Design, synthesis and evaluation of novel 2-oxoindoline-based acetohydrazides as antitumor agents

In our search for novel small molecules activating procaspase-3, we have designed and synthesized two series of novel (E)-N'-arylidene-2-(2-oxoindolin-1-yl)acetohydrazides (4) and (Z)-2-(5-substituted-2-oxoindolin-1-yl)-N'-(2-oxoindolin-3-ylidene)acetohydrazides (5). Cytotoxic evaluation revealed that the compounds showed notable cytotoxicity toward three human cancer cell lines: colon cancer SW620, prostate cancer PC-3, and lung cancer NCI-H23. Especially, six compounds, including 4f–h and 4n–p, exhibited cytotoxicity equal or superior to positive control PAC-1, the first procaspase-3 activating compound. The most potent compound 4o was three- to five-fold more cytotoxic than PAC-1 in three cancer cell lines tested. Analysis of compounds effects on cell cycle and apoptosis demonstrated that the representative compounds 4f, 4h, 4n, 4o and 4p (especially 4o) accumulated U937 cells in S phase and substantially induced late cellular apoptosis. The results show that compound 4o would serve as a template for further design and development of novel anticancer agents.

Normal cells in human body divide and die in a tightly regulated manner. Cell cycle and apoptosis are two processes linked to normal cellular growth and death. In case abnormality occurs, the cells keep dividing and are able to escape apoptosis, leading to the formation of extra mass tissue in the body, known as tumors. Malignant tumors, or cancer, remains one of the deadliest diseases nowadays since the cancer cells are able to spread throughout the body and metastasize to other organs, making the treatment extremely difficult 1 . Over decades, targeting cell cycle and apoptosis, especially apoptosis or programmed cell death process, are among the most common and effective approaches for anticancer drug development 2,3 .
With advances in molecular cell biology, many proteins involved in cellular apoptotic pathways, e.g. BIM, BAX, Bcl-2, p53, RIP, DED, Apo2L, and XIAP, to name a few, have been identified and employed as molecular targets for anticancer therapy 3 . As a result, a number of small molecules targeting these proteins have been discovered. For example, GDC-0152 (a XIAP's inhibitor), tenovin-1 (a p53 activator), or ABT-199 (an inhibitor of Bcl-2) have been demonstrated to effectively induce apoptosis and ultimately caused the death of cancer cells [4][5][6] .
Also played important roles in regulation of apoptotic pathways are caspases 7,8 . Currently, caspases, with at least fourteen members, are a large group of of cysteine proteases enzymes 7,8 . These enzymes are involved in both extrinsic and intrinsic pathways of the apoptotic machine 7,8 . Among these, caspase-3, known as the executioner caspase, is one of the key enzymes regulating apoptosis responses 7,8 . Caspase-3 exists as a low activity zymogen in cells, known as procaspase-3 7,8 , which has been found to be overexpressed in many types of human cancers 9 (e.g. neuroblastoma 10 , breast cancer 11 , lung carcinoma 12 , hepatocellular carcinoma 13 , lymphoma and Hodgkin's Disease 14 ). Due to their overexpression in cancer cells, it is well established that targeting caspases would be more advantageous over inhibiting other apoptotic proteins 9 . Great efforts of medicinal chemists have therefore placed on the development of novel caspase activators. Consequently, several small molecules as caspase activators have been reported [15][16][17][18] . In 2016, PAC-1, the first procaspase activating compound (Fig. 1), was granted as Orphan

Materials and methods
Chemistry. The reagents, solvents used in this work were purchased from commercially available vendors (mainly, Aldrich, Fluka Chemical Corp. (Milwaukee, WI, USA), or Merck) and used directly unless otherwise indicated. Thin layer chromatography (TLC) was performed in Whatman Silica Gel GF 250 . The TLC plate was visualized using 254 nm UV light. Gallenkamp (LabMerchant, UK) melting Point Apparatus was used for recording melting points of the compounds and are uncorrected. Re-crystallization in solvents or column chromatography on silica gel was used for purification of final compounds. Merck (silica gel 240 to 400 mesh) was used as stationary phase in column flash chromatography. 1 H NMR were analyzed on a 500 MHz spectrometer (Bruker). DMSO-d 6 was used as NMR solvent unless otherwise indicated. Chemical shifts are reported ppm. Mass spectra of the compounds were performed in PE Biosystems API2000 (electron ionization (EI), Perkin Elmer-USA) and Mariner (Electrospray ionization (ESI), Azco Biotech-USA) mass spectrometers, respectively. The elemental analyses (C, H, N) of the final compounds were recored on a Perkin Elmer elemental analyzer (model 2400).
Cytotoxicity assay. Three human cancer cell lines: colon cancer (SW620), prostate cancer (PC3), and lung cancer (NCI-H23) were used for sceening the cytotoxicity of the compounds. The cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA). Other reagents/media for cell culture were obtained from GIBCO (Grand Island, New York, USA). The testing cancer cells were culture in Dulbecco's Modified Eagle Medium until confluence. Then, they were trypsinized and suspended at the level of 3 × 10 4 cells/mL of cell culture medium. On day 0, cancer cells were seeded at a volume of 180 µL/well of 96-well plates and incu- and allowed to grow for 24 h. In our first experiment, we examined the effects of 4f, 4h, 4n, 4o, 4p, and PAC-1 on cell cycles at 50 µM. In the second experiment, we examined the dose-dependent effect of 4o at 5, 10, and 30 µM and PAC-1 at 30 µM on cell cycles. The cells were treated with compounds for 24 h, and then harvested. The harvested cells were washed twice with ice-cold PBS, fixed in 75% ice-cold ethanol, and stained with propidium iodide (PI) in the presence of RNase at room temperature for 30 min. The stained cells were analyzed for DNA content using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the data were processed using Cell Quest Pro software (BD Biosciences).
Apoptosis assay. The Annexin V-FITC/PI dual staining assay was used to determine the percentage of apoptotic cells. U937 cells (5 × 10 5 /mL per well) were plated in 6-well culture plates and allowed to grow for 24 h. In our first experiment, we examined the effects of 4f, 4h, 4n, 4o, 4p, and PAC-1 on apoptosis at 50 µM. In the second experiment, we examined the dose-dependent effect of 4o at 5, 10, and 30 µM and PAC-1 at 30 µM on apoptosis. The cells were treated with compounds for 24 h, and then harvested. The harvested cells were washed twice with ice-cold PBS and incubated in the dark at room temperature in 100 mL of 1 × binding buffer containing 1 µL Annexin V-FITC and 12.5 mL PI. After 15 min incubation, cells were analyzed for percentage undergoing apoptosis using a FACScalibur flow cytometer (BD Biosciences). The data were processed using Cell Quest Pro software (BD Biosciences).

(Z)-N'-(5-Methyl-2-oxoindolin-3-ylidene)-2-(2-oxoindolin-1-yl)acetohydrazide (5c
Next, we selected 5 representative compounds, including 4f, 4h, 4n, 4o and 4p, to investigate their effects on the caspase activity, cell cycle, and apoptosis. At first, we tried to use the extract of SW620, PC-3, and NCI-H23 in caspase activation assay, but these extracts did not show caspase activity. Thus, we used the extract of U937 cells in caspase-3 activation assay, referring to our previous study 31 . As reported, PAC-1 activated caspase-3 in 24-h-caspase-3 assay in U937 cells at 50 µM higher concentration than an IC 50 value of 8.47 µM in 48-h-cytotoxicity assay. However, our compounds unexpectedly were not recorded to activate caspase-3 activity. It was likely that the compounds might activate other caspases and eventually caused effects on the cell cycle and apoptosis. Next, we investigated the effect of PAC-1 and our chemicals on cell cycle and apoptosis by using U937 cells 31 . In the cell cycle analysis, U937 cells were treated with 50 µM of compounds for 24 h, stained with propidium iodide (PI) in the presence of RNase, and then analyzed for DNA content by using flow cytometry. PAC-1 was used in parallel as a positive control. The results illustrated in Fig. 3 indicate that the compounds tended to cause accumulation of cells in S phase, although PAC-1 caused the accumulation of cells in G0/G1 phase. Compound 4o inhibited cell cycles at 5 µM, which was close to IC 50 value (Fig. 4). In the Annexin V-FITC/ PI apoptotic analysis, compounds 4f, 4h, 4n, 4o and 4p also induced early, and more substantially, late apoptosis  www.nature.com/scientificreports/ (Fig. 5). The effects were more prominent with compounds 4h, 4o and 4p (Fig. 5). Compound 4o increased late apoptotic cell population at 5 µM, which was close to IC 50 value (Fig. 6). Regarding the effects of the compounds on cellular morphology, SW620 cells treated with PAC-1 and our compounds showed morphology of apoptotic cells (Figs. 7 and 8).

Conclusions
In conclusion, two series of novel (E)-N'-arylidene-2-(2-oxoindolin-1-yl)acetohydrazides (4a-p) and (Z)-2-(5substituted-2-oxoindolin-1-yl)-N'-(2-oxoindolin-3-ylidene)acetohydrazides (5a-f) were designed and synthesized. Biological results revealed that the significant cytotoxicity against three human cancer cell lines SW620, PC-3, and NCI-H23 of these compounds were obtained. Under our conditions, compounds 4f-h and 4n-p, exhibited cytotoxicity equal to superior to positive control PAC-1. In particular, compound 4o was the most potent with cytotoxicity up to three-to five-fold stronger than PAC-1 in three cancer cell lines tested. Cell cycle and apoptosis analysis showed that representative compounds 4f, 4h, 4n, 4o and 4p (especially 4o) accumulated U937 cells in the S phase and substantially induced late cell apoptosis. Collectively, the results show that compound 4o would serve as a template for further design and development of novel anticancer agents.