Bioorganic Chemistry 161 (2025) 108512

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Bioorganic Chemistry
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Precise structure manipulation of selective estrogen receptor modulators

led to first-in-class thiophene-3-benzamide derivatives as potential
ER-antagonists without uterotrophic activity
Bader Huwaimel a,b, Amr S. Abouzied a,b,* , Abdul-Hamid Emwas c , Al-Shaimaa F. Ahmed d,
Mohamed K.S. El-Nagar e, Hamdoon A. Mohammed f, Nader M. Alrashidi g ,
Marwan A. Alrashidi g , Rakan E. Alshammari g , Abdulelah Y. Albladi g , Saad Alqarni a,
Shaymaa E. Kassab h,**
a
Department of Pharmaceutical Chemistry, College of Pharmacy, University of Ha’il, Ha’il 81442, Saudi Arabia
b
Medical and Diagnostic Research Center, University of Ha’il, Hail 55473, Saudi Arabia
c
Core Labs, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
d
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
e
Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Sadat City, Menoufia 32897, Egypt
f
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Qassim 51452, Saudi Arabia
g
College of Pharmacy, University of Hail, Hail 81442, Saudi Arabia
h
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, El-Buhaira 22516, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: The present study introduces two sets of compounds: panel A, which features thiophene-3-benzamide, and panel
Thiophene-3-benzamide B, which includes pyrazole-4-benzamide. We designed these compounds to target estrogen receptors (ER) while
Antiestrogen minimizing uterotrophic activities. The chemical structures of the two panels have been derived from structural
Uterotrophic activity
modifications of the selective estrogen modulator, methyl-piperidinopyrazole (MPP). These modifications aim to
Methyl-piperidinopyrazole
Tamoxifen
reduce uterotrophic effects. In our design, we incorporated amide, amine, and ketone spacers between the three
phenyl rings, substituting the central thiophene and pyrazole rings in the target ER antagonists. This structural
strategy aims to alter the formation of tris-p-phenol metabolites, which are associated with the potential es­
trogenic activity of this class of compounds. The cytotoxicity of the compounds from panel A revealed significant
activity against MCF7 cells, which are estrogen-dependent breast cancer cells. Importantly, these compounds
demonstrated minimal cytotoxicity against other cell lines, including skin, osteosarcoma, and triple-negative
breast cancer. Among the compounds tested, 5-benzoyl-thiophene-3-carboxamide 5a and 5-(4-chlorobenzoyl)-
thiophene-3-carboxamide 5d exhibited the highest cytotoxicity against MCF7 cells, with IC50 values of 7.38 μM
and 8.50 μM, respectively. Furthermore, both 5a and 5d showed potential as antiestrogens, exhibiting no es­
trogenic activity in the uterine tissues of immature rats. In the dose-response experiment, the 5d antiestrogenic
potency (EC50 = 5.530 μM) was comparable to that of Tamoxifen (EC50 = 7.625 μM). Molecular Docking and
Molecular Dynamics simulations elucidated the antiestrogenic activity of 5d and inactivity of 5-fluorobenzoyl
counterpart, 5e. The inactive derivative 5e in the active site exhibited an unfavorable conformation and un­
stable drug-receptor complex formation.

1. Introduction accomplish this by accepting transcription coactivators [4] at the

coactivator binding site (CBS), resulting in transcription and diverse
Estrogen receptors (ER) are nuclear receptors activated by the es­ biological activities at the cellular level [5,6]. Estrogen receptor-induced
trogen hormone; they are essential for DNA binding [1–3]. They transcriptional signaling governs various physiological processes,

* Corresponding author at: Department of Pharmaceutical Chemistry, College of Pharmacy, University of Ha’il, Ha’il 81442, Saudi Arabia.
** Corresponding author.
E-mail addresses: [Link]@[Link] (A.S. Abouzied), [Link]@[Link] (S.E. Kassab).

[Link]
Received 17 February 2025; Received in revised form 21 April 2025; Accepted 22 April 2025
Available online 25 April 2025
0045-2068/© 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

including osteogenesis [7], cardiovascular function, reproductive ac­ potent estrogen receptor (ER) antagonists from MPP (the molecule
tivities in both sexes, and cellular proliferation [8–10]. The receptor under consideration) [12]. In that investigation, we revealed that the
stays dormant until activated by estrogen [11]. conformationally constrained analog (Fig. 1) had much lower utero­
Over the past few decades, the interaction between nonsteroidal li­ trophic activity than Tamoxifen (TMX). This time, we’re pursuing the
gands and estrogen receptors (ER) has opened new possibilities for same goal with a different strategy: modifying MPP’s pharmacodynamic
inducing either agonist or antagonist activities [12–15]. This interaction profile to achieve antagonism in both breast and uterine tissues.
has contributed to the discovery of receptor-binding domains and has Based on the chemical structure of MPP and its relationship to
helped clarify the binding patterns that lead to either receptor activation antiestrogenic and uterotrophic activities, we propose replacing the
or silencing [16,17]. centroid pyrazole ring with a thiophene bioisostere [42] for new target
Selective estrogen receptor modulators (SERMs) bind to a specific antiestrogens (Fig. 2, Panel A).
area of the protein called the ligand binding domain (LBD) [16]. Their Furthermore, we intend to incorporate one or two hydrogen bond
effects can differ, with agonist or antagonist qualities depending on the donors (HBD) or hydrogen bond acceptors (HBA) atom spacers between
target tissue where these modulators act [18–21]. the 2-, 3-, and 5-phenyl groups and the centroid heterocycle (Fig. 2,
Tamoxifen (TMX) was the inaugural approved drug for the treatment Panel A). The spacers will connect the three phenyl groups to the het­
of breast cancer. TMX is a triphenylethylene estrogen receptor partial erocycle via two deactivating amide and carbonyl groups, along with
agonist exhibiting antiestrogenic properties in breast tissue and estro­ one activating amine group. The inclusion of one or two atom spacers
genic activities in the endometrium [22]. Tamoxifen exhibits a weak serves to modulate metabolic oxidation and orient the hydroxyl group of
binding affinity for the estrogen receptor (ER) and is metabolized in the the resultant phenol to either the meta (m-) or para (p-) positions,
body to 4-hydroxytamoxifen (4-OH-TMX), which possesses a greater contingent upon whether the spacer functions as a deactivator or
binding affinity and selective estrogen-modulating activity [23,24]. The activator.
increased binding affinity of 4-OH-TMX results from its trans-biphenyl Transforming p-phenol to m-phenol inside a trans-like diphenol
configuration, characterized by the hydroxylation of one phenyl group configuration may diminish the probability of generating the tris-p-
[25]. The molecule’s antiestrogenic activity arises from its triphenyl­ phenol metabolite linked to MPP and PPT, which exhibit uterotrophic
ethylene structure. Concerns regarding the correlation between properties. Moreover, extending the branched phenyl groups from the
Tamoxifen and endometrial carcinoma [25–28] in the context of breast centroid heterocycle through these spacer atoms may promote the
cancer treatment, there has been a need to explore novel drugs with a establishment of a trans-like bisphenol configuration in the ligand-
similar structural framework that retain full estrogen receptor antago­ binding domain (LBD) of the active site, thereby enabling access to
nist activity in both the breast and uterus. diphenols from any of the triaryl branches. We propose that these triaryl
Clomiphene, another SERM in the triphenylethylene class of anties­ derivatives possess equivalent capacity to generate the intended trans-
trogens, exerts its antiestrogenic effects through non-hydroxylated trans- like model for the antagonists.
biphenyl groups [29,30]. Some studies have indicated that clomiphene’s We have also widened our structural development possibilities to
biotransformation into multiple p-phenol counterparts enhances its limit the formation of the triphenol metabolite by substituting different
antiestrogenic and antiproliferative potency [30,31]. substituents at the p-position of 5-benzoylthiophene. Finally, our
A recent report has revealed the complex crystal structure of research will concentrate on two chemical classes of structural analogs
clomiphene, which shows a binding pattern like that of other nonste­ to MPP: thiophene-3-benzamide and pyrazole-4-benzamide. We will test
roidal antiestrogens [13]. However, this binding does not form their specific cytotoxicity against estrogen receptor-dependent breast
hydrogen bonds with the ligand-binding domain (LBD) amino acids cancer, analyze their anti-uterotrophic and uterotrophic activities, and
Glu353 and His524, which typically interact with the trans-biphenolic confirm their potential as full antagonists in breast and uterine tissues.
groups found in other members of the triphenylethylene class [32–34]. We will also investigate the pyrazole ring as a centroid heterocycle
The development of the triphenylethylene class of antiestrogens led for a single derivative of the target antiestrogens (Fig. 2, Panel B). These
to the creation of 1,3,5-triphenylpyrazole by replacing the central heterocycle substitutions will help us assess the utility of the proposed
ethylene bridge with a pyrazole ring [35–37]. This modification gave strategy for the pyrazole ring.
rise to Methyl piperidinopyrazole (MPP), an intriguing new ligand in
this class [38]. The 1,3-diphenyl diol groups in MPP provide a trans-like 2. Results and discussion
biphenol structure, essential for binding affinity to estrogen receptors
(ER). Additionally, the 5-phenyl group has an amino ether side chain, 2.1. Chemistry
crucial for the antagonist activity associated with the triphenylpyrazole
modality [38]. In Scheme 1, the active methylene group of aceto-acetanilide (1)
Following the identification of propylpyrazole triol (PPT) as a underwent self-catalyzed aldol condensation with an equimolar quan­
possible ER agonist [39], the discovery of MPP inspired us to highlight tity of phenyl isothiocyanate (2) in the presence of anhydrous K₂CO₃ in
structural aspects to consider in the future design of new class members, dry DMF at room temperature. This reaction resulted in a non-isolable
notably for reducing uterotrophic activity. One important finding is that ketene N,S-acetal salt intermediate (3).
MPP is an antagonist in breast tissue but an agonist in uterine tissue We subsequently generated the target thiophene derivatives (5a-e)
[40]. by performing a nucleophilic substitution reaction between the corre­
Furthermore, a report indicated that replacing the ether bridge in the sponding phenacyl bromide derivatives (4a-e) and the N,S-acetal salt
amino ether side chain with a methylene group enhances the antagonist intermediate (3). This reaction resulted in the synthesis of S-alkylated
activity of MPP [41]. This observation, when combined with the agonist intermediates, followed by the nucleophilic addition of the carbanion
activity of PPT, suggests that MPP may metabolize in vivo into methyl­ from the CH₂ group to the carbonyl group of the benzoyl moiety,
pyrazole triol, which could reduce the enhanced antagonist activity eventually producing the corresponding thiophene derivatives (5a-e) in
provided by the methylene metabolic blocker [12]. good yields (Scheme 1).
The present study is part of an ongoing program to develop strategies The 1H NMR and 13C NMR spectra confirmed the target structures
to convert selective estrogen receptor modulators (SERMs) into full re­ (5a-e). In the 1H NMR of (5b), a singlet peak for the 3-methyl group
ceptor antagonists in endometrial and breast tissues [12]. This approach occurred at δH = 2.38 ppm, followed by two typical singlet peaks for the
seeks to minimize the risk of endometrial carcinoma associated with this CONH and aniline-NH groups at δH = 9.50 and 10.17 ppm, respectively.
class of antiestrogens. The 13C NMR spectra of the same compound revealed two distinct sig­
Previously, we proposed and tested a strategy for generating more nals corresponding to the CONH and CO groups at δC = 163.28 and

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B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 1. Structural features of some reported SERMs, related ER-agonists, and ER-antagonists.

Fig. 2. The molecular design strategy of the target panels A and B of ER-antagonists.

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Scheme 1. Synthesis of target ER-antagonist derivatives, thiophene-3-benzamides.

Reagents and conditions: (i) K2CO3/dry DMF, 2 h, r.t.; (ii) stirring, r.t., overnight.

187.77 ppm, respectively. 19F NMR spectra verified the structure of 5e 2.2. Biological activity
by showing a peak at δF = − 108.21 ppm for the 4-fluorobenzoyl group.
We started with the mercapto derivative (6) and acidified the in­ 2.2.1. In vitro cytotoxicity assay of the new ER antagonists against cancer
termediate (3) to synthesize the target ER-antagonist (8). The nucleo­ cell lines:
philic substitution process between (6) and phenylhydrazine yielded the We tested the target ER-antagonist thiophene-3-benzamide de­
hydrazine derivative (7), as shown in Scheme 2. We did not purify the rivatives (5a-e) and pyrazole-4-benzamide derivative (8) on four types
remaining product (7) before proceeding with the reaction. This reac­ of cancer cells: MCF-7, MDA-MB-231, A-431, and HOS. The use of these
tion involved an intramolecular nucleophilic addition to the ketonic cell lines, particularly MCF-7, allowed us to assess the compounds’ ef­
carbonyl group, yielding the pyrazole derivative (8) after continuous ficacy against MCF-7 and demonstrate on-target cytotoxicity resulting
stirring at room temperature. from their ER-antagonist activity.
The conversion to pyrazole-4-benzamide (8) was confirmed by 1H The cell lines were treated with a 10 μM dosage of test compounds for
NMR and 13C NMR spectra. The 1H NMR spectrum shows a singlet signal 48 h. We found that the thiophene-3-benzamide derivatives (5a-d)
for the 3-methyl group at δH = 3.35 ppm and two distinct singlet peaks inhibited cell proliferation the most, with values of 60.80 %, 60.40 %,
for the CONH and aniline-NH groups at δH 9.86 and 10.20 ppm, 58.40 %, and 66.80 %, respectively (Table 1). In contrast, the pyrazole-
respectively. The 13C NMR spectra of the same molecule showed two 4-benzamide derivative (8) was less effective against MCF-7 breast
characteristic signals for the CONH and C– –N groups at δC 166.94 and cancer cells. Notably, the compounds (5a-d) have no cytotoxic action
165.76 ppm, respectively. against the remaining cancer cell lines (Table 1).
To analyze their potencies, we calculated the IC50 values for the

Scheme 2. Synthesis of target ER-antagonist derivative, pyrazole-4-benzamide.

Reagents and conditions: (i) Conc. HCl, r.t., 30 min.; (ii) phenylhydrazine, TEA, reflux, 30 h. or MW/200 ◦ C, 10 min; (iii) stirring, r.t., 2 h.

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Table 1 the percentage increase in uterine weight of the treated animals in

In vitro cytotoxicity assay measured with MTT test of the ER-antagonists (5a-e) comparison to the control group, as illustrated in Table 3 and Fig. 4.
and (8) against different human cancer cell lines (ATCC). The outcomes of the antiestrogenic activity assessment for com­
Cpd Cancer cell lines pounds 5a and 5d (Fig. 3) aligned with the cytotoxicity assay results.
% inhibition at (10 μM) IC50 (μM) ± SD
The putative ER-antagonist activity of 5a and 5d was 61.3 % and 63.7 %,
respectively, in comparison to Tamoxifen’s 69.6 %. Furthermore, the
MCF-7 MDA-MB-231 A-431 HOS MCF-7
estrogenic activity of 5a and 5d was negligible (Fig. 4). This outcome
TMX – – – – 10.96 may corroborate the previously articulated hypothesis regarding the
4-OH-TMX – – – – 4.94 [43] metabolic alteration of the chemical structures of the target ER antag­
5a 60.8 % 6.3 % 23.5 % 32.5 % 7.38 ± 1.46
5b 60.4 % 3.7 % 26.4 % 34.2 % 11.23 ± 1.74
onists (5a-e) to inhibit the synthesis of the ER-agonist metabolite of
5c 58.4 % 16.2 % 33.7 % 42.3 % ND triphenol. The p-tolyl derivative 5b showed a reversal in activity: it
5d 66.8 % 18.3 % 27.5 % 34.5 % 8.50 ± 1.38 functioned as a possible ER antagonist in vitro, as indicated in Tables 1
5e 35.7 % 1.2 % 5.8 % 4.9 % ND and 2, yet exhibited substantial ER agonist activity in vivo (Table 3 and
8 45.0 % ND ND ND ND
Fig. 4).
IC50: Lethal concentration of the test sample that causes the death of 50 % of The alteration in the activity of compound 5b, in contrast to the other
cells, IC50 values are expressed as mean ± S.D. of triplicate measurements for evaluated compounds 5a, 5c, and 5d, implies that the presence of the p-
each concentration used., [4-OH-TMX; 4-hydroxytamoxifen the positive con­ methyl group may result from metabolic oxidation, potentially yielding
trol, MCF-7, MDA-MB-231; breast, A431; skin, HOS; Osteosarcoma, cell lines, hydroxymethyl or carboxylic acid derivatives. Conversely, the two ER
ND; not determined.
antagonists, 5a and 5d, lacked the potential for metabolic oxidation at
the para position. Concurrently, the p-nitro derivative 5c exhibited
drugs that inhibited at least 60 % (Table 1). The measurement involves neither antiestrogenic (Fig. 3) nor estrogenic (Fig. 4) activities.
treating the cell lines with eight doses: 0.780, 1.560, 3.125, 6.250,
12.500, 25, 50, and 100 μM for 72 h. We found that compounds (5a), 2.2.4. Anti-uterotrophic assay of ER-antagonist 5d
(5b), and (5d) have IC50 values of 7.38 μM, 11.23 μM, and 8.50 μM, Given that 5d demonstrated the highest percentage of inhibitory
respectively. The IC50 values for 5a (7.38 μM) and 5d (8.50 μM) were activity, we performed a dose-response experiment to confirm the dose-
comparable to the reported value for 4-OH-TMX (4.94 μM) [43], while dependent effect of this compound (Table 4).
5b’s IC50 (11.23 μM) was like that of TMX (10.96 μM), showing that 5b The findings indicated the potential efficacy of the p-chlorophenyl
has a poor binding affinity and activity toward the ER. derivative 5d (ER EC50 = 5.53 μM), demonstrating activity very similar
Despite the moderate cytotoxic effects found, we decided to include to TMX (ER EC50 = 7.625 μM) [12].
compounds 5b and 5c in the in vivo testing of possibly active compounds
5a and 5d to assess their uterotrophic and anti-uterotrophic activities. 2.3. Docking study
This inclusion will help assess whether these compounds can be con­
verted into active metabolites. In previous work [12], we described how the activating factor 2 (AF-
2) region of the estrogen receptor (ER) recruits coactivators to initiate
2.2.2. ER-α gene-expression assay the receptor transcriptional signaling function [49]. This process re­
In the ER-α gene expression assay [44–46], we assessed the efficacy quires helix 12 (H12), one of the twelve helices (H1− H12) of the re­
of the test drugs (5a-d) in suppressing ER-α gene expression [47] in ceptor structure [50], to be positioned freely above the hydrophobic
MCF-7 cancer cells. The expression levels of the ER-α gene in cells void of the ligand-binding domain (LBD) [16]. Any changes in the
exposed to the test compounds correlated with the cytotoxic potential of conformation of H12, caused by interactions with the amino acids in
these compounds (Table 2). In comparison to the negative control (CTL), helices H3 and H5 and the connecting loop between helices H11 and
compounds 5a (0.33) and 5d (0.26) exhibited the most significant H12, can result in the AF-2 region adopting an antagonist conformation
downregulation of ER-α gene expression. The least effective down­ [16,51,52]. This change prevents the accommodation of receptor
regulators were 5b (0.53) and 5c (0.71), which showed the lowest coactivators, ultimately halting the transcription process [52].
cytotoxicity. The results of the ER-α gene expression experiment Previous studies showed that blocking the estrogen receptor (ER) is
corroborated the categorization of compounds 5a and 5d as prospective essential by using a binder that forms hydrogen bonds with specific
ER antagonists. amino acids in the H3, H5, and H11–12 connecting loops [53–55]. To
study the binding modes of active and inactive compounds from a new
2.2.3. Anti-uterotrophic versus Uterotrophic activities of ER-antagonists chemical class, we docked compounds 5a, 5d, and 5e into the active site
(5a-d): of the ER-α crystal structure in a complex with DMERI-8 (PDB entry
Immature female rats received daily treatments of 10 μg/kg of 7RS4) [13].
estradiol for three days, alongside 20 mg/kg of tamoxifen (TMX) as a We first redocked the co-crystallized ligand DMERI-8 into the ER-α
reference standard or equimolar dosages of the synthesized derivatives active site to validate our docking process for the test compounds. The
(5a-d). The efficacy of the derivatives was assessed by quantifying the best pose achieved an alignment with the original ligand conformation,
weight disparity of the uterus in the treated animal groups relative to the resulting in a root mean square deviation (RMSD) of 1.05 Å. This pose
control groups [48], as illustrated in Table 3 and Fig. 3. Furthermore, we formed identical hydrogen bonding interactions with the active site
evaluated the uterotrophic activity of these compounds by determining amino acids, consistent with those of the co-crystallized ligand,
demonstrating no compromises in the structural integrity (Fig. 6).
Table 2 The original ligand antagonist DMERI-8 established hydrogen
Effect of the target antiestrogens (5a-d) on ER-α gene expression level in MCF-7. bonding interactions with the H3 amino acid (Arg394) through the ni­
Treatment ER-α gene expression ± SEM in MCF-7 cancer cells trogen atom of the benzimidazole ring (Fig. 6). It also formed bonds with
the H11-H12 connecting loop amino acid (Lys529) via the oxygen atoms
CTL (¡) 1 ± 0.04
DOX 0.44 ± 0.05 of the carboxylic acid. Additionally, it interacted with the H11 amino
5a 0.33 ± 0.03 acid (Glu353) through the NH group of the benzimidazole ring. The p-
5b 0.53 ± 0.04 fluorophenyl group interacted with the H3 amino acid (His524) to align
5c 0.71 ± 0.05 with the typical structural characteristics of most estrogen receptor
5d 0.26 ± 0.03
antagonists.

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Table 3
Anti-uterotrophic and uterotrophic activities of ER-antagonists (5a-d).
Anti-uterotrophic activity Uterotrophic activity

Treatment R Uterus weight (g) %Antiestrogenic Treatment Uterus weight (g) %estrogenic
(mean ± S.E.M)b (mean ± S.E.M)d

Estrogena – 0.092 ± 0.009 CTL (¡)c 0.133 ± 0.012

Tamoxifen – 0.134 ± 0.007 69.6 % Tamoxifen – 57 % [12]
5a H 0.150 ± 0.017 61.3 % 5a 0.087 ± 0.007 19.3 %
5b CH3 0.186 ± 0.060 16.08 % 5b 0.123 ± 0.005 68.5 %
5c NO2 0.266 ± 0.056 3.1 % 5c 0.062 ± 0.003 − 19.32 %
5d Cl 0.145 ± 0.013 63.7 % 5d 0.081 ± 0.014 − 11.08 %
a
Estrogen group (n = 6) that received 10 μg/kg/day estradiol in the vehicle medium.
b
Uterus weight difference values in g (Estrogen group-treated) are expressed as a mean ± SEM of three independent experiments.
c
CTL (− ) Control group (n = 6) that didn’t receive any treatment but the vehicle medium.
d
Uterus weight difference values in g (− ve CTL-treated) are expressed as a mean ± SEM of three independent experiments.

Fig. 3. Bar charts showing the effect of the target ER-antagonists (5a-d) on the uterus weight of estrogen-treated juvenile rats expressed as a normalized weight to the
body weight. Data are expressed as mean ± S.E.M., * denotes significance from the Estrogen group. Est; Estrogen, TMX; Tamoxifen.

Conversely, the docking solution for the phenyl derivative 5a (Fig. 6) narrower angle, resulting in its two aryl fragments being farther away
indicated that its optimal configuration within the active site closely from His524 and Glu353 than those in 5a and 5d.
mirrored that of the original ligand. It established hydrogen bonds with A contoured surface [56] was generated for the optimum confor­
Lys529 via the carbonyl oxygen of the benzoyl moiety. The optimum mation of the MMP of ER antagonist 5d, aligned with the inactive
structure of this compound revealed that the anilino and benzamido aryl compound 5e (Fig. 8). This alignment elucidates the positioning of the
moieties were suitably aligned with His524 and Glu353. Nonetheless, no anilino and benzamido fragments of 5d, which exhibit a significantly
hydrogen bonds were established with either His524 or Glu353, pre­ open dihedral angle (Fig. 8), facilitating proximity to His524 and
serving the original ligand’s configuration. Glu353 compared to 5e.
We also point out that the active p-chlorobenzoyl derivative 5d and Based on the results stated above, we conclude that the ER-antago­
the inactive p-fluorobenzoyl derivative 5e are matched molecular pairs nist’s active site layout should include diaryl fragments with a wide-
(MMP). Analyzing the docking solutions for both compounds (Fig. 6) open dihedral angle near His524 and Glu353. In addition, a third aryl
will help us understand why 5e exhibits reduced activity compared to ring should extend from the core scaffold to form hydrogen bonds with
5d despite only a minor structural difference. The docking analysis for the H3, H11-H12 connecting loop, and H5 amino acids.
5d revealed that its best position is like that of 5a (Fig. 7A), with
hydrogen bonds forming between the carbonyl oxygen and Lys529. 2.4. Molecular dynamics simulation
Controversially, the inactive compound 5e also occupied the active site
with a similar binding mode (Fig. 7B). To investigate the molecular basis for the observed difference in
Furthermore, we aligned the conformations of the best poses of the activity between the two structurally related ligands 5d and 5e, we
original ligand DMERI-8 and compounds 5a, 5d, and 5e to determine performed extensive molecular dynamics simulations on both protein-
why 5a and 5d are active while 5e is inactive. Surprisingly, the shapes of ligand complexes, followed by detailed root mean square deviation
the active compounds 5a and 5d matched closely with the original (RMSD), root mean square fluctuation (RMSF), and interaction
ligand (Fig. 7B), showing a wide-open angle between the aryl parts that profiling.
are near the LBD amino acids His524 and Glu353. In contrast, the
inactive compound 5e (Fig. 7A) exhibited a distinct conformation with a

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Fig. 4. Bar charts showing the effect of ER-antagonists (5a-d) on the uterus weight of non-treated juvenile rats expressed as a normalized weight to the body weight.
Data are expressed as mean ± S.E.M. * denotes significance from control.

2.4.3. Protein–ligand interaction patterns

Table 4
2D interaction maps derived from simulation data further underscore
In-vivo Dose-response experiment results of antiestrogenic
the distinction between the two compounds. The 5d ligand maintained
activity of compound 5d compared to the standard drug
TMX.
hydrophobic and hydrogen-bond interactions with critical residues,
including LEU384, PHE404, MET421, and VAL533, persisting for more than
Sample ER EC50 (μM) ± S.E.M
30 % of the simulation time (Fig. 12a). These contacts are spatially
5d 5.53 ± 1.67 complementary and structurally cohesive, helping anchor the ligand
TMX 7.625 [12] effectively within the active site. PHE404 appears to contribute to
π-stacking or hydrophobic stabilization.
2.4.1. Structural stability and conformational dynamics On the other hand, the 5e ligand demonstrated a less optimal
The compound 5d exhibited a well-stabilized trajectory, as indicated interaction network (Fig. 12b). Although it formed transient hydrogen
by the protein Cα RMSD plot (Fig. 9a). After an initial equilibration bonds with THR347 and GLY521 and hydrophobic contacts with LEU346,
period within the first 30–50 ns, the RMSD values converged and pla­ LEU387, and ALA350, many interactions involved water mediation or low
teaued in the 2.5–2.8 Å range, signifying structural equilibrium. occupancy over the simulation time. A water molecule bridged multiple
Simultaneously, the ligand’s RMSD, when fitted onto the protein, hydrogen bonds, suggesting a reliance on solvent for interaction stabi­
remained stable around 2.5–3.0 Å throughout the trajectory, suggesting lization—typically indicative of weaker binding.
persistent occupancy within the binding pocket and minimal confor­ The integrative data support a mechanistic rationale for the activity
mational drift. divergence between the two ligands. The 5d compound promotes a
In contrast, the 5e compound showed considerable structural fluc­ stable protein conformation and maintains enduring, direct interactions
tuations. The Cα RMSD of the protein exceeded 20 Å multiple times, with residues critical for binding, resulting in minimized structural drift
peaking above 40 Å, particularly around the 30 ns and 95 ns marks, and limited flexibility. These features correlate with strong binding af­
indicative of large conformational transitions or partial unfolding events finity and likely biological potency.
(Fig. 9b). The ligand’s RMSD profile mirrored this instability, with In contrast, the 5e compound induces pronounced protein mobility
pronounced spikes aligning with the periods of protein disruption. and demonstrates poor interaction fidelity, with a tendency toward high
Although it eventually resettled to ~4.5 Å, the fluctuations suggest a conformational entropy and water-mediated stabilization. This behavior
weak or transient interaction with the binding site. aligns with reduced binding strength and, by extension, a diminished
pharmacological effect.
2.4.2. Flexibility analysis
RMSF profiling of the 5d complex demonstrated that most residues 3. Conclusion
exhibited low-to-moderate fluctuations, particularly in the structured
core (Fig. 10a). Dynamic movement was primarily localized at the N- The suggested alterations to methyl-piperidinopyrazole, which are
and C-terminal regions, which is typical for protein dynamics. intended to provide a selective estrogen receptor modulator, signifi­
Conversely, the 5e complex revealed elevated RMSF values across cantly reduced uterotrophic activity, lowering the risk of uterine cancer.
multiple protein segments, with peaks reaching up to 13 Å (Fig. 10b). We obtained these findings by using amide, amine, and ketone spacers.
Such widespread flexibility is a hallmark of compromised structural Using phenyl-deactivating spacers likely inhibited the production of tris-
integrity and is often associated with weak ligand engagement. p-phenol metabolites from the thiophene-3-benzamide derivatives 5a
The 5d ligand’s RMSF remained below 2.5 Å across most atoms, and 5d, implying that they may have antiestrogenic effects without
reinforcing its restricted mobility and stable positioning (Fig. 11a). uterotrophic activities. However, making a comparable alteration to the
While the 5e ligand exhibited consistently high RMSF values (~4.0–4.8 pyrazole-4-benzamide derivative 8 eliminated its antiestrogenic effect.
Å), consistent with its unstable binding behavior (Fig. 11b). Docking studies of the active derivative 5-(4-chlorobenzoyl)-

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B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 6. Docking solutions of the original ligand (DMERI-8) and compounds (5a), (5c), and (5d) in the active binding site of ER-α (PDB entry 7RS4) showing the
corresponding docking scores. The amino acid residues of the active site are represented using a three-letter code. Atoms are colour-coded, with blue indicating
nitrogen, red for oxygen, yellow for sulfur, and grey for carbon. Hydrogen bonding interactions are illustrated with yellow dotted lines. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

thiophene-3-carboxamide 5d and the inactive derivative 5-(4-fluo­ 4. Experimental

robenzoyl)-thiophene-3-carboxamide 5e at the estrogen receptor’s
active site revealed that the diaryl fragments in the trans-like confor­ 4.1. Chemistry
mation play an important role. To successfully exert ER-antagonist ac­
tivity, they must maintain a wide dihedral angle and situate themselves All reagents and solvents were obtained from Sigma-Aldrich, Acros,
near His524 and Glu353. and Fisher Scientific companies. We measured the melting points using a
Digital SMP10 Stuart melting point apparatus, without any corrections.
The microwave-based reactions were conducted using the Milestone

8
B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 7. A. The overlay of the best docking pose of compounds 5a (blue), 5d (yellow), and 5e (purple) showcases the failure of 5e to align with the poses of sister
compounds 5a and 5d. B. The overlay of the best docking pose of compounds 5a (blue), 5d (yellow), 5e (purple), and the original ligand DMERI-8 (turquoise)
showcases the perfect alignment of 5a and 5d with the reference ER-antagonist DMERI-8. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

Fig. 8. Surface creation for the best docking poses of the MMP of compounds 5d and 5e. The overlay of the MMP poses shows the distinct molecular conformation
difference between the active 5d and inactive 5e.

Flexi-Wave microwave reactor. Infrared (IR) spectra were recorded on a by collecting 64 scans with a 5-s recycling delay. The 13C NMR spectra
PerkinElmer 1430 spectrophotometer using anhydrous potassium bro­ were recorded using a 1D sequence with power-gated decoupling using a
mide (KBr) discs, υ (cm− 1), at the Microanalytical Center, Faculty of 30-degree flip angle (zgpg30) adapted from Bruker standard pulses. The
Science, Cairo University. The NMR spectra were recorded using a spectral width was set to 240 ppm, digitized to 64 k points by collecting
Bruker 800 MHz AVANAC NEO NMR spectrometer equipped with a 5 2 k scans. Bruker Topspin 4.07 software (Bruker BioSpin, Rheinstetten,
mm triple resonance TCI Cryo probe (BrukerBioSpin, Rheinstetten, Germany) was used for data collection and spectral analysis. Reactions
Germany). Each 1H spectrum was recorded using the zg pulse program were checked using thin-layer chromatography (TLC) with Merck Silica

9
B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 9. RMSD analysis reveals stable binding and structural convergence of 5d (a), whereas 5e displays large fluctuations, indicating significant conformational
changes (b).

gel/TLC cards that have a fluorescent indicator, using a mix of Hexane 4.2.1. 5-Benzoyl-4-methyl-N-phenyl-2-(phenylamino)thiophene-3-
and Ethyl acetate or DCM and MeOH as the solvents, and the spots were carboxamide (5a)
seen with a Spectroline E series dual wavelength UV lamp at 254 nm. Yellow solid, (Dioxane) Yield: 91 %; m.p.: 207–209 ◦ C. IR (KBr)
ʋmax/cm¡1: 3287 (NH), 1676 (Ar-CO), 1618 (CONH). 1H NMR, (400
4.2. General method for synthesis of thiophene-3-benzamide derivatives MHz, DMSO‑‑d6): δ 2.37 (s, 3H, CH3), 7.02–7.12 (m, 2H, Ar–H),
(5a-e) 7.29–7.39 (m, 6H, Ar–H), 7.51 (t, J = 7.40 Hz, 2H, Ar–H), 7.59 (t, J =
7.27 Hz, 1H, Ar–H), 7.66 (d, J = 7.09 Hz, 2H, Ar–H), 7.70 (d, J = 7.95
To a stirred solution of anhydrous potassium carbonate (10 mmol) in Hz, 2H, Ar–H), 9.53 (s, 1H, CONH), 10.18 (s, 1H, NH). 13C NMR (100
DMF (30 mL), acetoacetanilide (10 mmol) was added. After stirring for MHz, DMSO‑‑d6): δ 187.33 (Ar-CO), 162.60 (CONH), 155.98, 144.88,
one hour, we dropped phenyl isothiocyanate (10 mmol) into the 141.23, 140.54, 138.91, 131.33, 129.32, 128.48, 128.34, 127.81,
mixture. The stirring continued for an additional 2 h. We added the 123.50, 123.28, 121.60, 119.76, 119.21, 118.96, 16.13 (2-CH3). HRMS
resulting mixture portion-wise to a cooled suspension of phenacyl bro­ (m/z): calcd.: 412.1245, found: {m/z: [M + H]+; 413.1318}.
mide derivatives (10 mmol) over 30 min. Once the addition was com­
plete, the reaction mixture was left to stir overnight. We then treated the 4.2.2. 4-Methyl-5-(4-methylbenzoyl)-N-phenyl-2-(phenylamino)
mixture with cold water (50 mL) containing five drops of 0.1 N HCl. The thiophene-3-carboxamide (5b)
resulting product was collected and crystallized from an appropriate Yellow crystal, (Dioxane), Yield: 88 %; m.p.: 210–212 ◦ C. IR (KBr)
solvent. ʋmax/cm¡1: 3286 (NH), 1685 (pHCO), 1624 (CONH). 1H NMR, (400
MHz, DMSO‑‑d6): δ 2.38 (br. s, 3H, CH3), 2.39 (br. s, 3H, CH3),
7.00–7.13 (m, 2H, Ar–H), 7.30–7.37 (m, 8H, Ar–H), 7.59 (d, J = 8.07
Hz, 2H, Ar–H), 7.71 (d, J = 7.82 Hz, 2H, Ar–H), 9.50 (s, 1H, CONH),

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B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 10. Protein RMSF plot indicates 5d’s moderate residue flexibility with peaks at loop regions (a), whereas 5e shows significantly higher fluctuations, suggesting
structural instability (b).

10.17 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑‑d6): δ 187.77(pH-CO), 129.11, 128.84, 128.15, 127.98, 127.74, 123.03, 122.90, 121.10,
163.28(CONH), 156.20, 144.97, 142.17, 141.87, 139.46, 138.27, 119.29, 118.81, 118.69, 118.45, 118.06, 16.74 (2-CH3). HRMS (m/z):
129.89, 129.79, 129.59, 129.42, 129.06, 128.71, 124.09, 123.73, calcd.: 446.0865, found: {m/z: [M + H]+; 447.0929 (35Cl), and
122.09, 120.38, 119.82, 119.64, 21.54 (Thiophene-CH3), 16.74 (Ph- 449.0909 (37Cl)}.
CH3). HRMS (m/z): calcd.: 426.1402, found: {m/z: [M + H]+;
427.1475}. 4.2.5. 5-(4-Fluorobenzoyl)-4-methyl-N-phenyl-2-(phenylamino)thiophene-
3-carboxamide (5e)
4.2.3. 4-Methyl-5-(4-nitrobenzoyl)-N-phenyl-2-(phenylamino)thiophene- Green crystal, (Ethanol/Dioxane), Yield: 89 %; m.p.: 162–164 ◦ C. IR
3-carboxamide (5c) (KBr) ʋmax/cm¡1: 3277 (NH), 1670 (Ph-CO), 1645 (CONH). 1H NMR,
Orange crystal, (Ethanol/Dioxane), Yield: 86 %; m.p.: 247–249 ◦ C. (400 MHz DMSO‑‑d6): δ 2.37 (s, 3H, CH3), 7.00–7.17 (m, 2H, Ar–H),
IR (KBr) ʋmax/cm¡1: 3268 (NH), 1685 (Ph-CO), 1624 (CONH). 1H 7.24–7.43 (m, 6H, ArH), 7.58 (d, J = 8.31 Hz, 2H, Ar–H), 7.70 (t, J =
NMR, (400 MHz, DMSO‑‑d6): δ 2.37 (s, 3H, CH3), 7.02–7.13 (m, 2H, 7.89 Hz, 4H, Ar–H), 9.59 (s, 1H, CONH), 10.20 (s, 1H, NH). 13C NMR
Ar–H), 7.26–7.40 (m, 8H, Ar–H), 7.70 (d, J = 7.70 Hz, 2H, Ar–H), (100 MHz, DMSO‑‑d6): δ 193.78 (Ar-CO), 185.51 (CONH), 164.85,
7.72–7.82 (m, 2H, Ar–H), 9.55 (s, 1H, CONH), 10.19 (s, 1H, NH). 13C 162.69, 162.14, 155.55, 144.48, 140.74, 138.44, 136.48, 136.47,
NMR (100 MHz, DMSO‑‑d6): δ 185.28 (Ph-CO), 162.33(CONH), 157.35, 130.26, 130.22, 129.13, 128.86, 128.17, 128.02, 119.33, 118.74,
148.59, 146.37, 146.17, 140.93, 138.88, 129.38, 128.91, 128.49, 118.70, 118.49, 114.96, 114.85, 107.07, 16.24 (2-CH3). 19F NMR (376
123.78, 123.67, 123.56, 121.70, 119.76, 119.71, 117.97, 16.24 (2-CH3). MHz, DMSO‑‑d6): δ − 108.21 (s, 4-F). HRMS (m/z): calcd.: 430.1151,
HRMS (m/z): calcd.: 457.1096, found: {m/z: [M + H]+; 458.1157}. found: {m/z: [M + H]+; 431.1224}.

4.2.4. 5-(4-Chlorobenzoyl)-4-methyl-N-phenyl-2-(phenylamino)thiophene- 4.2.6. Synthesis of 3-methyl-N,2-diphenyl-5-(phenylamino)-2,3-dihydro-

3-carboxamide (5demonstrated the highest percentage of inhibitory d) 1H-pyrazole-4-carboxamide (8)
Yellow crystal, (Dioxane), Yield: 90 %; m.p.: 223–225 ◦ C. IR (KBr) We prepared a cooled suspension of (3, 10 mmol), to which we
ʋ max/cm¡1: 3305 (NH), 1675 (Ph-CO). 1H NMR, (400 MHz, slowly added 10 drops of concentrated hydrochloric acid over 15 min
DMSO‑‑d6): δ 2.36 (s, 3H, CH3), 7.04–7.15 (m, 2H, Ar–H), 7.26–7.42 while stirring. Once the addition was complete, we added an appropriate
(m, 6H, Ar–H), 7.71 (d, J = 7.95 Hz, 2H, Ar–H), 7.88 (d, J = 8.68 Hz, amount of phenylhydrazine (20 mmol) with a few drops of triethyl­
2H, Ar–H), 8.34 (d, J = 8.56 Hz, 2H, Ar–H), 9.69 (s, 1H, CONH), 10.24 amine. We then heated the reaction mixture under reflux for 3 h or
(s, 1H, NH). 13C NMR (100 MHz, DMSO‑‑d6): δ 185.94(Ph-CO), 162.04 subjected it to microwave irradiation at 200 ◦ C for 10 min. After cooling
(CONH), 155.80, 144.78, 140.64, 138.67, 138.41, 135.61, 129.30, at room temperature, we filtered the resulting product, dried it, and

11
B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Fig. 11. Ligand RMSF plot shows 5d’s moderate atomic flexibility variation (a), while 5e exhibits consistently higher RMSF values across atoms (b).

Fig. 12. Schematic diagrams showing interactions that persist for over 30 % of the 200-ns simulation are displayed, highlighting key binding features of 5d (a) and
5e (b).

recrystallized it from ethanol and acetone. Ar–H), 7.07 (t, J = 6.90 Hz, 1H, Ar–H), 7.10–7.23 (m, 3H, Ar–H),
White crystals, Yield: 52 %; m.p.: 182–184 ◦ C. IR (KBr) ʋmax/cm¡1: 7.31 (t, J = 7.10 Hz, 3H, Ar–H), 7.58 (dd, J = 7.70 Hz, 3H, Ar–H), 7.79
3267 (NH), 1663 (CONH). 1H NMR, (400 MHz, DMSO‑‑d6): δ 3.35 (s, (s, 1H, Ar–H), 9.86 (s, 1H, CONH), 10.20 (s, 1H, NH). 13C NMR (100
3H, CH3), 6.71 (t, J = 7.00 Hz, 1H, Ar–H), 6.79 (d, J = 7.70 Hz, 3H, MHz, DMSO‑‑d6): δ 166.94(COHN), 165.76 (CN), 149.41, 139.25,

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B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

129.24, 129.12, 123.91, 119.61, 119.00, 112.66, 43.61 (2-CH3). HRMS Germany). Total RNA (5 μg) was combined with a master mix. The
(m/z): calcd.: 368.1637, found: {m/z: [M + H]+; 369.2287}. master mix contained 50 mM MgCl₂, 10× RT buffer, 10 mM of each
dNTP, 50 μM oligo-dT primer, 20 IU ribonuclease inhibitors, and 50 IU
MuLV reverse transcriptase. Each sample was mixed and centrifuged for
4.3. Biological activity
30 s at 1000 g before being transferred to the thermocycler. The RT
reaction was carried out at 25 ◦ C for 10 min, followed by 1 h at 42 ◦ C,
4.3.1. MTT cytotoxicity assay of the test ER-antagonists against human
and completed with a denaturation step at 99 ◦ C for 5 min [44]. The
cancer cell lines MCF-7, MDA-MB-231, HOS, and A-431
reaction tubes containing RT preparations were then flash-cooled in an
Tissue culture media MEM-α and DMEM:Hams F12, fetal calf serum
ice chamber before being utilized for cDNA amplification using the
(FCS), and trypsin-EDTA were procured from Invitrogen, Paisley, Scot­
quantitative Real-Time polymerase chain reaction (qRT-PCR).
land, and maintained at 4 ◦ C. Both media are augmented with L-gluta­
The cDNA copy number of the breast cell line was determined using
mine at a final concentration of 4 mM, sodium pyruvate at 1 mM,
the StepOne™ Real-Time PCR System from Applied Biosystems (Thermo
penicillin at 0.1 unit/mL, streptomycin at 100 ng/mL, a 100-fold dilu­
Fisher Scientific, Waltham, MA, USA). PCR reactions were produced in
tion of non-essential amino acid stock (Sigma cat# M7145), and fetal
25 μL mixes containing 12.5 μL of SYBR® Premix Ex TaqTM, 0.5 μL of a
calf serum (10 % v/v). The research employs multiple cancer cell lines,
sense primer, 0.5 μL of an antisense primer, 6.5 μL of distilled water, and
including MCF-7, MDA-MB-231 (breast), A-431 (skin epidermoid car­
5 μL of cDNA template. We separated the reaction program into three
cinoma), and HOS (bone osteosarcoma). [Link] We adhered to
steps. The first step lasted three minutes at 95.0 ◦ C. The second stage
the protocols for managing culture flasks and subculturing RPMI media
consisted of 40 cycles, each of which was broken into three steps: (a)
(Biowhittaker, Belgium), which incorporated fetal bovine serum (FBS)
95.0 ◦ C for 15 s, (b) 55.0 ◦ C for 30 s, and (c) 72.0 ◦ C for 30 s. The third
(GIBCo, USA), penicillin, and glutamine. [Link]
stage consisted of 71 cycles, beginning at 60.0 ◦ C and increasing by
The MTT cell viability experiment evaluated the cytotoxic effects of
0.5 ◦ C every 10 s until 95.0 ◦ C. Each experiment has distilled water
the examined drugs on several human cancer cell lines (Supplementary
control. Table 1 lists primer sequences for the estrogen receptor α (ERα)
Table S1). The assay was conducted thrice to determine the half-
gene. At the end of each qPCR, we performed a melting curve analysis at
maximal inhibitory concentration (IC50), defined as the concentration
95.0 ◦ C to confirm the quality of the primers used. We used the 2 −
required to eliminate 50 % of the cells (in μM), following a 24-h incu­
ΔΔCT method [45] to determine the target’s relative quantification to
bation of 10,000 cells in each well of a 96-well plate. A single test dose of
the reference.
10 μM was initially administered, followed by the evaluation of eight
distinct doses to determine the IC50 value utilizing a comprehensive
4.3.3. Percentage of anti-uterotrophic activity of the test ER-antagonists
array of cancer cell lines. The cells were subjected to treatment with the
To assess the anti-estrogenic activity [48], rats were randomly allo­
test substances for 48 h, conducted in triplicate. The cells were subjected
cated to groups (n = 6) for each group receiving daily subcutaneous
to treatment with test samples for 48 h in triplicate. 0.5 % DMSO served
injections of estradiol (10 μg/kg/day). All rats administered estrogen
as a negative control. We employed the MTT [3-(4,5-dimethylthiazol-2-
received an intraperitoneal injection of Tamoxifen (20 mg/kg/day) or
yl)-2,5-diphenyltetrazolium bromide] test, as delineated by Mosmann
equimolar dosages of the compounds under examination (5a-d) every
[57], to assess cytotoxicity.
day for three consecutive days, excluding the control group, which

( / )
%cell viability = mean absorbancetreated sample mean absorbancenegative control sample × 100%death rate = 100–(%cell viability)

received an equal volume of 10 μg/kg/day estrogen in the vehicle only.

Throughout the in vivo efficacy and pharmacodynamic investigations, no
The inhibitory concentration of 50 % (IC50) was measured from the obvious indicators of toxicity were found in animals treated with the
exponential curve of %cell viability against concentration (Dos­ tested compounds at the indicated doses. Body weight, behavior
e–Response Curve) [58] using the GraphPad Prism program (version 10; (including irritability or lethargy), bleeding, and overall physical con­
San Diego, CA, USA). dition were all equivalent to those in the vehicle control group, indi­
cating that the treatments were well tolerated. On the fourth day, all rats
μM = [ppm/compound molecular weight]x1000
were sacrificed via cervical dislocation, and the uteri were meticulously
removed from adipose tissue and weighed promptly.
4.3.2. Gene expression analysis
The suppression of uterine growth relative to the growth induced by
estradiol alone served as an indicator of the anti-uterotrophic impact.
[Link]. RNA isolation and reverse transcription (RT) reaction. Total RNA Results were presented as percentage inhibition derived from the for­
was isolated from the human breast cancer cell line (MCF-7) using the mula:
RNeasy Mini Kit (Qiagen, Hilden, Germany), supplemented with a
DNase I (Qiagen) digestion step, as per the manufacturer’s protocol. The Percentage of anti − uterotrophic activity
isolated total RNA was processed with one unit of RQ1 RNase-free = (Ws − Ws + t)/(Ws − Wv)* 100
DNase to break down any remaining DNA, mixed with DEPC-treated
water, and then measured at 260 nm with a spectrophotometer. The Wv represents the average uterine weights of animals administered
purity of total RNA was determined by a 260/280 nm ratio of 1.8 to 2.1. with the vehicle. Ws represents the average uterine weights of animals
Furthermore, formaldehyde-containing agarose gel electrophoresis [46] administered estradiol. Ws + t represents the average uterine weights of
with ethidium bromide-stain detection of the 28S and 18S bands animals administered a combination of estradiol and the test drug. The
ensured integrity [46]. Aliquots were utilized immediately for reverse dosages of the evaluated substances are determined on a molar basis.
transcription (RT) but were otherwise kept at − 80 ◦ C. Values are shown as mean ± standard error of the mean (SEM).
Poly(A) + RNA from breast cell lines was converted into cDNA in 20
μl using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas,

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B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

4.3.4. Percentage uterotrophic activity of the test ER-antagonists London dG as the main scoring method. We implemented a further
Wistar immature female rats (40–50 g) obtained from the animal refinement phase using the induced fit method [12,56] and the affinity
care facility at Nahda University, Beni Suef (NUB), were allowed to dG score function. The docking poses of the test drug were evaluated
acclimate to laboratory conditions for 3 days before the experiment, based on binding energy scores, RMSD values, and the alignment of
with free access to food and water. We conducted all experiments binding forces with the original ligand DMERI-8. We identified the
following the recommendations of the International Animal Care and optimal position, superimposed it with the original ligand for compar­
Use Committee. The research was approved by “The Commission on the ison, and saved the image as a JPEG file for export.
Ethics of Scientific Research,” Faculty of Pharmacy, Minia University
(No. 63/2019). We supplied food and water ad libitum. Estradiol was 4.5. Molecular dynamics simulation
diluted in olive oil and injected subcutaneously on the dorsal loose skin
at 10 μg/kg/d, while Tamoxifen (TMX) was previously tested at 20 mg/ All molecular dynamics simulations were performed using the free
kg/day [12]. We used all compounds in an equimolar dose of TMX. We academic Desmond simulation package [63,64] (Schrödinger Release
dissolved all compounds in a [Link] mixture of DMSO, Tween 20, and 2024-2, 2024). The software’s high-performance computing capabilities
saline, respectively, and administered them intraperitoneally. Rats were were employed to explore the dynamic stability and interaction
randomly assigned to groups (n = 6) for each group was subjected to behavior of two protein-ligand complexes—each featuring either an
daily S.C. injections of test compounds for three consecutive days, active or inactive ligand.
except for the control group, which received an equal volume of the Before the simulation, both protein-ligand systems were subjected to
vehicle. On the 4th day, all rats were sacrificed by cervical dislocation, thorough system preparation using the Desmond-maestro Protein
and the uteri were dissected free of fat and weighed immediately. Preparation Wizard, ensuring proper assignment of bond orders, ioni­
The relative gains in uterine weight were calculated as mg uterine zation states, and optimization of hydrogen-bonding networks.
weight/100 g body weight. We determined the percentage of utero­ Each complex was then embedded in an orthorhombic simulation
trophic activity based on the uterine weight using the following equa­ box with periodic boundary conditions and solvated using the TIP3P
tion: [48]. water model. An appropriate buffer distance of at least 10 Å was
maintained between the solute and the edges of the box to minimize
%Uterotrophic activity = (WT − WC/WS − WC)/WC* 100
edge effects. Neutralization was achieved by adding counter-ions, and
WT is the mean uterine weight of a rat treated with the test com­ physiological salt conditions (0.15 M NaCl) were replicated to approx­
pound, WC is the control group, and WS is the standard treatment group. imate the cellular environment.
WS = the mean uterine weight of a rat treated with the standard. WC Energy minimization of the system was performed to relieve steric
represents the mean uterine weight of the control rat. clashes and prepare the system for dynamics. Subsequently, equilibra­
We performed all statistical analyses using GraphPad Prism (version tion was carried out using a series of restrained MD steps under the NPT
6.0; San Diego, CA, USA). We used the Student t-test to compare two ensemble, gradually relaxing the system and stabilizing temperature and
different treatments. Value was expressed as the mean ± S.E.M. The pressure at 300 K and 1 atm, respectively, using the Nose-Hoover chain
level of significance was set at P < 0.05. thermostat and barostat [65,66].
The production MD simulations were executed under NPT conditions
4.3.5. EC50 of the test ER-antagonist (5d) as an anti-uterotrophic agent for 200 ns using Desmond’s default time step of 2 fs. OPLS_2005 force
We performed a dose-response experiment for the ER antagonist 5d field parameters were applied throughout to describe the interactions
to determine the EC50 value. Five dosages (1, 3, 10, 15, and 20 mg/kg/ between atoms accurately [67,68]. Trajectory data were recorded at 20
day) of test compound 5d were administered by injection for three days, ps intervals for downstream analysis.
accompanied by subcutaneous injection of estradiol. We slaughtered the Post-simulation analyses, including RMSD, RMSF, and protein-
animals on the fourth day, excised the uteri, and weighed them. We ligand interaction timelines, were performed using tools provided
conducted all statistical analyses using GraphPad Prism (version 6.0; within the Desmond Simulation Interaction Diagram (SID) and Maestro
San Diego, CA, USA). Values were presented as mean ± standard error of interface. Interaction frequencies were calculated over the 200 ns
the mean (S.E.M.). We deemed the results statistically significant if the simulation period to identify residues engaging in persistent binding
p-values were less than 0.05. contacts with each ligand.

CRediT authorship contribution statement

4.4. Docking studies
Bader Huwaimel: Writing – review & editing, Resources, Method­
We conducted molecular docking analyses using the Molecular ology, Formal analysis. Amr S. Abouzied: Writing – review & editing,
Operating Environment (MOE 2018.0802) software [59–61]. The RCSB Validation, Supervision, Resources, Methodology, Investigation, Fund­
Protein Data Bank website, [Link] provided the crystal ing acquisition, Formal analysis, Data curation. Abdul-Hamid Emwas:
structure of ER complexed with the receptor antagonist DMERI-8 (PDB Software, Resources, Methodology, Formal analysis. Al-Shaimaa F.
code 7RS4) [13]. Ahmed: Writing – review & editing, Visualization, Validation, Software,
We prepared the test compound by adding hydrogen, calculating Resources, Methodology, Investigation, Formal analysis, Data curation,
partial charges, and reducing energy using the MMFF94x Force Field at a Conceptualization. Mohamed K.S. El-Nagar: Writing – review & edit­
precision level of 0.01. ing, Writing – original draft, Visualization, Validation, Software, Re­
We conducted the protein preparation by eliminating the repetitive sources, Methodology, Formal analysis, Data curation. Hamdoon A.
sequences. We employed the MOE QuickPrep technique (RMSD Mohammed: Resources, Data curation. Nader M. Alrashidi: Method­
gradient = 0.1 kcal/mol, AMBER10: EHT field) to optimize structural ology, Data curation. Marwan A. Alrashidi: Methodology, Data cura­
issues and calculate partial charges [62]. tion. Rakan E. Alshammari: Writing – review & editing, Writing –
We employed the procedures and parameters of the MOE Dock original draft, Visualization, Validation, Software, Resources, Method­
protocol to identify optimal poses and binding score values. First, the co- ology, Formal analysis, Data curation. Abdulelah Y. Albladi: Method­
crystallized ligand was taken out of the active site and then put back in ology. Saad Alqarni: Writing – review & editing, Visualization,
to check if the docking process could be repeated successfully. Molecular Software, Resources, Methodology, Investigation, Formal analysis.
docking was then performed on the selected compounds using the Shaymaa E. Kassab: Writing – review & editing, Writing – original
standard settings of MOE, with the triangle matcher for positioning and draft, Visualization, Validation, Supervision, Software, Resources,

14
B. Huwaimel et al. Bioorganic Chemistry 161 (2025) 108512

Project administration, Methodology, Investigation, Formal analysis, [11] D.M. Lonard, B.W. O’Malley, Nuclear receptor coregulators: modulators of
pathology and therapeutic targets, Nat. Rev. Endocrinol. 8 (2012) 598–604,
Data curation, Conceptualization.
[Link]
[12] M.A. Ragab, M. Elagawany, H. Daabees, A.-S.F. Ahmed, E.M. Awad, C. Billon,
B. Elgendy, K.A.M. Abouzid, S.E. Kassab, Structure-based design and synthesis of
Funding
conformationally constrained derivatives of methyl-piperidinopyrazole (MPP) with
estrogen receptor (ER) antagonist activity, Bioorg. Chem. 119 (2022) 105554,
This research was funded by the Scientific Research Deanship at the [Link]
University of Ha’il—Saudi Arabia under project number (RG-24 152). [13] D.J. Hosfield, S. Weber, N.S. Li, M. Sauvage, C.F. Joiner, G.R. Hancock, E.
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