Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer

Abstract

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Introduction

The retinoic acid receptor-related orphan receptors (RORs) belong to a nuclear receptors subfamily that consists of RORα, RORβ, and RORγ [1, 2]. The RORγ exists in two isoforms: one isoform (RORγ or RORc1) is widely expressed in a variety of tissues, while the expression of the second isoform (RORγt or RORc2) is restricted to the thymus and cells of the immune system. Because the two isoforms of RORγ only differ in their N-terminal and share the same ligand-binding domain, ligands will inevitably target both isoforms [3, 4]. RORγt regulates the differentiation of CD4 + T cells into Th17 cells, and plays a pivotal role in the production of the pro-inflammatory cytokines, including IL-17 (interleukin 17) and IL-22. With a direct impact on the Th17/IL-17 pathway, RORγ has emerged as an attractive drug-target for the treatment of autoimmune disease, particularly psoriasis [5–7]. RORγ has also been found to be considered as potential therapeutic targets in many cancers, including prostate cancer, colon cancer, lung cancer and breast cancer [8–14]. In recent year, significant progress has been made in the development of small molecule RORγ inverse agonists [15–21]. For example, SR2211 (1, Fig. 1) [22] and GSK805 [23] are potent and selective RORγ inverse agonists. Several of these RORγ inverse agonists have been developed and evaluated in clinical trials, such as IMU-935 (2, Fig. 1), BI 730357 (3, Fig. 1), ARN-6039, ABBV-553, RTA-1701, and VTP-43742 [24–29].

Fig. 1. Structures of representative RORγ inverse agonists.

1 (SR2211), 2 (IMU-935), 3 (BI 730357), 4 (XY101), 5 (XY123) and 6 were shown below.

Prostate cancer (PC) is the most frequently diagnosed solid tumor and the second leading cause of cancer-related deaths among men worldwide [30, 31]. Since Huggins and Hodges made the observation over 70 years ago that the activation of androgen receptor (AR) signaling promote the growth and survival of prostate cancer, androgen deprivation therapy (ADT) has become the standard first-line approach of treatment for prostate cancers [32]. Unfortunately, after the initial efficacy of ADT, most patients with prostate cancer eventually acquire resistance and develop more aggressive castration-resistant prostate cancer (CRPC). These CRPC patients are subsequently treated with second-generation therapeutic drugs such as abiraterone acetate (a cytochrome P450 17A1 inhibitor) [33], enzalutamide [34], and apalutamide (AR antagonists) [35], but most patients eventually develop secondary resistance to these medications within a few years. In the past decades, research has proved the development of CRPC continues to be dependent on AR signaling through multiple mechanisms, including AR gene amplifications, point mutations, and expression of constitutively active AR splice variants [36–38]. Given the severity of this disease, there is a critical need to identify the new molecular targets for the treatment of drug resistant CRPC.

In the previous study, we discovered that RORγ is the driver of the AR, and targeting RORγ can be used in the anti-CRPC drug development [8]. In CRPC, RORγ is overexpressed and promotes the transcription of AR gene. RORγ inverse agonists XY101 (4, Fig. 1) [39] and XY123 (5, Fig. 1) [40] inhibited the expression of both AR and its variant AR-VS, and the growth of CRPC cell lines both in vitro and in vivo. In particular, IMU-935 has successfully progressed into phase I for the treatment of prostate cancer based on our previous study [8, 41]. RORγ has been gaining increasing interest as a potential therapeutic target for the treatment of CRPC. Although many RORγ inverse agonists have been reported, RORγ inverse agonists for CRPC are still limited and deficient. Therefore, potent and selective RORγ inverse agonists with different chemotypes are still in high demand to further verify the therapeutic potential of RORγ inhibition in CRPC.

In this article, we report the structure-based design, synthesis, and biological evaluation of a novel class of potent and selective small molecule RORγ inverse agonists, the 1,2,3,4-tetrahydroquinolines. The identified compounds could bring practical benefits in developing novel RORγ inverse agonists for the treatment of prostate cancer.

Materials and methods

Molecular docking studies

The crystal structures of RORγ in complex with the inverse agonists (PDB code: 4QM0) were utilized for the molecular docking study. All the ligand and protein preparations were performed in Maestro (version 9.4, Schrödinger, LLC, New York, NY, 2013), a component of the Schrödinger program. The proteins were prepared using the Protein Preparation Wizard. Hydrogens were added, bond orders were assigned, and missing side chains for some residues were added using Prime. The added hydrogens were subjected to energy minimization until the root-mean-square deviation (RMSD) relative to the starting geometry reached 0.3 Å. The Glide docking program in Maestro 9.4 was used for docking studies. For Glide docking, the grid was defined using a 20 Å box centered on the ligand, and the important water molecules around ligand were retained. All parameters were kept as default. The designed molecules were docked using Glide SP mode, and the predicted binding modes of all the compounds were ranked based to their glide scores.

General chemistry

All commercial reagents were used without further purification unless otherwise specified. Enzalutamide was purchased from Selleck. Final compounds were purified either by silica gel chromatography (300–400 mesh) or by recrystallization. ¹H-NMR and ¹³C-NMR spectra were recorded using a Bruker AV-400 or AV-500 spectrometer. Coupling constants (J) are expressed in hertz (Hz). NMR chemical shifts (δ) are reported in parts per million (ppm) units relative to the internal control (TMS). Low or high resolution ESI-MS was recorded using an Agilent 1200 HPLC-MSD mass spectrometer or an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer, respectively. Compound purities were determined by reverse-phase high-performance liquid chromatography (HPLC) with solvent A (H₂O) and solvent B (MeOH) as eluents. HPLC analysis was conducted on a Dionex Summit HPLC column (Inertsil ODS-SP, 5.0 μm, 4.6 mm × 250 mm (GL Sciences Inc.)), equipped with a UVD170U detector, a manual injector, and a P680 pump. The detection wavelength was set at 254 nm, and the flow rate is 1.0 mL/min. The purity of all the final compounds was determined by HPLC to be >95%.

7-Nitro-1,2,3,4-tetrahydro-quinoline (8)

1,2,3,4-Tetrahydroquinoline (7, 5 g, 37.54 mmol) was dissolved in 13 mL of concentrated sulfuric acid while cooled with an ice-bath. After stirring for 30 min, 1.7 mL (37.54 mmol) of concentrated nitric acid in 7 mL of sulfuric acid was added dropwise at 0 to 5 °C. The reaction mixture was stirred in the ice bath for 3 h and then poured onto ice. The solution was neutralized to pH 8 with sodium carbonate, and the aqueous mixture was extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with saturated brine, dried over Na₂SO₄ and evaporated. The title compound was obtained as a viscous brown oil (5.1 g, 76%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.26 (s, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 6.41 (s, 1H), 3.15–3.26 (m, 2H), 2.70–2.79 (m, 2H), 1.71–1.85 (m, 2H).

7-Nitro-1-((4-(trifluoromethyl)benzyl)sulfonyl)-1,2,3,4-tetrahydroquinoline (9f)

Compound 8 (400 mg, 2.24 mmol) and (4-(trifluoromethyl)phenyl)methanesulfonyl chloride (868.03 mg, 3.36 mmol) were added in pyridine. The reaction mixture was heated to 80 °C for 4 h. Hydrochloric acid was added, and the aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with saturated brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (4/1, v/v) to afford the target compound (233.2 mg, 26%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.79 (d, J = 7.8 Hz, 1H), 7.74 (s, 1H), 7.70 (d, J = 7.7 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 4.93 (s, 2H), 3.71 – 3.66 (m, 2H), 2.89 – 2.83 (t, J = 6.5 Hz, 2H), 1.91 – 1.84 (m, 2H).

1-((4-(Trifluoromethyl)benzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-amine (10f)

A reaction mixture of Fe power (162.4 mg, 2.9 mmol) and NH₄Cl (31.03 mg, 0.58 mmol) in AcOH (1 mL) and water (20 mL) was heated at 80 °C for 5 min. Compound 9f (233.2 mg, 0.58 mmol) was dissolved in DMF (15 mL) and added to the reaction mixture. After the reaction was completed, and the reaction mixture was cooled to rt. The solid was filtered off, and the filtrate was extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with saturated brine, dried over Na₂SO₄ and evaporated. The residue was purified by silica gel chromatography with petroleum and ether/ethyl acetate (4/1, v/v) to afford the title compound (184.75 mg, 86%)。¹H NMR (500 MHz, DMSO-d₆) δ 7.75 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H), 6.82 – 6.76 (m, 2H), 6.32 (d, J = 8.1 Hz, 1H), 4.97 (s, 2H), 4.66 (s, 2H), 3.43 (t, J = 5.3 Hz, 2H), 2.54 (t, J = 6.6 Hz, 2H), 1.72 – 1.62 (m, 2H).

2,4-Difluoro-N-(1-((4-(trifluoromethyl)benzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13e)

General procedure for syntheses of 6, 11a-11k, 13a-13j, 14b-14c. To a solution of compound 10f (100 mg, 0.27 mmol) in pyridine was added 2,4-difluorobenzenesulfonyl chloride (87.2 mg, 0.41 mmol). The reaction mixture was heated to 80 °C for 4 h. Diluted HCl was added, and the aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with saturated brine, dried with Na₂SO₄ and evaporated. The crude product was purified by silica gel chromatography with petroleum ether/ethyl acetate (4/1, v/v) to afford the title compound as a white solid (88.5 mg, 60%). ¹H NMR (500 MHz, DMSO-d₆) δ 10.61 (s, 1H), 7.92-7.83 (m, 1H), 7.73 (d, J = 7.7 Hz, 2H), 7.54 (t, J = 9.4 Hz, 1H), 7.44-7.36 (m, 3H), 7.25 (t, J = 8.2 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 4.60 (s, 2H), 3.50 – 3.43 (m, 2H), 2.59 (t, J = 6.1 Hz, 2H), 1.71 – 1.62 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.19, 166.09, 164.16, 164.07, 159.98, 159.87, 157.93, 157.82, 136.97, 135.04, 133.35, 132.49, 132.41, 131.48, 130.22, 129.35, 129.10, 128.85, 128.59, 125.25, 125.22, 124.73, 123.92, 123.78, 122.94, 115.88,112.89, 112.41, 112.23, 106.17, 105.96, 105.75, 55.71, 46.26, 25.83, 21.58. HRMS (ESI) m/z [M + Na]⁺ found 569.0586. HPLC analysis: MeOH − H₂O (85:15), t_R = 6.54 min, 99.28% purity.

2-(4-(Ethylsulfonyl)phenyl)-N-(1-((4-(trifluoromethyl)benzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)acetamide (14a)

General procedure for syntheses of 12a, 12b, 14a. To a solution of 2-(4-(ethylsulfonyl)phenyl)acetic acid (109.56 mg, 0.48 mmol) in DCM were added HATU (182.4 mg, 0.48 mmol) and DIPEA (124.08 mg, 0.96 mmol) and stirred for 10 min. Compound 10 f (120 mg, 0.32 mmol) was added, and the reaction was stirred at room temperature for overnight. Water was added, the aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the organic layer was washed with saturated brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (1/1, v/v) to give the title compound (70.6 mg, 38%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.26 (s, 1H), 7.85 (d, J = 7.6 Hz, 2H), 7.77 (s, 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.61 (d, J = 7.7 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 4.72 (s, 2H), 3.80 (s, 2H), 3.52 – 3.46 (m, 2H), 3.27 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 6.3 Hz, 2H), 1.77 – 1.69 (m, 2H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.07, 142.11, 137.19, 136.78, 136.70, 133.65, 131.70, 129.70, 129.00, 128.75, 127.79, 125.24, 123.73, 123.00, 115.06, 112.10, 56.03, 49.21, 46.41, 42.82, 25.93, 21.97, 7.12. HRMS (ESI), m/z [M + Na]⁺ found 603.1186. HPLC analysis: MeOH − H₂O (85:15), t_R = 7.05 min, 97.14% purity.

2,4-Difluoro-N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (6)

White solid, 25% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 7.90 – 7.81 (m, 1H), 7.59 – 7.51 (m, 1H), 7.51 – 7.45 (m, 3H), 7.34 (t, J = 8.8 Hz, 2H), 7.30 – 7.22 (m, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.84 (dd, J = 8.2, 2.1 Hz, 1H), 3.72 (t, J = 5.8 Hz, 2H), 2.34 (t, J = 6.6 Hz, 2H), 1.57 – 1.46 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.22, 166.12, 165.58, 164.19, 164.10, 163.57, 159.99, 159.88, 157.94, 157.83, 136.47, 135.05, 135.03, 134.90, 132.59, 132.51, 129.97, 129.65, 129.57, 126.36, 123.68, 123.60, 123.57, 117.18, 116.56, 116.38, 115.25, 112.35, 112.32, 112.17, 112.15, 106.20, 105.99, 105.79, 46.26, 25.42, 20.79. MS (ESI) m/z [M − 1]⁻ found 481.2. HPLC analysis: MeOH − H₂O (85:15), t_R = 7.11 min, 99.57% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (11a)

White solid, 63% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J = 7.6 Hz, 2H), 7.58 – 7.44 (m, 6H), 7.04 (t, J = 8.5 Hz, 2H), 6.95 – 6.86 (m, 2H), 6.76 (s, 1H), 3.75 (t, J = 6.0 Hz, 2H), 2.40 (t, J = 6.6 Hz, 2H), 1.64 – 1.55 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.56, 163.56, 139.39, 136.44, 135.75, 135.05, 135.03, 132.85, 129.91, 129.76, 129.69, 129.17, 126.67, 125.82, 116.85, 116.59, 116.41, 114.94, 46.29, 25.42, 20.85. MS (ESI) m/z [M − 1]⁻ found 444.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.12 min, 98.67% purity.

4-Fluoro-N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (11b)

White solid, 53% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 7.82 (dd, J = 8.8, 5.2 Hz, 2H), 7.52 (dd, J = 8.8, 5.2 Hz, 2H), 7.47 (d, J = 2.0 Hz, 1H), 7.41 (t, J = 8.8 Hz, 2H), 7.35 (t, J = 8.8 Hz, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.82 (dd, J = 8.2, 2.1 Hz, 1H), 3.72 (t, J = 5.8 Hz, 2H), 2.35 (t, J = 6.6 Hz, 2H), 1.58 – 1.48 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.59, 165.29, 163.58, 163.30, 136.47, 135.72, 135.70, 135.52, 135.07, 135.05, 129.97, 129.78, 129.72, 129.70, 129.64, 126.08, 117.14, 116.61, 116.47, 116.42, 116.29, 115.22, 46.26, 25.43, 20.85. MS (ESI) m/z [M − 1]⁻ found 462.8. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.15 min, 99.02% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-4-(trifluoromethyl)benzenesulfonamide (11c)

White solid, 53% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.52 (s, 1H), 8.00 – 7.95 (m, 4H), 7.52 – 7.42 (m, 3H), 7.33 (t, J = 8.8 Hz, 2H), 6.95 (d, J = 8.3 Hz, 1H), 6.85 (dd, J = 8.2, 2.1 Hz, 1H), 3.71 (t, J = 5.8 Hz, 2H), 2.35 (t, J = 6.6 Hz, 2H), 1.58 – 1.46 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.56, 163.55, 143.21, 136.51, 135.10, 135.00, 134.98, 130.07, 129.69, 129.61, 127.69, 126.52, 126.49, 126.41, 124.39, 122.22, 117.33, 116.56, 116.38, 115.40, 46.24, 25.43, 20.80. MS (ESI) m/z [M − 1]⁻ found 513.2. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.36 min, 99.22% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-4-(trifluoromethoxy)benzenesulfonamide (11d)

White solid, 67% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.39 (s, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.53 – 7.48 (m, 2H), 7.46 (d, J = 1.9 Hz, 1H), 7.34 (t, J = 8.7 Hz, 2H), 6.95 (d, J = 8.3 Hz, 1H), 6.84 (dd, J = 8.3, 2.0 Hz, 1H), 3.71 (t, J = 5.7 Hz, 2H), 2.35 (t, J = 6.5 Hz, 2H), 1.58 – 1.47 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.58, 163.58, 151.06, 138.27, 136.51, 135.32, 135.02, 135.00, 130.04, 129.71, 129.63, 129.32, 126.30, 121.37, 120.78, 118.72, 117.31, 116.58, 116.40, 115.38, 46.26, 25.44, 20.83. MS (ESI) m/z [M − 1]⁻ found 528.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.43 min, 99.81% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-4-nitrobenzenesulfonamide (11e)

White solid, 74% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 8.38 (d, J = 8.6 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.57 – 7.48 (m, 2H), 7.43 (s, 1H), 7.35 (t, J = 8.5 Hz, 2H), 6.96 (d, J = 7.9 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 3.71 (t, J = 5.2 Hz, 2H), 2.36 (t, J = 6.3 Hz, 2H), 1.59 – 1.45 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.60, 163.59, 149.81, 144.75, 136.56, 135.04, 135.02, 134.88, 130.15, 129.71, 129.64, 128.32, 126.61, 124.59, 117.53, 116.67, 116.49, 115.62, 46.22, 25.46, 20.80. MS (ESI) m/z [M − 1]⁻ found 490.0. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.15 min, 98.95% purity.

Methyl 4-(N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)sulfamoyl)benzoate (11f)

White solid, 58% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.44 (s, 1H), 8.10 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.54-7.48 (m, 2H), 7.46 (d, J = 1.8 Hz, 1H), 7.32 (t, J = 8.7 Hz, 2H), 6.93 (d, J = 8.4 Hz, 1H), 6.82 (dd, J = 8.0, 1.9 Hz, 1H), 3.84 (s, 3H), 3.71 (t, J = 5.6 Hz, 2H), 2.35 (t, J = 6.7 Hz, 2H), 1.57 – 1.48 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.55, 165.02, 163.54, 143.28, 136.49, 135.25, 135.04, 133.24, 130.02, 129.96, 129.72, 129.64, 127.16, 126.23, 117.24, 116.58, 116.40, 115.31, 52.53, 46.26, 25.45, 20.84. MS (ESI) m/z [M − 1]⁻ found 502.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.30 min, 95.46% purity.

Methyl 3-(N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)sulfamoyl)benzoate (11g)

White solid, 48% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.41 (s, 1H), 8.34 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.53 – 7.42 (m, 3H), 7.31 (t, J = 8.6 Hz, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 3.85 (s, 3H), 3.70 (t, J = 5.4 Hz, 2H), 2.33 (t, J = 6.4 Hz, 2H), 1.55 – 1.46 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.56, 164.88, 163.55, 140.03, 136.49, 135.26, 135.02, 134.99, 133.22, 131.09, 130.63, 130.02, 130.00, 129.69, 129.61, 127.23, 126.42, 117.45, 116.57, 116.39, 115.63, 52.55, 46.23, 25.42, 20.81. MS (ESI) m/z [M − 1]⁻ found 502.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.21 min, 98.66% purity.

4-Chloro-2-fluoro-N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-y-l)benzenesulfonamide (11h)

White solid, 49% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.09 – 8.01 (m, 1H), 7.72 (dd, J = 8.7, 2.5 Hz, 1H), 7.51 – 7.44 (m, 3H), 7.40 (td, J = 8.6, 2.5 Hz, 1H), 7.33 (t, J = 8.8 Hz, 2H), 6.96 – 6.91 (m, 1H), 6.84 (dd, J = 8.2, 2.0 Hz, 1H), 3.76 – 3.67 (t, J = 5.6 Hz, 2H), 2.34 (t, J = 6.6 Hz, 2H), 1.57 – 1.47 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.56, 165.06, 163.55, 163.03, 136.46, 135.07, 134.80, 134.15, 134.07, 132.95, 132.93, 132.71, 132.61, 129.99, 129.64, 129.56, 126.00, 119.44, 119.23, 116.67, 116.57, 116.39, 114.97, 114.79, 114.64, 46.28, 25.42, 20.79. MS (ESI) m/z [M + Na]⁺ found 521.6. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.28 min, 98.94% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)thiophene-2-sulfonamide (11i)

White solid, 60% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J = 3.6 Hz, 1H), 7.59 – 7.49 (m, 4H), 7.11 – 7.02 (m, 3H), 7.01 – 6.92 (m, 2H), 6.68 (s, 1H), 3.77 (t, J = 6.0 Hz, 2H), 2.44 (t, J = 6.6 Hz, 2H), 1.66 – 1.56 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.59, 163.58, 139.89, 136.47, 135.50, 135.05, 135.03, 133.32, 132.42, 129.92, 129.80, 129.72, 127.52, 126.23, 117.20, 116.64, 116.46, 115.40, 46.31, 25.48, 20.86. MS (ESI) m/z [M − 1]⁻ found 451.1. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.01 min, 99.43% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-5-methylthiophene-2-sulfonamide (11j)

White solid, 43% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.35 (s, 1H), 7.65 – 7.57 (m, 2H), 7.56 – 7.52 (m, 1H), 7.41 – 7.32 (m, 3H), 6.95 (d, J = 8.2 Hz, 1H), 6.90 – 6.80 (m, 2H), 3.74 (m, 2H), 2.43 (s, 3H), 2.38 (t, J = 6.5 Hz, 2H), 1.60 – 1.50 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.62, 163.61, 147.51, 136.81, 136.49, 135.67, 135.12, 135.10, 132.91, 129.94, 129.84, 129.76, 126.03, 125.96, 116.90, 116.66, 116.47, 115.03, 46.32, 25.49, 20.91, 15.06. MS (ESI) m/z [M − 1]⁻ found 464.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.20 min, 98.05% purity.

N-(1-((4-Fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide (11k)

White solid, 33% yield. ¹H NMR (400 MHz, DMSO-d₆) δ1H NMR (500 MHz, DMSO) δ 10.13 (s, 1H), 7.61–7.54 (m, 2H), 7.53 (d, J = 1.7 Hz, 1H), 7.35 (t, J = 8.8 Hz, 2H), 7.27 – 7.20 (m, 2H), 7.00 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.80 (dd, J = 8.2, 1.9 Hz, 1H), 4.29 – 4.19 (m, 4H), 3.73 (t, J = 5.8 Hz, 2H), 2.34 (t, J = 6.6 Hz, 2H), 1.56 – 1.48 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.60, 163.59, 147.14, 143.23, 136.45, 135.94, 135.08, 135.05, 131.80, 129.92, 129.82, 129.75, 125.70, 120.33, 117.46, 116.79, 116.60, 116.42, 115.80, 114.85, 64.31, 63.98, 46.30, 25.44, 20.87. MS (ESI) m/z [M + Na]⁺ found 527.0. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.20 min, 99.29% purity.

4-Fluoro-N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzamide (12a)

White solid, 36% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.93 – 7.86 (m, 2H), 7.84 (s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.68 – 7.59 (m, 2H), 7.16 – 7.02 (m, 4H), 6.99 (d, J = 8.2 Hz, 1H), 3.75 (t, J = 5.7 Hz, 2H), 2.42 (t, J = 6.4 Hz, 2H), 1.65 – 1.53 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 165.05, 164.40, 163.61, 163.07, 137.26, 136.18, 135.24, 135.21, 131.32, 131.30, 130.48, 130.40, 130.02, 129.95, 129.27, 125.67, 117.28, 116.69, 116.51, 115.58, 115.36, 115.19, 46.32, 25.54, 21.10. MS (ESI) m/z [M + Na]⁺ found 451.4. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.48 min, 99.27% purity.

2-(4-(Ethylsulfonyl)phenyl)-N-(1-((4-fluorophenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)acetamide (12b)

White solid, 40% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.92 (s, 1H), 7.86 (d, J = 7.8 Hz, 2H), 7.71 (t, J = 5.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.43 – 7.35 (m, 3H), 6.99 (d, J = 8.3 Hz, 1H), 3.80 (s, 2H), 3.74 (t, J = 4.3 Hz, 2H), 3.31 – 3.23 (m, 2H), 2.38 (t, J = 6.4 Hz, 2H), 1.61 – 1.53 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.09, 165.60, 163.59, 142.12, 137.20, 136.81, 136.22, 135.18, 135.15, 130.21, 129.95, 129.88, 129.43, 127.82, 125.30, 116.69, 116.51, 116.05, 114.33, 49.23, 46.27, 42.88, 25.46, 21.05, 7.14. MS (ESI) m/z [M + H]⁺ found 517.2. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.00 min, 99.42% purity.

N-(1-((4-(tert-Butyl)phenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-2,4-difluorobenzenesulfonamide (13a)

White solid, 26% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 7.91 – 7.83 (m, 1H), 7.62 – 7.52 (m, 2H), 7.47 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.25 (td, J = 8.6, 2.2 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.84 (dd, J = 8.2, 2.0 Hz, 1H), 3.69 (t, J = 5.7 Hz, 2H), 2.33 (t, J = 6.6 Hz, 2H), 1.55 – 1.44 (m, 2H), 1.26 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.21, 166.12, 164.19, 164.10, 160.01, 159.90, 157.96, 157.85, 156.40, 136.80, 135.95, 134.87, 132.69. 132.61, 129.95, 126.47, 126.08, 125.98, 123.71, 123.68, 123.60, 123.57, 116.86, 115.04, 112.34, 112.32, 112.16, 112.14, 106.19, 105.98, 105.77, 46.19, 34.82, 30.64, 25.50, 20.76. MS (ESI) m/z [M − 1]⁻ found 519.0. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.72 min, 96.76% purity.

Methyl 3-((7-((2,4-difluorophenyl)sulfonamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)benzoate (13b)

White solid, 47% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.98 (s, 1H), 7.90 – 7.80 (m, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.54 – 7.45 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H), 3.72 (t, J = 5.6 Hz, 2H), 2.31 (t, J = 6.4 Hz, 2H), 1.55 – 1.43 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.24, 166.14, 164.70, 164.22, 164.12, 160.03, 159.89, 157.94, 157.84, 139.39, 136.35, 135.03, 133.62, 132.61, 132.52, 130.77, 130.74, 130.15, 130.02, 126.92, 126.49, 123.64, 123.53, 117.31, 115.26, 112.32, 112.11, 106.20, 106.00, 105.79, 52.62, 46.36, 25.30, 20.81. MS (ESI) m/z [M − 1]⁻ found 520.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.25 min, 98.38% purity.

Methyl 2-((7-((2,4-difluorophenyl)sulfonamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)benzoate (13c)

White solid, 47% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.61 (s, 1H), 7.87 – 7.79 (m, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.56 – 7.45 (m, 2H), 7.38 (s, 1H), 7.23 (t, J = 8.5 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 3.68 (s, 3H), 3.69 – 3.63 (m, 2H), 2.40 (t, J = 6.7 Hz, 2H), 1.53 – 1.44 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 167.35, 166.22, 166.13, 164.20, 164.11, 159.98, 159.87, 157.93, 157.82, 136.85, 136.09, 134.82, 133.36, 132.66, 132.58, 132.29, 130.52, 129.92, 128.65, 127.96, 126.80, 123.65, 123.62, 123.54, 123.51, 117.56, 115.95, 112.34, 112.32, 112.16, 112.14, 106.17, 105.96, 105.75, 52.69, 45.60, 25.18, 20.85. MS (ESI) m/z [M + H]⁺ found 522.8. HPLC analysis: MeOH − H₂O (90:10), t_R = 6.98 min, 95.99% purity.

2,4-Difluoro-N-(1-((3-(methylsulfonyl)phenyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13d)

White solid, 37% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.67 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 7.90 – 7.82 (m, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.58 – 7.41 (m, 2H), 7.24 (t, J = 8.1 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 3.76 (t, J = 5.0 Hz, 2H), 3.25 (s, 3H), 2.32 (t, J = 6.0 Hz, 2H), 1.60 – 1.46 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.22, 166.13, 164.20, 164.11, 159.97, 159.86, 157.93, 157.82, 142.03, 139.84, 136.18, 135.10, 132.50, 131.74, 131.04, 130.91, 130.03, 126.42, 124.97, 124.81, 123.68, 123.57, 117.18, 115.07, 112.32, 112.15, 106.05, 46.40, 43.14, 25.24, 20.93. MS (ESI) m/z [M − 1]⁻ found 540.8. HPLC analysis: MeOH − H₂O (90:10), t_R = 6.79 min, 97.36% purity.

Methyl 4-(((7-((2,4-difluorophenyl)sulfonamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)methyl)benzoate (13f)

White solid, 78% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.98 – 7.81 (m, 3H), 7.53 (t, J = 9.2 Hz, 1H), 7.39 (s, 1H), 7.33 – 7.18 (m, 3H), 7.02 (d, J = 7.7 Hz, 1H), 6.79 (d, J = 7.6 Hz, 1H), 4.58 (s, 2H), 3.86 (s, 3H), 3.44 – 3.38 (m, 2H), 2.56 (t, J = 6.2 Hz, 2H), 1.65 – 1.56 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.20, 166.11, 165.81, 164.18, 164.09, 159.99, 159.89, 157.95, 157.84, 137.02, 135.04, 133.86, 132.53, 132.44, 131.02, 130.25, 129.65, 129.14, 124.68, 123.91, 123.88, 123.80, 123.77, 115.88, 112.81, 112.41, 112.39, 112.24, 112.21, 106.18, 105.97, 105.76, 55.97, 52.17, 46.33, 25.86, 21.58. MS (ESI) m/z [M + H]⁺ found 537.7. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.19 min, 97.34% purity.

2,4-Difluoro-N-(1-((4-fluorobenzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13g)

White solid, 52% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.59 (s, 1H), 7.91 – 7.82 (m, 1H), 7.53 (t, J = 8.9 Hz, 1H), 7.39 (s, 1H), 7.29 – 7.09 (m, 5H), 7.02 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 4.48 (s, 2H), 3.40 (t, J = 5.5 Hz, 2H), 2.57 (t, J = 6.5 Hz, 2H), 1.66 – 1.56 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.19, 166.10, 164.17, 164.07, 163.18, 161.23, 159.99, 159.88, 157.94, 157.84, 137.14, 135.10, 132.74, 132.68, 132.51, 132.43, 130.26, 124.89, 124.87, 124.47, 123.95, 123.92, 123.84, 123.81, 115.79, 115.45, 115.28, 112.67, 112.42, 112.39, 112.24, 112.21, 106.18, 105.97, 105.76, 55.28, 46.27, 25.93, 21.57. MS (ESI) m/z [M − 1]⁻ found 495.0. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.09 min, 97.61% purity.

2,4-Difluoro-N-(1-((3-fluorobenzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13h)

White solid, 39% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 7.91 - 7.83 (m, 1H), 7.57 – 7.47 (m, 1H), 7.44 – 7.34 (m, 2H), 7.28 – 7.17 (m, 2H), 7.02 (d, J = 8.3 Hz, 1H), 6.99 – 6.90 (m, 2H), 6.79 (dd, J = 8.2, 1.8 Hz, 1H), 4.51 (s, 2H), 3.43 (t, J = 5.6 Hz, 2H), 2.56 (t, J = 6.4 Hz, 2H), 1.67 – 1.57 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.20, 166.11, 164.18, 164.09, 162.74, 160.80, 159.99, 159.88, 157.95, 157.84, 137.07, 135.09, 132.52, 132.43, 131.11, 131.05, 130.41, 130.34, 130.26, 126.83, 126.81, 124.59, 123.92, 123.89, 123.81, 123.78, 117.46, 117.28, 115.86, 115.52, 115.36, 112.71, 112.41, 112.39, 112.23, 112.21, 106.18, 105.98, 105.77, 55.57, 46.31, 25.90, 21.55. MS (ESI) m/z [M − 1]⁻ found 495.0. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.11 min, 97.90% purity.

2,4-Difluoro-N-(1-((2-fluorobenzyl)sulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13i)

White solid, 40% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (s, 1H), 7.91 – 7.81 (m, 1H), 7.52 (t, J = 8.8 Hz, 1H), 7.46 – 7.39 (m, 1H), 7.37 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.26 – 7.17 (m, 3H), 7.02 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 4.51 (s, 2H), 3.40 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 1.69 – 1.60 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.18, 166.09, 164.16, 164.07, 161.72, 159.97, 159.86, 159.74, 157.92, 157.82, 137.01, 134.98, 132.92, 132.52, 132.44, 131.03, 130.97, 130.18, 124.60, 124.43, 124.40, 123.89, 123.86, 123.78, 123.75, 116.02, 115.90, 115.83, 115.52, 115.35, 112.78, 112.33, 112.30, 112.15, 112.12, 106.13, 105.92, 105.72, 49.94, 46.38, 25.84, 21.68. MS (ESI) m/z [M − 1]⁻ found 494.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.10 min, 95.91% purity.

Methyl 3-(((7-((2,4-difluorophenyl)sulfonamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)methyl)benzoate (13j)

White solid, 88% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 7.99 – 7.80 (m, 2H), 7.67 (s, 1H), 7.58 – 7.46 (m, 2H), 7.46 – 7.35 (m, 2H), 7.28 – 7.15 (m, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.79 (d, J = 7.1 Hz, 1H), 4.59 (s, 2H), 3.83 (s, 3H), 3.46 – 3.31 (m, 2H), 2.58 – 2.49 (m, 2H), 1.63 – 1.49 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.20, 166.10, 165.68, 164.17, 164.08, 159.97, 159.86, 157.93, 157.82, 137.05, 135.45, 135.10, 132.54, 132.45, 131.40, 130.29, 129.81, 129.35, 129.21, 128.93, 124.58, 123.83, 123.75, 115.83, 112.66, 112.38, 112.21, 106.17, 105.97, 105.76, 55.53, 52.15, 46.29, 25.89, 21.45. MS (ESI) m/z [M − 1]⁻ found 534.9. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.13 min, 97.50% purity.

Methyl 2-((7-(2-(4-(ethylsulfonyl)phenyl)acetamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)benzoate (14b)

White solid, 37% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.88 – 7.79 (m, 3H), 7.76 – 7.70 (m, 1H), 7.65 – 7.56 (m, 4H), 7.52 – 7.44 (m, 2H), 7.04 (d, J = 8.3 Hz, 1H), 3.79 (s, 2H), 3.74 – 3.66 (m, 5H), 3.31 – 3.23 (m, 2H), 2.44 (t, J = 6.6 Hz, 2H), 1.59 – 1.50 (m, 2H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.08, 167.49, 142.12, 137.10, 136.79, 136.56, 136.16, 133.36, 132.44, 130.76, 130.19, 129.34, 128.57, 128.42, 127.80, 125.72, 116.47, 115.10, 52.79, 49.22, 45.57, 42.86, 25.20, 21.04, 7.13. MS (ESI) m/z [M + Na]⁺ found 579.4. HPLC analysis: MeOH − H₂O (90:10), t_R = 6.92 min, 99.38% purity.

Methyl 3-(((7-(2-(4-(ethylsulfonyl)phenyl)acetamido)-3,4-dihydroquinolin-1(2H)-yl)sulfonyl)methyl)benzoate (14c)

White solid, 41% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 10.25 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.89 – 7.82 (m, 3H), 7.78 (s, 1H), 7.62 (d, J = 8.0 Hz, 2H), 7.55 – 7.46 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 4.71 (s, 2H), 3.85 (s, 3H), 3.80 (s, 2H), 3.46 (t, J = 5.5 Hz, 2H), 3.32 – 3.23 (m, 2H), 2.62 (t, J = 6.3 Hz, 2H), 1.70 – 1.61 (m, 2H), 1.10 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.08, 165.75, 142.14, 137.22, 136.79, 136.77, 135.67, 131.56, 130.23, 129.75, 129.70, 129.67, 129.10, 128.88, 127.80, 123.64, 114.98, 111.93, 55.87, 52.18, 49.22, 46.23, 42.84, 25.96, 21.87, 7.13. MS (ESI) m/z [M + Na]⁺ found 593.5. HPLC analysis: MeOH − H₂O (90:10), t_R = 7.00 min, 97.92% purity.

Expression and purification of the RORγ LBD

The human RORγ LBD (residues 262–507, wild type or C455E mutant) were expressed as His6-tag protein using the pET24a expression vector (Novagen). The protein expression and purification procedures were performed as previously described [42]. The purified protein was stored at −80 °C and used for the TSA assay, AlphaScreen assay, or cocrystal experiment.

Thermal stability shift assay (TSA)

Thermal stability shift assay was conducted using the Bio-Rad CFX96 Real-Time PCR system. The TSA assay was performed as previously described [8]. Briefly, protein and compounds were diluted using reaction buffer to obtain final concentrations of 10 μM protein and 200 μM compounds. Reactions were performed in a 10 μL final volume in 96-well PCR plates. SYPRO Orange (Sigma) was added as a fluorescence probe at a dilution of 1:1000 and incubated with the compounds on ice for 30 min. The fluorescence readings were recorded at a 0.5 °C interval. All experiments were performed in duplicate. The data were fitted to a Boltzmann sigmoid curve function. ΔT_m is the difference in T_m values calculated for reactions with and without compounds.

AlphaScreen assay

Ligands were evaluated for their ability to disrupt the interactions between RORγ LBD and the SRC1-4 coactivator peptide utilizing AlphaScreen technology (Perkin Elmer) using a histidine detection kit (Nickel Chelate) from Perkin Elmer. All reactions contained 200 nM receptor LBD bound to nickel acceptor beads (5 μg/mL) and 50 nM biotinylated SRC1-4 peptide bound to streptavidin donor beads (5 μg/mL) in the presence of the indicated amounts of candidate compounds. Compound concentrations varied from 10 nM to 20 μM in the dose-response assay. The AlphaScreen assay buffer contained 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and 0.1 mg/mL bovine serum albumin (BSA) at a pH of 7.4. The N-terminal biotinylated coactivator peptide SRC1-4 sequence was QKPTSGPQTPQAQQKSLLQQLLTE.

Cell transient transfection assays

293 T cells were plated into 96-well plates at 1.5 × 10⁴ per well (100 μL/well) and grown at 37 °C under a humidified 5% CO₂ atmosphere and maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) for 24 h. For Gal4-RORγ driven reporter assays, 293 T cells were transfected with 25 ng Gal4-RORγ-LBD plasmid, 25 ng pG5-luc reporter plasmid and 5 ng Renilla luciferase expression plasmid per well. For RORα, RORβ, LXRα, FXR, similar procedures were performed. The cells were transiently transfected in Opti-MEM medium using a DNA (μg) to Lipofectamine 2000 (Invitrogen) transfection reagent (μL) ratio of 1:3. Five hours after transfection, tested compounds were added. Cells were harvested after another 24 h for a luciferase assay using the dual-luciferase reporter assay system (Promega). Luciferase activities were normalized to Renilla activity, which was cotransfected as an internal control.

Crystallization, data collection, and structure determination

The RORγ LBD (C455E) protein was mixed with 5 equiv of ligand and incubated on ice for 40 min. Crystals of RORγ LBD (C455E) with compound 13e were grown at 20 °C in sitting drop vapor diffusion by mixing 2 μL of the protein (10 mg/mL) with 1 μL of reservoir solution containing 0.2 M (NH₄)₂SO₄, 20% PEG8000, 0.1 M BisTris, pH 7.2. Most crystals could be observed after 2 days of incubation and grew to full size approximately 2 weeks. The crystals were cryoprotected using the well solution supplemented with additional ethylene glycol and flash frozen in liquid nitrogen. X-ray diffraction data were collected on beamline BL19U1 at Shanghai Synchrotron Radiation Facilities (SSRF) at 100 K. All data were processed (indexing and integration) using the program MOSFLM [43] and scaled using Aimless from the Collaborative Computational Project 4 (CCP4) program suite [44]. Molecular replacement was performed with the CCP4 program Phaser [45] using RORγ LBD complex structure (PDB code: 3B0W) as a search model. The model was refined using CCP4 program REFMAC5 [46] and rebuilt with COOT [47]. The quality of the model was assessed using MolProbity [48]. PyMOL was utilized to prepare the figures. The statistics of data collection and the model refinement are summarized in Supporting Information Table S2. Crystal of 13e with RORγ LBD (C455E) diffracted to resolutions of 2.57 Å. The coordinates were deposited in the PDB with the code 7XQE.pdb.

Prostate cancer cell culture, cell viability, and cell colony formation assays

Prostate cancer cell lines LNCaP, C4-2B and 22Rv1 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified incubator containing 5% CO₂. For cell viability, cells were seeded in 384 opaque-walled plates (Corning, 3570) at 500–1000 cells per well (optimum density for growth) in 20 μL of culture medium. After 12 h, ten indicated concentrations ranging from 5 nM to 100 μM of the test compounds were added into the 384 opaque-walled plates. After incubation for 96 h, 25 μL of Cell-Titer GLO reagent (Promega) was added into each well, and plates were shaken for 10 min. Luminescence was measured on an EnSpire multimode plate reader (PerkinElmer), according to the manufacturer’s instructions. The in vitro half-maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 7 software.

For colony formation, C4-2B and 22Rv1 cells were seeded in 6-well plates with each well contained 1500 cells in 2 mL of the medium. Subsequently, cells were treated with vehicle or indicated concentrations of 13e and incubated for additional 14 days at 37 °C. The medium was removed and the plates were washed with 2 mL PBS for one time. The cell colonies were stained with 2.5% crystal violet in MeOH for 2 h at room temperature, washed crystal violet with water and allowed dishes to dry. Digital images were photographed with an HP scanner.

Analysis of mRNA expression in cells

LNCaP cells were seeded into 12-well plates at 3.0 × 10⁵ cells per well. After 24 h, compounds were added at the indicated doses and treated for 48 h. Subsequently, total RNA was isolated with TRIzol reagent (Invitrogen, 15596018). The obtained total RNA was further subjected to reversely transcription to obtain cDNA with the Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen, 11141ES60*) for qPCR. The quantitative PCR analyses were performed in triplicate using standard SYBR green reagents (Hieff® qPCR SYBR® Green Master Mix, Yeasen, 11201ES08*). The expression levels of full length AR-FL, PSA (KLK3), KLK2, TMPRSS2, C-MYC and FKBP5 gene were assessed by real-time PCR, normalizing to β-Actin. The primers used were as follows: AR-FL_fwd, ACA TCA AGG AAC TCG ATC GTA TCA TTG C; AR-FL_rev, TTG GGC ACT TGC ACA GAG AT; PSA_fwd, CAC AGG CCA GGT ATT TCA GGT; PSA_rev, GAG GCT CAT ATC GTA GAG CGG; KLK2_fwd, CAA CAT CTG GAG GGG AAA GGG; KLK2_rev, AGG CCA AGT GAT GCC AGA AC; TMPRSS2_fwd, CAA GTG CTC CAA CTC TGG GAT; TMPRSS2_rev, AAC ACA CCG ATT CTC GTC CTC; C-MYC_fwd, GGC TCC TGG CAA AAG GTC A; C-MYC_rev, CTG CGT AGT TGT GCT GAT GT; FKBP5_fwd, GGG AAG ATA GTG TCC TGG TTA G; FKBP5_rev, GCA GTC TTG CAG CCT TAT TC; β-Actin_fwd, GAG AAA ATC TGG CAC CAC ACC; β-Actin_rev, ATA CCC CTC GTA GAT GGG CAC.

PSA luciferase reporter gene assay

LNCaP cells were seeded into each well of 24-well plates and transiently transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. 200 ng PSA-Luc reporter plasmid and 10 ng renilla luciferase expression plasmid per well were cotransfected into LNCaP cells. After 24 h, cells were treated with vehicle or test compounds at the designated concentrations. After further 24 h, the cells were harvested for a luciferase assay using the dual-luciferase reporter assay system (Promega). Luciferase activities were normalized to Renilla activity, which was co-transfected as an internal control.

Western blotting

22Rv1 cells were treated with vehicle or indicated concentrations of tested compounds for 48 h. Subsequently, cells were harvested and lysed in RIPA buffer (Dingguo Changsheng Biotechnology Co.LTD, WB-0071) supplemented with 1% PMSF (phenylmethylsulfonyl fluoride) and 1% protease inhibitor cocktail. The extracted total proteins were separated by SDS–PAGE and transferred onto poly(vinylidene difluoride) (PVDF) membranes. The membranes were incubated for 1 h in blocking buffer (5% non-fat dry milk in 1 x TBST) and then reacted with primary antibodies (AR, Abclonal; A2053; β-action, Cell Signaling, 8H10D10) overnight at 4 °C. Membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h. Finally, the signal was detected using a Minichemi imager.

Cell apoptosis assay

22Rv1 cells (3 × 10⁵ cells/well) were seeded in six-well plates and exposed to 1, 13e, and 14a with different concentrations for 48 h. Subsequently, the cells were harvested by trypsinization and washed with cold PBS. After centrifugation and removal of the supernatant, the cells were resuspended in a 300 μL binding buffer and stained with 5 μL of Annexin V-FITC and 5 μL of PI. After 10 min incubation in the dark at room temperature, the stained cells were analyzed by a BD LSR Fortessa SORP flow cytometer (BD Biosciences, San Jose, CA, USA).

In vivo efficacy studies in 22Rv1 xenograft model in mice

Four-week old male NOD-SCID mice were purchased from the GemPharmatech Co., Ltd (Nanjing, China). All experiments were approved and carried out according to the guidelines of the Care and Use of Laboratory Animals of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (IACUC: 2019006). For establishing tumors, 3 × 10⁶ cells were suspended in total of 100 µL PBS and Matrigel (1:1), and implanted subcutaneously into the right flanks of male NOD/SCID mice. When the tumor volume reached approximately 100 mm³, mice were randomized into groups (n = 5 − 6 per group) and treated with 13e (50 mg/kg), 14a (50 mg/kg) or vehicle via intraperitoneal (ip) injection for 3 weeks. Compound 13e and 14a were dissolved in the solution containing 15% Cremophor EL (Calbiochem), 82.5% PBS and 2.5% DMSO. Tumor volumes were determined according to the formula π/6 (L × l²), by measuring tumor length (L) and width (l) with a caliper every day. Tumor growth inhibition (TGI) was calculated as 100 – 100 × ((T − T₀)/(C − C₀)). T and T₀ are the mean tumor volumes for the teat groups on the last day of treatment and on the first day of treatment, respectively. C and C₀ are the mean tumor volumes for the vehicle control group on the last day of treatment and on the first day of treatment, respectively. Animal activity and body weights were also monitored during the duration of the study to assess potential acute toxicity.

Results and discussion

Identification and validation of 1,2,3,4-tetrahydroquinolines scaffold by high-throughput screening

High throughput screening (HTS) techniques have been a powerful tool in small-molecule drug discovery. The goal of an HTS campaign is not to discover the optimal drug candidate for the target of interest but rather to provide a starting point for exploration by medicinal chemistry [49]. To identify novel scaffolds for targeting RORγ, we performed an HTS using a 12000-compound library purchased from ChemDiv and employed thermal shift assay (TSA) to detect. TSA proved quite effective as a method to identify the melting temperature (T_m) of a protein and the change in melting temperature (∆T_m) induced by the binding of ligands. We used TSA to evaluate RORγ inverse agonists for their ability to bind and stabilize proteins. At the end of the screening process, a total of 24 compounds stabilized RORγ by ≥ 2 °C (ΔT_m ≥ 2 °C), the criterion established for selection. Among them, compound 6 (Fig. 1) bearing a 1,2,3,4-tetrahydroquinolines scaffold showed the best activity with a thermal shift of 7.3 °C. A complete list of molecules identified is included as Supporting Information (Table S1). To further evaluate the inhibitory potential of compound 6 from the HTS, transactivation activity was assessed by the luciferase reporter assay. The luciferase reporter assay can test the activity of compounds on RORγ transcription. The results showed that compound 6 displayed low micromolar level activity with an IC₅₀ value of 1.45 μM.

To investigate the receptor-ligand interaction details, the binding mode of compound 6 bound to RORγ LBD was predicted by molecular docking, as demonstrated in Fig. 2a. In compound 6, the 1,2,3,4-tetrahydroquinoline core forms an edge-to-face π − π interaction with Phe378. The NH of the sulfonamide group forms a direct hydrogen bond with Phe377. The 2,4 difluorophenyl group in the left side of the 1,2,3,4-tetrahydroquinoline core extends toward the Arg367 pocket, which forms van der Waals with the hydrophobic residues of the central portion of the pocket. Besides, the 4-fluorophenyl group in the upper side of the 1,2,3,4-tetrahydroquinoline core occupies an existed vacant cavity between helix 11 (H11) and helix 11′ (H11′), and forms an edge-to-face π − π interaction with His479. These results demonstrated that compound 6 bearing 1,2,3,4-tetrahydroquinoline scaffold can be used as a promising starting point for the development of RORγ inverse agonists. To develop compounds with improved potency for RORγ, we focused on either the left side or upper side parts of the core, and performed an extensive SAR study in an effort to enhance the protein-ligand interactions.

Fig. 2. 3D presentation of binding modes of ligands with RORγ LBD (PDB code: 4QM0).

a Predicted binding mode of compound 6 (green) in complex with RORγ LBD. b Predicted binding mode of compound 12b (pink) in complex with RORγ LBD. H-bonds and π − π stacking interactions are shown as yellow and green dashed lines, respectively.

Chemistry

The syntheses of the 1,2,3,4-tetrahydroquinoline derivatives are illustrated in Scheme 1. 7-Nitro-1,2,3,4-tetrahydroquinoline (8) was prepared by the nitration reaction starting from the commercially available 1,2,3,4-tetrahydroquinoline (7). Compound 8 was treated with the different sulfonyl chlorides to generate final sulfonamides 9a-k, which were subsequently reduced with Fe to obtain the corresponding amines 10a-k. Compounds 10a-k were treated with the appropriate sulfonyl chlorides or carboxylic acids to generate final sulfonamides 11a-k, 13a-j and amides 12a-b, 14a-c.

Scheme 1.

Synthesis of 1,2,3,4-tetrahydroquinoline derivatives 11a-k, 13a-j, 12a-b, and 14a-c^a. ^aReagents and conditions: (a) H₂SO₄, HNO₃, 0 °C, 3 h, 76%; (b) R₁SO₂Cl or R₂SO₂Cl, pyridine, 80 °C, 4 h, 20%–90%; (c) Fe, AcOH, NH₄Cl, DMF/H₂O, 80 °C, 1 h, 80%–95%; (d) R₁COOH, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIPEA), dichloromethane (DCM), room temperature (rt), overnight, 35%–47%.

Structure-activity relationships of N-substituent derivatives at 7-position of the 1,2,3,4-tetrahydroquinoline scaffold

Our preliminary modeling suggested that the initial lead, 6, could nicely fit into the RORγ binding pocket and maintain the key interactions with RORγ LBD. The 2,4-difluorophenyl attached to the sulfonamide stretch to the polar region around Arg367, and there is still some space near Arg367 region for further optimization. In our initial efforts, we focused on the SAR at 7-position of the 1,2,3,4-tetrahydroquinoline scaffold. The influence of phenyl substitution at the sulfonyl moiety was first explored. As shown in Table 1, compound 11a, with a phenyl and compound 11b, with a 4-fluorophenyl showed similar activity as that of 6. The substitution at the para position of the phenyl group with trifluoromethyl (11c), trifluoromethoxy (11d) and nitro (11e) exhibited weaker potency than 11b. When the methyl ester group was introduced at the phenyl, compound 11f with a para-position substituent showed encouraging activity with an IC₅₀ value of 0.89 μM. Compound 11g, which bears a meta-position methyl ester group, was less potent than 11f. When two halogen atoms (11h) were presented as 2,4-substitutions, the activity was similar than that of 11g. Next, we explored replace the phenyl moiety in the left side with heteroaromatic rings. The resulting compounds 11i, 11j and 11k exhibited IC₅₀ values ranging from 0.74 μM to 1.68 μM and a thermal shift of over 5 °C. To obtain more potent compounds, we further explore the substitutions at the amide moiety. When the 4-fluorophenyl group was introduced, the resulting compound 12a showed decreased activity (IC₅₀ = 3.69 μM). Encouragingly, introduction of the ethyl sulfonyl at the para-position of the benzyl group (12b) led to a large increased potency with an IC₅₀ value of 0.03 μM and a thermal shift of 10.4 °C. Overall, 12b was the most potent compound in the first-round optimization.

Table 1.

SAR of R₁-group substitutions.

Compd	X	R1	Gal4-RORγ-LBDa	TSAb
			IC50 (μM)	ΔTm (°C)
6	SO2		1.45 ± 0.23	7.3
11a	SO2		1.05 ± 0.00	6.0
11b	SO2		1.08 ± 0.24	5.8
11c	SO2		3.94 ± 0.49	0
11d	SO2		3.69 ± 0.24	8.0
11e	SO2		6.85 ± 1.72	7.9
11f	SO2		0.89 ± 0.09	4.4
11g	SO2		2.61 ± 0.03	6.5
11h	SO2		2.86 ± 0.07	8.5
11i	SO2		1.68 ± 0.20	6.0
11j	SO2		0.74 ± 0.15	7.3
11k	SO2		0.79 ± 0.07	6.0
12a	CO		3.69 ± 0.01	2.3
12b	CO		0.03 ± 0.01	10.4

^aIC₅₀ values were calculated from the luciferase assay. The data are calculated as mean ± standard deviation (SD) from at least two independent experiments.

^bΔT_m values were calculated from the thermal shift assay.

To elucidate the improved potency of 12b at the molecular level, the molecular docking was performed (Fig. 2b). It was shown that the core structure and 4-fluorophenyl sulfonyl group of 12b adopted the same binding mode as that of 6. The most significant difference between these two structures is the left moiety of the core structure. Our docking study revealed that the ethyl sulfone moiety of 12b forms H-bond interactions with the residues Leu287 and Arg367, which may contribute to the improved potency of 12b.

Structure-activity relationships of N-substituent derivatives at 1-position of the 1,2,3,4-tetrahydroquinoline scaffold

Analysis of the predicted binding mode for compound 6 in complex with RORγ LBD revealed that the 4-fluorophenyl attached to the sulfonamide extends toward the His479 region. Subsequently, we turned our attention to explore the SAR of N-sulfonyl group at 1-position of the 1,2,3,4-tetrahydroquinoline scaffold and this data are summarized in Table 2. Replacement of the fluorine (6) with tert-butyl (13a) could not obviously affect the RORγ inhibitory potency. When the methyl ester group was introduced the phenyl ring, the ortho-substituted compound 13c (IC₅₀ = 0.16 μM) showed more potent inhibitory activity than the meta-substituted compound 13b (IC₅₀ = 1.15 μM). 13d, which bears a meta-position methylsulfonyl group, exhibited similar potency to the initial hit. Next, modification was focused on benzyl group and different substituents were introduced to the benzyl group (Table 2). Introducing the trifluoromethyl group at the para-position of the benzyl group resulted in compound 13e, which exhibited with an IC₅₀ value of 0.55 μM and a thermal shift of 8.1 °C. Replacing the trifluoromethyl group with the methyl ester group (13f) led to a 11.3-fold decrease of activity compared with 13e. When the fluoro was inserted at ortho, meta, and para positions of the benzyl group, the para-substituted derivative showed the best activity (13g > 13i > 13h, IC₅₀ = 0.75, 2.83, and 7.79 μM, respectively). Compound 13j (IC₅₀ = 0.77 μM), which bears a meta-position methyl ester group, demonstrated a similar potency compared with 13e.

Table 2.

SAR of R₂-group substitutions.

Compd	R2	Gal4-RORγ-LBDa	TSAb
		IC50 (μM)	ΔTm (°C)
6		1.45 ± 0.23	7.3
13a		2.36 ± 0.16	5.2
13b		1.15 ± 0.11	9.3
13c		0.16 ± 0.01	7.5
13d		1.17 ± 0.19	4.8
13e		0.55 ± 0.12	8.1
13f		6.20 ± 0.07	6.5
13g		0.75 ± 0.15	9.3
13h		7.79 ± 0.30	6.9
13i		2.83 ± 0.24	6.8
13j		0.77 ± 0.11	8.7

^aIC₅₀ values were calculated from the luciferase assay. The data are calculated as mean ± standard deviation (SD) from at least two independent experiments.

^bΔT_m values were calculated from the thermal shift assay.

To understand the structural basis for the high activity of 13e against RORγ, we successfully determined the cocrystal structure of 13e bound to the RORγ LBD (Fig. 3 and Supporting Information, Table S2). Examination of the X-ray co-crystal structure of 13e with RORγ revealed that binding mode of the 1,2,3,4-tetrahydroquinoline core and 2,4 difluorophenyl moiety were consistent with our initial molecular docking prediction. The central 1,2,3,4-tetrahydroquinoline ring resides in the hydrophobic region lined with Phe378, Phe388 and H323 and forms an edge-to-face π − π interaction with Phe388. The NH of the C-7 sulfonamide group makes an H-bond interaction with the backbone carbonyl of Phe377. The 4-trifluoromethylbenzyl occupies the hydrophobic cavity surrounded by residues His323, Leu324 and Met358, which may further push helix 12 (H12), contributing to the antagonistic conformation. H11 and H12 have not been resolved in this crystal structure.

Fig. 3. Cocrystal structure of compound 13e in complex with the RORγ LDB (PDB code: 7XQE).

a Compound 13e is well defined by the electron density and fits snugly in the ligand binding pocket. The electrostatic potential surface is shown. b 13e forms extensive interactions with the RORγ LDB. H-bonds and π − π stacking interactions are shown as yellow and green dashed lines, respectively.

We next combined the favorable modification at the C-7 position with the better substitutions at C-1 position, and designed and synthesized compounds 14a, 14b, 14c (Table 3). Compounds 14b and 14c showed good inhibitory activity with IC₅₀ values ranging from 42 nM to 70 nM and ΔT_m > 9 °C. Among these analogues, compound 14a stood out with nanomolar potency in the luciferase assay with an IC₅₀ of 4 nM, approximately 362-fold more potent than 6, and a thermal shift of 10.2 °C.

Table 3.

SAR of R₂-group substitutions.

Compd	R2	Gal4-RORγ-LBDa	TSAb
		IC50 (μM)	ΔTm (°C)
14a		0.004 ± 0.001	10.2
14b		0.07 ± 0.05	11.2
14c		0.042 ± 0.014	9.6

^aIC₅₀ values were calculated from the luciferase assay. The data are calculated as mean ± standard deviation (SD) from at least two independent experiments.

^bΔT_m values were calculated from the thermal shift assay.

To elucidate the potential binding mode, we predicted the binding mode of 14a to RORγ LBD (PDB code: 4QM0) by molecular docking (Fig. 4). The molecular docking analysis indicated that the two oxygen atoms of ethylsulfonyl group form H-bonding interactions with the residues Leu287 and Arg367. The rest of 14a adopts a similar binding mode to that of 13e.

Fig. 4. Predicted binding mode of compound 14a (yellow) in complex with RORγ LBD (PDB code: 4QM0).

H-bonds and π − π stacking interactions are shown as yellow and green dashed lines, respectively.

Nuclear receptor selectivity profile

To investigate the selectivity against other ROR isoforms and farnesoid X receptor (FXR), liver X receptor LXR-α, compounds 12b, 13e and 14a were assessed in transcriptional reporter assays. As shown in Table 4, all the tested compounds did not show any inhibitory activity against RORα, RORβ, FXR, and LXRα (IC₅₀ > 20 μM, data not shown). The results demonstrated that all the compounds showed excellent selectivity for RORγ versus the other nuclear receptors.

Table 4.

Potency and selectivity profiles.

Compd	Gal4-RORγ-LBDa IC50 (μM)					AlphaScreenb IC50 (μM)
	RORγ	RORα	RORβ	LXRα	FXR	RORγ
12b	0.03 ± 0.01	>20	>20	>20	>20	0.46 ± 0.17
13e	0.55 ± 0.12	>20	>20	>20	>20	0.26 ± 0.02
14a	0.004 ± 0.001	>20	>20	>20	>20	0.32 ± 0.07

^aTranscriptional activities were measured using reporter gene assays in 293 T cells with Gal4-RORγ-LBD, Gal4-RORα-LBD, Gal4-RORβ-LBD, Gal4-LXRα-LBD, and Gal4-FXR-LBD expression vectors.

^bIn vitro binding to the RORγ LBD was measured using AlphaScreen. IC₅₀ values are calculated as mean ± standard deviation (SD) from at least two independent experiments.

To further validate that the functional changes in gene transcription observed with compounds resulted from specific ligand interactions with RORγ, we characterized compounds 12b, 13e and 14a using an AlphaScreen assay (Table 4). The AlphaScreen assay monitors the ability of a ligand to disrupt the interaction of RORγ LBD and its coactivator peptide (SRC1-4). The results showed that all the tested compounds act as RORγ inverse agonist and potently disrupt the interaction between the RORγ LBD and SRC1-4, with IC₅₀ values ranging from 0.26 μM to 0.46 μM.

Inhibitory effects of inverse agonists on cell growth, apoptosis, migration, gene and protein expression in prostate cancer cells

To investigate the antitumor activities of the 1,2,3,4-tetrahydroquinoline analogues, compounds 12b, 13e and 14a were evaluated for their antiproliferative activities against a panel of human prostate cancer cell lines such as LNCaP, C4-2B and 22Rv1, which exhibit distinct expression levels of AR and AR variants. Compound 1 was included as reference. As shown in Table 5, all the compounds exhibited moderate growth inhibition efficacies against these AR-positive cell lines, with an IC₅₀ ranging from 3.55 μM to 12.78 μM. All of the tested compounds showed higher potency than the second-generation antiandrogen enzalutamide in cell growth inhibition against AR-positive prostate cancer cell lines.

Table 5.

Anti-proliferation effects of inverse agonists against the AR-positive prostate cancer cell lines LNCaP, C4-2B and 22Rv1.

Compd	Cell viability IC50 (μM)a
	LNCaP	C4-2B	22Rv1
Enzalutamide	33.84	20.77	42.31
1	8.93 ± 0.71	6.06 ± 0.08	6.74 ± 0.10
12b	9.35 ± 0.06	12.17 ± 0.77	10.70 ± 0.18
13e	6.42 ± 0.53	8.64 ± 2.73	6.78 ± 0.10
14a	3.55 ± 0.73	12.78 ± 0.95	8.47 ± 1.54

^aThe IC₅₀ was calculated from cell viability assay by Cell-Titer GLO (Promega). The data are calculated as mean ± standard deviation (SD) from at least two independent experiments.

The antiproliferative activities of compounds 12b and 14a were further evaluated in a wide range of cancer cell lines and normal cell lines (Supporting Information, Table S3). As shown in Table S3, compounds 12b and 14a showed weak inhibitory activities against Hs578T (triple negative breast carcinoma cell line), HT-29 (colon cancer cell line), A549 (non-small cell lung cancer cell line) and U2OS (osteosarcoma cell line). However, ompounds 12b and 14a were sensitive to MV4-11 (acute myeloid leukemia), with an IC₅₀ value of 5.58 and 3.99 μM, respectively. Importantly, both compounds did not show apparent inhibitory activities (IC₅₀ > 35 μM) against the normal cells HFL-1 (lung fibroblast cell line) and HL-7702 (hepatic epithelial cell line), which suggested no general toxicity. Overall, the compounds displayed preference for the AR-positive prostate cancer cell lines and acute leukemia cell line MV4-11.

The antitumor efficacy of compound 13e was further explored using a long-term survival assay that monitors colony-forming capacity in vitro. As shown in Fig. 5a, compound 13e dose-dependently inhibited colony formation significantly in C4-2B and 22Rv1 cells. Indeed, the number of cell clones progressively decreased and were almost absent in the presence of 4 μM 13e in both cell lines, consistent with its potency in cell viability assays.

Fig. 5. Compound 13e suppressed colony formation and expression of AR, AR-regulated gene and protein.

a Compound 13e inhibited the colony formations of C4-2B and 22Rv1 prostate cancer cells. Cells were cultured and treated with vehicle (DMSO), 2 μM, 4 μM, or 8 μM for 14 days followed by staining. b qRT-PCR analysis was performed to evaluate mRNA expression in LNCaP cells treated with vehicle (DMSO), 5 μM 1, 13e for 48 h. c PSA transcriptional activity was evaluated using luciferase reporter assays in LNCaP cells transfected with a PSA-luc reporter plasmid. Cells were treated with vehicle or with 0.4, 2, or 10 μM 13e. Data are expressed as the mean ± sem (n = 3): *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. d Western blot analysis of AR (AR-FL) in 22Rv1 cells after 48 h treatment with compound 13e at indicated concentrations. β-actin was used as the loading control.

We subsequently performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis to evaluate the ability of compound 13e to suppress the mRNA levels of AR, AR-regulated genes and other oncogenes in prostate cancer cell line LNCaP (Fig. 5b). Our data demonstrated that compound 13e effectively suppress the mRNA expression of the AR-regulated genes PSA (also known as KLK3), KLK2, FKBP5 and TMPRSS2 in LNCaP cells. In addition, compound 13e was also very effective in suppressing the expression of C-MYC gene in LNCaP cells. Besides, full-length AR (AR-FL) was suppressed at mRNA level following treatment with 13e in LNCaP cells.

Prostate-specific antigen (PSA) is a glycoprotein enzyme secreted by the prostate gland, and a powerful biomarker widely used in the diagnosis of prostate cancer. Therefore, we evaluated the effects of 13e on prostate-specific antigen (PSA) promoter in LNCaP cells using PSA luciferase assay. As shown in Fig. 5c, compound 13e dose-dependently inhibited the PSA reporter activity. Moreover, Western blotting analyses demonstrated that compound 13e effectively reduce AR-FL and AR variants (AR-VS) levels in 22Rv1 cells (Fig. 5d).

Further, a wound healing assay was carried out to determine whether 13e and 14a could inhibit the migration of 22Rv1 cells (Fig. 6). The results showed that 13e and 14a could significantly inhibit the migration of 22Rv1 cells in the wound healing assay after incubation for 48 h. Compound 1, a positive control, displayed lower migratory inhibitory potency than compounds 13e and 14a.

Fig. 6. Compounds 13e and 14a inhibited the migration of 22Rv1 cells.

a Representative images of the wound healing assay using 22Rv1 cells. which were treated with 10 μM DMSO, 1, 13e or 14a for 48 h. b Quantitative analysis of wound healing. The data are expressed as the mean ± sem (n = 3). *P < 0.05, ***P < 0.001 by two-tailed Student’s t test.

To investigate whether compounds 13e and 14a can lead to cell death by apoptosis, a flow cytometry based apoptosis assay was performed. 22Rv1 cells were treated with tested compounds, stained with annexin V-FITC and propidium iodide (PI), and subsequently analyzed by flow cytometer. Compound 1 was used as a reference compound. As shown in Fig. 7, 13e and 14a effectively induced the apoptosis of 22Rv1 cells in a dose-dependent manner. The percentage of apoptotic cells increased from 1.23% (control) to 34.80% (5 μM) and 61.70% (10 μM) after 13e treatment, respectively. Compound 14a induced the apoptosis of 22Rv1 cells with apoptotic rates of 21.95% at 5 μM and 45.6% at 10 μM. Both compounds displayed a higher level of apoptosis compared to compound 1.

Fig. 7. Compounds 13e and 14a induced apoptosis in 22Rv1 cells.

22Rv1 cells were treated with 1, 13e, or 14a at the indicated concentrations for 48 h. The apoptosis was examined by flow cytometer.

These results indicated that the RORγ inverse agonists 13e and 14a could effectively inhibit cell growth, migration, apoptosis and gene expression in prostate cancer. Therefore, targeting RORγ may represent an alternative strategy for the treatment of prostate cancer.

Compounds 13e and 14a reduce prostate tumor growth in vivo

To evaluate the antitumor efficacy of the present RORγ inverse agonists in vivo, we investigated the suppressive effects of 13e and 14a on prostate tumor growth in the 22Rv1 xenograft model in mice. Tumor-bearing mice were treated with 13e and 14a at dose of 50 mg/kg by intraperitoneal injection. Compared with vehicle control group, compound 13e at the dose of 50 mg/kg significantly suppressed the tumor growth during the treatment period (Fig. 8a), with tumor growth inhibition (TGI) value of 76.3%. Mice treated with compound 14a also demonstrated growth inhibition at a dose of 50 mg/kg when compared with the vehicle control group, with tumor growth inhibition (TGI) value of 43.4% (Fig. 8a). These data were further confirmed by the average tumor weight of the two treatment groups after the tumors were collected and weighed (Fig. 8b). In addition, both compounds did not cause obvious body weight loss or other signs of toxicity in this experiment (Fig. 8c).

Fig. 8. In vivo effect of compounds 13e and 14a on tumor volume, tumor weight, and body weight in xenograft model of 22Rv1.

a Antitumor efficacy of compounds 13e and 14a in a 22Rv1 prostate cancer xenograft mouse model. b Tumor weight of each treatment group at the end point of treatment. c Body weight change in 22Rv1 xenograft study. Mice were treated with intraperitoneal (ip) injections of vehicle or compounds 13e (50 mg/kg) and 14a (50 mg/kg) for 3 weeks and were monitored. Data are expressed as the mean tumor volume ± sem of the animals in each treatment group. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test.

Conclusions

In this study, we report the structure-based design, synthesis, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists starting from high throughput screening approach. Our efforts have yielded highly potent and efficacious RORγ inverse agonists. The represent compounds 13e (XY039) and 14a (XY077) effectively inhibited RORγ transcription activity and exhibited high selectivity for RORγ versus the other NRs in our selectivity panel. The X-ray crystal structure of RORγ in complex with 13e highlighted the structural determinants for potent inhibitory activity. In vitro, both of compounds potently inhibited cell growth, and expression of AR and AR regulated genes in AR positive prostate cancer cell lines. In vivo, 13e and 14a could effectively inhibited the tumor growth in the 22Rv1 xenograft model in mice, especially 13e. Taken together, our data demonstrated that the present RORγ inverse agonists are promising candidates for potential treatment of prostate cancer.

Author contributions

YX, XSW, YZ and XYL conceived and designed the research. XSW, XYL, CCL, XFZ, CZ, XSC, ZFL, TW, HHY, CP, QQH, HS conduct the research. XSW wrote the manuscript. YX, XSW, YZ and XYL revised the revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-024-01274-z.