Design, synthesis and apoptosis inducing activity of nonsteroidal flavone-methanesulfonate derivatives on MCF-7 cell line as potential sulfatase inhibitor

Article abstract of DOI:10.1007/s00044-021-02767-w

In recent years, focusing on new potent anticancer agents with selective activity is one of the greatest challenges in cancer therapy. Breast cancer is the most common cancer and the main cause of cancer deaths in women. The sulfatase enzyme plays an important role in converting the sulfated steroids into non-sulfate steroid hormones, which increases the growth and development of many hormone-dependent cancers, such as breast cancer. In this regard, structure-based optimization was conducted to design novel flavone-sulfonates pharmacophore as a new steroid sulfatase inhibitor. In the present work, the conventional methods for the synthesis of 4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate derivatives were reported. Their cytotoxicity was evaluated with MTT assay against a breast cancer cell line (MCF-7). The apoptosis inducing activity of the most cytotoxic compound 3c with an IC₅₀ value of 0.615 μM was evaluated in comparison to docetaxel in the presence of estradiol which is a crucial growth factor to survive the cancerous cells. The results of double staining Annexin V-FITC/PI analysis suggested that the cytotoxic activity of this compound 3c in MCF-7 cells occurs via apoptosis. Molecular docking studies were conducted to clarify the inhibition mode of the most promising compound (3c) over the sulfatase (1P49) binding site. The analysis revealed the role of hydrogen bond interaction with Gly181 and hydrophobic interactions through the 1P49 active site in the ligand-receptor complex as significant descriptors to rationalize the potential inhibition activity. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02767-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1677–1687
https://doi.org/10.1007/s00044-021-02767-w
ORIGINAL RESEARCH
Design, synthesis and apoptosis inducing activity of nonsteroidal
flavone-methanesulfonate derivatives on MCF-7 cell line as potential
sulfatase inhibitor
Mahdiyeh H. S. Javadi1 Aida Iraji2,3 Maliheh Safavi4 Hamed Montazeri5 Parastoo Tarighi6 Samane Eftekhari6
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●
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Latifeh Navidpour7 Seyedeh Sara Mirfazli
8
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Received: 23 April 2021 / Accepted: 8 July 2021 / Published online: 20 July 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
In recent years, focusing on new potent anticancer agents with selective activity is one of the greatest challenges in cancer
therapy. Breast cancer is the most common cancer and the main cause of cancer deaths in women. The sulfatase enzyme
plays an important role in converting the sulfated steroids into non-sulfate steroid hormones, which increases the growth and
development of many hormone-dependent cancers, such as breast cancer. In this regard, structure-based optimization was
conducted to design novel flavone-sulfonates pharmacophore as a new steroid sulfatase inhibitor. In the present work, the
conventional methods for the synthesis of 4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate derivatives were reported.
Their cytotoxicity was evaluated with MTT assay against a breast cancer cell line (MCF-7). The apoptosis inducing activity
of the most cytotoxic compound 3c with an IC50 value of 0.615 µM was evaluated in comparison to docetaxel in the presence
of estradiol which is a crucial growth factor to survive the cancerous cells. The results of double staining Annexin V-FITC/PI
analysis suggested that the cytotoxic activity of this compound 3c in MCF-7 cells occurs via apoptosis. Molecular docking
studies were conducted to clarify the inhibition mode of the most promising compound (3c) over the sulfatase (1P49)
binding site. The analysis revealed the role of hydrogen bond interaction with Gly181 and hydrophobic interactions through
the 1P49 active site in the ligand-receptor complex as significant descriptors to rationalize the potential inhibition activity.
Graphical Abstract
4
* Latifeh Navidpour
Department of Biotechnology, Iranian Research Organization for
Science and Technology, Tehran, Iran
navidpur@sina.tums.ac.ir
5
* Seyedeh Sara Mirfazli
Department of Pharmacognosy and Pharmaceutical
Biotechnology, School of Pharmacy, Iran University of Medical
Sciences, Tehran, Iran
mirfazli.s@iums.ac.ir
1
6
7
8
Department of Chemistry, North Tehran Branch, Islamic Azad
University, Tehran, Iran
Department of Medical biotechnology, Faculty of allied medicine,
Iran university of Medical sciences, Tehran, Iran
2
Stem Cells Technology Research Center, Shiraz University of
Medical Sciences, Shiraz, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
3
Central Research Laboratory, Shiraz University of Medical
Sciences, Shiraz, Iran
Department of Medicinal Chemistry, School of Pharmacy, Iran
University of Medical Sciences, Tehran, Iran

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Keywords Cancer Breast cancer Arylsulfatase Steroid sulfatase Flavone
Introduction
evaluation of piperazinyl-ureido sulfamates as STS inhibi-
tors recorded micromolar to low nanomolar potency in
which employing 4-chlorophenyl pendant on the structure
achieved the most potent compound with STS IC50 values
of 5.1 nM [13]. Grienke et al develop three ligand-based
pharmacophore models to identify STS inhibitors from
natural sources and lanostanetype triterpene was predicted
Cancer is known as a chronic and noncommunicable dis-
ease [1]. In more than hundred different kinds of cancers,
breast cancer is the most common cancer and the main
cause of cancer deaths in women [2]. The World Health
Organization (WHO) reports biologically active hormones,
including estrogens, as one of the most important factors to
develop breast cancer. It was reported that the steroid levels,
including estrone (E1), estradiol (E2), estrone sulfate (E1S),
and estradiol sulfate (E2S), are enhanced in breast tumors
compared to levels in the plasma and breast normal tissue
[3]. The importance of estrogen production in the patho-
genesis of breast carcinoma is supported by numerous stu-
dies [4, 5]. Modern targeted cancer therapy has now focused
on decreasing estrogen levels [6].
as STS inhibitors. In this context, piptolinic acid D (IC50
=
10.5 μM), pinicolic acid B (IC50 = 12.4 μM), and ganoderol
A (IC50 = 15.7 μM) being the most pronounced and first
natural STS inhibitors [14].
Results and discussion
The inhibitor design
Sulfatase is widely distributed throughout the body, and
it acts in physiological processes and pathological condi-
tions. Steroid sulfatase (STS) is known as a sulfatase
enzyme that catalyzes the conversion of sulfated steroid
(hormone precursors) to free steroid (active form) that sti-
mulates the growth of tumors in various tissues especially
the breast. STS has a mushroom-like shape, with two
hydrophobic antiparallel α-helices structures [7]. STS has
been found in the membranes of the endoplasmic reticulum
which anchoring the functional domain on the surface. The
active site of STS is located deep in a cavity of the rounded
part of the STS and rests near the membrane surface which
forms four potential and two functional glycosylation sites
[8]. The inhibition of STS is a novel therapeutic strategy for
the treatment of estrogen-dependent tumors. Some STS
inhibitors have entered clinical trials and their efficacy is
under investigation in postmenopausal women with breast
cancer [9]. Therefore, inhibition of this enzyme decreases
the level of the active hormone to target the breast, endo-
metrial, prostate, and other hormone-sensitive cancers [10].
In 2020, Maltais et al. reported the design of fluorescent
inhibitors of STS by adding a dansyl group on an estrane
scaffold. Inhibition assays on HEK-293 cells expressing
exogenous STS depicted an IC50 value of 69 nM for the
most potent compound [11]. Also, this compound process
fluorescent properties that provide an interesting tool to
evaluate the cellular changes. In the other study, a series of
3-aminocoumarin-7-O-sulfamates were designed and syn-
thesized. Structure-activity relationship showed that sub-
stitution on 3-position of coumarin improved better STS
inhibition. According to in vitro study 3-benzyl-amino-
coumarin-7-O-sulfamates inhibited human placenta STS
with IC50 of 0.13 μM in an irreversible manner [12]. In vitro
The STS inhibitors can be classified into four main cate-
gories; including steroid sulfamate-based inhibitors, non-
steroidal sulfamate inhibitors, steroid non-sulfamate based
inhibitors, and nonsteroid non-sulfamate based inhibitors.
The first steroidal STS inhibitors were designed based on
the similarity with the substrate parental structure. Hallmark
in this regard back to 1994 in which the replacement of OH
of the sulfate group (A, Fig. 1) by an NH2, generated
estrone-3-O-sulfamate known as EMATE (C, Fig. 1,) as an
irreversible steroidal inhibitor. This compound performed a
great activity in MCF-7 cells, with an IC50 value of 65 pM
[15]. The overall designing strategic was based on the
substitution at the steroidal 3-phenolic position with the
resistance group to hydrolyze by the enzyme [16]. There-
fore, the core aryl O-sulfamate pharmacophore was devel-
oped over the past few years [17]. Other steroidal inhibitors
of STS containing different functional groups, for example,
phosphonothioate (D), thiophosphate (E) and phosphate (F)
have been developed over time [18, 19]. Unexpectedly,
high estrogenicity was observed with estrogenic inhibitors
and thus unsuitable effects were seen as anticancer agents.
These results stimulate an intense search for orally active
and non-estrogenic STS inhibitors [20].
The coumarin derivatives (containing two-ring coumarin
aryl sulfamate) exhibits high activity against STS such as 4-
methylcoumarin-7-O-sulfamate (G, COUMATE) with an
IC50 value of 380 nM against placental microsomes. Com-
pound H demonstrated 71% STS inhibition in rat liver after
24 h single oral dose [21]. 667-COUMATE (I) as coumarin
tricyclic derivatives showed an IC50 value of 8 nM without
significant estrogenic side effects. Also, compound J (fla-
vone-based agents) efficiently inhibited the purified human

Medicinal Chemistry Research (2021) 30:1677–1687
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Fig. 1 Structures of most potent STS inhibitors and designed compound
STS with IC50 = 0.026 µM and Ki = 0.19 µM without
estrogenic side effects on MCF-7 cells and good profile for
the treatment of breast cancer [22, 23].
methanesulfonyl chloride in the presence of triethylamine
gave the corresponding products 3a–i (Scheme 1).
As a result, in this study, flavone structure as potent and
irreversible STS inhibitors was selected as the backbone
[7, 18]. The sulfamate group attached to the aryl ring seems
to blocks STS activity via providing nonbreaking bound.
Besides the structural derivatization of target compounds
mainly focused on the substitution of the methanesulfonate-
flavone at the various position of the B aryl ring (Table 1).
Cytotoxic evaluation
MCF-7 as a breast cancer cell line with the expression of
STS was selected for further study. The IC50s of all com-
pounds are shown in Table 2 comparing with Docetaxel as a
reference drug. The most active compound was 3c (X =
OCH3–C6H4) with an IC50 value of 0.615 μM.
●
Synthesis of inhibitors
The unsubstituted derivative (3a) showed relatively
good cytotoxicity with an IC50 value of 1.591 μM.
Methyl substitution as a small electron-donating group
●
Initially, for the synthesis of desired compounds, the ben-
zoyl chloride derivatives (1a–i) were prepared, then reacted
with 2,4-dihydroxyacetophenone, after baker-vankatarman
rearrangement and neutralized with HCl. The 7-hydroxy-2-
phenyl-4H-chromen-4-one derivatives (2a–i) were synthe-
sized in the presence of K2CO3 in acetone under reflux
conditions for 8 h. Finally, the reaction of 2a–i with
on phenyl ring significantly decreased the cytotoxic
potency (3b, IC50 = 2.0 μM) compared with 3a. How-
ever, the replacement of the methyl group with para-
methoxy one as a bulk-electron donating moiety led to
an improvement in the cytotoxicity (3c, IC50
=
0.615 μM) comparing with the unsubstituted one (3a).

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Table 1 Summary of yields for
synthesis of 2-phenyl-4-oxo-4H-
chromen-7-yl methanesulfonate
derivatives
Entry R1
R2
R3
Yield of step (ii) Melting point
Yield of step (iii) Melting point
(°C)
(°C)
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
H
H
23%
21%
>250
>250
>250
>250
>250
>250
>250
>250
>250
67%
69%
78%
71%
65%
43%
52%
48%
54%
120–121
184–185
208–212
201–203
229–230
218–220
215–216
219–221
224–226
CH3
OCH3 32%
Cl
Br
H
12%
16%
13%
OCH3 OCH3
3g
3h
3i
OCH3
H
H
OCH3 15%
OCH3 OCH3 17%
OCH3 OCH3 OCH3 26%
Scheme 1 Synthesis of 2-phenyl-4-oxo-4H-chromen-7-yl methanesulfonate, Reagents and conditions: (i) Thionyl chloride, reflux, 2 h; (ii) 1:
K2CO3, acetone, reflux, 8 h; 2: H2O and methanol (1:1), Reflux, 2 h; (iii) THF, MSC, Triethylamine, RT, 24 h
●
Similarly, the introduction of the electron-withdrawing
group into 3a, resulting in 3d (IC50 = 1.057, R = para-
Cl-C6H4) and 3e (IC50 = 1.303, R = para-Br-C6H4)
which led to an increase in the cytotoxic activity
compared to the unsubstituted derivative. However, the
smaller group (Cl) recorded better activity in compar-
ison with the Br counterpart.
For the multi-substituted compound possessing MeO, the
reduction of anticancer activity was afforded for two
substituted groups. The activity of these analogs changes
was in the following order: 2,3-diOMe (IC50 = 2.60 μM)
was seen in compound 3i containing 2,3,4-triOMe with
an IC50 value of 1.02 μM.
Inhibition of STS in a cell-based assay
Based on the results, compound 3c with the least IC50 value
(IC50 = 0.615 0.077 µM) was selected to measure STS
inhibitory activity. In this regard, proliferation assay was
done for two concentrations of the selected compound alone
and in the presence of 100 nM estradiol. In these experi-
ments, the well-known chemotherapy agent docetaxel (Doc)
was used as the toxicity positive control. Based on data
●
> 2,4-diOMe (IC50 = 3.21 μM) > 3,4-diOMe (IC50
=
5.67 μM). However, improvement in the cytotoxicity

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Table 2 The cytotoxic activity
of synthetic nonsteroidal
flavone-methanesulfonate
derivatives against a breast
cancer cell line (MCF-7) after
48 h treatment
Compound
3a
R
IC50 (µM)a
Compound
R
IC50 (µM)a
1.591 0.433
3f
2.598 0.32
3b
3c
2.359 0.177
0.615 0.077
3g
3h
3.215 0.070
5.669 0.050
3d
1.057 0.408
3i
1.024 0.300
0.500 0.108
3e
1.303 0.410
Docetaxel
–
aEach value represents the mean SD (n = 3–5)
which was exposed to the co-treatment of 3c and estradiol
indicated more cell viability in comparison with the 3c
confirming the decreased number of active steroids and the
inhibition of STS. The cell viability in cell receives 500 nM
of Doc were significantly decreased compared to the control
group. However, cells treated with Doc + E2 showed
approximately the same % viability compared to cells
exposed to Doc alone proposing the other mechanism of
anticancer activity, not STS inhibition. In other words,
estradiol treatment diminished the toxicity of 3c but not that
of Doc in MCF-7 cells. This is based on the experiment in
which, estradiol could compensate the 3c toxicity but not
Doc toxicity. This could be explained by different inhibitory
mechanisms of 3c and Doc in cell proliferation. Doc
induced anticancer activity via effects by tubulin poly-
merization inhibition while 3c seems to work as an STS
inhibitor.
Fig. 2 Viability of MCF-7 cells treated with compounds after 48 h.
Control; untreated cells, E2; cells treated with estradiol (100 nM), 3c;
cells treated with indicated concentration of c3, CE1; cells treated with
3c (615 nM) and E2 (100 nM), CE2; cells treated with 3c (750 nM)
and E2 (100 nM), Doc; cells treated with docetaxel (500 nM), Doc-E2;
cells treated with 500 nM Doc (500 nM) and E2 (100 nM). Absor-
bance values were normalized to the control group (Mean SE, n ≥ 3).
*p < 0.05, **p < 0.01
Cell apoptosis on MCF-7 cells
Analysis of the flow cytometry double staining Annexin V-
FITC/PI revealed that the synthetic compounds 3c reduced
cell viability and induced apoptosis in human breast cancer
cells. The flow cytometry analysis was used as a quantita-
tive method of determining early and late apoptosis in
treated cancer cells. Figure 3. summarizes the data and
showed an increase in the apoptotic index in MCF-7 cells
indicated in Fig. 2. The control group demonstrated around
100% of viability. As can be expected exposure to estradiol
increases viability to more than 100% (p value < 0.05). The
results of the assay defined that the 3c was able to sig-
nificantly decrease cell viability in two tested concentrations
to around 58.2% and 68.3%, respectively. Also, the group

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treated with synthetic compound compared with negative
control. The results of flow cytometric analysis showed that
exposure of the MCF-7 cell line to the IC50 concentration of
compound 3c induced early apoptosis in 16.3% of cells and
late apoptosis in 10% of treated cells. According to Fig. 3, it
was revealed that 13.5% of MCF-7 cells treated with
positive control were at the early stage of apoptosis and
8.11% of the cells were at the late stage of apoptosis after
24 h treatment. The results confirmed that the cytotoxic
activity of 3c breast cancer cells occurs via apoptosis.
Docking study of STS
Molecular docking was performed using smina in the Linux
platform. The most potent ligand 3c was subjected to dock
with the 3D structure of STS, 1P49. The interactions of the
best-docked confirmation of 3c with the active site residues
of STS are depicted in Fig. 4. The methanesulfonate group
is well placed near the entrance to the active site with
conventional hydrogen bond interaction with Gly181 (dis-
tance: 2.61 Å) and two carbon–hydrogen bonds with
Thr180 and Gly181. Pi-pi T-shaped interaction was also
observed between the flavone backbone of 3c and the
Phe178 residue (distance: 1.94 Å). The para-methoxy
pendant was surrounded with several lipophilic amino acids
and demonstrated two pi-alkyl interactions with Leu185 and
Val186 as well as two carbon–hydrogen bonds with Ile226
and Phe230 with 3.43 and 3.06 Å distance.
The binding energy of reference inhibitor Irosustat was
found to be −8.22 kcal/mol which is comparable with the
value of 3c −8.49 kcal/mol within the sulfatase. The hydro-
phobic interactions of the best-docked conformation of Irosu-
stat into active site residues of 1P49 are depicted in Fig. 5 (a).
The superimposed structure of Irosustat as a reported
reference sulfatase inhibitor and the most potent compound 3c
in the active site of steroidal sulfatase (PDB ID: 1P49) was
shown in Fig. 5 (b). The STS binding site is built up with
catalytically important hydrophobic amino acid residues and
represented a long lipophilic tunnel [24]. The orientation and
favorable interactions of 3c was similar to the reference
inhibitor. The binding mode of 3c and Irosustat over the 1P49
active site clearly demonstrated that chromenone backbone
elongated comfortably through the hydrophobic tunnel which
is in good consistency with their STS inhibitor despite the
absence of a steroidal scaffold in E2.
Conclusion
Fig. 3 Flow cytometric analysis of Annexin V-FITC/PI stained Mcf-7
cell line treated with compound 3c. The cells treated with a DMSO 1%
(negative control); b IC50 concentration of Doc; c IC50 concentration
of compound 3c
4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate deri-
vatives were designed, synthesized, and evaluated for their
inhibitory activity toward STS. This led to the identification
of the most potent cytotoxic agent of the series, compound

Medicinal Chemistry Research (2021) 30:1677–1687
1683
Fig. 4 Compound 3c was
docked to the binding pocket of
the STS (PDB: 1P49)
Fig. 5 Representation of
Irosustat pose over the STS
(1p49) active site (a), Irosustat
(burgundy) and most potent
compound 3c (blue)
superimposed in the active site
pocket (b)
3c, with IC50 of 0.615 µM. Compound 3c showed a
remarkable potency as an anticancer agent on breast can-
cerous cell lines in the presence of estradiol via inhibiting
cell proliferation through the proposed mechanism (block-
ing sulfatase enzyme). 3c significantly decreased the num-
ber of active estrogens by blocking STS, which can be
counterbalanced by estradiol treatment. This mechanism is
further authenticated by the lack of estradiol potential in
decreasing Doc toxicity that exerted its effects by tubulin
polymerization inhibition. Molecular modeling studies
confirmed the vital structural characteristic necessary for
STS inhibition and these results suggested that compound
3c accommodated well into the STS active site via forming
interaction with important residues.
Synthesis of 7-hydroxy-2-phenyl-4H-chromen-4-one
derivatives (2a–i)
Initially, 2,4-dihydroxyacetophenone (152 mg, 1 mmol) and
potassium carbonate (553 mg, 4 mmol) were dissolved in
dry acetone. Then the appropriate benzoyl chloride deriva-
tive (2 mmol) was added dropwise to the reflux solution for
5 min, and the resulting mixture was refluxed for 8 h. After
the completion of the reaction, the solvent was evaporated
under vacuum. Next, 5 ml of water and methanol (1:1) were
added and the mixture was refluxed for 2 h. The progression
of the reaction was monitored by TLC. Then, the mixture
was cooled to room temperature and was poured into ice
and then neutralized with 5% HCl solution. The precipitates
were filtered and purified by preparative TLC (hexane:
EtOAc, 1:1).
Material and method
7-Hydroxy-2-phenyl-4H-chromen-4-one (2a)
Synthesis of benzoyl chloride derivatives (1a–i)
White crystal, yield: 23%, mp: >250 ˚C, IR (KBr, cm−1):
3178 (OH), 1630; 1H NMR (500 MHz, DMSO-d6): δ 10.81
(s, 1H, OH), 8.07 (d, J = 8.7 Hz, 2H, H2’,6’), 7.89 (d, J =
8.7 Hz, 1H, H5), 7.55–7.62 (m, 3H, H3’,4’,5’), 7.01 (d, J =
2.1 Hz, 1H, H8), 6.93 (dd, J = 8.7, 2.1 Hz, 1H, H6), 6.91 (s,
1H, H3. (Anal. calcd. for C15H10O3: C, 76.62; H, 4.23; O,
20.15. Found: C, 75.98; H, 4.54; O, 20.01.
To the benzoic acid derivatives (1 mmol), thionyl chloride
(4 mmol, 0.28 mL) was added dropwise at room tempera-
ture and the resulting mixture was heated under reflux for
2 h. The extra thionyl chloride was evaporated under
vacuum and benzene was added 3 times to remove the
remaining of thionyl chloride.

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7-Hydroxy-2-(4-methylphenyl)-4H-chromen-4-one (2b)
2-(2,4-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2g)
White crystal, yield: 21%, mp: >250 ˚C, IR (KBr, cm−1):
3069 (OH), 1628; 1H NMR (500 MHz, DMSO-d6): δ 10.78
(s, 1H, OH), 7.96 (d, J = 8.7 Hz, 2H, H2’,6’), 7.88 (d, J =
8.7 Hz, 1H, H5), 7.38 (d, J = 8.7 Hz, 2H, H3’,5’), 7.0 (d, J =
2.1 Hz, 1H, H8), 6.92 (dd, J = 8.7, 2.1 Hz, 1H, H6), 6.85 (s,
1H, H3), 2.36 (s, 3H, CH3). Anal. calcd. for C16H12O3: C,
76.18; H, 4.79; O, 19.03. Found: C, 76.33; H, 4.52;
O, 18.79.
White crystal, yield:15%, mp: >250 ˚C, IR (KBr, cm−1):
2998 (OH), 1638; 1H NMR (500 MHz, DMSO-d6): δ 10.35
(s, 1H, OH), 8.11 (d, J = 8.7 Hz, 1H, H5), 7.84 (d, J =
8.7 Hz, 1H, H6’), 7.08 (s, 1H, H3), 7.01 (d, J = 8.7 Hz, 1H,
H6), 6.97 (s, 2H, H3’), 6.60 (d, J = 8.7 Hz, 1H, H5’), 6.52 (s,
1H, H8), 3.89 (s, 1H, OCH3), 3.86 (s, 1H, OCH3). Anal.
calcd. for C17H14O5: C, 68.45; H, 4.73; O, 26.82. Found: C,
68.76; H, 4.41; O, 27.12.
7-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (2c)
2-(3,4-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2h)
White crystal, yield: 32%, mp: >250 ˚C, IR (KBr, cm−1):
2981 (OH), 1685; 1H NMR (500 MHz, DMSO-d6): δ 10.93
(s, 1H, OH), 8.03 (d, J = 8.7 Hz, 2H, H2’,6’), 7.87 (d, J =
8.7 Hz, 1H, H5), 7.11 (d, J = 8.7 Hz, 2H, H3’,5’), 7.0 (d, J =
2.1 Hz, 1H, H8), 6.92 (dd, J = 8.7, 2.1 Hz, 1H, H6), 6.80 (s,
1H, H3), 3.86 (s, 3H, OCH3). Anal. calcd. for C16H12O4: C,
71.64; H, 4.51; O, 23.86. Found: C, 71.22; H, 4.82;
O, 24.01.
White crystal, yield:17%, mp: >250 ˚C, IR (KBr, cm−1):
3013 (OH), 1653; 1H NMR (500 MHz, DMSO-d6): δ 10.38
(s, 1H, OH), 7.94 (d, J = 8.7 Hz, 1H, H5), 7.70 (dd, J = 8.7,
2.1 Hz, 1H, H6’), 7.59 (s, 1H, H3), 7.37 (dd, J = 8.7, 2.1 Hz,
1H, H6), 7.11–7.14 (m, 2H, H2’,5’), 6.97 (s, 1H, H8), 3.89 (s,
1H, OCH3), 3.85 (s, 1H, OCH3). Anal. calcd. for C17H14O5:
C, 68.45; H, 4.73; O, 26.82. Found: C, 68.09; H, 4.98;
O, 26.86.
2-(4-chlorophenyl)-7-Hydroxy-4H-chromen-4-one (2d)
7-Hydroxy-2-(2,3,4-trimethoxy phenyl)-4H-chromen-4-one
(2i)
White crystal, yield:12%, mp: >250 ˚C, IR (KBr, cm−1):
3278 (OH), 1645; 1H NMR (500 MHz, DMSO-d6): δ 10.79
(s, 1H, OH), 8.10 (d, J = 8.7 Hz, 2H, H2’,6’), 7.89 (d, J =
8.7 Hz, 1H, H5), 7.64 (d, J = 8.7 Hz, 2H, H3’,5’), 7.01 (d, J
= 2.1 Hz, 1H, H8), 6.95 (s, 1H, H3), 6.93 (d, J = 2.1 Hz, 1H,
H6). Anal. calcd. for C15H9ClO3: C, 66.07; H, 3.33; O,
17.60. Found: C, 65.89; H, 3.62; O, 17.33.
White crystal, yield:26%, mp: >250 ˚C, IR (KBr, cm−1):
3014 (OH), 1629; 1H NMR (500 MHz, DMSO-d6): δ 10.80
(s, 1H, OH), 8.21 (d, J = 8.7 Hz, 1H, H5), 7.87 (d, J =
8.7 Hz, 1H, H6’), 7.56 (d, J = 8.7 Hz, 1H, H5’), 7.23 (s, 1H,
H3), 7.12–7.16 (m, 1H, H6), 6.78 (s, 1H, H8), 4.19 (s, 1H,
OCH3), 4.11 (s, 1H, OCH3), 3.91 (s, 1H, OCH3). Anal.
calcd. for C18H16O6: C, 65.85; H, 4.91; O, 29.24. Found: C,
65.30; H, 5.12; O, 29.40.
2-(4-bromophenyl)-7-Hydroxy-4H-chromen-4-one (2e)
White crystal, yield:16%, mp: >250 ˚C, IR (KBr, cm−1):
3301 (OH), 1607; 1H NMR (500 MHz, DMSO-d6): δ 10.69
(s, 1H, OH), 8.02 (d, J = 8.7 Hz, 2H, H2’,6’), 7.88 (d, J =
8.7 Hz, 1H, H5), 7.78 (d, J = 8.7 Hz, 2H, H3’,5’), 6.99 (d, J
= 2.1 Hz, 1H, H8), 6.94 (s, 1H, H3), 6.92 (dd, J = 8.7,
2.1 Hz, 1H, H6). Anal. calcd. for C15H9BrO3: C, 56.81; H,
2.86; O, 15.13. Found: C, 56.63; H, 3.07; O, 15.43.
Synthesis of 4-oxo-2-phenyl-4H-chromen-7-yl
methanesulfonate (3a–i)
To a solution of 2a–i (1 mmol) in dry tetrahydrofuran
(5 mL) methanesulfonyl chloride (1.5 mmol) and triethyla-
mine (2 mmol) was added dropwise in an ice bath and
stirred at room temperature overnight. Then the volatiles
were evaporated under vacuum. To this residue water was
added and it was extracted with ethyl acetate. The organic
phase was dried with sodium sulfate and the solvent was
evaporated under vacuum. The product was purified by
preparative TLC (hexane: EtOAc, 3:2).
2-(2,3-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2f)
White crystal, yield:13%, mp: >250 ˚C, IR (KBr, cm−1):
3005 (OH), 1627; 1H NMR (500 MHz, DMSO-d6): δ 10.42
(s, 1H, OH), 7.90 (d, J = 8.7 Hz, 2H, H5), 7.32 (dd, J = 8.7,
2.1 Hz, 1H, H6), 7.19 (dd, J = 8.7, 2.1 Hz, 1H, H4’), 6.94 (d,
J = 2.1 Hz, 1H, H8), 6.91–6.93 (m, 2H, H5’,6’), 6.61 (s, 1H,
H3), 3.88 (s, 1H, OCH3), 3.82 (s, 1H, OCH3). Anal. calcd.
for C17H14O5: C, 68.45; H, 4.73; O, 26.82. Found: C, 68.18;
H, 5.01; O, 26.65.
4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate (3a)
White crystal, yield:67%, mp:120–121 ˚C, IR (KBr, cm−1):
1638, 1335 (S = O), 1199; 1H NMR (500 MHz, DMSO-d6):
δ 8.17 (d, J = 8.4 Hz, 1H, H5), 8.15 (d, J = 8.4 Hz, 2H,

Medicinal Chemistry Research (2021) 30:1677–1687
1685
H2’,6’), 7.91 (d, J = 1.9 Hz, 1H, H8), 7.67–7.59 (m, 3H,
H3’,4’,5’), 7.50 (dd, J = 8.4, 1.9 Hz, 1H, H6), 7.13 (s, 1H,
H3), 3.55 (s, 3H, CH3SO2). Anal. calcd. for C16H12O5S: C,
60.75; H, 3.82; O, 25.29. Found: C, 60.81; H, 4.02;
O, 25.53.
δ 8.16 (d, J = 8.7 Hz, 1H, H5), 8.11 (d, J = 8.7 Hz, 1H,
H2’,6’), 7.92 (d, J = 2.2 Hz, 1H, H8), 7.83 (d, J = 8.7 Hz,
2H, H3’,5’), 7.50 (dd, J = 8.7, 2.2 Hz, 1H, H6), 7.17 (s, 1H,
H3), 3.55 (s, 3H, CH3SO2); MS: m/z 395 [M + 1]. Anal.
calcd. for C16H11BrO5S: C, 48.62; H, 2.81; O, 22.24.
Found: C, 48.43; H, 3.13; O, 22.56.
2-(4-methyl phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3b)
2-(2,3-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3f)
White crystal, yield:69%, mp:184–185 ˚C, IR (KBr, cm−1):
1642, 1342 (S = O), 1177; 1H NMR (500 MHz, DMSO-d6):
δ 8.15 (d, J = 8.7 Hz, 1H, H5), 8.06 (d, J = 8.7 Hz, 2H,
H2’,6’), 7.91 (d, J = 2.2 Hz, 1H, H8), 7.48 (dd, J = 8.7,
2.2 Hz, 1H, H6), 7.42 (dd, J = 8.7, 2.2 Hz, 1H, H3’,5’), 7.08
(s, 1H, H3), 3.55 (s, 3H, CH3SO2), 2.83 (s, 3H, CH3), 13C
NMR (125 MHz, DMSO-d6): 177.0 C4, 163.9 C2, 156.6
C8a, 152.9 C7, 142.88 C4’, 130.2 C3’,5’, 128.1 C5, 127.5 C1’,
126.8 C2’,6’, 122.5 C4a, 120.4 C6, 112.6 C8, 106.7 C3, 38.2
C–S, 21.4 CH3. Anal. calcd. for C17H14O5S: C, 61.81; H,
4.27; O, 24.22. Found: C, 61.48; H, 4.43; O, 24.18.
White crystal, yield: 43%, mp: 218–220 ˚C, IR (KBr, cm−1):
1648, 1368 (S = O), 1149;1H NMR (500 MHz, DMSO-d6):
δ 8.17 (d, J = 8.7 Hz, 1H, H5), 7.82 (d, J = 2.2 Hz, 1H, H8),
7.50 (dd, J = 8.9, 2.2 Hz, 1H, H6), 7.42 (d, J = 2.2 Hz, 1H,
H4’), 7.26–7.33 (m, 2H, H5’,6’), 6.83 (s, 1H, H3), 3.89 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.53 (s, 3H, CH3SO2);); 13C
NMR (125 MHz, DMSO-d6): 177.2 C4, 163.9 C2, 161.9 C8a,
161.0 C7, 157.0 C3’, 155.9 C2’, 131.1 C5, 120.8 C1’, 120.6
C5’, 117.7 C6’, 114.9 C4a, 112.5 C4’, 110.9 C3, 110.0 C6,
105.1 C8, 59.5 C–O2’, 58.5 C–O3’, 42.0 C–S. Anal. calcd.
for C18H16O7S: C, 57.44; H, 4.28; O, 29.76. Found: C,
57.73; H, 4.56; O, 30.03.
2-(4-methoxy phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3c)
2-(2,4-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3g)
White crystal, yield:78%, mp:208–212 ˚C, IR (KBr, cm−1):
1637, 1363 (S = O), 1179; 1H NMR (500 MHz, DMSO-d6):
δ 8.13 (d, J = 8.7 Hz, 1H, H5), 8.09 (d, J = 8.9 Hz, 2H,
H3’,5’), 7.85 (d, J = 2.2 Hz, 1H, H8), 7.46 (d, J = 8.7 Hz,
1H, H6), 7.13 (d, J = 8.9 Hz, 2H, H2’,6’), 6.98 (s, 1H, H3),
3.87 (s, 3H, OCH3), 3.52 (s, 3H, CH3SO2), 13C NMR
(125 MHz, DMSO-d6): 176.7 C4, 163.6 C2, 163 C4’, 156.6
C8a, 153 C7, 128.7 C2’,6’, 127.5 C5, 123.3 C1’, 122.8 C4a,
120.5 C6, 115.1 C3’,5’, 112.7 C8, 106.0 C3, 56.0 C–O, 37.4
C–S. Anal. calcd. for C17H14O6S: C, 58.95; H, 4.07; O,
27.72. Found: C, 59.22; H, 4.33; O, 28.10.
White crystal, yield: 52%, mp: 215–216 ˚C, IR (KBr, cm−1):
1659, 1349 (S = O), 1133; 1H NMR (500 MHz, DMSO-d6):
δ 8.06 (d, J = 8.7 Hz, 1H, H5), 7.94 (dd, J = 8.9, 2.2 Hz, 1H,
H6’), 7.62 (d, J = 2.2, 1H, H8), 7.56 (d, J = 2.2 Hz, 1H, H3’),
7.51 (dd, J = 8.9, 2.2 Hz, 1H, H6), 7.27 (dd, J = 8.9, 2.2 Hz,
1H, H ), 4.02 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.50 (s,
5’
3H, CH3SO2);); 13C NMR (125 MHz, DMSO-d6): 178.3 C4,
163.0 C2, 162.8 C4’, 160.4 C2’, 159.4 C8a, 157.9 C7, 130.2
C6’, 128.7 C5, 117.7 C4a, 114.4 C1’, 113.4 C3, 111.0 C6,
106.1 C5’, 105.1 C8, 101.2 C3’, 55.5 C–O2’, 55.4 C–O4’, 40.3
C–S. Anal. calcd. for C18H16O7S: C, 57.44; H, 4.28; O,
29.76. Found: C, 57.61; H, 4.55; O, 29.98.
2-(4-chloro phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3d)
White crystal, yield:71%, mp:201–203 ˚C, IR (KBr, cm−1):
1636, 1340 (S = O), 1179; 1H NMR (500 MHz, DMSO-d6):
δ 8.19 (d, J = 8.7 Hz, 2H, H2’,6’), 8.16 (d, J = 8.7 Hz, 1H,
H5), 7.92 (d, J = 2.2 Hz, 1H, H8), 7.69 (d, J = 8.7 Hz, 2H,
H3’,5’), 7.50 (dd, J = 8.7, 2.2 Hz, 1H, H6), 7.16 (s, 1H, H3),
3.55 (s, 3H, CH3SO2); MS: m/z 350 [M + 1]. Anal. calcd.
for C16H11ClO5S: C, 54.78; H, 3.16; O, 22.81. Found: C,
54.42; H, 3.33; O, 23.01.
2-(3,4-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3h)
White crystal, yield: 48%, mp: 219–221 ˚C, IR (KBr, cm−1):
1655, 1329 (S = O), 1169; 1H NMR (500 MHz, DMSO-d6):
δ 7.94 (d, J = 8.7 Hz, 1H, H5), 7.70 (d, J = 8.9 Hz, 1H, H6’),
7.59 (s, 1H, H8), 7.37 (d, J = 8.7 Hz, 1H, H6), 7.11–7.15 (m,
2H, H2’,5’), 6.98 (s, 1H, H3), 4.01 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 3.85 (s, 3H, CH3SO2); 13C NMR (125 MHz, DMSO-
d6): 178.9 C4, 165 C2, 160.0 C8a, 157.6 C7, 154.2 C3’, 149.3
C4’, 130.0 C5, 128.0 C1’, 127.5 C6’, 126.8 C4a, 112.5 C5’, 108
C6, 105.2 C2’, 104.5 C8, 103.9 C3, 56.9 C–O3’, 57.4 C–O4’,
42.4 C–S. Anal. calcd. for C18H16O7S: C, 57.44; H, 4.28; O,
29.76. Found: C, 57.09; H, 4.46; O, 29.88.
2-(4-bromo phenyl)-4-oxo-4H-chromen-7-yl
methanesulfonate (3e)
White crystal, yield: 65%, mp:229–230 ˚C, IR (KBr, cm−1):
1640, 1340 (S = O), 1178; 1H NMR (500 MHz, DMSO-d6):

1686
Medicinal Chemistry Research (2021) 30:1677–1687
4-oxo-2-(2,3,4-trimethoxy phenyl)-4H-chromen-7-yl
methanesulfonate (3i)
were assigned. The Irosustat and compound (3c) were
drawn using Marvin Sketch and subjected to energy mini-
mization using the steepest descent algorithm and then
Gasteiger charges were calculated by Open Babel. Binding
sites were determined automatically using the coordinates
of native co-crystallized ligands of the enzyme [26].
White crystal, yield: 54%, mp: 224–226 ˚C, IR (KBr, cm−1):
1680, 1328 (S = O), 1145; 1H NMR (500 MHz, DMSO-d6):
δ 8.15 (d, J = 8.7 Hz, 1H, H5), 7.81 (s, 1H, H8), 7.67 (d, J =
8.7 Hz, 1H, H6), 7.47 (d, J = 8.9 Hz, 1H, H6’), 7.03 (d, J =
8.9 Hz, 1H, H5’), 6.84 (s, 1H, H3), 3.89 (s, 3H, OCH3), 3.84
(s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.53 (s, 3H, CH3SO2);
13C NMR (125 MHz, DMSO-d6): 178.8 C4, 168.5 C2, 157.5
C8a, 157 C7, 154.6 C4’, 152.9 C2’, 141.9 C3’, 129.0 C5, 122.7
C6’, 119.5 C1’, 118.9 C4a, 110.3 C3, 109.0 C6, 107.5 C5’,
103.0 C8, 56.6 C–O2’, 54.9 C–O3’, 54.0 C–O4’, 38.4 C–S.
Anal. calcd. for C19H18O8S: C, 56.15; H, 4.46; O, 31.49.
Found: C, 56.55; H, 4.87; O, 31.73.
Funding This is to acknowledge any financial interest or benefit that
has arisen from the direct applications of this research.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Cell culture
References
A human breast cancer cell line, MCF-7, was obtained from
the Iranian Biological Resource Center (Tehran, Iran) and
maintained in RPMI 1640 medium (Biowest). The medium
was supplemented with 10% fetal bovine serum (FBS)
(Gibco, Carlsbad, CA, USA) and 1% antibiotics (Penicillin
and Streptomycin). The cells were incubated at 37 °C under
the standard condition of 95% humidity and 5% CO2 to
reach 70% cell confluency [25].
1. American Cancer Society. Cancer Facts & Figures 2018. Atlanta:
American Cancer Society; 2018.
2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM.
Estimates of worldwide burden of cancer in 2008: GLOBOCAN
2008. Int J Cancer. 2010;127:2893–917.
3. Pasqualini JR. The selective estrogen enzyme modulators in breast
cancer: a review. Biochim Biophys Acta. 2004;1654:123–43..
4. Deroo BJ, Korach KS. Estrogen receptors and human disease. J
Clin Investig. 2006;116:561–70.
5. Saha T, Makar S, Swetha R, Gutti G, Singh SK. Estrogen sig-
naling: an emanating therapeutic target for breast cancer treatment.
Eur J Med Chem. 2019;177:116–43.
Cell proliferation assay
6. Ireson CR, Chander SK, Purohit A, Parish DC, Woo LWL, Potter
BVL. Pharmacokinetics of the nonsteroidal steroid sulphatase
inhibitor 667 COUMATE and its sequestration into red blood
cells in rats. Br J Cancer. 2004;91:1399–404.
7. Reed MJ, Purohit A, Woo LWL, Newman SP, Potter BVL.
Steroid sulfatase: molecular biology, regulation, and inhibition.
Endocr Rev 2005;26:171–202.
8. Rižner TL. The Important Roles of Steroid Sulfatase and Sulfo-
transferases in Gynecological Diseases. Front Pharmacol
2016;7:30.
9. Geisler J, Sasano H, Chen S, Purohit A. Steroid sulfatase inhibi-
tors: Promising new tools for breast cancer therapy? J Steroid
Biochem Mol Biol 2011;125:39–45.
10. Thakur A, Singla R, Jaitak V. Coumarins as anticancer agents: a
review on synthetic strategies, mechanism of action and SAR
studies. Eur J Med Chem. 2015;101:476–95.
11. Maltais R, Ngueta Djiemeny A, Roy J, Barbeau X, Lambert JP,
Poirier D. Design and synthesis of dansyl-labeled inhibitors of
steroid sulfatase for optical imaging. Bioorg Med Chem.
2020;28:115368.
12. Hng Y, Lin MH, Lin TS, Liu IC, Lin IC, Lu YL, et al. Design
and synthesis of 3-benzylaminocoumarin-7-O-sulfamate deri-
vatives as steroid sulfatase inhibitors. Bioorg Chem
2020;96:103618.
13. Moi D, Foster PA, Rimmer LG, Jaffri A, Deplano A, Balboni G,
et al. Synthesis and in vitro evaluation of piperazinyl-ureido sul-
The cytotoxicity of compounds was assessed by MTT assay
which measures the percentage of viable cells. Cells were
seeded in a 96-well cell culture plate at 7 × 103 cells/well
and incubated for 48 h. Then, cells were exposed to fresh
medium containing different concentrations of compounds.
Subsequently, the medium was replaced with tetrazolium
salt (5 mg/ml of PBS) (MTT, (3-(4, 5-dimethylthiazol-2-yl)-
2, 5-diphenyltetrazolium bromide) (Sigma, St.Louis, MO,
USA) followed by additional 4 h incubation at 37 °C. The
formed formazan crystals were dissolved in 100 µl of
Dimethyl sulfoxide (DMSO) (Merck) and measured for
absorbance at 570 nm using a plate reader (BioRad, Model
680). Also finally, IC50 values were defined as the half-
maximal inhibitory concentration.
Docking analysis
To determine the possible binding modes of the compound,
docking analysis was carried out against the STS enzyme
using the smina molecular docking. The X-ray crystal
structures of STS enzyme (PID: 1P49) were extracted from
the PBD site and were prepared by removing solvent
molecules and the co-crystallized ligands. Polar hydrogen
atoms were added to the enzymes and the Kollmann charges
famates as steroid sulfatase inhibitors. Eur
2019;182:111614.
J Med Chem.
14. Grienke U, Kaserer T, Kirchweger B, Lambrinidis G, Kandel RT,
Foster PA, et al. Steroid sulfatase inhibiting lanostane triterpenes –

Medicinal Chemistry Research (2021) 30:1677–1687
1687
Structure activity relationship and in silico insights. Bioorg Chem
2020;95:103495.
21. Reed MJ, Potter BVL. Compounds that inhibit oestrone sulpha-
tase and/or aromatase and methods for making and using, 2003;
US6506792B1.
22. Nussbaumer P, Lehr P, Billich A. 2-Substituted 4-(thio)chrome-
none 6-O-sulfamates: potent inhibitors of human steroid sulfatase.
J Med Chem. 2002;45:4310–20.
23. Nussbaumer P, Winiski AP, Billich A. Estrogenic potential of 2-
alkyl-4-(thio) chromenone 6-O-sulfamates: potent inhibitors of
human steroid sulfatase. J Med Chem. 2003;46:5091–5094.
24. Ouellet C, Maltais R, Ouellet E, Barbeau X, Lagüe P, Poirier D.
Discovery of a sulfamate-based steroid sulfatase inhibitor with
intrinsic selective estrogen receptor modulator properties. Eur J
Med Chem. 2016;119:169–82.
15. Purohit A, Dauvois S, Parker MG, Potter BVL, Williams GJ,
Reed MJ. The hydrolysis of oestrone sulphate and dehy-
droepiandrosterone sulphate by human steroid sulphatase
expressed in transfected COS-1 cells. J Steroid Biochem Mol Biol
1994;50:101–4.
16. Purohit A, Williams GJ, Howarth NM, Potter BV, Reed MJ.
Inactivation of steroid sulfatase by an active site-directed inhibitor,
estrone-3-O-sulfamate. Biochemistry. 1995;34:11508–14.
17. Thomas MP, Potter BV. Discovery and Development of the Aryl
O-Sulfamate Pharmacophore for Oncology and Women’s Health.
J Med Chem. 2015;58:7634–58..
18. Kozak W, Daśko M, Masłyk M, Pieczykolan JS, Gielniewski B,
Rachon J, et al. Phosphate tricyclic coumarin analogs as steroid
sulfatase inhibitors: synthesis and biological activity. RSC Adv.
2014;5:44350–44358.
19. Demkowicz S, Kozak W, Daśko M, Masłyk M, Gielniewski B,
Rachon J. Synthesis of bicoumarin thiophosphate derivatives
assteroid sulfatase inhibitors. Eur J Med Chem. 2015;101:358–66.
20. Nussbaumer P, Billich A. Steroid sulfatase inhibitors. Med Res
Rev. 2004;24:529–76.
25. Edraki N, Iraji A, Firuzi O, Fattahi Y, Mahdavi M, Foroumadi A,
et al. 2-Imino 2H-chromene and 2-(phenylimino) 2H-chromene 3-
aryl carboxamide derivatives as novel cytotoxic agents: synthesis,
biological assay, and molecular docking study. J Iran Chem Soc.
2016;13:2163–71.
26. Saeedi M, Maleki A, Iraji A, Hariri R, Akbarzadeh T, Edraki N,
et al. Synthesis and bio-evaluation of new multifunctional
methylindolinone-1,2,3-triazole hybrids as anti-Alzheimer’s
agents. J Mol Struct. 2021;1229:129828.