Synthesis and biological evaluation of certain hydrazonoindolin-2-one derivatives as new potent anti-proliferative agents

Abstract

In connection with our research program on the development of novel indolin-2-one-based anticancer candidates, herein we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. The synthesised derivatives were in vitro evaluated for their anti-proliferative activity towards lung A-549, colon HT-29, and breast ZR-75 human cancer cell lines. Compounds 5b, 5c, 7b, and 10e emerged as the most potent derivatives with average IC₅₀ values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC₅₀ = 8.11 µM). Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC₅₀ = 16 µM. In addition, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).

Graphical Abstract

Introduction

Cancer is a major health burden worldwide and it is deemed to be the second leading cause of mankind mortality after cardiovascular diseases. Most of the clinically available anticancer chemotherapeutic agents are not able to discriminate between cancer cells and the rapidly dividing healthy cells¹. Moreover, the growing increase in drug resistance and undesired side effects of the clinically available cancer chemotherapeutic agents aroused the necessity to search for newer more potent and safer cancer chemotherapeutic candidates^2,3. Subsequently, there is an urgent need to pay much attention to modify and update drug leads from the point of view of medicinal chemistry and drug design to fulfill more effective and potent anticancer agents.

Isatin (1H-indole-2,3-dione) is a privileged scaffold that is present endogenously in both human and other mammalian fluids and tissues, in addition to its presence in diverse naturally occurring components such as marine natural products and alkaloids⁴. Isatin constitutes a leading class of heterocycles that is endowed with interesting biological activities^4–8, chiefly anticancer activity. Nintedanib (Ofev^®) I (Figure 1), an orally available triple angiokinase inhibitor, is approved in Europe in combination with docetaxel for patients with advanced non-small cell lung cancer of adenocarcinoma who have progressed to first-line chemotherapy⁹. Currently, Nintedanib is being investigated in patients with various solid tumors including phase III studies in colorectal cancer^10,11 and phase I studies in breast cancer¹². Sunitinib (Sutent^®) II (Figure 1), a 5-fluoro-3-substituted isatin derivative, has been approved by the FDA for the treatment of gastrointestinal stromal tumors (GIST) and advanced renal cell carcinoma (RCC)¹³. It is a multikinase inhibitor that targets VEGFR-1, VEGFR-2, PDGFRb, and c-Kit.

Figure 1.

Structures of the isatin-based approved anticancer drugs; Nintedanib I and Sunitinib II.

Recently, our research group has paid much attention to develop many novel small molecules based on the indolin-2-one core as potent anticancer agents^14–22. In 2017, we reported two studies^14,15 regarding development of hydrazonoindolin-2-one-based derivatives and evaluation of their anti-proliferative activity against A549 (lung), HT-29 (colon), and ZR-75 (breast) human cancer cell lines, beside evaluation of their pro-apoptotic activity. In the first study¹⁴, a series of hydrazonoindolin-2-ones were synthesized with general structure of III [3-((-benzylidene)hydrazono)indolin-2-one] (Figure 2). The SAR study concerning compounds III suggested that incorporation of lipophilic groups, as methoxy groups, enhances the anti-proliferative activity. Regarding the second study¹⁵, we adopted the molecular hybridisation approach to design and synthesise four different sets of isatin-quinazoline, isatin-phthalazine, and isatin-quinoxaline hybrids, where phthalazine hybrids IV (Figure 2) emerged as the most active anti-proliferative series in this study.

Figure 2.

Structures of some reported hydrazonoindoline-2-ones III & IV, and the target derivatives 3a-e, 5a-e, 7a-c, and 10a-l.

Based on the aforementioned findings and in connection with our research program on the development of indolin-2-one-based anticancer candidates^14–22, it was thought worthwhile to extend our investigations to probe certain hydrazonoindolin-2-ones displaying promising anti-proliferative activity. In this study, we reported the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l, adopting three distinctive strategies to develop such derivatives (Figure 2). The first one focused on grafting of large lipophilic ester or acetyl groups on the pendant phenyl ring of lead structure III to furnish target compounds 3a-e (Figure 2). In the second strategy, a bioisosteric tactic was utilised to replace the pendant substituted phenyl ring in compounds 3a-e with 2-thiazolyl ring, as in compounds 5a-e, or 5-pyrazolyl ring, as in compounds 7a-c (Figure 2), to carry out further elaboration of the hydrazonoindolin-2-ones scaffold and to investigate a valuable SAR. Finally, the hybridisation approach was adopted to conjugate 4-arylthiazoles with indolin-2-one moiety, compounds 10a-l (Figure 2).

The synthesised hydrazonoindolin-2-ones (3a-e, 5a-e, 7a-c, and 10a-l) were in vitro evaluated for their anti-proliferative activity towards three human cancer cell lines, namely lung cancer A-549, human colon cancer HT-29, and breast cancer ZR-75 cell lines. Furthermore, the most active anti-proliferative congeners were further evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cells. In addition, their effect on multidrug-resistant lung cancer NCI-H69AR cell line was assessed. Finally, the cytotoxic activities of the active members were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss 3t3) to estimate their selectivity for tumor cells.

Materials and methods

Chemistry

Melting points were measured with a Stuart melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and were uncorrected. Mass spectra were recorded using Agilent Quadrupole 6120 LC/MS with ESI (Electrospray ionisation) source (Agilent Technologies, Palo Alto, CA). NMR spectra were recorded on a Bruker NMR spectrometer (Bruker, Karlsruhe, Germany). ¹H spectra were run at 500 MHz and ¹³C spectra were run at 125 MHz in deuterated dimethyl sulfoxide (DMSO-d₆). Chemical shifts are expressed in values δ (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in Hertz. The abbreviations used are: “s” for singlet, “d” for doublet, and “m” for multiplet. Elemental analyses were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques.

General procedure for the synthesis of hydrazones 3a-e, 5a-e, and 7a-c

A mixture of equimolar quantities of previously reported hydrazones 1a-c¹⁹ (2 mmol) and aldehydes 2a,b, 4a,b, or 6 (2 mmol) in methyl alcohol (30 ml) containing catalytic amount of glacial acetic acid was refluxed for 4 h, and then cooled to room temperature. The precipitates formed were collected by filtration, dried and crystallised from EtOH/DMF to afford compounds 3a-e, 5a-e, and 7a-c, respectively.

3-((4-Acetylbenzylidene)hydrazono)indolin-2-one (3a)

Yield 73%, m.p. 228–230 °C; IR (KBr, ν cm⁻¹): 3184 (NH), 1722 and 1660 (2 × C=O), 1616 (C=N); ¹H NMR (DMSO-d₆) δ: 2.64 (s, 3H, CH₃), 6.91 (d, J = 7.5 Hz, 1H, H-7 isatin), 7.03 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.42 (t, J = 7.5 Hz, 1H, H-6 isatin), 7.80 (d, J = 7.5 Hz, 1H, H-4 isatin), 8.04–8.14 (m, 4H, of 4-COCH₃-C₆H₄), 8.64 (s, 1H, –CH=N), 10.91 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 27.4 (CH₃), 111.5, 116.7, 122.9, 129.1, 129.2, 129.3, 129.4, 134.5, 137.7, 139.4, 145.7, 150.4, 158.3, 164.8 (C=O), 198.1 (C=O); MS m/z: 291 [M⁺]; Anal. Calcd for C₁₇H₁₃N₃O₂ (291.30): C, 70.09; H, 4.50; N, 14.42; Found C, 69.88; H, 4.56; N, 14.51.

Methyl 4-(((2-oxoindolin-3-ylidene)hydrazono)methyl)benzoate (3b)

Yield 70%, m.p. 225–227 °C; IR (KBr, ν cm⁻¹): 3275 (NH), 1724 and 1675 (2 × C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 3.91 (s, 3H, CH₃), 6.92 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.01 (t, 1H, H-5 isatin, J = 8.0 Hz), 7.41 (t, J = 8.0 Hz, 1H, H-6 isatin), 7.51 (d, J = 7.5 Hz, 1H, H-4 isatin), 8.09–8.13 (m, 4H, of 4-COOCH₃-C₆H₄), 8.65 (s, 1H, –CH=N), 11.00 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 53.1 (COOCH₃), 116.2, 123.1, 128.8, 129.5, 130.4, 134.9, 137.4, 138.1 (2C), 145.2, 145.5, 150.4, 158.5, 164.10, 164.9, 166.1 (C=O); MS m/z: 307 [M⁺]; Anal. Calcd for C₁₇H₁₃N₃O₃ (307.30): C, 66.44; H, 4.26; N, 13.67; Found C, 66.69; H, 4.21; N, 13.52.

Methyl 4-(((5-chloro-2-oxoindolin-3-ylidene)hydrazono)methyl)benzoate (3c)

Yield 65%, m.p. 266–268 °C; IR (KBr, ν cm⁻¹): 3215 (NH), 1734 and 1680 (2 × C=O), 1616 (C=N); ¹H NMR (DMSO-d₆) δ: 3.91 (s, 3H, CH₃), 6.94 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.49 (d, J = 8.0 Hz, 1H, H-6 isatin), 7.74 (s, 1H, H-4 isatin), 8.08–8.15 (m, 4H, of 4-COOCH₃-C₆H₄), 8.68 (s, 1H, –CH=N), 11.06 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 52.9 (COOCH₃), 113.1, 117.6, 126.4, 128.4, 129.4, 130.5, 132.8, 133.9, 134.4, 137.7, 144.4, 149.8, 159.3, 163.8, 164.5, 166.1 (C=O); MS m/z: 341 [M⁺]; Anal. Calcd for C₁₇H₁₂ClN₃O₃ (341.75): C, 59.75; H, 3.54; N, 12.30; Found C, 60.02; H, 3.49; N, 12.18.

3-((4-Acetylbenzylidene)hydrazono)-5-bromoindolin-2-one (3d)

Yield 80%, m.p. 270–272 °C; IR (KBr, ν cm⁻¹): 3178 (NH), 1751 and 1718 (2 × C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 2.65 (s, 3H, CH₃), 6.88 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.59 (d, J = 7.5 Hz, 1H, H-6 isatin), 7.89 (s, 1H, H-4 isatin), 8.07 (d, J = 8.0 Hz, 2H, H-2 and H-6 of 4-COCH₃-C₆H₄), 8.14 (d, J = 8.5 Hz, 2H, H-3 and H-5 of 4-COCH₃-C₆H₄), 8.67 (s, 1H, –CH=N), 11.06 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 27.5 (CH₃), 113.5, 113.9, 118.3, 129.3, 129.5, 131.2, 136.6, 137.5, 139.5, 144.8, 145.9, 149.1, 159.4, 164.3 (C=O), 198.1 (C=O); MS m/z: 370 [M⁺]; Anal. Calcd for C₁₇H₁₂BrN₃O₂ (370.20): C, 55.15; H, 3.27; N, 11.35; Found C, 54.89; H, 3.33; N, 11.49.

Methyl 4-(((5-bromo-2-oxoindolin-3-ylidene)hydrazono)methyl)benzoate (3e)

Yield 85%, m.p. 283–285 °C; IR (KBr, ν cm⁻¹): 3157 (NH), 1705 and 1685 (2 × C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 3.91 (s, 3H, CH₃), 6.89 (d, J = 8.5 Hz, 1H, H-7 isatin), 7.60 (dd, J = 8.5, 2.0 Hz, 1H, H-6 isatin), 7.86 (d, J = 2.0 Hz, 1H, H-4 isatin), 8.08 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-COOCH₃-C₆H₄), 8.15 (d, J = 8.0 Hz, 2H, H-3 and H-5 of 4-COOCH₃-C₆H₄), 8.67 (s, 1H, –CH=N), 11.07 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 52.9 (COOCH₃), 113.5, 113.9, 118.3, 129.3 (2 × C), 130.4 (2 × C), 131.2, 132.7, 136.6, 137.7, 144.8, 149.6, 159.0, 164.3, 166.1 (C=O); MS m/z: 386 [M⁺]; Anal. Calcd for C₁₇H₁₂BrN₃O₃ (386.20): C, 52.87; H, 3.13; N, 10.88; Found, 53.10; H, 3.08; N, 10.72.

3-(((4-Methylthiazol-2-yl)methylene)hydrazono)indolin-2-one (5a)

Yield 70%, m.p. 238–240 °C; IR (KBr, ν cm⁻¹): 3199 (NH), 1732 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ: 2.45 (s, 3H, CH₃), 6.91 (dd, J = 7.5, 3.5 Hz, 1H, H-7 isatin), 7.03 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.42 (d × t, J = 8.0, 3.5 Hz, 1H, H-6 isatin), 7.68 (s, 1H, H-5 thiazole), 7.75 (d, J = 7.5 Hz, 1H, H-4 isatin), 8.69 (s, 1H, –CH=N), 10.93 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 17.2 (CH₃), 111.6, 116.6, 120.0, 122.9, 129.3, 134.8, 145.9, 151.0, 153.3, 155.4, 162.5, 164.6 (C=O); MS m/z: 270 [M⁺]; Anal. Calcd for C₁₃H₁₀N₄OS (270.31): C, 57.76; H, 3.73; N, 20.73; Found C, 57.65; H, 3.75; N, 20.68.

5-Chloro-3-((thiazol-2-ylmethylene)hydrazono)indolin-2-one (5b)

Yield 75%, m.p. 293–295 °C; IR (KBr, ν cm⁻¹): 3217 (NH), 1722 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ: 6.93 (d, J = 8.5 Hz, 1H, H-7 isatin), 7.49–755 (m, 2H, Ar-H), 7.79 (s, 1H, H-4 isatin), 8.19 (d, J = 3.0 Hz, 1H, Ar-H), 8.82 (s, 1H, –CH=N), 11.08 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 113.2, 117.6, 125.9, 126.5, 128.4, 134.1, 144.7, 146.0, 150.9, 154.9, 163.2, 164.4 (C=O); MS m/z: 290 [M⁺]; Anal. Calcd for C₁₂H₇ClN₄OS (290.73): C, 49.57; H, 2.43; N, 19.27; Found C, 49.70; H, 2.48; N, 19.16.

5-Chloro-3-(((4-methylthiazol-2-yl)methylene)hydrazono)indolin-2-one (5c)

Yield 77%, m.p. 290–292 °C; IR (KBr, ν cm⁻¹): 3230 (NH), 1718 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ: 2.45 (s, 3H, CH₃), 6.90 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.61 (d, J = 8.0, 2.0 Hz, 1H, H-6 isatin), 7.75 (s, 1H, H-5 thiazole), 7.81 (s, 1H, H-4 isatin), 8.73 (s, 1H, –CH=N), 11.10 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 17.2 (CH₃), 113.1, 117.6, 120.6, 126.4, 128.4, 128.9, 134.1, 144.7, 146.3, 155.1, 162.2, 164.4 (C=O); MS m/z: 304 [M⁺]; Anal. Calcd for C₁₃H₉ClN₄OS (304.75): C, 51.23; H, 2.98; N, 18.38; Found C, 51.39; H, 3.03; N, 18.23.

5-Bromo-3-((thiazol-2-ylmethylene)hydrazono)indolin-2-one (5d)

Yield 83%, m.p. 260–262 °C; IR (KBr, ν cm⁻¹): 3236 (NH), 1718 (C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 6.89 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.61 (d, J = 8.5 Hz, 1H, H-6 isatin), 7.93 (s, 1H, H-4 isatin), 8.16 (d, J = 3.0 Hz, 1H, H-5 thiazole), 8.20 (d, J = 3.0 Hz, 1H, H-4 thiazole), 8.81 (s, 1H, –CH=N), 11.08 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 118.3, 125.9, 130.4, 131.7, 136.9, 145.1, 145.3, 146.1, 150.9, 154.9, 163.2, 164.2 (C=O); MS m/z: 335 [M⁺]; Anal. Calcd for C₁₂H₇BrN₄OS (335.18): C, 43.00; H, 2.11; N, 16.72; Found C, 42.79; H, 2.14; N, 16.85.

5-Bromo-3-(((4-methylthiazol-2-yl)methylene)hydrazono)indolin-2-one (5e)

Yield 80%, m.p. 248–250 °C; IR (KBr, ν cm⁻¹): 3138 (NH), 1735 (C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 2.46, 2.48 (2s, 3H, CH₃), 6.88 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.60 (dd, J = 8.0, 2.0 Hz, 1H, H-6 isatin), 7.72, 7.83 (2s, 1H, H-5 thiazole), 7.90, 7.94 (2d, J = 2.0 Hz, 1H, H-4 isatin), 8.53, 8.75 (2 × s, 1H, –CH=N), 11.07, 11.11 (2 × s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 16.9, 17.2 (2 × CH₃), 113.6, 114.1, 115.7, 118.0, 118.4, 120.6, 121.4, 130.9, 131.7, 132.4, 135.4, 136.8, 136.9, 144.8, 145.0, 150.9, 152.3, 154.9 155.1, 155.6, 162.2, 164.1, 164.2 (C=O); MS m/z: 349 [M⁺]; Anal. Calcd for C₁₃H₉BrN₄OS (349.21): C, 44.71; H, 2.60; N, 16.04; Found C, 44.90; H, 2.54; N, 16.15.

3-(((1H-pyrazol-5-yl)methylene)hydrazono)indolin-2-one (7a)

Yield 74%, m.p. 283–285 °C; IR (KBr, ν cm⁻¹): 3215 (NH), 1708 (C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 6.93–7.05 (m, 3H, Ar-H), 7.39 (t, J = 7.5 Hz, 1H, H-6 isatin), 7.51 (d, J = 7.5 Hz, 1H, H-4 isatin), 7.96–8.00 (m, 1H, Ar-H), 8.55 (s, 1H, –CH=N), 11.01 (s, 1H, NH), 13.54 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 102.9, 111.2, 118.6, 122.8, 123.0, 131.7, 134.9, 142.0, 145.4, 145.9, 156.3, 165.0 (C=O); MS m/z: 239 [M⁺]; Anal. Calcd for C₁₂H₉N₅O (239.23): C, 60.25; H, 3.79; N, 29.27; Found C, 60.07; H, 3.82; N, 29.39.

3-(((1H-pyrazol-5-yl)methylene)hydrazono)-5-chloroindolin-2-one (7b)

Yield 77%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3221 (NH), 1735 (C=O), 1620 (C=N); ¹H NMR (DMSO-d₆) δ: 6.90 (d, J = 9.0 Hz, 1H, Ar-H), 6.94 (d, J = 8.0 Hz, 1H, Ar-H), 7.46 (dd, J = 8.0, 2.5 Hz, 1H, Ar-H), 7.74 (d, J = 2.5 Hz, 1H, Ar-H), 7.96 (d, J = 8.0 Hz, 1H, Ar-H), 8.59 (s, 1H, –CH=N), 11.00 (s, 1H, NH), 13.17 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 103.0, 112.5, 118.9, 125.7, 127.5, 135.9, 137.1, 140.5, 144.7, 147.1, 157.1, 164.2 (C=O); MS m/z: 273 [M⁺]; Anal. Calcd for C₁₂H₈ClN₅O (273.68): C, 52.66; H, 2.95; N, 25.59; Found C, 52.43; H, 3.00; N, 25.75.

3-(((1H-pyrazol-5-yl)methylene)hydrazono)-5-bromoindolin-2-one (7c)

Yield 85%, m.p. 264–266 °C; IR (KBr, ν cm⁻¹): 3209 (NH), 1734 (C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 6.82 (d, J = 8.5 Hz, 1H, Ar-H), 6.87 (d, J = 8.0 Hz, 1H, Ar-H), 7.58 (d, J = 8.5 Hz, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 8.10 (d, J = 8.0 Hz, 1H, Ar-H), 8.60 (s, 1H, –CH=N), 11.04 (s, 1H, NH), 13.62 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 104.76, 113.28, 118.65, 122.19, 127.95, 131.43, 136.39, 144.51, 147.30, 150.39, 156.64, 164.61 (C=O); MS m/z: 318 [M⁺]; Anal. Calcd for C₁₂H_7brN₅O (318.13): C, 45.31; H, 2.53; N, 22.01; Found C, 45.49; H, 2.55; N, 21.86.

General procedure for the synthesis of thiazoles 10a-l

A mixture of previously reported thiosemicarbazones 8a-c²³ (2 mmol) and bromoethanones (9a-d) (2 mmol) in absolute ethanol (20 ml) was heated under reflux for 3 h, then left to cool. The precipitated solid was collected by filtration, washed with ethanol, dried and recrystallised from EtOH/DMF to yield the corresponding thiazole derivatives 10a-l, respectively. Compounds 10a-e and 10g-l^24–29.

3–(2-(4-Phenylthiazol-2-yl)hydrazono)indolin-2-one (10a)

Yield 70%, m.p. 275–277 °C; IR (KBr, ν cm⁻¹): 3136 (NH), 1707 (C=O), 1616 (C=N); ¹H NMR (DMSO-d₆) δ:6.96 (dd, J = 8.0, 3.5 Hz, 1H, H-7 isatin), 7.09 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.30–7.54 (m, 6H, Ar-H), 7.64 (s, 1H, H-5 thiazole), 7.89 (d, J = 7.5 Hz, 1H, H-4 isatin), 11.26 (s, 1H, NH), 13.32 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 107.3, 111.6, 119.9, 120.4, 121.8, 122.9, 126.0, 128.5, 129.2, 130.9, 132.6, 134.4, 141.8, 148.8, 151.5, 163.7, 166.5 (C=O); MS m/z: 320 [M⁺]; Anal. Calcd for C₁₇H₁₂N₄OS (320.37) C, 63.73; H, 3.78; N, 17.49; Found, C, 63.91; H, 3.81; N, 17.49.

3–(2-(4–(4-Tolyl)thiazol-2-yl)hydrazono)indolin-2-one (10b)

Yield 67%, m.p. 284–286 °C; IR (KBr, ν cm⁻¹): 3315 (NH), 1707 (C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ: 2.38 (s, 3H, CH₃), 6.96 (dd, J = 8.0, 3.5 Hz, 1H, H-7 isatin), 7.08 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.20–7.38 (m, 3H, Ar-H), 7.53 (d, J = 8.0 Hz, 1H, H-4 isatin), 7.68 (s, 1H, H-5 thiazole), 7.79 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-CH₃-C₆H₄), 11.26 (s, 1H, NH), 13.33 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 21.3 (CH₃), 119.9, 120.3, 121.8, 122.9, 126.1, 128.3, 129.5, 129.8, 130.6, 130.9, 131.8, 132.5, 137.8, 141.75, 151.5, 163.5, 166.4 (C=O); MS m/z: 334 [M⁺]; Anal. Calcd for C₁₈H₁₄N₄OS (334.39): C, 64.65; H, 4.22; N, 16.75; Found, C, 64.43; H, 4.24; N, 16.87.

3–(2-(4–(4-Chlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (10c)

Yield 73%, m.p. 295–297 °C; IR (KBr, ν cm⁻¹): 3257 (NH), 1693 (C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 6.95 (d, J = 7.5 Hz, 1H, H-7 isatin), 7.06 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.32 (t, J = 7.5 Hz, 1H, H-6 isatin,), 7.45 (d, J = 8.5 Hz, 2H, H-3 and H-5 of 4-Cl-C₆H₄), 7.51 (d, J = 8.0 Hz, 1H, H-4 isatin), 7.67 (s, 1H, H-5 thiazole), 7.90 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-Cl-C₆H₄), 11.24 (s, 1H, NH), 13.33 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 107.9, 111.5, 120.2, 120.3, 122.9, 127.8, 127.9, 128.9, 129.1, 130.1, 130.9, 132.7, 133.3, 141.8, 150.3, 163.6, 166.7 (C=O); MS m/z: 354 [M⁺]; Anal. Calcd for C₁₇H₁₁ClN₄OS (354.81) C, 57.55; H, 3.12; N, 15.79; Found, C, 57.76; H, 3.17; N, 15.66.

3–(2-(4–(4-Bromophenyl)thiazol-2-yl)hydrazono)indolin-2-one (10d)

Yield 75%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3259 (NH), 1689 (C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 6.95 (d, J = 7.5 Hz, 1H, H-7 isatin), 7.09 (t, J = 7.5 Hz, 1H, H-5 isatin), 7.33 (t, J = 7.5 Hz, 1H, H-6 isatin), 7.52 (d, J = 7.5 Hz, 1H, H-4 isatin), 7.60 (d, J = 8.0 Hz, 2H, H-3 and H-5 of 4-Br-C₆H₄), 7.68 (s, 1H, H-5 thiazole), 7.84 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-Br-C₆H₄), 11.25 (s, 1H, NH), 13.34 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.1, 111.5, 120.2, 120.4, 121.5, 122.9, 128.0, 128.2, 131.0, 131.9, 132.1, 132.7, 133.6, 141.8, 150.3, 163.7, 166.2 (C=O); MS m/z: 399 [M⁺]; Anal. Calcd for C₁₇H₁₁BrN₄OS (399.26) C, 51.14; H, 2.78; N, 14.03; Found, C, 50.89; H, 2.81; N, 14.14.

5-Chloro-3–(2-(4-phenylthiazol-2-yl)hydrazono)indolin-2-one (10e)

Yield 80%, m.p. 292–294 °C; IR (KBr, ν cm⁻¹): 3169 (NH), 1707 (C=O), 1612 (C=N); ¹H NMR (DMSO-d₆) δ:6.95 (d, J = 7.5 Hz, 1H, H-7 isatin), 7.30–7.60 (m, 6H, Ar-H), 7.68 (s, 1H, H-5 thiazole), 7.84 (d, J = 7.5 Hz, 1H, H-4 isatin), 11.25 (s, 1H, NH), 13.34 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.1, 111.5, 120.2, 120.4, 121.5, 122.9, 128.19, 131.0, 132.1, 132.7, 133.6, 141.8, 150.3, 163.7, 166.7 (C=O); MS m/z: 354 [M⁺]; Anal. Calcd for C₁₇H₁₁ClN₄OS (354.81) C, 57.55; H, 3.12; N, 15.79; Found, C, 57.73; H, 3.07; N, 15.64.

5-Chloro-3–(2-(4-(p-tolyl)thiazol-2-yl)hydrazono)indolin-2-one (10f)

Yield 78%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3155 (NH), 1685 (C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 2.38, 2.40 (2s, 3H, CH₃), 6.85, 6.97 (2 × d, J = 8.5 Hz, 1H, H-7 isatin), 7.28–7.38 (m, 3H, Ar-H), 7.52, 7.58 (2s, 1H, H-5 thiazole), 7.71, 7.78 (2 × d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-CH₃-C₆H₄), 8.34, 8.41 (2 × d, J = 2.0 Hz, 1H, H-4 isatin), 10.62, 11.36 (2s, 1H, NH), 13.34 (s, br, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 21.3 (CH₃), 106.9, 111.5, 113.0, 119.8, 121.9, 126.0, 126.1, 127.0, 129.8, 129.9, 131.3, 131.7, 137.9, 140.3, 151.6, 163.5, 166.2 (C=O); MS m/z: 368 [M⁺]; Anal. Calcd for C₁₈H₁₃ClN₄OS (368.84) C, 58.61; H, 3.55; N, 15.19; Found, C, 58.77; H, 3.51; N, 15.07.

5-Chloro-3–(2-(4–(4-chlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (10g)

Yield 85%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3215 (NH), 1695 (C=O), 1620 (C=N); ¹H NMR (DMSO-d₆) δ: 6.98 (d, 1H, H-7 isatin, J = 8.5 Hz), 7.37 (dd, J = 8.5, 2.0 Hz, 1H, H-6 isatin), 7.48 (d, J = 8.5 Hz, 2H, H-3 and H-5 of 4-Cl-C₆H₄), 7.53 (d, J = 2.0 Hz, 1H, H-4 isatin), 7.74 (s, 1H, H-5 thiazole), 7.92 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-Cl-C₆H₄), 11.37 (s, 1H, NH), 13.34 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.5, 113.1, 119.8, 121.9, 127.1, 127.8, 127.9, 129.2, 129.5, 130.3, 131.6, 132.3, 133.2, 140.4, 150.3, 163.5, 166.5 (C=O); MS m/z: 389 [M⁺]; Anal. Calcd for C₁₇H₁₀Cl₂N₄OS (389.26): C, 52.45; H, 2.59; N, 14.39; Found, C, 52.67; H, 2.62; N, 14.53.

3–(2-(4–(4-Bromophenyl)thiazol-2-yl)hydrazono)-5-chloroindolin-2-one (10h)

Yield 90%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3250 (NH), 1690 (C=O), 1620 (C=N); ¹H NMR (DMSO-d₆) δ: 6.97 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.37 (dd, J = 8.5, 2.0 Hz, 1H, H-6 isatin), 7.53 (d, J = 2.0 Hz, 1H, H-4 isatin), 7.61 (d, J = 8.5 Hz, 2H, H-3 and H-5 of 4-Br-C₆H₄), 7.75 (s, 1H, H-5 thiazole), 7.85 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-Br-C₆H₄), 11.37 (s, 1H, NH), 13.34 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.5, 113.1, 120.0, 121.9, 122.0, 126.7, 128.1, 128.3, 130.6, 131.2, 132.1, 132.3, 133.6, 140.4, 150.4, 163.5, 166.7 (C=O); MS m/z: 433 [M⁺]; Anal. Calcd for C₁₇H₁₀BrClN₄OS (433.71) C, 47.08; H, 2.32; N, 12.92; Found, C, 46.87; H, 2.29; N, 13.11.

5-Bromo-3–(2-(4-phenylthiazol-2-yl)hydrazono)indolin-2-one (10i)

Yield 83%, m.p. 285–287 °C; IR (KBr, ν cm⁻¹): 3169 (NH), 1685 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ:6.91 (d, J = 8.0 Hz, 1H, H-7 isatin), 7.42–7.84 (m, 6H, Ar-H), 7.90 (d, J = 8.0 Hz, 1H, Ar-H), 8.53 (d, J = 8.0 Hz, 1H, H-4 isatin), 11.37 (s, 1H, NH), 13.32 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 107.8, 112.0, 113.5, 114.7, 122.4, 122.5, 126.1, 128.5, 129.2, 129.4, 131.3, 133.1, 134.4, 140.7, 151.6, 163.3, 166.3 (C=O); MS m/z: 399 [M⁺]; Anal. Calcd for C₁₇H₁₁BrN₄OS (399.26) C, 51.14; H, 2.78; N, 14.03; Found, C, 51.37; H, 2.72; N, 13.88.

5-Bromo-3–(2-(4–(4-tolyl)thiazol-2-yl)hydrazono)indolin-2-one (10j)

Yield 80%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3157 (NH), 1685 (C=O), 1618 (C=N); ¹H NMR (DMSO-d₆) δ: 2.38, 2.39 (2s, 3H, CH₃), 6.80, 6.92 (2 × d, J = 8.0 Hz, 1H, H-7 isatin), 7.18, 7.24 (2 × d, J = 7.5 Hz, 2H, H-3 and H-5 of 4-CH₃-C₆H₄), 7.41, 7.49 (2 × dd, J = 8.0, 2.0 Hz, 1H, H-6 isatin), 7.59 (s, 1H, H-5 thiazole), 7.63, 8.52 (2 × d, J = 2.0 Hz, 1H, H-4 isatin), 7.72, 7.78 (2 × d, J = 8.0 Hz, 2H, H-2 and H-6 of 4-CH₃-C₆H₄), 10.62, 11.37 (2s, 1H, NH), 12.97, 13.32 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 21.3 (CH₃), 106.9, 113.5, 114.7, 122.4, 122.5, 126.0, 126.1, 129.8, 129.9, 131.2, 131.7, 133.0, 137.9, 140.7, 151.7, 163.3, 166.2 (C=O); MS m/z: 413 [M⁺]; Anal. Calcd for C₁₈H₁₃BrN₄OS (413.29) C, 52.31; H, 3.17; N, 13.56; Found, C, 52.59; H, 3.14; N, 13.48.

5-Bromo-3–(2-(4–(4-chlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (10k)

Yield 84%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3159 (NH), 1687 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ: 6.92 (d, J = 8.5 Hz, 1H, H-7 isatin), 7.48–7.57 (m, 4H, Ar-H), 7.64 (s, 1H, H-5 thiazole), 7.92 (d, J = 8.5 Hz, 2H, H-2 and H-6 of 4-Cl-C₆H₄), 11.37 (s, 1H, NH), 13.32 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.8, 113.4, 115.1, 122.2, 122.5, 127.9 (2 × C), 129.2, 129.8, 131.5, 132.9, 133.1, 133.2, 140.9, 150.3, 163.4, 166.5 (C=O); MS m/z: 433 [M⁺]; Anal. Calcd for C₁₇H₁₀BrClN₄OS (433.71) C, 47.08; H, 2.32; N, 12.92; Found, C, 47.15; H, 2.35; N, 12.81.

5-Bromo-3–(2-(4–(4-bromophenyl)thiazol-2-yl)hydrazono)indolin-2-one (10l)

Yield 90%, m.p. >300 °C; IR (KBr, ν cm⁻¹): 3157 (NH), 1685 (C=O), 1614 (C=N); ¹H NMR (DMSO-d₆) δ: 6.93 (d, J = 8.5 Hz, 1H, H-7 isatin), 7.50–7.87 (m, 6H, Ar-H), 8.52 (s, 1H, H-4 isatin), 11.38 (s, 1H, NH), 13.33 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ: 108.7, 113.6, 114.6, 121.7, 122.3, 122.8, 128.1, 128.2, 131.4, 132.1, 133.1, 133.6, 140.9, 142.1, 150.3, 163.4, 166.5 (C=O); MS m/z: 478 [M⁺]; Anal. Calcd for C₁₇H₁₀Br₂N₄OS (478.16) C, 42.70; H, 2.11; N, 11.72; Found, C, 42.52; H, 2.09; N, 11.78.

Biological evaluation

In vitro anti-proliferative activity

The anti-proliferative activity of the title compounds 3a-e, 5a-e, 7a-c, and 10a-l towards cancer cell lines, normal cell lines, and multidrug-resistant lung cancer NCI-H69AR cell line was determined using CellTiter-Glo™ Luminescent cell viability assay according to the previously published procedures^14,15.

Cell cycle effects

Cell cycle effects of the synthesised compounds 7b and 10e were determined adopting the reported procedures^14,15.

Results and discussion

Chemistry

Our proposed synthetic strategy to the target hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l is depicted in Schemes 1 and 2. The synthesis of the 3-hydrazonoindolin-2-ones 1a-c was accomplished via refluxing indoline-2,3-diones with hydrazine hydrate in ethanol. Further condensation of hydrazones 1a-c with benzaldehydes (2a, b), thiazole-2-carbaldehydes (4a, b), or 1H-pyrazole-5-carbaldehyde (6) in methyl alcohol in the presence of a catalytic amount of glacial acetic acid afforded the target derivatives 3a-e, 5a-e, and 7a-c, respectively, in 65–85% yield (Scheme 1).

Scheme 1.

Synthesis of the hydrazonoindolin-2-ones 3a-e, 5a-e, and 7a-c. Reagents and conditions: (i) Methanol, glacial acetic acid (cat.), reflux, 4 h.

Scheme 2.

Synthesis of thiazoles 10a-l. Reagents and conditions: (i) Ethanol, reflux, 3 h.

The assigned structures of compounds 3a-e, 5a-e, and 7a-c were confirmed by IR, ¹H and ¹³C NMR spectra. Their IR spectra showed absorption bands around 3200 cm⁻¹ for the (NH) groups of the indolin-2-one moiety, in addition to the absorption bands of (C=O) groups in the region of 1660–1751 cm⁻¹. ¹H NMR spectra of compounds 3a-e, 5a-e, and 7a-c revealed the presence of a singlet signal in the region δ 10.93–11.11 corresponding to NH proton of isatin moiety, in addition to the singlet signal of the methine (–CH=N–) proton in the region δ 8.55–8.82. Also, compounds 7a-c displayed additional signal attributable to the NH proton of the pyrazole ring at δ 13.17–13.62. Moreover, the ¹³C NMR spectra of these compounds displayed signals resonating around δ 164 corresponding to the amidic (C=O) carbons.

On the other hand, the condensation of indoline-2,3-diones with thiosemicarbazide in refluxing ethanol containing a catalytic amount of acetic acid yielded the corresponding thiosemicarbazones 8a-c. The second targeted isatin-based derivatives 10a-l were obtained in good yields (67–90%) through the reaction of the key intermediates 8a-c with the appropriate phenacyl bromide 9a-d in refluxing absolute ethyl alcohol (Scheme 2).

IR spectra of the compounds 10a-l displayed the characteristic absorption bands in the regions 3136–3315 cm⁻¹ and 1685–1707 cm⁻¹ corresponding to the (NH) and (C=O) groups, respectively, while their ¹H NMR spectra were characterised by the presence of three singlets in the regions δ 11.24–11.38, 13.32–13.36, and 7.58–7.75 which were assigned to two NH groups and H-5 of the thiazole ring, respectively. Besides, ¹³C NMR spectra of these compounds showed characteristic signals resonating around δ 166 which was attributed to (C=O) carbon. It is worth noting that some derivatives are existing in E/Z geometrical isomerism in the DMSO-solution state, such as compounds 5e, 10f, and 10j¹⁷.

Biological evaluation

In vitro anti-proliferative activity

The cancer cell growth inhibitory activity of the synthesised 25 compounds 3a-e, 5a-e, 7a-c, and 10a-l was evaluated in vitro towards three human cancer cell lines, namely lung cancer A-549, human colon cancer HT-29, and breast cancer ZR-75 cell lines using CellTiter-Glo™ Luminescent cell viability assay³⁰. Sunitinib was included in this assay as a reference drug.

The prepared hydrazonoindolin-2-ones were first evaluated at preliminary anti-proliferative assay against the three cancer cell lines in quadruplicate at a single concentration of 30 µM. The data presented as percentage growth inhibition (GI%) caused by the test compounds (Table 1).

Table 1.

Anti-proliferative (cell growth inhibitory activity at 30 µM concentration) activity of the target compounds 3a-e, 5a-e, 7a-c, 10a-l, and Sunitinib towards HT-29, ZR-75, and A-549 cell lines.

	Growth inhibition %
Compound No.	A-549	ZR-75	HT-29	Average growth inhibition %
3a	12.7 ± 7.1	29.6 ± 24.4	53.6 ± 12.2	32.0
3b	68.1 ± 2.8	80.6 ± 7.0	92.3 ± 3.7	80.3
3c	93.5 ± 2.8	98.9 ± 2.1	95.9 ± 4.7	96.1
3d	17.3 ± 10.2	53.9 ± 14.4	56.8 ± 15.1	42.7
3e	−4.2 ± 9.3	55.9 ± 18.2	26.3 ± 24.4	41.1
5a	2.4 ± 5.0	40.8 ± 25.7	37.9 ± 26.3	27.0
5b	93.6 ± 5.4	95.0 ± 8.2	96.2 ± 4.5	94.9
5c	98.8 ± 1.5	98.0 ± 2.6	96.4 ± 4.8	97.7
5d	94.8 ± 2.5	96.4 ± 1.5	98.5 ± 3.1	96.6
5e	95.3 ± 3.1	96.9 ± 2.4	99.0 ± 1.1	97.1
7a	8.2 ± 15.1	60.1 ± 5.8	60.8 ± 13.1	43.0
7b	91.5 ± 1.3	82.3 ± 15.2	94.9 ± 1.8	89.6
7c	32.8 ± 8.9	19.2 ± 22.8	28.8 ± 17.3	26.9
10a	70.7 ± 5.7	76.9 ± 7.1	91.2 ± 8.3	79.6
10b	24.4 ± 14.1	62.1 ± 10.7	33.9 ± 21.2	40.1
10c	31.4 ± 9.1	77.1 ± 10.9	85.4 ± 7.0	64.6
10d	12.3 ± 11.3	53.8 ± 19.2	20.6 ± 26.9	28.9
10e	80.4 ± 6.0	89.3 ± 7.2	85.7 ± 10.2	85.1
10f	27.4 ± 5.8	50.3 ± 33.6	54.4 ± 19.5	44.0
10g	18.9 ± 3.2	52.8 ± 9.4	34.4 ± 23.6	35.4
10h	−2.6 ± 7.5	9.0 ± 19.2	−8.3 ± 43.8	9.0
10i	92.3 ± 6.2	97.5 ± 3.1	96.3 ± 5.9	95.4
10j	85.9 ± 9.1	73.1 ± 3.5	90.1 ± 5.3	83.0
10k	72.4 ± 3.5	50.6 ± 11.5	66.8 ± 19.1	63.3
10l	−20.1 ± 5.9	40.9 ± 16.7	27.8 ± 4.3	34.4
Sunitinib	59.5 ± 2.3	90.7 ± 4.5	85.7 ± 2.7	78.7

The tested hydrazonoindolin-2-ones displayed different levels of anti-proliferative activity and possessed a distinctive pattern of selectivity against the tested cancer cell lines with average growth inhibition in the range of 9.0–97.7%. Close examination of the GI% values in Table 1, revealed that compounds 3b, 3c, 5b-e, 7b, 10e, 10i, and 10j are the most active analogs in this study, showing broad spectrum activity toward the tested cell lines with average GI% values >80%. On the other hand, compounds 3a, 5a, 7c, 10d, 10h, and 10l displayed modest growth inhibitory activity (GI% values <35%). It is noteworthy to mention that compounds 3e and 10l stimulated the growth of the A-549 cell line and compound 10h stimulated the growth of both A-549 and HT-29 cell lines.

The quantitative inhibitory concentration 50% (IC₅₀) values were evaluated for the active members that displayed effective growth inhibition toward the three cancer cell lines (GI% values >35%) in the preliminary single high dose (30 µM) screening (Table 2). From the displayed results in Table 2, it was obvious that several of the synthesised hydrazonoindolin-2-ones possessed excellent to moderate growth inhibitory activity against the tested cancer cell lines.

Table 2.

Inhibitory concentration 50% (IC₅₀) of anti-proliferative activity of the tested compounds 3b-e, 5b-e, 7a, 7b, 10a-k, and Sunitinib toward HT-29, ZR-75, and A-549 cell lines.

	IC50 (µM)a
Compound No.	A-549	ZR-75	HT-29	Average IC50 (µM)
3b	11.25 ± 1.45	9.72 ± 2.52	21.05 ± 1.86	14.01
4c	6.07 ± 0.90	4.20 ± 0.57	8.49 ± 1.01	6.25
4d	26.09 ± 4.47	NAb	NAb	>28.69
3e	NAb	23.33 ± 0.0	NAb	>27.77
5b	4.87 ± 0.49	2.09 ± 0.12	6.15 ± 0.51	4.37
5c	2.15 ± 0.23	1.16 ± 0.39	4.29 ± 0.25	2.53
5d	12.53 ± 1.73	12.68 ± 2.40	15.32 ± 1.93	13.51
5e	3.54 ± 0.42	2.93 ± 0.46	8.51	7.49
7a	22.94 ± 3.51	NAb	NAb	>27.65
7b	2.29 ± 0.21	1.37 ± 0.23	2.75 ± 0.31	2.14
10a	9.41 ± 1.29	4.35	19.44 ± 1.03	11.07
10b	NAb	24.59	NAb	>28.19
10c	17.2 ± 2.56	4.89	NAb	>17.36
10e	4.79 ± 0.45	1.95 ± 1.03	7.25 ± 0.83	4.66
10f	22.72 ± 3.71	23.41	NAb	>25.38
10g	6.07 ± 7.41	1.87 ± 0.26	NAb	>12.65
10i	6.13 ± 0.76	1.96 ± 0.85	8.26 ± 0.86	5.45
10j	12.84 ± 1.40	10.91 ± 2.81	15.22 ± 1.10	12.99
10k	20.13 ± 3.81	3.61	19.75 ± 1.63	14.49
Sunitinib	10.14 ± 0.8	8.31 ± 2.4	5.87 ± 0.3	8.11

^aIC₅₀ values are the mean ± SD of four separate experiments.

^bNA: Compounds having IC₅₀ value >30 μM.

Investigations of the anti-proliferative activity against A-549 indicated that compounds 5b, 5c, 5e, 7b, and 10e (IC₅₀ = 4.87 ± 0.49, 2.15 ± 0.23, 3.54 ± 0.42, 2.29 ± 0.21, and 4.79 ± 0.45 µM, respectively) were found to be the most potent counterparts as they were 2, 4.7, 2.8, 4.4 and 2.1 times, respectively, more active than Sunitinib (IC₅₀ = 10.14 ± 0.8 µM). Besides, compounds 3c, 10a, 10g, and 10i possessed excellent anti-proliferative activity against A-549 cells (IC₅₀ = 6.07 ± 0.90, 9.41 ± 1.29, 6.07 ± 7.41, and 6.13 ± 0.76 µM, respectively) which are better than the used reference drug Sunitinib. On the other hand, compounds 3e and 10b showed weak activity with IC₅₀ >30 µM. Concerning activity against ZR-75 cells, compounds 3c, 5b, 5c, 5e, 7b, 10a, 10c, 10e, 10g, 10i, and 10k displayed excellent anti-proliferative activity superior to that of Sunitinib with IC₅₀ range 1.16–4.89 µM. Whereas, compounds 3d and 7a possessed weak activity with IC₅₀ >30 µM. Moreover, anti-proliferative evaluation in HT-29 cell line revealed that two members, 5c and 7b, only showed superior activity to Sunitinib (IC₅₀ = 4.29 ± 0.25 and 2.75 ± 0.31 µM, respectively). Also, compounds 3c, 5b, 5e, 10e, and 10i were moderately active with IC₅₀ range of 6.15–8.49 µM.

Compound 7b was the most active congener in the synthesised Schiff bases 3a-e, 5a-e, and 7a-c with an average IC₅₀ value of 2.14 µM toward the tested human cancer cell lines being nearly four-fold more potent than the positive control, Sunitinib (average IC₅₀ value =8.11 µM). Whereas, compound 10e was the most active candidate in the arylthizole derivatives 10a-l with an apparent average IC₅₀ value of 4.66 µM against the tested cell lines.

Structure activity relationship

Observing the results, we could deduce valuable data about the structure activity relationship (SAR). Firstly, investigation of the impact of the substitution on the pendant phenyl group in compounds 3a-e revealed that grafting an ester group (compound 3b; average IC₅₀= 14.01 µM) is more beneficial for activity than acetyl group (compound 3a; average IC₅₀> 30 µM). The effect of bioisosteric replacement of the pendant phenyl ring in compounds 3a-e with 2-thiazolyl ring, compounds 5a-e, or 5-pyrazolyl ring, compounds 7a-c was explored. Both 2-thiazolyl moiety (compound 5c; average IC₅₀ = 2.53 µM) and 5-pyrazolyl moiety (compound 7b; average IC₅₀= 2.14 µM) resulted in remarkable increase in the activity in comparison to compounds 3a-e (compound 3c; average IC₅₀= 6.25 µM). Also, substitution of the 2-thiazolyl ring in compounds 5a-e with 4-methyl group resulted in an increase of the anti-proliferative activity (compound 5b; average IC₅₀ = 4.37 µM vs. compound 5c; average IC₅₀ = 2.53 µM).

Regarding phenylthiazolyl series, compounds 10a-l, the impact of the substitution on the 4-phenyl group was examined. The decreased average IC₅₀ values of compounds 10a, 10e, and 10i (11.07, 4.66 and 5.45 µM, respectively) which incorporated an unsubstituted phenyl moiety than that of their corresponding members 10b, 10f, and 10j (>28.19, >25.38, and 12.99 µM, respectively), with 4-methyl substituted phenyl moiety, and their corresponding members 10c, 10g, and 10k (>17.36, >12.65, and 14.49 µM, respectively), with 4-chloro substituted phenyl moiety, indicated that unsubstitution of the phenyl moiety is advantageous. On the other hand, substitution with 4-bromo substituent (compounds 10d, 10h, and 10l) diminished the activity. Finally, we explored the effect of the substitution of the indolin-2-one moiety. Interestingly, grafting 5-chloro substituent was found to be more beneficial for the activity rather than unsubstituted or 5-bromo substituted indolin-2-one moieties throughout the study.

In conclusion, we can deduce that replacing the phenyl moiety of compounds 3a-e with 2-thiazolyl ring, 5-pyrazolyl ring, or its substitution with alipophilic ester group greatly enhances the anti-proliferative activity. In addition, hybridisation of the indolin-2-one moiety with 4-phenylthiazolylmoiety, compounds 10a-l seems to be a prosperous approach.

Cell cycle effects

The cell cycle consists of a number of complex biochemical pathways and controlled by numerous mechanisms to ensure correct cell division. An important role is played by cyclin/cyclin-dependent kinase (CDK) complexes, which are critical regulators of cell cycle progression and RNA transcription. Deregulation of the cell cycle underlies the aberrant cell proliferation which characterises loss of cell cycle checkpoint control and cancer^31,32. Retinoblastoma protein (pRb), a cell cycle regulatory molecule, plays a critical role in control of the G1-to-S phase checkpoint of the cell cycle. pRb suppresses the activity of E2F transcription factors thereby inhibiting transcription of cell cycle promoting genes, in its hypophosphorylated state. Upon phosphorylation, mainly via CDKs, phosphorylated pRb dissociates from E2F and permits cell cycle progression^33,34. In the current medical era, therapeutic strategies directed against critical cell cycle regulators have stood out as an attractive approach for discovering therapies for many human malignancies.

In this study, cancer cells derived from lung adenocarcinoma (A-549) were used to evaluate the effects of compounds 7b and 10e on various aspects of the cell cycle progression. Both immunofluorescent imaging of phosphorylated Rb protein and total DNA content of each cell were explored to determine the activity of compounds 7b and 10e over a range of concentrations less than 100 nM to 50 µM (Figures 3–5).

Figure 3.

Effects on number and cell cycle distribution. A-549 cells were treated with dose curve. Compound 7b (left) or 10e (right). Cell number per cell cycle was determined by DNA content by DAPI fluorescent labeling and imaging cytometry following 48 h treatment.

Figure 4.

Dose-dependent changes in total levels of phosphorylated Rb protein of 7b, 10e and Sunitinib.

Figure 5.

Immunofluorescent imaging of phosphorylated Rb protein for 7b, 10e, and Sunitinib.

Compound 7b caused a dose-dependent reduction in the total cell number after 48 h of treatment with IC₅₀ value =8.08 µM, while compound 10e caused a dose-dependent reduction in the total cell number after 24 h of treatment with IC₅₀ value =7.38 µM (Table 3).

Table 3.

IC₅₀ for reductions in the total cell number and cell cycle effects of compounds 7b, 10e, and Sunitinib.

	IC50 (µM) for reductions in the total cell number		IC50 (µM) for reduction in Rb phosphorylation
Compound No.	24 h	48 h	24 h	48 h	Cell cycle effects
7b	12.67 ± 13.6	8.08 ± 0.95	12.07 ± 0.6	13.56 ± 0.6	G1 decreased and G2/M-phases increased
10e	7.38 ± 1.04	7.27 ± 1.15	7.59 ± 0.38	12.46 ± 2.1	G1 increased and G2/M-phases decreased
Sunitinib	12.54 ± 9.82	3.48 ± 0.61	3.18 ± 0.07	14.01 ± 0.6	G1 decreased and G2/M-phases increased

Moreover, compound 7b caused a decrease in the percentage of cells in the G1 phase of the cell cycle with corresponding increase in the G2/M-phases. Arrest in G2 may represent a checkpoint blockade whereas mitotic arrest may, in some cases, lead to mitotic catastrophe and subsequent programed death of cells with multiple or aberrant nuclei. In contrast, compound 10e caused an increase in the percentage of cells in the G1 phase of the cell cycle with corresponding decreases in S- and G2/M phases. This suggests that part of the compound effects on growth may be attributable to a decreased rate of progression through the cell cycle and corresponding decreases in proliferation.

However, levels of phosphorylated Rb protein were significantly reduced in a dose-dependent manner by the control and the test compounds 7b and 10e (Figure 4). The IC₅₀ values were consistent with the IC₅₀ values for reductions in cell number caused by each compound after 24 h of treatment while the correlation is less apparent after 48 h of treatment (Table 3). This may support the hypothesis that inhibition of cyclin-dependent kinases by isatin compounds plays a vital role in their growth inhibitory activity.

Selectivity

The cell growth inhibitory activity of compounds 7b and 10e was examined towards three non-tumorigenic cell lines to estimate their selectivity for tumor cells. A-549 human non-small cell lung cancer (NSCLC) cell line was included in the assay for comparison. Cultures derived from human fibrocystic mammary tissue (MCF-10A) are non-tumorigenic and exhibit features of primary cultures of breast tissue including dome formation³⁵. IEC-6 cells derived from rat intestine show morphologic and karyotypic features of normal intestinal epithelial cells³⁶. Fibroblasts derived from embryonic tissue from mice (Swiss-3t3) are both non-tumorigenic and contact inhibited³⁷. Compounds 7b and 10e were tested quadruplicate at maximum concentrations of 25 µM, with subsequent 10 serially diluted concentrations.

The results were displayed in Figure 6, while the IC₅₀ values and mean selectivity index were listed in Table 4. Both compounds 7b and 10e displayed mean tumor selectivity index (1.6 and 1.8, respectively) higher than that of Sunitinib (1.4).

Figure 6.

Selectivity profile of 7b, 10e, and Sunitinib.

Table 4.

Selectivity for compounds 7b, 10e, and Sunitinib towards tumor and non-tumorigenic cell lines.

	IC50 (µM) ± SD
Compound No.	Intestine IEC-6	Breast MCF-10A	Fibroblast Swiss-3t3	NSCLC A-549	Mean tumor selectivity
7b	8.70 ± 4.32	3.42 ± 0.45	6.85 ± 0.76	3.84 ± 0.67	1.6
10e	8.98 ± 1.25	5.37 ± 0.61	8.51 ± 1.01	4.13 ± 0.61	1.8
Sunitinib	4.56 ± 0.54	4.43 ± 0.23	4.07 ± 0.75	3.06 ± 0.9	1.4

Multidrug resistant lung cancer cell line (NCI-H69AR)

The anti-proliferative activity of compounds 7b and 10e was evaluated towards multidrug resistant lung cancer cell line (NCI-H69AR), which expresses the ABCC1 efflux pump protein. Table 5 and Figure 7 revealed that both 7b and 10e displayed growth inhibitory activity towards NCI-H69AR cells with IC₅₀ values of 16.0 µM. Accordingly, the NCI-H69AR cells were about four-fold less sensitive than A-549 cells, suggesting that these compounds might be subjected to efflux by ABCC1. Furthermore, the H69AR cells manifested a lesser degree of resistance to the reference drug, Sunitinib (1.9-fold less sensitive).

Table 5.

Cancer cell growth inhibitory activity of compounds 7b, 10e, and Sunitinib towards sensitive (A-549) and resistant (NCI-H69AR) cell lines.

	IC50 (µM)
Compound No.	Sensitive A-549	Resistant NCI-H69AR	Fold resistant
7b	3.8 ± 0.67	16.0	4.2
10e	4.1 ± 0.61	16.6	4.0
Sunitinib	3.1 ± 0.9	5.8 ± 0.52	1.9

Figure 7.

Activity of 7b, 10e, and Sunitinib towards sensitive and resistant cancer cell lines.

Conclusion

In summary, herein, we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. All the prepared hydrazonoindolin-2-ones were examined for their anti-proliferative activity towards three cell lines, namely lung A-549, colon r HT-29, and breast ZR-75 human cancer cell lines using CellTiter-Glo™ Luminescent cell viability assay. Compounds 5b, 5c, 7b, and 10e were the most potent members with average IC₅₀ values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC₅₀ = 8.11 µM). The SAR study suggested that replacing the phenyl moiety of compounds 3a-e with 2-thiazolyl ring, 5-pyrazolyl ring, or its substitution with a lipophilic ester group greatly enhances their anti-proliferative activity. In addition, hybridisation of the indolin-2-one moiety with 4-phenylthiazolyl moiety, compounds 10a-l, seems to be a prosperous approach. Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 human cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC₅₀ = 16 µM. Finally, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).

Acknowledgements

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. RG-1438–083.

Disclosure statement

No potential conflict of interest was reported by the authors.