New Isatin–Indole Conjugates: Synthesis, Characterization, and a Plausible Mechanism of Their in vitro Antiproliferative Activity

Abstract

Background: Cancer remains the leading cause of human morbidity universally. Hence, we sought to assess the in vitro antiproliferative activity of new isatin-based conjugates (5a–s) against three human cancer cell lines.

Methods: The antiproliferative activities of compounds 5a–s were evaluated in vitro and their ADME (absorption, distribution, metabolism and excretion) was carried out using standard protocols. Subsequently, Western blot analysis was conducted to elucidate the potential antiproliferative mechanism of compounds 5a–s .

Results: The in vitro antiproliferative activities of compounds 5a–s against the tested cancer cell lines ranged from 20.3 to 95.9%. Compound 5m had an IC₅₀ value of 1.17 μM; thus, its antiproliferative potency was approximately seven-fold greater than that of sunitinib (IC₅₀ = 8.11 μM). In-depth pharmacological testing was conducted with compound 5m to gain insight into the potential antiproliferative mechanism of this class of compounds. Compound 5m caused an increase in the number of cells in the G1 phase, with a concomitant reduction of those in the G2/M and S phases. Additionally, compound 5m significantly and dose-dependently reduced the amount of phosphorylated retinoblastoma protein detected. Compound 5m enhanced expression of B cell translocation gene 1, cell cycle-associated proteins (cyclin B1, cyclin D1, and phosphorylated cyclin-dependent kinase 1), and a pro-apoptotic protein (Bcl-2-associated X protein gene), and activated caspase-3. ADME predictions exposed the oral liability of compounds 5a-s .

Conclusion: Herein, we revealed the antiproliferative activity and ADME predictions of the newly-synthesized compounds 5a–s and provided a detailed insight into the pharmacological profile of compound 5m . Thus, compounds 5a–s can potentially be exploited as new antiproliferative lead compounds for cancer chemotherapeutic.

Introduction

Cancer, the second major source of mortality in humans after cardiovascular diseases, remains a considerable health obstacle worldwide.¹ Although substantial advances have been made with cancer-diagnosis tools and many chemotherapeutic agents are currently available as treatments, successful management of cancer remains a medical challenge. Because current cancer-chemotherapeutic agents show a lack of selectivity and can elicit multiple-drug resistance, there exists a constant need to derive newer and safer anticancer agents.^2–4

Creating novel anticancer chemotherapeutic agents based on naturally occurring bioactive substances has been gaining increased interest.^5,6 2,3-Dioxindole (isatin, I, Figure 1) is a natural substance found in some plant species, which can also be present in humans, due to endogenous consumption. Compound I has a distinguished pharmacological profile and exhibits a wide spectrum of biological actions, including anti-oxidant,⁷ anti-inflammatory,⁸ anti-bacterial,⁹ and anticancer^10,11 activities. The United States Federal Drug Administration approved sunitinib and nintedanib as isatin-bearing anticancer drugs,^10,12 and many compounds with an isatin scaffold such as SU6668 and SU5416 have displayed outstanding anticancer activity.^13–15 Therefore, isatin-bearing drugs might exhibit extensive cytotoxicity against cancer cells.

Figure 1 Chemical structures of compounds I, II, and 5a–s.

Indole (1H-benzo[b]pyrrole, II, Figure 1) has a bicyclic heterocycle scaffold that is widely distributed in animals, microbial hormones, and plants.^16,17 It is considered to be a beneficial motif in drug-discovery programs because of its biodiversity, versatility, and anticancer activity.^18–21 Previous evidence suggests that myriad indole-based compounds exert anticancer activities through various mechanisms,^22–27 suggesting the possibility of using the indole scaffold in anticancer drugs. Notably, anticancer indole-based molecules target several pathways, such as those involving tubulin polymerization, DNA topoisomerase, histone deacetylase, proto-oncogenes, and serine/threonine (PIM) kinase.^28–31

Screening the literature exposed that a number of bioactive molecules bear carbohydrazide moiety. They display a wide spectrum of biological activities including anticancer activity.^32–34

Combining the structural features of two differing pharmacophores is an effective tactic in medicinal chemistry, as new bioactive, chemotherapeutic chemical entities characterized by a reduced risk of drug–drug interactions and improved anticancer profiles can be obtained.^35–37 Considering the structural characteristics of isatin and indole, merging these two bioactive moieties into a single compound through a carbohydrazide bridge is predicted to result in new promising anticancer leads. Accordingly, we aimed to synthesize and evaluate the in vitro antiproliferative activities of the molecular hybrids 5a–s. The most active antiproliferative candidate 5m was also examined in an in-depth pharmacological-profiling study to understand the potential mechanism whereby these compounds exert their antiproliferative effect.

Materials and Methods Chemistry

General The melting points of compounds 5a–s were measured using the Gallenkamp melting point equipment and are presented as uncorrected values. Compounds 5a–s were dissolved in DMSO-d₆ for nuclear magnetic resonance (NMR) measurements at 500 and 125 MHz for ¹H and ¹³C, respectively, using a Bruker NMR spectrometer (Bruker, Reinstetten, Germany) at the Research Center (College of Pharmacy, King Saud University, Saudi Arabia). TMS was used as an internal standard for the NMR measurements. The measured chemical shifts are presented as δ-values in parts per million (ppm). Compounds 5a–s were subjected to microanalyses at the Microanalysis Laboratory (Cairo University, Cairo, Egypt), and the results aligned well with the proposed structures (i.e., within ± 0.4% of the theoretical values). A 6120 LC/MS Agilent Quadrupole system with an electrospray-ionization(ESI) source (Agilent Technologies, Palo Alto, CA, USA) was used to obtain the mass spectra of compounds 5a–s. Compound 3 and compounds 4a–n were prepared as described previously.³⁸

General Procedure for Synthesizing Carbohydrazides 5a–s The acid hydrazide 3 (0.18 g, 1 mmol) was dissolved in absolute ethanol (15 mL), and the appropriate isatin derivative 4a–n (1 mmol) was added to the stirred ethanolic solution containing drops of glacial acetic acid. The reaction mixture was refluxed under continuous stirring for 4 h, and then the hot alcoholic reaction mixture was filtered. The collected solids were re-crystallized from the ethanol/DMF mixture (3:1) to yield the corresponding target carbohydrazides 5a–s with yields of 40 to 95%.

N′-[(3Z)-2-Oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5a): Yellow powder, melting point (m.p.) > 300 °C (yield 89%); ¹H NMR (DMSO-d₆) ppm: 6.95 (d, J = 7.5 Hz, 1H, H_ar.), 7.09–7.16 (m, 2H, H_ar.), 7.29 (d, J = 7.0 Hz, 1H, H_ar.), 7.42 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.61 (s, 1H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.05 (d, J = 7.5 Hz, 1H, H_ar.), 10.88 (s, 1H, NH), 11.69 (s, 1H, NH), 11.97 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 104.2, 111.1, 112.9, 116.1, 120.7, 122.3, 122.7, 123.2, 125.1, 127.2, 127.5, 129.3, 133.2, 137.6, 144.4 (C_ar., CH_ar., C=N), 160.9, 165.3 (2 × C=O); MS m/z: 303 [M-H]^–.

N′-[(3Z)-5-Bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5b): Yellow powder, m.p. > 300 °C (yield 76%); ¹H NMR (DMSO-d₆) ppm: 6.90 (d, J = 8.5 Hz, 1H, H_ar.), 7.09–7.14 (m, 1H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.59 (d, J = 8.0 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.73 (d, J = 8 Hz, 1H, H_ar.), 8.34 (s, 1H, H_ar.), 11.01 (s, 1H, NH), 11.87 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 104.8, 108.6, 112.9, 113.8, 117.5, 120.7, 121.0, 122.8, 125.2, 127.5, 129.2, 135.2, 136.4, 137.7, 143.5 (C_ar. and CH_ar., C=N), 160.7, 164.8 (2 × C=O); MS m/z: 381 [M-H]⁻, 383 [M-H+2]^–.

N′-[(3Z)-5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5c): Yellow powder, m.p. > 300 °C (yield 91%); ¹H NMR (DMSO-d₆) ppm: 6.95 (d, J = 8.5 Hz, 1H, H_ar.), 7.09–7.14 (m, 1H, H_ar.), 7.27–7.32 (m, 1H, H_ar.), 7.43–7.47 (m, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.22 (s, 1H, H_ar.), 11.00 (s, 1H, NH), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm:102.1, 109.3, 112.4, 112.9, 117.1, 120.7, 122.8, 125.2, 126.2, 126.6, 127.5, 132.4, 137.6, 138.9, 143.0 (C_ar. and CH_ar., C=N), 163.5, 165.1 (2 × C=O); MS m/z: 337 [M-H]⁻; 339 [M-H+2]^–.

N′-[(3Z)-5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5d): Yellow powder, m.p. > 300 °C (yield 95%); ¹H NMR (DMSO-d₆) ppm: 6.93 (dd, J = 4, 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 8.0 Hz, 1H, H_ar.), 7.25–7.30 (m, 2H, H_ar.), 7.50 (d, J = 8.5 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.02 (d, J = 8.0 Hz, 1H, H_ar.), 10.89 (s, 1H, NH), 11.79 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 108.4, 111.8, 112.8, 112.9, 113.1, 114.3, 120.7, 120.9, 122.7, 122.8, 125.3, 125.5, 137.5, 139.1 (C_ar., CH_ar., C=N), 157.9 (C_1ʹ-F, J = 235.0 Hz, C_ar.), 163.7, 165.4 (2 × C=O); MS m/z: 321 [M-H]^–.

N′-[(3Z)-5-Methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5e): Orange powder, m.p. > 300 °C (yield 75%); ¹H NMR (DMSO-d₆) ppm: 3.82 (s, 3H, OCH₃), 6.86 (d, J = 8.5 Hz, 1H, H_ar.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, H_ar.), 7.09 (d, J = 7.5 Hz, 1H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.63 (s, 1H, H_ar.), 7.70–7.74 (m, 2H, H_ar.), 10.69 (s, 1H, NH), 11.81 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 56.3 (OCH₃), 102.6, 111.6, 112.6, 112.9, 113.1, 116.4, 118.7, 120.7, 122.8, 125.1, 127.5, 137.5, 138.1, 154.9, 155.9 (C_ar., CH_ar., C=N), 163.8, 165.4 (2 × C=O); MS m/z: 333 [M-H]^–.

N′-[(3Z)-1-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5f): Yellow powder, m.p. 258–260 °C (yield 41%); ¹H NMR (DMSO-d₆) ppm: 3.23 (s, 3H, NCH₃), 7.13 (d, J = 8.0 Hz, 1H, H_ar.), 7.17–7.21 (m, 2H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.47–7.53 (m, 2H, H_ar.), 7.61 (s, 1H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.09 (d, J = 7.5 Hz, 1H, H_ar.), 11.76 (s, 1H, NH), 11.98 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 109.8, 110.5, 113.1, 121.0, 122.8, 125.1, 125.4, 126.8, 127.4, 129.3, 132.0, 133.1, 137.6, 144.1, 145.4 (C_ar., CH_ar., C=N), 161.7, 163.9 (2 × C=O); MS m/z: 317 [M-1]^–.

N′-[(3Z)-5-Bromo-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5g): Yellow powder, m.p. 291–293 °C (yield 63%); ¹H NMR (DMSO-d₆) ppm: 3.21 (s, 3H, NCH₃), 7.08–7.15 (m, 2H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.63–7.68 (m, 2H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.38 (s, 1H, H_ar.), 11.93 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 111.6, 112.9, 114.5, 120.6, 122.8, 123.3, 125.2, 127.5, 128.9, 134.1, 135.0, 137.5, 138.1, 143.1, 144.5 (C_ar., CH_ar., C=N), 161.4, 162.1 (2 × C=O); MS m/z: 395 [M-H]⁻, 397 [M-H+2]^–.

N′-[(3Z)-5-Chloro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5h): Yellow powder, m.p. 292–294 °C (yield 82%); ¹H NMR (DMSO-d₆) ppm: 3.22 (s, 3H, NCH₃), 7.10 (d, J = 8.0, 1H, H_ar.), 7.13 (d, J = 4.0 Hz, 1H, H_ar.), 7.27–7.31 (m,1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.54 (dd, J = 8.5, 2.0 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.73 (d, J = 8.0, 1H, H_ar.), 8.26 (s, 1H, H_ar.), 11.92 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 109.3, 111.1, 112.0, 112.9, 120.7, 122.8, 125.2, 126.3, 126.8, 127.5, 127.9, 132.2, 137.6, 142.7, 144.1 (C_ar., CH_ar., C=N), 159.4, 161.5 (2 × C=O); MS m/z: 351 [M-H]^–.

N′-[(3Z)-5-Fluoro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5i): Yellow powder, m.p. 283–285 °C (yield 54%); ¹H NMR (DMSO-d₆) ppm: 3.22 (s, 3H, NCH₃), 7.09–7.14 (m, 2H, H_ar.), 7.27–7.30 (m, 1H, H_ar.), 7.37 (ddd, J = 2.5, 9.0, 9.0, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.67 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.07 (d, J = 7.5 Hz, 1H, H_ar.), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.7 (NCH₃), 109.4, 110.6, 113.1, 114.2, 115.7, 118.9, 120.7, 122.8, 125.2, 125.4, 127.5, 129.0, 137.6, 141.8 (C_ar., CH_ar., C=N), 158.3 (C_1ʹ-F, J = 235.6 Hz, C_ar.), 161.8, 163.9 (2 × C=O);MS m/z: 335 [M-H]^–.

N′-[(3Z)-5-Methoxy-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5j): Orange powder, m.p. 279–281 °C (yield 86%); ¹H NMR (DMSO-d₆) ppm: 3.19 (s, 3H, NCH₃), 3.84 (s, 3H, OCH₃), 7.04 (d, J = 8.5 Hz, 1H, H_ar.), 7.08–7.12 (m, 2H, H_ar.), 7.27–7.30 (m, 1H, H_ar.), 7.63 (s, 1H, H_ar.), 7.66 (d, J = 8.0 Hz, 1H, H_ar.), 7.72–7.77 (m, 2H, H_ar.), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 56.4 (OCH₃), 110.2, 111.3, 112.9, 113.4, 115.8, 117.9, 120.7, 122.8, 125.1, 127.5, 129.2, 137.5, 139.2, 155.5 (C_ar., CH_ar., C=N), 161.7, 163.9 (2 × C=O); MS m/z: 347 [M-H]^–.

N′-[(3Z)-1-Benzyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5k): Yellow powder, m.p. 249–251 °C (yield 56%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.16–7.21 (m, 1H, H_ar.), 7.28–7.32 (m, 2H, H_ar.), 7.35–7.38 (m, 3H, H_ar.), 7.42–7.45 (m, 2H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.74 (d, J = 7.5 Hz, 1H, H_ar.), 8.14 (d, J = 7.5 Hz, 1H, H_ar.), 11.80 (s, 1H, NH), 12.00 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.2 (CH₂), 110.4, 113.0, 115.7, 120.7, 120.9, 121.3, 122.7, 123.0, 127.5, 127.7, 127.9, 128.0, 129.2, 134.1, 136.7, 137.7, 144.3, 146.8, 147.5 (C_ar., CH_ar., C=N), 162.4, 164.2 (2 × C=O); MS m/z: 393 [M-H]^–.

N′-[(3Z)-1-Benzyl-5-bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5l): Yellow powder, m.p. 268–270 °C (yield 51%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.01 (d, J = 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 7.5 Hz, 1H, H_ar.), 7.29 (d, J = 7.5 Hz, 2H, H_ar.), 7.36–7.38 (m, 3H, H_ar.), 7.42 (d, J = 7.5 Hz, 1H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.59 (s, 1H, H_ar.), 7.71–7.77 (m, 2H, H_ar.), 8.44 (s 1H, H_ar.), 11.99 (s, 1H, NH), 12.20 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 110.3, 112.1, 113.1, 114.8, 120.7, 127.7, 127.9, 128.2, 129.2, 134.1, 135.8, 136.4, 136.6, 137.6, 138.1, 142.1, 143.3, 148.7, 156.5 (C_ar., CH_ar., C=N), 161.5, 162.6 (2 × C=O); MS m/z: 471 [M-H]⁻; 473 [M-H+2]^–.

N′-[(3Z)-1-Benzyl-5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5m): Yellow powder, m.p. 268–270 °C (yield 54%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.5 Hz, 1H, H_ar.), 7.11–7.13 (m, 1H, H_ar.), 7.28–7.31 (m, 2H, H_ar.), 7.35–7.37 (m, 3H, H_ar.), 7.43 (d, J = 7.0 Hz, 1H, H_ar.), 7.46–7.47 (m, 1H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.71 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.32 (s, 1H, H_ar.), 11.97 (s, 1H, NH), 12.18 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 111.7, 112.5, 112.9, 113.1, 120.7, 121.6, 127.5, 127.7, 127.9, 128.0, 128.2, 129.2, 135.9, 136.4, 137.6, 138.1, 141.7, 144.4, 149.6 (C_ar., CH_ar., C=N), 159.7, 161.6 (2 × C=O); MS m/z: 427 [M-H]⁻; 429 [M-H+2]^–.

N′-[(3Z)-1-Benzyl-5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5n): Yellow powder, m.p. 251–253 °C (yield 49%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.04–7.06 (m, 1H, H_ar.), 7.09–7.13 (m, 1H, H_ar.), 7.28–7.32 (m, 3H, H_ar.), 7.34–7.39 (m, 4H, H_ar.), 7.29 (d, J = 8.5 Hz, 1H, H_ar.), 7.72 (s, 1H, H_ar.), 7.75 (d, J = 8.0 Hz, 1H, H_ar.), 8.13 (d, J = 7.5 Hz, 1H, H_ar.), 11.91 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 110.9, 111.1, 113.0, 114.4, 119.0, 120.7, 122.8, 125.3, 127.5, 127.7, 127.9, 128.0, 128.9, 129.2, 136.5, 137.6, 139.9, 140.6 (C_ar., CH_ar., C=N), 158.4 (C_1ʹ-F, J = 237.9 Hz, C_ar.), 161.9, 164.2 (2 × C=O); MS m/z: 411 [M-H]^–.

N′-[(3Z)-1-Benzyl-5-methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5o): Orange powder, m.p. 268–270 °C (yield 64%); ¹H NMR (DMSO-d₆) ppm: 3.81 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 6.95 (d, J = 8.5 Hz, 1H, H_ar.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 8.0 Hz, 1H, H_ar.), 7.26–7.31 (m, 2H, H_ar.), 7.34–7.38 (m, 4H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.68 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 7.78 (s, 1H, H_ar.), 11.92 (s, 1H, NH), 11.98 s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.1 (CH₂), 56.4 (OCH₃), 110.9, 113.0, 116.1, 120.7, 122.8, 125.2, 127.6, 127.7, 127.9, 128.0, 129.2, 129.3,136.7, 136.8, 137.7, 138.0, 138.1, 144.1, 155.6 (C_ar., CH_ar., C=N), 156.5, 164.1 (2 × C=O);MS m/z: 423 [M-H]^–.

N′-[(3Z)-1-(4-Chlorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5p): Yellow powder, m.p. 261–263 °C (yield 65%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.14 (m, 1H, H_ar.), 7.19–7.21 (m, 1H, H_ar.), 7.28–7.32 (m, 1H, H_ar.), 7.40–7.44 (m, 3H, H_ar.), 7.51 (d, J = 8.0 Hz, 2H, H_ar.), 7.65 (s, 1H, H_ar.), 7.72–7.75 (m, 1H, H_ar.), 7.77 (d, J = 8.0 Hz, 1H, H_ar.), 8.15 (d, J = 7.5 Hz, 1H, H_ar.), 11.81 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 42.6 (CH₂), 110.9, 113.0, 119.9, 123.1, 125.2, 125.4, 127.5, 129.2, 129.7, 129.9, 131.9, 132.6, 132.8, 135.2, 135.7, 137.7, 138.0, 142.9, 144.1 (C_ar., CH_ar., C=N), 161.8, 164.2 (2 × C=O); MS m/z: 427 [M-H]⁻; 429 [M-H+2]^–.

N′-[(3Z)-1-(4-Cyanobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5q): Yellow powder, m.p. 266–268 °C (yield 57%); ¹H NMR (DMSO-d₆) ppm: 5.13 (s, 2H, CH₂), 7.05 (d, J = 7.5 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.20 (ddd, J = 2.0, 7.5, 7.5 Hz, 1H, H_ar.), 7.39–7.43 (m, 1H, H_ar.), 7.56 (d, J = 8.5 Hz, 2H, H_ar.), 7.63–7.65 (m, 3H, H_ar.), 7.77 (d, J = 8.0 Hz, 1H, H_ar.), 7.84 (d, J = 3.0 Hz, 1H, H_ar.), 7.85 (d, J = 3.0 Hz, 1H, H_ar.), 8.17 (d, J = 7.5 Hz, 1H, H_ar.), 11.82 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 42.9 (CH₂), 110.8, 110.9, 113.0, 115.8, 119.9, 120.7, 122.7, 123.2, 125.4, 127.1, 127.5, 128.6, 128.8, 133.1, 133.2, 137.7, 138.0, 141.9, 142.5, 142.8 (C_ar., CH_ar., 2 × CN), 161.9, 164.2 (2 × C=O); MS m/z: 418 [M-H]^–.

N′-[(3Z)-1-(4-Methylbenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5r): Yellow powder, m.p. 256–258 °C (yield 40%); ¹H NMR (DMSO-d₆) ppm: 2.27 (s, 3H, CH₃), 4.97 (s, 2H, CH₂), 7.04 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.16 (d, J = 8.5 Hz, 2H, H_ar.), 7.27 (d, J = 8.5 Hz, 2H, H_ar.), 7.29–7.34 (m, 1H, H_ar.), 7.41–7.44 (m, 1H, H_ar.), 7.52 (d, J = 8.5 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.13 (d, J = 7.5 Hz, 1H, H_ar.), 11.80 (s, 1H, NH), 12.00 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 21.1 (CH₃), 43.0 (CH₂), 110.4, 113.0, 115.7, 120.7, 122.7, 123.0, 125.2, 127.0, 127.5, 127.7, 127.9, 129.1, 129.2, 129.8, 132.9, 133.6, 137.2, 137.7, 144.3 (C_ar., CH_ar., C=N), 162.5, 164.1 (2 × C=O); MS m/z: 407 [M-H]^–.

N′-[(3Z)-2-Oxo-1-phenyl-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5s): Yellow powder, m.p. 277–279 °C (yield 55%); ¹H NMR (DMSO-d₆) ppm: 6.84 (d, J = 8.0 Hz, 1H, H_ar.), 7.11–7.14 (m, 1H, H_ar.), 7.24–7.31 (m, 2H, H_ar.), 7.44–7.47 (m, 1H, H_ar.), 7.50–7.54 (m, 4H, H_ar.), 7.61–7.64 (m, 2H, H_ar.), 7.58 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.23 (d, J = 7.5 Hz, 1H, H_ar.), 11.89 (s, 1H, NH), 12.01 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 110.1, 113.0,115.7, 120.7, 122.8, 123.5, 125.2, 127.2, 127.4, 127.5, 128.9, 130.2, 133.1, 134.2, 135.5, 137.3, 137.7, 141.8, 145.1 (C_ar., CH_ar., C=N), 162.9, 163.4 (2 × C=O); MS m/z: 379 [M-H]^–.

Pharmacological Evaluation Pharmacological assessment of the title compounds 5a–s was carried out using previously reported protocols^39,40 and they are provided as Supporting materials. The human cancer cell lines were procured commercially from the American Type Culture Collection.

Western Blot Analysis

Materials Immunoblotting detection was performed with antibodies from Abcam against cyclin E1(clone number HE12, catalog number ab3927), cyclin D1 (clone number EP272Y, catalog number ab40754), cyclin B1 (clone number Y106, catalog number ab32053), β actin (clone number mAbcam8226, catalog number ab8226), BTG1 (clone number EPR8274(2), catalog number ab151740), BAX (clone number 2D2, catalog numberab77566), bcl-2 (clone number E17, catalog number ab32124), and caspase-3 (catalog number ab13847).

Methods A-549 non-small cell lung cancer (NSCLC) cells were plated in 25-cm² culture dishes at a seeding density of 5 × 10⁴/dish before treatment with compound 5m for 24, 48, and 72 h. Whole cell lysates were prepared using radioimmunoprecipitation assay buffer (Sigma-Aldrich, MO, USA). Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad, CA, USA). Twenty micrograms of treated and control lysates were loaded in each well of 4–20% Mini-Protean TGX Gels (Bio-Rad) for electrophoresis and then transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% (w/v) skimmed milk prepared in Tris-buffered saline and Tween 20, incubated with primary antibodies overnight at 4 °C, washed, and then incubated with a secondary, horseradish peroxidase-coupled anti-mouse or anti-rabbit antibody. Bands were visualized with a Western Bright ECL Kit for 5000 cm² Membrane and Blue Basic Autoradiography Film (BioExpress, CA, USA).

Results and Discussion Chemistry

The chemical structures of the title compounds 5a–s and their intermediates are shown in Scheme 1. The free carboxylic group of indole-2-carboxylic acid (1) was esterified, which resulted in the corresponding methyl ester 2.⁴¹ Subsequently, compound 2 was subjected to hydrazinolysis with hydrazine hydrate, thereby forming the acid hydrazide 3.⁴² The target indole–isatin conjugates 5a–s were respectively prepared by reacting the acid hydrazide 3 with the appropriate isatin derivatives 4a–n in ethanol-containing drops of acetic acid. The stereochemistry of the title conjugates 5a–s was assigned the (Z)-configuration, based on the previously documented X-ray results of structurally related analogous compounds.^43,44

Scheme 1 Preparation of compounds 5a–s. Reagents and conditions: (i) Few drops of concentrated sulfuric acid, absolute methanol, reflux, 4 h; (ii) H2N-NH2*H2O, absolute methanol, reflux, 2 h; (iii) Few drops of glacial acetic acid, absolute ethanol, reflux, 4 h.

Pharmacological Investigations

Antiproliferative Activity The cell growth-inhibitory activities of nineteen conjugates 5a–s were evaluated with human breast (ZR-75), colon (HT-29) and lung (A-549) tumor cells. Each test compound was assessed at 30 µM in quadruplicate (Table 1). The title compounds 5a–s displayed average antiproliferative activities against the ZR-75, HT-29, and A-549 cell lines of 20.3 to 95.9%.

Table 1 Antiproliferative Activities (Cell Growth-Inhibitory Activities at a Concentration of 30 µm) of Compounds 5a–s and Sunitinib Against the Indicated Cell Lines

Table 2 shows the IC₅₀ (inhibitory concentration 50%) values determined for compounds 5a–s against the tested human cancer cell lines. Compound 5m had an average IC₅₀ value of 1.17 µM against the tested human cancer cell lines, making it the most active candidate, with a potency approximately seven-fold greater than that of sunitinib (average IC₅₀ value of 8.11 µM). Consequently, further pharmacological profiling was conducted with compound 5m to better understand the potential antiproliferative mechanism of compounds 5a–s.

Table 2 The IC50 Values of Compounds 5a–s and Sunitinib Against the Tested Cell Lines

Structure Activity Relationship

The title compounds 5a–s are structurally classified into five groups namely, N–unsubstituted derivatives 5a–e, N–methyl derivatives 5f–j, N–benzyl derivatives 5k–o, N–4–substituted benzyl derivatives 5p–r, and N–phenyl derivative 5s. In the first group 5a–e, compound 5e, bearing a methoxy substituent in the five position of the isatin nucleus, is the most active congener with an average IC₅₀ value of 18.18 µM. Compound 5g, in the second group 5f–j, have a bromo substituent in the five position of the isatin ring and it showed an average IC₅₀ value of 2.06 µM being nearly four times more potent than sunitinib (average IC₅₀ value of 8.11 µM). The third group 5k–o seems to be the best group with an average IC₅₀ range of 1.17–2.79 µM in which compound 5m, bearing 5–chloroisatin fragment, is the most active congener with an average IC₅₀ value of 1.17 µM being about sevenfold more potent than sunitinib. Moreover, the in vitro antiproliferative activity of the fourth group 5p–r is in the following order 5r ˃5p˃5q with an average IC₅₀ range of 1.46–3.92 µM. Compound 5s, bearing N–phenylisatin scaffold displayed good in vitro antiproliferative activity with an average IC₅₀ value of 3.36 µM (Table 2). In conclusion, the N–benzyl moiety on the isatin residue seems to be the favorable substituent to get good in vitro antiproliferative activity of this class of compounds.

Effects on Cell-Cycle Progression

The influence of compound 5m on different aspects of cell-cycle progression was investigated with the A-549 cell line based on the total DNA content of each cell. Additionally, immunofluorescent imaging of the phosphorylated retinoblastoma (Rb) protein was performed over the concentration range, 0.05–100 µM. A significant dose-dependent decrease in the total cell number (Figure 2) was noted after treatment with compound 5m for 48 h (IC₅₀ value = 1.38 µM; Table 3).

Figure 2 Dose-dependent reductions in the numbers of adherent cells. A-549 NSCLC cells were treated for 24 or 48 h, as indicated, followed by fixation and addition of the fluorescent label. Values are presented as the average number of cells per 4 fields within each well.

Table 3 IC50 Values for the Reductions in Total Cell Numbers and the Effects of Compound 5m and Sunitinib on Cell-Cycle Progression

In addition, the percentage of cells in G1 phase increased and the percentages of cells in the S and G2/M phases concomitantly decreased after treatment with compound 5m. This finding suggests that the ability of compound 5m to influence cell proliferation could be ascribed to decreased cell-cycle progression. Conversely, sunitinib treatment resulted in a reduced percentage of cells in G1 phase and a concomitant increase in cells in S or G2/M phase. G2 arrest could be regarded as a checkpoint blockade. Mitotic arrest may, in some instances, result in mitotic catastrophe and subsequent programmed death of cells with multiple or aberrant nuclei.

Moreover, both sunitinib and 5m caused a substantial dose-dependent decrease in the extent of Rb protein phosphorylation (Figure 3), suggesting the possibility that the antiproliferative activity of compounds with isatin fragments can be attributed to their effect on cyclin-dependent kinases. Compound 5m had an IC₅₀ value of 2.52 µM after 24 h, indicating that it was ~1.7-fold more potent than sunitinib (Table 3). Treatment with compound 5m did not affect the extent of P-Tyr.

Figure 3 Dose-dependent reductions in Rb phosphorylation. A-549 NSCLC cells were treated for 24 or 48 h (as indicated), fixed, and subjected to quantitative indirect immunofluorescence and image analysis.

Western Blot Analysis

Data from a previous study revealed a correlation between the antiproliferative marker, B cell translocation gene 1 (BTG1), and the inhibition of proliferation in lung cancer, which suggests that compound 5m most likely induces cycle arrest during G1 phase.⁴⁵ Treatment with compound 5m increased BTG1 expression (Figure S1), confirming its potential to act as an antiproliferative agent. To confirm the influence of compound 5m on cell-cycle progression, the expression levels of different cell cycle-related proteins were examined using Western blot analysis. As depicted in Figure S2, compound 5m regulated cell cycle-related proteins and activated cyclin-dependent kinase 1 (cdc-2) via phosphorylation at Tyr 15.⁴⁶ Although cell-cycle analysis revealed an increase in the G1 population after treatment with compound 5m, the ability of 5m to upregulate the G2/M phase-related proteins, cyclin B1 and p-cdc2 (Figure S2), was interesting. Ultimately, these findings suggest that cell-cycle arrest after treatment with 5m can occur during both G1 and G2 phases.^47,48

Many anticancer agents are designed to cause DNA damage and, consequently, apoptosis.⁴⁹ Hence, the ability of compound 5m to inhibit cell proliferation by inducing apoptosis was also examined. Compound 5m activated caspase-3, with increased production of cleaved caspase-3 within 24 h of treatment (Figure S3). The expression levels of the pro-survival protein encoded by the B-cell lymphoma/leukemia-2 (Bcl-2) gene and the pro-apoptotic protein encoded by the Bcl-2-associated X protein (BAX) gene were also evaluated. Treatment with compound 5m increase BAX expression but had no obvious effect on Bcl-2 expression (Figure S3). The altered BAX/BCL-2 ratio after treatment with 5m suggests the involvement of the intrinsic apoptosis pathway.

Selectivity

The selectivity of compound 5m was tested using three non-tumorigenic cell lines. IEC-6 cells obtained from rat intestines manifested both morphologic and karyotypic properties of normal intestinal epithelial cells.⁵⁰ Cultures obtained from human fibrocystic mammary tissue (MCF-10A) are non-tumorigenic and display characteristics similar to primary cultures of breast tissue, including dome formation.⁵¹ Fibroblasts obtained from the embryonic tissue of mice (Swiss 3t3 fibroblasts) are both non-tumorigenic and contact-inhibited.⁵² A-549 NSCLC cells were included for comparison. The antiproliferative activities of compound 5m and sunitinib were assessed in quadruplicate at a maximum concentration of 25 µM and then at 10 serially diluted concentrations. Compound 5m inhibited cell growth (>50%) in both normal and tumor cell lines. However, compound 5m exhibited a 2.6-fold selectivity value, whereas sunitinib displayed a 1.4-fold selectivity value (Figure 4 and Table 4).

Figure 4 Tumor selectivity. A-549 NSCLC cells and non-tumorigenic cells derived from intestine, breast, and fibroblasts were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay.

Table 4 Selectivity of Compound 5m and Sunitinib Against Tumor and Non-Tumorigenic Cell Lines

Multidrug-Resistant Lung Cancer Cell Line

The antiproliferative activity of compound 5m was examined against a sensitive NSCLC cell line (A-549) and a multidrug-resistant lung cancer cell (NCI-H69AR) expressing the ABCC1 efflux pump protein. Both compound 5m and sunitinib were tested in quadruplicate at a maximum concentration of 25 µM, and then at 10 serially diluted concentrations. Compound 5m inhibited growth in both cell lines, with an IC₅₀ value of 1.33 µM in A-549 cells (Figure 5 and Table 5). The H69AR cells were ~19-fold less sensitive, as compound 5m could be effluxed by the ABCC1 pump protein. Additionally, a lesser degree of sunitinib resistance (1.9-fold less sensitivity) was found in H69AR cells.

Figure 5 Evaluation of susceptibility to efflux. A-549 NSCLC cells and NCI-H69AR cells expressing the ABCC1 (MRP1) multi-drug resistance efflux transporter were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay.

Table 5 Antiproliferative Activities of Compound 5m and Sunitinib Against Sensitive (a-549) and Resistant (NCI-H69AR) Cell Lines

Cell Viability Assay Using HEK 293 Cell Line

Cell viability assay for compound 5m showed no apparent toxicity towards human embryonic kidney (HEK) 293 cells following 24 hrs exposure to different 5m concentrations, indicated by percent viability ranging from 93.72±0.68 to 98.67±0.39. Furthermore, there was no significant change in viability after longer exposure time to 5m up to 72 hrs.

In Silico Predictions of ADME Properties of Compounds 5a-s

In silico ADME (absorption, distribution, metabolism and excretion) predictions as well as some pharmacokinetic characteristics of the target compounds 5a–s are presented in Table S1 which are calculated according to the literature methods.^53,54 All the title compounds 5a–s follow the Lipinski's rule of 5 (RO5) where log P values lie between 2.2–4.2 (<5), molecular weight range 304–473 (<500), hydrogen bond acceptor range 3–4 (<5), and hydrogen bond donor range 2–3 (<5), indicating that these compounds might not be expected to cause problems with oral bioavailability. In addition, compound 5a–s exhibited topological polar surface area (TPSA) range 77.6–101.4 Ų (<140 Ų), denoting good transport through cellular plasma membranes. In summary, compounds 5a–s displayed no violations to RO5 criteria suggesting their liability to be new orally bioavailable antiproliferative candidates.

Conclusion

Herein, we identified the newly synthesized isatin-indole molecular hybrids 5a–s with various spectroscopic approaches and estimated their potentials as antiproliferative agents in vitro. Compound 5m displayed promising in vitro antiproliferative potential against three human cancer cell lines. With an average IC₅₀ value of 1.17 µM, this compound had a seven-fold greater potency than sunitinib (IC₅₀ = 8.11 µM). By deriving its pharmacological profile, we sought to characterize the mechanisms underlying the antiproliferative activities of compounds 5a–s. We found reduced percentages of cells in S and G2/M phases, an increased percentage of cells in G1 phase, and a significant dose-dependent decrease in the extent of Rb protein phosphorylation. Furthermore, Western blot analysis confirmed the extents of BTG1, cyclin B1, cyclin D1, and p-cdc2 upregulation caused by compound 5m. Within 24 h of cell treatment, compound 5m increased BAX expression and activated caspase-3, as indicated by the observed increase in cleaved caspase-3 levels. ADME predictions for compounds 5a–s might help in further development of new anticancer agents with favorable oral bioavailability. Based on the findings presented herein, these lead compounds can be utilized in preclinical studies in the field of cancer chemotherapy.

Supporting Materials

Detailed protocols for the pharmacological evaluation of the title compounds 5a–s, Western blot results for compound 5m (Figures S1–S3), and ADME Table (Table S1) are provided as Supporting materials.

Acknowledgment

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research through Research Group Project Number RG-1440-140.

Disclosure

Dr Adam B Keeton reports support of this project through the Deanship of Scientific Research at King Saud University through Research Group Project Number RG-1440-140. The authors declare no other conflicts of interest.

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Introduction Cancer, the second major source of mortality in humans after cardiovascular diseases, remains a considerable health obstacle worldwide.¹ Although substantial advances have been made with cancer-diagnosis tools and many chemotherapeutic agents are currently available as treatments, successful management of cancer remains a medical challenge. Because current cancer-chemotherapeutic agents show a lack of selectivity and can elicit multiple-drug resistance, there exists a constant need to derive newer and safer anticancer agents.^2–4 Creating novel anticancer chemotherapeutic agents based on naturally occurring bioactive substances has been gaining increased interest.^5,6 2,3-Dioxindole (isatin, I, Figure 1) is a natural substance found in some plant species, which can also be present in humans, due to endogenous consumption. Compound I has a distinguished pharmacological profile and exhibits a wide spectrum of biological actions, including anti-oxidant,⁷ anti-inflammatory,⁸ anti-bacterial,⁹ and anticancer^10,11 activities. The United States Federal Drug Administration approved sunitinib and nintedanib as isatin-bearing anticancer drugs,^10,12 and many compounds with an isatin scaffold such as SU6668 and SU5416 have displayed outstanding anticancer activity.^13–15 Therefore, isatin-bearing drugs might exhibit extensive cytotoxicity against cancer cells. Figure 1 Chemical structures of compounds I, II, and 5a–s. [/RAW_REF_TEXT] Indole (1H-benzo[b]pyrrole, II, Figure 1) has a bicyclic heterocycle scaffold that is widely distributed in animals, microbial hormones, and plants.^16,17 It is considered to be a beneficial motif in drug-discovery programs because of its biodiversity, versatility, and anticancer activity.^18–21 Previous evidence suggests that myriad indole-based compounds exert anticancer activities through various mechanisms,^22–27 suggesting the possibility of using the indole scaffold in anticancer drugs. Notably, anticancer indole-based molecules target several pathways, such as those involving tubulin polymerization, DNA topoisomerase, histone deacetylase, proto-oncogenes, and serine/threonine (PIM) kinase.^28–31 Screening the literature exposed that a number of bioactive molecules bear carbohydrazide moiety. They display a wide spectrum of biological activities including anticancer activity.^32–34 Combining the structural features of two differing pharmacophores is an effective tactic in medicinal chemistry, as new bioactive, chemotherapeutic chemical entities characterized by a reduced risk of drug–drug interactions and improved anticancer profiles can be obtained.^35–37 Considering the structural characteristics of isatin and indole, merging these two bioactive moieties into a single compound through a carbohydrazide bridge is predicted to result in new promising anticancer leads. Accordingly, we aimed to synthesize and evaluate the in vitro antiproliferative activities of the molecular hybrids 5a–s. The most active antiproliferative candidate 5m was also examined in an in-depth pharmacological-profiling study to understand the potential mechanism whereby these compounds exert their antiproliferative effect. Materials and Methods Chemistry General The melting points of compounds 5a–s were measured using the Gallenkamp melting point equipment and are presented as uncorrected values. Compounds 5a–s were dissolved in DMSO-d₆ for nuclear magnetic resonance (NMR) measurements at 500 and 125 MHz for ¹H and ¹³C, respectively, using a Bruker NMR spectrometer (Bruker, Reinstetten, Germany) at the Research Center (College of Pharmacy, King Saud University, Saudi Arabia). TMS was used as an internal standard for the NMR measurements. The measured chemical shifts are presented as δ-values in parts per million (ppm). Compounds 5a–s were subjected to microanalyses at the Microanalysis Laboratory (Cairo University, Cairo, Egypt), and the results aligned well with the proposed structures (i.e., within ± 0.4% of the theoretical values). A 6120 LC/MS Agilent Quadrupole system with an electrospray-ionization(ESI) source (Agilent Technologies, Palo Alto, CA, USA) was used to obtain the mass spectra of compounds 5a–s. Compound 3 and compounds 4a–n were prepared as described previously.³⁸ General Procedure for Synthesizing Carbohydrazides 5a–s The acid hydrazide 3 (0.18 g, 1 mmol) was dissolved in absolute ethanol (15 mL), and the appropriate isatin derivative 4a–n (1 mmol) was added to the stirred ethanolic solution containing drops of glacial acetic acid. The reaction mixture was refluxed under continuous stirring for 4 h, and then the hot alcoholic reaction mixture was filtered. The collected solids were re-crystallized from the ethanol/DMF mixture (3:1) to yield the corresponding target carbohydrazides 5a–s with yields of 40 to 95%. N′-[(3Z)-2-Oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5a): Yellow powder, melting point (m.p.) > 300 °C (yield 89%); ¹H NMR (DMSO-d₆) ppm: 6.95 (d, J = 7.5 Hz, 1H, H_ar.), 7.09–7.16 (m, 2H, H_ar.), 7.29 (d, J = 7.0 Hz, 1H, H_ar.), 7.42 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.61 (s, 1H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.05 (d, J = 7.5 Hz, 1H, H_ar.), 10.88 (s, 1H, NH), 11.69 (s, 1H, NH), 11.97 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 104.2, 111.1, 112.9, 116.1, 120.7, 122.3, 122.7, 123.2, 125.1, 127.2, 127.5, 129.3, 133.2, 137.6, 144.4 (C_ar., CH_ar., C=N), 160.9, 165.3 (2 × C=O); MS m/z: 303 [M-H]^–. N′-[(3Z)-5-Bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5b): Yellow powder, m.p. > 300 °C (yield 76%); ¹H NMR (DMSO-d₆) ppm: 6.90 (d, J = 8.5 Hz, 1H, H_ar.), 7.09–7.14 (m, 1H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.59 (d, J = 8.0 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.73 (d, J = 8 Hz, 1H, H_ar.), 8.34 (s, 1H, H_ar.), 11.01 (s, 1H, NH), 11.87 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 104.8, 108.6, 112.9, 113.8, 117.5, 120.7, 121.0, 122.8, 125.2, 127.5, 129.2, 135.2, 136.4, 137.7, 143.5 (C_ar. and CH_ar., C=N), 160.7, 164.8 (2 × C=O); MS m/z: 381 [M-H]⁻, 383 [M-H+2]^–. N′-[(3Z)-5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5c): Yellow powder, m.p. > 300 °C (yield 91%); ¹H NMR (DMSO-d₆) ppm: 6.95 (d, J = 8.5 Hz, 1H, H_ar.), 7.09–7.14 (m, 1H, H_ar.), 7.27–7.32 (m, 1H, H_ar.), 7.43–7.47 (m, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.22 (s, 1H, H_ar.), 11.00 (s, 1H, NH), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm:102.1, 109.3, 112.4, 112.9, 117.1, 120.7, 122.8, 125.2, 126.2, 126.6, 127.5, 132.4, 137.6, 138.9, 143.0 (C_ar. and CH_ar., C=N), 163.5, 165.1 (2 × C=O); MS m/z: 337 [M-H]⁻; 339 [M-H+2]^–. N′-[(3Z)-5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5d): Yellow powder, m.p. > 300 °C (yield 95%); ¹H NMR (DMSO-d₆) ppm: 6.93 (dd, J = 4, 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 8.0 Hz, 1H, H_ar.), 7.25–7.30 (m, 2H, H_ar.), 7.50 (d, J = 8.5 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.02 (d, J = 8.0 Hz, 1H, H_ar.), 10.89 (s, 1H, NH), 11.79 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 108.4, 111.8, 112.8, 112.9, 113.1, 114.3, 120.7, 120.9, 122.7, 122.8, 125.3, 125.5, 137.5, 139.1 (C_ar., CH_ar., C=N), 157.9 (C_1ʹ-F, J = 235.0 Hz, C_ar.), 163.7, 165.4 (2 × C=O); MS m/z: 321 [M-H]^–. N′-[(3Z)-5-Methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5e): Orange powder, m.p. > 300 °C (yield 75%); ¹H NMR (DMSO-d₆) ppm: 3.82 (s, 3H, OCH₃), 6.86 (d, J = 8.5 Hz, 1H, H_ar.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, H_ar.), 7.09 (d, J = 7.5 Hz, 1H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.63 (s, 1H, H_ar.), 7.70–7.74 (m, 2H, H_ar.), 10.69 (s, 1H, NH), 11.81 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 56.3 (OCH₃), 102.6, 111.6, 112.6, 112.9, 113.1, 116.4, 118.7, 120.7, 122.8, 125.1, 127.5, 137.5, 138.1, 154.9, 155.9 (C_ar., CH_ar., C=N), 163.8, 165.4 (2 × C=O); MS m/z: 333 [M-H]^–. N′-[(3Z)-1-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5f): Yellow powder, m.p. 258–260 °C (yield 41%); ¹H NMR (DMSO-d₆) ppm: 3.23 (s, 3H, NCH₃), 7.13 (d, J = 8.0 Hz, 1H, H_ar.), 7.17–7.21 (m, 2H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.47–7.53 (m, 2H, H_ar.), 7.61 (s, 1H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.09 (d, J = 7.5 Hz, 1H, H_ar.), 11.76 (s, 1H, NH), 11.98 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 109.8, 110.5, 113.1, 121.0, 122.8, 125.1, 125.4, 126.8, 127.4, 129.3, 132.0, 133.1, 137.6, 144.1, 145.4 (C_ar., CH_ar., C=N), 161.7, 163.9 (2 × C=O); MS m/z: 317 [M-1]^–. N′-[(3Z)-5-Bromo-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5g): Yellow powder, m.p. 291–293 °C (yield 63%); ¹H NMR (DMSO-d₆) ppm: 3.21 (s, 3H, NCH₃), 7.08–7.15 (m, 2H, H_ar.), 7.28 (d, J = 7.5 Hz, 1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.63–7.68 (m, 2H, H_ar.), 7.73 (d, J = 8.0 Hz, 1H, H_ar.), 8.38 (s, 1H, H_ar.), 11.93 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 111.6, 112.9, 114.5, 120.6, 122.8, 123.3, 125.2, 127.5, 128.9, 134.1, 135.0, 137.5, 138.1, 143.1, 144.5 (C_ar., CH_ar., C=N), 161.4, 162.1 (2 × C=O); MS m/z: 395 [M-H]⁻, 397 [M-H+2]^–. N′-[(3Z)-5-Chloro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5h): Yellow powder, m.p. 292–294 °C (yield 82%); ¹H NMR (DMSO-d₆) ppm: 3.22 (s, 3H, NCH₃), 7.10 (d, J = 8.0, 1H, H_ar.), 7.13 (d, J = 4.0 Hz, 1H, H_ar.), 7.27–7.31 (m,1H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.54 (dd, J = 8.5, 2.0 Hz, 1H, H_ar.), 7.66 (s, 1H, H_ar.), 7.73 (d, J = 8.0, 1H, H_ar.), 8.26 (s, 1H, H_ar.), 11.92 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 109.3, 111.1, 112.0, 112.9, 120.7, 122.8, 125.2, 126.3, 126.8, 127.5, 127.9, 132.2, 137.6, 142.7, 144.1 (C_ar., CH_ar., C=N), 159.4, 161.5 (2 × C=O); MS m/z: 351 [M-H]^–. N′-[(3Z)-5-Fluoro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5i): Yellow powder, m.p. 283–285 °C (yield 54%); ¹H NMR (DMSO-d₆) ppm: 3.22 (s, 3H, NCH₃), 7.09–7.14 (m, 2H, H_ar.), 7.27–7.30 (m, 1H, H_ar.), 7.37 (ddd, J = 2.5, 9.0, 9.0, 1H, H_ar.), 7.51 (d, J = 8.0 Hz, 1H, H_ar.), 7.67 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.07 (d, J = 7.5 Hz, 1H, H_ar.), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.7 (NCH₃), 109.4, 110.6, 113.1, 114.2, 115.7, 118.9, 120.7, 122.8, 125.2, 125.4, 127.5, 129.0, 137.6, 141.8 (C_ar., CH_ar., C=N), 158.3 (C_1ʹ-F, J = 235.6 Hz, C_ar.), 161.8, 163.9 (2 × C=O);MS m/z: 335 [M-H]^–. N′-[(3Z)-5-Methoxy-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5j): Orange powder, m.p. 279–281 °C (yield 86%); ¹H NMR (DMSO-d₆) ppm: 3.19 (s, 3H, NCH₃), 3.84 (s, 3H, OCH₃), 7.04 (d, J = 8.5 Hz, 1H, H_ar.), 7.08–7.12 (m, 2H, H_ar.), 7.27–7.30 (m, 1H, H_ar.), 7.63 (s, 1H, H_ar.), 7.66 (d, J = 8.0 Hz, 1H, H_ar.), 7.72–7.77 (m, 2H, H_ar.), 11.86 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 26.6 (NCH₃), 56.4 (OCH₃), 110.2, 111.3, 112.9, 113.4, 115.8, 117.9, 120.7, 122.8, 125.1, 127.5, 129.2, 137.5, 139.2, 155.5 (C_ar., CH_ar., C=N), 161.7, 163.9 (2 × C=O); MS m/z: 347 [M-H]^–. N′-[(3Z)-1-Benzyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5k): Yellow powder, m.p. 249–251 °C (yield 56%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.16–7.21 (m, 1H, H_ar.), 7.28–7.32 (m, 2H, H_ar.), 7.35–7.38 (m, 3H, H_ar.), 7.42–7.45 (m, 2H, H_ar.), 7.51 (d, J = 8.5 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.74 (d, J = 7.5 Hz, 1H, H_ar.), 8.14 (d, J = 7.5 Hz, 1H, H_ar.), 11.80 (s, 1H, NH), 12.00 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.2 (CH₂), 110.4, 113.0, 115.7, 120.7, 120.9, 121.3, 122.7, 123.0, 127.5, 127.7, 127.9, 128.0, 129.2, 134.1, 136.7, 137.7, 144.3, 146.8, 147.5 (C_ar., CH_ar., C=N), 162.4, 164.2 (2 × C=O); MS m/z: 393 [M-H]^–. N′-[(3Z)-1-Benzyl-5-bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5l): Yellow powder, m.p. 268–270 °C (yield 51%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.01 (d, J = 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 7.5 Hz, 1H, H_ar.), 7.29 (d, J = 7.5 Hz, 2H, H_ar.), 7.36–7.38 (m, 3H, H_ar.), 7.42 (d, J = 7.5 Hz, 1H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.59 (s, 1H, H_ar.), 7.71–7.77 (m, 2H, H_ar.), 8.44 (s 1H, H_ar.), 11.99 (s, 1H, NH), 12.20 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 110.3, 112.1, 113.1, 114.8, 120.7, 127.7, 127.9, 128.2, 129.2, 134.1, 135.8, 136.4, 136.6, 137.6, 138.1, 142.1, 143.3, 148.7, 156.5 (C_ar., CH_ar., C=N), 161.5, 162.6 (2 × C=O); MS m/z: 471 [M-H]⁻; 473 [M-H+2]^–. N′-[(3Z)-1-Benzyl-5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5m): Yellow powder, m.p. 268–270 °C (yield 54%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.5 Hz, 1H, H_ar.), 7.11–7.13 (m, 1H, H_ar.), 7.28–7.31 (m, 2H, H_ar.), 7.35–7.37 (m, 3H, H_ar.), 7.43 (d, J = 7.0 Hz, 1H, H_ar.), 7.46–7.47 (m, 1H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.71 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.32 (s, 1H, H_ar.), 11.97 (s, 1H, NH), 12.18 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 111.7, 112.5, 112.9, 113.1, 120.7, 121.6, 127.5, 127.7, 127.9, 128.0, 128.2, 129.2, 135.9, 136.4, 137.6, 138.1, 141.7, 144.4, 149.6 (C_ar., CH_ar., C=N), 159.7, 161.6 (2 × C=O); MS m/z: 427 [M-H]⁻; 429 [M-H+2]^–. N′-[(3Z)-1-Benzyl-5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5n): Yellow powder, m.p. 251–253 °C (yield 49%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.04–7.06 (m, 1H, H_ar.), 7.09–7.13 (m, 1H, H_ar.), 7.28–7.32 (m, 3H, H_ar.), 7.34–7.39 (m, 4H, H_ar.), 7.29 (d, J = 8.5 Hz, 1H, H_ar.), 7.72 (s, 1H, H_ar.), 7.75 (d, J = 8.0 Hz, 1H, H_ar.), 8.13 (d, J = 7.5 Hz, 1H, H_ar.), 11.91 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.3 (CH₂), 110.9, 111.1, 113.0, 114.4, 119.0, 120.7, 122.8, 125.3, 127.5, 127.7, 127.9, 128.0, 128.9, 129.2, 136.5, 137.6, 139.9, 140.6 (C_ar., CH_ar., C=N), 158.4 (C_1ʹ-F, J = 237.9 Hz, C_ar.), 161.9, 164.2 (2 × C=O); MS m/z: 411 [M-H]^–. N′-[(3Z)-1-Benzyl-5-methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5o): Orange powder, m.p. 268–270 °C (yield 64%); ¹H NMR (DMSO-d₆) ppm: 3.81 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 6.95 (d, J = 8.5 Hz, 1H, H_ar.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, H_ar.), 7.10 (d, J = 8.0 Hz, 1H, H_ar.), 7.26–7.31 (m, 2H, H_ar.), 7.34–7.38 (m, 4H, H_ar.), 7.52 (d, J = 8.0 Hz, 1H, H_ar.), 7.68 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 7.78 (s, 1H, H_ar.), 11.92 (s, 1H, NH), 11.98 s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 43.1 (CH₂), 56.4 (OCH₃), 110.9, 113.0, 116.1, 120.7, 122.8, 125.2, 127.6, 127.7, 127.9, 128.0, 129.2, 129.3,136.7, 136.8, 137.7, 138.0, 138.1, 144.1, 155.6 (C_ar., CH_ar., C=N), 156.5, 164.1 (2 × C=O);MS m/z: 423 [M-H]^–. N′-[(3Z)-1-(4-Chlorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5p): Yellow powder, m.p. 261–263 °C (yield 65%); ¹H NMR (DMSO-d₆) ppm: 5.02 (s, 2H, CH₂), 7.06 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.14 (m, 1H, H_ar.), 7.19–7.21 (m, 1H, H_ar.), 7.28–7.32 (m, 1H, H_ar.), 7.40–7.44 (m, 3H, H_ar.), 7.51 (d, J = 8.0 Hz, 2H, H_ar.), 7.65 (s, 1H, H_ar.), 7.72–7.75 (m, 1H, H_ar.), 7.77 (d, J = 8.0 Hz, 1H, H_ar.), 8.15 (d, J = 7.5 Hz, 1H, H_ar.), 11.81 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 42.6 (CH₂), 110.9, 113.0, 119.9, 123.1, 125.2, 125.4, 127.5, 129.2, 129.7, 129.9, 131.9, 132.6, 132.8, 135.2, 135.7, 137.7, 138.0, 142.9, 144.1 (C_ar., CH_ar., C=N), 161.8, 164.2 (2 × C=O); MS m/z: 427 [M-H]⁻; 429 [M-H+2]^–. N′-[(3Z)-1-(4-Cyanobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5q): Yellow powder, m.p. 266–268 °C (yield 57%); ¹H NMR (DMSO-d₆) ppm: 5.13 (s, 2H, CH₂), 7.05 (d, J = 7.5 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.20 (ddd, J = 2.0, 7.5, 7.5 Hz, 1H, H_ar.), 7.39–7.43 (m, 1H, H_ar.), 7.56 (d, J = 8.5 Hz, 2H, H_ar.), 7.63–7.65 (m, 3H, H_ar.), 7.77 (d, J = 8.0 Hz, 1H, H_ar.), 7.84 (d, J = 3.0 Hz, 1H, H_ar.), 7.85 (d, J = 3.0 Hz, 1H, H_ar.), 8.17 (d, J = 7.5 Hz, 1H, H_ar.), 11.82 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 42.9 (CH₂), 110.8, 110.9, 113.0, 115.8, 119.9, 120.7, 122.7, 123.2, 125.4, 127.1, 127.5, 128.6, 128.8, 133.1, 133.2, 137.7, 138.0, 141.9, 142.5, 142.8 (C_ar., CH_ar., 2 × CN), 161.9, 164.2 (2 × C=O); MS m/z: 418 [M-H]^–. N′-[(3Z)-1-(4-Methylbenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5r): Yellow powder, m.p. 256–258 °C (yield 40%); ¹H NMR (DMSO-d₆) ppm: 2.27 (s, 3H, CH₃), 4.97 (s, 2H, CH₂), 7.04 (d, J = 8.0 Hz, 1H, H_ar.), 7.10–7.13 (m, 1H, H_ar.), 7.16 (d, J = 8.5 Hz, 2H, H_ar.), 7.27 (d, J = 8.5 Hz, 2H, H_ar.), 7.29–7.34 (m, 1H, H_ar.), 7.41–7.44 (m, 1H, H_ar.), 7.52 (d, J = 8.5 Hz, 1H, H_ar.), 7.65 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.13 (d, J = 7.5 Hz, 1H, H_ar.), 11.80 (s, 1H, NH), 12.00 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 21.1 (CH₃), 43.0 (CH₂), 110.4, 113.0, 115.7, 120.7, 122.7, 123.0, 125.2, 127.0, 127.5, 127.7, 127.9, 129.1, 129.2, 129.8, 132.9, 133.6, 137.2, 137.7, 144.3 (C_ar., CH_ar., C=N), 162.5, 164.1 (2 × C=O); MS m/z: 407 [M-H]^–. N′-[(3Z)-2-Oxo-1-phenyl-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5s): Yellow powder, m.p. 277–279 °C (yield 55%); ¹H NMR (DMSO-d₆) ppm: 6.84 (d, J = 8.0 Hz, 1H, H_ar.), 7.11–7.14 (m, 1H, H_ar.), 7.24–7.31 (m, 2H, H_ar.), 7.44–7.47 (m, 1H, H_ar.), 7.50–7.54 (m, 4H, H_ar.), 7.61–7.64 (m, 2H, H_ar.), 7.58 (s, 1H, H_ar.), 7.74 (d, J = 8.0 Hz, 1H, H_ar.), 8.23 (d, J = 7.5 Hz, 1H, H_ar.), 11.89 (s, 1H, NH), 12.01 (s, 1H, NH); ¹³C NMR (DMSO-d₆) ppm: 110.1, 113.0,115.7, 120.7, 122.8, 123.5, 125.2, 127.2, 127.4, 127.5, 128.9, 130.2, 133.1, 134.2, 135.5, 137.3, 137.7, 141.8, 145.1 (C_ar., CH_ar., C=N), 162.9, 163.4 (2 × C=O); MS m/z: 379 [M-H]^–. Pharmacological Evaluation Pharmacological assessment of the title compounds 5a–s was carried out using previously reported protocols^39,40 and they are provided as Supporting materials. The human cancer cell lines were procured commercially from the American Type Culture Collection. Western Blot Analysis Materials Immunoblotting detection was performed with antibodies from Abcam against cyclin E1(clone number HE12, catalog number ab3927), cyclin D1 (clone number EP272Y, catalog number ab40754), cyclin B1 (clone number Y106, catalog number ab32053), β actin (clone number mAbcam8226, catalog number ab8226), BTG1 (clone number EPR8274(2), catalog number ab151740), BAX (clone number 2D2, catalog numberab77566), bcl-2 (clone number E17, catalog number ab32124), and caspase-3 (catalog number ab13847). Methods A-549 non-small cell lung cancer (NSCLC) cells were plated in 25-cm² culture dishes at a seeding density of 5 × 10⁴/dish before treatment with compound 5m for 24, 48, and 72 h. Whole cell lysates were prepared using radioimmunoprecipitation assay buffer (Sigma-Aldrich, MO, USA). Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad, CA, USA). Twenty micrograms of treated and control lysates were loaded in each well of 4–20% Mini-Protean TGX Gels (Bio-Rad) for electrophoresis and then transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% (w/v) skimmed milk prepared in Tris-buffered saline and Tween 20, incubated with primary antibodies overnight at 4 °C, washed, and then incubated with a secondary, horseradish peroxidase-coupled anti-mouse or anti-rabbit antibody. Bands were visualized with a Western Bright ECL Kit for 5000 cm² Membrane and Blue Basic Autoradiography Film (BioExpress, CA, USA). Results and Discussion Chemistry The chemical structures of the title compounds 5a–s and their intermediates are shown in Scheme 1. The free carboxylic group of indole-2-carboxylic acid (1) was esterified, which resulted in the corresponding methyl ester 2.⁴¹ Subsequently, compound 2 was subjected to hydrazinolysis with hydrazine hydrate, thereby forming the acid hydrazide 3.⁴² The target indole–isatin conjugates 5a–s were respectively prepared by reacting the acid hydrazide 3 with the appropriate isatin derivatives 4a–n in ethanol-containing drops of acetic acid. The stereochemistry of the title conjugates 5a–s was assigned the (Z)-configuration, based on the previously documented X-ray results of structurally related analogous compounds.^43,44 Scheme 1 Preparation of compounds 5a–s. Reagents and conditions: (i) Few drops of concentrated sulfuric acid, absolute methanol, reflux, 4 h; (ii) H₂N-NH₂•H₂O, absolute methanol, reflux, 2 h; (iii) Few drops of glacial acetic acid, absolute ethanol, reflux, 4 h. [/RAW_REF_TEXT] Pharmacological Investigations Antiproliferative Activity The cell growth-inhibitory activities of nineteen conjugates 5a–s were evaluated with human breast (ZR-75), colon (HT-29) and lung (A-549) tumor cells. Each test compound was assessed at 30 µM in quadruplicate (Table 1). The title compounds 5a–s displayed average antiproliferative activities against the ZR-75, HT-29, and A-549 cell lines of 20.3 to 95.9%. Table 1 Antiproliferative Activities (Cell Growth-Inhibitory Activities at a Concentration of 30 µm) of Compounds 5a–s and Sunitinib Against the Indicated Cell Lines [/RAW_REF_TEXT] Table 2 shows the IC₅₀ (inhibitory concentration 50%) values determined for compounds 5a–s against the tested human cancer cell lines. Compound 5m had an average IC₅₀ value of 1.17 µM against the tested human cancer cell lines, making it the most active candidate, with a potency approximately seven-fold greater than that of sunitinib (average IC₅₀ value of 8.11 µM). Consequently, further pharmacological profiling was conducted with compound 5m to better understand the potential antiproliferative mechanism of compounds 5a–s. Table 2 The IC₅₀ Values of Compounds 5a–s and Sunitinib Against the Tested Cell Lines [/RAW_REF_TEXT] Structure Activity Relationship The title compounds 5a–s are structurally classified into five groups namely, N–unsubstituted derivatives 5a–e, N–methyl derivatives 5f–j, N–benzyl derivatives 5k–o, N–4–substituted benzyl derivatives 5p–r, and N–phenyl derivative 5s. In the first group 5a–e, compound 5e, bearing a methoxy substituent in the five position of the isatin nucleus, is the most active congener with an average IC₅₀ value of 18.18 µM. Compound 5g, in the second group 5f–j, have a bromo substituent in the five position of the isatin ring and it showed an average IC₅₀ value of 2.06 µM being nearly four times more potent than sunitinib (average IC₅₀ value of 8.11 µM). The third group 5k–o seems to be the best group with an average IC₅₀ range of 1.17–2.79 µM in which compound 5m, bearing 5–chloroisatin fragment, is the most active congener with an average IC₅₀ value of 1.17 µM being about sevenfold more potent than sunitinib. Moreover, the in vitro antiproliferative activity of the fourth group 5p–r is in the following order 5r ˃5p˃5q with an average IC₅₀ range of 1.46–3.92 µM. Compound 5s, bearing N–phenylisatin scaffold displayed good in vitro antiproliferative activity with an average IC₅₀ value of 3.36 µM (Table 2). In conclusion, the N–benzyl moiety on the isatin residue seems to be the favorable substituent to get good in vitro antiproliferative activity of this class of compounds. Effects on Cell-Cycle Progression The influence of compound 5m on different aspects of cell-cycle progression was investigated with the A-549 cell line based on the total DNA content of each cell. Additionally, immunofluorescent imaging of the phosphorylated retinoblastoma (Rb) protein was performed over the concentration range, 0.05–100 µM. A significant dose-dependent decrease in the total cell number (Figure 2) was noted after treatment with compound 5m for 48 h (IC₅₀ value = 1.38 µM; Table 3). Figure 2 Dose-dependent reductions in the numbers of adherent cells. A-549 NSCLC cells were treated for 24 or 48 h, as indicated, followed by fixation and addition of the fluorescent label. Values are presented as the average number of cells per 4 fields within each well. [/RAW_REF_TEXT] Table 3 IC₅₀ Values for the Reductions in Total Cell Numbers and the Effects of Compound 5m and Sunitinib on Cell-Cycle Progression [/RAW_REF_TEXT] In addition, the percentage of cells in G1 phase increased and the percentages of cells in the S and G2/M phases concomitantly decreased after treatment with compound 5m. This finding suggests that the ability of compound 5m to influence cell proliferation could be ascribed to decreased cell-cycle progression. Conversely, sunitinib treatment resulted in a reduced percentage of cells in G1 phase and a concomitant increase in cells in S or G2/M phase. G2 arrest could be regarded as a checkpoint blockade. Mitotic arrest may, in some instances, result in mitotic catastrophe and subsequent programmed death of cells with multiple or aberrant nuclei. Moreover, both sunitinib and 5m caused a substantial dose-dependent decrease in the extent of Rb protein phosphorylation (Figure 3), suggesting the possibility that the antiproliferative activity of compounds with isatin fragments can be attributed to their effect on cyclin-dependent kinases. Compound 5m had an IC₅₀ value of 2.52 µM after 24 h, indicating that it was ~1.7-fold more potent than sunitinib (Table 3). Treatment with compound 5m did not affect the extent of P-Tyr. Figure 3 Dose-dependent reductions in Rb phosphorylation. A-549 NSCLC cells were treated for 24 or 48 h (as indicated), fixed, and subjected to quantitative indirect immunofluorescence and image analysis. [/RAW_REF_TEXT] Western Blot Analysis Data from a previous study revealed a correlation between the antiproliferative marker, B cell translocation gene 1 (BTG1), and the inhibition of proliferation in lung cancer, which suggests that compound 5m most likely induces cycle arrest during G1 phase.⁴⁵ Treatment with compound 5m increased BTG1 expression (Figure S1), confirming its potential to act as an antiproliferative agent. To confirm the influence of compound 5m on cell-cycle progression, the expression levels of different cell cycle-related proteins were examined using Western blot analysis. As depicted in Figure S2, compound 5m regulated cell cycle-related proteins and activated cyclin-dependent kinase 1 (cdc-2) via phosphorylation at Tyr 15.⁴⁶ Although cell-cycle analysis revealed an increase in the G1 population after treatment with compound 5m, the ability of 5m to upregulate the G2/M phase-related proteins, cyclin B1 and p-cdc2 (Figure S2), was interesting. Ultimately, these findings suggest that cell-cycle arrest after treatment with 5m can occur during both G1 and G2 phases.^47,48 Many anticancer agents are designed to cause DNA damage and, consequently, apoptosis.⁴⁹ Hence, the ability of compound 5m to inhibit cell proliferation by inducing apoptosis was also examined. Compound 5m activated caspase-3, with increased production of cleaved caspase-3 within 24 h of treatment (Figure S3). The expression levels of the pro-survival protein encoded by the B-cell lymphoma/leukemia-2 (Bcl-2) gene and the pro-apoptotic protein encoded by the Bcl-2-associated X protein (BAX) gene were also evaluated. Treatment with compound 5m increase BAX expression but had no obvious effect on Bcl-2 expression (Figure S3). The altered BAX/BCL-2 ratio after treatment with 5m suggests the involvement of the intrinsic apoptosis pathway. Selectivity The selectivity of compound 5m was tested using three non-tumorigenic cell lines. IEC-6 cells obtained from rat intestines manifested both morphologic and karyotypic properties of normal intestinal epithelial cells.⁵⁰ Cultures obtained from human fibrocystic mammary tissue (MCF-10A) are non-tumorigenic and display characteristics similar to primary cultures of breast tissue, including dome formation.⁵¹ Fibroblasts obtained from the embryonic tissue of mice (Swiss 3t3 fibroblasts) are both non-tumorigenic and contact-inhibited.⁵² A-549 NSCLC cells were included for comparison. The antiproliferative activities of compound 5m and sunitinib were assessed in quadruplicate at a maximum concentration of 25 µM and then at 10 serially diluted concentrations. Compound 5m inhibited cell growth (>50%) in both normal and tumor cell lines. However, compound 5m exhibited a 2.6-fold selectivity value, whereas sunitinib displayed a 1.4-fold selectivity value (Figure 4 and Table 4). Figure 4 Tumor selectivity. A-549 NSCLC cells and non-tumorigenic cells derived from intestine, breast, and fibroblasts were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay. [/RAW_REF_TEXT] Table 4 Selectivity of Compound 5m and Sunitinib Against Tumor and Non-Tumorigenic Cell Lines [/RAW_REF_TEXT] Multidrug-Resistant Lung Cancer Cell Line The antiproliferative activity of compound 5m was examined against a sensitive NSCLC cell line (A-549) and a multidrug-resistant lung cancer cell (NCI-H69AR) expressing the ABCC1 efflux pump protein. Both compound 5m and sunitinib were tested in quadruplicate at a maximum concentration of 25 µM, and then at 10 serially diluted concentrations. Compound 5m inhibited growth in both cell lines, with an IC₅₀ value of 1.33 µM in A-549 cells (Figure 5 and Table 5). The H69AR cells were ~19-fold less sensitive, as compound 5m could be effluxed by the ABCC1 pump protein. Additionally, a lesser degree of sunitinib resistance (1.9-fold less sensitivity) was found in H69AR cells. Figure 5 Evaluation of susceptibility to efflux. A-549 NSCLC cells and NCI-H69AR cells expressing the ABCC1 (MRP1) multi-drug resistance efflux transporter were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay. [/RAW_REF_TEXT] Table 5 Antiproliferative Activities of Compound 5m and Sunitinib Against Sensitive (a-549) and Resistant (NCI-H69AR) Cell Lines [/RAW_REF_TEXT] Cell Viability Assay Using HEK 293 Cell Line Cell viability assay for compound 5m showed no apparent toxicity towards human embryonic kidney (HEK) 293 cells following 24 hrs exposure to different 5m concentrations, indicated by percent viability ranging from 93.72±0.68 to 98.67±0.39. Furthermore, there was no significant change in viability after longer exposure time to 5m up to 72 hrs. In Silico Predictions of ADME Properties of Compounds 5a-s In silico ADME (absorption, distribution, metabolism and excretion) predictions as well as some pharmacokinetic characteristics of the target compounds 5a–s are presented in Table S1 which are calculated according to the literature methods.^53,54 All the title compounds 5a–s follow the Lipinski’s rule of 5 (RO5) where log P values lie between 2.2–4.2 ( Conclusion Herein, we identified the newly synthesized isatin-indole molecular hybrids 5a–s with various spectroscopic approaches and estimated their potentials as antiproliferative agents in vitro. Compound 5m displayed promising in vitro antiproliferative potential against three human cancer cell lines. With an average IC₅₀ value of 1.17 µM, this compound had a seven-fold greater potency than sunitinib (IC₅₀ = 8.11 µM). By deriving its pharmacological profile, we sought to characterize the mechanisms underlying the antiproliferative activities of compounds 5a–s. We found reduced percentages of cells in S and G2/M phases, an increased percentage of cells in G1 phase, and a significant dose-dependent decrease in the extent of Rb protein phosphorylation. Furthermore, Western blot analysis confirmed the extents of BTG1, cyclin B1, cyclin D1, and p-cdc2 upregulation caused by compound 5m. Within 24 h of cell treatment, compound 5m increased BAX expression and activated caspase-3, as indicated by the observed increase in cleaved caspase-3 levels. ADME predictions for compounds 5a–s might help in further development of new anticancer agents with favorable oral bioavailability. Based on the findings presented herein, these lead compounds can be utilized in preclinical studies in the field of cancer chemotherapy. Supporting Materials Detailed protocols for the pharmacological evaluation of the title compounds 5a–s, Western blot results for compound 5m (Figures S1–S3), and ADME Table (Table S1) are provided as Supporting materials. Acknowledgment The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research through Research Group Project Number RG-1440-140. Disclosure Dr Adam B Keeton reports support of this project through the Deanship of Scientific Research at King Saud University through Research Group Project Number RG-1440-140. The authors declare no other conflicts of interest. References [RAW_REF_TEXT] 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi:10.3322/caac.v61:2 [/RAW_REF_TEXT] [RAW_REF_TEXT] 2. Jayashree B, Nigam S, Pai A, et al. Targets in anticancer research-a review. Indian J Exp Biol. 2015;53:489–507. [/RAW_REF_TEXT] [RAW_REF_TEXT] 3. Emami S, Dadashpour S. Current developments of coumarin-based anti-cancer agents in medicinal chemistry. Eur J Med Chem. 2015;102:611–630. doi:10.1016/j.ejmech.2015.08.033 [/RAW_REF_TEXT] [RAW_REF_TEXT] 4. Zwick E, Bange J, Ullrich A. Receptor tyrosine kinase signalling as a target for cancer intervention strategies. Endocr Relat Cancer. 2001;8(3):161–173. doi:10.1677/erc.0.0080161 [/RAW_REF_TEXT] [RAW_REF_TEXT] 5. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 2012;75(3):311–335. doi:10.1021/np200906s [/RAW_REF_TEXT] [RAW_REF_TEXT] 6. Luo F, Gu J, Chen L, et al. Systems pharmacology strategies for anticancer drug discovery based on natural products. Mol Biosyst. 2014;10(7):1912–1917. doi:10.1039/c4mb00105b [/RAW_REF_TEXT] [RAW_REF_TEXT] 7. Swathi K, Sarangapani M. Synthesis and screening of biologically significant 5 hydroxy isatin derivatives for antioxidant activity. Adv Exp Med Biol. 2015;822:129–137. [/RAW_REF_TEXT] [RAW_REF_TEXT] 8. Sharma PK, Balwani S, Mathur D, et al. Synthesis and anti-inflammatory activity evaluation of novel triazolyl-isatin hybrids. J Enzyme Inhib Med Chem. 2016;31(6):1520–1526. doi:10.3109/14756366.2016.1151015 [/RAW_REF_TEXT] [RAW_REF_TEXT] 9. Gil-Turnes MS, Hay ME, Fenical W. Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science. 1989;246(4926):116–118. doi:10.1126/science.2781297 [/RAW_REF_TEXT] [RAW_REF_TEXT] 10. Vine K, Matesic L, Locke J, et al. Cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from 2000-2008. Anticancer Agents Med Chem. 2009;9(4):397–414. doi:10.2174/1871520610909040397 [/RAW_REF_TEXT] [RAW_REF_TEXT] 11. Attia MI, Eldehna WM, Afifi SA, et al. New hydrazonoindolin-2-ones: synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into PLGA microspheres. PLoS One. 2017;12(7):e0181241. doi:10.1371/journal.pone.0181241 [/RAW_REF_TEXT] [RAW_REF_TEXT] 12. Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Eng J Med. 2007;356(2):115–124. doi:10.1056/NEJMoa065044 [/RAW_REF_TEXT] [RAW_REF_TEXT] 13. Griffith R, Brown M, McCluskey A, et al. Small molecule inhibitors of protein kinases in cancer-how to overcome resistance. Mini Rev Med Chem. 2006;6(10):1101–1110. doi:10.2174/138955706778560184 [/RAW_REF_TEXT] [RAW_REF_TEXT] 14. Garofalo A, Naumova E, Manenti L, et al. The combination of the tyrosine kinase receptor inhibitor SU6668 with paclitaxel affects ascites formation and tumor spread in ovarian carcinoma xenografts growing orthotopically. Clin Cancer Res. 2003;9(9):3476–3485. [/RAW_REF_TEXT] [RAW_REF_TEXT] 15. Krishnegowda G, Gowda AP, Tagaram HRS, et al. Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway. Bioorg Med Chem. 2011;19(20):6006–6014. doi:10.1016/j.bmc.2011.08.044 [/RAW_REF_TEXT] [RAW_REF_TEXT] 16. Lakhdar S, Westermaier M, Terrier F, et al. Nucleophilic reactivities of indoles. J Org Chem. 2006;71(24):9088–9095. doi:10.1021/jo0614339 [/RAW_REF_TEXT] [RAW_REF_TEXT] 17. Diss LB, Robinson SD, Wu Y, et al. Age-related changes in melatonin release in the murine distal colon. ACS Chem Neurosci. 2013;4(5):879–887. doi:10.1021/cn4000617 [/RAW_REF_TEXT] [RAW_REF_TEXT] 18. de Sa A, Fernando R, Barreiro EJ, et al. From nature to drug discovery: the indole scaffold as a ‘privileged structure’. Mini Rev Med Chem. 2009;9(7):782–793. doi:10.2174/138955709788452649 [/RAW_REF_TEXT] [RAW_REF_TEXT] 19. Ahmad A, A Sakr W, Wahidur Rahman K. Anticancer properties of indole compounds: mechanism of apoptosis induction androle in chemotherapy. Curr Drug Targets. 2010;11(6):652–666. doi:10.2174/138945010791170923 [/RAW_REF_TEXT] [RAW_REF_TEXT] 20. Akkoç MK, Yüksel MY, Durmaz İ, et al. Design, synthesis, and biological evaluation of indole-based 1, 4-disubstituted piperazines as cytotoxic agents. Turk J Chem. 2012;36(4):515–525. [/RAW_REF_TEXT] [RAW_REF_TEXT] 21. MacDonough MT, Strecker TE, Hamel E, et al. Synthesis and biological evaluation of indole-based, anti-cancer agents inspired by the vascular disrupting agent 2-(3′-hydroxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxybenzoyl)-6-methoxyindole (OXi8006). Bioorg Med Chem. 2013;21(21):6831–6843. doi:10.1016/j.bmc.2013.07.028 [/RAW_REF_TEXT] [RAW_REF_TEXT] 22. Almagro L, Fernández-Pérez F, Pedreño M. Indole alkaloids from catharanthus roseus: bioproduction and their effect on human health. Molecules. 2015;20(2):2973–3000. doi:10.3390/molecules20022973 [/RAW_REF_TEXT] [RAW_REF_TEXT] 23. Bradner W. Mitomycin C: a clinical update. Cancer Treat Rev. 2001;27(1):35–50. doi:10.1053/ctrv.2000.0202 [/RAW_REF_TEXT] [RAW_REF_TEXT] 24. Singh Sidhu J, Singla R, Jaitak V, Jaitak V. Indole derivatives as anticancer agents for breast cancer therapy: a review. Anticancer Agents Med Chem. 2016;16(2):160–173. doi:10.2174/1871520615666150520144217 [/RAW_REF_TEXT] [RAW_REF_TEXT] 25. Sravanthi T, Manju S. Indoles-a promising scaffold for drug development. Eur J Pharm Sci. 2016;91:1–10. doi:10.1016/j.ejps.2016.05.025 [/RAW_REF_TEXT] [RAW_REF_TEXT] 26. Chadha N, Silakari O. Indoles as therapeutics of interest in medicinal chemistry: bird’s eye view. Eur J Med Chem. 2017;134:159–184. doi:10.1016/j.ejmech.2017.04.003 [/RAW_REF_TEXT] [RAW_REF_TEXT] 27. Almutairi MS, Hassan ES, Keeton AB, et al. Antiproliferative activity and possible mechanism of action of certain 5-methoxyindole tethered C-5 functionalized isatins. Drug Des Devel Ther. 2019;13:3069. doi:10.2147/DDDT [/RAW_REF_TEXT] [RAW_REF_TEXT] 28. Sang Y-L, Zhang W-M, Lv P-C, et al. Indole-based, antiproliferative agents targeting tubulin polymerization. Curr Top Med Chem. 2016;17(2):120–137. doi:10.2174/1568026616666160530154812 [/RAW_REF_TEXT] [RAW_REF_TEXT] 29. Song Y-L, Dong Y-F, Yang T, et al. Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues. Bioorg Med Chem. 2013;21(24):7624–7627. doi:10.1016/j.bmc.2013.10.034 [/RAW_REF_TEXT] [RAW_REF_TEXT] 30. Giannini G, Marzi M, Di Marzo M, et al. Exploring bis-(indolyl) methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors. Bioorg Med Chem Lett. 2009;19(10):2840–2843. doi:10.1016/j.bmcl.2009.03.101 [/RAW_REF_TEXT] [RAW_REF_TEXT] 31. Haddach M, Michaux J, Schwaebe MK, et al. Discovery of CX-6258. A potent, selective, and orally efficacious pan-pim kinases inhibitor. ACS Med Chem Lett. 2012;3(2):135–139. doi:10.1021/ml200259q [/RAW_REF_TEXT] [RAW_REF_TEXT] 32. Xia Y, Fan C-D, Zhao B-X, et al. Synthesis and structure–activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against A549 lung cancer cells. Eur J Med Chem. 2008;43:2347–2353. doi:10.1016/j.ejmech.2008.01.021 [/RAW_REF_TEXT] [RAW_REF_TEXT] 33. Zhang D, Wang G, Zhao G, et al. Synthesis and cytotoxic activity of novel 3-(1H-indol-3-yl)-1H-pyrazole-5-carbohydrazide derivatives. Eur J Med Chem. 2011;46:5868–5877. doi:10.1016/j.ejmech.2011.09.049 [/RAW_REF_TEXT] [RAW_REF_TEXT] 34. Pieczonka AM, Strzelczyk A, Sadowska B, et al. Synthesis and evaluation of antimicrobial activity of hydrazones derived from 3-oxido-1H-imidazole-4-carbohydrazides. Eur J Med Chem. 2013;64:389–395. doi:10.1016/j.ejmech.2013.04.023 [/RAW_REF_TEXT] [RAW_REF_TEXT] 35. Gediya LK, Njar VC. Promise and challenges in drug discovery and development of hybrid anticancer drugs. Expert Opin Drug Discov. 2009;4(11):1099–1111. doi:10.1517/17460440903341705 [/RAW_REF_TEXT] [RAW_REF_TEXT] 36. Mishra S, Singh P, Singh P. Hybrid molecules: the privileged scaffolds for various pharmaceuticals. Eur J Med Chem. 2016;124:500–536. doi:10.1016/j.ejmech.2016.08.039 [/RAW_REF_TEXT] [RAW_REF_TEXT] 37. Kerru N, Singh P, Koorbanally N, et al. Recent advances (2015–2016) in anticancer hybrids. 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Z Krist-New Cryst St. 2017;232(3):431–432. [/RAW_REF_TEXT] [RAW_REF_TEXT] 42. Almutairi M, Zakaria A, Al-Wabli R, et al. Synthesis, spectroscopic identification and molecular docking of certain N-(2-{[2-(1H-indol-2-ylcarbonyl)hydrazinyl] (oxo)acetyl}phenyl)acetamides and N-[2-(2-{[2-(acetylamino)phenyl](oxo) acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides: new antimicrobial agents. Molecules. 2018;23(5):1043. [/RAW_REF_TEXT] [RAW_REF_TEXT] 43. Haress NG, Ghabbour HA, Almutairi MS, et al. Crystal structure of 5-methoxy-N′-[(3Z)-5-chloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide-DMSO (1/1), C₂₅H₁₈ClFN₄O₃· C₂H₆OS. Z Krist-New Cryst St. 2016;231(4):1021–1023. [/RAW_REF_TEXT] [RAW_REF_TEXT] 44. Almutairi MS, Ghabbour HA, Haress NG, et al. Crystal structure of 5-methoxy-N′-[(3Z)-1-benzyl-5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide-DMSO (1/1), C₂₇H₂₅FN₄O₄S. Z Krist-New Cryst St. 2016;231(4):1025–1027. [/RAW_REF_TEXT] [RAW_REF_TEXT] 45. Sun G, Lu Y, Cheng Y, et al. The expression of BTG1is downregulated in NSCLC and possibly associated with tumor metastasis. Tumor Biol. 2014;35(4):2949–2957. doi:10.1007/s13277-013-1379-6 [/RAW_REF_TEXT] [RAW_REF_TEXT] 46. Welburn JP, Tucker JA, Johnson T, et al. How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A. J Biol Chem. 2007;282(5):3173–3181. doi:10.1074/jbc.M609151200 [/RAW_REF_TEXT] [RAW_REF_TEXT] 47. Blajeski AL, Phan VA, Kottke TJ, et al. G 1 and G 2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. J Clin Invest. 2002;110(1):91–99. doi:10.1172/JCI13275 [/RAW_REF_TEXT] [RAW_REF_TEXT] 48. Liu X, Zhao P, Wang X, et al. Celastrol mediates autophagy and apoptosis via the ROS/JNK and Akt/mTOR signaling pathways in glioma cells. J Exp Clin Cancer Res. 2019;38(1):184. doi:10.1186/s13046-019-1173-4 [/RAW_REF_TEXT] [RAW_REF_TEXT] 49. Rixe O, Fojo T. 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References

1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi:10.3322/caac.v61:2

2. Jayashree B, Nigam S, Pai A, et al. Targets in anticancer research-a review. Indian J Exp Biol. 2015;53:489–507.

3. Emami S, Dadashpour S. Current developments of coumarin-based anti-cancer agents in medicinal chemistry. Eur J Med Chem. 2015;102:611–630. doi:10.1016/j.ejmech.2015.08.033

4. Zwick E, Bange J, Ullrich A. Receptor tyrosine kinase signalling as a target for cancer intervention strategies. Endocr Relat Cancer. 2001;8(3):161–173. doi:10.1677/erc.0.0080161

5. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 2012;75(3):311–335. doi:10.1021/np200906s

6. Luo F, Gu J, Chen L, et al. Systems pharmacology strategies for anticancer drug discovery based on natural products. Mol Biosyst. 2014;10(7):1912–1917. doi:10.1039/c4mb00105b

7. Swathi K, Sarangapani M. Synthesis and screening of biologically significant 5 hydroxy isatin derivatives for antioxidant activity. Adv Exp Med Biol. 2015;822:129–137.

8. Sharma PK, Balwani S, Mathur D, et al. Synthesis and anti-inflammatory activity evaluation of novel triazolyl-isatin hybrids. J Enzyme Inhib Med Chem. 2016;31(6):1520–1526. doi:10.3109/14756366.2016.1151015

9. Gil-Turnes MS, Hay ME, Fenical W. Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science. 1989;246(4926):116–118. doi:10.1126/science.2781297

10. Vine K, Matesic L, Locke J, et al. Cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from 2000-2008. Anticancer Agents Med Chem. 2009;9(4):397–414. doi:10.2174/1871520610909040397

11. Attia MI, Eldehna WM, Afifi SA, et al. New hydrazonoindolin-2-ones: synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into PLGA microspheres. PLoS One. 2017;12(7):e0181241. doi:10.1371/journal.pone.0181241

12. Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Eng J Med. 2007;356(2):115–124. doi:10.1056/NEJMoa065044

13. Griffith R, Brown M, McCluskey A, et al. Small molecule inhibitors of protein kinases in cancer-how to overcome resistance. Mini Rev Med Chem. 2006;6(10):1101–1110. doi:10.2174/138955706778560184

14. Garofalo A, Naumova E, Manenti L, et al. The combination of the tyrosine kinase receptor inhibitor SU6668 with paclitaxel affects ascites formation and tumor spread in ovarian carcinoma xenografts growing orthotopically. Clin Cancer Res. 2003;9(9):3476–3485.

15. Krishnegowda G, Gowda AP, Tagaram HRS, et al. Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway. Bioorg Med Chem. 2011;19(20):6006–6014. doi:10.1016/j.bmc.2011.08.044

16. Lakhdar S, Westermaier M, Terrier F, et al. Nucleophilic reactivities of indoles. J Org Chem. 2006;71(24):9088–9095. doi:10.1021/jo0614339

17. Diss LB, Robinson SD, Wu Y, et al. Age-related changes in melatonin release in the murine distal colon. ACS Chem Neurosci. 2013;4(5):879–887. doi:10.1021/cn4000617

18. de Sa A, Fernando R, Barreiro EJ, et al. From nature to drug discovery: the indole scaffold as a ‘privileged structure’. Mini Rev Med Chem. 2009;9(7):782–793. doi:10.2174/138955709788452649

19. Ahmad A, A Sakr W, Wahidur Rahman K. Anticancer properties of indole compounds: mechanism of apoptosis induction androle in chemotherapy. Curr Drug Targets. 2010;11(6):652–666. doi:10.2174/138945010791170923

20. Akkoç MK, Yüksel MY, Durmaz İ, et al. Design, synthesis, and biological evaluation of indole-based 1, 4-disubstituted piperazines as cytotoxic agents. Turk J Chem. 2012;36(4):515–525.

21. MacDonough MT, Strecker TE, Hamel E, et al. Synthesis and biological evaluation of indole-based, anti-cancer agents inspired by the vascular disrupting agent 2-(3′-hydroxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxybenzoyl)-6-methoxyindole (OXi8006). Bioorg Med Chem. 2013;21(21):6831–6843. doi:10.1016/j.bmc.2013.07.028

22. Almagro L, Fernández-Pérez F, Pedreño M. Indole alkaloids from catharanthus roseus: bioproduction and their effect on human health. Molecules. 2015;20(2):2973–3000. doi:10.3390/molecules20022973

23. Bradner W. Mitomycin C: a clinical update. Cancer Treat Rev. 2001;27(1):35–50. doi:10.1053/ctrv.2000.0202

24. Singh Sidhu J, Singla R, Jaitak V, Jaitak V. Indole derivatives as anticancer agents for breast cancer therapy: a review. Anticancer Agents Med Chem. 2016;16(2):160–173. doi:10.2174/1871520615666150520144217

25. Sravanthi T, Manju S. Indoles-a promising scaffold for drug development. Eur J Pharm Sci. 2016;91:1–10. doi:10.1016/j.ejps.2016.05.025

26. Chadha N, Silakari O. Indoles as therapeutics of interest in medicinal chemistry: bird’s eye view. Eur J Med Chem. 2017;134:159–184. doi:10.1016/j.ejmech.2017.04.003

27. Almutairi MS, Hassan ES, Keeton AB, et al. Antiproliferative activity and possible mechanism of action of certain 5-methoxyindole tethered C-5 functionalized isatins. Drug Des Devel Ther. 2019;13:3069. doi:10.2147/DDDT

28. Sang Y-L, Zhang W-M, Lv P-C, et al. Indole-based, antiproliferative agents targeting tubulin polymerization. Curr Top Med Chem. 2016;17(2):120–137. doi:10.2174/1568026616666160530154812

29. Song Y-L, Dong Y-F, Yang T, et al. Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues. Bioorg Med Chem. 2013;21(24):7624–7627. doi:10.1016/j.bmc.2013.10.034

30. Giannini G, Marzi M, Di Marzo M, et al. Exploring bis-(indolyl) methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors. Bioorg Med Chem Lett. 2009;19(10):2840–2843. doi:10.1016/j.bmcl.2009.03.101

31. Haddach M, Michaux J, Schwaebe MK, et al. Discovery of CX-6258. A potent, selective, and orally efficacious pan-pim kinases inhibitor. ACS Med Chem Lett. 2012;3(2):135–139. doi:10.1021/ml200259q

32. Xia Y, Fan C-D, Zhao B-X, et al. Synthesis and structure–activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against A549 lung cancer cells. Eur J Med Chem. 2008;43:2347–2353. doi:10.1016/j.ejmech.2008.01.021

33. Zhang D, Wang G, Zhao G, et al. Synthesis and cytotoxic activity of novel 3-(1H-indol-3-yl)-1H-pyrazole-5-carbohydrazide derivatives. Eur J Med Chem. 2011;46:5868–5877. doi:10.1016/j.ejmech.2011.09.049

34. Pieczonka AM, Strzelczyk A, Sadowska B, et al. Synthesis and evaluation of antimicrobial activity of hydrazones derived from 3-oxido-1H-imidazole-4-carbohydrazides. Eur J Med Chem. 2013;64:389–395. doi:10.1016/j.ejmech.2013.04.023

35. Gediya LK, Njar VC. Promise and challenges in drug discovery and development of hybrid anticancer drugs. Expert Opin Drug Discov. 2009;4(11):1099–1111. doi:10.1517/17460440903341705

36. Mishra S, Singh P, Singh P. Hybrid molecules: the privileged scaffolds for various pharmaceuticals. Eur J Med Chem. 2016;124:500–536. doi:10.1016/j.ejmech.2016.08.039

37. Kerru N, Singh P, Koorbanally N, et al. Recent advances (2015–2016) in anticancer hybrids. Eur J Med Chem. 2017;142:179–212. doi:10.1016/j.ejmech.2017.07.033

38. Al-Wabli R, Zakaria A, Attia M. Synthesis, spectroscopic characterization and antimicrobial potential of certain new isatin-indole molecular hybrids. Molecules. 2017;22(11):1958. doi:10.3390/molecules22111958

39. Abdel-Aziz HA, Eldehna WM, Keeton AB, et al. Isatin-benzoazine molecular hybrids as potential antiproliferative agents: synthesis and in vitro pharmacological profiling. Drug Des Devel Ther. 2017;11:2333. doi:10.2147/DDDT

40. Grauzdytė D, Pukalskas A, Viranaicken W, et al. Protective effects of phyllanthus phillyreifolius extracts against hydrogen peroxide induced oxidative stress in HEK293 cells. PLoS One. 2018;13(11):e0207672. doi:10.1371/journal.pone.0207672

41. Almutairi MS, Ghabbour HA, Attia MI. Crystal structure of methyl 1H-indole-2-carboxylate, C₁₀H₉NO₂. Z Krist-New Cryst St. 2017;232(3):431–432.

42. Almutairi M, Zakaria A, Al-Wabli R, et al. Synthesis, spectroscopic identification and molecular docking of certain N-(2-{[2-(1H-indol-2-ylcarbonyl)hydrazinyl] (oxo)acetyl}phenyl)acetamides and N-[2-(2-{[2-(acetylamino)phenyl](oxo) acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides: new antimicrobial agents. Molecules. 2018;23(5):1043.

43. Haress NG, Ghabbour HA, Almutairi MS, et al. Crystal structure of 5-methoxy-N′-[(3Z)-5-chloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide-DMSO (1/1), C₂₅H₁₈ClFN₄O₃· C₂H₆OS. Z Krist-New Cryst St. 2016;231(4):1021–1023.

44. Almutairi MS, Ghabbour HA, Haress NG, et al. Crystal structure of 5-methoxy-N′-[(3Z)-1-benzyl-5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide-DMSO (1/1), C₂₇H₂₅FN₄O₄S. Z Krist-New Cryst St. 2016;231(4):1025–1027.

45. Sun G, Lu Y, Cheng Y, et al. The expression of BTG1is downregulated in NSCLC and possibly associated with tumor metastasis. Tumor Biol. 2014;35(4):2949–2957. doi:10.1007/s13277-013-1379-6

46. Welburn JP, Tucker JA, Johnson T, et al. How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A. J Biol Chem. 2007;282(5):3173–3181. doi:10.1074/jbc.M609151200

47. Blajeski AL, Phan VA, Kottke TJ, et al. G 1 and G 2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. J Clin Invest. 2002;110(1):91–99. doi:10.1172/JCI13275

48. Liu X, Zhao P, Wang X, et al. Celastrol mediates autophagy and apoptosis via the ROS/JNK and Akt/mTOR signaling pathways in glioma cells. J Exp Clin Cancer Res. 2019;38(1):184. doi:10.1186/s13046-019-1173-4

49. Rixe O, Fojo T. Is cell death a critical end point for anticancer therapies or is cytostasis sufficient? Clin Cancer Res. 2007;13(24):7280–7287. doi:10.1158/1078-0432.CCR-07-2141

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