recrystalliza-
tion (EtOH) to give the title compound 18 as a yellow
powder (0.158 g, 21%); mp 234–236 °C; vmax (KBr)/cm–1
3237 (O–H), 1666 (C=O), 1605 (C=C); δH (400 MHz,
DMSO-d6), 4.86 (4H, s, OCH2), 7.03 (2H, t, J 9 Hz, H-5ʹ,
5ʺ), 7.10 (2H, dd, J 9, 2 Hz, H-6ʹ, 6ʺ), 7.26 (2H, dd, J 13,
2 Hz, H-2ʹ, 2ʺ), 7.55 (2H, s, CH=); δC (101 MHz,
DMSO-d6) 67.6 (OCH2), 117.9 (d, J 3 Hz, C-5ʹ, 5ʺ),
118.4 (d, J 18 Hz, C-2ʹ, 2ʺ), 125.8 (d, J 6 Hz, C-1ʹ, 1ʺ),
128.0 (d, J 2 Hz, C-6ʹ, 6ʺ), 131.7 (=Cquat), 134.0 (=CH),
146.6 (d, J 13 Hz, C–O), 150.7 (d, J 242 Hz, CF), 184.4
(C=O); δF (376 MHz, DMSO-d6), –131.9; HRMS (ESI–)
m/z found 343.0789; calculated for C19H13F2O4 (M–H+)
343.0787.
Synthetic ketones 2–19 were readily isolated as crys-
talline solids that had NMR, IR, and high resolution mass
spectra consistent with the expected structures. The
cyclopentanone derivative 2 did not show significant
inhibition of the growth of either ovarian cell line at
concentrations up to 10 μM (Table 1); cyclopentanone 4
was also ineffective against cisplatin-resistant cells.
However, all of the cyclohexanones 5–15 and heterocyclic
analogs 16–19, showed inhibition with IC50 < 10 μM,
including against cisplatin-resistant cells. This contrasts
with the results obtained using carboplatin 20, which is
used to treat ovarian cancer clinically, but is compara-
tively ineffective against cisplatin-resistant cells. It is
notable that the cyclohexanone and the 4-
ethylcyclohexanone derivatives 5 and 11 are much more
active than cyclopentanone 2, which has identically sub-
stituted aromatic rings. Alkyl substitution of the 4-
position of the cyclohexanone ring is well tolerated,
even with the bulky tert-butyl group in analogs 14 and 15.
It is also evident that the pyranones 16–18 and the thio-
pyranone 19 have high levels of activity against ovarian
cancer cells. These results should be compared with pre-
vious studies in which thiopyranone derivatives were
found to be highly active against both NB4 acute pro-
myelocytic leukemic cells and NB4-R1 retinoic acid
resistant cells (Tan et al. 2014), and a piperidin-4-one
Method of cell culture and viability assay
Human ovarian carcinoma cells sensitive to cisplatin,
A2780 and resistant to cisplatin, A2780-CP70 were grown
and maintained in RPMI 1640 medium supplemented with
10% fetal bovine serum at 37 °C, 5% CO2. The cells were
plated in 96-well culture plates at a density of 1 × 104 cells/
well and allowed to adhere at 37 °C for 24 h. Then the next
day the cells were treated with various doses of com-
pounds ranging from 0.1 nM to 10 μM or vehicle. Cell
viability assay, MTT, was performed following incubation
time of 96 h. In such experiments the supernatant was
removed and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was added for 4 h. The
ability of cells to form formazan crystals by active mito-
chondrial respiration was determined by using a micro-
plate reader after dissolving the crystals in DMSO.
Cytotoxicity was expressed as a relative percentage of the
absorbance measured at 540 nm in the control and drug-
treated cells. MTT assay (Mosmann 1983) measures the
conversion of the soluble tetrazolium salt (MTT) to an
insoluble purple formazan by succinate dehydrogenase in
the mitochondria of living cells. IC50 values are reported as
mean s.e. mean (n = 2–4 experiments). Carboplatin (20)
was used as a reference chemotherapeutic drug.

Medicinal Chemistry Research (2020) 29:935–941
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Table 1 Biological activity (IC50 in MTT assay) of synthetic curcumin analogs 2–19 against A2780 ovarian cancer cells and A2780-CP70
cisplatin-resistant ovarian cancer cells
Compound
X
R1
R2
IC50
IC50 (μM)
(μM) A2780
A2780-CP70
2
–
OH
OMe
F
OMe
OH
OH
OMe
OH
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OH
OMe
OH
OH
>10
>10
3
–
0.043 0.01
0.749 0.25
0.595 0.09
0.28 0.08
0.99 0.5
6.59 0.6
8.19 0.29
5.32 1.1
0.15 0.09
0.36 0.08
2.04 0.35
2.86 1.74
3.95 1.5
0.178 0.04
0.26 0.03
1.65 0.9
0.129 0.07
2.57 0.35
0.458 0.1
>10
4
–
5
CH2
CH2
CH2
CHMe
CHMe
CHEt
CHEt
CHEt
CHEt
CHtBu
CHtBu
O
OH
OMe
F
0.71 0.1
2.21 0.05
3.8 0.09
1.69 0.71
3.24 0.27
3.065 0.62
1.25 0.09
1.82 0.44
3.38 0.3
3.48 0.8
2.34 0.3
1.29 0.98
0.367 0.01
2.67 0.4
0.153 0.064
44.9 3.5
6
7
8
OMe
F
9
10
H
11
OH
OMe
F
12
13
14
OH
OMe
OH
OH
F
15
16
17
O
18
O
19
S
OMe
20 (carboplatin)
IC50 data for carboplatin 20 are shown for comparison
derivative was active against cisplatin-resistant ovarian
cancer cells (Selvendiran et al. 2007).
tetrahydropyranone 17 were mixed with a model thiol (3-
mercaptopropionic acid) in a 1:20 molar ratio. After a
few hours it was observed that the signal intensities due
to the C=CH protons diminished (Fig. 1). These signals
are singlets which occur downfield of the aromatic sig-
nals, with chemical shifts between 7.3 and 7.6 ppm. The
appearance of new signals between 6.6–7.0 and
3.7–4.0 ppm was consistent with Michael addition hav-
ing occurred in each case, with the cyclopentanone 2
clearly being less reactive than the six-membered ring
analogs. Analysis of data obtained at different times
indicated that the pseudo first order rate constant for
consumption of cyclohexanone 5 was 5.1 times greater
than that of the analogous cyclopentanone 2, whereas
that for the tetrahydropyranone 17 was 8.0 times greater
than that of 2. Thus, for these three compounds, the trend
in electrophilicity correlates with cytotoxicity.
With regard to the substituents on the aryl groups, we
observe that replacing R1 = OMe in 8 by R1 = F in 9 did not
improve activity, neither did replacement of R1 = OH in 16
to give 18, whereas substituting R1 = H in compound 10 by
R1 = F to give 13 was of limited benefit.
It is known that the enones such as these are able to
react as electrophiles, undergoing addition of soft
nucleophiles such as thiols to the β-carbon of the C=C
bond. It has previously been shown that such reactions
are responsible for the induction of phase 2 enzymes
such as quinone reductase (Dinkova-Kostova et al.
2001). To compare the electrophilicity of representative
compounds from the present study, we ran a series of
1H NMR spectra on samples in which individual
samples of cyclopentanone 2, cyclohexanone 5, and

940
Medicinal Chemistry Research (2020) 29:935–941
1
Fig. 1 400 MHz H NMR
spectra of mixtures of individual
ketones (1.00 × 10–5 mol) with
3-mercaptopropanoic acid
(2.00 × 10–4 mol) in DMSO-d6
(0.60 mL) at 21 °C. Spectra
shown were recorded 0.5 h (left)
and 4.0 h (right) after sample
preparation. Ketones used were
cyclopentanone 2 (top spectra),
cyclohexanone 5 (middle
spectra), and tetrahydropyranone
17 (bottom spectra)
The fact that cisplatin-resistant cells were sensitive to
the cytotoxicity induced by these compounds also indi-
cates that the mechanism(s) of action of these compounds
are likely to be independent of nucleus events associated
with the DNA repair cascade as platinum compounds
primarily affect the DNA within the nucleus. Organic
compounds that target DNA, such as nitrogen mustards
and anthraquinones, show strong cross-resistance with
cisplatin (Hamaguchi et al. 1993). However, other non
DNA-related damage can also lead to apoptosis or cell
death. For example, it has been shown that changes in the
mitochondrial function following the release of cyto-
chrome c into the cytosol resulted in caspase activation,
which in turn leads to apoptosis (Liu et al. 1996). This
indicated that apoptosis can also be initiated in the cell
cytosol and not just the nucleus (Cory and Adams 2002).
Considering that many patients do develop resistance to
platinum drugs would highlight the importance of
developing analogues that at least initially activate the
apoptotic pathway in the cytoplasm of the cells, which
bypasses the resistance pathway. Further experiments are
required to substantiate the mechanism of action of these
compounds.
Conclusions
Most of the synthetic curcumin analogues that were stu-
died showed significant inhibition of ovarian cancer cell
growth, including inhibition of cisplatin-resistant cells.
The ability of the enone C=C groups in these analogues
react electrophilically with thiol groups of biological
molecules is likely to be relevant to their activity.
The derivatives of the heterocyclic ketones stand out
as being particularly active and worthy of further
investigation.

Medicinal Chemistry Research (2020) 29:935–941
941
Acknowledgements We are grateful to the EPSRC NMSF at Swansea
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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