Substitutional Diversity-Oriented Synthesis and In Vitro Anticancer Activity of Framework-Integrated Estradiol-Benzisoxazole Chimeras

Hybridization of steroids and other pharmacophores often modifies the bioactivity of the parent compounds, improving selectivity and side effect profile. In this study, estradiol and 3′-(un)substituted benzisoxazole moieties were combined into novel molecules by structural integration of their aromatic rings. Simple estrogen starting materials, such as estrone, estradiol and estradiol-3-methylether were used for the multistep transformations. Some of the heterocyclic derivatives were prepared from the estrane precursor by a formylation or Friedel–Crafts acylation—oximation—cyclization sequence, whereas others were obtained by a functional group interconversion strategy. The antiproliferative activities of the synthesized compounds were assessed on various human cervical, breast and prostate cancer cell lines (HeLa, MCF-7, PC3, DU-145) and non-cancerous MRC-5 fibroblast cells. Based on the primary cytotoxicity screens, the most effective cancer-selective compounds were selected, their IC50 values were determined and their apoptosis-inducing potential was evaluated by quantitative real-time PCR. Pharmacological studies revealed a strong structure–function relationship, where derivatives with a hydroxyl group on C-17 exhibited stronger anticancer activity compared to the 17-acetylated counterparts. The present study concludes that novel estradiol-benzisoxazole hybrids exert remarkable cancer cell-specific antiproliferative activity and trigger apoptosis in cancer cells.


Introduction
Naturally occurring and synthetic benzisoxazoles [1], particularly their 3-substituted representatives [2], are important pharmacophores and serve as valuable tools for drug design and discovery, having a high number of positive hits in biological screens. Because of their versatile properties and potential as selective ligands for a variety of macromolecular targets, these bicyclic aromatic ring systems constitute the essential structural motif in a wide range of pharmacologically active compounds, including a number of potential anticancer agents (Figure 1) [3][4][5][6][7][8][9]. Furthermore, the benzisoxazole scaffold is often used as a bioisosteric replacement for the benzoyl group of biologically active molecules [10].
Chemical modification of natural steroids with different heterocycles provides a way to alter the function of the parent compound, and several derivatives have been demonstrated to be effective in the prevention and treatment of many types of cancers [11]. Although there are no examples in the literature for the synthesis of steroidal benzisoxazoles, the incorporation of the five-membered isoxazole ring into a sterane backbone in either a connected [12][13][14] or a condensed manner [15] led to some effective antiproliferative agents (I-VIII, Figure 1). Nevertheless, the phenolic A-ring of estrogens offers the possibility to synthesize aromatic ring-integrated benzisoxazole hybrids and this modification may have beneficial outcomes in several aspects. First, molecular hybridization of steroids with other  [3][4][5][6][7][8] and steroidal isoxazoles (VII,VIII) [13,15] with anticancer activity and the proposed E2-benzisoxazole hybrids.
As a continuation of our ongoing research for designing steroidal A-ring integrated chimeras with anticancer activity [18,[23][24][25][26], a benzisoxazole scaffold containing different substituents at the C-3 position of the heteroring was hybridized with the aromatic ring of E2. According to a comprehensive analysis of the chemical structure of marketed anticancer agents, the most abundant functional groups of these drugs are OH, COOH, COOR, NH 2 and F; moreover, 43.4% of them contain both aromatic and non-aromatic rings as part of their structure [27]. These structural features were taken into account in designing the synthesis of novel compounds that show diversity in the substitution pattern of the N,O-heterocyclic ring. Virtual screening of the pharmacokinetic parameters using ChemAxon's Chemicalize software [28] showed that almost all the molecules designed to be produced meet the drug-like criteria defined by Lipinski and Veber [29,30] (Supplementary Material p50).
Various protocols for the synthesis of 3-substituted benzisoxazoles have been reported so far [31]. Among others, catalytic cyclizations of 2-hydroxyaryl aldoximes and ketoximes [32] leading to the N-O bond formation through intramolecular Mitsunobu reaction [33] or by conversion of the hydroxyl group of the oximes to good, leaving groups followed by base-catalyzed ring-closure, are well-known procedures [3,34]. However, due to the necessity of a strong base or high temperature, these methods often involve the formation of other products; e.g., Beckmann rearrangement of the oxime and subsequent cyclization can lead to isomeric benzoxazoles. A new route using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)/PPh 3 at room temperature (RT) was reported to overcome these disadvantages [34]. Since the phenolic substructure is present in estrogens, the 2-hydroxyaryl aldehyde or ketone precursors of oximes can be obtained by regioselective formylation or Friedel-Crafts acylation. Although some drawbacks of these approaches could be found, such as the need for 3-4 reaction steps from E2 to benzisoxazoles, we tried to optimize each step in order to achieve high conversions without the formation of undesirable byproducts.
The newly synthesized benzisoxazole derivatives were evaluated for their in vitro antiproliferative activities on DU-145 and PC3 (both prostate cancer), HeLa (cervical cancer) and MCF-7 (breast cancer) cell lines. For comparison, the cytotoxicity of the compounds was tested on MRC-5 non-cancerous lung fibroblast cells. According to the results of the initial screening, the most potent anticancer agents were selected, their IC 50 values were determined and their apoptosis-inducing potential was examined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) measurements.

Syntheses
Based on the literature background, the synthesis of estradiol-A-ring-integrated unsubstituted benzoisoxazole and those containing diverse functional groups at C-3 position of the heteroring were designed (Figure 1). For the multistep transformations estrone (E1), estradiol (E2) and estradiol-3-methylether (E2Me) were used as starting materials. Some of the reactions (if R = H, Me, Et, i Pr, Ph, COOMe, CF 3 , OH, OMe) were initiated by regioselective formylation or Friedel-Crafts acylation at the C-2 position of the corresponding estrane precursor (1 or E2). The following oxime formation and activation of the oxime-OH offered the possibility of cyclization with the phenolic OH group to isoxazole under appropriate conditions. In other cases (if R = COOH, CONH 2 , CH 2 OH, CH 2 F, CHF 2 ), a common 3 -methylcarboxylated benzisoxazole intermediate served as a precursor for additional functional group interconversion (FGI).
Next, condensation reactions of ketones 2b-g were carried out with hydroxylamine to afford the corresponding oximes as a single isomer (3b, 3c and 3g) [37] or mixtures of E and Z forms (3d-f) [38] (Scheme 1). Subsequent DDQ/PPh 3 -initiated cyclization of the oximes in dichloromethane (DCM) under mild conditions afforded the desired E2-benzisoxazole hybrids (4b-g). It is worth mentioning that lower yields (25% and 40%) were obtained for 4e and 4f. Because of stereoelectronic reasons, only one of the oxime isomers was able to cyclize to isoxazole with the bulky adduct formed from DDQ and PPh 3 [34], whereas the other remained unchanged during the reaction, as confirmed by thin-layer chromatography (TLC) monitoring. Additionally, the presence of the Ph group in 3e activated the compound towards Beckmann rearrangement, leading to the formation of 2-phenylbenzoxazole as the main product. In order to enhance the yield, an alternative route was carried out for the transformation of 2e, involving an imine formation (5e) with ammonia and the following chlorination/dehydrohalocyclization by N-chlorosuccinimide (NCS)/K 2 CO 3 [39]. In this case, steric factors do not impede the cyclization to 4e, and a higher yield (60%) was obtained (Scheme 1). Unfortunately, this latter method did not work for the conversion of the trifluoromethyl derivative 2f.
For the synthesis of two additional heterocyclic derivatives (4h and 4i), E1 was used as a starting material (Scheme 2). After regioselective Friedel-Crafts acylation with TFAA and a subsequent haloform reaction of 6a [40], an E2-salicylic acid domain-integrated hybrid (6b) was produced. This compound was next converted to a hydroxamic acid derivative of E2 (7) by a three-step sequence involving C-17 ketone reduction, methylester formation of the acid moiety [41] and the final nucleophilic acyl substitution with NH 2 OH [42]. Mitsunobutriggered heterocyclization in anhydrous THF at RT [43] afforded 3 -hydroxybenzisoxazole 4h in 89% yield, whereas its O-methylation furnished 4i in 82% yield (Scheme 2). was able to cyclize to isoxazole with the bulky adduct formed from DDQ and PPh3 [34], whereas the other remained unchanged during the reaction, as confirmed by thin-layer chromatography (TLC) monitoring. Additionally, the presence of the Ph group in 3e activated the compound towards Beckmann rearrangement, leading to the formation of 2-phenylbenzoxazole as the main product. In order to enhance the yield, an alternative route was carried out for the transformation of 2e, involving an imine formation (5e) with ammonia and the following chlorination/dehydrohalocyclization by N-chlorosuccinimide (NCS)/K2CO3 [39]. In this case, steric factors do not impede the cyclization to 4e, and a higher yield (60%) was obtained. (Scheme 1). Unfortunately, this latter method did not work for the conversion of the trifluoromethyl derivative 2f.
For the synthesis of two additional heterocyclic derivatives (4h and 4i), E1 was used as a starting material (Scheme 2). After regioselective Friedel-Crafts acylation with TFAA and a subsequent haloform reaction of 6a [40], an E2-salicylic acid domain-integrated hybrid (6b) was produced. This compound was next converted to a hydroxamic acid derivative of E2 (7) by a three-step sequence involving C-17 ketone reduction, methylester formation of the acid moiety [41] and the final nucleophilic acyl substitution with NH2OH [42]. Mitsunobu-triggered heterocyclization in anhydrous THF at RT [43] afforded 3′hydroxybenzisoxazole 4h in 89% yield, whereas its O-methylation furnished 4i in 82% yield (Scheme 2). In the following, additional benzisoxazole derivatives were synthesized by FGI of the pre-formed substituted heteroring (Scheme 3). Reduction of the previously obtained 17-O-protected 3′-methyl ester (4g) with NaBH4 in MeOH gave 4j. Deacetylation of 17-OH led to 4k, whereas conversion of 4j with diethylaminosulfur trifluoride (DAST) in DCM and subsequent deprotection afforded the fluoromethylated product 4m in high yield. On the other hand, 4j was mildly oxidized with Dess-Martin periodinane (DMP) to 4n, which was then transformed to 4p by nucleophilic fluorination followed by deacetylation of 4o. Interestingly, treatment of 4n with LiOH or NaOH initiated not only deacetylation but also formic acid elimination accompanied by heteroring opening to give 2-cyano-E2 (8). The same product (8) was also obtained from 4a by Kemp elimination [33] or from 4q by decarboxylation under the influence of heat and/or basic medium. Otherwise, the latter product (4q) was prepared by alkaline hydrolysis of the two ester functionalities in 4g at In the following, additional benzisoxazole derivatives were synthesized by FGI of the pre-formed substituted heteroring (Scheme 3). Reduction of the previously obtained 17-O-protected 3 -methyl ester (4g) with NaBH 4 in MeOH gave 4j. Deacetylation of 17-OH led to 4k, whereas conversion of 4j with diethylaminosulfur trifluoride (DAST) in DCM and subsequent deprotection afforded the fluoromethylated product 4m in high yield. On the other hand, 4j was mildly oxidized with Dess-Martin periodinane (DMP) to 4n, which was then transformed to 4p by nucleophilic fluorination followed by deacetylation of 4o. Interestingly, treatment of 4n with LiOH or NaOH initiated not only deacetylation but also formic acid elimination accompanied by heteroring opening to give 2-cyano-E2 (8). The same product (8) was also obtained from 4a by Kemp elimination [33] or from 4q by decarboxylation under the influence of heat and/or basic medium. Otherwise, the latter product (4q) was prepared by alkaline hydrolysis of the two ester functionalities in 4g at RT. Starting from 4q, two additional derivatives (4r and 4s) were also synthesized by FGI. The preparation of the 3 -amino-benzisoxazole-E2 hybrid (4t) proved to be the most difficult challenge. Since the DDQ/PPh 3 -induced cyclization of amidoxime 9 obtained from 8 failed and 2-amino-oxazole 10 was formed instead of the desired isoxazole 4t by Tiemann rearrangement, the Curtius rearrangement of the acyl azide available from 4q with diphenylphosphorylazide (DPPA) in refluxing toluene was tried to carry out. Nevertheless, unfortunately, this reaction did not lead to success either. The reaction did not proceed even to the formation of the azide, so the preparation of the amino-substituted derivative (4t) was discarded.
Structural determination of the novel steroidal A-ring-fused isoxazoles (4a-s) was accomplished using 1 H NMR, 13 C NMR (J-mod) and MS measurements. The fact of the cyclization was confirmed by the disappearance of the proton signal of the phenolic OH Molecules 2022, 27, 7456 6 of 26 group in the proton spectra, and in the case of the 3 -substituted derivatives (4b-s) by the negative peak of the three hydrogen-free, sp 2 -hybridized carbon atoms (C-2, C-3, and C-3 ). For the unsubstituted isoxazole 4a, the peak of 3 -H was detected at 8.60 ppm, whereas C-3 was observed as a positive signal at 146.2 ppm. The measured molecular masses were in good agreement with those calculated from the structures.
Molecules 2022, 27, x FOR PEER REVIEW 6 of RT. Starting from 4q, two additional derivatives (4r and 4s) were also synthesized by FG The preparation of the 3′-amino-benzisoxazole-E2 hybrid (4t) proved to be the mo difficult challenge. Since the DDQ/PPh3-induced cyclization of amidoxime 9 obtaine from 8 failed and 2-amino-oxazole 10 was formed instead of the desired isoxazole 4t b Tiemann rearrangement, the Curtius rearrangement of the acyl azide available from 4 with diphenylphosphorylazide (DPPA) in refluxing toluene was tried to carry ou Nevertheless, unfortunately, this reaction did not lead to success either. The reaction di not proceed even to the formation of the azide, so the preparation of the amino-substitute derivative (4t) was discarded. Structural determination of the novel steroidal A-ring-fused isoxazoles (4a-s) wa accomplished using 1 H NMR, 13 C NMR (J-mod) and MS measurements. The fact of th cyclization was confirmed by the disappearance of the proton signal of the phenolic O group in the proton spectra, and in the case of the 3′-substituted derivatives (4b-s) by th negative peak of the three hydrogen-free, sp 2 -hybridized carbon atoms (C-2, C-3, and C 3′). For the unsubstituted isoxazole 4a, the peak of 3′-H was detected at 8.60 ppm, wherea

Pharmacological Studies
With the newly synthesized estradiol-A-ring-integrated benzisoxazole derivatives in our hand, we set off to investigate their in vitro anticancer activity. First, all compounds were solubilized in cell culture grade dimethyl sulfoxide (DMSO) at a final concentration of either 2.5, 5 or 10 mM, respectively, depending on the solubility. Then, each compound was subjected to a preliminary toxicity screen on prostate cancer (DU-145, PC3), cervical cancer (HeLa) and MCF-7 breast cancer cell lines. The non-cancerous MRC-5 cells were also incorporated into the tests to determine the cancer-selective antiproliferative effect of the synthesized molecules. Cells of each cell line were incubated for 72 h with the compounds applied at 2.5 µM concentration, then MTT cell viability assays were carried out. The results of the viability screen (Supplementary Material p51) are shown as a heatmap ( Figure 2). of either 2.5, 5 or 10 mM, respectively, depending on the solubility. Then, each compound was subjected to a preliminary toxicity screen on prostate cancer (DU-145, PC3), cervical cancer (HeLa) and MCF-7 breast cancer cell lines. The non-cancerous MRC-5 cells were also incorporated into the tests to determine the cancer-selective antiproliferative effect of the synthesized molecules. Cells of each cell line were incubated for 72 h with the compounds applied at 2.5 μM concentration, then MTT cell viability assays were carried out. The results of the viability screen (Supplementary Material p51.) are shown as a heatmap ( Figure 2).  The cytotoxicity screen resulted in a great number of positive hits, whereas the estradiol derivatives exhibited strong antiproliferative activity on cancer cells, in particular, HeLa cells were highly sensitive to the treatments. Furthermore, most compounds presented outstanding cancer cell-selective performance by showing remarkable activity on cancerous cell lines, but no or negligible effect on non-cancerous fibroblasts. To validate these findings, we selected the three most promising derivatives, namely 4b, 4c and 4d (3 -methyl, 3ethyl, and 3 -isopropyl substituted steroidal benzisoxazoles), and examined their anticancer efficiency more thoroughly. We assessed the minimal inhibitory concentration (IC 50 ) of the compounds by treating DU-145, HeLa, MCF-7 and MRC-5 cells with either 4b, 4c or 4d in 1, 2, 3, 4, 5, 6, 8 and 10 µM concentrations for 72 h and for the viability data, dose-response curves were fitted (Supplementary Material, p52-53) and IC 50 values were calculated accordingly (Table 1). For comparison, the IC 50 values of cisplatin on the same cell lines are also included [24].
These results agreed with the primary cytotoxicity screen (Figure 2), as the obtained IC 50 concentrations verified that the tested compounds were selectively effective and very potent on every cancer cell line involved in the examination. The IC 50 values of the molecules were at least one but sometimes even two magnitudes higher on non-cancerous MRC-5 cells than on malignant cells. Interestingly, each estradiol-benzisoxazole hybrid was more effective on the three cancer cell lines than the classic chemotherapy drug cisplatin. In fact, cisplatin affected the viability of non-cancerous fibroblasts similarly to steroids; nevertheless, it exhibited significantly weaker antiproliferative capacity on cancer cells than the steroidal chimeras. In summary, potent and tumour cell-selective compounds were found on breast, cervical and prostate cancer cell lines among these E2benzisoxazole hybrids with 3 -methyl (4b), 3 -ethyl (4c) and 3 -isopropyl (4d) substitution, i.e., this alkyl substitution of the heterocycle proved to be favourable in terms of biological effect and selectivity. Cisplatin values according to our publication [24].
Lastly, to delineate the possible mechanism of cytotoxicity induced by these novel steroidal heterocycles, we evaluated the apoptosis-inducing potential of the selected three compounds. Most cancer cells try to evade apoptosis and avoid undergoing this form of programmed cell death, which is often the reason behind the ineffectiveness of chemotherapy. Thus, examination of the apoptosis-triggering capacity of newly synthesized anticancer agents is imperative. For this, DU-145, HeLa, and MCF-7 cells were treated with either 4b, 4c or 4d in different concentrations for 72 h. After treatments, total RNA was isolated, reverse transcribed into cDNA and the relative expression levels of some key apoptotic marker genes (BAX, Casp-3, p21, p53) were measured by real-time qPCR (Figure 3). Since MCF-7 cells are known to be deficient in functional caspase-3 due to a deletion in exon 3 of the gene [44], we did not examine the relative transcript levels of this gene in MCF-7 breast cancer cells. In accordance with the cytotoxicity data, we found that exposure of cancer cells to any of the three E2-benzisoxazole hybrids induced significant alterations in the expression profile of the examined pro-apoptotic genes (Figure 3.). The most sensitive marker was p21 since massive upregulation of p21 expression was observable on each cancer cell line In accordance with the cytotoxicity data, we found that exposure of cancer cells to any of the three E2-benzisoxazole hybrids induced significant alterations in the expression profile of the examined pro-apoptotic genes ( Figure 3). The most sensitive marker was p21 since massive upregulation of p21 expression was observable on each cancer cell line following treatments. Of the three cancerous cell types, DU-145 cells were the most affected, as increased transcript levels of every pro-apoptotic marker, i.e., p21, BAX, Casp-3 as well as p53 were also visible in the case of DU-145 cells.

General
Chemicals, reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich and Alfa Aesar) and used without further purification. Amylene-stabilized dichloromethane was used for the Friedel-Crafts acylations, cyclizations and the Dess-Martin oxidation as solvent. Melting points (Mps) were determined on an SRS Optimelt digital apparatus and are uncorrected. The transformations were monitored by TLC using 0.25 mm thick Kieselgel-G plates (Si 254 F, Merck). The compound spots were detected by spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid. Purifications by column chromatograpy (CC) were carried out on silica gel 60, 40-63 µm (Merck) using flash mode. Elementary analysis data were obtained with a PerkinElmer CHN analyzer model 2400. NMR spectra were recorded with a Bruker DRX 500 instrument at RT in CDCl 3 or DMSO-d 6 using residual solvent signals as an internal reference. Chemical shifts are reported in ppm (δ scale) and coupling constants (J) are given in Hz. Multiplicities of the 1 H signals are indicated as a singlet (s), a broad singlet (bs), a doublet (d), a doublet of doublets (dd), a doublet of triplets (dt), a triplet (t), a triplet of doublets (td), a quartet (q) or a multiplet (m). 13 C NMR spectra are 1 H-decoupled and the J-MOD pulse sequence was used for multiplicity editing. In this spin-echo type experiment, the signal intensity is modulated by the different coupling constants J of carbons depending on the number of attached protons. Both protonated and unprotonated carbons can be detected (CH 3 and CH carbons appear as positive signals, whereas CH 2 and C carbons appear as negative signals). The purified derivatives were dissolved in high purity acetonitrile and introduced with an Agilent 1290 Infinity II liquid chromatography pump to an Agilent 6470 tandem mass spectrometer equipped an electrospray ionization chamber. Flow rate was 0.5 mL·min −1 and contained 0.1% formic acid or 0.1% ammonium hydroxide to help facilitate ionization. The instrument operated in MS1 scan mode with 135 V fragmentor voltage, and the spectra were recorded from 300 to 500 m/z, which were corrected with the background.

General Procedure for the Synthesis of Oximes 3a-g
To a solution of 2-substituted estradiol derivative (2a-g, 1.0 mmol) in EtOH (10 mL), hydroxylamine hydrochloride and a base were added in excess and the mixture was stirred at ambient temperature for a certain period. The solvent was removed under reduced pressure, and the residue was suspended in water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic phase was washed with NH 4 Cl (1 M, 10 mL), water (10 mL) and brine (10 mL), and then dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The crude product was purified by CC.

Three-Step Synthesis of Compound 7
Synthesis of 3,17β-dihydroxyestra-1,3,5(10)-triene-2-carboxylic acid (6c) by the reduction of 6b: To a solution of 6b (1.05 g, 3.34 mmol) in EtOH (30 mL), NaBH 4 (631 mg, 5 equiv.) was added in small portions over a 10 min period and stirring was continued for 30 min at RT. The mixture was neutralized with HCl (6 M) and the EtOH was removed under reduced pressure. To the residue HCl (1 M, 10 mL) was added and extracted with EtOAc (3 × 10 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The crude product was purified by CC (EtOAc/DCM = 20:80 with 1% AcOH additive to reduce tailing). Yield (6c) Synthesis of 3,17β-dihydroxyestra-1,3,5(10)-triene-2-carboxylic acid methyl ester (6d) by esterification of 6c: To a solution of 6c (950 mg, 3.00 mmol) in DMF (10 mL), Na 2 CO 3 (382 mg, 1.2 equiv.) and MeI (280 µL, 1.5 equiv.) were added and the mixture was stirred at 50 • C for 2 h. Then, it was repeatedly concentrated under vacuum with the addition of toluene, and then water (10 mL) was added and extracted with EtOAc (3 × 10 mL). The combined organic phase was washed with aq. NaHCO 3 (10 wt. %, 2 × 10 mL), water (10 mL) and brine (10 mL), and then dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The crude product was purified by CC (EtOAc/DCM = 5:95  3.70-3.77 (1H, t, J 8.5, 17-αH) antiproliferative activity of the obtained compounds provided numerous positive hits, several derivatives exhibited strong anticancer performance, and most estradiol-benzisoxazole hybrids showed remarkable cancer cell selectivity. The three most promising compounds, the 3 -methyl, 3 -ethyl and 3 -isopropyl substituted steroidal benzisoxazoles showed a high degree of cytotoxicity on all tested cancerous cell lines, whereas treatment of non-cancerous cells with these derivatives resulted in no, or minimal change in cell viability. The minimal inhibitory concentrations (IC 50 ) of the three compounds were determined on cancerous and non-cancerous cell lines. Interestingly, the IC 50 values of each molecule were one or two magnitudes lower for the cancerous cell lines compared to the values obtained on non-cancerous fibroblasts. The IC 50 values of the most potent derivatives were compared to that of cisplatin, a clinically available drug, where we found that unlike cisplatin, estradiolbenzisoxazole hybrids show an outstanding cancer cell specificity. Lastly, we found that each of the three compounds exhibit strong apoptosis inducing potential, which could be the underlying cause of their impressive anticancer performance. Based on our findings, estradiol-benzisoxazole hybridization seems to be exceptionally advantageous for securing excellent anticancer activity; therefore, this structural motif should be considered in rational drug design and future synthesis approaches for clinical cancer therapy. The mechanism of action of the most potent steroidal hybrids as well as their hormone receptor binding will be further investigated.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27217456/s1, p2-49: 1 H NMR and 13 C NMR spectra of the synthesized compounds, Table in p50: Predicted pharmacokinetic parameters of synthesized compounds, Table in