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Original Research

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

Reem I Al-Wabli1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0002-7836-2496
ORCID Icon,
Aliyah A Almomen1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0002-0181-6330
ORCID Icon,
Maha S Almutairi1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
,
Adam B Keeton2 Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
,
Gary A Piazza2 Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604-1405, USA
&
Mohamed I Attia1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;3 Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), Giza 12622, EgyptCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-5019-6086
ORCID Icon
Pages 483-495 | Published online: 03 Feb 2020

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.Citation1 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.Citation2–Citation4

Creating novel anticancer chemotherapeutic agents based on naturally occurring bioactive substances has been gaining increased interest.Citation5,Citation6 2,3-Dioxindole (isatin, I, ) 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,Citation7 anti-inflammatory,Citation8 anti-bacterial,Citation9 and anticancerCitation10,Citation11 activities. The United States Federal Drug Administration approved sunitinib and nintedanib as isatin-bearing anticancer drugs,Citation10,Citation12 and many compounds with an isatin scaffold such as SU6668 and SU5416 have displayed outstanding anticancer activity.Citation13–Citation15 Therefore, isatin-bearing drugs might exhibit extensive cytotoxicity against cancer cells.

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

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

Indole (1H-benzo[b]pyrrole, II, ) has a bicyclic heterocycle scaffold that is widely distributed in animals, microbial hormones, and plants.Citation16,Citation17 It is considered to be a beneficial motif in drug-discovery programs because of its biodiversity, versatility, and anticancer activity.Citation18–Citation21 Previous evidence suggests that myriad indole-based compounds exert anticancer activities through various mechanisms,Citation22–Citation27 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.Citation28–Citation31

Screening the literature exposed that a number of bioactive molecules bear carbohydrazide moiety. They display a wide spectrum of biological activities including anticancer activity.Citation32–Citation34

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.Citation35–Citation37 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.Citation38

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, Har.), 7.09–7.16 (m, 2H, Har.), 7.29 (d, J = 7.0 Hz, 1H, Har.), 7.42 (d, J = 7.5 Hz, 1H, Har.), 7.51 (d, J = 8.5 Hz, 1H, Har.), 7.61 (s, 1H, Har.), 7.73 (d, J = 8.0 Hz, 1H, Har.), 8.05 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.09–7.14 (m, 1H, Har.), 7.28 (d, J = 7.5 Hz, 1H, Har.), 7.51 (d, J = 8.0 Hz, 1H, Har.), 7.59 (d, J = 8.0 Hz, 1H, Har.), 7.65 (s, 1H, Har.), 7.73 (d, J = 8 Hz, 1H, Har.), 8.34 (s, 1H, Har.), 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 (Car. and CHar., 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, Har.), 7.09–7.14 (m, 1H, Har.), 7.27–7.32 (m, 1H, Har.), 7.43–7.47 (m, 1H, Har.), 7.51 (d, J = 8.0 Hz, 1H, Har.), 7.66 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.22 (s, 1H, Har.), 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 (Car. and CHar., 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, Har.), 7.10 (d, J = 8.0 Hz, 1H, Har.), 7.25–7.30 (m, 2H, Har.), 7.50 (d, J = 8.5 Hz, 1H, Har.), 7.66 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.02 (d, J = 8.0 Hz, 1H, Har.), 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 (Car., CHar., C=N), 157.9 (C1ʹ-F, J = 235.0 Hz, Car.), 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, Har.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, Har.), 7.09 (d, J = 7.5 Hz, 1H, Har.), 7.28 (d, J = 7.5 Hz, 1H, Har.), 7.51 (d, J = 8.5 Hz, 1H, Har.), 7.63 (s, 1H, Har.), 7.70–7.74 (m, 2H, Har.), 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 (Car., CHar., 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, Har.), 7.17–7.21 (m, 2H, Har.), 7.28 (d, J = 7.5 Hz, 1H, Har.), 7.47–7.53 (m, 2H, Har.), 7.61 (s, 1H, Har.), 7.73 (d, J = 8.0 Hz, 1H, Har.), 8.09 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.28 (d, J = 7.5 Hz, 1H, Har.), 7.51 (d, J = 8.5 Hz, 1H, Har.), 7.63–7.68 (m, 2H, Har.), 7.73 (d, J = 8.0 Hz, 1H, Har.), 8.38 (s, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.13 (d, J = 4.0 Hz, 1H, Har.), 7.27–7.31 (m,1H, Har.), 7.51 (d, J = 8.5 Hz, 1H, Har.), 7.54 (dd, J = 8.5, 2.0 Hz, 1H, Har.), 7.66 (s, 1H, Har.), 7.73 (d, J = 8.0, 1H, Har.), 8.26 (s, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.27–7.30 (m, 1H, Har.), 7.37 (ddd, J = 2.5, 9.0, 9.0, 1H, Har.), 7.51 (d, J = 8.0 Hz, 1H, Har.), 7.67 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.07 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., C=N), 158.3 (C1ʹ-F, J = 235.6 Hz, Car.), 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, Har.), 7.08–7.12 (m, 2H, Har.), 7.27–7.30 (m, 1H, Har.), 7.63 (s, 1H, Har.), 7.66 (d, J = 8.0 Hz, 1H, Har.), 7.72–7.77 (m, 2H, Har.), 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 (Car., CHar., 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, Har.), 7.10–7.13 (m, 1H, Har.), 7.16–7.21 (m, 1H, Har.), 7.28–7.32 (m, 2H, Har.), 7.35–7.38 (m, 3H, Har.), 7.42–7.45 (m, 2H, Har.), 7.51 (d, J = 8.5 Hz, 1H, Har.), 7.65 (s, 1H, Har.), 7.74 (d, J = 7.5 Hz, 1H, Har.), 8.14 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.10 (d, J = 7.5 Hz, 1H, Har.), 7.29 (d, J = 7.5 Hz, 2H, Har.), 7.36–7.38 (m, 3H, Har.), 7.42 (d, J = 7.5 Hz, 1H, Har.), 7.52 (d, J = 8.0 Hz, 1H, Har.), 7.59 (s, 1H, Har.), 7.71–7.77 (m, 2H, Har.), 8.44 (s 1H, Har.), 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 (Car., CHar., 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, Har.), 7.11–7.13 (m, 1H, Har.), 7.28–7.31 (m, 2H, Har.), 7.35–7.37 (m, 3H, Har.), 7.43 (d, J = 7.0 Hz, 1H, Har.), 7.46–7.47 (m, 1H, Har.), 7.52 (d, J = 8.0 Hz, 1H, Har.), 7.71 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.32 (s, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.09–7.13 (m, 1H, Har.), 7.28–7.32 (m, 3H, Har.), 7.34–7.39 (m, 4H, Har.), 7.29 (d, J = 8.5 Hz, 1H, Har.), 7.72 (s, 1H, Har.), 7.75 (d, J = 8.0 Hz, 1H, Har.), 8.13 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., C=N), 158.4 (C1ʹ-F, J = 237.9 Hz, Car.), 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, Har.), 7.02 (dd, J = 2.0, 8.5 Hz, 1H, Har.), 7.10 (d, J = 8.0 Hz, 1H, Har.), 7.26–7.31 (m, 2H, Har.), 7.34–7.38 (m, 4H, Har.), 7.52 (d, J = 8.0 Hz, 1H, Har.), 7.68 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 7.78 (s, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.10–7.14 (m, 1H, Har.), 7.19–7.21 (m, 1H, Har.), 7.28–7.32 (m, 1H, Har.), 7.40–7.44 (m, 3H, Har.), 7.51 (d, J = 8.0 Hz, 2H, Har.), 7.65 (s, 1H, Har.), 7.72–7.75 (m, 1H, Har.), 7.77 (d, J = 8.0 Hz, 1H, Har.), 8.15 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.10–7.13 (m, 1H, Har.), 7.20 (ddd, J = 2.0, 7.5, 7.5 Hz, 1H, Har.), 7.39–7.43 (m, 1H, Har.), 7.56 (d, J = 8.5 Hz, 2H, Har.), 7.63–7.65 (m, 3H, Har.), 7.77 (d, J = 8.0 Hz, 1H, Har.), 7.84 (d, J = 3.0 Hz, 1H, Har.), 7.85 (d, J = 3.0 Hz, 1H, Har.), 8.17 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.10–7.13 (m, 1H, Har.), 7.16 (d, J = 8.5 Hz, 2H, Har.), 7.27 (d, J = 8.5 Hz, 2H, Har.), 7.29–7.34 (m, 1H, Har.), 7.41–7.44 (m, 1H, Har.), 7.52 (d, J = 8.5 Hz, 1H, Har.), 7.65 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.13 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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, Har.), 7.11–7.14 (m, 1H, Har.), 7.24–7.31 (m, 2H, Har.), 7.44–7.47 (m, 1H, Har.), 7.50–7.54 (m, 4H, Har.), 7.61–7.64 (m, 2H, Har.), 7.58 (s, 1H, Har.), 7.74 (d, J = 8.0 Hz, 1H, Har.), 8.23 (d, J = 7.5 Hz, 1H, Har.), 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 (Car., CHar., 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 protocolsCitation39,Citation40 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 . The free carboxylic group of indole-2-carboxylic acid (1) was esterified, which resulted in the corresponding methyl ester 2.Citation41 Subsequently, compound 2 was subjected to hydrazinolysis with hydrazine hydrate, thereby forming the acid hydrazide 3.Citation42 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.Citation43,Citation44

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.

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 (). 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

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 (). 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 () was noted after treatment with compound 5m for 48 h (IC50 value = 1.38 µM; ).

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.

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 (), 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 (). 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.

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.Citation45 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.Citation46 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.Citation47,Citation48

Many anticancer agents are designed to cause DNA damage and, consequently, apoptosis.Citation49 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.Citation50 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.Citation51 Fibroblasts obtained from the embryonic tissue of mice (Swiss 3t3 fibroblasts) are both non-tumorigenic and contact-inhibited.Citation52 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 ( and ).

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.

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 ( and ). 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.

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.Citation53,Citation54 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 Å2 (<140 Å2), 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.

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.

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.

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