Design, synthesis and pharmacological characterization of N-(3-ethylbenzo[d]isoxazol-5-yl) sulfonamide derivatives as BRD4 inhibitors against acute myeloid leukemia

Abstract

BRD4 plays a key role in the regulation of gene transcription and has been identified as an attractive target for cancer treatment. In this study, we designed 26 new compounds by modifying 3-ethyl-benzo[d]isoxazole core with sulfonamides. Most compounds exhibited potent BRD4 binding activities with ΔT_m values exceeding 6 °C. Two crystal structures of 11h and 11r in complex with BRD4(1) were obtained to characterize the binding patterns. Compounds 11h and 11r were effective for BRD4(1) binding and showed remarkable anti-proliferative activity against MV4-11 cells with IC₅₀ values of 0.78 and 0.87 μM. Furthermore, 11r (0.5−10 μM) concentration-dependently inhibited the expression levels of oncogenes including c-Myc and CDK6 in MV4-11 cells. Moreover, 11r (0.5−10 μM) concentration-dependently blocked cell cycle in MV4-11 cells at G₀/G₁ phase and induced cell apoptosis. Compound 11r may serve as a new lead compound for further drug development.

Introduction

Bromodomain and extra-terminal (BET) family consists of four members including BRD2, BRD3, BRD4, and BRDT. All the BET proteins contain two structurally similar bromodomains (BD1 and BD2), which can bind to acetyl-lysine (K_Ac) residues on histone tails of chromatin. Thus, the bromodomain-containing proteins act as epigenetic “readers” by binding to K_Ac via the bromodomain to regulate gene expression [1]. Each bromodomain has two loop regions (ZA and BC loops) [2] and a hydrophobic area named as “WPF shelf” [3]. The entire structure constitutes the K_Ac binding site.

BRD4 is a well-studied member of BET family. It can recruit the positive transcription elongation factor b (P-TEFb) to the promoter and activate RNA polymerase II [4, 5]. In addition, BRD4 can promote the transcription of key genes (Bcl-2, c-Myc and CDK6) [6,7,8], which plays an essential role in the proliferation and cell cycle progression of tumor cells. Therefore, the expression of key oncogenes can be inhibited through the displacement of BRD4 from chromatin. Accumulative evidence indicates that BRD4 protein has been implicated in various human diseases, including acute myeloid leukemia (AML) [8,9,10], prostate cancer [11, 12], breast cancer [13,14,15], gastrointestinal stromal tumor (GIST) [16], neuroblastoma [17], pancreatic cancer [18, 19], cholangiocarcinoma [20] as well as inflammations [21, 22].

Multiple BRD4 inhibitors have been reported by researchers, some of which are undergoing clinical trials for cancer therapy. (+)-JQ1 (1, Fig. 1) was the first potent BRD4 bromodomain inhibitor with a triazolothienodiazepine skeleton [23], which was widely used as a probe to explore the biological function of BET proteins in treatment of various human diseases. The structurally related derivatives OTX-015 (Supplementary Fig. S1a) [24] and I-BET762 (Supplementary Fig. S1b) [25] have completed the phase 1 clinical trials for malignancies. 3,5-Dimethylisoxazole was another preferred scaffold, which was firstly reported by Hewings [26]. The scaffold has been used for the design of different BRD4 inhibitors (Supplementary Fig. S1c and S1d) [27, 28]. The representative inhibitor I-BET151 (2, Fig. 1) discovered by GSK was reported for the treatment of MLL-fusion leukemia [6]. In addition, several BRD4 inhibitors with different scaffolds, such as PFI-1 (3, Fig. 1) [29, 30], 2-thiazolidinone derivative (Supplementary Fig. S1e) [31], ABBV-075 (Supplementary Fig. S1f) [32] and CPI-0610 (Supplementary Fig. S1g) [33], are undergoing clinical trials for solid tumor and lymphoma with encouraging data. We previously reported two class of BET bromodomain inhibitors containing a benzo[cd]indol-2(1H)-one structure (Supplementary Fig. S1h) [34] and a 3-methyl-benzo[d]isoxazole scaffold (Y06036, 4, Fig. 1) [35], respectively. Compound 4 demonstrated potent and selective binding affinities to BET proteins with good in vitro and in vivo efficacy against prostate cancer [35]. The X-ray diffraction of 4-BRD4 crystal structure also provided solid evidence for further structure-based optimization. Despite the structural differences of various inhibitors, they share similar warheads that can mimic the K_Ac.

Fig. 1: Chemical structures of representative BRD4 inhibitors.

The listed inhibitors were (+)-JQ1 (1), I-BET151 (2), PFI-1 (3) and Y06036 (4).

BRD4 inhibitors are undoubtedly potential anticancer drugs. However, the existing inhibitors are often accompanied by side effects of thrombocytopenia, fatigue, and diarrhoea [36]. None of the BRD4 inhibitors received approval in the past decade, which meant there was no obvious “low-hanging fruit” of their development. Besides, BRD inhibitors are not the “magic bullet” for treatment of all cancers as we expected and they are likely to work in selective cancer types [36]. Different chemical structures may cause nuances for BRD inhibition and show different effect in tumor types or toxicity profile. Enriching the structural library of inhibitors and performing rational combinations may be a potential direction for the research of BET inhibitors in the future. Thus, the development of new BRD4 inhibitors still attracts attentions. The fine structure-activity relationships (SARs) are needed to explore for providing inhibitors with different cellular pharmacological profiles and clinical application. In this work, we report the design and evaluation of a class of new BRD4 inhibitors with a 3-ethyl-benzo[d]isoxazole scaffold for potential treatment of AML. The biological functions of the compounds were evaluated by thermal shift assay, crystallography analysis, cell viability assay, RT-PCR technology, Western bolt and flow cytometry.

Materials and methods

Chemistry

The reagents and solvents were purchased from commercial sources and used without further purification. ¹H NMR and ¹³C NMR spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) with DMSO-d₆ or CDCl₃ as the solvent. Chemical shifts are given in ppm throughout. Coupling constants (J) are expressed in hertz (Hz). NMR chemical shifts (δ) are reported in parts per million (ppm) units. The high-resolution mass spectra were performed on a UPLC G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). TLC analysis was performed on GF254 silica gel plates (Qingdao Haiyang Chemical, China) under ZF-20D UV light (254 nm) (Yuhua instrument, Gongyi, China) or I₂ in silica gel for monitoring all reactions. Flash column chromatography was performed on silica gel (300–400 mesh). Melting point was measured (uncorrected) on X-6 melting point apparatus (Beijing Tech, China). Purity of all compounds was determined by LC-20AT prominence high-performance liquid chromatography (HPLC) (SHIMADZU, Kyoto, Japan) using an InertSustain C18 column (150 mm × 4.6 mm, 5 μm) with the 65% solvent A (MeOH) and 35% solvent B (H₂O) as eluents. The flow rate was set as 0.8 mL/min and the signals were monitored by a SPD-20A prominence UV/VIS detector at 254 nm. The purity of all the final compounds was determined by HPLC to be >97%.

N-(4-hydroxy-2-methoxy-5-propionylphenyl)acetamide (7)

N-(2, 4-dimethoxyphenyl) acetamide 6 (13.0 g, 66.6 mmol, synthesized according to previous procedure [35]) and propionyl chloride (18.5 g, 199.8 mmol) was dissolved in DCM (40 mL). Anhydrous AlCl₃ (35.5 g, 266.4 mmol) was added in portions with vigorous stirring under ice-cooling. The reaction temperature was raised to 43 °C and stirred for 3 h. After the reaction was completed, the mixture was slowly added dropwise to crushed ice with stirring. The organic layer was separated, and the solvent was evaporated under reduced pressure. Dilute hydrochloric acid was added dropwise to the residue and the mixture was stirred for 0.5 h. The solid was filtered and washed with hydrochloric acid and water, and then dried to obtain the target compound as a pale green solid (12.8 g, 81.0% yield). ¹H NMR (400 MHz, DMSO-d₆), δ: 12.57 (s, 1H, OH), 9.19 (brs, 1H, NH), 8.30 (s, 1H, ArH), 6.58 (s, 1H, ArH), 3.87 (s, 3H, OCH₃), 2.95 (q, J = 7.2 Hz, 2H, CH₂CH₃), 2.05 (s, 3H, COCH₃), 1.09 (t, J = 7.2 Hz, 3H, CH₂CH₃); MS (ESI), m/z: 238.1 [M + H]⁺.

N-(4-hydroxy-5-(1-(hydroxyimino)propyl)-2-methoxyphenyl)acetamide (8)

Compound 7 (11.0 g, 46.4 mmol), hydroxylamine hydrochloride (6.4 g, 92.7 mmol) and anhydrous sodium acetate (7.6 g, 92.7 mmol) was dissolved in 130 mL of a mixed solvent of ethanol and water (anhydrous ethanol: water = 7: 3, v/v). The mixture was warmed to 80 °C and stirred under reflux for 2 h. After the reaction was completed, the mixture was cooled to room temperature. The mixture was concentrated under reduced pressure to precipitate a solid. Water (100 mL) was then added, and the solid was filtered, washed and dried to obtain the title compound as a pale green solid (10.1 g, 86.4% yield). ¹H NMR (400 MHz, DMSO-d₆), δ: 11.80 (s, 1H, OH), 11.31 (s, 1H, OH), 9.07 (brs, 1H, NH), 7.87 (s, 1H, 3-ArH), 6.55 (s, 1H, 6-ArH), 3.80 (s, 3H, OCH₃), 2.70 (q, J = 7.5 Hz, 2H, CH₂CH₃), 2.03 (s, 3H, COCH₃), 1.08 (t, J = 7.5 Hz, 3H, CH₂CH₃); MS (ESI), m/z: 253.1 [M + H]⁺.

N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)acetamide (9)

Compound 8 (10.5 g, 41.6 mmol) was dissolved in 1, 4-dioxane (45 mL). Next, 24 mL of N, N-dimethylformamide dimethyl acetal (DMF-DMA) was added slowly to the mixture with vigorous stirring. The temperature was raised to 100 °C and stirred for 10 min. After the reaction was completed, dilute hydrochloric acid (50 mL) and water (180 mL) was added to the mixture with stirring to precipitate a solid. The solid was washed and dried to obtain the target compound as a pale green solid (5.5 g, 56.4% yield). ¹H NMR (400 MHz, DMSO-d₆), δ: 9.29 (brs, 1H, NH), 8.27 (s, 1H, 7-ArH), 7.37 (s, 1H, 3-ArH), 3.93 (s, 3H, OCH₃), 2.91 (q, J = 7.6 Hz, 2H, CH₂CH₃), 2.11 (s, 3H, COCH₃), 1.30 (t, J = 7.6 Hz, 3H, CH₂CH₃); MS (ESI), m/z: 235.1 [M + H]⁺.

3-Ethyl-6-methoxybenzo[d]isoxazol-5-amine (10)

Compound 9 (5.0 g, 21.3 mmol) was added to hydrochloric acid (120 mL, 3 mol/L). The reaction mixture was stirred at 90 °C for 3 h. After the reaction was completed, the sodium hydroxide solution was added to adjust the pH to 7–9, which allow the precipitation of a solid. The product was filtered, and the cake was washed with water, dried to obtain the target compound as a brown solid (3.8 g, 92.6% yield). ¹H NMR (400 MHz, CDCl₃), δ: 6.92 (s, 1H, 7-ArH)), 6.82 (s, 1H, 4-ArH), 3.92 (s, 3H, OCH₃), 3.85 (brs, 2H, NH₂), 2.89 (q, J = 7.6 Hz, 2H, CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 159.00, 158.75, 151.19, 133.75, 113.77, 102.82, 91.52, 55.91, 18.90, 12.19; MS (ESI), m/z: 193.1 [M + H]⁺.

General procedure for the synthesis of 3-ethyl-benzo[d]isoxazol-containing sulfonamides 11

To a solution of DCM (6 mL) was added compound 10 (65 mg, 0.34 mmol), sulfonyl chloride (0.41 mmol) and pyridine (0.3 mL). The resulting mixture was stirred at 43 °C for 3–12 h. The reaction was monitored by TLC (ethyl acetate/petroleum ether (1:1, v/v)). On completion of the reaction, the mixture was diluted with 1 mol/L HCl (8 mL) and water (15 mL) and extracted with ethyl acetate (3 × 20 mL). The separated organic phase was washed with water and brine, dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel to give the title compound as a solid.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)ethanesulfonamide (11a)

White solid; yield: 54.9%; m.p.: 122–123 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H, 7-ArH), 7.05 (s, 1H, 4-ArH), 6.78 (brs, 1H, SO₂NH), 3.99 (s, 3H, OCH₃), 3.04 (q, J = 7.4 Hz, 2H, SO₂CH₂CH₃), 2.96 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.41 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃), 1.34 (t, J = 7.4 Hz, 3H, SO₂CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.35, 159.85, 152.37, 123.45, 114.51, 112.75, 92.22, 56.51, 45.51, 18.84, 12.11, 8.12; HRMS (ESI) m/z calcd for C₁₂H₁₆N₂O₄S [M + H]⁺: 285.0831; found: 285.0912. HPLC, t_R = 4.278 min, 98.91% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)propane-1-sulfonamide (11b)

White solid; yield: 39.4%; m.p.: 108–109 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 1H, 7-ArH), 7.05 (s, 1H, 4-ArH), 6.76 (s, 1H, SO₂NH), 3.99 (s, 3H, OCH₃), 3.05 – 2.90 (m, 4H, SO₂CH₂, 3-CH₂CH₃), 1.88 – 1.76 (m, 2H, SO₂CH₂CH₂), 1.41 (t, J = 7.5 Hz, 3H, 3-CH₂CH₃), 0.99 (t, J = 7.4 Hz, 3H, CH₂CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.35, 159.87, 152.40, 123.49, 114.53, 112.75, 92.22, 56.52, 52.94, 18.85, 17.20, 12.92, 12.11; HRMS (ESI) m/z calcd for C₁₃H₁₈N₂O₄S [M + H]⁺: 299.0987; found: 299.1067. HPLC, t_R = 5.636 min, 98.36% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)butane-1-sulfonamide (11c)

White solid; yield: 48%; m.p.: 100–101 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 1H, 7-ArH), 7.05 (s, 1H, 4-ArH), 6.76 (s, 1H, SO₂NH), 3.99 (s, 3H, OCH₃), 3.10 – 2.85 (m, 4H, SO₂CH₂, 3-CH₂CH₃), 1.85 – 1.75 (m, 2H, SO₂CH₂CH₂), 1.51 – 1.28 (m, 5H, 3-CH₂CH₃, SO₂CH₂CH₂CH₂), 0.88 (t, J = 7.0 Hz, 3H, CH₂CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.36, 159.88, 152.39, 123.49, 114.53, 112.79, 92.21, 56.48, 50.94, 25.41, 21.44, 18.85, 13.56, 12.11; HRMS (ESI) m/z calcd for C₁₄H₂₀N₂O₄S [M + H]⁺: 313.1144; found: 313.1224. HPLC, t_R = 8.027 min, 99.03% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11d)

White solid; yield: 47.8%; m.p.: 169–170 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H, ArH), 7.74 – 7.56 (m, 2H, phenyl ArH), 7.56 – 7.30 (m, 3H, phenyl ArH), 6.91 (brs, 1H, SO₂NH), 6.81 (s, 1H, 4-ArH), 3.62 (s, 3H, OCH₃), 2.97 (d, J = 5.5 Hz, 2H, 3-CH₂CH₃), 1.42 (s, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.74, 159.90, 153.16, 138.80, 133.05, 128.77 (2×C), 127.22 (2×C), 122.92, 114.87, 114.36, 91.94, 56.12, 18.88, 12.121; HRMS (ESI) m/z calcd for C₁₆H₁₆N₂O₄S [M + H]⁺: 333.0831; found: 333.0916. HPLC, t_R = 6.890 min, 97.76% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-fluorobenzenesulfonamide (11e)

White solid; yield: 54.4%; m.p.: 179–180 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 7.4 Hz, 2H, 6’-ArH, 7-ArH), 7.58 – 7.43 (m, 1H, 4’-ArH), 7.29 (brs, 1H, SO₂NH), 7.20 – 7.07 (m, 2H, 3’, 5’-ArH), 6.85 (s, 1H, 4-ArH), 3.77 (s, 3H, OCH₃), 2.94 (q, J = 7.5 Hz, 2H, 3-CH₂CH₃), 1.39 (t, J = 7.5 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.60, 160.36 (d, J = 257.6 Hz, F-2’-ArC), 159.83, 157.81 (d, J = 257.6 Hz, F-2’-ArC), 153.03, 135.54 (d, J = 9.1 Hz, F-4’-ArC), 135.45 (d, J = 9.1 Hz, F-4’-ArC), 130.90, 126.83 (d, J = 13.1 Hz, F-1’-ArC), 126.70 (d, J = 13.1 Hz, F-1’-ArC), 124.17 (d, J = 4.0 Hz, F-6’-ArC), 124.13 (d, J = 4.0 Hz, F-6’-ArC), 122.48, 116.87 (d, J = 21.2 Hz, F-3’-ArC), 116.66 (d, J = 21.2 Hz, F-3’-ArC), 114.26, 114.23, 91.90, 56.18, 18.82, 12.09; HRMS (ESI) m/z calcd for C₁₆H₁₅FN₂O₄S [M + H]⁺: 351.0737; found: 351.0817. HPLC, t_R = 6.538 min, 98.90% purity.

2-Chloro-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11f)

White solid; yield: 54.4%; m.p.: 121–122 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.93 (dd, J = 7.9, 1.3 Hz, 1H, 6’-ArH), 7.70 (s, 1H, 7-ArH), 7.55 (brs, 1H, SO₂NH), 7.51 – 7.46 (m, 1H, 3’-ArH), 7.43 (td, J = 7.7, 1.4 Hz, 1H, 4’-ArH), 7.30 – 7.24 (m, 1H, 5’-ArH), 6.85 (s, 1H, 4-ArH), 3.78 (s, 3H, OCH₃), 2.93 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.45, 159.78, 152.83, 136.24, 134.10, 132.06, 131.81, 131.61, 126.81, 122.62, 114.19, 113.57, 91.93, 56.13, 18.81, 12.10; HRMS (ESI) m/z calcd for C₁₆H₁₅ClN₂O₄S [M + H]⁺: 367.0441; found: 367.0522. HPLC, t_R = 8.824 min, 98.63% purity.

2-Bromo-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11g)

White solid; yield: 64.7%; m.p.: 142–143 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.00 – 7.93 (m, 1H, 3’-ArH), 7.73 – 7.67 (m, 2H, 7-ArH, 6’-ArH), 7.64 (brs, 1H, SO₂NH), 7.38 – 7.28 (m, 2H, 4’, 5’-ArH), 6.85 (s, 1H, 4-ArH), 3.78 (s, 3H, OCH₃), 2.93 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.46, 159.78, 152.84, 137.96, 135.14, 134.04, 132.04, 127.39, 122.63, 120.31, 114.18, 113.61, 91.94, 56.08, 18.82, 12.11; HRMS (ESI) m/z calcd for C₁₆H₁₅BrN₂O₄S [M + H]⁺: 410.9936 & 412.9915; found: 411.0012 & 412.9994. HPLC, t_R = 10.020 min, 98.18% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-methoxybenzenesulfonamide (11h)

White solid; yield: 71.7%; m.p.: 142–143 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 7.6 Hz, 1H, 6’-ArH), 7.72 (s, 1H, 7-ArH), 7.59 (brs, 1H, SO₂NH), 7.45 (t, J = 7.7 Hz, 1H, 4’-ArH), 7.00 – 6.90 (m, 2H, 3’, 5’-ArH), 6.86 (s, 1H, 4-ArH), 3.96 (s, 3H, 2’-OCH₃), 3.83 (s, 3H, 6-OCH₃), 2.91 (q, J = 7.5 Hz, 2H, 3-CH₂CH₃), 1.36 (t, J = 7.5 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 160.91, 159.81, 156.45, 152.21, 135.05, 130.94, 126.20, 123.72, 120.27, 114.21, 111.97, 111.80, 91.81, 56.35, 56.10, 18.79, 12.10; HRMS (ESI) m/z calcd for C₁₇H₁₈N₂O₅S [M + H]⁺: 363.0936; found: 363.1018. HPLC, t_R = 7.302 min, 97.86% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-(trifluoromethoxy)benzenesulfonamide (11i)

White solid; yield: 67.9%; m.p.: 92–93 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J = 7.9, 1.5 Hz, 1H, 6’-ArH), 7.73 (s, 1H, 7-ArH), 7.55 (td, J = 8.4, 1.6 Hz, 1H, 4’-ArH), 7.38–7.30 (m, 2H, SO₂NH, 3’-ArH), 7.26 (td, J = 8.0, 0.8 Hz, 1H, 5’-ArH), 6.87 (s, 1H, 4-ArH), 3.79 (s, 3H, OCH₃), 2.93 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.43, 159.82, 152.61, 146.22, 134.92, 131.69, 130.09, 125.90, 124.16 (q, J = 262.6 Hz, OCF₃), 122.56, 121.56 (q, J = 262.6 Hz, OCF₃), 118.97 (q, J = 262.6 Hz, OCF₃), 118.95, 116.37 (q, J = 262.6 Hz, OCF₃), 114.24, 113.24, 91.89, 56.13, 18.81, 12.10; HRMS (ESI) m/z calcd for C₁₇H₁₅F₃N₂O₅S [M + H]⁺: 417.0654; found: 417.0735. HPLC, t_R = 11.895 min, 99.20% purity.

3-Chloro-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11j)

White solid; yield: 53.5%; m.p.: 152–153 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H, 7-ArH), 7.73 (s, 1H, 2’-ArH), 7.51 (d, J = 7.8 Hz, 1H, 6’-ArH), 7.47 (d, J = 8.1 Hz, 1H, 4’-ArH), 7.30 (t, J = 7.9 Hz, 1H, 5’-ArH), 6.95 (brs, 1H, SO₂NH), 6.85 (s, 1H, 4-ArH), 3.68 (s, 3H, OCH₃), 2.98 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.43 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.91, 159.89, 153.22, 140.49, 135.01, 133.12, 130.00, 127.35, 125.39, 122.34, 115.35, 114.45, 92.06, 56.19, 18.87, 12.12; HRMS (ESI) m/z calcd for C₁₆H₁₅ClN₂O₄S [M + H]⁺: 367.0441; found: 367.0518. HPLC, t_R = 12.235 min, 98.62% purity.

3-Bromo-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11k)

White solid; yield: 70.7%; m.p.: 160–161 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (s, 1H, 2’-ArH), 7.77 (s, 1H, 7-ArH), 7.62 (d, J = 7.8 Hz, 1H, 6’-ArH), 7.55 (d, J = 7.7 Hz, 1H, 4’-ArH), 7.23 (t, J = 8.0 Hz, 1H, 5’-ArH), 6.96 (brs, 1H, SO₂NH), 6.85 (s, 1H, 4-ArH), 3.68 (s, 3H, OCH₃), 2.98 (q, J = 7.5 Hz, 2H, 3-CH₂CH₃), 1.43 (t, J = 7.5 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.93, 159.88, 153.25, 140.61, 136.00, 130.20, 130.19, 125.82, 122.71, 122.31, 115.45, 114.44, 92.06, 56.21, 18.87, 12.13; HRMS (ESI) m/z calcd for C₁₆H₁₅BrN₂O₄S [M + H]⁺: 410.9936 & 412.9915; found: 411.0017 & 412.9996. HPLC, t_R = 13.004 min, 97.46% purity.

5-Chloro-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-methoxybenzenesulfonamide (11l)

White solid; yield: 71.4%; m.p.: 157–158 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J = 2.4 Hz, 1H, 6’-ArH), 7.72 (s, 1H, 7-ArH), 7.57 (brs, 1H, SO₂NH), 7.40 (dd, J = 8.8, 2.4 Hz, 1H, 4’-ArH), 6.94 – 6.84 (m, 2H, 4-ArH, 3’-ArH), 3.95 (s, 3H, 2’-OCH₃), 3.84 (s, 3H, 6-OCH₃), 2.94 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.07, 159.85, 155.03, 152.24, 134.64, 130.52, 127.69, 125.57, 123.21, 114.34, 113.20, 112.46, 91.96, 56.50, 56.39, 18.81, 12.10; HRMS (ESI) m/z calcd for C₁₇H₁₇ClN₂O₅S [M + H]⁺: 397.0547; found: 397.0625. HPLC, t_R = 13.742 min, 98.46% purity.

5-Bromo-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-methoxybenzenesulfonamide (11m)

White solid; yield: 43.5%; m.p.: 167–168 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 2.4 Hz, 1H, 6’-ArH), 7.72 (s, 1H, 7-ArH), 7.60 – 7.50 (m, 2H, SO₂NH, 4’-ArH), 6.89 (s, 1H, 4-ArH), 6.83 (d, J = 8.8 Hz, 1H, 3’-ArH), 3.94 (s, 3H, 2’-OCH₃), 3.84 (s, 3H, 6-OCH₃), 2.94 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.39 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.07, 159.87, 155.53, 152.21, 137.59, 133.31, 128.05, 123.21, 114.38, 113.63, 112.43 (2×C), 91.98, 56.46, 56.40, 18.84, 12.15; HRMS (ESI) m/z calcd for C₁₇H₁₇BrN₂O₅S [M + H]⁺: 441.0042 & 443.0021; found: 441.0125 & 443.0107. HPLC, t_R = 15.044 min, 99.11% purity.

5-Bromo-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2,3-dihydrobenzofuran-7-sulfonamide (11n)

White solid; yield: 84.0%; m.p.: 152–153 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (s, 1H, 7-ArH), 7.64 (d, J = 1.5 Hz, 1H, 6’-ArH), 7.41 (brs, 1H, SO₂NH), 7.39 (d, J = 1.5 Hz, 1H, 4’-ArH), 6.90 (s, 1H, 4-ArH), 4.70 (t, J = 8.8 Hz, 2H, OCH₂CH₂), 3.84 (s, 3H, OCH₃), 3.21 (t, J = 8.8 Hz, 2H, OCH₂CH₂), 2.94 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.39 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.11, 159.87, 156.13, 152.23, 132.86, 131.98, 130.16, 123.08, 121.76, 114.26, 112.09, 111.71, 91.98, 73.60, 56.36, 28.88, 18.83, 12.16; HRMS (ESI) m/z calcd for C₁₈H₁₇BrN₂O₅S [M + H]⁺: 453.0042 & 455.0021; found: 453.0123 & 455.0105. HPLC, t_R = 13.697 min, 98.42% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-4-fluorobenzenesulfonamide (11o)

White solid; yield: 61.0%; m.p.: 141–142 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H, 7-ArH), 7.73 – 7.64 (m, 2H, 2’, 6’-ArH), 7.10 – 7.00 (m, 2H, 3’, 5’-ArH), 6.91 (brs, 1H, SO₂NH), 6.85 (s, 1H, 4-ArH), 3.67 (s, 3H, OCH₃), 2.97 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.42 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 166.52, 163.98, 161.85 (d, J = 199.0 Hz, F-4’-ArC), 159.88 (d, J = 199.0 Hz, F-4’-ArC), 153.18, 134.89 (d, J = 4.0 Hz, F-1’-ArC), 134.85 (d, J = 4.0 Hz, F-1’-ArC), 130.03 (d, J = 9.1 Hz, F-2’, 6’-ArC), 129.94 (d, J = 9.1 Hz, F-2’, 6’-ArC), 122.64, 116.14 (d, J = 22.2 Hz, F-3’, 5’-ArC), 115.92 (d, J = 22.2 Hz, F-3’, 5’-ArC), 115.19, 114.46, 92.02, 56.16, 18.87, 12.11; HRMS (ESI) m/z calcd for C₁₆H₁₅FN₂O₄S [M + H]⁺: 351.0737; found: 351.0819. HPLC, t_R = 8.228 min, 98.80% purity.

4-Chloro-N-(3-ethyl-6-methoxybenzo[d]isoxazol-5-yl)benzenesulfonamide (11p)

White solid; yield: 55.3%; m.p.: 177–178 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H, 7-ArH), 7.60 (d, J = 7.4 Hz, 2H, 2’, 6’-ArH), 7.35 (d, J = 7.4 Hz, 2H, 3’, 5’-ArH), 6.92 (brs, 1H, SO₂NH), 6.85 (s, 1H, 4-ArH), 3.66 (s, 3H, OCH₃), 2.98 (d, J = 7.1 Hz, 2H, 3-CH₂CH₃), 1.43 (d, J = 6.7 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.84, 159.86, 153.15, 139.62, 137.31, 129.07 (2×C), 128.66 (2×C), 122.51, 115.18, 114.45, 92.07, 56.18, 18.88, 12.11; HRMS (ESI) m/z calcd for C₁₆H₁₅ClN₂O₄S [M + H]⁺: 367.0441; found: 367.0521. HPLC, t_R = 12.146 min, 97.73% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-4-nitrobenzenesulfonamide (11q)

White solid; yield: 71.4%; m.p.: 168–169 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 8.8 Hz, 2H, 3’, 5’-ArH), 7.87 (d, J = 8.8 Hz, 2H, 2’, 6’-ArH), 7.82 (s, 1H, 7-ArH), 7.02 (brs, 1H, SO₂NH), 6.86 (s, 1H, 4-ArH), 3.66 (s, 3H, OCH₃), 2.99 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.44 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 162.02, 159.83, 153.11, 150.24, 144.62, 128.52 (2×C), 123.99 (2×C), 121.77, 115.71, 114.62, 92.26, 56.24, 18.88, 12.10; HRMS (ESI) m/z calcd for C₁₆H₁₅N₃O₆S [M + H]⁺: 378.0682; found: 378.0764. HPLC, t_R = 8.050 min, 98.64% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-4-methoxybenzenesulfonamide (11r)

White solid; yield: 85.0%; m.p.: 145–146 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H, 7-ArH), 7.61 (d, J = 8.9 Hz, 2H, 2’, 6’-ArH), 6.92 (brs, 1H, SO₂NH), 6.85 – 6.79 (m, 3H, 4-ArH, 3’, 5’-ArH), 3.79 (s, 3H, 6-OCH₃), 3.68 (s, 3H, 4’-OCH₃), 2.97 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.42 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 163.13, 161.58, 159.88, 153.01, 130.33, 129.41 (2×C), 123.24, 114.29, 114.22, 113.89 (2×C), 91.91, 56.18, 55.59, 18.86, 12.13; HRMS (ESI) m/z calcd for C₁₇H₁₈N₂O₅S [M + H]⁺: 363.0936; found: 363.1019. HPLC, t_R = 7.687 min, 98.97% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-4-(trifluoromethoxy)benzenesulfonamide (11s)

White solid; yield: 83.1%; m.p.: 127–128 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H, 7-ArH), 7.71 (d, J = 8.8 Hz, 2H, 2’, 6’-ArH), 7.21 (d, J = 8.4 Hz, 2H, 3’, 5’-ArH), 6.88 (brs, 1H, SO₂NH), 6.84 (s, 1H, 4-ArH), 3.62 (s, 3H, OCH₃), 2.98 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.43 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 162.00, 159.89, 153.38, 152.33, 137.19, 129.38 (2×C), 124.03 (q, J = 260.6 Hz, OCF₃), 122.33, 121.45 (q, J = 260.6 Hz, OCF₃), 120.64 (2×C), 118.87 (q, J = 260.6 Hz, OCF₃), 116.29 (q, J = 260.6 Hz, OCF₃), 115.91, 114.52, 92.03, 56.04, 18.88, 12.09; HRMS (ESI) m/z calcd for C₁₇H₁₅F₃N₂O₅S [M + H]⁺: 417.0654; found: 417.0732. HPLC, t_R = 18.620 min, 98.50% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2,4-dimethoxybenzenesulfonamide (11t)

White solid; yield: 78.2%; m.p.: 151–152 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.80 – 7.72 (m, 1H, 6’-ArH), 7.70 (s, 1H, 7-ArH), 7.53 (brs, 1H, SO₂NH), 6.87 (s, 1H, 4-ArH), 6.45 – 6.35 (m, 2H, 3’, 5’-ArH), 3.90 (s, 3H, 2’-OCH₃), 3.86 (s, 3H, 6-OCH₃), 3.78 (s, 3H, 4’-OCH₃), 2.91 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.36 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 165.12, 160.80, 159.82, 157.94, 152.08, 132.80, 124.03, 118.28, 114.19, 111.47, 103.99, 99.21, 91.78, 56.37, 56.05, 55.68, 18.80, 12.13; HRMS (ESI) m/z calcd for C₁₈H₂₀N₂O₆S [M + H]⁺: 393.1042; found: 393.1124. HPLC, t_R = 7.618 min, 98.26% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2-methoxy-4-nitrobenzenesulfonamide (11u)

White solid; yield: 57.7%; m.p.: 215–216 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 8.7 Hz, 1H, 6’-ArH), 7.85 – 7.77 (m, 2H, 3’, 5’-ArH), 7.74 (s, 1H, 7-ArH), 7.59 (brs, 1H, SO₂NH), 6.89 (s, 1H, 4-ArH), 4.10 (s, 3H, 2’-OCH₃), 3.84 (s, 3H, 6-OCH₃), 2.93 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.38 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.28, 159.76, 157.03, 152.38, 151.73, 132.38, 131.82, 122.60, 115.17, 114.46, 113.21, 107.04, 92.12, 56.99, 56.49, 18.81, 12.08; HRMS (ESI) m/z calcd for C₁₇H₁₇N₃O₇S [M + H]⁺: 408.0787; found: 408.0870. HPLC, t_R = 8.697 min, 97.27% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-3-fluoro-4-methoxybenzenesulfonamide (11v)

White solid; yield: 74.6%; m.p.: 203–204 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 1H, 7-ArH), 7.55 – 7.33 (m, 2H, 2’, 6’-ArH), 6.94 (brs, 1H, SO₂NH), 6.91 – 6.80 (m, 2H, 4-ArH, 5’-ArH), 3.88 (s, 3H, 4’-OCH₃), 3.73 (s, 3H, 6-OCH₃), 2.97 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.42 (t, J = 7.4 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.68, 159.88, 152.96, 152.70 (d, J = 252.5 Hz, F-3’-ArC), 151.74 (d, J = 10.1 Hz, F-4’-ArC), 151.64 (d, J = 10.1 Hz, F-4’-ArC), 150.20 (d, J = 252.5 Hz, F-3’-ArC), 130.65 (d, J = 6.1 Hz, F-1’-ArC), 130.59 (d, J = 6.1 Hz, F-1’-ArC), 124.73 (d, J = 4.0 Hz, F-5’-ArC), 124.69 (d, J = 4.0 Hz, F-5’-ArC), 122.82, 115.39 (d, J = 21.2 Hz, F-2’-ArC), 115.18 (d, J = 21.2 Hz, F-2’-ArC), 114.40, 112.38, 112.36, 92.05, 56.40, 56.26, 18.88, 12.13; HRMS (ESI) m/z calcd for C₁₇H₁₇FN₂O₅S [M + H]⁺: 381.0842; found: 381.0923. HPLC, t_R = 7.802 min, 97.97% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-3,4-dimethoxybenzenesulfonamide (11w)

White solid; yield: 57.9%; m.p.: 144–145 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H, 7-ArH), 7.28 (dd, J = 8.5, 2.1 Hz, 1H, 6’-ArH), 7.14 (d, J = 2.1 Hz, 1H, 2’-ArH), 6.95 (brs, 1H, SO₂NH), 6.84 (s, 1H, 4-ArH), 6.77 (d, J = 8.5 Hz, 1H, 5’-ArH), 3.86 (s, 3H, 6-OCH₃), 3.76 (s, 3H, 3’-OCH₃), 3.69 (s, 3H, 4’-OCH₃), 2.96 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.41 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.58, 159.83, 153.05, 152.78, 148.79, 130.32, 123.27, 121.36, 114.25, 114.21, 110.10, 109.48, 91.94, 56.22, 56.15, 56.10, 18.87, 12.17; HRMS (ESI) m/z calcd for C₁₈H₂₀N₂O₆S [M + H]⁺: 393.1042; found: 393.1123. HPLC, t_R = 5.211 min, 98.56% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)-2,3-dihydrobenzofuran-5-sulfonamide (11x)

White solid; yield: 58.3%; m.p.: 207–208 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.75 (s, 1H, 7-ArH), 7.54 – 7.46 (m, 2H, 2’, 6’-ArH), 6.92 (brs, 1H, SO₂NH), 6.86 (s, 1H, 4-ArH), 6.70 (d, J = 8.2 Hz, 1H, 3’-ArH), 4.61 (t, J = 8.8 Hz, 2H, OCH₂CH₂), 3.15 (t, J = 8.8 Hz, 2H, OCH₂CH₂), 2.97 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.41 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 164.09, 161.47, 159.87, 152.87, 130.32, 128.94, 128.00, 124.57, 123.39, 114.29, 113.72, 109.28, 91.93, 72.28, 56.23, 28.93, 18.88, 12.15; HRMS (ESI) m/z calcd for C₁₈H₁₈N₂O₅S [M + H]⁺: 375.0936; found: 375.1017. HPLC, t_R = 6.877 min, 98.15% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)thiophene-2-sulfonamide (11y)

White solid; yield: 71.4%; m.p.: 171–172 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.82 (s, 1H, 7-ArH), 7.50 (dd, J = 5.0, 1.2 Hz, 1H, thiophen 5-H), 7.40 (dd, J = 3.7, 1.2 Hz, 1H, thiophen 3-H), 7.04 (brs, 1H, SO₂NH), 6.96 (t, J = 4.0 Hz, 1H, thiophen 4-H), 6.88 (s, 1H, 4-ArH), 3.72 (s, 3H, OCH₃), 2.98 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.43 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 161.79, 159.93, 153.21, 139.10, 132.70, 132.51, 127.16, 122.79, 114.66, 114.35, 91.98, 56.24, 18.87, 12.14; HRMS (ESI) m/z calcd for C₁₄H₁₄N₂O₄S₂ [M + H]⁺: 339.0395; found: 339.0477. HPLC, t_R = 5.799 min, 98.60% purity.

N-(3-Ethyl-6-methoxybenzo[d]isoxazol-5-yl)pyridine-3-sulfonamide (11z)

White solid; yield: 69.2%; m.p.: 172–173 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.91 (s, 1H, pyridine 2-H), 8.72 (d, J = 4.0 Hz, 1H, pyridine 4-H), 7.92 (d, J = 8.1 Hz, 1H, pyridine 6-H), 7.81 (s, 1H, 7-ArH), 7.38 – 7.30 (m, 1H, pyridine 5-H), 7.12 (s, 1H, SO₂NH), 6.84 (s, 1H, 4-ArH), 3.63 (s, 3H, OCH₃), 2.98 (q, J = 7.6 Hz, 2H, 3-CH₂CH₃), 1.43 (t, J = 7.6 Hz, 3H, 3-CH₂CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 162.09, 159.85, 153.33, 153.20 (2×C), 147.92, 135.06, 123.41, 121.88, 116.21, 114.57, 92.18, 56.19, 18.86, 12.09; HRMS (ESI) m/z calcd for C₁₅H₁₅N₃O₄S [M + H]⁺: 334.0783; found: 334.0865. HPLC, t_R = 4.186 min, 97.89% purity.

Docking studies

Preparation of the receptor

The protein (PDB ID: 5Y8Y) were prepared using the Protein Preparation Wizard within Maestro (Schrödinger, New York, NY, USA). The hydrogen atoms were added, bond orders were assigned, and missing side chains for some residues were added using Prime. The water orientations were optimized to assign H-bond using PROPKA program with the pH parameter of 7. The added hydrogens were subjected to energy minimization using OPLS3 force field.

Preparation of the ligands and molecular docking

The ligands were prepared using the LigPrep module with parameter set as no ionization. The OPLS3 force field was selected for energy minimization. The Glide docking program was used for docking studies. The grid was defined as a 20 Å box centered on the ligand. The important water molecules were kept in the binding pocket. All parameters were kept as default. No constraints were applied to all compounds except for 12. The H-bond constraint to Asn140 was applied for the docking of scaffold 12.

TSA assays

A 10 μL reaction mixture was added to 96-well plate. Each biochemical reaction was consisted of 1 μL of 100 μM protein, 4 μL of 500 μM compound, 1 μL of 10× fluorescent dye of SYPRO(R) orange protein gel stain (Sigma-Aldrich, Saint Louis, MO, USA), 1 μL of 10× buffer (100 mM HEPES, 1500 mM NaCl, 50% glycerin and deionized water, pH of 7.5) and 3 μL of deionized water. Total DMSO concentration was restricted to 1% or less. The 96-well plate was filmed and centrifuged at 1000 r/min for 1 min at room temperature, and then incubated on ice for 30–60 min in the dark. The plate was submitted for detection using the Bio-Rad CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The parameter of temperature range was set as 30–80 °C with a step of 0.3 °C per minute. The excitation and emission filters of the SYPRO orange dye were set at 465 nm and 590 nm, respectively. The melting temperature (T_m) was calculated by fitting the melting curve to Boltzmann equation using GraphPad Prism 5. ΔT_m represents the difference of T_m values for the tested reactions and the blank reaction. The experiments were performed in triplicates. The expression and purification of BRD4 BD1 proteins were carried out as previously described [34, 35].

Alphascreen assay

The experiment was performed according to the protocol in the previous literature [34, 35].

X-ray crystallography

Crystallization

The concentrated BRD4(1) was incubated with a 5-fold excess of compounds. A mosquito micro protein crystallizer (TTP Labtech, Royston, UK) was used for crystallization. Crystal of 11h–BRD4(1) was grown by mixing 200 nL of the protein (12 mg/mL and 3.64 mM final ligand concentration) with 200 nL of reservoir solution containing 0.2 M ammonium sulfate, 0.1 M MES monohydrate pH 6.5, 30% w/v polyethylene glycol monomethyl ether 5,000. Crystal of 11r–BRD4(1) was grown by mixing 200 nL of the protein (7 mg/mL and 2.12 mM final ligand concentration) with 200 nL of reservoir solution containing 0.1 M TRIS hydrochloride pH 8.5, 8% w/v polyethylene glycol 8000.

Data collection and structure solution

Data collection and structure solution were performed using a protocol from previous studies [34, 35]. Data collection and refinement statistics can be found in Table 1. The coordinates have been deposited with PDB accession codes: 7V1U for 11h–BRD4(1), 7V2J for 11r–BRD4(1).

Table 1 Data collection and refinement statistics for ligands and BRD4(1) complexes.

Cellular anti-proliferative assay

MV4-11 cells (ATCC, Rockville, MD, USA) were cultured in IMDM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified incubator containing 5% CO₂. In the cell growth assay, cells were seeded in 384-well plates at 1000 cells per well in 20 μL of culture medium and cultured for 12 h. Different concentrations of diluted compounds or DMSO control were added into the wells in a volume of 10 µL with the final concentrations from 5 nM to 100 μM and cultured for 120 h. Then, 25 μL CellTiter-GLO reagent (Promega, Madison, WI, USA) was added in each well and mixed on an orbital shaker for 10 min to induce cell lysis. The lysates were incubated for another 10 min and centrifuged for 1 min. Luminescence was measured on an Enspire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA), according to the manufacturer’s instructions. Each concentration point was performed in triplicate. The fluorescence signals were normalized to the DMSO-treated cells, and the inhibitory curves and IC₅₀ values were calculated by nonlinear regression and dose-response inhibition equation analysis using GraphPad Prism 5 software.

Quantitative real-time PCR (qRT-PCR)

Cells in the logarithmic growth phase were seeded in a 96-well plate with 5000 cells per well in 90 μL of medium, cultured for 12 h and then treated with 10 μL of different concentrations of compound. Three replicates per well for each concentration. After cultured for 48 h, the cells were harvested and washed with PBS. The total RNA was extracted by TRIzol Reagent kit (Beyotime, Shanghai, China). A 20 μL system was used for cDNA synthesis including 2 μL of RNA, 4 μL of 5× iScript reaction mix (Bio-Rad), 1 μL of iScript reverse transcriptase (Bio-Rad), and 13 μL of Nuclease-free water. The Applied Biosystems Veriti^® 96-Well Thermal Cycler instrument (Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription with reaction at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min to obtain cDNA. Then, the qRT-PCR assay was performed on the LightCycler 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA) with a 20 μL reaction system, including 1 μL of cDNA, 7.4 μL of Nuclease-free water, 10 μL of SsofastEvaGreenSupermix (Bio-Rad), 0.8 μL of forward primer, 0.8 μL of reverse primer, respectively. The target mRNA expression was quantified using the 2^−∆∆Ct method and normalized to GAPDH expression. One-way ANOVA and Tukey’s test were used for analysis of the statistical differences between test groups. P values < 0.05 were considered to be significantly different. The primer sequences for qPCR used are as follows: c-Myc_fwd, 5’-CACTAACATCCCACGCTCTGA-3’; c-Myc_rev, 5’-AAATCATCGCAGGCGGAACA-3’; CDK6_fwd, 5’-CCGACTGACACTCGCAGC-3’; CDK6_rev, 5’-TCCTCGAAGCGAAGTCCTCA-3’; GAPDH_fwd, 5’-AATGGGCAGCCGTTAGGAAA-3’; GAPDH_rev, 5’-GCGCCCAATACGACCAAATC-3’.

Western blotting assay

After treatment with different concentrations of compound for 48 h, MV4-11 cells were harvested, washed by PBS, and then lysed in 400 μL of RIPA buffer (Beyotime) containing protease inhibitors PMSF (10 μL PMSF per 1 mL of RIPA) for 30 min on ice. The lysate was transferred to 1.5 mL eppendorf centrifuge tube and separated from the cell debris by centrifugation at 12,000 r/min for 5 min at 4 °C. The protein concentrations were measured using BCA Protein Assay Kit (Beyotime). Other lysate was mixed with 5× loading buffer and heated for 10 min for denaturation. The samples were separated by 8% SDS-PAGE separating gels and 4% SDS-PAGE stacking gels and then blotted into PVDF membranes (Millipore, Schwalbach, Germany). Subsequently, the PVDF membrane was washed with TBST, immersed in blocking solution (TBST solution with 5% fat-free milk) and shook at room temperature for 1 h to block the non-specific protein binding sites of PVDF membrane. The membrane was washed with TBST and then transferred to a ziplock bag. The primer antibody of anti-c-Myc antibody (ab32072, Abcam, Cambridge, UK) was diluted in a ratio of 1:1000 and incubated with the PVDF membrane at 4 °C overnight. Then, the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab205718, Abcam) was diluted in a ratio of 1:50000 and incubated with the PVDF membrane at room temperature for 1 h. The chemical luminescence reagent BeyoECL Plus A and B (Beyotime) was mixed in equal volume and added to the PDVF membrane. After 5 min, Tanon 6600 luminescence imaging workstation (Tanon, Shanghai, China) was used for detection. Image Pro Plus 6.0 software was used to analyze the optical density value. The tested protein levels were relative to the internal reference of GAPDH and analyzed using GraphPad Prism 5 software. One-way ANOVA and Tukey’s test were used for analysis of the statistical differences between test groups. P values < 0.05 were considered to be significantly different.

Cell cycle analysis

Cells were seeded in 96-well plates at the density of 5 × 10³ per well and incubated for 12 h. The gradient concentrations of compound 11r or DMSO were added to the plates and incubated for 48 h at 37 °C. The cells were then harvested and washed with pre-cooled PBS, and then centrifuged at 10,000 r/min for 5 min at 4 °C. Cells were fixed with 500 μL 70% ethanol (v/v, 30% PBS) at 4 °C overnight and collected at 800 r/min for 15 min. Cells were washed with PBS and re-suspended in 0.4 mL PBS, and then transferred to a tube at a density of 10⁶/mL. 3 μL of RNase-A was added to the tube with the final concentration of about 50 μg/mL and digested at 37 °C for 30 min. 50 µL of PI was added to the tube with the final concentration of about 65 µg/mL, and incubated on ice bath in the dark for 30 min. The cell cycle distribution was analyzed by DxFLEX flow cytometry (Beckman Coulter, Brea, CA, USA) within 1 h. The data were analyzed using Modfit LT 5.0 software and quantified by GraphPad Prism 5 software. One-way ANOVA and Tukey’s test were used for analysis of the statistical differences between test groups. P values < 0.05 were considered to be significantly different.

Apoptosis analysis

Cells were collected and washed with 4 °C pre-cooled PBS, and then centrifuged at 10,000 r/min for 5 min at 4 °C. The cells was re-suspended in 1× Annexin binding buffer with the density of 10⁶/mL. 5 μL Alexa Fluor 488 Annexin V and 1 μL 100 μg/mL propidium iodide (PI) working solution (Invitrogen, Carlsbad, CA, USA) were added to each 100 μL cell suspension and incubated at room temperature for 15 min in dark and analyzed by flow cytometry. The data were analyzed using the workstation for flow cytometry (Beckman) and quantified by GraphPad Prism 5 software. One-way ANOVA and Tukey’s test were used for analysis of the statistical differences between test groups. P values < 0.05 were considered to be significantly different.

Results

Chemistry

The target compounds were synthesized in a tandem six steps with the commercially available 2,4-dimethoxyaniline as the starting material (Scheme 1). In the first step, the amino group of compound 5 was acetylated with acetic anhydride to obtain compound 6. Next, a propionyl group was introduced on the benzene ring of 6 via anhydrous AlCl₃ mediated Friedel-Crafts reaction, which afforded compound 7. The methoxy group at the ortho position of the carbonyl group was selectively removed to give the hydroxyl group. Compound 7 was reacted with hydroxylamine hydrochloride to generate the oxime intermediate 8. The intermediate 8 was submitted to cyclization at high temperature in the presence of DMF-DMA to give isoxazole derivative 9, which was then hydrolyzed by hydrochloric acid to give the scaffold 10. The target compounds 11a–z were finally synthesized in a one-plot step through the reaction of compound 10 with different sulfonyl chlorides.

Scheme 1

The synthetic route of 3-ethyl-benzo[d]isoxazole containing sulfonamide derivatives. Reagents and conditions: (a) Acetic anhydride, triethylamine, DCM, rt, 88%; (b) Propionyl chloride, AlCl₃, DCM, 43 °C, 81%; (c) Hydroxylamine hydrochloride, sodium acetate, EtOH/H₂O, 80 °C, 86%; (d) DMF-DMA, 1,4-dioxane, 100 °C, 56%; (e) HCl/H₂O, 90 °C, 93%; (f) Sulfonyl chlorides, pyridine, DCM, 43 °C, 39%–85%.

Biological activity

SAR studies against BRD4

The X-ray crystallographic structure of 4-BRD4(1) complex (PDB: 5Y8Y) previously reported by our laboratory provided solid evidence for the structure-based optimization in this work. We initially docked the scaffolds 10 (3-ethyl-6-methoxybenzo[d]isoxazol-5-amine) and 12 (3-methyl-6-methoxybenzo[d]isoxazol-5-amine) into the bromodomain of BRD4. Both scaffolds resided into the K_Ac binding site and the 3-ethyl on scaffold 10 could occupy more space in the sub-pocket than 3-methyl group (12) (Fig. 2). The binding advantage was also supported by the two docking scores (−7.2 & −39 vs −6.6 & −35) (Supplementary Table S1). Next, many compounds containing the 3-ethyl-6-methoxybenzo[d]isoxazol-5-amine core were designed and submitted to molecular docking before synthesis (Supplementary Table S1). The two scoring models such as docking score and glide emodel score were used as the preliminary screening reference indicators. Compounds with docking score < −7.1 and glide emodel score < −55 would be synthesized and tested.

Fig. 2: Structure–based design of new benzo[d]isoxazole derivatives.

Green sticks: compound 10, yellow sticks: compound 12.

All the synthesized small molecules were evaluated by the TSA screening method against BRD4(1) (Table 2). Firstly, an ethyl group (11a) was introduced at R position, which showed moderate binding activity to BRD4. Extending the alkyl chain to 3 carbon atoms (11b) or 4 carbon atoms (11c) led to increased activities with ΔT_m values of 6.5 °C and 7.2 °C, respectively.

Table 2 Binding activities of compounds 11a-n against BRD4.

Next, phenyl groups were introduced at R position. The unsubstituted phenyl compound 11d exhibited moderate activity. The introduction of small groups such as F (11e), Cl (11f), Br (11g), OCH₃ (11h), OCF₃ (11i) to the 2’ position of benzene ring led to increased activities. Among which, the 2’-OCH₃ substituted compound 11h showed the most potent binding activity with thermal shift value of 8.0 °C. The 2’-OCF₃ also showed increased activity compared with 11e–g and slightly decreased activity versus 11h. Parallel results showed that the introduction of small groups at the 3’ position of benzene ring still maintained slightly stronger activities (11f vs 11j; 11g vs 11k). Compounds with 2’ and 3’ multi-substitutions (11l–n) were also designed and synthesized. The compounds showed ΔT_m values of 6.0–6.6 °C, which were slightly lower than that of 11h.

To further detail the structure-activity relationships, we investigated the impact of substitutions at the 4’ position of the benzene ring (Table 3). The results for compounds 11o–p indicated that 4’-F and 4’-Cl were tolerated on the phenyl ring, with similar ΔT_m values against BRD4 compared to 2’ substitution (11e & 11f). The introduction of the strongly electron-withdrawing nitro group in compound 11q showed an increased activity. The compound 11r with electron-donating 4’-OCH₃ group displayed similar activity compared to 11q. Analog 11s showed a decreased activity versus 11r.

Table 3 Binding activities of compounds 11o-z against BRD4.

To identify more potent inhibitors, multi-substituted compounds at 2’ and 4’ positions or at 3’ and 4’ positions were synthesized. Since the 2’- or 4’- methoxy groups showed better activities, we retained the above groups in the synthesis of 11t-x. Among them, compounds 11t, 11u, 11x exhibited potent activities with ΔT_m values of 6.5, 7.5, 6.0 °C, respectively. We also introduced two heterocyclic motifs as R groups, which afforded 11y and 11z with good binding activities.

The IC₅₀ values for several representative compounds were further determined by alphascreen method. Most compounds exhibited nanomolar activities (Fig. 3). Among which, 11h exhibited the most potent activity with an IC₅₀ value of 0.094 μM.

Fig. 3: The inhibitory effect of compounds against BRD4(1).

The inhibitory curves and the half-maximum inhibitory concentration (IC₅₀) values for all compounds against BRD4(1) were obtained from the alphascreen assay.

We also successfully obtained two crystal structures. The binding modes of compounds 11h and 11r in complex with BRD4(1) were determined by X-ray crystallography and illustrated in Fig. 4. Multiple hydrogen bond interactions were observed between the compounds and the protein or the water network. The 3-ethyl-benzo[d]isoxazole cores shared the similar binding pattern in both compounds with the oxygen forming a hydrogen bond to Asn140 and the nitrogen forming a hydrogen bond to the NO.1 conserved water molecule (Fig. 4a). The 2’-OCH₃ of 11h formed a hydrogen bond to NO.9 solvent water molecule, which further bound to Leu92 (Fig. 4b). The 4’-OCH₃ of 11r formed a hydrogen bond to NO.8 water molecule, which further bound to Ile146 (Fig. 4c). The benzene rings attached to sulfonamides in both compounds were not fully overlapped with each other (Fig. 4d). The 4’ carbon atom of 11r shifted 1.3 Å toward the orientation of No. 8 water molecule compared with 11h, probably to form a more stable H-bond (Supplementary Fig. S2a). This shift of 11r further induced the nearby amino residues such as Asp145 and Trp81 to shift 0.8 Å and 0.5 Å, respectively.

Fig. 4: X-ray crystal structures of representative compounds 11h (green sticks) and 11r (magentas sticks) in complex with BRD4(1) (PDB ID: 7V1U for 11h, 7V2J for 11r; protein, gray surface; key residues, thin sticks; water molecules, red spheres; hydrogen bonds, yellow dashed lines; electron density, blue grid).

a Superimposed crystal structures of 11h and 11r in complex with BRD4(1). The 3-ethyl-benzo[d]isoxazole cores of compounds share the similar poses in the KAc binding pocket. b X-ray crystal structure of 11h bound to BRD4(1). c X-ray crystal structure of 11r bound to BRD4(1). d Superimposed crystal structures of 11h (cyan thin sticks for protein residues) and 11r (gray thin sticks for protein residues) bound to BRD4(1), with the phenyls occupying on the WPF shelf. e Superimposed crystal structures of 11h and 3 (PDB ID: 4E96, yellow stick) in complex with BRD4(1). f Superimposed crystal structures of 11r and 4 (PDB ID: 5Y8Y, cyan stick) in complex with BRD4(1). The vacuum electrostatic surface of BRD4(1) was generated by pymol.

The crystal structures of 11h, 11r, 3 and 4 were also superimposed. All the scaffolds in the four compounds shared similar binding modes to mimic the K_Ac (Supplementary Fig. S2b). The benzene rings of 11h and 3 were overlapped well with each other in the WPF region (Fig. 4e, Supplementary Fig. S2c). Besides, the benzene rings of 11r and 4 were also completely overlapped (Fig. 4f, Supplementary Fig. S2c).

Selectivity evaluation

TSA assay was performed for representative compounds against 11 bromodomain-containing proteins. All compounds exhibited binding activities with ΔT_m of 6.2–8.0 °C for BRD4(1) and 3.8–5.8 °C for BRD3(1) (Table 4). Besides, the compounds also showed activities with ΔT_m of 1.5–4.5 °C for BRD2(2) and BRDT(1). No thermal shifts were observed in other proteins.

Table 4 Selectivity evaluation for representative compounds against 11 bromodomain-containing proteins by thermal shift assay.

Inhibitory activity against AML cells

Several compounds were selected for evaluation of the inhibitory effect against human AML cells MV4-11. Compound 4 was included as a positive control. After the treatment with compounds for 120 h, the proliferation of MV4-11 cells were inhibited in a dose-dependent manner (Fig. 5). Compounds 11h and 11r showed nanomolar inhibitory activities with EC₅₀ values of 0.78 μM and 0.87 μM. Other selected compounds also exhibited good inhibitory activities with EC₅₀ values ranging from 2.5 μM to 5.5 μM.

Fig. 5: Inhibition of cell proliferation of compounds against the MV4-11 cell lines.

CellTiter-Glo was used to detect the viability after the treatment of the indicated concentrations of compounds for 120 h.

Inhibition on the expression of BRD4 downstream genes

To further confirm the anti-proliferative mechanism of inhibitors, the real-time quantitative PCR and western blot assays were performed to detect the level of target genes in MV4-11 cells effected by representative compounds. As the research on the biological mechanism of 11h was still in progress, only the results of 11r were reported here. The mRNA expression of c-Myc and CDK6 in MV4-11 decreased significantly in the compound 11r treatment group (1 μM, 5 μM and 10 μM) compared with the control group (Fig. 6). In addition, a remarkable dose-dependent decrease of c-Myc expression was observed at the protein level (Fig. 7).

Fig. 6: Effect of the mRNA expressions of c-Myc and CDK6 in MV4-11.

The mRNA levels of c-Myc (a) and CDK6 (b) were detected by RT-PCR. The mRNA levels of c-Myc and CDK6 were normalized to control. The results were presented as mean ± SD (n = 3). **P < 0.01, vs. control group.

Fig. 7: The level of c-Myc expression in MV4-11.

a The representative bands for different treatment groups were detected by Western blot assay. b The quantification of the expression levels of c-Myc. The levels of c-Myc were normalized to control. The results were presented as mean ± SD (n = 3).

Flow cytometry analysis

To further investigate the mechanism of anti-proliferative effects, we employed flow cytometry analysis to evaluate the ability of compound 11r for induction of cell cycle arrest and apoptosis in the MV4-11 cell lines. In cell cycle analysis assay, compound 11r could induce cell cycle arrest in G₀/G₁ phase in a dose-dependent manner (Fig. 8). The apoptosis rate of 11r (1 μM, 5 μM and 10 μM) treated groups was significantly increased compared with the control group (Fig. 9).

Fig. 8: MV4-11 cells were arrested at G₁ phase by 11r.

a Flow cytometry analysis of the distribution of cell cycle in MV 4-11 cells after treatment of DMSO and the indicated concentrations of compound 11r. Propidium iodide was used for staining of MV4-11. b The quantification of the analysis of cell cycle phase. Results were mean ± SD for 3 individual experiments which, for each condition, were repeated 3 times. **P < 0.01, vs. control group.

Fig. 9: Compound 11r induced apoptosis in MV4-11 cells.

a MV4-11 cells were treated with different concentrations of 11r, then apoptosis in MV4-11 cells was assessed by flow cytometer analysis after staining with AnnexinV and PI. b The quantification of the apoptosis rate in MV4-11 cells. Results were mean ± SD for 3 individual experiments which, for each condition, were repeated 3 times. **P < 0.01, vs. control group.

Discussion

We previously reported a class of BET inhibitors containing a 3-methyl-6-methoxybenzo[d]isoxazol scaffold [35]. Molecular docking studies revealed similar binding advantage for 3-ethyl-substituted backbone (Fig. 2). This led to the design of compounds containing the 3-ethyl-6-methoxybenzo[d]isoxazol-5-amine core, which we would like to explore the chemical space and investigate the more detailed structure-activity relationships against BRD4.

As the sulfonamide is an effective linker for attaching the substituent to occupy the WPF shelf region [29, 34, 35], we initially retained the sulfonamide while changing the R groups. The results were consistent with the previous data [35] that lipophilic substituent of three to five heavy atoms was acceptable for occupying the WPF region. Since the benzene ring is also a lipophilic substituent, the substituted or unsubstituted phenyl groups were introduced at R position. The data suggested that the 2’ and 3’ positions of the benzene were tolerated for substitution. Besides, the 4’ position of the phenyl ring was also tolerated for modification, which was different from the previous SAR studies [35]. Moreover, the heterocycle was also tolerated for substitution and can be further modified to generate potent inhibitors.

The TSA assay evaluates the binding affinity under the condition of excess compound to protein concentration ratio (20:1). In order to investigate the binding ability of the compound to the protein at different concentrations, alphascreen assays were performed for representative compounds. The results further confirmed the potent activities of all the selected compounds (Fig. 3).

In the early stage of the research, molecular docking technology was used for the guidance of drug design. However, the real binding modes of small molecules and proteins were still worthy of exploration. Two crystal structures of compounds 11h and 11r in complex with BRD4(1) were obtained. Both compounds exhibited similar binding mode with the 3-ethyl-benzo[d]isoxazole core residing into the K_Ac binding pocket (Fig. 4a). The 9 water molecules and proteins formed a water network as previously reported [35]. The electron density maps of both compounds showed excellent shape complementary with the protein binding pocket. The crystal structures of 11h and 11r were also superimposed with the two structural related compounds 3 and 4 for comparison. It was noteworthy that the 3-ethyl on the scaffolds of 11h and 11r extended deeper to inner sub-pocket as we expected compared with 4 (Supplementary Fig. S2b). However, the four compounds adopted two sets of binding modes with nuance in the WPF region. The results indicated that substitutions at the 4’ or 5’ positions of the benzene ring would push the benzene ring to the side away from Trp81. We originally thought that the benzene ring was bound at the same position and then the binding potency may be improved by adding substituents to the benzene ring. The results may explain why the activities of multi-substituted compounds were not improved.

The crystal structures were also overlaid with the docked structures for comparison. The crystal structure of 11h exhibited the similar binding mode compared to the docked structure (Supplementary Fig. S2d). However, the crystal structure of 11r was obviously different from the docked structure in the poses of the phenyl substitution (Supplementary Fig. S2e). We deemed that the 4’-substituted phenyl compounds would cause the surrounding Asp145 to shift flexibly when they bound to the protein. However, Asp145 was not shifted flexibly to accommodate the small molecules in conventional molecular docking process, which generated the inaccurate binding mode. Overall, the obtained crystal structures further confirmed the high binding affinities of small molecules to proteins.

The bromodomains are structurally conserved modules and are present in a variety of proteins. To evaluate the selectivity profile of compounds, 11 bromodomain-containing proteins from different families were selected for testing. The results indicated that all compounds exhibited excellent selectivity for BET family of bromodomains over other protein members (Table 4). Although the reference compound 4 showed weak activity for EP300 with ΔT_m of 1.25 °C, other synthesized compounds showed less activity for this protein.

Much evidence showed that BRD4 played important roles in the development of acute myeloid leukemia [6, 8, 9]. Therefore, we evaluated the anti-proliferative effect of representative compounds against AML cells MV4-11. Compounds 11h (0.78 μM) and 11r (0.87 μM) showed similar inhibitory activities compared to positive compound 4 (0.85 μM). Combined data indicated that the cellular inhibitory activities were mainly caused by the inhibition of BET bromodomains. As an epigenetic reader of acetyl lysine, BRD4 participated in the regulation of specific gene expression including c-Myc and CDK6 in AML [6, 37]. Compound 11r concentration-dependently inhibited the expression levels of oncogenes including c-Myc and CDK6 in MV4-11 cells. The results showed that the compound inhibited the expression of downstream oncogenes by inhibiting the BET bromodomains. Moreover, flow cytometry analysis indicated that 11r concentration-dependently blocked cell cycle in MV4-11 cells at G₀/G₁ phase and induced cell apoptosis.

Conclusions

In summary, based on our previously reported BRD4 inhibitors, a new series of 3-ethyl-benzo[d]isoxazole derivatives were designed and synthesized as BRD4 inhibitors under the guidance of molecular docking studies. The detailed SARs indicated that 3-ethyl was acceptable for the sub-pocket. Further exploration also indicated that the 4’ position of the phenyl ring was tolerated for modification. The actual binding modes of potent compounds 11h and 11r were disclosed by the X-ray cocrystal structures. Among the 26 synthesized compounds, compound 11h and 11r were potent inhibitors against BRD4 and showed strong anti-proliferative effect against human leukemia cell lines MV4-11. In the RT-PCR and Western blot assay, compound 11r could decrease the transcription or expressions of c-Myc and CDK6 in MV4-11 cell. In addition, 11r was able to induce cell cycle arrest at G₀/G₁ phase and induce cell apoptosis. The results indicated that N-(3-ethylbenzo[d]isoxazol-5-yl)sulfonamide derivative 11r is a promising lead compound and can be used to further explore drug candidate.