Synthesis, Characterization and Anti-Cancer Activity of Hydrazide Derivatives Incorporating a Quinoline Moiety

Identification of the novel (E)-N′-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propanehydrazide scaffold 18 has led to the development of a new series of biologically active hydrazide compounds. The parent compound 18 and new quinoline derivatives 19–26 were prepared from the corresponding quinoline hydrazones and substituted carboxylic acids using EDC-mediated peptide coupling reactions. Further modification of the parent compound 18 was achieved by replacement of the quinoline moiety with other aromatic systems. All the newly synthesized compounds were evaluated for their anti-cancer activity against the SH-SY5Y and Kelly neuroblastoma cell lines, as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Analogues 19 and 22 significantly reduced the cell viability of neuroblastoma cancer cells with micromolar potency and significant selectivity over normal cells. The quinoline hydrazide 22 also induced G1 cell cycle arrest, as well as upregulation of the p27kip1 cell cycle regulating protein.


Introduction
Cancer is a heterogeneous type of genetic diseases, resulting from the alteration of many key genetic and molecular pathways [1]. Treatment regimens are often harsh, with conventional chemotherapies often not being effective, also inducing side-effects and encountering drug-resistance [2]. An important class of chemotherapeutics that effectively induce cell death, but can become ineffective due to drug-resistance, are the histone deacetylase inhibitors (HDACi) [3]. These molecules exert efficacy by regulating transcription of genes that suppress cell proliferation and angiogenesis, induce cell differentiation and promote apoptosis [4,5]. In particular, the HDACi suberoylanilide hydroxamic acid (SAHA, Figure 1) has shown potent cytotoxic effects towards a number of tumour types with low toxicity towards normal cells [6,7]. As SAHA targets only a few In order to provide a basis for the discovery of novel SAHA enhancers, a subset (10,560 compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify molecules that can act synergistically with a clinical dose of SAHA to overcome resistance in SAHA-resistant MDA-MB-231 breast cancer cells, as previously reported [11]. From this screening, we identified the quinoline-hydrazide scaffold (E)-N′-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anti-cancer lead compound, which reduced the MDA-MB-231 cell viability by <30% in the absence of SAHA, >60% in the presence of SAHA and also displayed a differential of >55% between these conditions. Quinoline is a pharmacologically valuable scaffold that is prevalent in a variety of biologically active synthetic and natural compounds [12]. Throughout the 20th century, the chemistry of quinolines has been the subject of intense study and different interesting bioactivity such as antibacterial, antifungal, anti-inflammatory, antimalarial and anticancer activities [13][14][15][16][17][18]. This anticancer activity is quite broad, with quinoline derivatives having been used against many cancers, such as those of the breast, prostate, gastrointestinal tract, colon and liver [19][20][21][22]. Importantly, a number of quinoline-based anticancer drugs have also been used clinically, including camptothecin and its analogues (irinotecan and topotecan) [23,24] and bosutinib ( Figure 3) [25,26]. The hydrazide-hydrazone moiety (-(C=O)NHN=CH) has been identified as an important fragment for various biological activities including antibacterial, antifungal, analgesic, antiinflammatory, antidepressant and anticancer activities [27][28][29]. When used in conjunction with a quinoline In order to provide a basis for the discovery of novel SAHA enhancers, a subset (10,560 compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify molecules that can act synergistically with a clinical dose of SAHA to overcome resistance in SAHA-resistant MDA-MB-231 breast cancer cells, as previously reported [11]. From this screening, we identified the quinoline-hydrazide scaffold (E)-N 1 -((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anti-cancer lead compound, which reduced the MDA-MB-231 cell viability by <30% in the absence of SAHA, >60% in the presence of SAHA and also displayed a differential of >55% between these conditions. types with low toxicity towards normal cells [6,7]. As SAHA targets only a few signalling pathways, single agent treatment with SAHA has been found to be ineffective against several cancers [8,9]. Cytotoxic drugs are found to be most effective when given in combination to achieve additive or synergistic effects. The combination of SAHA with other chemotherapeutic agents that possess different mechanisms of action has been considered to be a more promising approach to reduce drug resistance and minimize overlapping toxicities [8,10]. In order to provide a basis for the discovery of novel SAHA enhancers, a subset (10,560 compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify molecules that can act synergistically with a clinical dose of SAHA to overcome resistance in SAHA-resistant MDA-MB-231 breast cancer cells, as previously reported [11]. From this screening, we identified the quinoline-hydrazide scaffold (E)-N′-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anti-cancer lead compound, which reduced the MDA-MB-231 cell viability by <30% in the absence of SAHA, >60% in the presence of SAHA and also displayed a differential of >55% between these conditions. Quinoline is a pharmacologically valuable scaffold that is prevalent in a variety of biologically active synthetic and natural compounds [12]. Throughout the 20th century, the chemistry of quinolines has been the subject of intense study and different interesting bioactivity such as antibacterial, antifungal, anti-inflammatory, antimalarial and anticancer activities [13][14][15][16][17][18]. This anticancer activity is quite broad, with quinoline derivatives having been used against many cancers, such as those of the breast, prostate, gastrointestinal tract, colon and liver [19][20][21][22]. Importantly, a number of quinoline-based anticancer drugs have also been used clinically, including camptothecin and its analogues (irinotecan and topotecan) [23,24] and bosutinib ( Figure 3) [25,26]. The hydrazide-hydrazone moiety (-(C=O)NHN=CH) has been identified as an important fragment for various biological activities including antibacterial, antifungal, analgesic, antiinflammatory, antidepressant and anticancer activities [27][28][29]. When used in conjunction with a quinoline Quinoline is a pharmacologically valuable scaffold that is prevalent in a variety of biologically active synthetic and natural compounds [12]. Throughout the 20th century, the chemistry of quinolines has been the subject of intense study and different interesting bioactivity such as antibacterial, antifungal, anti-inflammatory, antimalarial and anticancer activities [13][14][15][16][17][18]. This anticancer activity is quite broad, with quinoline derivatives having been used against many cancers, such as those of the breast, prostate, gastrointestinal tract, colon and liver [19][20][21][22]. Importantly, a number of quinoline-based anticancer drugs have also been used clinically, including camptothecin and its analogues (irinotecan and topotecan) [23,24] and bosutinib ( Figure 3) [25,26]. types with low toxicity towards normal cells [6,7]. As SAHA targets only a few signalling pathways, single agent treatment with SAHA has been found to be ineffective against several cancers [8,9]. Cytotoxic drugs are found to be most effective when given in combination to achieve additive or synergistic effects. The combination of SAHA with other chemotherapeutic agents that possess different mechanisms of action has been considered to be a more promising approach to reduce drug resistance and minimize overlapping toxicities [8,10]. In order to provide a basis for the discovery of novel SAHA enhancers, a subset (10,560 compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify molecules that can act synergistically with a clinical dose of SAHA to overcome resistance in SAHA-resistant MDA-MB-231 breast cancer cells, as previously reported [11]. From this screening, we identified the quinoline-hydrazide scaffold (E)-N′-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anti-cancer lead compound, which reduced the MDA-MB-231 cell viability by <30% in the absence of SAHA, >60% in the presence of SAHA and also displayed a differential of >55% between these conditions. Quinoline is a pharmacologically valuable scaffold that is prevalent in a variety of biologically active synthetic and natural compounds [12]. Throughout the 20th century, the chemistry of quinolines has been the subject of intense study and different interesting bioactivity such as antibacterial, antifungal, anti-inflammatory, antimalarial and anticancer activities [13][14][15][16][17][18]. This anticancer activity is quite broad, with quinoline derivatives having been used against many cancers, such as those of the breast, prostate, gastrointestinal tract, colon and liver [19][20][21][22]. Importantly, a number of quinoline-based anticancer drugs have also been used clinically, including camptothecin and its analogues (irinotecan and topotecan) [23,24] and bosutinib ( Figure 3) [25,26]. The hydrazide-hydrazone moiety (-(C=O)NHN=CH) has been identified as an important fragment for various biological activities including antibacterial, antifungal, analgesic, antiinflammatory, antidepressant and anticancer activities [27][28][29]. When used in conjunction with a quinoline The hydrazide-hydrazone moiety (-(C=O)NHN=CH) has been identified as an important fragment for various biological activities including antibacterial, antifungal, analgesic, antiinflammatory, antidepressant and anticancer activities [27][28][29]. When used in conjunction with a quinoline system, this combination has afforded compounds with antimicrobial [30], antimycobacterial [31], anti-tubercular [32,33], anticonvulsant [34] and cytotoxic activity [35]. These biological activities are related to the many interesting and important properties of the hydrazidehydrazone moiety, such as their relatively higher metabolic stability towards proteases than amides and their tuneable, labile nature in acidic pH. The anticancer activity of quinoline-based compounds is exerted through many mechanisms, such as apoptosis [36], inhibition of angiogenesis [37], receptor inhibition and DNA intercalation [38][39][40][41]. Many compounds also exert their activity through cell cycle arrest, particularly at the G 2 /M phase [42][43][44][45] or the S phase [46][47][48]. However, a few examples have been reported where cell cycle arrest occurs at the G 0 /G 1 phase [49,50]. p27 kip1 is involved in the regulation of G 0 to S phase transitions by regulating the activity of cyclin-dependent kinases [51,52]. While p27 is rarely mutated or deleted in human cancers, it is frequently deregulated through reduced expression or mislocalization to the cytoplasm [53]. Currently, p27 expression levels in cancer serve prognostic purposes [54], as they can correlate to treatment response in either a positive or inverse fashion depending upon the disease model [55][56][57][58][59][60]. However, therapeutics targeting p27 have also been investigated in numerous cancers to enforce apoptotic consequences [61,62]. In this paper, we report the quinoline-hydrazide (E)-N 1 -((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anticancer lead compound. We further report the synthesis, characterization and in vitro biological evaluation of a series of quinoline-based and non-quinoline-based analogues, including their effects on cell cycle distribution and the expression of the cell cycle-related p27 kip1 protein.

Synthesis of Quinoline-Based Hydrazide-Hydrazones
In order to generate a diverse array of analogues, a convergent synthesis was envisaged, whereby the targeted hydrazide-hydrazone structure could be formed by an amide-coupling reaction between an alkyl carboxylic acid and aryl hydrazone (Scheme 1). system, this combination has afforded compounds with antimicrobial [30], antimycobacterial [31], anti-tubercular [32,33], anticonvulsant [34] and cytotoxic activity [35]. These biological activities are related to the many interesting and important properties of the hydrazidehydrazone moiety, such as their relatively higher metabolic stability towards proteases than amides and their tuneable, labile nature in acidic pH. The anticancer activity of quinoline-based compounds is exerted through many mechanisms, such as apoptosis [36], inhibition of angiogenesis [37], receptor inhibition and DNA intercalation [38][39][40][41]. Many compounds also exert their activity through cell cycle arrest, particularly at the G2/M phase [42][43][44][45] or the S phase [46][47][48]. However, a few examples have been reported where cell cycle arrest occurs at the G0/G1 phase [49,50]. p27 kip1 is involved in the regulation of G0 to S phase transitions by regulating the activity of cyclin-dependent kinases [51,52]. While p27 is rarely mutated or deleted in human cancers, it is frequently deregulated through reduced expression or mislocalization to the cytoplasm [53]. Currently, p27 expression levels in cancer serve prognostic purposes [54], as they can correlate to treatment response in either a positive or inverse fashion depending upon the disease model [55][56][57][58][59][60]. However, therapeutics targeting p27 have also been investigated in numerous cancers to enforce apoptotic consequences [61,62]. In this paper, we report the quinoline-hydrazide (E)-N′-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2) as a novel anticancer lead compound. We further report the synthesis, characterization and in vitro biological evaluation of a series of quinoline-based and non-quinoline -based analogues, including their effects on cell cycle distribution and the expression of the cell cycle-related p27 kip1 protein.

Synthesis of Non-Quinoline Hydrazide-Hydrazones
In order to elaborate on the structure-activity relationship and identify other heterocyclic motifs with potential cytotoxic activity, the quinoline motif was replaced with naphthalene, indole and benzaldehyde. Naphthalene was selected in order to investigate the necessity of the quinoline nitrogen, which might participate in target interaction. Indole was selected not only for its known biological activities, but also to determine if the ring size is essential for activity. Benzene was selected in order to gain insight into the necessity for a system containing a pendant ring. The aldehydes 20-22 were subjected to the same synthetic protocol described above, firstly being converted to the corresponding hydrazones 23-25, followed by EDC-coupling to acid 11 to give the targeted hydrazide-hydrazones 26-28 (Scheme 2). The alkyl acids 1-4 were prepared following literature methods [63]. Similarly, the substituted 2-chloro-quinoline-3-carbaldehydes 5-7 were isolated in yields of 47%-60% following Vilsmeier cyclisation of the corresponding acetanilides 8-10, according to literature methods (Scheme 1) [64,65]. Following aqueous work-up, carbaldehydes 5-7 were then treated with neat hydrazine hydrate at room temperature in ethanol to generate the quinoline hydrazones 11-13 in 74%-87% yields (Scheme 1). Finally, the desired hydrazide-hydrazones 14-19 were obtained by EDC-coupling of the hydrazones (11)(12)(13) to the appropriate carboxylic acids 1-4 using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), which gave hydrazide-hydrazones 14-19 in yields of 62%-68% (Scheme 1).

Synthesis of Non-Quinoline Hydrazide-Hydrazones
In order to elaborate on the structure-activity relationship and identify other heterocyclic motifs with potential cytotoxic activity, the quinoline motif was replaced with naphthalene, indole and benzaldehyde. Naphthalene was selected in order to investigate the necessity of the quinoline nitrogen, which might participate in target interaction. Indole was selected not only for its known biological activities, but also to determine if the ring size is essential for activity. Benzene was selected in order to gain insight into the necessity for a system containing a pendant ring. The aldehydes 20-22 were subjected to the same synthetic protocol described above, firstly being converted to the corresponding hydrazones 23-25, followed by EDC-coupling to acid 11 to give the targeted hydrazide-hydrazones 26-28 (Scheme 2). The new hydrazide-hydrazones 14-19, 26-28 were characterized by physical and spectral data (IR, 1 H-NMR, 13 C-NMR and EIMS). The IR spectra of the derivatives showed the azomethine C-H stretching doublet absorption band at 2850-2950 cm -1 , the C=N absorption band at 1550-1600 cm -1 , a carbonyl stretch at 1620-1680 cm -1 and a strong N-H stretch at 3180-3220 cm -1 . In the 1 H-NMR spectra of the hydrazides, the azomethine proton appeared at 8.22-8.90 ppm as a sharp singlet, whereas the corresponding amide N-H proton appeared as a broad singlet at 9.18-9.37 ppm. The protons of the heterocyclic systems and carboxylic acid nucleus showed resonances at the expected chemical shift values.
As stereochemistry is an essential factor for biological activity, single crystals of 28 were examined by X-ray diffraction in order to determine the relative stereochemistry of the C=N bond ( Figure 4). The X-ray diffraction study clearly indicated an E conformation for the C=N bond, as well as showing the alkyl chain in a bent conformation. Theoretically, this orientation might impact on the potential binding of the hydrazide-hydrazone motif to transition metals, which is a mechanism seen in multiple quinoline-hydrazone compounds [66][67][68][69]. Furthermore, it could also affect any selectivity between larger binding pockets, as opposed to shallower grooves.
The new hydrazide-hydrazones 14-19, 26-28 were characterized by physical and spectral data (IR, 1 H-NMR, 13 C-NMR and EIMS). The IR spectra of the derivatives showed the azomethine C-H stretching doublet absorption band at 2850-2950 cm -1 , the C=N absorption band at 1550-1600 cm -1 , a carbonyl stretch at 1620-1680 cm -1 and a strong N-H stretch at 3180-3220 cm -1 . In the 1 H-NMR spectra of the hydrazides, the azomethine proton appeared at 8.22-8.90 ppm as a sharp singlet, whereas the corresponding amide N-H proton appeared as a broad singlet at 9.18-9.37 ppm. The protons of the heterocyclic systems and carboxylic acid nucleus showed resonances at the expected chemical shift values.
As stereochemistry is an essential factor for biological activity, single crystals of 28 were examined by X-ray diffraction in order to determine the relative stereochemistry of the C=N bond ( Figure 4). The X-ray diffraction study clearly indicated an E conformation for the C=N bond, as well as showing the alkyl chain in a bent conformation. Theoretically, this orientation might impact on the potential binding of the hydrazide-hydrazone motif to transition metals, which is a mechanism seen in multiple quinoline-hydrazone compounds [66][67][68][69]. Furthermore, it could also affect any selectivity between larger binding pockets, as opposed to shallower grooves.

Cell Viability Assays
The newly synthesized compounds were evaluated for their ability to inhibit the growth of SH-SY5Y and Kelly neuroblastoma, and MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines using the Alamar blue (Resazurin) assay [70]. Briefly, the cells were allowed to attach for 24 h in a 96-well culture plate before being exposed to the ligands at a concentration of 10 µM in DMSO for 72 h, either in the presence or absence of SAHA. Comparative values for cell viability in each well were determined by a Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments. The anticancer activity of the compounds was evaluated by comparison to a negative (DMSO) control (Table 1 and Figure 5).

Cell Viability Assays
The newly synthesized compounds were evaluated for their ability to inhibit the growth of SH-SY5Y and Kelly neuroblastoma, and MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines using the Alamar blue (Resazurin) assay [70]. Briefly, the cells were allowed to attach for 24 h in a 96-well culture plate before being exposed to the ligands at a concentration of 10 µM in DMSO for 72 h, either in the presence or absence of SAHA. Comparative values for cell viability in each well were determined by a Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments. The anticancer activity of the compounds was evaluated by comparison to a negative (DMSO) control (Table 1 and Figure 5).

Cell Viability Assays
The newly synthesized compounds were evaluated for their ability to inhibit the growth of SH-SY5Y and Kelly neuroblastoma, and MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines using the Alamar blue (Resazurin) assay [70]. Briefly, the cells were allowed to attach for 24 h in a 96-well culture plate before being exposed to the ligands at a concentration of 10 µM in DMSO for 72 h, either in the presence or absence of SAHA. Comparative values for cell viability in each well were determined by a Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments. The anticancer activity of the compounds was evaluated by comparison to a negative (DMSO) control (Table 1 and Figure 5).   In general, it was found that these compounds displayed similar levels of activity whether in the presence or absence of SAHA. It was also observed that the hydrazide-hydrazone compounds had a greater cytotoxic effect on the neuroblastoma cells (SH-SY5Y and Kelly) compared to the breast cancer cell lines (MCF-7 and MDA-MB-231). MCF-7 cells were found to be the most resistant towards treatment with the hydrazide compounds, with reductions in cell viability ranging from 5%-33%. The compounds were generally more effective in the MDA-MB-231 breast adenocarcinoma cell line, with cell viability values being up to 20% lower than those in the MCF-7 cell line. Similarly, these compounds were also observed to have a greater effect on the cell viability of the Kelly neuroblastoma cell line than the SH-SY5Y cell line, again with up to 20% lower cell viability.
Notably, compound 17, bearing an additional methoxy group, was found to be the most active agent against both breast adenocarcinoma cell lines, with cell reductions of 33% and 38% in MCF-7 and MDA-MB-231 cells, respectively. Further, this compound was also the most active agent against the neuroblastoma cell lines, with reductions in cell viability of 82% and 96% in the SH-SY5Y and Kelly cell lines, respectively. The importance of the methoxy groups was further demonstrated by the observation that compound 14, which bears no methoxy substituents, had lower activity than the lead compound 15. While compound 14 showed no activity towards breast adenocarcinoma cell lines, the highest activity was observed with the 20% reduction in cell viability of Kelly cells.
It was also found that replacing the sulfane linkage with an ether linkage increased the potency of the synthesized compounds. The ether 16, which was found to be the second most active compound of those screened, displayed an enhanced potency of 42% and 40% against the SH-SY5Y and Kelly cell lines, respectively, compared to the corresponding sulfane lead compound 15. However, only 15% and 6% of additional reductions were observed for the MDA-MB-231 and MCF-7 cell lines respectively. The ether 19 showed 7% and 25% greater reductions in cell viability towards the SH-SY5Y and Kelly cell lines, respectively, than was observed for the corresponding sulfane 18. Similarly, 7% and 22% of additional reductions were observed for the MCF-7 and MDA-MB-231 cell lines respectively.
Hydrazides with shorter chains were also found to be more potent than their longer chain counterparts. With respect to compounds 15 (n = 1) and 18 (n = 4), bearing sulfane linkages, it was observed that compound 15 reduced viability of Kelly cells by up to 37% more than compound 18. In the case of SH-SY5Y cell, 18% of additional reduction was observed. Similarly, the shorter chain analogue 16 reduced cell viability by up to 53% more than its longer chain analogue 19 for both Kelly and SH-SY5Y neuroblastoma cell lines.
Additionally, the replacement of the quinoline system with other aromatic groups generally led to a reduction in activity. Replacement of the quinoline (compound 15) with naphthalene (compound 26) resulted in similar levels of cytotoxicity across all cell lines and exhibited up to a 54% of reduction in the case of Kelly cells. MCF-7 was found to be the most resistant cell with only 7% reduction. However, the indole (compound 27) and phenyl (compound 28) derivatives showed significant decrease in activity compared to 15, with 28 typically having lower activity than 27. This suggests that the size of this heterocycle is important for its cytotoxic activity, with bicyclic systems more favourable than the tested monocyclic counterpart.
Dose response experiments were also conducted for the most potent compounds 16 and 17. These compounds were tested against the neuroblastoma and breast adenocarcinoma cell lines at concentrations ranging from 0.1-25.0 µM, with their IC 50 values determined ( Table 2)

Toxicity Study on Normal Human Cells
Compounds 16 and 17 were then further tested against the MRC-5 and WI-38 normal human lung fibroblast cell lines (Figure 6). At a concentration of 10 µM, 16 reduced MRC-5 and WI-38 cell viability by only 10% and 5% respectively, while 17 reduced cell viability by about 30% in the two normal lung fibroblast cell lines. Notably, these two compounds were significantly less toxic towards the normal lung fibroblast cells compared to neuroblastoma cells. Taken together, the data suggests that these novel quinoline hydrazides warrant further investigation as potential anti-cancer agents, particularly for the treatment of neuroblastoma. that the size of this heterocycle is important for its cytotoxic activity, with bicyclic systems more favourable than the tested monocyclic counterpart. Dose response experiments were also conducted for the most potent compounds 16 and 17. These compounds were tested against the neuroblastoma and breast adenocarcinoma cell lines at concentrations ranging from 0.1-25.0 µM, with their IC50 values determined ( Table 2)

Toxicity Study on Normal Human Cells
Compounds 16 and 17 were then further tested against the MRC-5 and WI-38 normal human lung fibroblast cell lines (Figure 6). At a concentration of 10 µM, 16 reduced MRC-5 and WI-38 cell viability by only 10% and 5% respectively, while 17 reduced cell viability by about 30% in the two normal lung fibroblast cell lines. Notably, these two compounds were significantly less toxic towards the normal lung fibroblast cells compared to neuroblastoma cells. Taken together, the data suggests that these novel quinoline hydrazides warrant further investigation as potential anti-cancer agents, particularly for the treatment of neuroblastoma.

Effects on Cell Cycle and the Expression of Related Proteins
In order to determine if the synthesized compounds 16 and 17 play an active role in cell cycle progression, we analysed their effect on the cell cycle progression of SH-SY5Y cells after 72 h treatment. It was found that cells treated with 10 µM of compound 16 showed no significant difference in the proportions of cells across the cell cycle, compared to the DMSO control ( Figure 7). Interestingly, cells treated with 10 µM of compound 17 showed an increased proportion of cells in G0/G1, with 69% of cells in G0/G1, compared with the solvent control treated cells with 61% of cells in G0/G1 (Figure 7). Furthermore, treatment with compound 17 also reduced the number of cells in the S phase and G2/M phase.

Effects on Cell Cycle and the Expression of Related Proteins
In order to determine if the synthesized compounds 16 and 17 play an active role in cell cycle progression, we analysed their effect on the cell cycle progression of SH-SY5Y cells after 72 h treatment. It was found that cells treated with 10 µM of compound 16 showed no significant difference in the proportions of cells across the cell cycle, compared to the DMSO control ( Figure 7). Interestingly, cells treated with 10 µM of compound 17 showed an increased proportion of cells in G 0 /G 1 , with 69% of cells in G 0 /G 1 , compared with the solvent control treated cells with 61% of cells in G 0 /G 1 ( Figure 7). Furthermore, treatment with compound 17 also reduced the number of cells in the S phase and G 2 /M phase. On the other hand, p27 kip1 was highly up-regulated in SH-SY5Y cells treated with 10 µM of compound 17, in comparison with the solvent control ( Figure 8). However, two other cell cycle regulatory proteins, E2F1 and pRb, were unchanged by treatment with compound 17. Since p27 kip1 is a protein that prevents cell progression from the G1 to S phase, the results suggest that compound 17 induces cell cycle arrest by up-regulating p27 kip1 .

General Information
Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich (Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for all compounds as general methods and appropriate references have been given for known compounds. 1 H (300 MHz) and 13 C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX 300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected. Infrared spectra were recorded on a Avatar Series FTIR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated On the other hand, p27 kip1 was highly up-regulated in SH-SY5Y cells treated with 10 µM of compound 17, in comparison with the solvent control ( Figure 8). However, two other cell cycle regulatory proteins, E2F1 and pRb, were unchanged by treatment with compound 17. Since p27 kip1 is a protein that prevents cell progression from the G1 to S phase, the results suggest that compound 17 induces cell cycle arrest by up-regulating p27 kip1 .

General Information
Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich (Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for all compounds as general methods and appropriate references have been given for known compounds. 1 H (300 MHz) and 13 C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX 300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected. Infrared spectra were recorded on a Avatar Series FTIR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated On the other hand, p27 kip1 was highly up-regulated in SH-SY5Y cells treated with 10 µM of compound 17, in comparison with the solvent control ( Figure 8). However, two other cell cycle regulatory proteins, E2F1 and pRb, were unchanged by treatment with compound 17. Since p27 kip1 is a protein that prevents cell progression from the G 1 to S phase, the results suggest that compound 17 induces cell cycle arrest by up-regulating p27 kip1 .

General Information
Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich (Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for all compounds as general methods and appropriate references have been given for known compounds. 1 H (300 MHz) and 13 C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX 300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected. Infrared spectra were recorded on a Avatar Series FTIR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε) in cm´1 M´1. High-resolution [ESI] mass spectra were recorded by the UNSW Bioanalytical Mass Spectrometry Facility, on an Orbitrap LTQ XL (Thermo Scientific, Waltham, MA, USA) ion trap mass spectrometer using a nanospray (nano-electrospray) ionization source.
3.1.1. General Procedure for the Synthesis of Substituted Carboxylic Acids 1-4 To a solution of thiophenol or phenol (10 mmol) in aqueous 10% KOH (25 mL) and EtOH (30 mL) was added 3-bromopropionic acid or 6-bromohexanoic acid (11 mmol) in a saturated solution of K 2 CO 3 (10 mL, 5.5 mmol). The resulting mixture was heated at reflux for 5 h. The reaction mixture was neutralized with 18% HCl solution and the resulting precipitate was collected via filtration, washed with water and crystallized from petroleum ether to give the product as a white solid.

General Procedure for the Synthesis of Acetylated Anilines 8-10
A mixture of the appropriate aniline (50 mmol) and dimethoxyaminopyridine (DMAP, 50 mmol) in dichloromethane (100 mL) was stirred for 10 min at room temperature. Acetic anhydride (50 mmol) was added dropwise and the resulting mixture was stirred for a further 4 h at room temperature. Water (25 mL) was added and the resulting precipitate was thoroughly washed with ethanol followed by water to remove unreacted acetic anhydride and acetic acid. The desired product was dried at 50˝C under vacuum to yield the acetyl derivatives as a white solid. N,N-Dimethylformamide (0.025 mol) was cooled in an ice-salt bath and treated with phosphoryl chloride (0.070 mol) for 20 min. The resulting mixture was added dropwise to a cooled solution of the appropriate acetylated aniline (8-10) (0.010 mol) in N,N-dimethylformamide (10 mL) with stirring. The mixture was stirred at 90˝C for 12 h. A small amount of crushed ice was added and the mixture was basified to pH 14 with 5 M NaOH. After stirring at ambient temperature for 1 h, the precipitate was filtered, washed with water, and dried to give the title compound.

General Procedure for the Preparation of Hydrazones 11-13, 23-25
A mixture of the appropriate aldehyde 5-7, 20-22 (5 mmol) and excess hydrazine hydrate was stirred overnight in ethanol. After completion of the reaction, the solvent was evaporated. Water was added to the residue and the resulting mixture was extracted with ethyl acetate. The organic extract was dried with anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give the title compound.   The appropriate hydrazone 11-13, 23-25 (4 mmol) and substituted carboxylic acid 1-4 (4 mmol) and EDC (4.4 mmol) were dissolved in dry N-N-dimethylformamide. Triethylamine (4.4 mmol) was added and the solution was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate (300 mL) and washed with 0.5 M hydrochloric acid solution (100 mL) and saturated NaHCO 3 solution (100 mL). The organic phase was dried over MgSO 4 , filtered and concentrated under reduced pressure to give the title compound.

Cell Biology Techniques
The SH-SY5Y and Kelly human neuroblastoma cell lines were generously donated by Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). The MDA-MB-231 and MCF-7 breast cancer cell lines were purchased from the American Type Culture Collection. All cell lines were cultured under standard conditions at 37˝C in 5% CO 2 as an adherent monolayer in Dulbecco's modified Eagle's medium supplemented with L-glutamine (DMEM) (Invitrogen, Waltham, MA, USA) and 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA).

Method for Cell Viability Assays
Cell viability was measured by the standard Alamar blue assay, as previously described [34]. Briefly, cells were allowed to attach for 24 h in 96-well culture plates. The cells were then continuously exposed to serial dilutions of the hydrazide-hydrazone derivatives for 72 h, either in the presence or absence of SAHA (0.5 or 1 µM), with five replicate wells for each determination. Cell viability was determined by the addition of 22 µL of Alamar blue reagent, recorded at comparative 0 h and 5 h values, using a Wallac 1420 Victor III spectrophotometer (GMI, Ramsey, MN, USA), which measured light absorbance in each well at 570 nm. The cell viability of each plate was calculated as a percentage compared to matched DMSO controls (0.5%). The mean (+/´SEM) is shown for three independent experiments.

Cell Cycle Study by Propidium Iodide Staining
The cell cycle phase distribution of a cell population was determined by measuring cell DNA content using flow cytometry with propidium iodide (PI) (Sigma Aldrich) staining. The cells were trypsinized and resuspended at a concentration of 1ˆ10 6 in a complete medium, then pelleted by cold centrifugation at 1200 r.p.m., rinsed with PBS and recollected as a cell pellet. The cells were then resuspended in 0.1 mL cold PBS and were fixed in 80% cold ethanol by adding 1 mL on top of the cell suspension and incubated at 4˝C for 30 min. Prior to flow cytometric analysis, the cells were pelleted and the entire supernatant carefully discarded. The samples were treated with RNase A (Sigma, Sydney, NSW, Australia) for 30 min at room temperature and then stained with propidium iodide (50 µg/mL, final concentration) for 45 min at room temperature. The samples were analysed using a BD Caliber Flow cytometer (BD, San Jose, CA, USA). The cell cycle analysis was performed in FlowJo. Cell populations were divided into three subsets of cells that represented the sub G 0 /G 1 , G 0 /G 1 phase, S phase and G 2 /M (as determined from comparison with untreated cell control gating).

Statistical Analysis
Results of the cell viability studies were statistically analyzed using the two-tailed, unpaired Student's t-test. Results are expressed as mean values with 95% confidence intervals.

Conclusions
The quinoline-hydrazide-hydrazone motif was identified in a library screen as a promising lead scaffold for anti-cancer activity against neuroblastoma cells. This work describes the synthesis of a series of related quinoline hydrazide-hydrazones as well as three derivatives containing different aromatic systems, via an EDC-mediated coupling. The methodology involved the simple reaction of hydrazones with substituted carboxylic acids under EDC-mediated conditions. Preliminary SAR investigations revealed that the presence of a bicyclic system comprised of 6-membered rings is favoured for activity. Furthermore, the presence of methoxy groups was found to increase activity, as was exchange of the use of an ether linkage, rather than a sulfane. The two most potent analogues 16 and 17 showed low micromolar potency against neuroblastoma cells. Furthermore, these two compounds displayed significant selectivity for neuroblastoma cancer cells over normal human lung fibroblast cells. It was also found that compound 17 induced upregulation of the cell cycle mediator p27 kip1 , with a corresponding cell cycle arrest at G 1 in SH-SY5Y neuroblastoma cells.