Antiestrogenic activity and possible mode of action of certain new nonsteroidal coumarin-4-acetamides

Article abstract of DOI:10.3390/molecules25071553

The preparation of certain 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamides IIIa-h was planned as a step in the development of new modified nonsteroidal antiestrogens. The purity of target compounds IIIa-h was checked by thin-layer chromatography (TLC), and their structures were confirmed using various spectroscopic tools including IR, ¹H-NMR, ¹³C-NMR, and MS spectroscopy. Viability tests were applied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effect of the synthesized compounds against two breast cancer cell lines, MCF-7 and MDA-MB-231. Compound IIIb proved the most active against MCF-7 cells, with an IC₅₀ value of 0.32 μM. The results of an analysis of in vitro antiestrogenic activity indicated that only compound IIIb exhibited antiestrogenic activity; its IC₅₀ value of 29.49 μM was about twice as potent as that of the reference compound, MIBP. The aromatase activity was evaluated for the synthesized target compounds IIIa-g and the intermediates Ib and IIa. A significant aromatase inhibition was observed for the intermediate Ib and compound IIIe, with IC₅₀ values of 14.5 and 17.4 μM, respectively. Compound IIIb, namely 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one, could be used as an antiestrogen and/or cytotoxic agent with selective activity against tumor cells.

Full text of DOI:10.3390/molecules25071553

molecules
Article
Antiestrogenic Activity and Possible Mode of Action
of Certain New Nonsteroidal Coumarin-4-acetamides
Maha S. Almutairi 1,
*
, Areej N. Al Suwayyid 1,2, Amal Aldarwesh 3 , Omaima M. Aboulwafa 4
and Mohamed I. Attia 1,5,
*
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia; asuwayyid@gmail.com
Saudi Food and Drug Authority, Riyadh 13513, Saudi Arabia
Department of Optometry and Visual Sciences, College of Applied Medical Sciences, King Saud University,
Riyadh 12372, Saudi Arabia; aaldarweesh@ksu.edu.sa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21500, Egypt; omaimawafa@yahoo.com
Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre (ID: 60014618), El Bohooth Street, Dokki, Giza 12622, Egypt
Correspondence: malmutbiri@ksu.edu.sa (M.S.A.); mattia@ksu.edu.sa (M.I.A.); Tel.: +966-180-56161 (M.I.A.)
2
3
4
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 February 2020; Accepted: 27 March 2020; Published: 28 March 2020
Abstract: The preparation of certain 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamides IIIa–h was
planned as a step in the development of new modified nonsteroidal antiestrogens. The purity of
target compounds IIIa–h was checked by thin-layer chromatography (TLC), and their structures were
confirmed using various spectroscopic tools including IR, 1H-NMR, 13C-NMR, and MS spectroscopy.
Viability tests were applied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay to evaluate the cytotoxic effect of the synthesized compounds against two breast cancer
cell lines, MCF-7 and MDA-MB-231. Compound IIIb proved the most active against MCF-7 cells, with
an IC50 value of 0.32
µM. The results of an analysis of in vitro antiestrogenic activity indicated that only
compound IIIb exhibited antiestrogenic activity; its IC50 value of 29.49
µM was about twice as potent
as that of the reference compound, MIBP. The aromatase activity was evaluated for the synthesized
target compounds IIIa–g and the intermediates Ib and IIa. A significant aromatase inhibition was
observed for the intermediate Ib and compound IIIe, with IC50 values of 14.5 and 17.4 µM, respectively.
Compound IIIb, namely 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one, could be
used as an antiestrogen and/or cytotoxic agent with selective activity against tumor cells.
Keywords: coumarin-4-acetamide; nonsteroidal antiestrogens; SERMs; aromatase inhibitors
1. Introduction
Breast cancer is one of the most common and devastating cancers in women worldwide.
According to researchers in the United States, the number of new cases of female breast cancer
is 124.9 per 100,000 women per year
Arabia, 708 patients (13.08%) died [
related to estrogen receptors (ER) [3,4
[
2
1
]. In 2018, out of 5411 breast cancer cases diagnosed in Saudi
]. The first prognostic and predictive factor of breast cancer is
]. The development of ER antagonists has enabled successful
treatment of postmenopausal women with hormone-dependent breast cancers [ ]. Increased levels of
estrogens are associated with tumor growth in endocrine-dependent tissues [6].
5
The coumarin (benzopyran-2-one, chromen-2-one,) ring system was used in research over 200 years
ago. The name coumarin is derived from Coumarouna odorata Aube, from which it was isolated for the
first time in 1820 [7]. Coumarins exhibit relatively low toxicity and participate in a remarkable array
Molecules 2020, 25, 1553; doi:10.3390/molecules25071553
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1553
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of biological activities [
8
–
13]. One of the most interesting among the diverse biological activities of
coumarins is their anti-breast cancer activity.
During the identification of potent coumarin-based selective estrogen receptor modulators (SERM)
compounds, the goal was to find a potent estrogen inhibitor with an interleukin-6 (IL-6) inhibitory
activity [14]. The presence of an amine side-chain in compound SP500263 (Figure 1) plays a key
role in determining its SERM activity; however, this compound also showed undesirable agonist
activity in the MCF-7 proliferation assay [15,16]. The core structures of SERMs are diverse, including
triphenylethylene [17
,18], benzothiophene, chromene (benzopyran) [19
–23], naphthalene, indole, and
steroid derivatives [24].
O
N
OH
HO
O
N
O
HO
O
O
SP500263
acolbifene
O
N
O
R
O
IIIa-h
R = 6-OCH3 or 7-OCH3
Figure 1. Chemical structures of SP500263, acolbifene, and the target compounds IIIa-h.
Unfortunately, anti-breast cancer drugs like tamoxifen (TAM) [18] achieve significant clinical
results in only 30–40% of patients, because drug resistance usually develops after one or two years
of treatment [25]. Among the fourth generation of SERMs, benzopyrans are potent antiestrogenic
compounds with high oral bioavailability that have recently been tested in vitro and in vivo. These
compounds display a 1.5–2.9-fold greater affinity than 17β-estradiol (E2) for the estrogen receptors in
human breast cancer and normal uterine cytosol [19]. These compounds have no agonistic estrogenic
activity in the in vitro human breast cancer models and in vivo in nude mice [19]. The nonsteroidal
antiestrogen acolbifene (Figure 1) is the most potent antiestrogen in terms of inhibition of both ER
and ER 20 26]. This compound has a number of advantages over all other antiestrogens and
should be investigated for the treatment of ER-positive breast cancer and other estrogen-sensitive
malignancies [20 21]. In addition, aromatase enzyme is involved in the last step of the estrogen
α
β [7, ,
,
biosynthetic pathway [27]. A number of coumarin derivatives bearing an imidazole ring at position
four have been designed and synthesized as strong and selective aromatase inhibitors (AIs) [28].
From the practical point of view, the compounds of interest contain substituted acetamide
functionalities attached to the four position of coumarin nuclei, while maintaining all functionalities at
their relative positions in the designed compounds. This pattern might dramatically affect the binding
to the ER and subsequently the enhancement of their biological activity as anti-breast cancer agents.
2. Results and Discussion
2.1. Chemistry
Coumarin (2H-Chromen-2-one) has been synthesized by several methods, including
Pechman–Duisberg condensation [29]. According to Scheme 1, the intermediates methyl (6-methoxy-2-
oxo-2H-chromen-4-yl)acetate (Ia), methyl (7-methoxy-2-oxo-2H-chromen-4-yl) acetate (Ib), (6-methoxy-

Molecules 2020, 25, 1553
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2-oxo-2H-chromen-4-yl)acetic acid (IIa), and (7-methoxy-2-oxo-2H-chromen-4-yl)acetic acid (IIb) were
required for the synthesis of the target compounds IIIa–h. The reaction sequence to prepare the title
compounds IIIa–h is outlined in Scheme 1.
Compound No.
R
Compound No.
R
IIIa
IIIb
IIIc
IIId
6-OCH3
7-OCH3
C6H5
IIIe
IIIf
IIIg
IIIh
3-OH, 4-OCH3-C6H3
C6H5
4-Br-C6H4
3-OH, 4-OCH3-C6H3
4-Br-C6H4
Reagents and conditions: (i) Dimethylacetone-1,3-dicarboxylate, H2SO4, 0 °C, 24 h; (ii) Piperidine, p-
TSOH, toluene, reflux, 4 h; (iii) Citric acid, H2SO4 at 0 °C, 24 h; (iv) Ethanol, 0.5%NaOH, reflux 3 h; (v)
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), dimethyformamide (DMF), appropriate
alkyl- or arylamine, 0 °C, 24 h; (vi) Methanol, H2SO4, reflux, 5 h.
Scheme 1. Synthesis of the target compounds IIIa–h.
The 1H-NMR spectra of compounds Ia,b exhibited a singlet peak at 3.77 ppm, which was assigned
to methyl ester (COOCH3) protons, while the disappearance of the characteristic peak at 12.81 ppm
for the carboxylic acid proton was observed. 13C-NMR spectroscopy indicated the presence of a new
carbon peak at 52.68 ppm, which was assigned to be the methyl ester carbon (COOCH3).
The direct reaction of a carboxylic acid with an amine proceeds smoothly with aliphatic secondary
amines like piperidine producing the target compounds IIIa,b. On the other hand, the best method
for the preparation of compounds IIIc was the use of a 1-ethyl-3-[3-(dimethylamino)propyl]
–
h
carbodiimide HCl (EDCI.HCl) reagent, which was added to a mixture of the appropriate aromatic
amine and the acids IIa,b in dimethyformamide (DMF).
The structures of compounds IIIa–h were confirmed by their IR spectra, in which the appearance
of C–N bands at 1026 cm−1 and 1024 cm−1 indicates compounds IIIa and IIIb, respectively. The
disappearance of broad –OH bands suggested the formation of a new amide bond for compounds

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1
IIIc–h. The H-NMR spectra of compounds IIIa
,
b
did not show –NH amide protons as they are
tertiary amides. In addition, the 1H-NMR spectra of compounds IIIc–h were characterized by the
appearance of –NH peaks that resonate at 10.08–10.53 ppm as singlets. Moreover, the 13C-NMR spectra
of compounds IIIc–h were characterized by the presence of six new aromatic carbons in the range
of 108.2–147.9 ppm. The 13C-NMR and DEPT spectra of the compound IIIa were well resolved and
confirmed the presence of all carbon atoms in the molecule. On other hand, the HSQC spectrum of the
compound IIIa specified its proton–carbon coupling.
2.2. Biological Evaluation
2.2.1. In Vitro Cytotoxicity
The biological investigation started with the evaluation of the cytotoxic activity of the intermediates
Ia–b and IIa–b (Table 1). Compounds Ib and IIa had a highly cytotoxic effect against the MCF-7 cell
line, with IC50 values of 1.44 and 1.00
cytotoxic to the MDA-MB-231 cell line, with its IC50 value of 1.00
potent than the reference standard camptothecin (IC50 value of 19.24
µ
M, respectively. However, only compound Ib was highly
M being about 19 times more
M, Table 1 and Figure 2).
µ
µ
Methyl (7-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ib) was highly cytotoxic towards both breast
cancer cell lines.
Table 1. Cytotoxicity of the intermediates Ia,b, IIa,b and camptothecin.
IC50 (µM) ± S.E.M
IC50 (µM) ± S.E.M
MDA-MB-231
Compound No.
MCF-7
Ia
Ib
IIa
13.90 ± 1.15
1.44 ± 0.02
1.00 ± 0.03
8.08 ± 0.25
4.41 ± 0.28
47.45 ± 2.51
1.00 ± 0.04
80.18 ± 4.23
216.35 ± 13.15
19.24 ± 1.14
IIb
Camptothecin
Figure 2. Cytotoxicity of the intermediates Ia,b, IIa,b, and camptothecin.
The cytotoxic activity of the synthesized compounds IIIa–h was evaluated in vitro against the
human breast adenocarcinoma MCF7 (ER+ breast cancer cell line) and the MDA-MB-231 (triple-negative

Molecules 2020, 25, 1553
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breast cancer cell line, TNBC), using camptothecin as a pyranone-bearing reference standard. Treatment
of the ER+ MCF-7 and MDA-MB-231 cell lines with 0.39, 1.56, 6.25, 25, and 100 µM concentrations of
the synthesized target compounds IIIa–h resulted in a significant cytotoxic effect. The most active
compounds against the MCF-7 breast cancer cell line were subjected to in vitro antiestrogenic activity
and in vitro aromatase inhibition activity.
Considering the cytotoxicity of N-substituted coumarin-4-acetamides IIIa–h (Table 2 and Figure 3),
compound IIIb is most active against MCF-7 cells with an IC50 value of 0.32
µM, as compared with the
reference cytotoxic compound, camptothecin (IC50 = 4.41 M). In addition, compound IIIe is the most
µ
active candidate against the MDA-MB-231 cell line with an IC50 value of 2.14 µM, while camptothecin
displayed an IC50 value of 19.24 µM against the same cell line.
Table 2. Cytotoxicity of the synthesized N-substituted coumarin-4-acetamides IIIa–h and camptothecin.
IC50 (µM) ± S.E.M.
IC50 (µM) ± S.E.M.
MDA-MB-231
Compound No.
MCF-7
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
IIIh
1.82 ± 0.03
0.32 ± 0.04
10.92 ± 0.99
2.80 ± 0.04
15.50 ±1.24
5.69 ± 0.08
0.72 ± 0.02
5096.02 ± 241
4.41 ± 0.28
38.21 ±1.52
12.90 ±1.17
4.60 ± 0.32
22.77 ±1.54
2.14 ± 0.06
6.94 ± 0.41
90.80 ± 3.84
14.06 ± 0.61
19.24 ± 1.14
Camptothecin
Figure 3. Cytotoxicity of N-substituted coumarin-4-acetamides IIIa–h and camptothecin.
In general, 7-methoxycoumarin-4-acetamide derivatives IIIb and IIIf–h showed a greater in vitro
cytotoxic effect against MCF-7 cells as compared with their positional isomers 6-methoxycoumarin
derivatives IIIa and IIIc–e. The most active compounds are in the following order: IIIb
IIId, with IC50 values of 0.32, 0.72, 1.82, and 2.80 M, respectively. On the other hand, the results
of the cytotoxic evaluation of the acetamides IIIa–h against MDA-MB-231 human breast cancer cells
> IIIg > IIIa
>
µ

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indicated that compounds IIIc
,
e, and
f
had a highly cytotoxic effect, with IC50 values of 4.6, 2.1, and
6.9 µM, respectively (Table 2 and Figure 3).
2.2.2. In Vitro Antiestrogenic Activity
The in vitro antiestrogenic activity of the selected target compounds IIIa,b, IIId, and IIIf,g was
studied through an estrogen-dependent human breast cancer MCF-7 cell proliferation assay in the
presence of 17- -estradiol. The ability of the tested compounds to inhibit cell proliferation induced
by 17- -estradiol was determined (Table 3 and Figure 4). One of the disadvantages of the compound
SP500263 was that it had an undesirable agonist activity in the MCF-7 proliferation assay. However,
β
β
among the synthesized compounds, compound IIIb is the most active, with its IC50 value of 29.49 µM
being about twice as potent as the reference compound, MIBP.
Table 3. In Vitro antiestrogenic activity of tested synthesized compounds IIIa,b, IIId, IIIf,g, and MIBP.
IC50 (µM) ± S.E.M.
Compound No.
MCF-7
IIIa
IIIb
IIId
IIIf
IIIg
62.01 ± 4.02
29.49 ± 3.25
213.86 ± 12.1
99.61 ± 6.39
64.40 ± 4.12
46.38 ± 3.14
Monoisobutyl phthalate (MIBP)
Figure 4. In Vitro antiestrogenic activity of the selected synthesized compounds and MIBP.
2.2.3. In Vitro Aromatase Inhibition
One of the most successful targeted breast cancer therapies is the inhibition of the AR enzyme,
which catalyzes the rate-limiting final step of estrogen biosynthesis by modulating ER. To ensure
cytotoxicity against the MCF-7 breast cancer cell line, aromatase enzymatic activity was assayed using
the aromatase inhibitor (AI), letrozole, as a reference standard. Compound Ib was equipotent to
letrozole (IC50 = 15.03 µM) aromatase inhibitory activity with an IC50 value of 14.5 µM (Table 4 and
Figure 5).

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Table 4. Aromatase inhibition activity of the selected synthesized compounds and letrozole.
Compound No.
IC50 (µM) ± S.E.M.
Ib
IIa
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
14.50 ± 1.32
32.20 ± 2.39
22.46 ± 3.40
89.06 ± 6.20
1104.00 ± 52.36
69.95 ± 3.91
17.38 ± 1.37
332.07 ± 18.20
27.11 ± 1.38
15.03 ± 1.8
Letrozole
Figure 5. Aromatase inhibition activity of the tested synthesized compounds and letrozole.
Moreover, acetamides IIIa–g showed low or no aromatase inhibition activity (Table 4 and Figure 5),
except compound IIIe, which showed moderate inhibitory activity against aromatase (IC50 = 17.38 µM),
alongside its promising activity towards the MDA-MB-231 human breast cancer cell line (IC50 value of
2.14 µM).
3. Experimental
3.1. Chemistry
3.1.1. General
Melting points were determined in open glass capillaries on an electrothermal melting point
apparatus and are uncorrected. Infrared (IR) spectra were recorded for potassium bromide discs
ν
(cm−1) on an IR affinity-1s Fourier transform infrared spectrophotometer. The 1H-NMR and 13C-NMR
spectra were determined on a Bruker (700 MHz) (Coventry, Germany) and Agilent Technologies
1
(600 MHz) (Palo Alto, CA, USA) spectrometer. Correlation spectroscopy: H, 13C, HSQC, and DEPT
spectra were recorded on a Bruker (700 MHz) spectrometer. Chemical shifts are expressed as
δ values
(ppm), using tetramethylsilane (TMS) as an internal reference. Signals are indicated by the following
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Mass spectra
(MS) were obtained on a GCMS QP 2010 Ultra/SE (Ver. 4.20), GCMS-TQ Series (Ver. 4.20) Shimadzu
apparatus (Kyoto, Japan). Elemental analyses were carried out at Research Center, College of Pharmacy,

Molecules 2020, 25, 1553
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King Saud University, Saudi Arabia, and the results agreed favorably with the proposed structures
within 0.4% of the theoretical values. Follow-up of the reaction and checking on the homogeneity of
±
the compounds were made by performing thin-layer chromatography (TLC) on pre-coated (0.25 mm)
(GF 254) silica gel plates. Routinely used developing solvents were volume to volume: C6H14:EtOAc
(6:4); C6H14:EtOAc (5:5); MeOH:CHCl3 (1:9). Visualization of the spots was performed by exposure to
a UV lamp at 254 nm or to iodine vapors. All statistical analyses were carried out using GraphPad
Prism (San Diego, CA, US) version 6.0 software. Statistical analysis was conducted using one-way
ANOVA, followed by multiple Tukey–Kramer post hoc tests at p < 0.05, which was considered a marker
of statistical significance.
3.1.2. General Procedure for the Synthesis of Methyl 2-(2-oxo-2H-chromen-4-yl)acetates Ia,b
Route A: To a stirred solution of dimethyl-acetone-1,3-dicarboxylate (1.83 mL, 2.2 g, 10 mmol),
the appropriate portions of methoxyphenol (1.24 g, 10 mmol) and concentrated H2SO4 (1.12 mL, 1 g,
11 mmol) were added, each in three equal portions, in a cold environment (temperature not exceeding
10 ◦C). The resulting reaction mixture was stored at 0 ◦C overnight, then poured into ice-cold water
(40 mL). The white precipitate that formed was filtered off and re-crystallized from methanol to give
the respective methyl 2-(2-oxo-2H-chromen-4-yl)acetate derivatives Ia,b [30].
Route B: A few drops of H2SO4 were added to a solution of 2-(7-methoxy-2-oxo-2H-
chromen-4-yl)acetic acid (IIb, 1 mg, 4 mmol) in MeOH (10 mL). The resulting solution was refluxed
for 5 h. After completion of the reaction, as indicated by TLC (ethylacetate/hexane, 4/6), MeOH was
evaporated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL), washed with
NaHCO3 (20 mL), dried over anhydrous Na2SO4, and filtered. Ethyl acetate was removed in vacuo to
produce compound Ib [31].
Methyl (6-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ia):
Yield (route A): 0.5 g (20%); White glittery crystals m.p.: 153–154 ◦C; IR (KBr):
ν
(cm−1): 1724
(C=O, lactone), 1734 (C=O, ester), 2941 (C–H, aliphatic), 3088 (C–H, aromatic); 1H-NMR (DMSO-d6)
ppm (600 MHz): 3.62 (s, 3H, ester CH3), 3.77 (s, 3H, Ar OCH3), 4.01 (s, 2H, CH2CO), 6.48 (s, 1H, H-3),
7.11 (s, 1H, H-5), 7.20 (s, 1H, H-7), 7.34 (s, 1H, H-8); 13C-NMR (DMSO-d6)
ppm (150 MHz): 36.9
δ
δ
(CH2CO), 52.7 (ester CH3), 56.2 (OCH3), 108.8 (C-5), 117.3 (C-3), 118.2 (C-7), 119.5 (C-4a), 119.7 (C-8),
147.8 (C-8a), 149.5 (C-4), 155.9 (C-6), 160.2 (C-2), 170.1 (C=O); MS m/z (% relative abundance): [M]+
248 (3.82).
Methyl (7-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ib):
Yield by route A: 1.2 g (48%) and by route B: 0.7 g (70%); White crystals m.p.: 116–118 ◦C; IR (KBr):
ν
(cm−1):1724 (C=O, lactone), 1734 (C=O, ester), 2966 (C–H, aliphatic), 3093 (C–H, aromatic); 1H-NMR
(DMSO-d6):
δ
ppm (600 MHz) 3.62 (s, 3H, ester CH3), 3.84 (s, 3H, ArOCH3), 3.97 (s, 2H, CH2CO), 6.30
ppm
(s, 1H, H-3), 6.94 (s, 1H, H-6), 7.00 (s, 1H, H-8) and 7.87 (s, 1H, H-5); 13C-NMR (DMSO-d6):
δ
(150 MHz) 37.1 (CH2CO), 52.7 (ester CH3), 56.4 (OCH3), 101.4 (C-8), 112.8 (C-6), 113.5 (C-3), 114.8
(C-4a), 127.0 (C-5), 149.9 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.9 (C-2), 170.1 (C=O); MS m/z (% relative
abundance): [M]+ 248 (0.33).
3.1.3. General Procedure for the Synthesis of Coumarin-4-Acetic acid Derivatives IIa,b
Route A: A mixture of citric acid monohydrate (4.2 g, 20 mmol) and concentrated H2SO4 (5.6 mL)
was stirred at room temperature for 60 min, then slowly heated (rate of heating governed by foaming)
to 70 ◦C. After 35 min at this temperature, with stirring throughout, the evolution of carbon monoxide
had slackened, and the clear solution was rapidly cooled to 0 ◦C. Then the appropriate methoxyphenol
(2 g, 16.1 mmol) and concentrated H2SO4 (2.24 mL) were added, each in three equal portions, to the
stirred solution at such a rate that the internal temperature did not exceed 10 ◦C. The resulting reaction
mixture was stored at 0 ◦C for 16 h, poured into ice cold water (40 mL), and the resulting crystalline
precipitate filtered off and washed thoroughly with H2O. The collected solid was dissolved under
stirring in 1N Na2CO3 solution (20 mL), heated for 15 min at 65 ◦C, and the insoluble matter was

Molecules 2020, 25, 1553
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filtered off and washed with water (2
×
10 mL). The combined filtrate and washings were acidified
with concentrated HCl to give the respective coumarin-4-acetic acid derivatives IIa,b [32].
Route B: A solution of methyl (6-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ia, 1 g, 4 mmol) in
ethanol (10 mL) and 0.5% NaOH (100 mL) was refluxed for 2 h. It was then cooled to room temperature,
acidified with concentrated HCl to pH = 2, and cooled to 0 ◦C. The precipitated solid was filtered off,
washed thoroughly with ethanol, and dried to give compound IIa, which was used for the next step
without further purification [33,34].
(6-Methoxy-2-oxo-2H-chromen-4-yl)acetic acid (IIa):
Yield by route A: 0.23 g (6.1%) and by route B: 0.84 g (89%); light brown fluffy powder m.p.:
178–179 ◦C; IR (KBr):
ν
(cm−1): 1174–1255 (C–O), 1670 (C=O, carboxylic acid), 1724 (C=O, lactone),
2924 (C–H, aliphatic), 3088 (C–H, aromatic), 3200(OH); 1H-NMR (DMSO-d6):
(s, 3H, ArOCH3), 3.92 (s, 2H, CH2CO), 6.48 (s, 1H, H-3), 7.15 (s, 1H, H-5), 7.22 (s, 1H, H-7), 7.35 (s, 1H,
H-8), 12.81 (s, 1H, COOH); 13C-NMR (DMSO-d6):
ppm (150 MHz) 36.9 (CH2CO), 56.2 (OCH3), 109.0
δ ppm (600 MHz) 3.78
δ
(C-5), 114.9 (C-3), 117.1 (C-7), 118.1 (C-4a), 119.4 (C-8), 147.8 (C-8a), 150.2 (C-4), 155.9 (C-6), 160.2 (C-2),
171.1 (C=O); MS m/z (% relative abundance): [M]+ 234 (0.58), [M + 1] 235 (12.06).
7-Methoxy-2-oxo-2H-chromen-4-yl)acetic acid (IIb):
Yield (route A): 3.2 g (85%); White crystals m.p.: 186–187 ◦C (literature: 186 ◦C [35]; IR (KBr):
ν
(cm−1): 1664 (C=O, carboxylic acid), 1718 (C=O, lactone), 2926 (C–H, aliphatic), 3439 (br, OH); 1H-NMR
(DMSO-d6): ppm (600 MHz) 3.82 (s, 5H, CH2CO &ArOCH3), 6.27 (s, 1H, H-3), 6.93 (s, 1H, H-6),
6.97 (s, 1H, H-8), 7.58 (s, 1H, H-5), 12.81 (s, 1H, COOH); 13C-NMR (DMSO-d6):
ppm (150 MHz) 37.7
δ
δ
(CH2CO), 56.3 (OCH3), 101.3 (C-8), 112.7 (C-6), 112.9 (C-3), 113.3 (C-4a), 127.0 (C-5), 150.6 (C-8a), 155.4
(C-4), 160.5 (C-7), 162.8 (C-2), 171.1 (C=O); MS m/z (% relative abundance): [M]+ 234 (1.89), [M + 1]
235 (4.09).
3.1.4. General Procedure for the Synthesis of 4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one
derivatives IIIa,b
The appropriate methyl (2-oxo-2H-chromen-4-yl)acetate Ia,b (0.248 g, 1 mmol) and piperidine
(1 mL, 0.85 g, 1 mmol) was heated to reflux in toluene (10 mL) in the presence of a catalytic amount of
p-toluene sulfonic acid (p-TSOH) for 4 h. The solvent was concentrated under reduced pressure and
the precipitated solid was filtered off, washed with toluene, and dried to yield the respective target
compounds IIIa,b [36].
6-Methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one (IIIa):
Yield: 0.21 (70%); Fluffy pale yellow powder m.p.: 167–168 ◦C; IR (KBr):
ν
(cm−1): 1026 (C–N), 1718
(C=O, amide), 1734 (C=O, lactone), 2939 (C–H, aliphatic), 3020 (C–H, aromatic); 1H-NMR (DMSO-d6):
ppm (700 MHz) 1.46 (br s, 2H, CH2 piperdine), 1.57 (br s, 2H, CH2-piperdine), 1.62 (br s, 2H,
δ
CH2-piperdine), 3.47 (br s, 2H, CH2-piperdine), 3.53 (br s, 2H, CH2-piperdine), 3.81 (s, 3H, OCH3),4.03
(s, 2H, CH2), 6.38 (s, 1H, H-3), 7.11 (s, 1H, H-5), 7.24 (d, J = 9.1 Hz, 1H, H-7), 7.39 (d, J = 9.1 Hz, 1H,
H-8); 13C-NMR (DMSO-d6):
δ ppm (175 MHz) 24.3 (CH2-piperdine), 25.9 (CH2-piperdine), 26.7 (CH2
piperdine), 36.7 (CH2CO), 42.5 (CH2-piperdine), 46.9 (CH2-piperdine), 56.2 (OCH3), 109.4 (C-5), 116.5
(C-3), 117.9 (C-7), 119.1 (C-8), 120.3 (C-4a), 147.7 (C-8a), 152.1 (C-4), 155.8 (C-6), 160.3 (C-2), 166.7 (C=O);
MS m/z (% relative abundance): [M]+ 301 (0.48).
7-Methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one (IIIb):
Yield: 0.21 g (70%); White crystals m.p.: 135–136 ◦C; IR (KBr):
amide), 1734 (C=O, lactone), 2949 (C–H, aliphatic), 3095 (C–H, aromatic); H-NMR (DMSO-d6)
ν
(cm−1): 1024 (C–N), 1718 (C=O,
1
δ
ppm (700 MHz):1.44–1.47 (m, 2H, CH2-piperdine), 1.56–1.57 (m, 2H, CH2-piperdine),1.61–1.63 (m, 2H,
CH2-piperdine), 3.45 (t, J = 5.6 Hz, 2H, CH2-piperdine), 3.51 (t, J = 5.6 Hz, 2H, CH2-piperdine), 3.87
(
s, 3H, OCH3), 3.98 (s, 2H, CH2), 6.19 (s, 1H, H-3), 6.96 (dd, J = 9.1, 2.8 Hz, 1H, H-6), 7.01(d, J = 2.8 Hz,
1H, H-8), 7.57 (d, J = 9.1 Hz, 1H, H-5); 13C-NMR (DMSO-d6):
ppm (175 MHz) 24.4 (CH2-piperdine),
25.8 (CH2-piperdine), 26.5 (CH2-piperdine), 31.2 (CH2CO), 42.7 (CH2-piperdine), 46.8 (CH2-piperdine),
δ

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56.4 (OCH3), 101.1 (C-8), 112.6 (C-6), 125.9 (C-3), 127.3 (C-4a), 128.5 (C-5), 152.6 (C-8a), 155.3 (C-4),
160.6 (C-7), 162.7 (C-2), 166.7 (C=O); MS m/z (% relative abundance): [M]+ 301 (10.33).
3.1.5. General Procedure for the Synthesis of 2-(2-oxo-2H-chromen-4-yl)-N-phenylacetamides IIIc-h
1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide HCl (EDCI.HCl, 0.47 g, 3 mmol) was slowly
added to a stirred solution containing the appropriate 2-oxo-2H-chromen-4-yl)acetic acid derivative
IIa,b
(0.234 g, 1 mmol) and the proper arylamine (1.2 mmol) in DMF (15 mL) at 0 ◦C. After 2 h, the
reaction mixture was warmed to room temperature and stirring was continued for an additional 24 h.
Thereafter, the reaction mixture was poured into H2O (22 mL). The separated solid was filtered off
under suctioning, washed repeatedly with H2O, and dried to give compounds IIIc–h.
2-(6-Methoxy-2-oxo-2H-chromen-4-yl)-N-phenylacetamide (IIIc):
Yield: 0.27 g (87%); White powder m.p.: 219–220 ◦C; IR (KBr):
ν
(cm−1) 1043 (C–N), 1078–1213
(C–O), 1660 (C=O, amide), 1734 (C=O, lactone), 2960 (C–H, aliphatic), 3059 (C–H, aromatic), 3286 (NH,
secondary amine); 1H-NMR (DMSO-d6):
δ ppm (700 MHz) 3.80 (s, 3H, OCH3), 3.99 (s, 2H, CH2), 6.54
(s, 1H, H-3), 7.08 (t, J = 7.7 Hz, 1H, H-40), 7.26 (dd, J = 9.1, 2.8 Hz, 1H, H-7), 7.32–7.34 (m, 3H, H-5 and
H-30 & H-50), 7.40 (d, J = 8.4 Hz, 1H, H-8), 7.71 (d, J = 8.4 Hz, 2H, H-20 & H-6”), 10.42 (s, 1H, NH);
13C-NMR (DMSO-d6):
δ ppm (175 MHz) 31.3 (CH2CO), 56.3 (OCH3), 108.8 (C-5), 117.2 (C-3), 118.2
(C-7), 119.4 (C-4a), 119.7 (C-20 & C-60), 120.1 (C-8), 124.1 (C-40), 129.3 (C-30 &C-50), 139.2 (C-10), 147.9
(C-8a), 150.8 (C-4), 156.0 (C-6), 160.3 (C-2), 167.0 (C=O); MS m/z (% relative abundance): [M]+ 309 (0.73).
N-(4-Bromophenyl)-2-(6-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIId):
Yield: 0.19 g (48%); Silver powder m.p.: 202–203 ◦C; IR (KBr):
ν
(cm−1) 600 (C-Br), 1031 (C–N),
1070–1246 (C–O), 1653 (C=O, amide), 1730 (C=O, lactone), 2978 (C–H, aliphatic), 3088 (C–H, aromatic),
1
3228 (NH, secondary amine); H-NMR (DMSO-d6):
δ ppm (700 MHz) 3.82 (s, 3H, OCH3), 4.05
(
d, J = 7.0 Hz, 2H, CH2), 6.53 (s, 1H, H-3), 7.16 (d, J = 2.8 Hz, 1H, H-5), 7.26 (dd, J = 9.1, 2.8 Hz, 1H,
H-7), 7.39 (d, J = 9.1 Hz, 1H, H-8), 7.51 (d, J = 9.1 Hz, 2H, H-20 &H-6”), 7.56 (d, J = 9.1 Hz, 2H, H-30 &
H-50), 10.53 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ ppm (175 MHz) 31.2 (CH2CO), 56.2 (OCH3), 108.8
(C-5), 115.7 (C-3), 117.3 (C-7), 118.2 (C-20 &C-60), 119.4 (C-4a), 119.7 (C-40), 121.7 (C-8), 132.2 (C-30
&C-50), 138.6 (C-10), 147.9 (C-8a), 149.7 (C-4), 156.0 (C-6), 160.2 (C-2), 169.6 (C=O); MS m/z (% relative
abundance): [M]+ 388 (0.48).
N-(3-Hydroxy-4-methoxyphenyl)-2-(6-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIIe):
Yield: 0.21 g (59%); Brown powder m.p.: 173–174 ◦C; IR (KBr):
ν
(cm−1): 1028 (C–N), 1043–1257
(C–O), 1685 (C=O, amide), 1734 (C=O, lactone), 2935 (C–H, aliphatic), 3083 (C–H, aromatic), 3253 (NH,
secondary amine), 2446–3527 (OH); 1H-NMR (DMSO-d6):
δ
ppm (700 MHz) 3.72 (s, 3H, C-40-OCH3),
3.80 (s, 3H, OCH3), 3.92 (s, 2H, CH2), 6.51 (s, 1H, H-3), 6.84 (d, J = 8.4 Hz, 1H, H-50), 6.93 (dd, J = 8.4,
2.8 Hz, 1H, H-60), 7.13 (d, J = 2.1 Hz, 1H, H-20), 7.25 (dd, J = 9.1, 2.8 Hz, 1H, H-7), 7.32 (d, J = 2.8 Hz,
1H, H-5), 7.40 (d, J = 9.1 Hz, 1H, H-8), 9.10 (s, 1H, OH), 10.15 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ
ppm (175 MHz) 31.2 (CH2CO), 56.3 (OCH3), 108.3 (C-20), 108.9 (C-5), 110.5 (C-3), 112.9 (C-50), 117.1
(C-60), 118.2 (C-7), 119.4 (C-4a), 120.1 (C-8), 132.7 (C-10), 144.5 (C-30), 146.9 (C-8a), 147.8 (C-40), 151.0
(C-4), 156.0 (C-6), 160.3 (C-2), 166.4 (C=O); MS m/z (% relative abundance): [M]+ 355 (0.59), [M + 1] 356
(35.71).
2-(7-Methoxy-2-oxo-2H-chromen-4-yl)-N-phenylacetamide (IIIf):
Yield: 0.2 g (65%); Yellow-brown powder m.p.: 217–218 ◦C; IR (KBr):
ν
(cm−1): 1028 (C–N), 1647
(C=O, amide), 1728 (C=O, lactone), 2978 (C–H, aliphatic), 3074 (C–H, aromatic), 3265 (NH, secondary
1
amine); H-NMR (DMSO-d6):
δ ppm (700 MHz) 3.87 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 6.34 (s, 1H,
H-3), 7.00 (dd, J = 9.1, 2.8 Hz, 1H, H-6), 7.03 (d, J = 2.8 Hz, 1H, H-8), 7.07 (t, J = 7.7 Hz, 1H, H-40), 7.32
t, J = 7.7 Hz, 2H, H-30 & H-50), 7.58 (d, J = 7.7 Hz, 2H, H-20& H-60), 7.75 (d, J = 8.4 Hz, 1H, H-5), 10.35
(
(s, 1H, NH); 13C-NMR (DMSO-d6):
δ ppm (175 MHz) 39.6 (CH2CO), 56.4 (OCH3), 101.4 (C-8), 112.7
(C-6), 113.1 (C-3), 113.4 (C-4a), 119.7 (C-20 & C-60), 124.1 (C-5), 127.0 (C-40), 129.3 (C-30 & C-50), 139.3
(C-10), 151.3 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.9 (C-2), 167.0 (C=O); MS m/z (% relative abundance):
[M + 1] 310 (33).

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N-(4-Bromophenyl)-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIIg):
Yield: 0.26 g (51%); Beige powder m.p.: 212–213 ◦C; IR (KBr): (cm−1): 500 (C-Br), 1066 (C–N),
1653 (C=O, amide), 1728 (C=O, lactone), 2935 (C–H, aliphatic), 3111 (C–H, aromatic), 3248 (NH,
ν
secondary amine); 1H-NMR (DMSO-d6):
δ ppm (700 MHz) 3.87 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 6.34
(s, 1H, H-3), 7.00 (dd, J = 8.4, 2.8 Hz, 1H, H-6), 7.04 (d, J = 2.8 Hz, 1H, H-8), 7.51 (d, J = 9.1 Hz, 2H, H-20
& H-60), 7.55 (d, J = 9.1 Hz, 2H, H-30 & H-50), 7.73 (d, J = 9.1 Hz, 1H, H-5), 10.48 (s, 1H, NH); 13C-NMR
(DMSO-d6):
δ ppm (175 MHz) 31.2 (CH2CO), 56.4 (OCH3), 101.4 (C-8), 112.7 (C-6), 113.1 (C-3), 113.5
(C-4a), 115.6 (C-20 & C-60), 121.6 (C-40), 127.0 (C-5), 132.1 (C-30 & C-50), 139.6 (C-10), 151.1 (C-8a), 155.4
(C-4), 160.5 (C-7), 162.9 (C-2), 167.3 (C=O); MS m/z (% relative abundance): [M]+ 388 (1.39), [M + 1] 389
(2.09), [M+2] 390 (1.29).
N-(3-Hydroxy-4-methoxyphenyl)-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIIh):
Yield: 0.26 g (73%); Brown powder m.p.: 188–189 ◦C; IR (KBr):
ν
(cm−1): 1028 (C–N), 1653 (C=O,
amide), 1707 (C=O, lactone), 2985 (C–H, aliphatic), 3100 (C–H, aromatic), 3228 (NH, secondary amine),
3327 (br OH); 1H-NMR (DMSO-d6):
δ
ppm (700 MHz) 3.72 (s, 3H, C-40-OCH3), 3.88 (d, J = 7.0 Hz,
5H, OCH3 & CH2), 6.31 (s, 1H, H-3), 6.84 (d, J = 8.4 Hz, 1H, H-50), 6.94 (dd, J = 9.1, 2.8 Hz, 1H, H-60),
7.00 (dd, J = 9.1, 2.8 Hz, 1H, H-6), 7.02 (d, J = 2.8 Hz, 1H, H-8), 7.12 (d, J = 2.8 Hz, 1H, H-20), 7.74
(
d, J = 9.1 Hz, 1H, H-5), 9.09 (s, 1H, OH), 10.08 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ ppm (175 MHz)
31.2 (CH2CO), 56.3 (2
×
OCH3), 101.4 (C-8), 108.2 (C-20), 110.4 (C-6), 112.7 (C-3), 112.9 (C-50), 113.1
(C-4a), 113.3 (C-60), 127.0 (C-5), 132.8 (C-10), 144.5 (C-30), 146.9 (C-40), 151.5 (C-8a), 155.4 (C-4), 160.6
(C-7), 162.9 (C-2), 166.4 (C=O); MS m/z (% relative abundance): [M + 2] 357 (27.25).
3.2. Biological Evaluation
3.2.1. Cytotoxicity Assay (MTT Assay)
The cytotoxicity of compounds Ia,b, IIa,b, and IIIa–h against the MCF-7 cell line (ER+ breast
cancer cell line) and MDA-MB-231 (triple-negative breast cancer cell line, TNBC) was determined using
camptothecin as a pyranone-bearing reference standard [37]. The detailed experimental procedures
are provided in the Supplementary Materials.
3.2.2. Antiestrogenic Activity
The antiestrogenic activity of the test compounds was examined by performing a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of the MCF-7 cell line.
In this experiment, MCF-7 cells were treated with 17
The effect of various concentrations of the tested compounds on cell proliferation in the presence
of 17 -estradiol was measured [37]. The detailed experimental procedures are provided in the
β-estradiol (+ve cell proliferation compound).
β
Supplementary Materials.
3.2.3. Aromatase Inhibition
Sandwich enzyme immunoassay was adopted for aromatase inhibition assessment [38]. The
detailed experimental procedures are provided in the Supplementary Materials.
4. Conclusions
In conclusion, 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamide derivatives IIIa–h have been
prepared, characterized, and tested for their in vitro cytotoxic and antiestrogenic, as well as aromatase
inhibition, activities. The target compounds IIIa–h showed variable cytotoxic activity against two
breast cancer cell lines, MCF-7 and MDA-MB-231. 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-
chromen-2-one (IIIb) was the most potent cytotoxic compound against MCF-7, being about 14-fold
more potent than the reference standard, camptothecin. It also manifested high in vitro antiestrogenic
activity (IC50 = 29.49
µM). These findings indicate that a cyclic aliphatic lipophilic substitution in
compound IIIb produced obvious antiestrogenic and cytotoxic activities. Thus, it might have high

Molecules 2020, 25, 1553
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affinity to ER. Unfortunately, the tested compounds show moderate to low aromatase inhibition activity,
except for compound IIIe, which showed moderate inhibitory activity against aromatase with an IC50
value of 17.38 µM.
Supplementary Materials: Detailed experimental procedures for the biological evaluation of the tested compounds
are provided in the Supplementary Materials. (MTT assay: file:///C:/Users/hp/Desktop/Areej-392/Biology%
20Result-392/MTT%20protocol%20SIGMA%20kit.pdf; Anti-estrogen assay: https://www.jstage.jst.go.jp/article/
bpb/26/8/26_8_1219/_pdf and aromatase inhibition assay: file:///C:/Users/hp/Desktop/Areej-392/Biology%20Result-
392/Aromatase%20eia%20kit%20582701.pdf).
Author Contributions: M.S.A., O.M.A., and M.I.A. suggested the study, supervised the synthesis, and prepared
the manuscript for publication. A.N.A.S. prepared and identified the title compounds. A.A. carried out the
biological investigations. All authors discussed the contents of the manuscript. All authors have read and agreed
to the published version of the manuscript.
Funding: “This research was funded by THE DEANSHIP OF SCIENTIFIC RESEARCH, KING SAUD UNIVERSITY,
through the Research Group Project grant number RG-1440–140” and “The APC was funded by Research Group
Project grant number RG-1440–140”.
Acknowledgments: The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through Research Group Project no. RG-1440–140.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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