Synthesis and Biological Evaluation of Bicalutamide Analogues for the Potential Treatment of Prostate Cancer

The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease progresses toward androgen-independent or castration-resistant forms. In this study, a series of 15 bicalutamide analogues (sulfide, deshydroxy, sulfone, and O-acetylated) were prepared and their antiproliferative activity evaluated against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap). Bicalutamide and enzalutamide were used as positive controls. Seven of these compounds displayed remarkable enhancement in anticancer activity across the four PC cell lines. The deshydroxy analogue (16) was the most active compound with IC50 = 6.59–10.86 µM. Molecular modeling offers a plausible explanation of the higher activity of the sulfide analogues compared to their sulfone counterparts.


Introduction
The androgen receptor (AR) has essential anabolic and reproductive roles in men and women. Additionally, AR signaling plays a crucial function in tumorigenesis and metastasis of different cancer types, including prostate, bladder, kidney, lung, breast, and liver [1][2][3]. AR is a member of the nuclear receptor family and consists of three main functional domains: a variable N-terminal domain, a highly conserved DNA-binding domain (DBD), and a conserved ligand-binding domain (LBD) [4]. Binding of testosterone and dihydrotestosterone (DHT) to the LBD induces AR conformational changes then translocation into the nucleus to interact with DNA and modulate prostate-specific antigen (PSA) levels [5]. AR antagonists (antiandrogens) inhibit these processes and are used for the treatment of advanced prostate cancer (PC) [6,7]. A variety of nonsteroidal antiandrogens (NSAA) are approved for the treatment of PC. The first generation NSAAs include flutamide, hydroxyflutamide, and bicalutamide ( Figure 1). However, these antiandrogens eventually fail to inhibit the AR upon long-term treatment, switching from being AR antagonists to AR agonists with the development of castration-resistant prostate cancer (CRPC), an aggressive form of the disease with poor prognosis. Similarly, resistance to the more recent second-generation antiandrogens (enzalutamide, apalutamide) is developing in PC patients via the upregulation of AR expression [8]. More recently, darolutamide (ODM-201) has been recently approved and clinically used in patients with nonmetastatic CRPC [9]. New AR antagonists are continuously needed to improve the efficacy of the clinically used compounds.
In this paper, we present the design and synthesis of a series of new bicalutamide analogues, building on our previous work [10][11][12][13] to offer new therapeutic options for combating the resistance observed in the clinical use of AR antagonists.

Results and Discussion
Generally, minor chemical changes in the chemical structure of nonsteroidal AR ligands can result in major pharmacological outcomes [14,15]. Previously, we published extensive SAR studies on bicalutamide analogues [10][11][12][13]. Introduction of fluorinated groups into the chemical structure of molecules provides a combination of electrostatic, steric and lipophilic impacts on the physicochemical properties and biological activities [16][17][18].
Here we are exploring the impact of additional chemical structure modifications on the antiproliferative activity in prostate cancer cell lines. The three main areas of modification are ring A, ring B, and linker area C as illustrated in Figure 2. Fifteen bicalutamide derivatives were prepared and their antiproliferative activity was evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap).

Results and Discussion
Generally, minor chemical changes in the chemical structure of nonsteroidal AR ligands can result in major pharmacological outcomes [14,15]. Previously, we published extensive SAR studies on bicalutamide analogues [10][11][12][13]. Introduction of fluorinated groups into the chemical structure of molecules provides a combination of electrostatic, steric and lipophilic impacts on the physicochemical properties and biological activities [16][17][18].
Here we are exploring the impact of additional chemical structure modifications on the antiproliferative activity in prostate cancer cell lines. The three main areas of modification are ring A, ring B, and linker area C as illustrated in Figure 2. Fifteen bicalutamide derivatives were prepared and their antiproliferative activity was evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap).

Results and Discussion
Generally, minor chemical changes in the chemical structure of nonsteroidal AR ligands can result in major pharmacological outcomes [14,15]. Previously, we published extensive SAR studies on bicalutamide analogues [10][11][12][13]. Introduction of fluorinated groups into the chemical structure of molecules provides a combination of electrostatic, steric and lipophilic impacts on the physicochemical properties and biological activities [16][17][18].
Here we are exploring the impact of additional chemical structure modifications on the antiproliferative activity in prostate cancer cell lines. The three main areas of modification are ring A, ring B, and linker area C as illustrated in Figure 2. Fifteen bicalutamide derivatives were prepared and their antiproliferative activity was evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap).

Sulfone Analogues of Bicalutamide
Bicalutamide sulfone (SO 2 ) derivatives (19)(20)(21)(22) were prepared by oxidation of the corresponding sulfide derivatives using two equivalents of m-chloroperbenzoic acid (mCPBA) over a reaction time of 24 h at room temperature, resulting in the complete oxidation into sulfone analogues [19], as outlined in Scheme 3.

Sulfone Analogues of Bicalutamide
Bicalutamide sulfone (SO2) derivatives (19)(20)(21)(22) were prepared by oxidation of the corresponding sulfide derivatives using two equivalents of m-chloroperbenzoic acid (mCPBA) over a reaction time of 24 h at room temperature, resulting in the complete oxidation into sulfone analogues [19], as outlined in Scheme 3.  Table 3.   An in silico docking study was performed to compare the putative binding modes of the sulfide (S) compound 16 (IC50 = 6.59-10.86 µ M) and its sulfone (SO2) analogue 21 (IC50 = 24.64-43.04 µ M). Both compounds share key interactions including an H-bond between the nitro group (NO2) and the guanidine group of Arg 752 of helix 5. Another H-bond was observed between the amide (NH) and the side chain S-methyl thioether (SCH3) group of Met 742 amino acid residue. However, only compound 16 shows an H-bond between its nitro group (NO2) and the side chain amide (NH2) group of Gln 711 ( Figure 4A), which may explain its higher activity compared to its sulfone analogue (21) which lacks such interaction ( Figure 4B). In addition, hydrophobic interactions were observed between the 4-trifluoromethyl phenyl moiety and the surrounding hydrophobic pocket formed of residues, Trp 741, Met 745, Leu 712, and Met 787 in both compounds, Figure 4.  An in silico docking study was performed to compare the putative binding modes of the sulfide (S) compound 16 (IC 50 = 6.59-10.86 µM) and its sulfone (SO 2 ) analogue 21 (IC 50 = 24.64-43.04 µM). Both compounds share key interactions including an H-bond between the nitro group (NO 2 ) and the guanidine group of Arg 752 of helix 5. Another H-bond was observed between the amide (NH) and the side chain S-methyl thioether (SCH 3 ) group of Met 742 amino acid residue. However, only compound 16 shows an H-bond between its nitro group (NO 2 ) and the side chain amide (NH 2 ) group of Gln 711 ( Figure 4A), which may explain its higher activity compared to its sulfone analogue (21) which lacks such interaction ( Figure 4B). In addition, hydrophobic interactions were observed between the 4-trifluoromethyl phenyl moiety and the surrounding hydrophobic pocket formed of residues, Trp 741, Met 745, Leu 712, and Met 787 in both compounds, Figure 4.

O-Acetylated Analogues of Bicalutamide
The acetyl derivatives (27, 28) were prepared according to the route outlined in Scheme 4. The 4-trifluoromethyl thiophenol (13) was reacted with methyl 2-methylglycidate (23) followed by saponification using sodium hydroxide solution to form propionic acid derivative (24), which was then acetylated using acetic anhydride to afford (25). Coupling with aniline derivatives (2, 26) was achieved using dimethylaminopyridine (DMAP) to afford 27 and 28 [21]. Sulfoxide derivatives (29 and 30) were prepared from 27 and 28, respectively, using one equivalent of mCPBA at 0 °C for 15-30 min while the sulfone derivative (31) was prepared from 27 using two equivalents of mCPBA at room temperature for 24 h.

O-Acetylated Analogues of Bicalutamide
The acetyl derivatives (27, 28) were prepared according to the route outlined in Scheme 4. The 4-trifluoromethyl thiophenol (13) was reacted with methyl 2-methylglycidate (23) followed by saponification using sodium hydroxide solution to form propionic acid derivative (24), which was then acetylated using acetic anhydride to afford (25). Coupling with aniline derivatives (2, 26) was achieved using dimethylaminopyridine (DMAP) to afford 27 and 28 [21]. Sulfoxide derivatives (29 and 30) were prepared from 27 and 28, respectively, using one equivalent of mCPBA at 0 • C for 15-30 min while the sulfone derivative (31) was prepared from 27 using two equivalents of mCPBA at room temperature for 24 h.

O-Acetylated Analogues of Bicalutamide
The acetyl derivatives (27, 28) were prepared according to the route outlined in Scheme 4. The 4-trifluoromethyl thiophenol (13) was reacted with methyl 2-methylglycidate (23) followed by saponification using sodium hydroxide solution to form propionic acid derivative (24), which was then acetylated using acetic anhydride to afford (25). Coupling with aniline derivatives (2, 26) was achieved using dimethylaminopyridine (DMAP) to afford 27 and 28 [21]. Sulfoxide derivatives (29 and 30) were prepared from 27 and 28, respectively, using one equivalent of mCPBA at 0 °C for 15-30 min while the sulfone derivative (31) was prepared from 27 using two equivalents of mCPBA at room temperature for 24 h.  Table 4.  Table 4. The predicted docking mode of compound 27 within the hAR-LBD showed H-bond interactions between the nitro group (NO 2 ) and the guanidine group of Arg 752. In addition, hydrophobic interactions were observed between the 4-trifluoromethyl phenyl moiety and the surrounding hydrophobic pocket, π-π stacking between the terminal phenyl ring and the indole side chain of Trp 741. Also, the acetyl moiety of compound 27 seemed to occupy a small hydrophobic subpocket, Figure 5A. The binding mode of bicalutamide is shown in Figure 5B.  The predicted docking mode of compound 27 within the hAR-LBD showed H-bond interactions between the nitro group (NO2) and the guanidine group of Arg 752. In addition, hydrophobic interactions were observed between the 4-trifluoromethyl phenyl moiety and the surrounding hydrophobic pocket, π-π stacking between the terminal phenyl ring and the indole side chain of Trp 741. Also, the acetyl moiety of compound 27 seemed to occupy a small hydrophobic subpocket, Figure 5A. The binding mode of bicalutamide is shown in Figure 5B.

General Method for the Preparation of Intermediates 4-5
Methacryloyl chloride 3 (8.4 mL, 85.96 mmol) was added over the course of 10 min to a stirring solution of the appropriate trifluoromethylaniline 1-2 (10.75 mmol) in N,Ndimethylacetamide (10 mL) at room temperature for 24 h. After the reaction was complete, the mixture was diluted with ethyl acetate (100 mL) and extracted with saturated NaHCO 3 solution (2 × 50 mL), then cold brine (2 × 50 mL). The combined organic layer was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. The crude oil residue was purified by flash column chromatography eluting with chloroform-ethyl acetate 95:5 v/v to obtain the titled compounds.

General Method for the Preparation of Compounds 16-18
To a mixture of 60% sodium hydride (NaH) in mineral oil (2.36 mmol) in 5 mL of anhydrous tetrahydrofuran at 0 • C under anhydrous THF under nitrogen atmosphere was added dropwise the corresponding thiophenol 10-13 (2.05 mmol). This mixture was stirred at room temperature for 20 min. A solution of the intermediate 5 (1.57 mmol in 5 mL anhydrous tetrahydrofuran) was added slowly to the thiophenol mixture and stirred at room temperature for 24h. The mixture was concentrated under vacuum, then diluted with 30 mL ethyl acetate washed with 20 mL brine and 30 mL water, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude residue was purified by column chromatography eluting with chloroform-ethyl acetate gradually increasing from 95:5 to 90:10 v/v [12]. To a stirring solution of the different sulfide 10, 11, 16, 17, 27 (0.7 mmol) in 5 mL anhydrous dichloromethane (DCM) was added m-chloroperbenzoic acid (mCPBA) (1.4 mmol). The solution was stirred at room temperature for 24 h. The reaction mixture was neutralized with 1M sodium hydroxide. Distilled water (50 mL) was added to the reaction mixture and was extracted with 2 × 50 mL of dichloromethane. The combined organic layers were washed, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by column chromatography, preparative TLC, or crystallization from methanol. N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-3-(pyridin-2-ylsulfonyl) propanamide (19), yield 76%, 1  Multimode Plate Reader (excitation l = 530 nm, emission l = 620 nm) to quantify the amount of attached viable cells. IC 50 values were calculated by four-parameter nonlinear curve fit using Oncotest Warehouse Software. For calculation of mean IC 50 values, the geometric mean was used [23].

Docking Studies
The X-ray crystal structure of the human androgen receptor ligand-binding domain hAR-LBD was downloaded from the Protein Data Bank (PDB code; 3RLJ) [24] and prepared for docking using the MOE (Molecular Operating Environment) [25] protein preparation tools. The chemical structures of our compounds were constructed, rendered, and minimized with the MMFF94x force-field in MOE. The docking simulations were performed using the Glide SP within Maestro software using the default settings (Glide, version 9.5, Schrodinger) [26]. The docking output database was saved as a mol2 file, and the visual inspection of the docking modes was performed in MOE.

Conclusions
Fifteen bicalutamide derivatives were prepared and their antiproliferative activity was evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap). These modifications offer an insight into the SAR of various propionanilide analogues. Bicalutamide and enzalutamide were used as positive controls. The results summarized in Tables 1-4 indicated that seven compounds, sulfide (12), deshydroxy (16,17), sulfone (21), and acetylated (27) derivatives, have better antiproliferative activity than the positive controls, bicalutamide (IC 50 = 45.20-51.61 µM) and enzalutamide (IC 50 = 11.47-53.04 µM). The deshydroxy analogue (16) was the most active compound with IC 50 = 6.59-10.86 µM, followed by the acetylated derivative (27) with IC 50 = 4.69-15.03 µM across the four prostate cancer cell lines. Molecular modeling was used to find a plausible explanation of the drop in activity upon oxidation of the sulfide analogue (16) into sulfone counterpart (21). The dose response curves of compounds (16,17) and their oxidized analogues (21,22) are represented in Figure 3. Retention and enhancement of bicalutamide antiproliferative activity were observed in some compounds. Displaying antiproliferative activity in the 22Rv1 and DU-145 cell lines suggests that other antiproliferative mechanisms could be involved. This possible off-target effect was noticed as well in the parent bicalutamide, which showed similar IC 50 values across the four cell lines. These findings may serve as a useful starting point for developing novel AR modulators. Funding: Welsh Government Academic Expertise for Business (A4B) scheme.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.