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

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][3][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 antibacterial, 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][14][15] Therefore, isatin-bearing drugs might exhibit extensive cytotoxicity against cancer cells.
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][19][20][21] Previous evidence suggests that myriad indole-based compounds exert anticancer activities through various mechanisms, [22][23][24][25][26][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][29][30][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][33][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][36][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-d 6 for nuclear magnetic resonance (NMR) measurements at 500 and 125 MHz for 1 H and 13 C, 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 electrosprayionization(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/

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.

Methods
A-549 non-small cell lung cancer (NSCLC) cells were plated in 25-cm 2 culture dishes at a seeding density of 5 × 10 4 /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 Trisbuffered saline and Tween 20, incubated with primary antibodies overnight at 4°C, washed, and then incubated with a secondary, horseradish peroxidase-coupled antimouse or anti-rabbit antibody. Bands were visualized with a Western Bright ECL Kit for 5000 cm 2 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 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 2 shows the IC 50 (inhibitory concentration 50%) values determined for compounds 5a-s against the tested human cancer cell lines. Compound 5m had an average IC 50 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 IC 50     Abbreviation: IC 50 , inhibitory concentration 50%. 8.11 µM). Consequently, further pharmacological profiling was conducted with compound 5m to better understand the potential antiproliferative mechanism of compounds 5a-s.
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 IC 50 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 IC 50 value of 2.06 µM being nearly four times more potent than sunitinib (average IC 50 value of 8.11 µM). The third group 5k-o seems to be the best group with an average IC 50 range of 1.17-2.79 µM in which compound 5m, bearing 5-chloroisatin fragment, is the most active congener with an average IC 50 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 IC 50 range of 1.46-3.92 µM. Compound 5s, bearing N-phenylisatin scaffold displayed good in vitro antiproliferative activity with an average IC 50 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 (IC 50 value = 1.38 µM; Table 3). 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 cyclindependent kinases. Compound 5m had an IC 50 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.

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 prosurvival 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 nontumorigenic 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).

Multidrug-Resistant Lung Cancer Cell Line
The antiproliferative activity of compound 5m was examined against a sensitive NSCLC cell line (A-549) and a multidrugresistant 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 IC 50 value of 1.33 µM in A-549 cells ( Figure 5 and Table 5).  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  (<140 Å 2 ), 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 IC 50 value of 1.17 µM, this compound had a seven-fold greater potency than sunitinib (IC 50 = 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.  Abbreviation: IC 50 , inhibitory concentration 50%.