Antiestrogenic Activity and Possible Mode of Action of Certain New Nonsteroidal Coumarin-4-acetamides.

The preparation of certain 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamides IIIa-h was planned as a step in the development of new modified nonsteroidal antiestrogens. The purity of target compounds IIIa-h was checked by thin-layer chromatography (TLC), and their structures were confirmed using various spectroscopic tools including IR, 1H-NMR, 13C-NMR, and MS spectroscopy. Viability tests were applied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effect of the synthesized compounds against two breast cancer cell lines, MCF-7 and MDA-MB-231. Compound IIIb proved the most active against MCF-7 cells, with an IC50 value of 0.32 μM. The results of an analysis of in vitro antiestrogenic activity indicated that only compound IIIb exhibited antiestrogenic activity; its IC50 value of 29.49 μM was about twice as potent as that of the reference compound, MIBP. The aromatase activity was evaluated for the synthesized target compounds IIIa-g and the intermediates Ib and IIa. A significant aromatase inhibition was observed for the intermediate Ib and compound IIIe, with IC50 values of 14.5 and 17.4 μM, respectively. Compound IIIb, namely 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one, could be used as an antiestrogen and/or cytotoxic agent with selective activity against tumor cells.


Introduction
Breast cancer is one of the most common and devastating cancers in women worldwide. According to researchers in the United States, the number of new cases of female breast cancer is 124.9 per 100,000 women per year [1]. In 2018, out of 5411 breast cancer cases diagnosed in Saudi Arabia, 708 patients (13.08%) died [2]. The first prognostic and predictive factor of breast cancer is related to estrogen receptors (ER) [3,4]. The development of ER antagonists has enabled successful treatment of postmenopausal women with hormone-dependent breast cancers [5]. Increased levels of estrogens are associated with tumor growth in endocrine-dependent tissues [6].
The coumarin (benzopyran-2-one, chromen-2-one,) ring system was used in research over 200 years ago. The name coumarin is derived from Coumarouna odorata Aube, from which it was isolated for the first time in 1820 [7]. Coumarins exhibit relatively low toxicity and participate in a remarkable array compounds with high oral bioavailability that have recently been tested in vitro and in vivo. These compounds display a 1.5-2.9-fold greater affinity than 17β-estradiol (E2) for the estrogen receptors in human breast cancer and normal uterine cytosol [19]. These compounds have no agonistic estrogenic activity in the in vitro human breast cancer models and in vivo in nude mice [19]. The nonsteroidal antiestrogen acolbifene (Figure 1) is the most potent antiestrogen in terms of inhibition of both ERα and ERβ [7,20,26]. This compound has a number of advantages over all other antiestrogens and should be investigated for the treatment of ER-positive breast cancer and other estrogen-sensitive malignancies [20,21]. In addition, aromatase enzyme is involved in the last step of the estrogen biosynthetic pathway [27]. A number of coumarin derivatives bearing an imidazole ring at position four have been designed and synthesized as strong and selective aromatase inhibitors (AIs) [28].
From the practical point of view, the compounds of interest contain substituted acetamide functionalities attached to the four position of coumarin nuclei, while maintaining all functionalities at their relative positions in the designed compounds. This pattern might dramatically affect the binding to the ER and subsequently the enhancement of their biological activity as anti-breast cancer agents. Unfortunately, anti-breast cancer drugs like tamoxifen (TAM) [18] achieve significant clinical results in only 30-40% of patients, because drug resistance usually develops after one or two years of treatment [25]. Among the fourth generation of SERMs, benzopyrans are potent antiestrogenic compounds with high oral bioavailability that have recently been tested in vitro and in vivo. These compounds display a 1.5-2.9-fold greater affinity than 17β-estradiol (E2) for the estrogen receptors in human breast cancer and normal uterine cytosol [19]. These compounds have no agonistic estrogenic activity in the in vitro human breast cancer models and in vivo in nude mice [19]. The nonsteroidal antiestrogen acolbifene (Figure 1) is the most potent antiestrogen in terms of inhibition of both ERα and ERβ [7,20,26]. This compound has a number of advantages over all other antiestrogens and should be investigated for the treatment of ER-positive breast cancer and other estrogen-sensitive malignancies [20,21]. In addition, aromatase enzyme is involved in the last step of the estrogen biosynthetic pathway [27]. A number of coumarin derivatives bearing an imidazole ring at position four have been designed and synthesized as strong and selective aromatase inhibitors (AIs) [28].
From the practical point of view, the compounds of interest contain substituted acetamide functionalities attached to the four position of coumarin nuclei, while maintaining all functionalities at their relative positions in the designed compounds. This pattern might dramatically affect the binding to the ER and subsequently the enhancement of their biological activity as anti-breast cancer agents.
The direct reaction of a carboxylic acid with an amine proceeds smoothly with aliphatic secondary amines like piperidine producing the target compounds IIIa,b. On the other hand, the best Scheme 1. Synthesis of the target compounds IIIa-h.
The 1 H-NMR spectra of compounds Ia,b exhibited a singlet peak at 3.77 ppm, which was assigned to methyl ester (COOCH 3 ) protons, while the disappearance of the characteristic peak at 12.81 ppm for the carboxylic acid proton was observed. 13 C-NMR spectroscopy indicated the presence of a new carbon peak at 52.68 ppm, which was assigned to be the methyl ester carbon (COOCH 3 ).
The direct reaction of a carboxylic acid with an amine proceeds smoothly with aliphatic secondary amines like piperidine producing the target compounds IIIa,b. On the other hand, the best method for the preparation of compounds IIIc-h was the use of a 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide HCl (EDCI.HCl) reagent, which was added to a mixture of the appropriate aromatic amine and the acids IIa,b in dimethyformamide (DMF).
The structures of compounds IIIa-h were confirmed by their IR spectra, in which the appearance of C-N bands at 1026 cm −1 and 1024 cm −1 indicates compounds IIIa and IIIb, respectively. The disappearance of broad -OH bands suggested the formation of a new amide bond for compounds IIIc-h. The 1 H-NMR spectra of compounds IIIa,b did not show -NH amide protons as they are tertiary amides. In addition, the 1 H-NMR spectra of compounds IIIc-h were characterized by the appearance of -NH peaks that resonate at 10.08-10.53 ppm as singlets. Moreover, the 13 C-NMR spectra of compounds IIIc-h were characterized by the presence of six new aromatic carbons in the range of 108.2-147.9 ppm. The 13 C-NMR and DEPT spectra of the compound IIIa were well resolved and confirmed the presence of all carbon atoms in the molecule. On other hand, the HSQC spectrum of the compound IIIa specified its proton-carbon coupling.

In Vitro Cytotoxicity
The biological investigation started with the evaluation of the cytotoxic activity of the intermediates Ia-b and IIa-b (Table 1). Compounds Ib and IIa had a highly cytotoxic effect against the MCF-7 cell line, with IC 50 values of 1.44 and 1.00 µM, respectively. However, only compound Ib was highly cytotoxic to the MDA-MB-231 cell line, with its IC 50 value of 1.00 µM being about 19 times more potent than the reference standard camptothecin (IC 50 value of 19.24 µM, Table 1 and Figure 2). Methyl (7-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ib) was highly cytotoxic towards both breast cancer cell lines.  Considering the cytotoxicity of N-substituted coumarin-4-acetamides IIIa-h (Table 2 and Figure  3), compound IIIb is most active against MCF-7 cells with an IC50 value of 0.32 μM, as compared with the reference cytotoxic compound, camptothecin (IC50 = 4.41 μM). In addition, compound IIIe is the most active candidate against the MDA-MB-231 cell line with an IC50 value of 2.14 μM, while camptothecin displayed an IC50 value of 19.24 μM against the same cell line. Table 2. Cytotoxicity of the synthesized N-substituted coumarin-4-acetamides IIIa-h and Considering the cytotoxicity of N-substituted coumarin-4-acetamides IIIa-h (Table 2 and Figure 3), compound IIIb is most active against MCF-7 cells with an IC 50 value of 0.32 µM, as compared with the reference cytotoxic compound, camptothecin (IC 50 = 4.41 µM). In addition, compound IIIe is the most active candidate against the MDA-MB-231 cell line with an IC 50 value of 2.14 µM, while camptothecin displayed an IC 50 value of 19.24 µM against the same cell line.  In general, 7-methoxycoumarin-4-acetamide derivatives IIIb and IIIf-h showed a greater in vitro cytotoxic effect against MCF-7 cells as compared with their positional isomers 6methoxycoumarin derivatives IIIa and IIIc-e. The most active compounds are in the following order: IIIb > IIIg > IIIa > IIId, with IC50 values of 0.32, 0.72, 1.82, and 2.80 μM, respectively. On the other hand, the results of the cytotoxic evaluation of the acetamides IIIa-h against MDA-MB-231 human breast cancer cells indicated that compounds IIIc, e, and f had a highly cytotoxic effect, with IC50 values of 4.6, 2.1, and 6.9 μM, respectively (Table 2 and Figure 3).

In Vitro Antiestrogenic Activity
The in vitro antiestrogenic activity of the selected target compounds IIIa,b, IIId, and IIIf,g was indicated that compounds IIIc, e, and f had a highly cytotoxic effect, with IC 50 values of 4.6, 2.1, and 6.9 µM, respectively (Table 2 and Figure 3).

In Vitro Antiestrogenic Activity
The in vitro antiestrogenic activity of the selected target compounds IIIa,b, IIId, and IIIf,g was studied through an estrogen-dependent human breast cancer MCF-7 cell proliferation assay in the presence of 17-β-estradiol. The ability of the tested compounds to inhibit cell proliferation induced by 17-β-estradiol was determined (Table 3 and Figure 4). One of the disadvantages of the compound SP500263 was that it had an undesirable agonist activity in the MCF-7 proliferation assay. However, among the synthesized compounds, compound IIIb is the most active, with its IC 50 value of 29.49 µM being about twice as potent as the reference compound, MIBP.

In Vitro Aromatase Inhibition
One of the most successful targeted breast cancer therapies is the inhibition of the AR enzyme, which catalyzes the rate-limiting final step of estrogen biosynthesis by modulating ER. To ensure cytotoxicity against the MCF-7 breast cancer cell line, aromatase enzymatic activity was assayed using the aromatase inhibitor (AI), letrozole, as a reference standard. Compound Ib was equipotent to letrozole (IC50 = 15.03 μM) aromatase inhibitory activity with an IC50 value of 14.5 μM (Table 4 and Figure 5).

In Vitro Aromatase Inhibition
One of the most successful targeted breast cancer therapies is the inhibition of the AR enzyme, which catalyzes the rate-limiting final step of estrogen biosynthesis by modulating ER. To ensure cytotoxicity against the MCF-7 breast cancer cell line, aromatase enzymatic activity was assayed using the aromatase inhibitor (AI), letrozole, as a reference standard. Compound Ib was equipotent to letrozole (IC 50 = 15.03 µM) aromatase inhibitory activity with an IC 50 value of 14.5 µM (Table 4 and Figure 5).

General
Melting points were determined in open glass capillaries on an electrothermal melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded for potassium bromide discs ν (cm −1 ) on an IR affinity-1s Fourier transform infrared spectrophotometer. The 1 H-NMR and 13 C-NMR spectra were determined on a Bruker (700 MHz) (Coventry, Germany) and Agilent Technologies (600 MHz) (Palo Alto, CA, USA) spectrometer. Correlation spectroscopy: 1 H, 13 9). Visualization of the spots was performed by exposure to a UV lamp at 254 nm or to iodine vapors. All statistical analyses were carried out using GraphPad Prism (San Diego, CA, US) version 6.0 software. Statistical analysis was conducted using one-way ANOVA, followed by multiple Tukey-Kramer post hoc tests at p < 0.05, which was considered a marker of statistical significance. Moreover, acetamides IIIa-g showed low or no aromatase inhibition activity (Table 4 and Figure 5), except compound IIIe, which showed moderate inhibitory activity against aromatase (IC 50 = 17.38 µM), alongside its promising activity towards the MDA-MB-231 human breast cancer cell line (IC 50 value of 2.14 µM).

General
Melting points were determined in open glass capillaries on an electrothermal melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded for potassium bromide discs ν (cm −1 ) on an IR affinity-1s Fourier transform infrared spectrophotometer. The 1 H-NMR and 13 C-NMR spectra were determined on a Bruker (700 MHz) (Coventry, Germany) and Agilent Technologies (600 MHz) (Palo Alto, CA, USA) spectrometer. Correlation spectroscopy: 1 H, 13 1:9). Visualization of the spots was performed by exposure to a UV lamp at 254 nm or to iodine vapors. All statistical analyses were carried out using GraphPad Prism (San Diego, CA, US) version 6.0 software. Statistical analysis was conducted using one-way ANOVA, followed by multiple Tukey-Kramer post hoc tests at p < 0.05, which was considered a marker of statistical significance.

General Procedure for the Synthesis of Coumarin-4-Acetic acid Derivatives IIa,b
Route A: A mixture of citric acid monohydrate (4.2 g, 20 mmol) and concentrated H 2 SO 4 (5.6 mL) was stirred at room temperature for 60 min, then slowly heated (rate of heating governed by foaming) to 70 • C. After 35 min at this temperature, with stirring throughout, the evolution of carbon monoxide had slackened, and the clear solution was rapidly cooled to 0 • C. Then the appropriate methoxyphenol (2 g, 16.1 mmol) and concentrated H 2 SO 4 (2.24 mL) were added, each in three equal portions, to the stirred solution at such a rate that the internal temperature did not exceed 10 • C. The resulting reaction mixture was stored at 0 • C for 16 h, poured into ice cold water (40 mL), and the resulting crystalline precipitate filtered off and washed thoroughly with H 2 O. The collected solid was dissolved under stirring in 1N Na 2 CO 3 solution (20 mL), heated for 15 min at 65 • C, and the insoluble matter was filtered off and washed with water (2 × 10 mL). The combined filtrate and washings were acidified with concentrated HCl to give the respective coumarin-4-acetic acid derivatives IIa,b [32].
Route B: A solution of methyl (6-methoxy-2-oxo-2H-chromen-4-yl)acetate (Ia, 1 g, 4 mmol) in ethanol (10 mL) and 0.5% NaOH (100 mL) was refluxed for 2 h. It was then cooled to room temperature, acidified with concentrated HCl to pH = 2, and cooled to 0 • C. The precipitated solid was filtered off, washed thoroughly with ethanol, and dried to give compound IIa, which was used for the next step without further purification [33,34]. The appropriate methyl (2-oxo-2H-chromen-4-yl)acetate Ia,b (0.248 g, 1 mmol) and piperidine (1 mL, 0.85 g, 1 mmol) was heated to reflux in toluene (10 mL) in the presence of a catalytic amount of p-toluene sulfonic acid (p-TSOH) for 4 h. The solvent was concentrated under reduced pressure and the precipitated solid was filtered off, washed with toluene, and dried to yield the respective target compounds IIIa,b [36].

Cytotoxicity Assay (MTT Assay)
The cytotoxicity of compounds Ia,b, IIa,b, and IIIa-h against the MCF-7 cell line (ER+ breast cancer cell line) and MDA-MB-231 (triple-negative breast cancer cell line, TNBC) was determined using camptothecin as a pyranone-bearing reference standard [37]. The detailed experimental procedures are provided in the Supplementary Materials.

Antiestrogenic Activity
The antiestrogenic activity of the test compounds was examined by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of the MCF-7 cell line. In this experiment, MCF-7 cells were treated with 17β-estradiol (+ve cell proliferation compound). The effect of various concentrations of the tested compounds on cell proliferation in the presence of 17β-estradiol was measured [37]. The detailed experimental procedures are provided in the Supplementary Materials.

Aromatase Inhibition
Sandwich enzyme immunoassay was adopted for aromatase inhibition assessment [38]. The detailed experimental procedures are provided in the Supplementary Materials.

Conclusions
In conclusion, 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamide derivatives IIIa-h have been prepared, characterized, and tested for their in vitro cytotoxic and antiestrogenic, as well as aromatase inhibition, activities. The target compounds IIIa-h showed variable cytotoxic activity against two breast cancer cell lines, MCF-7 and MDA-MB-231. 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2Hchromen-2-one (IIIb) was the most potent cytotoxic compound against MCF-7, being about 14-fold more potent than the reference standard, camptothecin. It also manifested high in vitro antiestrogenic activity (IC 50 = 29.49 µM). These findings indicate that a cyclic aliphatic lipophilic substitution in compound IIIb produced obvious antiestrogenic and cytotoxic activities. Thus, it might have high affinity to ER. Unfortunately, the tested compounds show moderate to low aromatase inhibition activity, except for compound IIIe, which showed moderate inhibitory activity against aromatase with an IC 50 value of 17.38 µM.