Synthesis and antiproliferative evaluation of novel 3,5,8-trisubstituted coumarins against breast cancer

Abstract

Aim: This study focused on designing and synthesizing novel derivatives of 3,5,8-trisubstituted coumarin. Results: The synthesized compounds, particularly compound 5, exhibited significant cytotoxic effects on MCF-7 cells, surpassing staurosporine, and reduced toxicity toward MCF-10A cells, highlighting potential pharmacological advantages. Further, compound 5 altered the cell cycle and significantly increased apoptosis in MCF-7 cells, involving both early (41.7-fold) and late stages (33-fold), while moderately affecting necrotic signaling. The antitumor activity was linked to a notable reduction (4.78-fold) in topoisomerase IIβ expression. Molecular modeling indicated compound 5‘s strong affinity for EGFR, human EGF2 and topoisomerase II proteins. Conclusion: These findings highlight compound 5 as a multifaceted antitumor agent for breast cancer.

Plain language summary

Summary points.

• Novel derivatives of 3,5,8-trisubstituted coumarin and azacoumarin were synthesized and comprehensively characterized.

• Evaluation of the synthesized compounds against MCF-7 breast cancer cells through diphenyltetrazolium bromide assay revealed significant cytotoxic activity.

• Compound 5 demonstrated potent antiproliferative activity (IC₅₀ = 11.36 ± 0.55 μM) against MCF-7 cells, exceeding staurosporin, and exhibited lower toxicity (IC₅₀ = 39.70 μM) toward normal breast MCF-10A cells, suggesting pharmacological advantages.

• Flow cytometric analysis of the MCF-7 cell cycle highlighted alterations in cell population distribution, reducing cells in the G₀/G₁ and S phases while significantly increasing those in the G₂/M phase.

• Annexin V-FITC/propidium iodide analysis revealed substantial increases in both early and late-stage apoptosis in MCF-7 cells induced by compound 5, with a moderate impact on necrosis.

• Molecular mechanism investigation identified a significant reduction (4.78-fold) in the expression levels of topoisomerase IIβ in MCF-7 cells, associating it with compound 5's antitumor activity.

• Molecular modeling and dynamic studies showcased compound 5's considerable affinity for the binding pockets of EGFR, human EGF2 and topoisomerase II proteins.

Breast and cervical cancers are widely recognized as the predominant gender-specific malignancies, accounting for approximately 33.1% of the total spectrum of cancers affecting the female population [1]. Patients with breast cancer have access to a wide range of treatment options [2], including invasive and noninvasive biopsy methods [3], radiotherapy procedures [4] and molecular-based therapies [5], all alongside the traditional chemotherapy approach [6]. Chemotherapy is a well-established and highly effective treatment approach that is widely acknowledged for its efficacy in addressing localized breast and liver malignancies [7]. It achieves this by interfering with the molecular processes of these malignancies [8], ultimately leading to the induction of programmed cell death (apoptosis) [9], tissue death (necrosis) [10] and cellular self-degradation (autophagy) [11]. Nevertheless, it is important to note that a significant limitation of current medications lies in their lack of specificity, so there are several difficulties to overcome, such as the well-documented side effects of cancer therapy. Furthermore, a more widespread concern in the field of medicine, apart from the side effects of therapeutic drugs, is the growing problem of multidrug resistance [12], which presents a significant barrier to successful therapeutic interventions [13].

Coumarin, known by the names 1,2H-chromen-2-one and 2H-1-benzopyran-2-one, are naturally occurring compounds with a remarkably diverse range of biological effects, including cell cycle arrest [14], angiogenesis inhibition [15], kinase inhibition [16], telomerase inhibition [17], antimitotic activity [18], monocarboxylate transporter inhibition [19], carbonic anhydrase inhibition [20], aromatase inhibition [21] and sulfatase inhibition [22]. The coumarin structure possesses the ability to participate in noncovalent interactions [23], including pi–pi interactions, hydrophobic interactions, electrostatic interactions, hydrogen bonding, metal coordination and Van der Waals forces, with the active sites of a wide variety of proteins. Coumarin derivatives have piqued the interest of many medicinal chemists due to their easy synthetic accessibility, low toxicity and elevated selectivity, making them a compelling choice for targeting drug-resistant cancers in a selective manner [24,25]. A wide range of both natural and synthetic drugs possessing the coumarin scaffold have gained significant medical importance (Figure 1) [26]. Coumarin derivatives such as warfarin, aesculetin, scopoletin and umbelliferone exhibit substantial antitumor activity toward breast, prostate, gastric and bladder cancer cell lines, respectively [27–29]. Hymecromone, also known as 4-methylumbelliferone (a), has been employed in various studies as a choleretic and antispasmodic agent [30]. Hymecromone has been observed to exhibit interactions with a variety of signal proteins, such as nuclear factor-κB, cytochrome c, caspase-3, caspase-9, IL-6, and aryl hydrocarbon receptor. Consequently, it has been implicated in the modulation of apoptotic and proapoptotic proteins, leading to both upregulation and downregulation of these crucial cellular components [31]. The cytotoxic potential of hymecromone has been demonstrated in various studies, particularly in relation to its activity against skin cancer. Additionally, hymecromone has exhibited cytotoxic effects on three distinct human cancer cell lines, namely lung adenocarcinoma (MGC-803), nasopharyngeal carcinoma (human KB) and colorectal carcinoma (HCT-116) [32]. Scopoletin, also known as 6-methoxy-7-hydroxycoumarin (b), exhibits a range of noteworthy biological activities, including antioxidant, hepatoprotective, anti-inflammatory and antifungal properties [33]. Scopoletin increases cell cycle arrest in prostate, cervical, cholangiocarcinoma and breast cancer cells [34]. Scopoletin contributes to G₂ and G₀/G₁ cellular accumulation. It also modulates the PI3K/AKT/mTOR pathway, affecting vital cellular functions. It also regulates the MAPK pathway, which is crucial to cellular signaling [34]. Carbochromen (c) has been reported to exhibit a favorable impact on coronary disease such as angina due to its vasodilation activity [35]. Phenprocoumon (d) and warfarin (e) are widely recognized anticoagulant agents that operate by acting as antagonists to vitamin K. Armillarisin A (f) is a compound that has various biological activities by inhibiting SPHK1 and DYRK1A enzymes [36]. Coumarin (1,2-benzopyrone) and 7-hydroxycoumarin (g) were found to have strong in vitro cytotoxic and cytostatic activity against clear cell renal carcinoma with IC₅₀ of 88 ± 8 and 180 ± 20 nM in monolayer and multicellular tumor spheroid [37]. Both compounds cytotoxically affected various human cancer cell lines when investigated in different in vitro models. In vitro investigations showed that 6-nitro-7-hydroxycoumarin (h) selectively inhibits proliferation in renal cell carcinoma cell lines, A-498 [38], human skin malignant melanocytes, SK-MEL-31 [39] and breast cancer [40].

Figure 1.

Representative reported coumarin-based bioactive probes and design of novel coumarin analogues.

(A) Representative coumarin-based bioactive natural compounds with clinical applications. (B) Representative structural features in the coumarin scaffold explored in this study.

The complex conformational arrangements of DNA, encompassing both twisting and folding, play a pivotal role in facilitating its optimal functionality, particularly in fundamental biological processes [41]. The hindrance in cell development causes DNA damage, delayed replication, unrepaired DNA strand breakage and cell death [42]. Structural modifications in DNA content are due to the influence of enzymatic catalysts such as topoisomerases (I, II) [43], which assume a pivotal function in the mitigation of tension and supercoiling phenomena that may arise during various DNA-related processes [44]. Topoisomerase II has two forms, Topo IIα and Topo IIβ, and the latter has been found to have a more crucial role in breast cancer treatment [45,46]. Breast cancer, a complex ailment, exhibits multifactorial etiology and risk factors [47]. A notable proportion, specifically a third, of individuals diagnosed with breast cancer exhibit characteristics indicative of postmenopausal status [48,49]. These patients are distinguished by the presence of tumors stimulated by estrogen receptor (ER). The utilization of ER antagonists inhibits estrogen production and its action has proven to be efficacious in the management of breast cancer [50]. Novobiocin, aesculin, daphnetin, aesculetin, furanocoumarin, bergapten and angelicin all are coumarin derivatives inhibiting the ATPase activity of topoisomerase II (Topo II) and topoisomerase I (Topo I), and hence actively play their role in breast cancer treatment [51].

Based on the above-mentioned facts and the authors' continuous efforts in discovering novel bioactive probes [52–57], the present study aimed to design and synthesize a set of 3,5,8-trisubstituted coumarin and azacoumarin derivatives. The envisioned analogues were designed to investigate the effect of different structural features on the bioactivity of the substituted-(azo)coumarin scaffold. In this regard, the authors envisioned exploring the effect of halogenation at 5-position, the azo/oxo analogues and benzamide/N-phenylacetamide substitution at 3-position (Figure 1). The antiproliferative activity of the designed coumarin analogues was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against the growth of MCF-7 cells. The authors extensively explored the mode of action for the antiproliferative activity of this class of compounds by assessing cell cycle arrest and programmed cell death by flow cytometric analysis. Further, they extended their investigations to assess the compounds' potential to target the activity of Topo II enzyme. Finally, they performed detailed molecular modeling and molecular simulation studies to evaluate the binding affinity of this class of coumarin compounds toward a set of targeted pharmacological proteins in breast cancer, including GFR, HER2 and Topo II.

Materials & methods

Chemicals, instruments & analysis

The melting points of all substances were assessed utilizing an electrothermal apparatus, with measurements conducted in open capillary tubes. Elemental analyses were carried out at the microanalytical unit of the Central Services Laboratory at the National Research Centre in Dokki, Cairo, Egypt. The Vario Elemental instrument from Berlin, Germany, was utilized for these analyses. Infrared spectra were acquired using a Jasco FT/IR.6100 Fourier transform infrared spectrometer, manufactured by Jasco in Tokyo, Japan. The KBr disc technique was employed for sample preparation and analysis. The proton NMR (¹H-NMR) and carbon-13 NMR (¹³C-NMR) spectra were acquired using a Bruker 400 DRX spectrometer (MA, USA) operating at a frequency of 300 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR. The Avance NMR spectrometer was utilized in conjunction with the Finnigan MATSSQ-7000 mass spectrometer (WA, USA) at the Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt, for the purpose of measurement. The reagents utilized in this study were procured from the esteemed Aldrich Chemical company and employed in their original form without any modifications.

Synthesis of 3-(benzoyl) amino-8-methoxy coumarin (2)

A combination of 3-methoxy-2-hydroxybenzaldehyde 1 (0.01 mol), N-benzoyl glycine (0.01 mol) and fused sodium acetate (0.03 mol) was subjected to fusion on a hot plate with the presence of acetic anhydride (2 ml) for 2–3 min. The resulting mixture underwent reflux for 8 h on a water bath, followed by cooling and pouring into water with continuous stirring. The resulting solid was isolated through filtration, washed with water and dried, and ultimately the product was crystallized from ethanol, yielding compound 2 in the form of yellow crystals, yield 71%, melting point (m.p.) 235°C. IR (KBr) ν_max: 3221 (NH), 1720 C=O lactone, 1693 (C=O) amide, 1605, 1582 (C=C), 1128, 1062 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.83, 3.93 (s, 3H, OCH₃ of two isomers), 7.24–8.15 (m, 8H, Ar-H), 8.60 (s, 1H, H-4 of coumarin ring), 9.67 (s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 169.0, 167.1, 166.4, 164.4, 162.9, 158.1, 151.5, 151.2, 146.8, 139.9, 139.8, 135.3, 134.5, 133.9, 132.8, 129.9, 129.2, 128.7, 128.1, 127.5, 127.4, 125.6, 125.4, 124.8, 123.5, 122.1, 120.5, 120.4, 119.8, 119.7, 115.9, 113.1 (carbons of coumarin and aromatic ring of two isomers), 56.6, 56.5 (OCH₃ of two isomers). MS: m/z (%); 295 (M⁺, 22.49), 294 (M⁺-1, 6.22), 162 (1.56), 161 (2.88), 119 (1.91), 117 (2.37), 106 (1.99), 105 (100), 104 (12.13), 103 (2.26), 102 (1.16), 77 (19.02), 76 (3.87), 51 (1.89) (Supplementary Figure 1). Anal. calcd. for C₁₇H₁₃NO₄ (M.wt = 295): C, 69.15; H, 4.41; N, 4.75. Found: C, 68.92; H, 4.21; N, 4.54.

Synthesis of ethyl-3-carboxylate 8-methoxy coumarin (3)

Diethylmalonate (0.012 mol) was added to 3-methoxy-2-hydroxybenzaldehyde 1 (0.01 mol) in the presence of piperidine following the method reported in the literature to provide compound 3 as colorless crystals, yield 86%, m.p. 92°C. IR (KBr) ν_max: 1755, 1735 (C=O), 1605, 1591 (C=C), 1201, 1069 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 1.32 (t, 3H, CH₃), 3.91 (s, 3H, OCH₃), 4.32 (q, 2H, OCH₂), 7.31–7.44 (m, 3H, Ar-H), 8.70 (s, 1H, H-4 of coumarin ring). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 164.5, 163.0 (C=O), 157.2, 154.4 (C–O), 149.1, 148.8, 134.9, 134.7, 130.7, 130.6, 125.3, 125.3, 118.8, 118.4, 118.2, 118.1, 116.6, 116.5, 61.7 (OCH₃), 56.9, 56.6 (OCH₃ of two isomers), 14.5 (CH₃). Anal. calcd. for C₁₃H₁₂O₂ (M.wt = 248): C, 62.90; H, 4.84. Found: C, 62.76; H, 8.66.

Synthesis of N-phenyl 8-methoxycoumarin-3-carboxamide (4)

A mixture comprising ester coumarin 3 (0.01 mol) and aniline (0.011 mol) in dimethylformamide (20 ml) underwent reflux for 16 h. After cooling, the reaction mixture was poured into water with continuous stirring. The resulting precipitate was isolated through filtration and subjected to crystallization from ethanol, yielding compound 4 in the form of pale yellow crystals, yield 83%, m.p. 262°C. IR (KBr) ν_max: 3225 (NH), 1736, 1693 (C=O), 1605, 1593 (C=C), 1127, 1056 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.96 (s, 3H, OCH₃) 7.15–7.75 (m, 8H, Ar-H), 8.91 (s, 1H, H-4 of coumarin ring), 10.69 (s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 160.7, 160.3 (C=O), 148.1, 146.9 (C–O), 143.6 (C-4 of coumarin), 138.4, 129.5, 125.7, 124.8, 121.7, 120.6, 120.4, 119.5, 116.7 (aromatic-C and coumarin ring-C3), 56.7 (OCH₃). MS: m/z (%); 295 (M⁺, 8.37), 204 (1.38), 203 (73.119), 119 (18.90), 117 (2.19), 116 (1.21), 104 (2.63), 103 (1.21), 94 (1.03), 93 (26.06), 92 (35.49), 91 (18.05), 90 (3.40), 89 (40.77), 88 (5.82), 77 (82.76), 76 (60.54), 75 (14.93), 65 (100), 64 (26.14), 63 (40.77), 62 (9.45), 51 (44.00), 50 (27.76) (Supplementary Figure 2). Anal. calcd. for C₁₇H₁₃NO₄ (M.wt = 295): C, 69.15; H, 4.41; N, 4.75. Found: C, 69.01; H, 4.11; N, 4.44.

Synthesis of 3-(N-benzoyl) amino-8-methoxy-azacoumarin (8)

A combination of N-benzoylglycine ester 7 (0.011 mol) and 3-methoxy-2-aminobenzaldehyde (0.01 mol) in the presence of piperidine (2 ml) was subjected to fusion on a hot plate for 2–3 min. Subsequently, ethanol (50 ml) was introduced to the reaction mixture, and the solution was heated under reflux for 8 h. After cooling, the reaction mixture was poured into ice water, neutralized with dilute hydrochloric acid (2%), and the resulting solid was collected by filtration, washed with water and dried. The final product was obtained by crystallization from ethanol, yielding 8 in the form of pale yellow crystals, yield 76%, m.p. 218°C. IR (KBr) ν_max: 3222 (NH), 1705–1685 (C=O), 1606, 1593 (C=C), 1128, 1071 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.94 (s, 3H, OCH₃), 7.25 (t, 1H, Ar-H), 7.31 (d, 2H, Ar-H), 7.57 (t, 2H, Ar-H), 7.64 (d, 1H, Ar-H), 7.98 (d, 2H, Ar-H) 8.61 (s, 1H, H-4 of coumarin ring), 9.68 (s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 166.4, 158.0, 146.8, 139.9, 133.9, 132.8, 129.2, 128.1, 127.5, 125.6, 124.8, 120.4, 119.8, 113.1 (carbons of coumarin and aromatic ring), 56.5 (OCH₃). MS: m/z (%); 294 (M⁺, 18.61), 203 (8.89), 162 (2.42), 105 (78.63), 104 (100), 103 (7.17), 92 (3.19), 91 (1.19), 78 (1.41), 77 (26.74) (Supplementary Figure 3). Anal. calcd. for C₁₇H₁₄N₂O₃ (M.wt = 294): C, 69.39; H, 4.76; N, 9.52. Found: C, 69.19; H, 4.44; N, 9.33.

General procedure for the synthesis of compounds 5, 6 & 9

In 20 ml of glacial acetic acid, coumarin derivatives 2, 4 and 8 (0.01 mol) were dissolved, followed by the addition of 10 ml of bromine (0.011 mol) dropwise with stirring at 60°C. After 5–10 min, the bromine color disappeared, leaving a yellow solution. At this stage, 0.5–1.0 ml of bromine–AcOH solution was added, and the resulting reaction mixture was stirred for 12–16 h at room temperature. The reaction mixture was then poured into water with continuous stirring, and the formed solid was filtered off, washed with water and dried. Finally, the product was crystallized from a suitable solvent to yield 5, 6 and 9.

Synthesis of 3-(benzoyl amino)-5-bromo-8-methoxy coumarin (5)

Bromination of compound 2 produced pale yellow crystals of 3-(benzoyl amino)-5-bromo-8-methoxy coumarin (5), yield 68%, m.p. 255°C. IR (KBr) ν_max: 3227 (NH), 1733, 1691 (C=O), 1605, 1588 (C=C), 1126, 1081 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.94 (s, 3H, OCH₃), 7.19–7.99 (m, 7H, Ar-H), 8.87 (s, 1H, H-4 of coumarin ring), 9.79 (br.s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 166.5, 155.2 (C=O), 146.2, 133.9, 131.4, 129.3, 128.8, 128.4, 126.8, 123.4, 115.8, 110.1 (carbons of coumarin and aromatic ring), 56.7 (OCH₃). MS: m/z (%); 373 (M⁺, unstable), 204 (1.42), 203 (51.46), 202 (32.19), 172 (1.36), 170 (1.55), 119 (3.45), 117 (1.40), 106 (1.42), 105 (48.65), 104 (27.18), 103 (2.10), 91 (13.59), 90 (1.46), 89 (22.61), 88 (4.06), 77 (100), 76 (62.92), 75 (23.56), 74 (10.88), 65 (15.12), 64 (27.87), 63 (88.40), 62 (17.68), 53 (12.50), 51 (59.59), 50 (33.97) (Supplementary Figure 4). Anal. calcd. for C₁₇H₁₂NBrO₄ (M.wt = 373): C, 54.69; H, 3.22; N, 3.75. Found: C, 54.34; H, 3.02; N, 3.43.

Synthesis of 5-bromo-phenyl-8-methoxycoumarin-3-carboxamide (6)

Pale yellow crystals of N-phenyl-5-bromo-8-methoxycoumarin-3-carboxamide (6) were prepared from compound 4 after bromination, yielding 77%, m.p. 283°C. IR (KBr) ν_max: 3265 (br, NH), 1733, 1692 (C=O), 1611, 1587 (C=C), 1125, 1082 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.94, 3.96 (s, 3H, OCH₃ of two isomers), 7.27–8.09 (m, 7H, Ar-H), 8.59, 8.89 (s, 1H, H-4 of coumarin ring of two isomers), 10.75 (s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 163.5, 162.3 (C=O), 148.6, 147.4 (C–O), 147.2 (C-4 of coumarin ring), 132.4, 126.8, 123.8, 121.9, 120.8, 115.7 (C-aromatic and C-3 of coumarin ring), 56.9, 56.8. MS: m/z (%); 375 (M⁺+2, 2.21), 373 (M⁺, 1.71), 333 (5.53), 331 (16.80), 330 (2.90), 329 (17.05), 328 (18.56), 327 (8.11), 326 (16.82), 283 (7.93), 282 (1.13), 281 (5.85), 257 (8.25), 256 (43.58), 255 (23.27), 254 (98.40), 241 (8.99), 239 (9.15), 288 (7.99), 227 (3.70), 226 (10.93), 225 (3.61), 213 (26.95), 212 (6.44), 211 (22.76), 210 (3.39), 203 (100), 199 (9.02), 197 (8.54), 185 (16.40), 184 (4.01), 183 (17.31), 176 (2.97), 175 (19.55), 171 (9.08), 170 (10.88), 169 (8.15), 168 (5.90), 157 (10.13), 156 (2.68), 155 (10.74), 147 (9.68), 119 (9.38), 118 (4.02), 117 (3.39), 104 (3.70), 103 (10.32), 102 (1.58), 91 (5.83), 90 (5.64), 89 (7.76), 88 (3.23), 77 (2.45), 76 (10.81), 75 (11.56), 74 (8.90), 63 (4.16), 62 (3.29), 50 (2.43) (Supplementary Figure 5). Anal. calcd. for C₁₇H₁₂NBrO₄ (M.wt = 373): C, 54.69; H, 3.22; N, 3.75. Found: C, 54.41; H, 2.48; N, 3.56.

Synthesis of 3-(benzoyl) amino-5-bromo-8-methoxy-azacoumarin (9)

Pale yellow crystals of 3-(benzoyl) amino-5-bromo-8-methoxy-azacoumarin (9) were obtained after bromination of compound 8, yield 73%, m.p. 254°C. IR (KBr) ν_max: 3265 (br, NH), 1703, 1695 (br, C=O), 1605, 1596 (C=C), 1265, 1083 (C–O) cm^-1. ¹H-NMR (300 MHz, DMSO-d₆, p.p.m.) δ: 3.93 (s, 3H, OCH₃), 7.20 (d, 1H, Ar-H), 7.56–7.68 (m, 5H, Ar-H), 7.98 (d, 1H, Ar-H), 8.83, 8.87 (s, 1H, H-4 of coumarin ring of two isomers), 9.76, 9.86 (br.s, 1H, NH of two isomers). ¹³C-NMR (100 MHz, DMSO-d₆, p.p.m.) δ: 166.5, 157.2 (C=O), 146.2, 140.6, 133.4, 132.2, 131.5, 129.9, 128.9, 128.4, 128.3, 126.9, 123.4, 123.2, 119.4, 113.3, 110.8 (carbons of coumarin and aromatic ring), 61.8, 56.7. MS: m/z (%); 374 (M⁺+2, 1.85), 372 (M⁺, 1.28), 271 (5.07), 269 (5.76), 204 (6.86), 203 (57.13), 202 (100.00), 201 (12.09), 189 (4.25), 175 (4.12), 119 (9.31), 118 (5.63), 117 (7.32), 116 (2.17), 105 (62.50), 104 (10.00), 91 (4.44), 90 (2.41), 89 (7.23), 77 (11.35), 76 (4.76), 63 (1.73) (Supplementary Figure 6). Anal. calcd. for C₁₇H₁₃N₂BrO₃ (M.wt = 372): C, 54.84; H, 3.49; N, 7.53. Found: C, 54.65; H, 3.29; N, 7.28.

Evaluation of cytotoxic activity

The study employed the MTT assay to assess the in vitro cytotoxic activity of 3,5,8-trisubstituted coumarins (2–6 and 8,9). Cells were seeded in a 96-well multiwell plate at a density of 10⁵ cells per well, allowing for a 24 h incubation period to facilitate cell adhesion before initiating the compound treatment. The test compounds were dissolved in DMSO. Various concentrations of the compound under investigation, specifically 10, 25, 50 and 100 μM, were applied to the cell monolayer. Three wells were carefully prepared for each concentration. The monolayer cells were subjected to a 48 h incubation at 37°C in an atmosphere with 5% carbon dioxide. After this incubation period, the cells underwent various steps, including fixation, thorough washing and subsequent staining. Staining involved adding 40 μl of MTT solution to each well, which was prepared with 5 mg/ml of MTT in a 0.9% NaCl medium. The staining process continued for an extra 4 h under carefully controlled incubation conditions. MTT crystals were dissolved in acidified isopropanol (180 μl/well), and the plate underwent gentle agitation at room temperature for optimal solubilization. After completion of the experiment, absorbance at 570 nm was measured using an ELISA reader for accurate quantification. The IC₅₀ value, representing the molar concentration needed for a 50% decrease in cell viability, was calculated and compared with the standard staurosporine after the experimental procedure [58–62]. The quantification of the surviving fractions was conducted, and subsequently the resulting data were represented as mean values along with their standard deviations.

Assessment of cell cycle distribution

MCF-7 cells were initially seeded at a density of 3.0 × 10⁵ cells per well and incubated at 37°C for 12 h. Following cell attachment, the specified cell types were treated with compound 5 at a dosage equivalent to its IC₅₀, and this treatment was followed by a 48 h incubation period. After completion of the therapeutic intervention, cell samples were collected and fixed in 75% ethanol at 20°C overnight. Following a thorough phosphate-buffered saline wash, centrifugation was employed to separate debris. A solution containing RNAse (Sigma, CA, USA) and propidium iodide (PI; Sigma) at a concentration of 5 mg/ml was added, and the cells were incubated for 15 min. The cells were then prepared for examination via flow cytometry, conducted using a BD Bioscience USA FACS Calibur cytometer and BD Bioscience's Cellquest software [63–65].

Annexin V-FITC/PI analysis

MCF-7 cells were exposed to compound 5 for a 48 h period at a concentration equivalent to its IC₅₀ value. Following completion of the treatment, cells were harvested and underwent two washes with phosphate-buffered saline using a centrifuge (180× g for 10 min at 4°C). After removal of the binding buffer, the cells were resuspended in 100 μl, and 5 μl of Annexin V-FITC was added to each well. After 10 min of incubation at room temperature, an additional 400 μl of binding buffer was added, bringing the total volume to 500 μl. Just prior to the start of the measurement process, the cellular specimens were stained with PI. The cells were analyzed using the Becton and Dickinson's advanced FACS Calibur Flow cytometer (Heidelberg, Germany). Data analysis was performed using Cell-Quest software developed by Becton and Dickinson. The Bio-Vision Annexin V-FITC apoptosis detection kit (cat. no. K101-25) was utilized. The experiment was repeated three-times, and the results were recorded as mean ± standard deviation [63–65].

Evaluation of Topo II activity

The human DNA Topo IIβ ELISA kit, specifically MBS#942146, was employed to evaluate the impact of compound 5 on Topo IIβ. Samples, standards and working reagents were prepared following the instructions provided by the kit manufacturer. Both standard and sample volumes were maintained at 100 μl, and the plates were incubated at 37°C for 2 h. After an initial 1-h incubation period at 37°C, 120 μl of biotin antibody was added to each well, followed by three washes with washing buffer and aspiration of the material. Subsequently, the wells were incubated for an additional 1 h at 37°C with 100 μl of horseradish peroxidase–avidin, involving a total of five rounds of washing. After the addition of 90 μl of 3,3′,5,5′-tetramethylbenzidine substrate to each well, the plates were incubated at 37°C, 5% CO₂, in the dark for 15–30 min. Optical density measurements at 450 nm were taken using a Robonik P2000 ELISA reader after adding 50 μl of stop solution [64]. The experiment was repeated three-times, and the results were recorded as mean ± standard deviation.

Molecular docking simulations

For the molecular docking simulation analysis, the authors used the Protein Data Bank (PDB) to download the 3D crystal structures for the EGFR tyrosine kinase receptor, the human topoisomerase II as PDB ID 1M17 and 5GWK, respectively, available at www.rcsb.org/. The 3D crystal structure chosen for the HER2 protein was identified by PDB ID 3POZ. Subsequently, molecular docking simulations were executed using the Glide software module developed by Schrodinger, LLC, in the year 2023. Ligand structures were generated using MarvinSketch^® 5.8.3 (2012) and ChemAxon tools and subsequently imported into Schrödinger's Maestro workflow. The LigPrep (Schrodinger, LLC, NY, USA) module was used for ligand preparation. The original 2D structures were imported to the Maestro platform. The grid dimensions were selected as per the previous literature and all calculations of docking were run at XP settings. Finally, for interactive visualization, the authors used Discovery Studio 4.5 [54,66–68].

Molecular dynamics simulation study

To carry out the molecular dynamics simulations, the authors simulated three complexes, labeled 5a:1M17, 5a:3POZ and 5a:5GWK, for a period of 100 ns each. For this, they used explicit solvent model with TI3P water molecules. The force field parameters were the OPLS-2005 force field. 10 Å × 10 Å × 10 Å simulation box was chosen and the authors further maintained a charge balance of 0.15 M, sodium ions (Na⁺) were introduced into the system. Moreover, NaCl solutions were incorporated to emulate physiological conditions. The specific details of these simulation parameters were derived from reference literature [69].

Results & discussion

Chemistry

Synthesis of coumarin derivatives

The synthesis of envisioned 3,5,8-trisubstituted coumarin analogues (2–6) was conducted following the approach illustrated in Figure 2. The synthesis was commenced from the commercially available 3-methoxy-2-hydroxybenzaldehyde (1). Thus, cyclocondensation of 3-methoxy-2-hydroxybenzaldehyde with N-benzoyl glycine in the presence of sodium acetate and acetic anhydride afforded the intermediate 3-(benzoyl) amino-8-methoxycoumarin (2) in a satisfied yield (71%). Further, 3-methoxy-2-hydroxybenzaldehyde was reacted with diethylmalonate in the presence of piperidine to afford 8-methoxycoumarin-carboxylate (3; 86% yield). The latter was subsequently reacted with aniline in dimethylformamide under reflux to provide N-phenyl-8-methoxycoumarin-3-carboxamide (4) with an impressive yield of 83%. To further explore the effect of halogen substitution at 5-position, compounds 2 and 4 were subjected to halogenation utilizing bromine as the halogenation reagent for the reaction. The experimental procedure was conducted under carefully controlled conditions utilizing glacial acetic acid at a temperature range of 40–60°C. Thus, compound 2 was subjected to a bromination reaction, leading to the synthesis of 3-(benzoyl) amino-5-bromo-8-methoxycoumarin (5) with a noteworthy yield of 68%. Similarly, compound 4 was subjected to a conversion reaction, resulting in the successful synthesis of N-phenyl-5-bromo-8-methoxycoumarin-3-carboxamide (6) with a satisfactory yield of 77%. The synthetic efforts were also extended to design a synthetic route for the synthesis of 3,5,8-trisubstituted azacoumarins (8 and 9). As shown in Figure 2, the experimental procedure initiates with the cyclocondensation reaction of N-benzoylglycine ester and 3-methoxy-2-aminobenzaldehyde in the presence of base catalyst (piperidine) to successfully provide 3-(N-benzoyl)-amino-8-methoxy-azacoumarin (8) in 76% yield. In the subsequent synthetic reaction, compound 8 was subjected to a bromination reaction utilizing bromine as a halogenating agent and glacial acetic acid as a solvent under controlled conditions of 60°C to afford the desired compound, 3-(N-benzoyl) amino-5-bromo-8-methoxy-azacoumarin (9) in 73% yield. The deliberate choice of bromine as the halogenating reagent and glacial acetic acid as the solvent was made to achieve the desired chemical conversion. The precise control of the reaction temperature at 60°C was of paramount importance to enhance the efficacy of the desired halogenation process.

Figure 2.

Synthesis of 3,5,8-trisubstituted coumarins (2–6) and azacoumarins (8–9).

(A) Reagents and reaction conditions: i) N-benzoylglycine, AcONa, Ac₂O, reflux, 8 h; ii) diethylmalonate, piperidine, EtOH, reflux, 12 h; iii) aniline, dimethylformamide, reflux, 16 h; iv) bromine, AcOH, 60°C–room temperature, 16 h. (B) Reagents and reaction conditions: i) N-benzoylglycine, piperidine, EtOH, reflux, 8 h; ii) bromine, AcOH, 60°C–room temperature, 12 h.

Evaluation of inhibitory activity toward the viability of MCF-7 cells

The current investigations aimed to evaluate the effects of synthesized 3,5,8-trisubstituted coumarins and azacoumarins on the survival of MCF-7, which serves as a model for breast cancer cells. To evaluate the antitumor properties of the compounds, the authors conducted the commonly utilized MTT colorimetric assay [70]. This assay is a well-established method for assessing cytotoxicity and cell viability. In this study, staurosporine (STU) was incorporated into the authors’ assessments as a reference and positive control. The cytotoxicity assessment was conducted on the MCF-7 cells employing synthesized 3,5,8-trisubstituted coumarins and azacoumarins in a dose-dependent manner. As depicted in Figure 3, the synthesized compounds (2, 4, 5, 6, 8, 9b and STU) exhibited a considerable and dose-dependent antiproliferative activity on the viability of MCF-7 cells. Analysis revealed that the introduction of the 3-(benzoyl)-amino moiety to the 8-methoxycoumarin scaffold resulted in a compound with considerable cytotoxic activity (2; IC₅₀ = 23.38 μM). The integration of a halogen atom, specifically bromine, at the 5-position of the 3-(benzoyl) amino-8-methoxy coumarin (2) structure induced alterations in the chemical properties of the compound, resulting in a compound with substantial antiproliferative activity (compound 5; IC₅₀ = 11.36 ± 0.55 μM). The incorporation of a bromine (Br) group at the 5-position of the coumarin ring in 3-(benzoyl) amino-8-methoxy coumarin (2) resulted in a notable increase in cytotoxic activity. The marked enhancement in cytotoxic activity suggesting the possible role of the bromine, as an element with a larger atomic size and considerable electronegativity, to improve the affinity of this scaffold to the target protein(s) [71]. Further, the incorporation of a Br atom resulted in a modification of the compound's lipophilicity, thereby impacting its capacity to traverse cell membranes and its mode of interaction with intracellular targets [72]. To gain more insights into the role of the N-benzoyl-amino moiety, the authors assessed the activity of the 3-N-phenyl-carboxamide analogue (compound 4), in which the position of the amide group was inverted. Interestingly, the shifting in the amide group position resulted in a substantial impairment in the inhibitory activity of the resulting compound (compound 4; IC₅₀ = 39.35 μM), suggesting the contribution of the amide group to the antiproliferative efficacy of the N-substituted-8-methodycoumarine scaffold. Similarly, incorporation of the Br at the 5-position demonstrated a significant increase in the inhibitory activity of the resulting compound (compound 6; IC₅₀ = 16.24 μM), affirming the critical role of the halogen substitution at 5-position in the antiproliferative activity of the 8-methoxycoumarin scaffold. To further explore the structural features in the 8-methodycoumarine scaffold, the cytotoxic activity of the corresponding (aza)–coumarin analogues (compounds 8 and 9) was evaluated. The findings revealed that shifting the oxo–coumarin into aza–analogue significantly impaired the inhibitory activity of the compound (compound 8; IC₅₀ = 68.9 μM). These results indicated that the variation in electron density and hydrogen bonding capacity of N and O atoms plays a crucial role in the reactivity and binding property of the 8-methoxycoumarin scaffold. Finally, incorporation of Br at 5-position into the 8-methoxy(aza)coumarin scaffold led to a substantial improvement in the cytotoxic activity, as indicated by the IC₅₀ value of compound 9 (IC₅₀ = 26.35 μM). Among the examined compounds, the analyses revealed that compound 5 demonstrated the highest antiproliferative activity against the viability of MCF-7 cells, displaying an IC₅₀ value of 11.36 μM, compared with that of STU anticancer drug (5.25 μM).

Figure 3.

Evaluation of the inhibitory potential of compounds 2–9 toward the viability of MCF-7 cells.

(A) The inhibitory activity of compounds 2–9 toward the proliferation of MCF-7 cells in a dose-dependent manner. (B) The IC₅₀ (μM) values of compounds 2–9 and staurosporine against the proliferation of MCF-7 cells. The data described was originated from independent triplicate and expressed as the mean ± standard error of the mean.

STU: Staurosporine.

Based on the promising antiproliferative activity of compound 5, the authors extended their investigations to explore its cytotoxic activity toward normal MCF-10A breast cells. Evaluating the selectivity of potential antitumor compounds is essential, as it aims to reduce the potential harm to normal cells. These data revealed that compound 5 displays a nonconsiderable antiproliferative effect toward MCF-10A cells (IC₅₀ = 39.70 μM), when assessed to the STU drug (IC₅₀ = 26.72 μM). Taken together, these results highlight the potential of 3,5,8-trisubstituted coumarins, specifically compound 5, as candidates for the development of anticancer agents targeting breast cancer. The significant antiproliferative effect observed in compound 5 on MCF-7 cells, coupled with its relatively reduced toxicity toward normal MCF-10A cells, underscores its potential pharmacological benefits.

Assessment of MCF-7 cell cycle

Encouraged by these findings, the authors conducted flow cytometric analysis to evaluate the influence of compound 5 on the MCF-7 cell cycle distribution. In this regard, compound 5 was applied to the MCF-7 cells at the IC₅₀ concentration and the cellular phases were carefully conducted and compared with the untreated MCF-7 cells. The results described in Figure 4 elucidate notable alterations in the cellular cycle distribution subsequent to the administration of compound 5, as demonstrated by the alteration in the cellular population within the cellular phases, including S, G₂/M and G₀/G₁ phases. The observed data revealed a prominent reduction in the ratio of cells residing in the G₀/G₁ phase, as indicated by a decline from an initial value of 55.81% to a final value of 49.83%. Similarly, it was observed that the proportion of cells present in the S phase experienced a diminished growth from 29.66 to 24.32%. Additionally, the results indicated a significant increase of cells in the G₂/M phase (14.53 to 25.85%).

Figure 4.

Impact of compound 5 on the MCF-7 cell cycle distribution.

Flow cytometry evaluation of cellular stages in (A) untreated MCF-7 cells and (B) treated cells with compound 5. Histogram of the cell cycle exploration of untreated and treated MCF-7 cells (C). The data described was originated from independent triplicate and expressed as the mean ± standard error of the mean.

These results underscore the complex and multifaceted nature of compound 5‘s impact on cell regulation. The observed decrease in G₀/G₁ phase population implies that compound 5 might be influencing cell cycle progression, possibly by modulating regulatory checkpoints or inducing cell cycle arrest. In addition, the decline in the proportion of cells in the S phase, from 29.66 to 24.32%, indicates potential interference with DNA synthesis and replication. The S phase is crucial for duplication of DNA, and the observed reduction highlights the influence of compound 5 on cellular replication and genomic stability. On the other hand, the notable increase in the G₂/M phase population indicates that compound 5 may be influencing the transition of cells from the S phase to G₂/M phase. Such alterations in G₂/M phase distribution could signify disruptions in cell division processes, potentially leading to cell cycle arrest or aberrant mitotic events. In conclusion, the data imply that compound 5 has a considerable impact on the cell cycle, promoting cell cycle progression, while concurrently interfering with DNA synthesis. Furthermore, the empirical findings suggest that compound 5 may induce programmed cell death in MCF-7 cells by enhancing the population of cells that are halted in the G₂/M phase of the cell cycle. Overall, these findings indicate that compound 5 holds considerable promise as an anticancer therapeutic agent.

Annexin V-FITC/PI assessments

Next, the authors accomplished a comprehensive investigation employing Annexin V-FITC/PI screening to assess the mode of action of compound 5 on triggering programmed cell death in MCF-7 cells. This analysis was employed to evaluate and discriminate cellular subpopulations exhibiting apoptotic and necrotic characteristics [73,74]. Based on the data depicted in Figure 5, it is apparent that the administration of compound 5 (at a concentration of IC₅₀) resulted in a notable upregulation in the overall proportion of programmed cell death. Upon exposure of MCF-7 cells to compound 5, a notable and statistically significant increase (17.5-fold) in the incidence of programmed cell death was observed. Exclusively, the proportion of cells undergoing apoptosis increased from 1.99% in the control cell to 34.82% in the treated cells. Further, the administration of compound 5 to MCF-7 cells resulted in a notable augmentation of apoptosis levels, encompassing both the early and late stages. As shown in Figure 5, compound 5 administration demonstrated a considerable rise (41.7-fold) in the percentage of early apoptosis in cells, escalating from 0.61 to 25.41% in the untreated and treated groups, respectively. Additionally, the findings revealed that compound 5 treatment significantly upregulates the late-stage apoptosis (33-fold increase), as indicated by the elevation of the percentage from 0.24% in the control group to 7.96%. On the other hand, treatment with compound 5 revealed a moderate influence on the necrotic signaling cascade within cells. Upon administration of compound 5, the frequency of cellular necrosis experienced a 1.27-fold elevation, escalating from 1.14 to 1.45% in untreated and treated cells, respectively. These results imply that compound 5 has a substantial potency to trigger the cell death in MCF-7 cells by initiating mainly the apoptotic signaling cascades. Annexin V-FITC, a well-established marker, exhibits a specific affinity for phosphatidylserine and selectively binds to it [75]. Phosphatidylserine, a type of phospholipid, has been observed to be prominently displayed on the cell membrane during the initial phases of apoptosis [76]. PI, a dye, has the ability to permeate cells with compromised membranes, a characteristic commonly observed in the later stages of apoptosis and necrosis [77]. The present study showed that compound 5 exhibited a substantial cytotoxic effect toward MCF-7 cells via induction of apoptosis. The significant impact of compound 5 in triggering both early and late apoptosis, suggesting that compound 5 may be able to initiate the apoptotic cascade at many points, resulting in cell death cascade in MCF-7 cells. The experimental results further suggest that compound 5 has a moderate effect on the necrosis cascade. Overall, these findings imply that compound 5 exhibits a therapeutic potential as an antitumor agent, most likely due to its ability to induce apoptosis in MCF-7 cells.

Figure 5.

Effect of compound 5 administration on the apoptotic and necrotic cascades in MCF-7 cells.

(A) The Annexin V-FITC analysis of control MCF-7 cells. (B) The Annexin V-FITC analysis of compound 5-treated MCF-7 cells. (C) A graphical analysis representing the impact of compound 5 administration on the cell death cascade in MCF-7 cells. The data described was originated from independent triplicate and expressed as the mean ± standard error of the mean.

PI: Propidium iodide.

Evaluation of Topo IIβ activity

The investigations were further extended to explore the capability of compound 5 to inhibit Topo IIβ activity in MCF-7 cells. The topoisomerase enzyme family, also known as DNA gyrase, is responsible for maintaining DNA's 3D structure [78]. DNA replication, transcription and recombination all rely on topoisomerases because of their ability to control DNA replication and repair breaks of DNA double strands [79]. The two isoforms of topoisomerases discovered so far are Topo I and Topo II. Both Topo I and Topo II aid in cancer treatment; however, their respective mechanisms vary depending on their locations. Topo II helps in DNA synthesis and the separation of chromosomes during mitosis, while Topo I is essential for topological maintenance, transcriptional control and DNA repair. As a result, pharmacological inhibition of Topo II may hinder transcription and DNA repair in cancer cells, leading to cell death [80,81]. To explore whether the antitumor activity of compound 5 toward MCF-7 cells is linked to its ability to target Topo II activity, the authors evaluated the activity of Topo IIß in MCF-7 cells. As represented in Figure 6, MCF-7 cells exhibited high expression levels of Topo IIß (505 pg/ml), indicating the crucial role of Topo IIß in the viability of cancer cells. Interestingly, treatment of MCF-7 with compound 5 at 11.36 μM significantly attenuated the expression level of Topo IIß by 4.78-fold, as compared with control cells. These results imply that compound 5 may impede the efficacy of DNA replication, transcription and repair mechanisms in MFC-7 cells. As a result, this interference has the potential to induce cell cycle arrest, ultimately culminating in cellular demise. These findings further support the antitumor potential of compound 5 toward MCF-7 cancer cells. The pronounced ability of compound 5 to inhibit Topo IIß activity in MCF-7 cells provides corroborative evidence for the observed capability of compound 5 to induce the apoptotic cascade in MCF-7 cells.

Figure 6.

Assessment of DNA topoisomerase IIß levels in MCF-7 cells.

The data described was originated from independent triplicate and expressed as the mean ± standard error of the mean.

Cont.: Control.

Molecular docking simulations

The pursuit of therapeutic targets in cancer treatment is of paramount importance. In the current study, the authors extended their exploration to assess the binding ability of compound 5 to three key targets in cancer progression. First, the EGFR tyrosine kinase plays a pivotal role in cell proliferation and survival, making it a critical focus for targeted therapies [82]. Second, the kinase domain of the HER2 protein is another key target, as HER2 overexpression is implicated in aggressive breast cancers, making drugs that target this domain crucial for treatment success [83]. Third, human Topo II is essential for DNA replication and repair, rendering it an attractive target for chemotherapy [84]. These targets hold the promise of improved cancer therapies, offering hope for better patient outcomes and enhanced quality of life. For the current study, the authors used molecular docking analysis to identify the possible binding modes for compound 5 toward the three selected targets, EGFR tyrosine kinase (PDB ID 1M17; resolution: 2.60 Å) [85], the kinase domain of HER2 protein (PDB ID 3POZ; resolution: 1.50 Å) [86] and human Topo II (PDB ID 5GWK; resolution: 3.15 Å) [87]. The receptor grid dimensions were chosen by keeping the centers of their respective, cocrystallized ligands, AQ4 [6,7-bis(2-methoxy-ethoxy)quinazoline-4-yl]-(3-ethynylphenyl)amine), 03P (N-{2-[4-({3-chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}amino)-5H-pyrrolo(3,2d)pyrimidin-5-yl]ethyl}-3-hydroxy-3-methyl-butanamide) and EVP ([(5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3d][1,3]dioxol-5-yl4,6-O-[(1R)-ethylidene]-β-D-glucopyranoside, respectively. In the case of EGFR target, the authors noticed that compound 5 had a higher docking score, extra precision (-9.53 kcal/mol), than the AQ4 (-6.11 kcal/mol) and the standard, STU (-7.32 kcal/mol). Compound 5 was interacted with target EGFR via amino acid residues Met742 (π–cation), Asp831 (Van der Waals interactions), Thr830 (H bonding; 1.98 Å), Phe832 (alkyl interactions), Val702 (alkyl interactions), Leu694 (alkyl interactions), Leu820 (alkyl interactions), Ala719 (alkyl interactions), Lys721 (π–cation) and Thr766 (H bonding; 2.67 Å; Figure 7A & B). AQ4 (cocrystallized ligand) represented a conventional H bonding interaction with Thr766 (Supplementary Figure 7). Other interactions included donor–donor interaction with amino acid residue Gln767 and acceptor–acceptor interaction with Lys721. Met742, Ala719 and Leu820 denoted π–sulfur, π–alkyl and π–sigma interactions, respectively (Supplementary Figure 7). STU was shown to interact with five amino acids (Val702, Lys721, Leu694, Ala719 and Leu820) via π–alkyl interactions (Supplementary Figure 7). The authors noticed one H bond interaction with amino acid residue Gln767. Met742 presented a π–sulfur interaction type. These findings point to the fact that compound 5 possesses a substantial affinity to targeting EGFR.

Figure 7.

3D binding modes and 2D binding interactions for compound 5.

(A & B) Toward the binding pocket of EGFR tyrosine kinase (PDB ID: 1M17). (C & D) Kinase domain of HER2 protein (PDB ID: 3POZ) and (E & F) human topoisomerase II (PDB ID: 5GWK).

PDB: Protein Data Bank.

The same trend was noticed for HER2 protein (docking score: 5; -7.76 kcal/mol; 03P: -7.21 kcal/mol and STU: -6.98 kcal/mol). Similarly, varieties of interacting amino acids were observed for compound 5 toward HER2 protein, including Asn842 (H bonding; 1.56 Å), Asp837 (Van der Waals interactions), Asp855 (Van der Waals interactions), Lys745 (H bonding; 1.06 Å), Val726 (alkyl interactions) and Arg841 (π–cation; Figure 7C & D). The cocrystallized ligand 03P denoted amino acid interactions with Lys745, Asn842 and Arg841 via H bonding. The authors also observed π–sigma interactions with Thr790 and Leu844. Val 726, Lys745, Leu788 and Ala743 showed a π–alkyl type of interaction (Supplementary Figure 8). The STU showed interactions with Leu718, Cys797, Ala743, Lys745 and 844 via π–alkyl interactions. Further, Gln791 and Met793 depicted conventional H bonds (Supplementary Figure 8). In summary, the results indicate that compound 5 exhibits a high affinity toward the kinase domain of HER2 protein.

Furthermore, in order to assess better binding analysis of compound 5, we explored the affinity of compound 5 toward human Topo II protein. Topo II contains six binding sites: N-terminal domain, core catalytic domain, ATP-binding sites, tower domain, C-terminal domain and magnesium ion-binding sites. These binding sites have been identified by x-ray crystallography and cryo-electron microscopy in the 3D structure of Topo II . Analysis showed that compound 5 possesses the best docked (docking score: -10.91 kcal/mol) candidate compared with cocrystallized ligand EVP (docking score: -8.33 kcal/mol). In the case of compound 5, two hydrogen bonds were observed for amino acids Ser464 and Ala465; π–alkyl interaction was observed for Ala465 amino acid residue (Figure 7E & F). The cocrystallized ligand EVP represented two H bonding interactions with amino acid residues Ser464 and Asp463. Furthermore, it also interacted with Asp541 and Ala465 via alkyl and π-anionic types of interactions, respectively (Supplementary Figure 9).

Molecular dynamics simulations

EGFR

After docking calculations, the authors simulated a complex 5:1m17 for the period of 100 ns to check the stability of the complex (Figure 8A). The root mean square deviation (RMSD) is the parameter used to assess the changes in displacement of atoms. The RMSD analysis for the complex revealed a stability of complex, as indicated by RMSD below 4.2 Å (Supplementary Figure 10A). Another parameter, root mean square fluctuation (RMSF), explains local changes among the chains. From RMSF, the authors noticed minor fluctuations (Supplementary Figure 10B). Supplementary Figure 10C indicates the ligand torsion profile, which is a summary of conformational changes for each rotatable bond. From Figure 8, the authors noticed hydrophobic interactions with amino acids such as Leu694, Val702, Ala719, Lys721, Met742, Leu764, Leu768, Pro770 and Leu820. Amino acid residues Lys721, Thr766, Gln767, Met769, Thr830 and Asp831 represented water bridges. Met769 depicted H bonding interaction. Proteins' secondary structure (SS) during the simulation is depicted in Supplementary Figure 10D & E. A total of 39.35% SS elements were found. Figure 8A denotes a protein–ligand contact plot. The authors observed 11% π–cationic-type interactions between arene moiety and Lys721. Met769 presented 97% H bonding interaction with –C=O– functionality of coumarin. Similarly, NH– functionality of carboxamide linkage exhibited more than 90% H bonding interaction with Gln767 and Thr766. Figure 8A provides an idea about the time line representation of the interactions and contacts.

Figure 8.

Molecular dynamics simulation analysis.

(A) The complex of compound 5 with EGFR tyrosine kinase (5:1M17), (B) kinase domain of HER2 protein (5:3POZ) and (C) human topoisomerase II (5:5GWK) showing the protein–ligand contact profile, protein–ligand contact plot and time line representation of the interactions and contacts.

HER2 protein

After docking calculations, the authors simulated a complex 5:3POZ for the period of 100 ns to check the stability of the complex (Figure 8B). The RMSD analysis for the complex revealed a stability of complex, as indicated by RMSD below 4.0 Å (Supplementary Figure 11A). From RMSF assessments, the authors noticed minor fluctuations (Supplementary Figure 11B). Supplementary Figure 11C indicates a ligand torsion profile, which is a summary of conformational changes for each rotatable bond. The bond between the arene moiety and C=O functionality of carboxamide exhibited majority rotations between -90° and 180°. Major rotations were observed for the Ar–O–CH₃ bond (-180–180°). As in Figure 8B, the protein–ligand (5) contacts were observed. They noticed hydrophobic interactions with amino acids such as Leu718, Val726, Met766, Leu777, Leu788, Leu792, Leu844, Phe856, Leu858 and Leu1001. Amino acid residues Lys728, Gly729, Gln787, Leu788, Thr790, Gln791, Thr854 and Asp855 represented water bridges. Cys775, Met793, Thr854, and Asp855 depicted H bonding interactions. Proteins’ SS during the simulation is depicted in Supplementary Figure 11D & E. A total of 40.70% SS elements were found. Figure 8B denotes a protein-ligand contact plot. The authors observed 57% hydrophobic interactions between the arene moiety and Phe856. Figure 8B also affords an idea about the time line representation of the interactions and contacts.

Topo II enzyme

Considering the docking result for compound 5, the authors then simulated it with Topo II, denoted as 5:5GWK, for the period of 100 ns. For this, they used the Desmond module from Schrodinger, LLC. The whole system of simulation had 98,917 atoms for 5:5GWK (28,727 water molecules; Figure 8C). From simulation results, the authors noticed that the Cα-RMSD backbone values were below 5.4 Å; also, minor fluctuations were noticed from the RMSF plot (Supplementary Figure 12). Water bridges were observed with Lys728, Gly729, Gln787, Thr790, Gln791 and Thr854. Met762 and Met766 represented major hydrophobic interactions for a complex 5a:5GWK. An H bond was observed only for amino acid Ser763. The figures present a ligand-protein contact plot, highlighting interactions that persisted for over 5.0% of the simulation time within the selected trajectory (0.00–100.00 ns) for compound 5. Furthermore, the ligand torsion plots succinctly summarize conformational changes in each rotatable bond within the ligand over the entire simulation duration (0.00–100.00 ns) for 5 toward the target Topo II (Supplementary Figure 12). In conclusion, these results substantiate the stable conformations of the ligand–protein complexes over the 100 ns simulation period.

Conclusion

In this comprehensive study, novel derivatives of 3,5,8-trisubstituted coumarin and azacoumarin were synthesized and characterized. The evaluation of these synthesized compounds against the proliferation of MCF-7 breast cancer cells through MTT assay revealed substantial cytotoxic activity, with compound 5 emerging as particularly noteworthy, boasting significant antiproliferative activity with an IC₅₀ value of 11.36 ± 0.55 μM compared with that of the reference compound, STU (IC₅₀ = 5.25 ± 0.25 μM). Importantly, compound 5 exhibited lower toxicity toward normal breast MCF-10A cells (IC₅₀ = 39.70 μM), underscoring its potential pharmacological advantages. Flow cytometric analysis of the MCF-7 cell cycle further illuminated the impact of compound 5, showcasing alterations in cell population distribution across phases, notably reducing cells in the G₀/G₁ and S phases while significantly increasing those in the G₂/M phase. Annexin V-FITC/PI analysis provided insights into the apoptotic effects of compound 5, revealing substantial increases in both early and late-stage apoptosis in MCF-7 cells, with a moderate impact on necrosis. Delving into the molecular mechanisms, the study uncovered that compound 5‘s antitumor activity is associated with a remarkable reduction in the expression levels of Topo IIβ (4.78-fold) in MCF-7 cells. Molecular modeling and molecular dynamics studies further elucidated the compound's molecular interactions, showcasing a considerable affinity for the binding pockets of EGFR, human EGF2 and Topo II proteins. Collectively, this study positions compound 5 as a promising antitumor agent for breast cancer, offering a multifaceted approach to combating breast cancer by reducing cancer cell viability, inducing apoptosis, causing cell cycle arrest and targeting Topo II activity. The detailed molecular insights provided underscore its potential as a valuable candidate for further exploration in breast cancer therapeutics.

Author contributions

Conceptualization: MG Salem, SN Mali, EM Saied and MF Youssef; methodology: MG Salem, SN Mali, RD Jawarkar, SZ Alshawwa, E Al-Olayan, EM Saied and MF Youssef; software: AM Alqahtani, SN Mali, HA Alshwyeh, RD Jawarkar, AS Altamimi, EM Saied and MF Youssef; validation: MG Salem, AM Alqahtani, SN Mali, RD Jawarkar, AS Altamimi, SZ Alshawwa, E Al-Olayan, EM Saied and MF Youssef; formal analysis: MG Salem, SN Mali, HA Alshwyeh, RD Jawarkar, AS Altamimi, E Al-Olayan and MF Youssef; investigation: AM Alqahtani, HA Alshwyeh, RD Jawarkar, AS Altamimi, E Al-Olayan, EM Saied and MF Youssef; data curation: MG Salem, AM Alqahtani, SN Mali, HA Alshwyeh, RD Jawarkar, SZ Alshawwa and MF Youssef; writing – original draft preparation: MG Salem, AM Alqahtani, SN Mali, RD Jawarkar, EM Saied and MF Youssef; writing – review and editing: MG Salem, AM Alqahtani, SN Mali, HA Alshwyeh, RD Jawarkar, AS Altamimi, SZ Alshawwa, E Al-Olayan, EM Saied and MF Youssef; visualization: MG Salem, AM Alqahtani, HA Alshwyeh, AS Altamimi, EM Saied and MF Youssef; supervision: MG Salem, SN Mali, EM Saied and MF Youssef; project administration: MG Salem, SZ Alshawwa, E Al-Olayan, EM Saied and MF Youssef; funding acquisition: MG Salem, SZ Alshawwa, E Al-Olayan and EM Saied. All authors have read and agreed to the published version of the manuscript.

Financial disclosure

This research was funded by the Faculty of Pharmacy, Port-Said University, and Faculty of Science, Suez Canal University, Egypt. The authors also extend their appreciation to Princess Nourah bint Abdulrahman University for funding this work through the Researchers Supporting Project number PNURSP2023R165, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This study was also supported by Researchers Supporting Project number RSP2024R111, King Saud University, Riyadh, Saudi Arabia.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.