Multistep Synthesis and In Vitro Anticancer Evaluation of 2-Pyrazolyl-Estradiol Derivatives, Pyrazolocoumarin-Estradiol Hybrids and Analogous Compounds

Although the hormone independent cytotoxic activity of several estradiol derivatives endowed with a simple substituent at C-2 has been reported so far, 2-heterocyclic and 2,3-condensed analogs are less investigated from both synthetic and pharmacological points of view. Therefore, novel A-ring-connected 2-pyrazoles of estradiol and, for comparison, their structurally simplified non-steroidal pairs were synthesized from estradiol 3-methyl ether and 6-methoxy-1,2,3,4-tetrahydronaphthalene. Friedel-Crafts acetylation of the protected phenolic compounds and subsequent O-demethylation led to ortho-substituted derivatives regioselectively, which were converted to arylhydrazones with phenylhydrazine, 4-tolylhydrazine and 4-chloro-phenylhydrazine, respectively, under microwave conditions. The hydrazones were subjected to cyclization with the Vilsmeier-Haack reagent immediately after preparation and the ring closure/formylation sequence resulted in steroidal and non-steroidal 4′-formylpyrazoles in moderate to good yields. During reductive transformations, 4-hydroxymethyl-pyrazoles were obtained, while oxidative lactonization of the 4-formylpyrazole moiety with the phenolic OH in the presence of the Jones reagent afforded A-ring-integrated pyrazolocoumarin hybrids and related analogs. Steroidal pyrazoles, which were produced as C-17 acetates due to acetylation of C-17 OH during the primary Friedel-Crafts reaction, underwent deacetylation in alkaline methanol to furnish 2-heterocyclic estradiol derivatives. Pharmacological studies revealed the overall and cancer cell-specific cytotoxicity of the derivatives and the half maximal inhibitory concentrations were obtained for the most promising compounds.


Introduction
Natural steroids play an important role in several fields of medicine, including contraception, treatment of inflammation, asthma, cardiovascular disease, osteoporosis, cancer, and other disorders [1,2]. During chemical modifications, steroids are often combined with other drugs or relevant pharmacophores via covalent bonds either acting as an effective agent or inactive carrier, or through domain integration of key structural units [3,4]. These multifunctional hybrid drugs or In recent years, considerable attention has been devoted to the synthesis of sex hormone-derived pyrazoles, which have proved to be the most promising of all heterocyclic derivatives for inhibiting one of the enzymes of steroidogenesis or influencing the cell cycle and inducing apoptosis in tumor cells [16,[25][26][27][28][29][30]. The introduction of such a structural element into the 2-position of estradiol may open new perspectives in the research of anticancer agents, as the pyrazole ring is a very common building block in non-steroidal anticancer compounds as well [31,32]. In addition, the incorporation of a formyl functionalized pyrazole moiety allows intramolecular cyclization with the phenolic 3-OH group of the A-ring, leading to domain-integrated pyrazolocoumarin-estradiol hybrid compounds. Both coumarins and pyrazolocoumarins are valuable heterocyclic units in a number of marketed drugs and experimental agents with anticancer, antimicrobial, and anti-inflammatory activities [33][34][35].
Given the aforementioned literature background, the aim of the current study was to develop an efficient synthetic route for the preparation of novel pyrazole-containing estradiol derivatives by modifying the phenolic A-ring, and thus to extend the chemical space towards the synthesis and investigation of a less studied family of compounds. Structurally similar molecules without C-and D-rings of the sterane core starting from 5,6,7,8-tetrahydro-2-naphthol were also synthesized for pharmacological comparison. To evaluate the anticancer efficiency of the new derivatives, their cytotoxic activity was screened in vitro on two prostate (DU145, PC-3) and two gynecological (HeLa, MCF-7) cell lines as well as on non-cancerous fibroblasts (MRC-5) using MTT assays [36]. Several compounds showing cancer cell-specific cytotoxicity were identified and the half maximal inhibitory concentrations for the most promising compounds were finally determined.

Syntheses
For the synthesis of A-ring-modified pyrazole derivatives, 2-acetyl estradiol 17β-acetate (4) was first prepared as starting material. Friedel-Crafts regioselective ortho-acetylation of the aromatic ring In recent years, considerable attention has been devoted to the synthesis of sex hormone-derived pyrazoles, which have proved to be the most promising of all heterocyclic derivatives for inhibiting one of the enzymes of steroidogenesis or influencing the cell cycle and inducing apoptosis in tumor cells [16,[25][26][27][28][29][30]. The introduction of such a structural element into the 2-position of estradiol may open new perspectives in the research of anticancer agents, as the pyrazole ring is a very common building block in non-steroidal anticancer compounds as well [31,32]. In addition, the incorporation of a formyl functionalized pyrazole moiety allows intramolecular cyclization with the phenolic 3-OH group of the A-ring, leading to domain-integrated pyrazolocoumarin-estradiol hybrid compounds. Both coumarins and pyrazolocoumarins are valuable heterocyclic units in a number of marketed drugs and experimental agents with anticancer, antimicrobial, and anti-inflammatory activities [33][34][35].
Given the aforementioned literature background, the aim of the current study was to develop an efficient synthetic route for the preparation of novel pyrazole-containing estradiol derivatives by modifying the phenolic A-ring, and thus to extend the chemical space towards the synthesis and investigation of a less studied family of compounds. Structurally similar molecules without C-and D-rings of the sterane core starting from 5,6,7,8-tetrahydro-2-naphthol were also synthesized for pharmacological comparison. To evaluate the anticancer efficiency of the new derivatives, their cytotoxic activity was screened in vitro on two prostate (DU145, PC-3) and two gynecological (HeLa, MCF-7) cell lines as well as on non-cancerous fibroblasts (MRC-5) using MTT assays [36]. Several compounds showing cancer cell-specific cytotoxicity were identified and the half maximal inhibitory concentrations for the most promising compounds were finally determined.

Syntheses
For the synthesis of A-ring-modified pyrazole derivatives, 2-acetyl estradiol 17β-acetate (4) was first prepared as starting material. Friedel-Crafts regioselective ortho-acetylation of the aromatic ring of estradiol 3-methyl ether (2), obtained from its precursor 1 by simple reduction, was carried out under mild conditions using a one-pot procedure, further developing the available literature method [37] (Scheme 1). Estradiol was previously reported to undergo diacetylation during treatment with acetyl chloride (AcCl), and AlCl 3 was found not to be a strong enough Lewis acid to initiate a Fries rearrangement of the resulting diacetate to compound 4; thus, the more expensive and moisture-sensitive ZrCl 4 had to be used [38,39]. However, it was also demonstrated by Bubert et al. that the acetylation could be performed in the presence of AlCl 3 without difficulty when the phenolic OH of estradiol was protected as a methyl ether (2) [37]. Thanks to the strong ortho-directing effect of the OMe group and because of the sterically less hindered character of C-2 over C-4, the aromatic electrophilic substitution occurred regioselectively to furnish a 3-protected 2-acetyl derivative (3) in good yield. After purification, the aromatic methyl ether (3) was cleaved in boiling dichloromethane (DCM) using a chloroaluminate ionic liquid reagent [37]. According to our observation, however, the acetylation/deprotection sequence can be carried out from 2 with AlCl 3 in a single step without isolating the 2-acetyl-3-methyl ether intermediate (3) (Scheme 1). Apart from the key role of low reaction temperature during acetylation in order to access high regioselectivity, the amount of the Lewis acid used was found to be of crucial importance; i.e., a twofold molar excess of AlCl 3 compared to the steroid favored the rapid formation and isolation of 3, while a fourfold excess was needed for complete conversion of 2 to 4 within 3 h at 0→25 • C. In contrast to the two-step transformation, which required two purification steps and an elevated temperature during deprotection, leading to the desired product (4) in a ca. 74% yield, our one-pot method proceeded under milder conditions in a shorter time, resulting in 4 in 80% yield after recrystallization.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 20 of estradiol 3-methyl ether (2), obtained from its precursor 1 by simple reduction, was carried out under mild conditions using a one-pot procedure, further developing the available literature method [37] (Scheme 1). Estradiol was previously reported to undergo diacetylation during treatment with acetyl chloride (AcCl), and AlCl3 was found not to be a strong enough Lewis acid to initiate a Fries rearrangement of the resulting diacetate to compound 4; thus, the more expensive and moisturesensitive ZrCl4 had to be used [38,39]. However, it was also demonstrated by Bubert et al. that the acetylation could be performed in the presence of AlCl3 without difficulty when the phenolic OH of estradiol was protected as a methyl ether (2) [37]. Thanks to the strong ortho-directing effect of the OMe group and because of the sterically less hindered character of C-2 over C-4, the aromatic electrophilic substitution occurred regioselectively to furnish a 3-protected 2-acetyl derivative (3) in good yield. After purification, the aromatic methyl ether (3) was cleaved in boiling dichloromethane (DCM) using a chloroaluminate ionic liquid reagent [37]. According to our observation, however, the acetylation/deprotection sequence can be carried out from 2 with AlCl3 in a single step without isolating the 2-acetyl-3-methyl ether intermediate (3) (Scheme 1). Apart from the key role of low reaction temperature during acetylation in order to access high regioselectivity, the amount of the Lewis acid used was found to be of crucial importance; i.e., a twofold molar excess of AlCl3 compared to the steroid favored the rapid formation and isolation of 3, while a fourfold excess was needed for complete conversion of 2 to 4 within 3 h at 0→25 °C . In contrast to the two-step transformation, which required two purification steps and an elevated temperature during deprotection, leading to the desired product (4) in a ca. 74% yield, our one-pot method proceeded under milder conditions in a shorter time, resulting in 4 in 80% yield after recrystallization. After optimization, the synthesis of the 2-acetylestradiol derivative (4) was repeated on a larger scale without significant changes to serve as starting material for heterocycle formation. Since the hydrazones of methyl ketones are suitable precursors of pyrazole-4-carbaldehydes upon treatment with the Vilsmeier-Haack reagent [26,40], compound 4 was next reacted with three arylhydrazines, namely phenylhydrazine, 4-tolylhydrazine, and 4-chlorophenylhydrazine, respectively, differing in After optimization, the synthesis of the 2-acetylestradiol derivative (4) was repeated on a larger scale without significant changes to serve as starting material for heterocycle formation. Since the hydrazones of methyl ketones are suitable precursors of pyrazole-4-carbaldehydes upon treatment with the Vilsmeier-Haack reagent [26,40], compound 4 was next reacted with three arylhydrazines, namely phenylhydrazine, 4-tolylhydrazine, and 4-chlorophenylhydrazine, respectively, differing in the electron demand of their substituents on the aromatic ring (Scheme 1). The reagents were liberated from their stable hydrochloride salts with NaOAc, and the ethanolic mixtures were irradiated by microwave (MW) at 100 • C. Since the carbonyl-C of the acetophenone moiety has a diminished reactivity toward nucleophilic attack due to steric and electronic reasons, hydrazone formation was found to be very sluggish under conventional heating, and only moderate conversion was achieved during 5 h of reflux. However, the MW-assisted condensations took place within 30 min and the crystalline hydrazones (6a-c) could be filtered off from the mixtures in yields around 75% (Table 1, entries 1-3); higher conversions could not be gained in these cases even by increasing the reaction time. Although the nucleophilicity of the terminal nitrogen of the arylhydrazines is definitely affected by the electron-donating CH 3 group or electron-withdrawing Cl atom on the aromatic ring, a substituent effect influencing the yield of the products could not be observed under MW conditions. Table 1. Synthesis of arylhydrazones from steroidal and non-steroidal methyl ketones and their conversion to 4-formylpyrazoles.

Entry
Starting The crude hydrazones were next treated with the Vilsmeier-Haack reagent generated in situ from dimethylformamide (DMF) and POCl 3 at 0 • C to afford 1-arylpyrazole-4-carbaldehydes (7a-c) in 72-78% yields via ring closure and simultaneous incorporation of two carbon atoms from the reagent [40] (Scheme 1 and Table 1, entries 1-3). The transformations were carried out at 60 • C for 3 h for 6a and 6b, while 4 h were needed for the conversion of 6c. Considering the proposed mechanism of the Vilsmeier-Haack reaction of hydrazones [41], the lower reactivity of 6c may be attributed to the electron-withdrawing effect of the Cl atom, which endows a molecule with reduced nucleophilic character against electrophilic attack. Thereafter, oxidative lactonization of 7a-c with the Jones reagent in acetone occurred rapidly to give pyrazolocoumarin steroid hybrids 9a-c, while reductive transformations led to 10a-c in good yields. Moreover, intramolecular ether formation was also tried for 10a under different conditions in order to obtain pyrazolopyran 12a; however, all synthetic efforts failed. Contrarily, 17-OH analogs of 4, 7a-c, and 10a-c were synthesized without difficulty in alkaline methanol. Since similar deacetylation of lactone 9a did not result in a single product due to partial ring-opening and parallel methyl ester formation, the idea of preparing 17-OH derivatives was discarded.
To gain insights into the synthetic and pharmacological differences and to find structure-activity relationships, the reaction sequence described above was also performed with 5,6,7,8-tetrahydro-2-naphthol (13), a simplified molecule that structurally mimics the A-and B-rings of the estrane backbone (Scheme 2). Friedel-Crafts acetylation of the protected starting material (14) occurred regioselectively to furnish 15 [42] in 69% yield after purification. In contrast to the similar reaction of the acetylated steroid 4, the subsequent condensation reactions with arylhydrazines did not result in isolable hydrazones, although high conversions were observed by TLC monitoring; therefore, the intermediates (16a-c) were subjected to 4-formylpyrazole formation under Vilsmeier-Haack conditions. Consequently, the yield of products 17a-c could only be calculated from the starting material (15) for the two consecutive steps. Although both transformations proceeded well, lower pyrazole yields (34-41%) were obtained compared to the reactions on steroids (54-61% for 4) due to the impossibility of removing excess reagents after the first step, the difficulty of purification, and the formation of byproducts due to the longer reaction times needed for heterocyclizations (Table 1, entries 4-6). Reduction of the formyl group in the heteroaromatic ring of 17a-c with NaBH 4 in EtOH furnished hydroxymethyl-substituted derivatives 18a and 18b in good yields (>80%), while the presence of the electron-withdrawing Cl atom in 17c allowed the formation of 18c only in a moderate yield (66%). A similar substituent effect, albeit to a lesser extent, was also observed upon conversion of 7c to 10c. The behavior of 17a-c under oxidative conditions was also found to differ from that of the analogous steroidal compounds (7a-c). The low solubility of 17a-c in acetone at room temperature required heating of the mixtures, which led to the formation of unidentified byproducts, with reduced yields of the desired lactones 19a-c.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 20 entries 4-6). Reduction of the formyl group in the heteroaromatic ring of 17a-c with NaBH4 in EtOH furnished hydroxymethyl-substituted derivatives 18a and 18b in good yields (>80%), while the presence of the electron-withdrawing Cl atom in 17c allowed the formation of 18c only in a moderate yield (66%). A similar substituent effect, albeit to a lesser extent, was also observed upon conversion of 7c to 10c. The behavior of 17a-c under oxidative conditions was also found to differ from that of the analogous steroidal compounds (7a-c). The low solubility of 17a-c in acetone at room temperature required heating of the mixtures, which led to the formation of unidentified byproducts, with reduced yields of the desired lactones 19a-c. Structural analyses of all synthesized compounds were carried out by NMR and ESI-MS measurements. Hydrazones 6a-c and 16a-c were not characterized but were transformed immediately after formation due to their instability and/or difficulty of isolation. However, the signals in the deshielded region (>6.8 ppm) of the 1 H NMR spectra of 7a-c and 17a-c confirmed the 2,3-disubstituted character of the condensed aromatic A-ring (two singlets for 1-H and 4-H and the appearance of the phenolic OH), the heterocyclization (a singlet for 5′-H at around 8.5 ppm), and the incorporation of a formyl group into the pyrazole moiety (4′-CHO peak at around 10.2 ppm) under the Vilsmeier-Haack conditions ( Table 2). The 13 C NMR spectra and the determined molecular weights also provided evidence for the chemical structures. Reduction of the formyl group by NaBH4 led to derivatives (10a-c and 18a-c) containing a hydroxymethyl group instead of a formyl group on their heteroaromatic ring, so instead of a signal of formyl proton, a singlet corresponding to two equivalent protons (CH2OH) could be observed around 4.86 ppm in the 1 H NMR spectra. Oxidative lactonization is associated with the disappearance of both CHO and phenolic OH proton signals in 9a-c and 19a-c, while a negative carbonyl signal around 158.5 ppm in the 13 C NMR spectra (J-MOD) confirmed the cyclic ester formation. Structural analyses of all synthesized compounds were carried out by NMR and ESI-MS measurements. Hydrazones 6a-c and 16a-c were not characterized but were transformed immediately after formation due to their instability and/or difficulty of isolation. However, the signals in the deshielded region (>6.8 ppm) of the 1 H NMR spectra of 7a-c and 17a-c confirmed the 2,3-disubstituted character of the condensed aromatic A-ring (two singlets for 1-H and 4-H and the appearance of the phenolic OH), the heterocyclization (a singlet for 5 -H at around 8.5 ppm), and the incorporation of a formyl group into the pyrazole moiety (4 -CHO peak at around 10.2 ppm) under the Vilsmeier-Haack conditions ( Table 2). The 13 C NMR spectra and the determined molecular weights also provided evidence for the chemical structures. Reduction of the formyl group by NaBH 4 led to derivatives (10a-c and 18a-c) containing a hydroxymethyl group instead of a formyl group on their heteroaromatic ring, so instead of a signal of formyl proton, a singlet corresponding to two equivalent protons (CH 2 OH) could be observed around 4.86 ppm in the 1 H NMR spectra. Oxidative lactonization is associated with the disappearance of both CHO and phenolic OH proton signals in 9a-c and 19a-c, while a negative carbonyl signal around 158.5 ppm in the 13 C NMR spectra (J-MOD) confirmed the cyclic ester formation.

Evaluation of In Vitro AnticancerActivity
Cytotoxicity screens were performed to assess the in vitro anticancer activity of the synthesized A-ring-linked 2-pyrazoles of estradiol, the pyrazolocoumarin-estradiol hybrids, and their structurally simplified non-steroidal analogs on various cancerous cell lines such as MCF-7, PC-3, DU145, and HeLa and on non-cancerous MRC-5 fibroblasts. The 2-acetyl-phenol starting materials (4, 5, and 15) were also included for comparison. All compounds were applied in 2.5 µ M concentration for 72 h. The obtained cell viability data were used to construct a heat map (Figure 2) in order to select compounds that exert cancer cell line-specific cytotoxicity. Overall, 10-15 potential molecules (steroids and non-steroids) were found to be competent against cisplatin-resistant PC-3 and a similar number of compounds were active on HeLa cells ( Figure 2). Furthermore, some compounds discriminated only DU145 cells and could eliminate these type of prostate cancer cells.

Evaluation of In Vitro AnticancerActivity
Cytotoxicity screens were performed to assess the in vitro anticancer activity of the synthesized A-ring-linked 2-pyrazoles of estradiol, the pyrazolocoumarin-estradiol hybrids, and their structurally simplified non-steroidal analogs on various cancerous cell lines such as MCF-7, PC-3, DU145, and HeLa and on non-cancerous MRC-5 fibroblasts. The 2-acetyl-phenol starting materials (4, 5, and 15) were also included for comparison. All compounds were applied in 2.5 µM concentration for 72 h. The obtained cell viability data were used to construct a heat map (Figure 2) in order to select compounds that exert cancer cell line-specific cytotoxicity. Overall, 10-15 potential molecules (steroids and non-steroids) were found to be competent against cisplatin-resistant PC-3 and a similar number of compounds were active on HeLa cells ( Figure 2). Furthermore, some compounds discriminated only DU145 cells and could eliminate these type of prostate cancer cells.
Based on the heat map, two steroidal 4-formylpyrazoles (7c and 8a), the reduced derivative of the latter compound (11a), and some tetrahydronaphthol-derived molecules (15, 17a, 17c, 18b, and 19a) were identified that exhibited a minimum of 40% cytotoxicity on at least one cancer cell line and had no or very mild effect on non-cancerous fibroblasts. To understand structure-function relationships, we also included the structural analogs of the selected compounds, namely 7a (C-17 acetate of 8a), 8c (C-17 OH counterpart of 7c), and 9a (steroidal pair of 19a) into subsequent analyses. All these 11 compounds (7a, 7c, 8a, 8c, 9a, 11a, 15, 17a, 17c, 18b, and 19a) were further examined to determine their IC 50 concentrations on all the cell lines previously mentioned and their efficacy was compared to the reference drug cisplatin. For this, compounds were applied on MCF-7, PC-3, DU145, and HeLa and on non-cancerous MRC-5 fibroblasts in various concentrations for 72 h or were treated with cisplatin at different concentrations. On the viability data, dose-response curves were fitted ( Figure S1) and IC 50 values were calculated accordingly (Table 3). In agreement with the primary cytotoxicity screen (Table S1), the obtained IC 50 concentrations clearly indicated which compound was selectively effective on one or more cancer cell lines. Non-steroid small molecules 15 and 19a were very potent and selective on HeLa or DU145 cells, respectively; however, the steroidal compounds 8a and 8c exhibited a similar selective effect on PC-3 and on HeLa cells, respectively. On the other hand, we identified compounds, like 7c, which were effective on three cancer cell lines, i.e., on PC-3, DU145, and MCF-7 cells, in significantly lower concentrations than cisplatin, albeit affecting non-cancerous fibroblasts as well. When the activity of structurally related molecules was compared, we realized that often the simplified small molecules themselves were able to induce significant toxicity (17a, 17c); however, when the same structural motif was incorporated into an estrane backbone, the resulting compounds were either more effective on the cancer cells (8a, PC-3) or were able to target a different cell line (8c, HeLa). Typically, acetylated steroidal pyrazoles were less favorable than deacetylated counterparts due to reduced cancer cell selectivity (7c, 10a). As a whole, potent and tumor cell-selective compounds were found on cervical and prostate cancer cell lines (9a-HeLa, 11a-PC-3, and 19a-DU145), each of which is a phenyl-containing derivative on the pyrazole ring, i.e., the substitution of the aromatic ring proved to be unfavorable in terms of biological effect or selectivity. Based on the heat map, two steroidal 4-formylpyrazoles (7c and 8a), the reduced derivative of the latter compound (11a), and some tetrahydronaphthol-derived molecules (15, 17a, 17c, 18b, and  19a) were identified that exhibited a minimum of 40% cytotoxicity on at least one cancer cell line and had no or very mild effect on non-cancerous fibroblasts. To understand structure-function relationships, we also included the structural analogs of the selected compounds, namely 7a (C-17 acetate of 8a), 8c (C-17 OH counterpart of 7c), and 9a (steroidal pair of 19a) into subsequent analyses. All these 11 compounds (7a, 7c, 8a, 8c, 9a, 11a, 15, 17a, 17c, 18b, and 19a) were further examined to determine their IC50 concentrations on all the cell lines previously mentioned and their efficacy was compared to the reference drug cisplatin. For this, compounds were applied on MCF-7, PC-3, DU145, and HeLa and on non-cancerous MRC-5 fibroblasts in various concentrations for 72 h or were treated with cisplatin at different concentrations. On the viability data, dose-response curves were fitted ( Figure S1) and IC50 values were calculated accordingly (Table 3). In agreement with the primary cytotoxicity screen (Table S1), the obtained IC50 concentrations clearly indicated which compound was selectively effective on one or more cancer cell lines. Non-steroid small molecules 15 and 19a were very potent and selective on HeLa or DU145 cells, respectively; however, the steroidal compounds 8a and 8c exhibited a similar selective effect on PC-3 and on HeLa cells, respectively. On the other hand, we identified compounds, like 7c, which were effective on three cancer cell lines, i.e., on PC-3, DU145, and MCF-7 cells, in significantly lower concentrations than cisplatin, albeit affecting non-cancerous fibroblasts as well. When the activity of structurally related molecules was compared, we realized that often the simplified small molecules themselves were able to induce significant toxicity (17a, 17c); however, when the same structural motif was incorporated into an estrane backbone, the resulting compounds were either more effective on the cancer cells (8a, PC-3) or were

General
Chemicals, reagents, and solvents were purchased from commercial suppliers ( Automated flow injection analyses were performed with an HPLC/MSD system. System accessories: a micro-well plate autoinjector, an Agilent 1100 micro vacuum degasser (Agilent Technologies, Santa Clara, CA, USA), a quaternary pump, and a 1946A MSD equipped with an electrospray ion source (ESI) operated in positive ion mode. ESI parameters were: nebulizing gas N 2 , at 35 psi; drying gas N 2 , at 350 • C and 12 L/min; capillary voltage 3000 V; fragmentor voltage 70 V. The MSD was operated with a mass range of m/z 60−620 in scan mode. Samples (0.2 µL) were injected directly into the solvent flow (0.3 mL/min) of acetonitrile/H 2 O = 70:30 (v/v) with the simultaneous addition of 0.1% formic acid with an automated needle wash. Agilent LC/MSD Chemstation (C.01.08, Agilent Technologies Inc., Santa Clara, CA, USA) was used as software to control the system.

General Procedure for the Friedel-Crafts Acetylation/Demethylation of Estradiol 3-Methyl Ether
(2) or 6-Methoxy-1,2,3,4-Tetrahydronaphthalene (14) Anhydrous AlCl 3 (10.5 g, 78.6 mmol) was suspended in dry CH 2 Cl 2 (75 mL) under N 2 atmosphere, and the mixture was cooled to 0 • C. Acetyl chloride (3.1 mL, 43.6 mmol) was added slowly, then the mixture was stirred for 15 min. A solution of 2 (5 g, 17.5 mmol) or 14 (2.6g, 17.6 mmol) in dry CH 2 Cl 2 (25 mL) was added dropwise to the mixture over a period of 10 min, then stirred for 15 min at 0 • C, after which it was allowed to warm to room temperature and stirred for another 3 h. The reaction mixture was poured onto crushed ice and stirred vigorously for 10 min. The organic layer was separated, and the remaining aqueous layer was extracted with CH 2 Cl 2 (2 × 50 mL). The combined organic layers were washed with brine, dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. 2-Acetyl-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (4). The crude product was recrystallized from MeOH. Yield To a solution of 4 (713 mg, 2.0 mmol) or 15 (380 mg, 2.0 mmol) in EtOH (5 mL), anhydrous NaOAc (246 mg, 3.0 mmol) and (p-substituted) phenylhydrazine hydrochloride (3.0 mmol) were added, and the mixture was irradiated at 100 • C for 30 min in a closed tube. Hydrazones 6a-c can be obtained by chilling the reaction vessel, filtering off the yellow precipitate and washing it with ice-cold methanol. 16a-c could not be isolated; thus, the reaction mixture was evaporated to dryness and was used as-is in the next step. POCl 3 (10.73 mmol, 1.0 mL) was added to DMF (10 mL) cooled to 0 • C. After stirring for 15 min, a solution of 6a-c or 16a-c (obtained in the previous step) in DMF (5 mL) was added dropwise to the reaction mixture and was then heated to 60 • C and stirred for another 3 h (7a-c) or 16 h (17a-c). The mixture was then poured onto crushed ice, extracted with EtOAc (3 × 25 mL), and the combined organic phases were washed with brine, dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude products were purified by column chromatography with EtOAc/CH 2 Cl 2 = 1:99 (7a-c) or hexane/CH 2 Cl 2 = 2:8 (17a-c).

Pharmacology
To evaluate the in vitro pharmacological effects of the synthesized molecules, each compound was first dissolved in cell culture grade DMSO (Sigma) to a final concentration of 10 mM. Using MTT colorimetric assay [36], the cytotoxicity of these compounds was evaluated on cancerous (MCF-7, HeLa, DU145, PC-3) and non-cancerous (MRC-5) cell lines. Briefly, 5000 cells/well of each cell line were seeded in 96-well plates and were left to grow overnight in a 5% CO 2 atmosphere at 37 • C. On the next day, cells were treated with each derivative separately, in case of the primary screen, or only with the selected compounds upon IC 50 determination. Cells were incubated with the test compounds for 72 h in a 5% CO 2 atmosphere at 37 • C. End point was measured by incubating cells for 1 h with 0.5 mg/mL of MTT reagent (Serva) dissolved in serum free culture medium. The formazan crystals formed by metabolically active cells were later dissolved in DMSO. Optical density of the obtained solutions was measured at 570 nm using a Synergy HTX plate reader. For the overall screen, all compounds were used at 2.5 µM concentration; for the determination of IC 50 values, the selected compounds were applied in 1, 2, 3, 4, 5, 6, and 7 µM concentrations, and the positive control cisplatin was applied for 24 h in 20, 40, 80, 160, and 330 µM concentrations. The analysis and conclusions were made based on three independent experiments. Normalized data were analyzed and the heat map was constructed using GraphPad Prism 7 software.

Conclusions
In summary, an AlCl 3 -induced one-pot process has been developed for the regioselective ortho-acetylation/demethylation of O-protected tetracyclic steroidal and bicyclic non-steroidal phenols under mild conditions. The acetylated phenols were then successfully subjected to MW-assisted arylhydrazone formation with three arylhydrazines having different p-substituted groups, which were then treated with the Vilsmeier-Haack reagent to give pyrazolyl estradiol and tetrahydronaphthol derivatives via cyclization and subsequent formylation. The resulting 4-formylpyrazoles were further converted by reduction to give 4-hydroxymethylpyrazoles, while pyrazolocoumarins were obtained by lactonization under oxidative conditions. Significant differences were observed during the similar reactions of the steroid and the smaller analogue modelling the A-and B-rings of estradiol, and a substituent effect mainly due to the presence of an electron-withdrawing Cl atom was also noticed in certain reaction steps. Among the synthesized molecules (steroid and non-steroid), we found 10-15 potent cancer cell-selective molecules, many of them proved to be competent against HeLa, DU145, or cisplatin-resistant PC-3 cells. Structure-activity considerations suggested that several simplified small molecules were able to induce significant toxicity; however, when the same structural motif was incorporated into an estrane framework, the resulting compounds were either more effective on the cancer cells or were able to target a different cell line. Moreover, acetylated steroidal pyrazoles proved to be less advantageous than their deacetylated counterparts due to reduced cancer cell selectivity. The unsubstituted phenyl ring on the pyrazole moiety proved to be the most favorable for selective antiproliferative effect.
Supplementary Materials: The following are available online, 1 H NMR and 13 C NMR spectra of the synthesized compounds, Table S1: Mean ± SD values of primary growth inhibitory screen (given as cell viability) used to construct the heat map, Figure S1: Dose-response curves used to evaluate IC 50 concentrations of selected compounds.