Synthesis, cytotoxicity and in silico study of some novel benzocoumarin-chalcone-bearing aryl ester derivatives and benzocoumarin-derived arylamide analogs

Article abstract of DOI:10.1515/znb-2020-0204

The development of new prostate cancer protein receptor cytochrome P450 17A1 inhibitors offers the possibility of generating structures of increased potency. To this end, the chalcone analogs 7 and 8 were prepared from treatment of methyl 3-oxo-3H-benzocoumarin-2-carboxylate (4) with aryl aldehydes. Treatment of 7 and 8 with three anti-inflammatory drugs, flurbiprofen, ketoprofen and ibuprofen, in the presence of POCl3/DMAP gave the ester analogs 9-12. Analogously, treatment of ethyl 3-oxo-3H-benzocoumarin-2-carboxylate (15), prepared previously from 2-hydroxy-1-naphthaldehyde (13) and dimethylmalonate (14), with various arylamines: 4-bromoaniline, 2-amino-6-methylpyridine, amino-antipyrine and 2-amino-5-nitrothiazole, in the presence of potassium tert-butoxide gave the benzocoumarine-3-arylamide analogs. The in vitro cytotoxic activities of 9-12 and 16-19 were evaluated against human prostate cancer cell lines (PC-3) and normal human liver epithelia (WRL-68) by MTT assay. Compounds 10 and 17 were the most active cytotoxic agents among the series against PC-3 cells with IC50 values of 71.35 and 78.25 μg mL-1 with SI values of 3.0 and 4.2, respectively (calculated from the cytotoxicity effects of 10 and 17 on the normal human liver epithelia [WRL-68]). Furthermore, compounds 11 and 12 were tested against breast cancer (HER2 cell lines), prostate cancer (DU-135 cell lines) and MCF-7 but were inactive. Molecular docking studies between the protein receptor CYPP450 17A1 and compounds 10 and 17 revealed that these compounds primarily form hydrophobic interactions with the receptor.

Full text of DOI:10.1515/znb-2020-0204

Z. Naturforsch. 2021; 76(3–4)b: 201–210
Nabeel A. Abdul-Ridha, Afraah D. Salmaan, Rita Sabah, Bahjat Saeed and
Najim A. Al-Masoudi*
Synthesis, cytotoxicity and in silico study of some
novel benzocoumarin-chalcone-bearing aryl ester
derivatives and benzocoumarin-derived arylamide
analogs
https://doi.org/10.1515/znb-2020-0204
Received December 19, 2020; accepted March 1, 2021;
published online April 3, 2021
docking studies between the protein receptor CYPP450 17A1
and compounds 10 and 17 revealed that these compounds
primarily form hydrophobic interactions with the receptor.
Keywords: amide analogs; benzocoumarin; breast cancer;
cytotoxicity; ester derivatives; molecular docking study.
Abstract: The development of new prostate cancer protein
receptor cytochrome P450 17A1 inhibitors offers the possibility
of generating structures of increased potency. To this end, the
chalcone analogs 7 and 8 were prepared from treatment of
methyl 3-oxo-3H-benzocoumarin-2-carboxylate (4) with aryl
aldehydes. Treatment of 7 and 8 with three anti-inflammatory
drugs, flurbiprofen, ketoprofen and ibuprofen, in the presence
of POCl3/DMAP gave the ester analogs 9–12. Analogously,
treatment of ethyl 3-oxo-3H-benzocoumarin-2-carboxylate
(15), prepared previously from 2-hydroxy-1-naphthaldehyde
(13) and dimethylmalonate (14), with various arylamines:
4-bromoaniline, 2-amino-6-methylpyridine, amino-antipyrine
and 2-amino-5-nitrothiazole, in the presence of potassium tert-
butoxide gave the benzocoumarine-3-arylamide analogs. The
in vitro cytotoxic activities of 9–12 and 16–19 were evaluated
against human prostate cancer cell lines (PC-3) and normal
human liver epithelia (WRL-68) by MTT assay. Compounds 10
and 17 were the most active cytotoxic agents among the series
against PC-3 cells with IC50 values of 71.35 and 78.25 μg mL–1
with SI values of 3.0 and 4.2, respectively (calculated from the
cytotoxicity effects of 10 and 17 on the normal human liver
epithelia [WRL-68]). Furthermore, compounds 11 and 12 were
tested against breast cancer (HER2 cell lines), prostate cancer
(DU-135 cell lines) and MCF-7 but were inactive. Molecular
1 Introduction
Benzocoumarin compounds target a variety of biological pro-
cesses and therefore are potential candidates for the treatment
of a large group of diseases. Benzocoumarins have been re-
ported to exhibit several biological activities including anti-
dyslipidemia and lipid [1–3], antioxidant ([3–5], anti-
inflammatory [6], antibacterial [7–9], antithrombotic [10],
antituberculosis [11], antitumor [5, 12, 13] agents as well as
selective inhibitors of alkaline phosphatase [14]. Some benzo-
coumarin analogs exhibited potential antiviral activity such
bis-benzocoumarin-thiadiazine analog (1; Figure 1) and was
found to block the function of viral neuraminidases of the
influenza virus, by preventing its reproduction by budding
from the host cell [15]. Meanwhile methylimidazole-
methylimidazole-benzocoumarin (2; Figure 1) [16] exhibi-
ted promising anti-HCV activity with EC50 values of 5.5 μm.
Further, several benzocoumarin derivatives exhibited sig-
nificant antitumor activity e.g. E-4-(3,4-dimethoxystyryl)-
2H-benzo[h]chromene-2-one (3; Figure 1) [17], which showed
maximum antiproliferative activity in breast cancer cell
lines (MDA-MB-231 and 4T1) and decreased the tumor size in
the in-vivo 4T1 cell-induced orthotopic syngeneic mouse
breast cancer model. Alternariol (3,7,9-1-methyl-6H-benzo
[c]chromen-6-one) is reported to inhibit cholinesterase en-
zymes [18], meanwhile, it is reported to be a full androgen
agonist in an in vitro assay [19]. In addition, it exhibited
antifungal and phytotoxic activity [20]. 3,4-Benzocoumarin,
a AOH (alternariol)‐like compound, has the sensibility of the
antibody with an IC50 of 919.2 ng mL−1 [21].
*Corresponding author: Najim A. Al-Masoudi, Department of
Chemistry, College of Science, University of Basrah, Basrah, Iraq; and
Am Tannenhof 8, 78464 Konstanz, Germany, E-mail: najim.al-
masoudi@gmx.de
Nabeel A. Abdul-Ridha, Department of Chemistry, College of Science,
University of Qadisiya, Diwaniyaa, Iraq
Afraah D. Salmaan, Department of Chemistry, College of Education,
University of Qadisiya, Diwaniyaa, Iraq
Rita Sabah, Department of Pharmaceutical Chemistry, College of
Pharmacy, University of Basrah, Basrah, Iraq
Fluorescence Imaging (FI) is one of the most popular
imaging modes in biomedical sciences for the visualization
Bahjat Saeed, Department of Chemistry, College of Education for Pure
Science, University of Basrah, Basrah, Iraq

202
N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
Figure 1: Some potentially active
benzocoumarin analogs.
of cells and tissues both in vitro and in vivo [22]. Benzocou- conditions following Murthi and Basak [30] method
marin analogs are considered as highly fluorescent imaging by treatment of 2-hydroxy naphthaldeyde with ethyl
agents with high emission yield, excellent photostability and acetoacetate. Treatment of 4 with 4-hydroxy- (5) and
extended spectral range [23]. As well, they constitute the 4-hydroxy-3-methoxybenzaldehyde (6) in the presence of
largest class of laser dyes in the blue-green region. These piperidine afforded after chromatographic purification
types of dyes have been employed as labels for fluorescent the desired chalcone analogs 7 and 8 in 86 and 79% yield,
energy transfer experiments. Murata et al. [24] reported the respectively.
syntheses and fluorescence properties of benzocoumarin
Next, a mild and efficient method is developed for the
derivatives as novel fluorophores emitting in the longer synthesis of esters following Chen et al. [31] method by
wavelength region and their application to analytical conversion of carboxylic acids into esters through carboxyl
reagents, while Rajisha et al. [25] have synthesized new activation by the reagent combination of POCl3 and DMAP.
benzocoumarinoxadiazolyls as strong blue-green fluorescent Thus, reaction of 8 with the anti-inflammatory drugs,
brighteners with good bathochromic shifts.
flurbiprofen, ketoprofen and ibuprofen, in the presence of
Chalcones derivatives possess various biological, POCl3 and catalytic amount of DMAP as a base to furnish
pharmacological and biocidal properties [26]. Various after chromatographic purification the desired esters 9–11
substitutions on both aryl rings (A and B) of the chalcones, in 80, 75 and 78% yield, respectively. Similarly, treatment
depending upon their positions in the aryl rings, appear to of 7 with ibuprofen under the same condition afforded the
influence anticancer activity by interfering with various bio- ester 12 in 80% yield, respectively (Scheme 1).
1
logical targets [27, 28], meanwhile chalcone molecules with a
The structures of 7–12 were confirmed by their IR, H
trimethoxyphenyl unit has been reported to be the most and 13C NMR and mass spectra. The benzocoumarin pro-
1
cytotoxic (IC50 = 0.21 nM) derivatives synthesized so far [29]. tons showed a similar pattern. In the H NMR spectra of
Considering the biological significance of benzocou- 7–12, H-4 appeared as a singlet in the range δ = 8.74–
marin and chalcone derivatives, herein we report the syn- 8.59 ppm, while H-5 of 7–10 resonated as doublet
thesis of new substituted chalcone-derived benzocoumarin (J = 7.5 Hz) or broad singlet in the range δ = 8.50–8.40 ppm.
scaffold, as well as their cytotoxicity on prostate cancer H-5 of compounds 11 and 12 overlapped with H-8 at
(PC-3 and DU-135 cells), breast cancer (MCF and HER2 cells) δ = 8.39 ppm, while H-6–H-10 of compounds 7–12 reso-
and an in silico molecular docking study.
nated as multiplet in the range δ = 7.80–7.13 ppm. The
olefinic protons H-12 and H-13 of 7 and 8 appeared in the
range δ = 6.88–6.67 ppm (multiplet) and δ = 7.85–7.71 ppm
(doublet, J12,
= 16.0 Hz), respectively, whereas these
13
2 Results and discussion
protons were overlapped with other aromatic protons of
compounds 9–12. The aliphatic protons were fully
analyzed (c.f. Section 4). In the 13C NMR spectra of 7–12, the
carbonyl carbon atoms of benzocoumarin backbone C2=O
and C11=O resonated in the ranges δ = 162.1–159.0 and
192.1–190.0 ppm, respectively, while the resonances in the
range δ = 175.8–175.3 ppm assigned to the carbonyl carbon
2.1 Chemistry
In the present investigation we report the syntheses
of some new benzocoumarin derivatives having
antitumor activity. 2-Acetyl-benzocoumarin precursor
(4) was prepared via Knoevenagel reaction in basic

N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
203
Scheme 1: Synthesis of new benzocoumarin-
chalcone-derived substituted ester
derivatives.
1
atoms C15=O of compounds 9–12. Compound 10 showed
The structures of 16–19 were identified by H and 13C
signals at δ = 197.4 ppm was attributed to the carbonyl and mass spectral data, which showed similar patterns of
carbon atom C17=O. The resonances at the ranges protons and carbon atoms as those of the benzocoumarin
1
δ = 138.7–132.8, 150.8–144.5 and 117.9–115.8 ppm were precursor. In the H NMR spectra, H-4 of the benzocou-
assigned to C-3, C-4 and C-4a, respectively. Carbon atoms marin scaffold resonated as a singlet in the range δ = 8.92–
C-5+C-9, C-5a and C-6 were resonated in the ranges 8.72, while H-5 of 16–18 appeared as a broad singlet at
δ = 123.1–122.7, 131.8–128.1 and 123.0–117.9 ppm, respec- δ = 8.78, 7.98 and 7.89 ppm, respectively. H-5 of compound
tively. In addition, the resonances at δ = 117.4–124.6 ppm 19 appeared as a doublet at δ = 7.91 ppm (J5, 6 = 7.5 Hz),
were assigned to C-10. The olefinic carbon atoms C-12 and whereas H-8 of 16–18 resonated as a broad singlet at
C-13 appeared in the ranges δ = 126.2–124.6 and 145.3 and δ = 8.65, 7.83 and 7.68 ppm, respectively. However, the
140.5 ppm, respectively. Carbon atom C–F of compound 10 doublet at δ = 7.69 ppm (J7, 8 = 7.5) was attributed to H-8 of
was appeared as a doublet at δ = 158.1 (JC, F = 270 Hz). The 19. In the 13C NMR spectra of 16–19, the resonances at the
other aromatic and aliphatic substituents carbon atoms regions δ = 158.0–157.0 and 163.6–163.0 ppm were
were fully analyzed (c.f. Section 4).
assigned for the carbonyl carbon atoms (C-2 and C-11),
We have selected ethyl 3-oxo-3H-benzocoumarin- respectively, whereas the carbonyl carbon atom (C-3) of
2-carboxylate (15), prepared previously from 2-hydroxy- pyrazole moiety (compound 18) appeared at δ = 62.2 ppm.
1-naphthaldehyde (13) and dimethyl malonate (14) (Vahini Atoms C-3–C4a resonated in the regions δ = 135.4–130.0,
et al. 2010) [32], as precursor for the synthesis of 146.0–135.1 and 118.0–116.6 ppm, respectively, while the
benzocoumarin-derived amide derivatives aiming to resonances in the regions δ = 125.1–120.1, 131.8–130.8,
examine their cytotoxicity against prostate cancer. Thus, 127.3–124.1 and 125.1–120.1 ppm were assigned to the car-
treatment of 15 with the arylamines 4-bromoaniline, bon atoms (C-5–C-7), respectively. However, C-8–C-10a
2-amino-6-methylpyridine, amino-antipyrine and 2-amino- were appeared at the regions δ = 129.1–125.1, 131.8–130.9,
5-nitrothiazole, in the presence of potassium tert-butoxide 116.6–114.7 and 149.4 ppm, respectively. The resonances at
at room temperature for 30–60 min, following the method δ = 61.4, 144.0 and 135.1 ppm were attributed to the carbon
of Kim et al. [33], afforded after purification the desired atoms (C-2, C-4 and C-5) of the thiazole moiety of 19. The
amide analogs 16–19 in 80, 74, 60 and 65% yield, respec- other aromatic aliphatic carbon atoms have been identified
tively (Scheme 2).
(c.f. Section 4).
Scheme 2: Synthesis of new benzocoumarin-
derived substituted arylamide derivatives.
Reagents and conditions: (i) dimethyl malo-
nate, EtOH-piperidine, reflux, 3 h; (ii) aryl-
amines, THF, potassium tert-butoxide, r.t.,
30–60 min.

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N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
was tested against breast cancer (HER2 cell lines) and
prostate cancer (DU-135 cell lines) with IC50 values of 66.39
and 119.8 μg mL−1, respectively. The cytotoxicity effects of
compounds 10 and 12 were further evaluated for their
toxicity against normal human liver epithelia (WRL-68)
with IC50 = 158.4 and 254.2 μg mL−1, resulting in SI values of
2.4 and 2.1, respectively (SI of doxorubicin = 9 and 14.5,
respectively). Compound 12 was tested against breast
cancer (MCF-7 cell lines) and noncancer cells WRL68 with
IC50 values of 146.8 and 157.4 μg mL−1, respectively,
resulting of SI = 1.1 (SI of doxorubicin = 9) (Figures S3,
Supplementary information). Therefore, compounds 10
and 12 showed no activity against both breast cancer
(MCF-7 and HER2) and prostate cancer (DU-135).
2.2 Cytotoxic activity in vitro
The anticancer activities of the new benzocoumarin-
chalcone-derived anti-inflammatory drugs 9–12 and
16–19 were determined in vitro by MTT assay [34] on cancer
cell lines, human prostate carcinoma (PC-3 cell lines) and
normal human liver epithelia (WRL-68), where doxoru-
bicin was used as a positive control. The results are sum-
marized in Table 1, and the cytotoxic effect expressed as
IC50 values in μg mL–1. After 72 h, all the complexes showed
no or relatively low cytotoxicity (Figures S1 and S2, Sup-
plementary information available online). Among the
tested compounds, 10 and 17 showed the strongest dose-
and time-dependent and significant cytotoxicity effect in/
on PC-3 cells with IC50 value of 71.35 and 78.25 μg mL−1
(Figure 2). However, the IC50 values for doxorubicin were
found to 17.5 μg mL−1 against PCa cell lines (PC-3) with
selective indices (SI) of 12 and 18, respectively.
2.3 Computational modeling study
The selective index (SI) is defined as the ratio of the
cytotoxicity of the tested compound with respect to normal
cells (IC50 WRL68) versus cancer cell and used to determine
the criterion of effectiveness of the compound. The SI
values higher than 1.0 indicate the compounds of consid-
erable anticancer specificity, and SI much greater than 1.0
highly selective ones. Therefore, we can conclude that
10 and 17, of the benzocoumarin-chalcone bearing keto-
profen and 6-methyl pyridine scaffolds are excellent se-
lective inhibitors against PCa (PC-3 cell lines) with SI = 3.0
and 4.2, respectively.
The prostate cancer protein receptor cytochrome P450 17A1
(P450c17) has been exploited as one of the therapeutic
target for PCa. In our search for new lead analogs as human
CYP450 17A1 inhibitor, we have selected compounds 10
and 17 for molecular docking study. Thus, molecular
docking of these two analogs into the three-dimensional
P450c17 receptor structure (PDB ID: 3RUK) was performed
using the Autodock program [35]. The prospective ligands
were ranked according to the highest energy of the best
conforms. Thus, the calculated energy scores for com-
pounds 10 and 17 are −6.98 and −9.85, respectively, indi-
cating selectivity and potency profiles of these analogs to
bind strongly the cytochrome P450 receptor. Figure 3(A)
In addition, compounds 10 and 12 have been selected
for further screening with other cell lines. Compound 10
Figure 2: Effect of concentration of
compound 10 and 17 on PC-3 and normal
human liver epithelia WRL-68 cells (n = 3)
for 72 h. Cell viability was evaluated by the
MTT colorimeter assay.

N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
205
Table : In vitro cytotoxic effectsa of some new benzocoumarin
demonstrated a π-π stacking interaction between the aro-
matic ring (f) of compound 10 and the pyrrole ring of
HEM600. Additionally, it showed the same stacking be-
tween the aromatic ring (e) and Asp298. Further, aliphatic
hydrophobic interactions with CYP450 17A1 receptors-
binding residues including Val482, Met369, Leu370 and
analogs given as ICb in μg mL−
.
Compd.
Cell line
PC-
WRL
SI

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.







Doxo
. Val483 were observed.
.
According to the cytochrome CYP450 receptor docking
.
.
.
.
.
results, Figure 3(B) demonstrated that compound 17 was
able to show three π -H interactions, one between the ar-
omatic ring (a) and Ala302, while the other interaction was
observed between the pyridine residue and Ala302. The
third π−H interaction was displayed between the aromatic
ring (c) and Ile205. Moreover, compound 17 showed three
C–H interactions of Me group of the pyridine ring with
Ala367, Val366 and Val483. In addition, a π -π interaction
aCytotoxic effects were expressed as IC for each cell line, obtained
by MTT assay after  h of treatment. bData represent the mean values
of three independent determinations. Doxo, Doxorubicin.
Figure 3: The interactions mode of compounds 10 (A) and 17 (B) with the active site amino acids of the human cytochrome P450 17A1 (PDB ID:
3RUK).

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N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
between pyrrole groups of HEM600 and pyridine ring was organic extracts were washed with brine, dried over
observed. Besides these interactions, nonbonding amino Na2SO4 and evaporated in vacuo. The residue was purified
acids acid residues were surrounded by compounds 10 and on a short SiO2 column using the eluent hexane-EtOAc (3:2)
17 which further enhances their inhibitory activity.
as eluent to give the desired chalcone.
4.2.1 2-(3-(4-Hydroxyphenyl)acryloyl)-3H-
benzozocoumarin-3-one (7)
3 Conclusion
In the present work, new benzocoumarin-chalcone
bearing substituted ester derivatives 9–12 as well as
benzocoumarin-derived arylamide analogs 16–19 have
been synthesized. The structures of the new synthesized
benzocoumarin analogs were confirmed by the spectral
and mass data. The synthesized analogs were evaluated for
their activity against prostate cancer (PC-3) and the results
showed that 10 and 17 were the most active analogs of the
series with IC50 values of 71.35 and 78.25 μg mL−1 (SI = 3.0
and 4.2, respectively, based on the effect of 10 and 17 on the
noncancer prostate cells WRL68). Molecular docking
simulations showed that analogs 10 and 17 can potentially
inhibit protein receptor CYP450. Therefore, these com-
pounds seem to be the most promising antiproliferative
agents against protein receptor cytochrome P450 17A1
dependent prostatic cancer cells.
From 4-hydroxybenzaldeyde 5 (61 mg). Yield: 147 mg,
(86%) as orange crystals, m.p.: 270–272 °C, Rf = 0.78. – IR
(KBr): ν = 3082 (Ar–H), 2939 (C–Haliphatic), 1730 (C=Olactone),
1680 (C=Ochalcone), 1608, 1552 (C=C) cm−1. – 1H NMR (DMSO-
d6): δ = 9.76 (s,1H, C–OH), 8.59 (s, 1H, H-4), 8.48 (d, 1H,
J
5, 6 = 7.5 Hz, H-5), 8.30 (d, 1H, J7, 8 = 7.5 Hz, H-8), 7.71 (d, 1H,
J
12, 13 = 15.6 Hz, H-13), 7.80 (m, 1H, H-6), 7.74 (m, 1H, H-7),
7.70–7.35 (m, 4H, H-9+H-10+H-2′+H-6′), 6.97–6.81 (m, 3H,
H-12+H-3′+H-5′). – 13C NMR (DMSO-d6): δ = 190.0 (C11=O),
162.1 (C2=O) 155.8 (C–OH), 155.0 (C-10a), 147.1 (C-4), 145.3
(Carom.-3′+C-13), 134.5 (C-3), 131.8 (Carom.-2″+Carom.-6″),
128.1 (C-5a+C-9), 127.8 (C-8a), 127.4 (C-8), 126.2 (Carom.
-
1″+C-12), 125.7 (Carom.-2′), 122.7 (C-5+C-6), 118.5 (C-7), 116.1
(C-4a+C-10), 115.5 (Carom.-3′+Carom.-5′). – MS ((+)-EI) for
C22H14O4: m/z (%) = 342 (85) [M]+.
4.2.2 2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-3H-
benzocoumarin-3-one (8)
4 Experimental section
4.1 General
From vanillin 6 (76 mg). Yield: 147 mg, (79%) as a brown
crystals, m.p.: 255–256 °C, Rf = 0.73. – IR (KBr): ν = 3085
(Ar–H), 2942 (C–Haliphatic), 1724 (C=Olactone), 1681
Melting points were determined on Stuart SMP3 melting
point apparatus and are uncorrected. The IR spectra were
recorded, on FT-IR Spectrophotometer (Shimaduz), using
KBr discs. 1H and 13C NMR spectra were recorded on Varian
500 (1H) and 125.65 MHz (13C) spectrometers, using DMSO-
d6 solvent containing tetramethylsilane as an internal
standard (chemical shifts in δ in ppm). Mass spectra (EI,
70 eV, and FAB) were recorded on MAT 8200 spectrometers
(Finnegan MAT, USA). The reactions were monitored by
thin layer chromatography, (eluent: hexane-EtOAc 4: 1),
and the spots were visualized by iodine and U.V.
1
(C=Ochalcone), 1610, 1572 (C=C) cm–1. – H NMR (DMSO-
d6): δ = 9.72 (s,1H, C–OH), 8.59 (s, 1H, H-4), 8.40 (d, 1H, J5,
= 7.4 Hz, H-5), 8.21 (br s, 1H, H-8), 7.85 (d, 1H,
6
J
12, 13 = 16.0 Hz, H-13), 7.70 (m, 1H, H-6), 7.65 (m, 1H, H-7),
7.62–7.34 (m, 3H, H-9+H-10+H-2′), 6.88 (m, 3H,
H-12+H-5′+H-6′), 3.82 (s, 3H, OMe). – 13C NMR (DMSO-d6):
δ = 190.4 (C11=O), 161.1 (C2=O), 154.8 (C-10a), 151.1 (C–
OMe), 150.8 (C-4+C-OH), 143.5 (C-13), 132.8 (C-3), 131.2
(C-9), 129.1 (C-5a), 125.2 (C-8a+C-12), 124.3 (C-8+Carom.
-
1″), 123.1 (C-12+Carom.-6′), 122.7 (C-5+C-6), 117.9
(C-4a+C-7), 116.1 (C-10), 112.7 (Carom.-2′), 45.4 (OMe). – MS
((+)-EI) for C23H16O5: m/z (%) = 373 (90) [M+H]+.
4.2 General procedure for the synthesis of
benzocoumarin-chalcone derivatives
(7 and 8)
4.3 General procedure for the synthesis of
benzocoumarin-chalcone bearing aryl
ester derivatives (9–12)
To a stirred solution of 4 (119 mg, 0.50 mmol) in EtOH
(25 mL) was added arylbenzaldehyde (0.50 mmol) and aq.
solution of 2 m NaOH (7 mL). After stirring at ambient
temperature for 24 h, the mixture was neutralized with 1 M To a mixture of 7 or 8 (0.60 mmol) and suitable carboxylic
HCl and partitioned with EtOAc (3 × 20 mL). The combined acid (0.5 mmol) in CH2Cl2 (10 mL) containing Et3N (101 mg,

N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
207
1.2 mmol) were added POCl3 (77 mg, 0.5 mmol) and DMAP 2′), 47.3 (OMe), 45.1 (C-16), 18.5 (Me-17). – MS ((+)-EI) for
(19 mg, 0.15 mmol) and the mixture was stirred at room C39H28O7: m/z (%) = 607 (65) [M+H]+.
temperature for 2–3 h. After completion the reaction via
monitoring by TLC, the mixture was evaporated to dryness
and the residue was partitioned between CHCl3 (3 × 10 mL)
and water (10 mL). The combined organic extracts were dried
(Na2SO4), filtered and evaporated to dryness. The residue
was poured on a short column of silica gel (5 g), using
hexane-EtOAc (3:2) as eluent to give the pure desired ester
4.3.3 2-Methoxy-4-(3-oxo-3-(3-oxo-3H-benzocoumarin-
2-yl)prop-1-en-1-yl)phenyl-2-(4-isobutyl phenyl)
propanoate (11)
From 8 (171 mg) and ibuprofen (103 mg) Yield: 218 mg,
(78%) as a purple powder, m.p.: 60–62 °C, Rf = 0.50. – IR
(KBr): ν = 3018 (Ar–H), 2955 (C–Haliphatic), 1732 (C=Oester),
1720 (C=Olactone), 1656 (C=Ochalcone), 1606, 1514 (C=C)
cm−1. – 1H NMR (DMSO-d6): δ = 8.68 (s, 1H, H-4), 8.39 (br
s, 2H, H-5+H-8), 7.85 (d, 1H, J12, 13 = 15.5 Hz, H-13), 7.75–
7.13 (m, 12H, Ar–H+H-12), 3.72 (m, 4H, OMe+H-16), 2.95
(m, 2H, CH2-18), 1.78 (m, 1H, H-19), 1.48 (br s, 3H, Me-17),
0.88 (br s, 6H, Me-20+Me-21). – 13C NMR (DMSO-d6):
δ = 192.0 (C11=O), 175.8 (C15=O), 159.8 (C2=O), 152.5
4.3.1 2-Methoxy-4-(3-oxo-3-(3-oxo-3H-benzocoumarin-
2-yl)prop-1-en-1-yl)phenyl-2-(2-fluoro-
[1,1′-biphenyl]-4-yl)propanoate (9)
From 8 (171 mg) and flurbiprofen (122 mg). Yield: 239 mg,
(80%) as a purple powder, m.p.: 73–75 °C, Rf = 0.49. – IR
(KBr): ν = 3061 (Ar–H), 2987 (C–Haliphatic), 1722 (C=Oester),
1699 (C=Olactone), 1656 (C=Ochalcone), 1606, 1514 (C=C),
(C-10a+C3′-OMe), 147.0 (C-4), 143.4 (C-13), 141.5 (Carom.
-
4′), 138.7 (C-3+Carom.-4″), 129.6, 128.5, 125.8
(Carom.+C-5a + C-6+C-8+C-8a + C-9+Carom.-1′), 124.7
(C-12), 122.9 (C-5+C-7+Carom.-6′), 115.8 (C4a + C-10+-
1
1419 (C–F) cm−1. – H NMR (DMSO-d6): δ = 8.71 (s, 1H,
H-4), 8.45 (d, 1H, J5, 6 = 7.5 Hz, H-5), 8.25 (m, 1H, H-8), 7.78
(d, 1H, J12, 13 = 16.0 Hz, H-13), 7.72–7.18 (m, 17H, Ar–
H+H-12), 3.82 (s, 3H, OMe), 2.91 (br s, 1H, H-16), 1.47 (br s,
3H, Me). – 13C NMR (DMSO-d6): δ = 192.1 (C11=O), 175.3
(C15=O), 159.0 (C2=O), 158.1 (d, JC, F = 270 Hz, C–F), 154.3
C
arom.-3′+Carom.-5′), 113.7 (Carom.-2′), 50.8 (OMe), 45.1
(C-16+C-18), 30.2 (C-19), 22.8 (Me-20+Me-21), 18.9 (Me-
17). – MS ((+)-EI) for C36H38O6: m/z (%) = 560 (75) [M]+.
(C-10a), 151.1 (C3′-OMe), 144.5 (C-4+C-13), 139.8 (Carom.
-
4.3.4 4-(3-Oxo-3-(3-oxo-3H-benzocoumarin-2-yl)prop-
1-yl)phenyl-2-(4-isobutylphenyl) propianoate (12)
4′), 135.0 (C-3+Carom.-1″+Carom.-1‴), 130.9
(C-5a+C-9+Carom.-1′), 129.0, 128.5, 128.0, 127.6
(Carom.+C-6+C-8+C-8a), 125.9 (C-12+Carom.-5′+Carom.-6″),
122.5 (C-5+C-7+Carom.-6′), 116.3 (C4a + C-10+Carom.-4″),
113.0 (Carom.-2′), 47.1 (OMe), 44.3 (C-16), 18.8 (Me-17). –
MS ((+)-EI) for C39H27FO7: m/z (%) = 597/599 (95) [M]+.
From 7 (171 mg) and ibuprofen (103 mg). Yield: 212 mg
(80%) as a purpule powder, m.p.: 84–86 °C, Rf = 0.53. – IR
(KBr): ν = 3061 (Ar–H), 2987 (C–Haliphatic), 1722 (C=Oester),
1699 (C=Olactone), 1656 (C=Ochalcone), 1606, 1514 (C=C), 1419
(C–F) cm−1. – 1H NMR (DMSO-d6): δ = 8.68 (s, 1H, H-4), 8.39
(br s, 2H, H-5+H-8), 7.85 (d, 1H, J12, 13 = 15.5 Hz, H-13), 7.75–
7.13 (m, 12H, Ar–H+H-12), 3.72 (m, 3H, H-16), 2.95 (m, 2H,
CH2-18), 1.78 (m, 3H, H-19), 1.48 (br s, 3H, Me-17), 0.88 (br s,
3H, Me-20+Me-21). – 13C NMR (DMSO-d6): δ = 191.4 (C11=O),
175.7 (C15=O), 158.8 (C2=O), 152.9 (C-10a+C-4′), 147.6 (C-4),
4.3.2 3-Methoxy-4-(3-oxo-3-(3-oxo-3H-benzocoumarin-
2-yl)prop-1-en-1-yl)phenyl-2-(3-benzoyl phenyl)
propanoate (10)
From 8 (171 mg) and ketoprofen (127 mg). Yield: 228 mg,
(75%) as a purpule powder, m.p.: 65–66 °C, Rf = 0.57. – IR
(KBr): ν = 2937 (C–Haliphatic), 1722 (C=Oester), 1656
(C=Olactone), 1657 (C=Ochalcone), 1600, 1512 (C=C) cm−1. – 1H
NMR (DMSO-d6): δ = 8.74 (s, 1H, H-4), 8.50 (br s, 1H, H-5),
8.34 (br s, 1H, H-8), 7.80–7.20 (m, 19H, Ar–H+H-12+H-13),
3.92 (br s, 3H, OMe+H-16), 1.49 (br s, 3H, Me-17). – 13C NMR
(DMSO-d6): δ = 197.4 (C17=O), 191.8 (C11=O), 175.6 (C15=O), 4.4 Ethyl 3-oxo-3H-benzocoumarin-
160.5 (C2=O), 153.8 (C-10a), 150.3 (C-OMe), 147.1 (C-4), 143.4
(C-13+Carom.-4′), 138.8 (C-3+Carom.-1″+Carom.-4″+Carom.-1‴),
140.5 (C-13), 139.5 (Carom.-4″), 133.2 (C-3+Carom.-1′+Carom.
-
1″), 129.5, 128.6, 127.2, 126.5, 124.1, (Carom.), 117.8
(C4a + C-10), 113.9 (Carom.-2′), 45.0 (C-16+C-18), 30.1 (C-19),
22.7 (Me-20+Me-21), 18.9 (Me-17). – MS ((+)-EI) for C35H30O5:
m/z (%) = 531 (95) [M+H]+.
2-carboxylate (15)
131.8 (C-5a+C-9+Carom.-1′), 130.6, 130.1, 129.4, 128.4, 126.3
(Carom.+C-6+C-8+C-8a), 125.3 (C-12+Carom.-5′+Carom.-6″),
This compound was prepared previously following Knoe-
venagel condensation reaction by refluxing 2-hydroxy-
1-naphthaldehyde (13) (172 mg, 1.0 mmol) and dimethyl
122.2 (C-5+C-7+Carom.-6′), 117.4 (C4a + C-10), 114.3 (Carom.
-

208
N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
malonate (14) (160 mg, 1.0 mmol) in EtOH (10 mL) in the (s, 1H, H-4), 7.98 (br s, 2H, H-5), 7.83 (br s, 1H, H-8), 7.25 (m,
presence of piperidine to furnish after work-up the ester 15 7H, Ar–H), 6.70 (br s, 1H, Hpyridine-5′), 2.15 (Me). – 13C NMR
(227 mg, 80%). Mp and mixed melting point (114–116 °C) (DMSO-d6) δ 163.2 (C11=O), 157.0 (C2=O), 155.0 (Cpyridine-2′),
together with the other spectral data were identical with 151.9 (Cpyridine-6′), 148.4 (C-10a), 146.0 (Cpyridine-4′+C-4),
those of the authentic sample prepared previously [32].
134.0 (C-3), 131.8 (C-9+C-5a), 125.1 (C-6+C-8), 123.2 (C-8a),
120.1 (C-5+C-7), 118.0 (C-4a), 116.6 (Cpyridine-3′), 114.7 (C-10),
112.5 (Cpyridine-5′). – MS ((+)-EI) for C20H14N2O3: m/z
(%) = 331 (80) [M+H]+.
4.5 General procedure for synthesis
benzocoumarin-chalcone-derived
substituted amide derivatives (16–19)
4.5.3 N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-3-oxo-3H-benzocoumarin-
2-carboxamide (18)
To a mixture of 15 (268 mg, 1.0 mmol) and arylamine
(1.0 mmol) in THF (20 mL) was added potassium tert-but-
oxide (224 mg, 2.0 eq) and the mixture was stirred at room
temperature for 30–60 min in the presence of air. The re-
action progress was monitored by TLC. After the comple-
tion of reaction, the solution was filtered and evaporated to
dryness. The residue was portioned between CHCl3
(3 × 15 mL) and water (15 mL) and the combined extracts
were dried (Na2SO4), filtered and evaporated to dryness.
The residue was purified on a short column of silica gel
(5 g), using hexane-EtOAc 2:1 to give the pure desired
amide.
From
4-amino-1,5-dimethyl-2-phenyl-pyrazole-3-one
(amino-antipyrine) (203 mg). Yield: 255 mg (60%) as a
colorless solid, m.p.: 125–126 °C, Rf = 0.48. – IR (KBr):
ν = 3433 (NHamide), 3064 (Ar–H), 2980 (C–Haliphatic), 1774
(C=Oantipyrine), 1763 (C=Oamide), 1682, 1562 (C=Olactone),
1610, 1562 (C=C) cm−1. – 1H NMR (DMSO-d6): δ = 9.68 (s, 1H,
NH), 8.72 (s, 1H, H-4), 7.89 (br s,1H, H-5), 7.68 (br s, 1H,
H-8), 7.48 (m, 4H, Ar–H). – 13C NMR (DMSO-d6): δ = 163.6
(C11=O), 162.2 (Cpyrazole-3′), 156.8 (C2=O), 149.0 (C-10a),
136.5 (C-4), 134.5 (C-3+Carom.-1″), 130.9 (C-5a+C-9+Carom.
-
3″+Carom.-5″), 129.1 (C-8+C-8a), 125.1 (C-6), 123.5 (C-5+C-7),
121.8 (Carom.-2″+Carom.-4″+Carom.-6″), 117.0 (C-4a), 115.3
(C-10), 103.4 (Cpyrazol-4′), 35.1 (N1′-Me), 14.9 (Cpyrazol-Me). –
MS ((+)-EI) for C25H19N3O4: m/z (%) = 425 (78) [M+H]+.
4.5.1 N-(4-Bromophenyl)-3-oxo-3H-benzocoumarin-
2-carboxamide (16)
From 4-bromoaniline (172 mg). Yield: 315 mg (80%) as a
greenish-white solid, m.p.: 92–94 °C, Rf = 0.44. – IR (KBr):
ν = 3452 (NHamide), 3063 (Ar–H), 2978 (C–Haliphatic), 1768,
1689 (C=Olactone+C=Oamide), 1610, 1562 (C=C), 1030 (C–Br)
cm−1. – 1H NMR (DMSO-d6): δ = 10.70 (s, 1H, NH), 8.92 (s, 1H,
H-4), 8.78 (br s, 1H, H-5), 8.65 (br s, 1H, H-8), 8.29–7.69 (m,
7H, Ar–H), 7.35 (br s, 1H, H-10). – 13C NMR (DMSO-d6):
δ = 163.6 (C11=O), 157.2 (C2=O), 149.0 (C-10a), 142.5 (C-4),
4.5.4 N-(5-Nitrothiazol-2-yl)-3-oxo-3H-benzocoumarin-
2-carboxamide (19)
From 2-amino-5-nitrothiazole (145 mg). Yield: 238 mg
(65%) as an orange solid, m.p.: 95–97 °C, Rf = 0.40. – IR
(KBr): ν = 3456 (NHamide), 3064 (Ar–H), 2980 (C–Haliphatic),
1764 (C=Oamide), 1693 (C=Olactone), 1610, 1562 (C=C), 1492
(NO2) cm−1. – 1H NMR (DMSO-d6): δ = 11.89 (s, 1H, NH), 9.46
(s, 1H, Hthiazole-4′), 8.74 (s, 1H, H-4), 7.91 (br s, 1H, H-5), 7.69
(br s, 1H, H-8), 7.48 (m, 4H, Ar–H). – 13C NMR (DMSO-d6):
δ = 163.5 (C11=O), 161.4 (Cthiazole-2′), 156.7 (C2=O), 149.2
(C-10a), 144.0 (Cthiazole-4′), 135.1 (C-3+C-4+Cthiazole-NO2),
131.8 (C-5a + C-9), 129.0 (C-8+C-8a), 124.1 (C-6), 122.0 (C-7),
120.7 (C-5), 117.1 (C-4a), 115.8 (C-10). – MS ((+)-FAB) for
C17H9N3O5S: m/z (%) = 390 (90) [M+Na]+.
135.0 (Carom.-1′), 133.4 (C-3), 130.8 (C-3+C-5a+C-9+Carom.
-
3′+Carom.-5′), 127.3 (C-6+C-8+C-8a), 125.1 (C-5+C-7), 120.9
(C-Br), 119.2 (Carom.-2′+Carom.-6′), 117.5 (C4a), 116.6 (C-10). –
MS ((+)-FAB) for C20H12BrNO3: m/z (%) = 416/418 (90)
[M+Na]+.
4.5.2 N-(6-Methylpyridin-2-yl)-3-oxo-3H-
benzocoumarin-2-carboxamide (17)
From 2-amino-6-methylpyridine (108 mg). Yield: 244 mg
(74%) as a colorless solid, m.p.: 87–89 °C, Rf = 0.42. – IR 4.6 Cytotoxicity assay
(KBr): ν = 3380 (NHamide), 3063 (Ar–H), 2961 (C–Haliphatic),
1743, 1699 (C=Olactone+C=Oamide), 1610, 1564 (C=C), 1296 Ninety µL containing 1.0 × 104 cells of cell line suspen-
(CN) cm−1. – 1H NMR (DMSO-d6): δ = 10.92 (s, 1H, NH), 8.82 sion (PC-3, MCF-7, HER2, DU-135 and WRL68 cells) were

N.A. Abdul-Ridha et al.: Benzocoumarin-derived ester and arylamide analogs
209
seeded into wells of a 96-well plate. After 24 h of incu- Lamarkian Genetic Algorithm (LGA) was used for pose
bation, 10 µL of crude extracts dissolved in 0.01% sampling and the number of energy simulations was set to
ethanol were added to final concentration 500 or 2500000. The default scoring function was used for
1,000 μg mL−1 and incubated for another 48 h. The calculating the docking scores. Autogrid was used to pre-
cytotoxicity test was determined by MTT (3- pare the maps. The results of molecular docking were
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- visualized in BIOVIA DISCOVERY STUDIO 2020 [38] and then
mide) assay [34]. In brief, 50 µL of MTT was added to the analyzing the docking results. All docking simulations
wells, the cells were cultured for additional 4 h at 37 °C. performed to validate the method, using the ligands pre-
Then 100 µL of 10% SDS was added to the wells. The sent in crystal structures, were able to reproduce the
solubilized formazan was measured at 595 nm using ligand-protein interaction geometries. The image of the
microplate spectrophotometer. Each treatment was native ligand for 3RUK against the redocked native ligand
assayed in triplicate. Three independent preparations of with AUTODOCK is shown in Figure S7 (Supplementary in-
the synthesized ligand and MTT assays were performed. formation), meanwhile the root-mean-square deviation of
Doxorubicin and 0.01% ethanol (vehicle control) were atomic positions (RMSD) was 0.405 Å.
used as positive and negative controls, respectively. The
cytotoxicity of the new ligands was expressed as IC50
value (concentration exhibited 50% cytotoxicity).
5 Supplementary information
Cytotoxic effects of 9, 10, 12 and 16–19 on both PC-3 and
WRL-68 cells (Figure S1), cytotoxic effects of 9–12 and 16–
19 and doxorubicin on PC-3 cells (Figure S2), cytotoxic ef-
fects of 9 on the prostate cancer (DU-135) and breast cancer
(HER2) cells as well as 12 on the breast cancer (MCF-7) cells
(Figure S3) together with the image of the native ligand for
3RUK against the redocked native ligand (Figure S4) and
other supporting data (1H and 13C NMR spectra of com-
pounds 7 (Figure S5, S6), 8 (Figure S7–9), 10 (Figure S12,
S13), 11 (Figure S14, S15), 12 (Figure S17), 16 (Figure S18,
S19), 17 (Figure S20, S21), 18 (Figure S22, S23) and 19
(Figure S24, S25) associated with this article can be found
as supplementary material in the online version (https://
doi.org/10.1515/znb-2020-0204).
4.7 Dock and virtual screening
4.7.1 Preparations of ligands and proteins
The structures of ligands 10 and 17 were prepared by AVOGADRO
(v. 1.0.1) [36] software and saved as PDB file formate. Then,
the two ligands were prepared selecting torsions and the
structures which were converted from PDB formate to PDBQT.
The PDBQT files for the proteins and the ligands, united atom
Kollman charges, fragmental volumes, and solvation pa-
rameters were performed by the MGLTools software. Ligand
structures were energy minimized with the MMFF94 force
field. The native ligands and crystallographic water mole-
cules were removed from the PDB structures and the polar
hydrogens were added before docking.
Acknowledgment: The authors thank the Department of
Chemistry, College of Education, University of Al-
Qadisiyah for providing necessary facilities.
4.7.2 Grid map calculations
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Conflict of interest statement: The authors declare no
conflicts of interest regarding this article.
AUTODOCK grid maps were calculated for each compound
using AUTOGRID4, based on the active site coordinates of
each protein crystal structure. The size of all grid boxes
60 × 60 × 60 xyz points with a grid spacing of 0.375 Å. The
grid center dimensions were 26.18, −5.07, and 38.31 for x, y
and z, respectively. Maps were calculated for each atom
type in each ligand along with an electrostatic and des-
olavation map using dielectric value of −0.1465.
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Supplementary Material: The online version of this article offers
supplementary material (https://doi.org/10.1515/znb-2020-0204).