Novel thiazole derivatives incorporating phenyl sulphonyl moiety as potent BRAFV600E kinase inhibitors targeting melanoma

Abstract

Novel thiazole derivatives possessing phenyl sulfonyl moiety were designed and synthesized as B-RAFV600E kinase inhibitors based on the clinically-approved anticancer drug, dabrafenib. All target compounds showed significant inhibition of B-RAFV600E kinase enzyme at nanomolar levels. Compounds 7b and 13a revealed excellent B-RAFV600E inhibitory activity, superior to that of dabrafenib with IC₅₀ values of 36.3 ± 1.9, 23.1 ± 1.2, and 47.2 ± 2.5 nM, respectively. Moreover, the title compounds were much more selective toward B-RAFV600E kinase than B-RAF wild type. In addition, the most potent compounds were further evaluated for their anticancer activity against B-RAFV600E-mutated and wild type melanoma cells. A positive correlation between the cytotoxic activity and selectivity for B-RAF V600E over B-RAF wild type was clearly observed for compounds 7b, 11c, 13a, and 17. All the screened compounds potently inhibited the growth of WM266.4 melanoma cells with IC₅₀ values in the range from 1.24 to 17.1 μM relative to dabrafenib (IC₅₀ = 16.5 ± 0.91 μM). Compounds 7b, 11a and 11c, 13a, and 17 were much more potent than dabrafenib against B-RAFV600E-mutated WM266.4 melanoma cells. Furthermore, compound 7b suppressed the phosphorylation of downstream ERK1/2 from WM266.4 cells. Also, the docking study revealed the proper orientation and well-fitting of the title compounds into the ATP binding site of B-RAFV600E kinase.

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1. Introduction

Ras/Raf/Mek/ERK kinase cascade, also known as MAPK pathway, plays a pivotal role in inter and intracellular communication, which controls the fundamental cellular processes such as cell growth, survival, differentiation, and proliferation.^1,2 This pathway is frequently activated or overexpressed in various disease conditions, particularly cancer.^3,4

RAF kinases are the key components of Ras/Raf/Mek/ERK pathway. Since their discovery in 1983, RAF kinases have been associated with cancer. Three isoforms (A-RAF, B-RAF, and C-RAF) are known for the RAF kinase family, showing different degrees of biochemical potencies (B-RAF > C-RAF > A-RAF).⁵ Being the most frequently mutated isoform in human cancers, B-RAF is the major activating kinase for the MEK/ERK pathway. Numerous activating mutations of B-RAF have been identified by several research groups. Accounting for more than 90% of B-RAF mutations in cancer, B-RAFV600E point mutation in which valine is substituted by glutamic acid in codon 600, is the most common, particularly in melanoma.⁶ The overexpression of B-RAFV600E kinase is associated with the proliferation, aggressiveness, and poor prognosis of malignant tumors. In melanoma, B-RAFV600E mutation accounts for 500 times activation relative to the wild-type B-RAF kinase.⁶ Therefore, B-RAFV600E kinase is considered as a research hotspot for the discovery of new anticancer agents, and great efforts have been devoted to the development of B-RAFV600E kinase inhibitors.^7,8 As a consequence of this effort, clinically approved B-RAFV600E kinase inhibitors (Fig. 1) have been introduced into the market, resulting in a dramatic change in the treatment of B-RAFV600E-driven cancers, especially melanoma.^9,10 Given that the B-RAF enzyme is crucial for normal cell processes, extensive efforts are currently being made by scientists to obtain potent and safe medications that selectively inhibit B-RAFV600E without interfering with the B-RAF wild-type.^11–13

Fig. 1 Clinically-approved B-RAFV600E inhibitors.

On the other hand, the thiazole nucleus has attracted significant attention in medicinal chemistry for the discovery and development of biologically active compounds, particularly anticancer agents.^14–16 Several thiazole-containing compounds have been reported as potent B-RAFV600E kinase inhibitors (Fig. 2).^8,17–23 Moreover, dabrafenib (Fig. 1), a thiazole derivative developed by GlaxoSmithKline as a potent and selective B-RAFV600E inhibitor, was approved by FDA in 2013 for the treatment of B-RAFV600E-driven tumors.¹⁹

Fig. 2 Reported thiazoles as B-RAFV600E inhibitors.

In the light of these facts and in continuation of our effort toward the discovery of potent anticancer agents,^24–36 herein, novel thiazole derivatives were designed and synthesized as potent B-RAFV600E kinase inhibitors with potential anticancer activities, based on the clinically-approved B-RAFV600E inhibitor, dabrafenib. The design of our target compounds is illustrated in Fig. 3. In this work, while conserving the thiazole core of dabrafenib, different moieties were incorporated into the thiazole nucleus, namely, arylazo 7a–c, arylhydrazono 11a–c, and aryl groups 13a, b, 15, 19. Motivated by the reported anticancer activity of phenyl sulphonyl derivatives,³⁰ the 2,6-difluorophenylsulphonamide group of dabrafenib was also replaced by the phenylsulphonylmethyl (PhSO₂CH₂) moiety. In addition, the fluorophenyl moiety in dabrafenib was replaced by the arylidenehydrazine moiety in the target compounds. Moreover, the substituent effect (R1–R4) on B-RAFV600E kinase inhibitory activity was also explored. On surveying the literature, it was found that bis vemurafenib compounds, reported by Grasso et al.,³⁷ possessed potent B-RAFV600E inhibitory activity in both cell-free and cell-based assays. Also, the anticancer and kinase inhibitory activity of bisthiazole compounds is well established.^38,39 Inspired by these findings, bisthiazole compound 15 was also synthesized to study the effect of dimerization on B-RAFV600E inhibition.

Fig. 3 Design of the target compounds.

In this work, all target compounds were screened in vitro for their ability to inhibit the kinase activity of B-RAFV600E. Furthermore, to evaluate the selectivity of the target compounds, the most active derivatives were tested in vitro against B-RAF wild type. The most potent compounds were also screened for their anticancer activity against B-RAFV600E-mutated and B-RAF wild type melanoma cells. Also, a cell-based assay was performed to measure the blocking effect of the title compounds on the phosphorylation of downstream ERK. Finally, a docking study of the most promising compounds was conducted to predict their binding modes within the active site of the B-RAFV600E kinase.

2. Experimental

2.1. Chemistry

The instruments utilized for recording the spectral data are illustrated in the ESI [see ESI† for more details]. The used hydrazonoyl chlorides were prepared by the same method cited in the literature reports.^40,41

2.1.1. Synthesis of 2-benzenesulfonyl-1-p-(substituted phenyl)-ethanone thiosemicarbazone (3 and 17). As usual, in our previous work^24,42 related to the synthesis of thiosemicarbazone derivatives, we synthesized derivatives 3 and 17 through the condensation reaction under reflux of phenylsulfone derivatives 1 and 16 (0.005 mol) with thiosemicarbazide 2 (0.005 mol) in 15 mL ethanol with drops of conc. HCl. Through the reflux of the reaction, the yellow solid of the two derivatives were precipitated after 10 min, and then the reflux was completed for 2 h. The yellow solid of the two derivatives were collected and crystallized from dioxane.
2.1.1.1. 2-Benzenesulfonyl-1-p-tolyl-ethanone thiosemicarbazone (3). Pale yellow solid (86% yield), mp 160–162 °C; IR (KBr) ν_max 3417, 3248, 3163 (NH and NH₂), 3055, 3001 (sp² C–H), 2916 (sp³ C–H), 1612 (C=N), 1496, 1458, 1427, 1303, 1141, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.28 (s, 3H, CH₃), 5.28 (s, 2H, CH₂), 7.07 (d, J = 8.1 Hz, 2H, Ar–H), 7.55–7.85 (m, 5H, Ar–H), 7.86 (d, J = 8.1 Hz, 2H, Ar–H), 7.93 (s, 1H, NH), 8.38 (s, 1H, NH), 10.52 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 14.2, 51.8, 122.6, 127.9, 129.0, 129.2, 130.7, 134.1, 134.8, 135.3, 139.0, 179.0. MS m/z (%) 348 (M⁺ + 1, 0.36), 347 (M⁺, 1.3), 192 (9), 147 (7), 117 (100), 115 (40), 105 (17), 91 (35), 77 (60), 65 (21). Anal. calcd for C₁₆H₁₇N₃O₂S₂ (347.46): C, 55.31; H, 4.93; N, 12.09. Found: C, 55.26; H, 4.81; N, 11.94%.
2.1.1.2. 2-Benzenesulfonyl-1-(4-fluorophenyl)-ethanone thiosemicarbazone (17). White crystals (88% yield), mp 205–207 °C; IR (KBr) ν_max 3387, 3317, 3263 (NH and NH₂), 3062, (sp² C–H), 2970, 2900 (sp³ C–H), 1604 (C=N), 1504, 1473, 1435, 1303, 1234, 1157, 1141, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 5.33 (s, 2H, CH₂), 7.05 (t, J = 9 Hz, 2H, Ar–H), 7.55–7.94 (m, 7H, Ar–H), 8.02 (s, 1H, NH), 8.41 (s, 1H, NH), 10.60 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ 52.9 (CH₂), 115.2, 115.4 (d, ²JCF, 20 Hz), 128.5, 129.7, 130.1, 130.2 (d, ³JCF, 8 Hz), 133.2, 134.7, 135.7, 139.7, 162.3, 164.0 (d, ¹JCF, 246 Hz), 179.7 (C=S). MS m/z (%) 353 (M⁺ + 2, 1.2), 352 (M⁺ + 1, 2.5), 351 (M⁺, 11), 210 (5), 196 (48), 151 (43), 135 (6), 121 (100), 109 (28), 101 (47), 95 (22), 77 (68). Anal. calcd for C₁₅H₁₄FN₃O₂S₂ (351.42): C, 51.27; H, 4.02; N, 11.96. Found: C, 51.05; H, 3.94; N, 11.86%.

2.1.2. Synthesis of thiazole derivatives 7a–c, 11a–c, 13a and 13b, 15 and 19. In a 100 mL round-bottom flask, substituted phenylsulfone–thiosemicarbazone derivatives 3 or 17 (0.002 mol), according to the listed reactions in Schemes 2–5, were added with the selected hydrazonoyl chlorides 4a–c or 8a–c, phenacyl bromide derivatives 12a and 12b, 14, or 18 (0.002 mol) in dioxane were refluxed after the addition of Et₃N (0.3 mL) for 5 h. After the reactions were completed, as checked by monitoring with TLC, the colored solids were collected and purified through crystallization from dioxane to give the thiazole derivatives 7a–c, 11a–c, 13a and 13b, 15, and 19, respectively.

Scheme 1 Synthesis of the thiosemicarbazone–phenylsulfone derivative 3.

Scheme 2 Reaction of thiosemicarbazone–phenylsulfone derivative 3 with hydrazonoyl chlorides 4a–c.

Scheme 3 Reaction of thiosemicarbazone–phenylsulfone derivative 3 with hydrazonoyl chloride 8a–c.

Scheme 4 Reaction of thiosemicarbazone–phenylsulfone derivative 3 with phenacyl bromide derivatives 12a, 12b and 14.

Scheme 5 Synthesis of thiosemicarbazone–phenylsulfone derivative 17 and thiazole derivative 19.

2.1.2.1. N-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-N′-(4-methyl-5-phenylazo-thiazol-2-yl)-hydrazine (7a). Dark red solid (82% yield), mp 173–174 °C; IR (KBr) ν_max 3255 (NH), 3055, 3001 (sp² C–H), 2924 (sp³ C–H), 1589 (C=N), 1535, 1489, 1411, 1373, 1311, 1242, 1157, 1072 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.35 (s, 3H, CH₃), 2.56 (s, 3H, CH₃), 5.26 (s, 2H, CH₂), 6.65–7.86 (m, 14H, Ar–H), 10.67 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 14.0, 32.1, 53.3, 120.4, 124.2, 127.9, 129.1, 129.5, 131.4, 131.5, 133.8, 134.6, 139.2, 141.9, 152.4, 152.5, 164.2, 166.7, 171.6. MS m/z (%) 490 (M⁺ + 1, 0.6), 489 (M⁺, 1.6), 347 (10), 231 (2.7), 143 (2), 117 (74), 105 (6), 92 (12), 91 (37), 77 (100). Anal. calcd for C₂₅H₂₃N₅O₂S₂ (489.6): C, 61.3; H, 4.7; N, 14.3. Found: C, 61.2; H, 4.6; N, 14.2%.
2.1.2.2. N-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-N′-(4-methyl-5-m-tolylazo-thiazol-2-yl)-hydrazine (7b). Dark red solid (89% yield), mp 148–150 °C; IR (KBr) ν_max 3425 (NH), 3032 (sp² C–H), 2916 (sp³ C–H), 1589 (C=N), 1535, 1489, 1373, 1311, 1265, 1188, 1072 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.27 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 2.54 (s, 3H, CH₃), 5.25 (s, 2H, CH₂), 6.78–7.86 (m, 13H, Ar–H), 10.64 (s, 1H, NH); MS m/z (%) 504 (M⁺ + 1, 1.3), 503 (M⁺, 4), 362 (12), 161 (2), 142 (11), 132 (10), 117 (64), 106 (31), 91 (100), 77 (89). Anal. calcd for C₂₆H₂₅N₅O₂S₂ (503.6): C, 62.0; H, 5.0; N, 13.9. Found: C, 61.9; H, 4.9; N, 13.8%.
2.1.2.3. N-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-N′-(4-methyl-5-p-tolylazo-thiazol-2-yl)-hydrazine (7c). Red solid (90% yield), mp 180–182 °C; IR (KBr) ν_max 3441 (NH), 3032 (sp² C–H), 2978, 2924 (sp³ C–H), 1589 (C=N), 1535, 1481, 1411, 1381, 1311, 1265, 1188, 1072 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.28 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 2.55 (s, 3H, CH₃), 5.25 (s, 2H, CH₂), 6.79–7.85 (m, 13H, Ar–H), 10.65 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 8.98 (CH₃), 21.56 (CH₃), 21.79 (CH₃), 45.99, 112.33, 115.35, 116.02, 122.89, 128.40, 128.65, 129.54, 129.63, 130.18, 133.01, 134.28, 139.19, 139.81, 143.87, 155.07. MS m/z (%) 504 (M⁺ + 1, 1.9), 503 (M⁺, 5.5), 362 (18), 161 (3), 142 (8), 132 (8), 117 (65), 106 (28), 91 (100), 77 (88). Anal. calcd for C₂₆H₂₅N₅O₂S₂ (503.6): C, 62.0; H, 5.0; N, 13.9. Found: C, 62.0; H, 5.1; N, 13.9%.
2.1.2.4. 2-[N′-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-hydrazino]-5-(phenyl-hydrazono)-thiazol-4-one (11a). Yellow solid (72% yield), mp 90–92 °C; IR (KBr) ν_max 3425, 3263 (2NH), 3032 (sp² C–H), 2985 (sp³ C–H), 1705 (C=O), 1612 (C=N), 1543, 1496, 1311, 1226, 1157, 1072 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.33 (s, 3H, CH₃), 5.21 (s, 2H, CH₂), 6.95–7.83 (m, 14H, Ar–H), 10.46 (s, 1H, NH), 12.40 (s, 1H, NH); MS m/z (%) 491 (M⁺, 10), 350 (8), 120 (14), 117 (91), 105 (13), 92 (39), 91 (55), 77 (100). Anal. calcd for C₂₄H₂₁N₅O₃S₂ (491.6): C, 58.6; H, 4.3; N, 14.3. Found: C, 58.5; H, 4.3; N, 14.1%.
2.1.2.5. 2-[N′-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-hydrazino]-5-[(4-chloro-phenyl)-hydrazono]-thiazol-4-one (11b). Yellow solid (71% yield), mp 100–102 °C; IR (KBr) ν_max 3417, 3248 (2NH), 2978, 2924 (sp³ C–H), 1705 (C=O), 1612 (C=N), 1489, 1458, 1311, 1234, 1157, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.34 (s, 3H, CH₃), 5.28 (s, 2H, CH₂), 7.06–7.88 (m, 13H, Ar–H), 10.56 (s, 1H, NH), 12.40 (s, 1H, NH); MS m/z (%) 528 (M⁺ + 2, 1), 527 (M⁺ + 1, 3), 526 (M⁺, 10), 142 (11), 129 (13), 117 (91), 115 (41), 111 (25), 91 (44), 83 (24), 77 (100). Anal. calcd for C₂₄H₂₀ClN₅O₃S₂ (526.0): C, 54.8; H, 3.8; N, 13.3. Found: C, 54.7; H, 3.7; N, 13.2%.
2.1.2.6. 2-[N′-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-hydrazino]-5-[(4-nitro-phenyl)-hydrazono]-thiazol-4-one (11c). Orange solid (65% yield), mp 180–181 °C; IR (KBr) ν_max 3417, 3248 (2NH), 3062 (sp² C–H), 2993, 2931 (sp³ C–H), 1705 (C=O), 1612 (C=N), 1496, 1450, 1327, 1311, 1234, 1149, 1111, 1072 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.29 (s, 3H, CH₃), 5.27 (s, 2H, CH₂), 7.07–8.40 (m, 13H, Ar–H), 10.52 (s, 1H, NH), 11.18 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 21.3 (CH₃), 46.1 (CH₂), 124.5, 126.3, 127.8, 128.0, 128.2, 128.5, 128.5, 128.6, 129.1, 129.5, 129.7, 133.9, 134.6, 136.8, 139.8, 180.0. MS m/z (%) 536 (M⁺, 2.36), 423 (3), 306 (3), 130 (6), 117 (100), 105 (11), 91 (45), 84 (13), 77 (82). Anal. calcd for C₂₄H₂₀N₆O₅S₂ (536.58): C, 53.7; H, 3.8; N, 15.7. Found: C, 53.6; H, 3.5; N, 15.5%.
2.1.2.7. N-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-N′-[4-(4-bromo-phenyl)-5H-thiazol-2-ylidene]-hydrazine (13a). Pale yellow solid (92% yield), mp 120–121 °C; IR (KBr) ν_max 3032 (sp² C–H), 2954, 2916 (sp³ C–H), 1604 (C=N), 1489, 1442, 1365, 1311, 1149, 1111, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.31 (s, 3H, CH₃), 5.16 (s, 2H, CH₂), 5.18 (s, 2H, CH₂), 7.13 (d, J = 7.8 Hz, 2H, Ar–H), 7.54–7.71 (m, 9H, Ar–H), 7.80 (d, J = 7.8 Hz, 2H, Ar–H); MS m/z (%) 527 (M⁺ + 1, 0.22), 385 (11), 124 (15), 117 (100), 91 (32), 77 (77). Anal. calcd for C₂₄H₂₀BrN₃O₂S₂ (526.5): C, 54.8; H, 3.8; N, 8.0. Found: C, 54.7; H, 3.7; N, 7.8%.
2.1.2.8. N-(2-Benzenesulfonyl-1-p-tolyl-ethylidene)-N′-(4-p-tolyl-5H-thiazol-2-ylidene)-hydrazine (13b). Pale orange solid (90% yield), mp 178–180 °C; IR (KBr) ν_max 3032 (sp² C–H), 2954, 2916 (sp³ C–H), 1604 (C=N), 1504, 1442, 1365, 1311, 1149, 1118, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.31 (s, 3H, CH₃), 2.36 (s, 3H, CH₃), 5.16 (s, 2H, CH₂), 5.19 (s, 2H, CH₂), 7.13 (d, J = 8 Hz, 2H, Ar–H), 7.15 (d, J = 8 Hz, 2H, Ar–H), 7.29 (d, J = 8 Hz, 2H, Ar–H), 7.52–7.65 (m, 5H, Ar–H), 7.73 (d, J = 8 Hz, 2H, Ar–H); MS m/z (%) 462 (M⁺ + 1, 0.7), 461 (M⁺, 2.2), 320 (14), 176 (12), 147 (8), 142 (17), 117 (70), 105 (3), 94 (16), 91 (27), 77 (100). Anal. calcd for C₂₅H₂₃N₃O₂S₂ (461.6): C, 65.1; H, 5.0; N, 9.1. Found: C, 65.0; H, 5.0; N, 9.0%.
2.1.2.9. 1,1′-[1,4-Butanediylbis(oxy)]bis[4-(2-{[2-(benzenesulfonyl)-1-(p-tolyl)ethylidene]-hydrazino}thiazol-4-yl)benzene] (15). Buff solid (78% yield), mp 185–187 °C; IR (KBr) ν_max 3420 (NH), 2939 (sp³ C–H), 1604 (C=N), 1550, 1512, 1303, 1242, 1157, 1049 cm⁻¹; ¹H NMR (DMSO-d₆) δ 1.91 (br.s, 4H, 2CH₂), 2.30 (s, 6H, CH₃), 4.10 (br.s, 4H, 2CH₂), 5.18 (s, 4H, CH₂), 6.99 (d, J = 8 Hz, 4H, Ar–H), 7.13 (d, J = 8 Hz, 4H, Ar–H), 7.61 (s, 2H, 2thiazole-H), 7.54–7.89 (m, 18H, Ar–H), 11.60 (br.s, 2H, 2NH); MS m/z (%) 982 (M⁺ + 1, 0.52), 981 (M⁺, 1), 256 (2), 139 (5), 117 (21), 110 (21), 98 (22), 91 (20), 84 (42), 77 (31). Anal. calcd for C₅₂H₄₈N₆O₆S₄ (981.2): C, 63.7; H, 4.9; N, 8.6. Found: C, 63.4; H, 4.9; N, 8.4%.
2.1.2.10. N-[2-Benzenesulfonyl-1-(4-fluorophenyl)-ethylidene]-N′-[4-(4-methoxyphenyl)thiazol-2-yl]-hydrazine (19). Green solid (86% yield), mp 145–147 °C; IR (KBr) ν_max 3271 (NH), 3062 (sp² C–H), 2939 (sp³ C–H), 1604 (C=N), 1558, 1512, 1442, 1311, 1242, 1157, 1080 cm⁻¹; ¹H NMR (DMSO-d₆) δ 3.79 (s, 3H, OCH₃), 5.23 (s, 2H, CH₂), 6.98–7.88 (m, 14H, Ar–H and thiazole-H), 11.75 (s, 1H, NH); MS m/z (%) 483 (M⁺ + 2, 0.36), 481 (M⁺, 1), 339 (14), 310 (10), 142 (10), 121 (100), 101 (44), 95 (19), 77 (82). Anal. calcd for C₂₄H₂₀FN₃O₃S₂ (481.6): C, 59.9; H, 4.2; N, 8.7. Found: C, 59.7; H, 4.1; N, 8.7%.

2.2. Biological activity

2.2.1. B-RAF kinase assay. The kinase inhibitory activity of the B-RAF enzyme was measured using B-RAF (wild-type) and B-RAF(V600E) Kinase Assay Kits (BIOSCIENCE). The B-RAF Assay Kit measures the B-RAF kinase activity using Kinase-Glo® MAX as a detection reagent.^43,44 The percentage of inhibition was calculated by equating the investigated phenylsulfone–thiazole derivatives to the control. The values of IC₅₀ were calculated from the curve of concentration-inhibition response with (n = 3) compared to the reference (dabrafenib) drug.

2.2.2. Cytotoxicity assay. The cytotoxic activity of the phenylsulfone–thiazole derivatives was estimated using the MTT assay.⁴⁵ Dabrafenib was used as the reference drug. In the laboratory MTT test group, Sigma was used. In summary, WM266.4 and Sk-mel-23 melanoma cells were grown in DMEM with the added penicillin, FBS, and streptomycin, and were retained with less than 5% CO₂ at 37 °C. Different concentrations of the phenylsulfone–thiazole derivatives to the cells were incubated at 37 °C for 48 h, followed by the addition of the MTT reagent to the cells and re-incubation. At a wavelength = 590 nm, the absorbance was recorded using a plate reader and the cell viability was calculated. IC₅₀ was extracted from the concentration inhibition response curve (n = 3).

2.2.3. Cell-based B-RAFV600E kinase assay. The blocking effect of title compounds, represented by compound 7b, on the phosphorylation of downstream ERK in B-RAFV600E-mutated WM266.4 melanoma cells was measured using the ELISA kit (Abcam's ERK1/2 (pT202/Y204) and ERK1/2 (Total) in vitro Simple Step ELISA™ Kit). This kit employs an affinity tag-labeled capture antibody and a reporter-conjugated detector antibody, which immunocaptured the sample analyte in solution. Briefly, the cells in the culture medium were treated with compound 7b and dabrafenib dissolved in DMSO. The expression of phosphorylated ERK (pERK) in the samples was measured (ng mL⁻¹) as duplicate determinations, and the data were compared with dabrafenib as the standard B-RAFV600E kinase inhibitors (for more details, see ESI†).

3. Results and discussion

3.1. Chemistry

The synthesis of the unreported thiosemicarbazone 3 was achieved by the acid-catalyzed condensation of the phenacyl phenyl sulfone derivative 1 with thiosemicarbazide 2 in acidic ethanol, as shown in Scheme 1. The synthesized phenyl sulfone–thiosemicarbazone derivative 3 was further analyzed using spectroscopic techniques. The standard NMR techniques were utilized for assigning all the protons as well as carbons in its structural frame (Scheme 1).

The ¹H NMR spectrum for phenyl sulfone–thiosemicarbazone 3 in dimethylsulfoxide-d₆ is described in Fig. 4. The ¹H NMR spectrum of thiosemicarbazone 3 revealed the presence of the open-chain thioamide-tautomer with the NH₂ protons being partially affected due to the outspread conjugation as well as the presence of the phenylsulfonylmethylene moiety. It was observed that the two protons of the amino group (NH₂) were magnetically non-equivalent, displaying two different chemical shift values at δ = 7.9 and 8.4 ppm. This observation could be explained in terms of intramolecular hydrogen bond that restricted or slowed down rotation around the N–C bond.^46,47 It is worth mentioning that the chemical shift values observed for all the NH protons in the presence of phenylsulfonylmethylene group showed a downfield shift of approximately +0.40 to +1.3 ppm compared to the previously reported analog lacking phenylsulfonylmethylene group.⁴⁸ Also, the ¹³C NMR spectrum of the carbothioamide derivative 3 showed a signal at δ = 179 ppm assigned to the C=S group, in addition to another signal at δ = 139 ppm corresponding to C=N.

Fig. 4 ¹H NMR spectrum and intramolecular hydrogen-bonding of compound 3.

Thiosemicarbazone 3 was reacted with hydrazonoyl halides 4a–c as cyclization reagents in dioxane/Et₃N to afford the corresponding substituted phenylsulfone–thiazoles 7a–c pendant to the arylazo substituents (Scheme 2). The IR spectra of derivatives 7a–c revealed the demise of the NH₂ absorption bands of the starting thiosemicarbazone as well as the carbonyl (C=O) group of the reacted hydrazonoyl chlorides. Compared to the ¹H NMR spectrum of the starting phenylsulfone–thiosemicarbazone 3, thiazole derivative 7a (taken as an example) revealed the disappearance of the magnetically-nonequivalent protons of NH₂. Also, the ¹H NMR spectrum of arylazothiazole derivative 7a displayed remarkable four singlet signals at δ = 2.38, 2.59, 5.28, and 10.86 corresponding to two CH₃, CH₂, and NH protons, respectively, in addition to fourteen aromatic protons in the δ range of 7.27–7.87 ppm (Scheme 2 and Fig. 5).

Fig. 5 Comparison of the downfield region of the ¹H NMR spectra of the reactant thiosemicarbazone 3 and its product 7a.

In a similar manner, the reactivity of thiosemicarbazone 3 toward C-1-(ethoxycarbonyl)-N-4-arylhydrazonoyl chlorides 8a–c was investigated. Thus, the reaction of (3) with halogenated reagents 8a–c in dioxane afforded the corresponding arylazothiazolone derivatives 11a–c, as shown in Scheme 3. The suggested structures of compounds 11a–c were confirmed based on the data extracted from their spectral analyses (Scheme 3).

The reaction of 2-(2-(phenylsulfonyl)-1-(p-tolyl)ethylidene)hydrazine-1-carbothioamide (3) with α-bromocarbonyl derivatives 12a and 12b in dioxane/Et₃N furnished the corresponding phenylsulfone–thiazoles 13a and 13b, as illustrated in Scheme 4. The ¹H NMR spectrum of derivative 13a showed three singlet signals at δ 2.31, 5.16, and 5.18 ppm assigned to the methyl and two methylene protons, respectively. Moreover, multiplet signals of the thirteen aromatic protons appeared in the region of δ 7.13–7.80 ppm. Molecular weights (m/z) of phenylsulfone–thiazole derivatives 13a and 13b, extracted from their mass spectra, were identical with the calculated values (Scheme 4).

The treatment of 2-(2-(phenylsulfonyl)-1-(p-tolyl)ethylidene)hydrazine-1-carbothioamide (3) with the interesting bis-α-bromocarbonyl derivative 14 in dioxane/Et₃N afforded the corresponding bis-phenylsulfone–thiazole derivative 15, as depicted in Scheme 4. The ¹H NMR spectrum of 15 revealed two singlet signals at δ 2.30 and 5.18 ppm assigned to the methyl and methylene protons, respectively. The protons of the four methylene (CH₂) groups of the aliphatic ether spacer were confirmed at δ = 1.90 and 4.10 ppm. In addition, the aromatic protons as well as the thiazole-5H signals were detected in the region of δ 6.99–7.89 and 7.61 ppm, respectively.

Prompted by the role of fluorine atom in enhancing the biological activity of previously reported fluorinated compounds,^49,50 p-fluorophenacyl sulfone 16 was reacted with thiosemicarbazide 2 in ethanolic solution containing HCl to obtain the corresponding thiosemicarbazone derivative 17 (Scheme 5). The reaction of the latter with p-methoxyphenacyl bromide 18 afforded the corresponding fluorinated thiazole product 19, as depicted in Scheme 5. Based on the spectroscopic data (experimental part), the structure of compound 19 was confirmed to be the N-[2-Benzenesulfonyl-1-(4-fluorophenyl)-ethylidene]-N′-[4-(4-methoxyphenyl)thiazol-2-yl]-hydrazine 19 (Scheme 5).

3.2. Biological activity

3.2.1. B-RAFV600E kinase assay. In this work, all target compounds were screened in vitro for their B-RAFV600E kinase inhibitory activity employing dabrafenib as the reference standard.

As presented in Table 1 and Fig. 6, all the tested compounds effectively inhibited the kinase activity of the B-RAFV600E enzyme with IC₅₀ values at the nanomolar level in the range from 23.1 to 205 nM, which is equated to the reference drug dabrafenib (IC₅₀ = 47.2 ± 2.5 nM). Among the screened compounds, derivatives 7b and 13a revealed excellent B-RAFV600E inhibitory activity, superior to that of dabrafenib with IC₅₀ values of 36.3 ± 1.9, 23.1 ± 1.2, and 47.2 ± 2.5 nM, respectively. Furthermore, as expected, the bisthiazole derivative 15 showed potent B-RAFV600E inhibition (IC₅₀ = 51.9 ± 2.7 nM), nearly equipotent to that of dabrafenib. In addition, 70% of the activity of dabrafenib toward B-rafV600E was observed for compound 17 (IC₅₀ = 68.2 ± 3.5 nM). Meanwhile, compounds 3, 7c, 11a and 11c, and 19 displayed almost half the potency of dabrafenib with the IC₅₀ values ranging from 84.9 ± 4.4 to 116.3 ± 8 nM. In this investigation, derivatives 7a, 11b, and 13b were the least potent against B-rafV600E kinase, exhibiting IC₅₀ values of about 200 nM.

Table 1 Kinase assay of B-RAF (V600E and wild-type) and cytotoxicity assay of the target compounds

Compound no.	B-RAF (V600E) (nM)	B-RAF (wild type) (nM)	WM266.4^a (μM)	Sk-mel-23^b (μM)
a B-RAFV600E-mutated WM266.4 melanoma cells.b B-RAF wild type Sk-mel-23 melanoma cells.c Not determined.
3	115.3 ± 6	__^c	__	__
7a	198.6 ± 10	__	__	__
7b	36.3 ± 1.9	123.5 ± 6.6	3.18 ± 0.17	7.87 ± 0.40
7c	108.4 ± 5.6	__	__	__
11a	84.9 ± 4.4	361.1 ± 19.4	6.18 ± 0.34	0.43 ± 0.02
11b	185.2 ± 9.6	__	__	__
11c	95.4 ± 5	229 ± 12.3	1.53 ± 0.08	22.3 ± 1.14
13a	23.1 ± 1.2	97.9 ± 5.2	4.52 ± 0.25	50.2 ± 2.56
13b	205.6 ± 11	__	__	__
15	51.9 ± 2.7	251.2 ± 13.5	17.1 ± 0.94	17.6 ± 0.89
17	68.2 ± 3.5	153 ± 8.2	1.24 ± 0.07	12.4 ± 0.63
19	116.3 ± 8	__	__	__
Dabrafenib	47.2 ± 2.5	231.2 ± 12.4	16.5 ± 0.91	9.03 ± 0.46

---

Fig. 6 In vitro B-RAFV600E assay curves of compounds 7b, 11c, 13a, 15, 17, and dabrafenib.

A brief investigation of the structure activity relationships revealed that, in general terms, arylazo thiazoles 7a–c showed higher B-RAFV600E kinase inhibitory activity than aryl hydrazono thiazole derivatives 11a–c. In addition, the tolyl derivatives 7b and 7c were much more potent than their unsubstituted phenyl analogue 7a (IC₅₀ = 198.6 ± 10 nM). Besides, exceeding the activity of dabrafenib, the 3-tolyl analogue 7b (IC₅₀ = 36.3 ± 1.9 nM) was 3-fold more active than its corresponding 4-tolyl derivative 7c (IC₅₀ = 108.4 ± 5.6 nM). On the other hand, the unsubstituted phenyl 11a and 4-nitrophenyl 11c thiazolone derivatives possessed equipotent activity against B-RAFV600E kinase that was 2-fold higher than that elicited by their 4-chlorophenyl analogue 11b.

Regarding compounds 13a and 13b, bearing the aryl moiety at the 4-position of thiazole nucleus, interestingly, the 4-bromophenyl analogue 13a (IC₅₀ = 23.1 ± 1.2 nM) was the most potent among the tested compounds, displaying double the activity of dabrafenib. Conversely, the replacement of bromine atom in 13a by a methyl group in derivative 13b resulted in a dramatic decrease in the activity with compound 13b (IC₅₀ = 205.6 ± 11 nM), being the least potent among the screened compounds.

3.2.2. B-RAF wild-type kinase assay. To evaluate the selectivity of this series, the most potent compounds in B-RAFV600E kinase assay, namely, 7b, 11a and 11c, 13a, 15, and 17 were further tested in vitro against B-RAF wild-type kinase. The results are depicted in Table 1 and Fig. 7. Noticeably, all the representative compounds showed a remarkable selectivity toward B-RAFV600E kinase, ranging from 2- to 5-fold, over the wild-type kinase. Compounds 7b, 11a, and 13a were approximately four times more selective to B-RAFV600E kinase rather than the B-RAF wild-type kinase, while two-fold selectivity for B-RAFV600E kinase was observed for compounds 11c and 17. Among the tested compounds, derivative 15 inhibited B-RAF wild-type kinase at IC₅₀ = 251.2 ± 13.5 nM versus 51.9 ± 2.7 nM for the B-RAFV600E kinase, displaying 5-fold selectivity for B-RAFV600E kinase, similar to that elicited by the reference drug dabrafenib.

Fig. 7 In vitro B-RAF wild type assay curves of compounds 7b, 11c, 13a, 15, 17, and dabrafenib.

3.2.3. Cytotoxicity against B-RAFV600E-mutated melanoma cells. To investigate the anticancer activity of the title compounds, the most potent derivatives, 7b, 11a and 11c, 13a, 15, and 17 were screened against B-RAFV600E-mutated WM266.4 melanoma cells employing dabrafenib as the reference drug. All screened compounds potently inhibited the growth of WM266.4 melanoma cells with IC₅₀ values in the range from 1.24 to 17.1 μM, relative to dabrafenib (IC₅₀ = 16.5 ± 0.91 μM). Apart from compound 15 that was equipotent to dabrafenib, the rest of the tested compounds including 7b, 11a and 11c, 13a, and 17 were much more potent than the reference drug dabrafenib. Analogues 7b, 11a, and 13a possessed remarkable anticancer activity against WM266.4 melanoma cells, three to five times more potent than that of dabrafenib. Moreover, with IC₅₀ values less than 2 μM, compounds 11c and 17 revealed a significant cytotoxic effect toward WM266.4 melanoma cells, exceeding (11-fold) that of dabrafenib (Table 1 and Fig. 8).

Fig. 8 Cytotoxicity assay curve of compounds 7b, 11a and 11c, 13a, 15, 17, and dabrafenib in B-RAFV600E-mutated WM266.4 melanoma cells.

3.2.4. Cytotoxicity against B-RAF wild type melanoma cells. The most potent derivatives, 7b, 11a and 11c, 13a, 15, and 17 were further evaluated against human melanoma cells Sk-mel-23 (B-RAF wild type). As presented in Table 1 and Fig. 9, a positive correlation between the cytotoxic activity and selectivity for B-RAF V600E over B-RAF wild type was clearly observed for compounds 7b, 11c, 13a, and 17. Compound 7b was approximately two times more potent against WM266.4 (B-RAFV600E-mutated) rather than Sk-mel-23 (B-RAF wild type) melanoma cells. Moreover, compounds 11c, 13a, and 17 were exceptionally more cytotoxic (10- to 15-fold) toward WM266.4 (B-RAFV600E-mutated) rather than Sk-mel-23 (B-RAF wild type) melanoma cells. Despite possessing high selectivity for B-RAFV600E kinase over B-RAF wild type, bis compound 15 revealed equal potency against the two tested melanoma cells. On the other hand, among the tested compounds, compound 11a was much more potent against Sk-mel-23 rather than WM266.4 melanoma cells.

Fig. 9 Cytotoxicity assay curve of compounds 7b, 11a and 11c, 13a, 15, 17, and dabrafenib in B-RAF wild type Sk-mel-23 melanoma cells.

3.2.5. Cell-based B-RAFV600E kinase assay. Furthermore, to link the B-RAFV600E kinase inhibitory activity with cell growth inhibition, an in vitro cell-based B-RAFV600E kinase assay was conducted. Thus, the blocking effect of compound 7b was chosen as a representative of the most promising compounds on the phosphorylation of downstream ERK1/2 from WM266.4 (B-RAFV600E-mutated) melanoma cell line. As shown in Table 2 and Fig. 10, the treatment of WM266.4 cells with compound 7b resulted in approximately 51% reduction in the level of phosphorylated ERK1/2 versus 71.9% reduction for dabrafenib compared to untreated (control) cells.

Table 2 The effect of compound 7b on the phosphorylation of ERK in the WM266.4 cell line

Compound no.	pERK 1/2^a (ng mL^−1)	% inhibition^b
a Data were expressed as mean ± standard error (S.E.) of two experiments.b Percentage of inhibition as compared with the control cancer cells.
7b	1.339 ± 0.035	51
Dabrafenib	0.769 ± 0.018	71.9
Control	2.732 ± 0.045	0

---

Fig. 10 The effect of compound 7b on the phosphorylation of ERK in the WM266.4 cell line.

3.3. Docking study

To explain the potent B-RAFV600E kinase inhibitory activity elicited by the title compounds on a molecular basis, a docking study of the most promising compounds in this work was performed using MOE (2014.0901). A high-resolution B-RAFV600E kinase enzyme co-crystallized with dabrafenib was retrieved form protein data bank (PDB code: 4XV2) to be used in the docking study.

Examining the docking results (Fig. 11–13) revealed good fitting and proper orientation of the docked compounds 7b, 11a and 11c, 13a, 15, and 17 into the ATP binding site of B-RAFV600E kinase enzyme with docking energy scores of −8.99, −8.74, −8.80, −7.88, −8.68, and −5.01 kcal mol⁻¹, respectively, compared to the co-crystallized ligand dabrafenib (−9.95 kcal mol⁻¹). Despite showing more potent inhibitory activity against the B-RAF V600E kinase, the docking score of compound 13a was relatively higher than that of compounds 7b, 11a, 11c, and 15. It is worth mentioning that inconsistencies between the inhibitory potency (experimental IC₅₀ values) and calculated docking scores have been reported.^51–53 Noticeably, all the analyzed compounds were involved in interactions with Cys532, a key residue in the hinge region of the enzyme active site. In addition, extensive hydrophobic interactions were observed between the docked compounds and Val471, Lys483, Leu505, Leu514, Phe583, and Phe595 residues, suggesting that the introduction of the hydrophobic groups (e.g., aryl and arylazo) into the thiazole core enhanced the affinity of the target compounds to the ATP binding site of the B-RAFV600E enzyme. The previous molecular docking results combined with the data of the enzyme assay confirm that the title phenylsulfone–thiazole derivatives are potential inhibitors of BRAFV600E kinase (see the ESI† for more details).

Fig. 11 The docked model of compound 7b into the active site of the B-RAFV600E kinase; (A) 2D, (B) 3D.

Fig. 12 The docked model of compound 13a into the active site of the B-RAFV600E kinase; (A) 2D, (B) 3D.

Fig. 13 (A) The 2D docked model of compound 11c into the active site of the B-RAF V600E kinase, (B); overlay of the docked compounds into the active site of the B-RAF V600E kinase.

4. Conclusion

To sum up, novel thiazole derivatives incorporating phenyl sulfonyl moiety were synthesized as the B-RAFV600E kinase inhibitors. All the target compounds inhibited the kinase catalytic activity of B-RAFV600E at nanomolar concentrations. Compounds 7b, 13a, and 15 revealed excellent B-RAFV600E inhibitory activities, superior or equipotent to that of the reference drug dabrafenib. Furthermore, the title compounds were remarkably more selective toward B-RAFV600E kinase than the B-RAF wild-type. In addition, the newly synthesized compounds potently inhibited the growth of B-RAFV600E-mutated melanoma cells, showing more selectivity toward B-RAF V600E-mutated over B-RAF WT melanoma cells. A docking study also displayed hydrogen bonding and hydrophobic interactions of the title compounds with key residues within the active site of the B-RAFV600E kinase.

Conflicts of interest

There are no conflicts of interest to declare.

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Footnote

† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra03624j

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