Discovery of Novel Quinoline-Chalcone Derivatives as Potent Anti-tumor Agents with Microtubule Polymerization Inhibitory Activity

A series of novel quinoline-chalcone derivatives were designed, synthesized and evaluated for their antiproliferative activity. Among them, compound 24d exhibited the most potent activity with IC50 values ranging from 0.009 to 0.016 μM in a panel of cancer cell lines. Compound 24d also displayed a good safety profile with LD50 value of 665.62 mg/kg by intravenous injection, and its hydrochloride salt 24d-HCl significantly inhibited tumor growth in H22 xenograft models without observable toxic effects, which was more potent than that of CA-4. Mechanism studies demonstrated that 24d bound to the colchicine site of tubulin, arrested cell cycle at the G2/M phase, induced apoptosis, depolarized mitochondria and induced reactive oxidative stress (ROS) generation in K562 cells. Moreover, 24d has potent in vitro anti-metastasis, in vitro and in vivo anti-vascular activities. Collectively, our findings suggest that 24d deserves to be further investigated as a potent and safe anti-tumor agent for cancer therapy. INTRODUCTION Microtubules provides a dynamic scaffold for maintenance of cell structure, protein trafficking, chromosomal segregation, and mitosis. They are long, hollow structures that are mainly composed of αand β-tubulin dimers. 2-4 Microtubule-targeting agents (MTAs) including microtubule stabilizers or destabilizers can interfere with microtubule dynamics, leading to mitotic blockade and cell apoptosis. In recent decades, MTAs that bind to the colchicine site have been attracting the interest of medicinal chemists because of their advantages over other site binders; these MTAs have simpler structures, improved aqueous solubility, broad therapeutic index, and reduced multidrug resistance (MDR) effects as comparing with other site binders. Notably, colchicine binding site inhibitors (CBSIs) can induce morphological changes in endothelial cells, thus provoking a rapid disrupt of existing tumor vasculature, and thus are commonly designated as vascular disrupting agents (VDAs). 14 Chalcones that bear an α, β-unsaturated ketone moiety represents a key structural motif in the plethora of biologically active molecules including synthetic and natural products. They have shown a broad range of biological activities such as antioxidant, antibacterial, antifungal, anti-HIV, anti-leishmanial, antimalarial, anti-inflammatory, and anticancer properties. In the development process of CBSIs, the α, β-unsaturated ketone moiety of chalcones was recognized as a privileged structure. Representative anti-tubulin chalcones, presented as compounds 1 and 2 (Figure 1a), showed remarkable antiproliferative activities. The more preferential s-trans conformation adopted by 2, which could interact with tubulin more easily, led to more potent cytotoxicity than 1. Another chalcone compound reported by Li’s group was compound 3, which exhibited excellent antipoliferative activity with IC50 values ranging from 3 to 9 nM against a panel of cancer cell lines. Our previous work on the modification of compound 1 also led to a novel chalcone analog 4, which displayed both potent anti-vascular and anti-tumor activities. Nitrogenous molecules, such as pyridines and quinolines, have numerous advantages over other non-nitrogenous molecules. The introduction of nitrogen atom greatly improves the basicity of molecules due to its basic characteristic, and nitrogen atom may form a strong hydrogen bond with the targets. Another important property is the polarity which can be used as a mean of reducing the lipophilic character, improving water solubility and oral absorption. The quinoline motif is frequently found in natural alkaloids that exhibit a wide range of biological activities. The quinoline ring system-containing drugs, such as quinine, chloroquine, mefloquine and amodiaquine, are used as efficient treatments of malaria. Quinoline analogs also exhibit anticancer activities with different mechanisms, including alkylating agents, tyrosine kinase inhibitors and tubulin inhibitors. Considering the poor aqueous solubility that impeded the clinical development of some CBSIs, incorporation of nitrogenous heterocycles, which could be salified with acids, may improve water solubility. Some examples of tubulin inhibitors bearing a quinoline skeleton are listed in Figure 1b, such as compounds 5, 6 and 7 11. Recently, the work performed by Alami et al. also proved that a quinoline ring as shown in compound 12 can replace the 3,4,5-trimethoxyphenyl moiety and provide compounds with more potent activities. Their docking studies predicted that the N-1 atom of quinoline formed a hydrogen bond with the critical residue Cys 241, which was further supported by the later disclosed crystal structure of tubulin in complex with 11. Thus, the quinoline moiety might be a surrogate of the 3,4,5-trimethoxyphenyl moiety when binding to the colchicine site. Our group has concentrated on discovering and developing novel anticancer agents targeting the tubulin-microtubule system. 36, 37 In our continuing works on the structural modification of the parent compound 1 around the 3,4,5-trimethoxyphenyl, which led to the discovery of a series of novel quinoline-chalcone derivatives (Figure 2). Meanwhile, given previous studies showed that the indole moiety was an alternative structure of the isovanillic ring. 39 Thus, new quinoline-chalcones that contain an indole moiety were also designed and synthesized. Herein, we would like to report their synthesis and anti-tumor activities in vitro and in vivo. In addition, the underlying cytotoxic mechanisms of the representative compound 24d are also


INTRODUCTION
Microtubules provides a dynamic scaffold for maintenance of cell structure, protein trafficking, chromosomal segregation, and mitosis. 1 They are long, hollow structures that are mainly composed of αand β-tubulin dimers. [2][3][4] Microtubule-targeting agents (MTAs) including microtubule stabilizers or destabilizers can interfere with microtubule dynamics, leading to mitotic blockade and cell apoptosis. 5 In recent decades, MTAs that bind to the colchicine site have been attracting the interest of medicinal chemists because of their advantages over other site binders; these MTAs have simpler structures, improved aqueous solubility, broad therapeutic index, and reduced multidrug resistance (MDR) effects as comparing with other site binders. [6][7][8][9][10][11][12] Notably, colchicine binding site inhibitors (CBSIs) can induce morphological changes in endothelial cells, thus provoking a rapid disrupt of existing tumor vasculature, and thus are commonly designated as vascular disrupting agents (VDAs). 13,14 Chalcones that bear an α, β-unsaturated ketone moiety represents a key structural motif in the plethora of biologically active molecules including synthetic and natural products. They have shown a broad range of biological activities such as antioxidant, antibacterial, antifungal, anti-HIV, anti-leishmanial, antimalarial, anti-inflammatory, and anticancer properties. 15 In the development process of CBSIs, the α, β-unsaturated ketone moiety of chalcones was recognized as a privileged structure. [16][17][18][19][20] Representative anti-tubulin chalcones, presented as compounds 1 and 2 (Figure 1a), showed remarkable antiproliferative activities. 21 The more preferential s-trans conformation adopted by 2, which could interact with tubulin more easily, led to more potent cytotoxicity than 1. Another chalcone compound reported by Li's group was compound 3, which exhibited excellent antipoliferative activity with IC50 values ranging from 3 to 9 nM against a panel of cancer cell lines. 22 Our previous work on the modification of compound 1 also led to a novel chalcone analog 4, which displayed both potent anti-vascular and anti-tumor activities. 23 Nitrogenous molecules, such as pyridines and quinolines, have numerous advantages over other non-nitrogenous molecules. The introduction of nitrogen atom greatly improves the basicity of molecules due to its basic characteristic, and nitrogen atom may form a strong hydrogen bond with the targets. Another important property is the polarity which can be used as a mean of reducing the lipophilic character, improving water solubility and oral absorption. 24 The quinoline motif is frequently found in natural alkaloids that exhibit a wide range of biological activities. The quinoline ring system-containing drugs, such as quinine, chloroquine, mefloquine and amodiaquine, are used as efficient treatments of malaria. 25 Quinoline analogs also exhibit anticancer activities with different mechanisms, including alkylating agents, tyrosine kinase inhibitors and tubulin inhibitors. 26 Considering the poor aqueous solubility that impeded the clinical development of some CBSIs, 27 incorporation of nitrogenous heterocycles, which could be salified with acids, may improve water solubility. Some examples of tubulin inhibitors bearing a quinoline skeleton are listed in Figure 1b, such as compounds 5 28 , 6 29 and 7 -11 [30][31][32][33][34] . Recently, the work performed by Alami et al. also proved that a quinoline ring as shown in compound 12 can replace the 3,4,5-trimethoxyphenyl moiety and provide compounds with more potent activities. 35 Their docking studies predicted that the N-1 atom of quinoline formed a hydrogen bond with the critical residue Cys 241, which was further supported by the later disclosed crystal structure of tubulin in complex with 11. 34 Thus, the quinoline moiety might be a surrogate of the 3,4,5-trimethoxyphenyl moiety when binding to the colchicine site.
Our group has concentrated on discovering and developing novel anticancer agents targeting the tubulin-microtubule system. 23,36,37 In our continuing works on the structural modification of the parent compound 1 around the 3,4,5-trimethoxyphenyl, which led to the discovery of a series of novel quinoline-chalcone derivatives ( Figure   2). Meanwhile, given previous studies showed that the indole moiety was an alternative structure of the isovanillic ring. 38,39 Thus, new quinoline-chalcones that contain an indole moiety were also designed and synthesized. Herein, we would like to report their synthesis and anti-tumor activities in vitro and in vivo. In addition, the underlying cytotoxic mechanisms of the representative compound 24d are also elucidated.

RESULTS AND DISCUSSION
Chemical Synthesis. In Alami's previous work, 35 the effects of different substitutions at the C-2 position of quinoline were investigated in detail with a methyl group being the most active. Thus, 2-methylquinoline was first chosen to replace the 3,4,5-trimethoxyphenyl moiety of compound 1. The synthetic route of key intermediates acetylquinoline 16 and propionylquinoline 21 was outlined in Scheme 1.
Disappointingly, this method was not applicable for the synthesis of propionylquinoline 21 due to the overreaction of 21 with ethylmagnesium bromide (C2H5MgBr) resulting in a great decreased yield. Thus, we attempted a tedious but effective route to synthesize 21. 2-Methylquinoline-4-carbaldehyde (19) was prepared by the oxidation of 18 according to the previous report [40] . Then, 19 underwent nucleophilic attack by CH3CH2MgBr to obtain 20, which was subsequently oxidized to produce the propionylquinoline 21.
The designed target compounds 24a-o containing a 2-methylquinoline moiety were synthesized using acetylquinoline 16 or propionylquinoline 21 with various commercially available aldehydes via aldol condensation reactions (Scheme 2).
Compound 24a and 24b bearing amino groups were synthesized using 3-nitro-4-methoxybenzaldehyde (22) in the condensation steps, after which the nitro group was reduced to amino group to afford 24a and 24b. Similarly, the MEM protective group was used to synthesize 24c and 24d bearing an isovanillic ring. The The synthetic route of target compounds 29c-n was outlined in Scheme 5.

BIOLOGY
Antiproliferative Activities and the structure activity relationships (SARs).
The in vitro antiproliferative efficacy of target compounds 24a-q with a 2-methylquinoline moiety were first assessed by MTT assays using human chronic myelogenous leukemia cell K562 and compared to the reference compound CA-4. As shown in Table 1, except for compounds 24j-k and 24n-o, which had indole moieties as ring B, all of the newly synthesized compounds exhibited decent antiproliferative activities in a nanomolar range. Among which, compounds 24b and 24d, featuring 3-amino-4-methoxyphenyl or 3-hydroxy-4-methoxyphenyl moieties, displayed the most potent activity with IC50 values of 0.011 and 0.009 μM, respectively, which were comparable to that of CA-4 (IC50 = 0.011 μM) and were approximately 6-fold more potent than the parent compound 1 (IC50 = 0.060 μM). The methyl substituent at the α-position of the unsaturated carbonyl group improved the activity (24a vs. 24b, 24c vs. 24d and 24k vs. 24l), which was similar to the results in previous reports [21,22] .
Additionally, different substituted indole derivatives 24i-q were synthesized and evaluated for their antiproliferative activity. However, most of this series displayed lower activities (IC50 > 1 μM) than the phenyl counterparts, except compounds 24l and 24m, of which the unsaturated double bonds were substituted at the C-5 position on indole moiety. Besides, the methyl substituent at the N-1 position of the indole (24m) increased the activity for approximately 5-fold when compared to the non-substituted counterpart 24l.
The effects of substitutions at the C-2 position on the quinoline moiety on activity were further investigated with both the isovanillic ring and methyl substituted α, β-unsaturated ketone retained. Thus, compounds 29a-n with different substituted quinolines were synthesized and evaluated for their antiproliferative efficacy. As shown in Table 1, all compounds 29a-n displayed decent activities except 29m, which had a lactam rather than the quinoline ring. Steric hindrance of the groups at the C-2 position on the quinoline moiety seemed to exert a critical influence on the activity, as compounds with smaller substitutions such as CH3 (24d, IC50 = 0.009 μM), NHCH3 (29g, IC50 = 0.018 μM), OCH3 (29k, IC50 = 0.030 μM), and H (29l, IC50 = 0.015 μM) were more active than other compounds with larger groups. Interestingly, the CH3 substituted compound 24d exhibited a slightly more potent activity than the correspond non-substituted counterpart 29l, though the methyl group has a larger steric hindrance than hydrogen.  Table 2, which indicated that the IC50 values of selected compounds against these four cancer cell lines were in nanomolar ranges. The K562 cell was the most sensitive cell line among the five cancer cell lines tested, and the most active compound 24d exhibited comparable activity to the reference compound CA-4 with IC50 values ranging from 0.009 to 0.016 μM. Notably, 24d displayed an approximately 6-fold improvement in activity compared with the parent compound 1. The SARs of the newly synthesized compounds were summarized in Figure 3.

Compound 24d Selectively Inhibited Cancer Cell Growth In Vitro.
Nonselective cytotoxicity is one of the main factors limiting the clinical use of anticancer drugs. 41 To obtain insights into the cytotoxic potential of these new compounds on normal human cells, the effects of compounds 24b, 24d, 29g, and 29l were evaluated in the normal human liver cell line L-O2 with CA-4 as the reference, which were compared with the IC50 values against human hepatocellular carcinoma cells (HepG2). As shown in Table 3 Thus, 24d was chosen for further biological studies.

Compound 24d Inhibited Tubulin Polymerization and Colchicine Binding
Effects. To investigate whether the antiproliferative activity of compound 24d was related to interactions with microtubule systems, 24d was evaluated for in vitro microtubule polymerization activity. The typical microtubule depolymerization agent (MDA) colchicine was employed as the reference. As shown in Figure 4, compound 24d displayed a concentration-dependent inhibition of tubulin polymerization, indicating that the mechanism of 24d was consistent with colchicine as an MDA.
Moreover, 24d exhibited more potent tubulin polymerization inhibitory activity (IC50 = 1.71 μM) than CA-4 (IC50 = 2.53 μM) (      Mitosis in eukaryotic cells is regulated by the activation of Cdc2 kinase, which is controlled by several steps including cyclin B1 binding and cdc25c phosphorylation. 44 Thus, to obtain insight into the mechanism of 24d in K562 cell cycle arrest, the expression of cell cycle regulatory proteins was investigated. As shown in Figure 7b and 7d, 24d decreased cdc2, cyclin B1 and cdc25c protein levels in a concentration-dependent manner. The results suggested that the 24d-induced G2/M arrest may be correlated with a change of expression of cdc2/cyclin B1 and cdc25c.

Compound 24d Induced Apoptosis via Regulating of Apoptosis-related Protein
Expressions. Mitotic arrest of tumor cells by microtubule targeting agents is generally associated with cellular apoptosis. 45 Hoechst 33342 staining was first used to assess morphology changes of K562 cells, as shown in Figure  Increasing evidence has indicated that the regulation of the Bcl-2 family of proteins is involved in the signaling pathways, 46 including pro-apoptotic (e.g., Bax and Bad) and anti-apoptotic proteins (e.g., Bcl-2 and Bcl-xl). As shown in Figure 8c and 8e, 24d upregulated Bad and Bax and downregulated Bcl-2 and Bcl-xl protein levels in a concentration-dependent manner. Thus, as described above, compound 24d induced cell apoptosis by interfering with the expression of apoptosis-related proteins.  Accumulating evidence reveals that increased levels of reactive oxygen species (ROS) is often associated with promoting cancer cell growth, 47 and mitochondrial membrane depolarization is related to mitochondrial production of ROS. 48 Thus, the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to evaluate the intracellular ROS levels after incubation with 24d. As shown in Figure 9b and 9c, 24d induced intracellular ROS generation with a dose-dependent manner, while the increased ROS was inhibited by pre-incubation with 2.5 mM of the ROS scavenger, N-acetyl cysteine (NAC).  Compound 24d Exhibited Potent Anti-Vascular Activity. Most microtubule binding agents possess potent vascular disrupting activity, which are contributed to the disruption of microtubule dynamics to induce endothelial cell shape change. 51 As HUVEC migration is the key step to generate new blood vessels, 52 wound healing assay was applied to assess the ability of 24d to inhibit HUVEC migration. As shown in Figure 11a and 11c, the untreated cells migrated to fill the area that was initially scraped after 24 h, while 24d significantly inhibited HUVEC migration at the dose of 20 nM.
We also evaluated the effect of 24d in a tube formation assay, which are based on the ability of HUVECs to form tubular and cord-like networks on Matrigel. In contrast to the tube-like networks of the control, the capillary-like tubes of HUVECs exposed to 24d at doses of 5, 10, and 20 nM for 6 h could be interrupted at different levels ( Figure 11b). These results showed that 24d effectively inhibited the tube formation of HUVECs.
The antiproliferative activity of 24d against HUVECs was also determined by an MTT assay to exclude the possibility that the anti-vascular activity of 24d was due to a cytotoxic action of 24d. The calculated IC50 value of 24d against HUVECs after a 24-h treatment was 0.250 ± 0.06 μM, which is higher than the concentration of 10 nM required for the obvious inhibition of cell migration and tube formation. These results indicate that 24d exhibited possessed anti-vascular activity.
Physicochemical Properties of 24d. To evaluate the drug-likeness of 24d, the physicochemical properties of 24d were predicted with compound 1 and CA-4 as the references. 53 As shown in Table 5, compound 24d conformed to Lipinski's rule of five. The aqueous solubility in phosphate buffer (pH 7.4) was also determined at 20 °C by HPLC. 54 As shown in Table 5, the solubility of 24d was approximately 5and 16-fold greater than compounds 1 and CA-4, respectively. Moreover, the hydrochloride salt of 24d (24d-HCl) could be easily prepared by the reaction of 24d with hydrogen chloride in ethyl acetate, which was soluble in PBS (solubility > 1000 μg/mL). The improvement of the aqueous solubility of 24d is most likely attributable to the quinoline moiety, which is more water-soluble than trimethylphenyl ring in compounds 1 and CA-4.  The Safety Profile of 24d in Mice. To investigate the safety profiles of 24d, the acute toxicity was determined in ICR mice treated with 409.6, 512, 640, 800, and 1000 mg/kg (iv, n = 10 per group) for 14 days. As shown in Table 6, treatment with 24d at doses of 409.6 mg/kg only caused one death in ten mice, while treatment with 24d at 1000 mg/kg killed nine mice. Finally, the median lethal dose (LD50) value of 24d was calculated to be 665.62 mg/kg, indicating the low toxicity of 24d and its safety as an anti-tumor agent.  Furthermore, we also observed the impact of 24d on tumor microvessels in vivo by immunohistochemistry. Tumor vessels were stained with CD31, which is a prominent endothelial marker that binds specifically to blood microvessels. As shown in Figure   12e, 24d-treated group at the dose of 20 mg/kg displayed considerably lower tumor microvessel density (MVD) than the control group, indicating that 24d exhibited potent in vivo anti-vascular activity.
Additionally, H&E staining of the heart, liver, spleen, lung and kidney collected at the end of the study also suggested no observable major organ-related toxicities ( Figure 13). Overall, these data indicated that compound 24d was efficacious in inhibiting the growth of cancer in vivo with no observable toxicity. It deserves further evaluation as a safe anti-tumor agent.

1-(2-Methylquinolin-4-yl)ethan-1-one (16). 2-Methylquinoline-4-carboxylic
acid (14) was synthesized according to literature report. 56    were then washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo to afford the crude products, which was purified by column chromatography with petroleum/ethyl acetate (2:1) to give 24a or 24b.   was added in one portion. After reaction completed, the mixture was extracted with EtOAc (3 × 20 mL), and the combined organic layers were then washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo to afford the crude products, which was purified by column chromatography with petroleum/ethyl acetate (4:1) to give products as colorless oil. Then, the products were dissolved in 5 mL EtOH, 1 mL 10% HCl aqueous was added followed by refluxing for 30 min. Then, the mixture was extracted with EtOAc (3 × 20 mL), and the combined organic layers were then washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo to afford the crude products, which was purified by column chromatography with petroleum/ethyl acetate (2:1) to give products 24c or 24d.

Acute Toxicity Determination. Five-week-old male Institute of Cancer
Research (ICR) mice were purchased from Shanghai SLAC Laboratory Animals Co.
Ltd. An acute toxicity study by intravenous injection was conducted according to the guidelines of the Organization for Economic Co-operation and Development. The animals were weighed and at random divided into five groups of ten animals. Then, the mice were intravenous injected with 24d (409.6, 512, 640, 800, and 1000 mg/kg) in a vehicle of 10% DMF/2% Tween 80/88% saline. The animals were observed continuously for 14 days. 2.18 Immunohistochemistry assay. Slides from mouse tissues (24d-treated group at 20 mg/kg) were embedded in paraffin were cut to a section of 4 μm, deparaffinized, and treated with citrate buffer. Then, they were blocked with avidin/biotin for 20 min.
The slides were incubated with CD31 overnight at 4 °C. Next, the slides were treated with secondary antibody with horseradish peroxidase goat anti-rabbit for 1-3 h and developed with 3, 3-diaminobenzidine. Finally, the slides were counterstained with hematoxylin. The mean microvessel density (MVD) was measured by calculating the CD31-positive cells in randomly selected areas in each section using image analysis software and then analyzed with OriginPro 8.0 software.

SUPPORTING INFORMATION AVAILABLE
1 H and 13 C NMR spectra for key intermediates and all target compounds, HPLC trace for compound 24d and Molecular Formula Strings are available free of charge via the internet at http://pubs.acs.org.
The statement for Molecular modeling (PDB) file: Authors will release the atomic coordinates and experimental data upon article publication.