Novel N-Substituted Amino Acid Hydrazone-Isatin Derivatives: Synthesis, Antioxidant Activity, and Anticancer Activity in 2D and 3D Models In Vitro

A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide and (N′,N‴)-1,1′-(methylenebis(4,1-phenylene))bis(5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid.


Introduction
Cancer is a malignant disease characterized by rapid and uncontrolled cell proliferation [1]. The early cancer treatment strategies were based on non-specific chemotherapeutic agents that block DNA synthesis in replicating cells [2], act as antimetabolites [3], or induce cell cycle arrest by other mechanisms [4]. Almost all existing anticancer cytostatic drugs have severe side effects due to low selectivity of the antiproliferative action and allow tumors to develop resistance to multiple chemotherapeutic drugs. The search for new effective anticancer agents with superior selectivity towards cancer cells is still of crucial importance [5]. The more recent strategies based on the targeted therapies aim at the identification and targeting of biomarkers specific for cancer cells, such as deregulated, mutated, or overexpressed proteins [6]. Protein kinases constitute important molecular targets for the development of novel anticancer agents, and a number of kinase inhibitors have been developed. Some are already in clinical use [7][8][9].
Oxindole derivatives have been widely recognized in cancer therapeutics as multikinase inhibitors. The progress towards synthetic oxindole derivatives accelerated with the FDA approval of sunitinib for the treatment of metastatic renal cell cancer [17], which acts as an inhibitor of several tyrosine kinases at nanomolar concentrations [18]. Optimization of the substituents around the oxindole nucleus led to the development of several other oxindole-based kinase inhibitors, including toceranib, the only dog-specific anti-cancer drug [19], and nintedanib, which has been approved for use in the United States to treat idiopathic pulmonary fibrosis with a progressive phenotype [20].
Kinase inhibitors semaxanib and orantinib bear an oxindole moiety with a substituent at the C3 position [21]. Semaxanib has reached phase III clinical trials in the treatment of advanced colorectal cancer [22]; and orantinib is in phase II clinical trials for the treatment of breast cancer and in phase III clinical trials for the treatment of hepatocellular carcinoma [23].
Wang et al. have reported syntheses of isatin derivatives bearing an α,β-unsaturated ketone moiety as the only substituent of an oxindole moiety at C3, and they have promising anticancer properties which are dependent on the electron-donating substituents on the benzyl ring [24].
Hydrazone derivatives, whose biological activity is associated with the presence of the active azomethine pharmacophore, constitute another significant class of biologically active compounds in medicinal and pharmaceutical chemistry [25]. These compounds, in combination with various heterocyclic scaffolds, produce diverse biological results, including antioxidant and anticancer activities [26][27][28].
A hydrazone moiety has been used as a linker in the construction of bis-isatin compounds with vast structural variety and numerous biological activities [15]. Ibrahim et al. have reported syntheses of variously substituted bis(2-oxoindolin-3-ylidene)-1H-pyrrole-2,4-dicarbohydrazide derivatives, which have shown promising anticancer activity against HepG2 (liver), MCF-7 (breast), and HCT-116 (colon) human cancer cell lines [30]. 5-Oxopyrrolidine is yet another scaffold incorporated in natural and synthetic biologically active compounds. The 2-pyrrolidinone rich fraction of Brassica oleracea var. capitatahas has been shown to exhibit antioxidant and in vitro anticancer activities [31]. Recently, the antifungal and anticancer properties exhibited by several compounds embedded with pyrrolidine-hydrazone and pyrrolidine-oxindole moieties have been reported [32,33].
As a continuation of our interest in further searching for the nitrogen-containing heterocyclic compounds possessing anticancer and antioxidant activities [34][35][36], we report herein the syntheses of a series of derivatives bearing one or two 2-oxindole-hydrazone moieties, and evaluations of their anticancer and antioxidant activities. Two human cell lines of different origins, namely, malignant melanoma (A375) and human colon adenocarcinoma (HT-29), were selected to test the proliferation inhibition and colonyforming inhibition of the synthesized compounds. Both cell lines are considered to be derived from very aggressive types of tumors, and resistance develops quite often during treatment with drugs [37,38]. In order to combat this resistance, combinations of drugs with different mechanisms of action are used. Kinase inhibitors such as dabrafenib [39] and dasatinib are often used [40]. Many more compounds are being studied at different stages of preclinical research. We decided to also explore the activity of novel compounds in tumor spheroids (3D cultures)-as they mimic the real tumor microenvironment much better than cell monolayers [41]-and identify the most promising hydrazone-isatin derivatives for further development.
In the 1 H NMR spectra of hydrazones 12-17, the proton of the secondary amine group adjacent to the phenyl ring (CH 2 NH) resonated as a singlet in the region of 5.26-5.97 ppm (Figures S1, S4, S7, S10, S13 and S16 in Supplementary Material). As expected, the amidegroup proton gave a singlet at lower field at 11.12 ppm in the 1 H NMR spectra of 12 and 16 (Figures S1 and S13 in Supplementary Material). Proton resonance at 10.78 ppm has been attributed to the secondary amine group in the 2-oxindole moiety, thereby-along with the increased intensity of the proton resonances in the aromatic region-confirming the presence of this moiety in the novel compounds. The 1 H NMR spectra of hydrazones 13-15 and 17 display double sets of resonances of the CO-NH group protons, and a 2-oxindole NH proton with signal intensity ratio 0.7:0.3 due to the restricted rotation around the amide bond ( Figures S4, S7, S10 and S16 in Supplementary Material). This splitting of the proton resonances indicates that in DMSO-d 6 , hydrazones exist as a mixture of Z/E isomers with respect to the hindered rotation around the amide bond. Usually, the Z isomer predominates [34,35,42]. In the 1 H NMR spectra, the NH protons of Z isomers resonate at a lower field with respect to the resonances attributed to E isomers [43]. In the 13 C NMR spectra for 12-17, two carbonyl group carbon resonances in the range of 162-175 ppm confirm the presence of a 2-oxindole moiety along with the amide group ( Figures S2, S5, S8, S11, S14 and S17 in Supplementary Material). When dihydrazides 7-11 were treated with isatin in the molar ratio 1:2.4, hydrazones 18-22 bearing two 2-oxindole moieties were obtained. In this case, yields of the target compounds were 36-68%. The 1 H NMR spectra for 18, 19 and 22 display the double sets of amide-group-proton and 2-oxindole-amine-proton resonances, indicating mixtures of Z/E isomers in DMSO-d 6 solutions (Figure S19, S22 and S31 in Supplementary Material).
With the aim of introducing a 2-oxindole fragment into a structure of a molecule containing the pyrrolidin-2-one moiety, reactions of 5-oxo-1-substituted phenylpyrrolidine- (30) was obtained from a corresponding hydrazide 29 in the reaction with isatin (molar ratio 1:1.2). In the 1 H NMR spectrum for 30, two distinct proton sets at 10.78-10.88 ppm and 11.14-11.24 ppm attributed to the amine group proton in 2-oxindole fragment and amide proton are visible, indicating the formation of Z/E isomers in DMSO-d6 solutions ( Figure S43 in Supplementary Material).

Scheme 2.
Syntheses of hydrazones 26-28 and 30. Azomethine group is marked in green colour and 2-oxindole moiety is represented in purple colour. (30) was obtained from a corresponding hydrazide 29 in the reaction with isatin (molar ratio 1:1.2). In the 1 H NMR spectrum for 30, two distinct proton sets at 10.78-10.88 ppm and 11.14-11.24 ppm attributed to the amine group proton in 2-oxindole fragment and amide proton are visible, indicating the formation of Z/E isomers in DMSO-d 6 solutions ( Figure S43 in Supplementary Material).

Anticancer Activity
The synthesized compounds 12-22, 26-28, 30, 32, 35 and 39 showed different levels of activity against human malignant melanoma (A375) and colon adenocarcinoma (HT-29) cell lines at 100 µM. This concentration was chosen based on our previous experience and the observations from other studies. It has been shown that 100 µM allows distinguishing the most active compounds from a group better than 10 µM [44]. A 100 µM concentration is suggested for primary screenings of anticancer agents [45]. It is also included in experiments as the highest concentration when establishing EC 50 values [46,47]. In general, our compounds showed relatively low activity against the A375 cell line used in the screening experiments ( Figure 1). Malignant melanoma is usually characterized as a cancer that is poorly responsive to many available chemotherapeutic agents due to different drug-resistance mechanisms [48]. HT-29 cells were more sensitive to the majority of the compounds tested. However, they did not have highly cytotoxic effects against this cell line. HT-29 cells are typically resistant to the majority of available drugs due to stemness [37], high expression of the MRP-1 P-glycoprotein that enhances drug efflux [49], and other mechanisms. Among mono-N-substituted N -(2-oxoindolin-3-ylidene)propanehydrazides 12-17, compound 17 bearing the 2-methyl-5-nitrobenzene moiety was identified as the most active one, whereas bis(hydrazone-isatins) 18 (bearing an unsubstituted benzene ring) and 20 (bearing the electron-donating methoxy group in benzene ring) were the most active among the compounds with two N -(2-oxoindolin-3-ylidene)propanehydrazide "arms." Another compound, which was selected for more thorough testing was diphenylmethane derivative 35 bearing two 5-oxo-N -(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide "arms" at p-positions of benzene rings. These four compounds reduced cell viability below 10%. The effective concentrations that reduced cell viability by 50% (EC 50 values) were determined ( Figure 2). The mono-hydrazone-isatin 17, bearing an electron-withdrawing nitro group at the o-position of the benzene ring, was the least active out of the four compounds selected, and it did not possess any selectivity towards cancer cell lines (EC 50 = 46.7 ± 2.0 µM against A375, and EC 50 = 44.6 ± 2.0 µM against HT-29) compared to fibroblasts (EC 50 = 50.2 ± 4.8 µM). The cytotoxic effects of bis(hydrazone-isatins) 20 and 35 were similar against both cell lines; however, compound 35 showed less cytotoxicity toward fibroblasts, and this makes it a more promising one.
As a comparison, dacarbazine, which is a chemotherapeutic drug approved for melanoma treatment, is far less active and inhibits A375 cell survival only at high concentrations of 25-100 µM [50]. However, the clinically approved BRAF inhibitor dabrafenib reduces A375 cell viability by 50% at a nanomolar concentration after 72 h of incubation [51]. 5-Fluorouracil, which is approved as a chemotherapeutic agent against colon cancer, possesses a rather low cytotoxic effect after 72 h of incubation, and usually five or more days are needed for it to reach its EC 50 value, which is >100 µM [52]. The kinase inhibitor regorafenib achieves 50% HT-29 cell viability reduction at 0.5 µM concentration after 6 h of incubation [53].
A clonogenic assay was used to evaluate each compound's impact on the ability of a single cell to form a colony. Numbers of colonies (ability of single cells to survive) and the areas of colonies (ability to form a colony and proliferate) were determined. The most active compounds, 17, 18, 20 and 35, were tested for their activity against human melanoma (A375) and colon adenocarcinoma (HT-29) cell lines at 50% of their EC 50 values.
The synthesized compounds showed higher activity against HT-29 colony formation ( Figure 3). Derivatives 17 and 18 did not affect A375 colony number or colony area, compared to the control (p > 0.05), although both compounds slowed down the colony proliferation. Compound 20 was identified as the most active compound; its inhibitory effect on the ability to proliferate and form colonies was the highest. For instance, in A375 and HT-29 cell lines, the colony areas dropped down to 78.9% and 60.6%, respectively, and reached their lowest values in comparison to the control cells ( Figure 3). The activity of diphenylmethane derivative 35 was similar to that of bis(hydrazone-isatin) 20 bearing the electron-donating methoxy group at p-position of benzene ring.
Malignant melanoma is one of the most aggressive tumor types, and A375 is characterized by high proliferation and migration rates, and high invasiveness potential [38]. This suggests the need to identify novel molecular targets and new therapeutic strategies. The combination strategy is one of the possible solutions that can overcome the resistance of melanoma cells to conventional therapy. Li and Han [54] have shown that a combination of dacarbazine and all-trans retinoic acid, loaded in lipid nanoformulations, is able to reduce B16F10's colony formation ability, whereas dacarbazine alone shows a limited ability to inhibit colony formation. In several studies, the colony-forming effect of kinase inhibitors was assessed by clonogenic assay. Sinik et al. [55] determined that MERTK inhibitor UNC2025 decreased colony formation and cell density in most tested BRAF mutant and BRAF wild-type cell lines at 300-500 nM. Ross et al. [39] have determined that the antifolate methotrexate sensitizes resistant malignant melanoma cells to the kinase inhibitor dabrafenib, and their combination reduces colony formation by up to 42.5% relative to dabrafenib alone. Similarly, colon cancer is considered to be one of the most aggressive cancers worldwide, and it gains resistance to drugs quite often [37]. In order to combat the resistance, the approach of pretreating cancer cells with other compounds before adding chemotherapeutic agents is widely studied. By using clonogenic assays, it has been shown that the kinase inhibitor dasatinib could have a favorable synergistic effect with oxaliplatin, which is an approved drug for colon adenocarcinoma treatment [40]. A combination of 100 nM dasatinib and 2.5 µM oxaliplatin significantly reduced HT-29 (but not KM12-L4) colony growth after 14 days of incubation. In summary, the effects of different compounds on colony-forming ability depends a lot on the cell line used, and kinase inhibitors could sensitize cells to cytotoxic drugs. In our case, the hydrazone-isatin derivatives bear kinase inhibitor fragments and are supposed to have a similar mechanism of action. However, deeper analysis of mechanisms of action is needed. Nowadays, 3D cell culture models are becoming popular among cancer biologists due to the spatial arrangement of cells, and they enable the formation of hypoxia inside and a gradient of tested substances [41,56]. Such a model resembles a real tumor microenvironment and is more reminiscent of a real tumor than conventional cell monolayers. Thus, the effects of 20 and 50 µM solutions of bis(hydrazone-isatins) 18, 20, and 35 on melanoma A375 and colon adenocarcinoma HT-29 cell spheroid growth ( Figure 4) were studied. As shown in Figure 4C, all tested compounds reduced A375 spheroid growth by~120% at the higher concentration of 50 µM in comparison to the control group. Diphenylmethane derivative 35 showed an inhibitory effect on A375 spheroid growth at a concentration of 20 µM, too. Meanwhile, the effect on HT-29 3D culture growth was lower. Only compound 35 statistically significantly inhibited colon cancer cell spheroid growth (by~105%) in comparison with the control ( Figure 4C). While taking into account that spheroid size does not always necessarily correlate with viability [57], we decided to study cell viability in all groups at the end of the experiment ( Figure 4D). Interestingly, cell viability was from 2.5-fold to 10-fold lower compared to the control for both types of spheroids. This could be explained by cells at the cores of the spheroids being more affected by the compound, thereby becoming more hypoxic or even necrotic, denser, and less viable. Some spheroids at the end of the experiment became looser and irregular in shape, and started to disintegrate ( Figure 4A,B). However, Golas et al. [58] determined that SKI-606, an inhibitor of Src and Abl kinases at the concentration of 2.5 µM, reduced the HT-29 spheroid size after the 6 days of incubation and concluded that SKI-606 strengthens cell-cell interactions. Cell viability was not measured at the end of this experiment, but the hypothesis regarding cell-cell interaction protection against SKI-606 treatment has been formed. Another study by Folkesson et al. [59] has revealed that MEK and TAK1 inhibitors strongly reduce cell viability both in 2D and 3D cell cultures, and combined treatments with a MEK inhibitor and conventional drugs produce synergistic effects in 3D models. Meanwhile, MEK inhibitors combined with STAT3 small-molecule inhibitors can cause cell death in 3D cultures [60]. This means that kinase inhibitors could be beneficial in reducing cell viability and inhibiting the growth of cells in 2D models.
In summary, compounds 20 and 35 have been identified as the most promising anticancer agents out of the series of hydrazone-isatin derivatives. Though their cytotoxicity against both cancer cell lines was not very high (EC 50 values were in the range of 22-30 µM), they were more active than dacarbazine 5-fluorouracil and reforafenib, which are used to treat malignant melanoma and colon cancer. It is worth mentioning that the selectivity of the most promising compounds towards cancer cells (in comparison to fibroblasts) was not high. Compound 35 alone was 1.6 times more active against A375 and 1.4 times more selective against HT-29 compared to fibroblasts. However, the selectivity studies were performed in cell monolayers, and this is only the first step toward identifying the most active scaffolds for further development of more selective and more active anticancer agents. It should be worth exploring the accumulation of modified compounds in more sophisticated models, e.g., the three-dimensional models combining both fibroblasts and cancer cells, and thereby evaluating the fractions and types of surviving cells after compound treatment. Furthermore, the selectivity towards tumors is a major problem for many (even clinically-available) cytotoxic agents. Currently, many different approaches are used to improve selectivity, such as packing molecules into nanoformulations [61] and conjugating cytotoxic agents to antibodies [62].

Antioxidant Activity
Reactive oxygen species (ROS) and reactive nitrogen species (NOS) are well known as both harmful and beneficial species [63]. However, imbalances between endogenous antioxidant defense and ROS oxidative stress have been related to an extensive range of diseases, including cardiovascular, inflammatory, neurodegenerative, and autoimmune ones. Overproduction of ROS can be responsible for damage to vital cell components, especially to DNA, lipids, and proteins. It is known that the use of antioxidants is beneficial in the prevention or delaying of numerous diseases associated with oxidative stress, including cancer, Alzheimer's and other neurodegenerative diseases, and atherosclerosis [64][65][66][67]. Antioxidants are believed to prevent and treat various types of malignancies [68]. The compounds 12-22, 26-28, 30, 32, 35 and 39 were designed to contribute to radical-scavenging (to be antioxidants) [69,70].
Ferric ion (Fe 3+ ), which is a relatively biologically inactive form of iron, can be reduced to the active Fe 2+ depending on the conditions, particularly pH, and oxidized back through a Fenton-type reaction with the production of hydroxyl radicals or a Haber-Weiss reaction with the generation of superoxide anions [71,72]. Reducing power measures the reductive ability of an antioxidant, and it is evaluated by the transformation of Fe 3+ to Fe 2+ by donation of an electron in the presence of the tested compound [73].
As seen from the results presented in Figure 5, compounds 32, 35 and 39 possessed the highest capacity to reduce Fe(TPTZ) 3+ to Fe(TPTZ) 2+ in comparison with the positive control, ascorbic acid (103.41 µM). It is interesting to note that compounds 32 and 35 are bis(hydrazone-isatins), and compound 39 has just one 5-oxo-N -(2-oxoindolin-3ylidene)pyrrolidine-3-carbohydrazide "arm." The relatively high ferric reducing antioxidant power of 39 proves the expectation that substitution of diphenylmethane with a diphenylamine moiety bearing a secondary amine group [74] enhances the reducing power of compound 39. Another group of compounds which have been identified as possessing high antioxidant activity comprises bis(hydrazone-isatins) 19-21, whose structures differ by a substituent at the p-position of benzene ring. Compound 20 bearing the electron-donating methoxy group in the benzene ring has shown higher radical scavenging ability than 19 and 21. Compounds 19-21 have been found to be considerably more active than their analogues bearing just one N -(2-oxoindolin-3-ylidene)propanehydrazide "arm": 13-15. In this group, the ethoxy substituent in 15 had a more significant positive effect on the activity of the molecule. In another group of compounds, 26-28, bearing a pyrrolidin-2-one ring as a linker between the hydrazine-oxindole moiety and the p-substituted benzene ring, compound 26, containing an electron-donating methyl group, was identified as the least active one. The position of the methoxy substituent in the benzene ring had no significant influence on the antioxidant activity of 27 and 28.  3-((4-Methoxyphenyl)amino)-N -(2-oxoindolin-3-ylidene)propanehydrazide (14) Prepared as described in [35].

General Procedure for Synthesis of Compounds 18-22
To a solution of corresponding hydrazide (5 mmol) in methanol (25 mL), isatin (12 mmol) and glacial acetic acid (5 drops) were added. The reaction mixture was stirred at 65 • C for 15 min. Precipitate was filtered off, washed with methanol, and recrystallized from DMF/H 2 O mixture.

Cell Viability Assay
The effects of the isatin derivatives on cell viability were studied using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St Louis, MO, USA) assays, as described elsewhere [35,89]. Briefly, A375, HT-29, and HF cells were seeded in 96-well plates (Corning) in triplicate repeats at a volume of 100 µL (5 × 10 3 cells/well). After 24 h of incubation, the cells were treated with 100 µM of tested compounds. After 72 h, the cells were exposed to the reagent MTT for 4 h. Then the medium was aspirated and the formed formazan crystals were dissolved in 100 µL DMSO (Sigma-Aldrich Co.). The absorbance was measured at 570 and 630 nm using a multi-detection microplate reader. The compound's effect on cell viability was calculated using a formula: where A-mean of absorbance of tested compound; A 0 -mean of absorbance of blank (no cells, positive control); A NC -mean of absorbance of negative control (only cells, no treatment). The EC 50 values of the most active compounds, 17, 18, 20, and 35, were established by the same MTT procedure; only the compound serial dilutions from 50 to 1.56 µM were made in a medium and added to the cells in triplicate repeats. Each EC 50 value, which represents the concentration of a compound causing a 50% reduction of cancer cells' metabolic activity, has been calculated using the Hill equation.

Colony Formation Assay
Clonogenic assays were used to evaluate the inhibitory effects of the most active compounds, 17, 18, 20, and 35, on cell survival and proliferation via forming colonies, as described elsewhere [90]. Briefly, melanoma A375 and colon adenocarcinoma HT-29 cells were seeded into 12-well plates in triplicate repeats (2 × 10 2 cells/well) and grown in the previously described cell culture medium at 37 • C in a humidified atmosphere containing 5% CO 2 . After 24 h, the fresh media containing compounds 17, 18, 20, and 35 at concentrations representing 50% of calculated EC 50 values were added to cells. Then, cells were incubated at 37 • C in a humidified atmosphere containing 5% CO 2 for the next 7-8 days. Cells treated with medium containing 0.5% DMSO served as a negative control. After incubation, the colonies were stained with a 0.1% crystal violet (Sigma-Aldrich Co.) solution. First, the media were removed from cells, and the cells were washed with sterile PBS. Then, the cells were fixed in a 4% formaldehyde (Thermo Scientific) solution and washed with PBS two times to remove the fixative, and stained with crystal violet for 20 min. After the stain had been removed, the remaining stain residues were washed three times with sterile water. The plates were dried overnight and imaged with the SYNGENE G:BOX gel doc system, using Gen Sys software. Quantification was performed using Gene tools software.

Compound Activity in Spheroids
Cancer cell spheroids were formed by using the magnetic 3D Bioprinting method, as described elsewhere [91]. Briefly, melanoma (A375) and adenocarcinoma (HT-29) cells and human fibroblasts at 70% confluency in a 6-well plate were incubated with Nanoshuttle (n3D Biosciences, Inc.) for 7-8 h at 37 • C in a humidified atmosphere containing 5% CO 2 . After nanoparticles were taken up by cells, they were trypsinized, centrifuged, and seeded into ultra-low attachment 96-well plate in a volume of 100 µL (1.5 × 10 3 cancer cells and 1.5 × 10 3 human fibroblasts/well). The plate was placed on a magnetic drive and incubated for 2 days at 37 • C in a humidified atmosphere containing 5% CO 2 . Then the fresh medium containing 20 or 50 µM of tested compound was added to the wells. The spheroids were captured every two days using the Olympus IX73 inverted microscope (OLYMPUS CORPORATION). The quantitative analysis of compound anticancer activity in spheroids was performed using ImageJ (National Institutes of Health) and Microsoft Office Excel software).

Statistical Analysis
All biological experiments were repeated at least three times. The mean and standard deviation are reported. The data were processes using Microsoft Office Excel 2016 software (Microsoft Corporation, Redmond, WA, USA). Statistical analysis was performed by using Student's t-tests. The level of significance was set as p < 0.05.

Conclusions
In conclusion, a series of novel hydrazone-isatin derivatives were synthesized and evaluated for their anticancer and antioxidant properties. In the anticancer activity assay, the colon adenocarcinoma HT-29 cell line appeared to be more sensitive to the treatment with the hydrazone-isatin derivatives, compared to the malignant melanoma A375 cell line. Bis(hydrazone-isatins) were found to be more active than their mono analogues. Bis(N -(2-oxoindolin-3-ylidene)propanehydrazide) 20, bearing the electron-donating methoxy group at the p-position of the benzene ring, and diphenylmethane derivative 35, bearing two 5-oxo-N -(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide "arms" at p-positions of the benzene rings, were the most active among all compounds synthesized. These compounds reduced the colony-forming abilities of both cell lines, and also inhibited the growth and viability of colon cancer and melanoma spheroids.
The same bis(hydrazone-isatins) possessed the highest ferric reducing antioxidant power. These promising results suggest that variously substituted bis(hydrazone-isatins) based on an N-substituted β-amino acid scaffold could be developed further as a new class of anticancer agents against aggressive malignant melanoma and colon adenocarcinoma tumors.