Synthesis and SAR studies of novel 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives for anticancer activity
Yi-Fong Chen 1,2, Yi-Chien Lin 2, Susan L. Morris-Natschke 3, Chen-Fang Wei 2, Ting- Chen Shen 2, Hui-Yi Lin 2, Mei-Hua Hsu 2, Li-Chen Chou 2, Yu Zhao 3, Sheng-Chu Kuo
1,2, Kuo-Hsiung Lee 3,4*, Li-Jiau Huang 1,2*
1 The Ph.D. Program for Cancer Biology and Drug Discovery, China Medical University and Academia Sinica, No.91 Hsueh-Shih Road, Taichung, Taiwan
2 School of Pharmacy, China Medical University, No.91 Hsueh-Shih Road, Taichung, Taiwan
3 Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
4 Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
Correspondence:
Li-Jiau Huang, School of Pharmacy, China Medical University, no.91 Hsueh-Shih Road, Taichung, 40402, Taiwan. E-mail: [email protected] or Kuo-Hsiung Lee, Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA. E-mail:
*Both authors contributed equally to this work.
BACKGROUND AND PURPOSE
Our previous studies indicated that certain 4-phenylquinolin-2(1H)-one (4-PQ) derivatives can induce apoptosis in cancer cells. In the present study, we focused on further discovery of new 4-PQ analogs as apoptosis stimuli to obtain more effective and less toxic antitumor agents.
EXPERIMENTAL APPROACH
Forty-five 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives were designed and synthesized. Anti-proliferative activities were evaluated using an MTT assay, and structure-activity relationship (SAR) correlations were established in this new compound series. Compounds 9b, 9c, 9e and 11e were also evaluated against the National Cancer Institute (NCI)-60 human cancer cell line panel. To determine cancer cell specificity, subpanel-selectivity ratios were calculated from GI50 values.
Furthermore, compound 11e was found to induce apoptosis of COLO 205 cells by Hoechst 33258 and Annexin V-FITC/ PI staining assays. The fluorescence microscopy experiments revealed that 11e inhibited microtubules polymerization in a similar manner to colchicine. 11e-treatment influenced the expression of cell cycle- and apoptosis-related proteins in COLO 205 cells, causing G2/M arrest and multi- nucleation, increased cyclin B and CDK1, phospho-CDK1, and decreased aurora B,
phosphor-aurora B, phosphor-aurora A, and phosphor-histon H3. 11e increased levels of active caspase-3, caspase-8, and caspase-9 forms, cleaved PARP, AIF, Endo G, Apaf-1, cytochrome c, DR5, TRAIL, t-Bid, Bax, Bad, and P-JNK, but reduced levels of procaspase-3, procaspase-8, procaspase-9, PARP, Bid, Bcl-xL, and Bcl-2.
KEY RESULTS
New 6,7,8-substituted-4-substituted-benzyloxyquinolin-2(1H)-one derivatives were synthesized, characterized and evaluated for in vitro anticancer activity. Nine compounds (7e, 8e, 9b, 9c, 9e, 10c, 10e, 11c and 11e) displayed high potency against HL-60, Hep3B, H460 and COLO 205 cancer cells (IC50 <1 μM) without affecting Detroit 551 normal human cells (IC50 > 50 μM). Compounds 9b, 9c, 9e and 11e showed remarkable broad-spectrum antitumor activities against the NCI-60 tumor cell line panel. Particularly, compound 11e exhibited nanomolar potency against COLO 205 cancer cells. Mechanistic studies indicated that 11e exerts anti-cancer effects by disrupting microtubule assembly, and inducing G2/M arrest, polyploidy, and apoptosis via intrinsic and extrinsic signaling pathways in COLO 205 cells. Activation of JNK might play in TRAIL-induced COLO 205 cells apoptosis. Further investigation of its potential use as an anti-colon cancer agent is warranted.
CONCLUSION AND IMPLICATIONS
New quinolone derivatives were identified as potential pro-apoptotic agents.
Compound 11e can be used as a promising lead compound for future development of new antitumor agents.
Abbreviations
SAR, structure-activity relationship; NCI, National Cancer Institute; 4-PQ, 4- phenylquinolin-2(1H)-one; CDK, cyclin dependent kinase; CDKI, CDK inhibitors;
BG, benzylguanine; AGT, alkylguanine-DNA alkyltransferase; pDOS, privileged- substructure-based diversity-oriented synthesis; PPA, polyphosphoric acid.
Keywords: 4-Benzyloxyquinolin-2(1H)-one derivatives, NCI-60 screening panel,
Antitumor agents, Apoptosis, Structure-activity relationships
Introduction
Cancer is presently a worldwide health problem and leading cause of death in the United States and other developed countries . Cancer is a formidable disease caused by disordered cell growth and invasion of tissues and organs. While various therapies and strategies have been developed to treat cancer, most of them have limitations. Thus, new anticancer drugs are continually needed. The main challenge facing clinical cancer therapy is to find a specific approach that kills malignant cells with no or few adverse effects on normal tissues, and considerable attempts have been made to develop innovative, safe and effective methods to defeat cancer. While scientists have discovered many agents with cytostatic action against cancer cells , increasing comprehension of the biological processes involved in cancer cell survival has led to the design and discovery of better targeted, novel therapeutic anticancer drugs. For several chemotherapeutic agents, a direct correlation has been found between antitumor efficacy and ability to induce apoptosis . Thus, approaches aimed at promoting apoptosis in cancer cells have gained paramount importance in future cancer research .
Hetero-bicycles are indispensable structural units in compounds with a broad range
of biological activities. Among various nitrogen-containing fused heterocyclic skeletons, quinoline and quinolone structures are important components prevalent in a vast array of biological systems. Compounds with a quinoline nucleus exhibit various pharmacological properties, including antioxidant , anti-inflammatory , antibacterial , anti-HIV , anti-malarial , anti-tuberculosis , anti-Alzheimer’s disease , anticancer , etc.
Accordingly, Soloman and Lee described quinoline-containing subunits as ‘privileged structures’ for drug development . 2-Quinolone [quinolin-2(1H)-one], also called 1-aza coumarin or carbostyril, and 4-quinolone are structural isomers. The 2-quinolone skeleton is a fertile source of biologically active compounds, including a wide spectrum of alkaloids investigated for antitumor activity . In our previous investigation, 6,7-methylenedioxy-4-substituted phenylquinolin-2(1H)-one derivatives (4-phenylquinolin-2(1H)-ones; 4-PQs) were identified as novel apoptosis-inducing agents (Fig. 1) . Recently, Arya and co-workers reported that 4-hydroxyquinolin- 2(1H)-one derivatives, prepared efficiently through microwave irradiation, showed strong photo-antiproliferative activity . Thus, we have directed our focus onto 4-PQ analogs as apoptosis stimuli. In our current study, we targeted the 2-quinolone structure as a basic scaffold of new derivatives with different substituents.
Purine-based compounds such as olomoucine and roscovitine (Fig. 1), which contain
other hetero-bicyclic ring systems, are known ATP-binding site competitive inhibitors of cyclin dependent kinase (CDK) and are useful cell proliferation inhibitors in the treatment of cancer . Structure–activity relationship (SAR) studies on CDK inhibitors (CDKIs) demonstrated that a small hydrophobic group such as a non-polar benzyl group at the O6- or N6-position of the hetero-bicycle maximized CDK inhibition . In addition, numerous CDKI-related compounds that contain benzyl or arylmethyl groups on different core scaffolds, such as pyrazolo[1,5-a]pyrimidines , quinazolin-4-amines , pyrimidine and aminopurine (Fig. 1), have been studied. Furthermore, a series of 6- (benzyloxy)-2-(aryldiazenyl)-9H-purine derivatives were reported to act as prodrugs of O6-benzylguanine (O6-BG; Fig. 1), which selectively targets O6-alkylguanine-DNA alkyltransferase (AGT) in hypoxic tumor cells . The AGT protein plays a critical role in DNA repair, which can be exploited in chemotherapeutic treatment of neoplastic cells . Alkylation of AGT with the benzyl group of O6-benzylguanine (O6-BG) results in complete depletion of the alkyltransferase protein. Consequently, numerous O6-BG analogs have been developed as AGT inhibitors . In 2008, Ruiz et al. reported that a family of quinolinone compounds acted as novel non-nucleosidic AGT inhibitors.
These quinolinones could reach catalytic residue Cys145 buried deep within the binding groove, occupy the catalytic cleft of human DNA repair AGT protein, and act
as substrate mimics of the O6-guanine moiety.
Furthermore, the activity of biologically proven anticancer pharmacophores can be enhanced by introducing appropriate substitutions on the chemical scaffolds. In medicinal chemistry, shortening or lengthening chain length is a useful tactic to improve the affinity of target-binding. Some literature reports have demonstrated that the pro-apoptotic (antitumor) activity of certain compounds was dramatically improved by slightly changing the length and spacing of lateral branches, such as benzyl and other alkyl-aromatic side chains, on core skeletons . Such exploration and utilization of chemical diversity relative to pharmacological space is an on-going drug discovery strategy, referred to as privileged-substructure-based diversity-oriented synthesis (pDOS) . Based on this strategy, as well as the structures shown in Fig. 1, we proposed addition of a substituted benzyl (C ring) side chain linked at the O4-position of 4- hydroxyquinolin-2(1H)-one (2-quinolone scaffold) as a possible strategy for discovering new leads with pro-apoptotic bioactivity. The flexibility of the benzyl moiety might have some advantages to obtain better antitumor activity compared with our prior 4-PQ derivatives (Fig. 1). Therefore, we designed a series of 4- benzyloxyquinolin-2(1H)-one analogs 7a–e~15a–e, with the general structures of target compounds depicted in Fig. 1. To the best of our knowledge, this is the first
report on anticancer evaluation of 2-quinolone analogs bearing an O4-benzyl moiety.
The goal of current study is to discover more effective and less toxic antitumor agents, and contribute to the SAR profile of 2-quinolones with anti-proliferative activity and apoptotic induction in cancer cells.
Methods
Materials and physical measurements
All solvents and reagents were obtained commercially and used without further purification. The progress of all reactions was monitored by TLC (thin layer chromatography) on 2 × 6 cm pre-coated silica gel 60 F254 plates of thickness 0.25 mm (Merck). The chromatograms were visualized under UV at 254–366 nm. Column chromatography was performed using silica gel 60 (Merck, particle size 0.063–0.200 mm). Melting points (mp) were determined with a Yanaco MP-500D melting point apparatus and are uncorrected. IR spectra were recorded on Shimadzu IR-Prestige-21 spectrophotometers as KBr pellets. The 1D nuclear magnetic resonance (NMR, 1H and
13C) spectra were obtained on a Bruker Avance DPX-200 FT-NMR spectrometer at room temperature. The 2D NMR spectra were obtained on a Bruker Avance DPX-400 FT-NMR spectrometer, and chemical shifts expressed in parts per million (ppm, δ).
The following abbreviations are used: s, singlet; d, doublet; t, triplet; dd, double doublet; and m, multiplet. Mass spectra were performed at the Instrument Center of National Science Council at National Chung Hsing University, (Taichung City, Taiwan
R.O.C.), using a Finnigan ThermoQuest MAT 95 XL (EI-MS).
General procedure for the synthesis of 4-hydroxyquinolin-2(1H)-one derivatives (5a-i) 4-Hydroxyquinolin-2(1H)-one derivatives 5a–i were prepared by “one-pot” cyclization in polyphosphoric acid (PPA). A mixture of the appropriate substituted aniline 1a–i (1 equiv) and diethylmalonate (2) (1.2 equiv) was heated with 5 – 6 times by weight PPA at 130 °C for 2 – 6 h (TLC monitoring). Then, the mixture was cooled and diluted with water. A gum solidified upon standing overnight, and the precipitate was filtered, washed with water, and air-dried to provide 5a–i with sufficient purity for the next reaction. Physical and spectroscopic data for 5a are given below; the data for the remaining compounds are provided as Supplementary Material.
4-Hydroxyquinolin-2(1H)-one (5a)
Compound 5a (3.48 g, 21.59 mmol)was obtained from aniline (1a) (3.82 g, 41.01 mmol) and diethylmalonate (2) (7.88 g, 49.20 mmol); yield: 53%; light-yellow solid;
mp: 276–278 °C; IR(KBr) ν (cm － 1):1660 (C ＝O); 1H NMR (200 MHz, DMSO-
H–8), 7.47 (t, J = 7.8 Hz, 1H, H–7), 7.77 (d, J = 8.0 Hz, 1H, H–5), 11.28 (br. s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 98.56, 115.48, 115.63, 121.61, 123.11, 131.33, 139.55, 163.05, 164.18; MS (EI, 70 eV) m/z: 161.1[M]+; HRMS (EI) m/z:
calculated for C9H7NO2: 161.0477; found: 161.0472.
General procedure for the synthesis of 6,7,8-substituted-4-substituted
benyloxyquinolin-2(1H)-one derivatives (7a–e, 8a–e, 9a–e, 10a–e, 11a–e, 12a–e, 13a–
e, 14a–e, 15a–e)
A mixture of 4-hydroxyquinolin-2(1H)-one derivatives 5a–i (1 equiv) and K2CO3 (2 equiv) in DMF (10–20 mL) was heated at 90 °C for 1–2 h. The appropriate benzyl chloride or bromide (6a–e, 1–1.4 equiv) was added, and the mixture was heated at 80–
90 °C for 1–6 h. Reaction completion was confirmed by TLC monitoring. The mixture was poured into ice water (200 mL), and the precipitated solid was collected by filtration and then washed with water. The residue was treated with EtOAc and purified by recrystallization. If no solid formed solids after addition of ice water, then the reaction mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous MgSO4 before evaporation of solvent in vacuo. The residue was isolated by column chromatography (silica gel, EtOAc as eluate), and then recrystallized to give the corresponding pure products, 4-benzyloxyquinolin-2(1H)-one
derivatives 7a–e, 8a–e, 9a–e, 10a–e, 11a–e, 12a–e, 13a–e, 14a–e and 15a–e. Physical and spectroscopic data for 11e are given as examples below; the data for the remaining
compounds are provided as Supplementary Material.
4-(3',5'-Dimethoxybenzyloxy)-6-methoxyquinolin-2(1H)-one (11e)
Compound 11e (0.70 g, 2.05 mmol)was obtained from 5e (1.12 g, 5.86 mmol) and 3,5- dimethoxybenzyl bromide (1.48 g, 6.40 mmol); yield: 35%; white cotton crystal; mp:
217–219 °C; IR(KBr) ν (cm － 1): 1674 (C ＝ O); 1H NMR (200 MHz, DMSO-d6) δ (ppm): 3.74 (s, 6H, 3’, 5’–OCH3), 3.76 (s, 3H, 6–OCH3), 5.20 (s, 2H, –O–CH2–), 5.94 (s, 1H, H–3), 6.47 (dd, J = 2.2,2.2 Hz, 1H, H–4’), 6.66 (d, J = 2.2 Hz, 2H, H–2’, H–
6’), 7.14-7.26 (m, 3H, H–5,7,8), 11.32 (br. s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 55.63 (2C), 55.78, 70.05, 98.71, 100.04, 104.17, 105.62 (2C), 115.50, 117.18, 120.50, 133.55, 138.76, 154.43, 161.07 (2C), 161.89, 163.23; MS (EI, 70 eV) m/z:
341.0 [M]+; HRMS (EI) m/z: calculated forC19H19NO5: 341.1263; found: 341.1257.
MTT assay for anti-proliferative activity
Human tumor cell lines of the cancer screening panel were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO/BRL), penicillin (100 U/mL)/streptomycin (100 g/mL) (GIBCO/BRL) and 1% L-glutamine (GIBCO/BRL)
at 37 °C in a humidified atmosphere containing 5% CO2. Human hepatoma Hep 3B and normal skin Detroit 551 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (GIBCO/BRL), penicillin (100 U/mL)/streptomycin (100
g/mL) (GIBCO/BRL) and 1% L-glutamine (GIBCO/BRL) at 37 C in a humidified atmosphere containing 5% CO2. Logarithmically growing cancer cells were used for all experiments. The human tumor cell lines were treated with vehicle or test compounds for 48 h. Cell growth rate was determined by MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide] reduction assay . After 48 h treatment, cell growth rate was measured on an ELISA reader at a wavelength of 570 nm and the IC50 values of test compounds were calculated.
In vitro NCI-60 human tumor cell line (HTCL) panel
In vitro cytotoxic activities were evaluated through the Developmental Therapeutic Program (DTP) of the National Cancer Institute (NCI) . For more information on the
anticancer screening protocol, please see:
http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
Cell morphology and Hoechst 33258 staining
COLO 205 cells were plated at a density of 2.5×105 cells per well in 12-well plates, and then incubated with 50 nM of compound 11efor 12 h to 48 h. Cells were directly
examined and photographed under a contrast-phase microscope. Nuclei were stained with Hoechst 33258 (bis-benzimide, Sigma) to detect chromatin condensation or nuclear fragmentation, features of apoptosis. After 0, 12, 24, 36, and 48 h, 11e-treated cells were stained with 5 μg/mL Hoechst 33258 for 10 min. After washing twice with PBS, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at 25 °C.
Fluorescence of the soluble DNA (apoptotic) fragments was measured in a Varian Fluorometer at an excitation wavelength of 365 nm and emission wavelength of 460
nm.
Apoptosis studies
Determination of apoptotic cells by fluorescent staining was done as described previously . The Annexin V-FITC Apoptosis Detection Kit was obtained from Strong Biotech Corporation (Strong Biotech, Taiwan). The COLO 205 cells (2×105 cells/well) were fluorescently labeled for detection of apoptotic and necrotic cells by adding 100 μL of binding buffer, 2 μL of annexin V-FITC, and 2 μL of PI to each sample. Samples were mixed gently and incubated at room temperature in the dark for 15 min. Binding buffer (300 μL) was added to each sample immediately before flow cytometric
analysis. A minimum of 10,000 cells within the gated region was analyzed.
Flow cytometric analysis for cell cycle
COLO 205 cells were added to 50 nM of 11e for 0 h, 12 h, 24 h, 36 h, and 48 h. Cells were fixed in 70% ethanol overnight, washed twice, and re-suspended in PBS containing 20 μg/mL PI, 0.2 mg/ml RNase A, and 0.1% Triton X-100 in the dark room. After 30 min incubation at 37 °C, cell cycle distribution was analyzed using ModFit LT Software (Verity Software House, Topsham, USA) in a BD FACSCanto
flow cytometer (Becton Dickinson, San Jose, CA).
Molecular modeling
The crystal structure of microtubules in complex with N-deacetyl-N-(2- mercaptoacetyl)-colchicine (DAMA-colchicine) was downloaded from the Protein Data Bank (PDB entry 1SA0 : http://www.rcsb.org/pdb/home/home.do) . Docking studies were performed for proposed 11e in the colchicine binding site of tubulin. The AutoDock Vina was used to perform docking calculations . The final results were prepared with PyMOL (v. 1.3) in Windows 7. After removing the ligand and solvent molecules, hydrogen atoms were added to each amino acid atom. The 3D structure of compound were obtained from ChemBioDraw ultra 12.0 followed by MM2 energy minimization. Docking was carried out by AutoDock Vina in the colchicine binding pocket. Grid map in AutoDock 4.0 was used to define the interaction of protein and
ligand in the binding pocket. For compound binding into the colchicine binding site, a grid box size of 25×25×25 points in x, y and z directions was built and the grid center was located in x = 116.909, y = 89.688, and z = 7.904.
Localization of microtubules
After treatment, cells were fixed with 4% paraformaldehyde (PFA) in PBS, blocked with 2% bovine serum albumin, stained with anti-tubulin monoclonal antibody, and then with FITC conjugated anti-mouse IgG antibody. PI was used to stain the nuclei.
Cells were visualized using a Leica TCS SP2 Spectral Confocal System.
Mitochondrial membrane potential analysis
Cells were plated on 6 well at 1.0×106 cells/well and treated with 50 nM 11e for 6-24 h. Mitochondrial membranes were stained with 0.5 ml JC-1 working solution (BD MitoScreen Kit) to each sample. Samples were incubated for 10-15 min at 37 °C in the dark. Mitochondrial membrane potential was measured using the BD FACSCanto flow
cytometer (Becton Dickinson, San Jose, CA).
Western blot assay
The treated cells (1×107 cells/10 ml in 10 cm dish) were collected and washed with PBS. After centrifugation, cells were lysed in a lysis buffer. The lysates were incubated
on ice for 30 min and centrifuged at 12000 g for 20 min. Supernatants were collected, and protein concentrations were then determined using Bradford Assay. After adding a 5× sample loading buffer containing 625 mM Tris-HCl, pH = 6.8, 500 mM dithiothreitol, 10% SDS, 0.06% bromophenol blue, and 50% glycerol, protein samples were electrophoresed on 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. Immunoreactivity was detected using the Western blot
chemiluminescence reagent system (PerkinElmer, Boston, MA).
Statistical analysis
Statistical analysis was performed with an analysis of variance (ANOVA) followed by the Turkey's test. All data were expressed as mean ± SEM. *P < 0.001 was indicative of a significant difference.
Results
Chemistry
The synthetic procedures for the new 4-substituted benzyloxyquinolin-2(1H)-ones (7a–
e~15a–e) are illustrated in Scheme 1. A general synthetic approach to the key
intermediate 4-hydroxyquinolin-2(1H)-one is the Knorr quinoline synthesis, which involves cyclization and dehydration of a transient β-ketoanilide, formed by
condensation of a β-ketoester and aniline at relatively high temperature. More specific synthetic approaches include cyclization of N-acetylanthranilic acid derivatives , condensation of malonates/malonic acid with anilines using ZnCl2 and POCl3 , Ph2O , and cyclization of malonodianilides with polyphosphoric acid (PPA) , CH3SO3H/P2O5 , p-toluenesulfonic acid . In our study, 4-hydroxyquinolin-2(1H)-one derivatives (5a–i)
were synthesized by treatment of a substituted aniline (1a–i) with diethylmalonate (2) in one-flask , followed by cyclization of the formed monoanilide (3a–i) or malondianilide (4a–i) precursors in the presence of PPA. The target 4- benzyloxyquinolin-2(1H)-one derivatives 7a–e, 8a–e, 9a–e, 10a–e, 11a–e, 12a–e, 13a–e, 14a–e and 15a–e were synthesized by reaction of the intermediate 4-
hydroxyquinolin-2(1H)-one derivatives 5a–i with various benzyl halide 6a–e in the presence of K2CO3 and DMF . All synthetic products were characterized by IR, 1H and
13CNMR, and mass spectroscopy.
Previous literature has reported that 2-quinolones have a minor tautomeric structure (2-hydroxyquinoline) due to protonation of the carbonyl oxygen . Deprotonation of the 2-quinolonewould cause ring resonance and electron shifting within the N-1, O-2, C-3 and O-4 positions of the 4-hydroxyquinolin-2(1H)-one derivatives (Fig. 2A) . Consequently, previous reports have indicated that 4-hydroxyquinolin-2(1H)-ones
could be alkylated at the 1-NH, 2-OH, 4-OH, or 3-CH position . Therefore, we confirmed the structures of our synthesized compounds using NMR spectroscopic analyses. The 1H NMR spectrum of 4-benzyloxyquinolin-2(1H)-one derivatives 7a–
e~15a–e featured a singlet for O-linked C(9)-H2 methylene protons between 5.13–5.27 ppm, a singlet for a C(3)-H proton between 5.80–6.09 ppm, and a broad singlet for an exchangeable NH group between 10.47–11.54 ppm. The chemical shifts for the benzylic CH2 were consistent with O-alkylation rather than N-alkylation . The 13C shifts for O-alkylated compounds are typically downfield (higher ppm value; 52.7–
68.4) compared with N-alkylated compounds (lower ppm value; 28.6–45.0) . The 13C NMR spectra of 7a–e~15a–e included a O-linked methylene carbon between 65.74–
70.74 ppm, which again indicated O-alkylation. Furthermore, regioselective alkylation at the 4-OH position was confirmed by two-dimensional NMR study via heteronuclear multiple-quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectroscopy experiments that disclose the relationship between 1H–13C coupling. In the case of compound 11e, as shown in Fig. 2B, the 4-O-linkage was supported by observation of 3J-HMBC correlations between C(9)-H methylene protons (δH 5.20) on the 3',5'-dimethoxybenzyloxy moiety with the carbon at C(4) position (δc
161.89) of the 2-quinolone core, which shows a further correlation with the C(5)-H
proton (δH 7.14–7.26, overlapped). In other words, O4-alkylation was determined through the observation of H9/C4 and H5/C4 crosspeaks. These data proved that 3',5'- dimethoxybenzyloxy moiety is attached to the 4-O-position of the 2-quinolone core- structure. Furthermore, the IR spectra of 7a–e~15a–e possessed a characteristic absorption band for an amido C ＝ O group (1633–1674 cm–1).
Biological evaluation and structure-activity relationship (SAR) analysis
All newly synthesized target compounds (7a–e, 8a–e, 9a–e, 10a–e, 11a–e, 12a–e, 13a–e, 14a–e and 15a–e) were assayed for growth inhibitory activity against Detroit
551 (human normal skin fibroblast) and four cancer cell lines, including HL-60 (leukemia), Hep 3B (hepatoma), H460 (non-small-cell-lung carcinoma) and COLO 205 (colorectal adenocarcinoma). Cells were treated with compounds for 48 h, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide (MTT) assay. The antiproliferative activity of each compound was presented as the concentration of compound that achieved 50% inhibition (IC50) of cancer cell growth. The results are summarized in Table 1. Collectively, the present series of novel 4-benzyloxyquinolin-2(1H)-one derivatives exhibited diverse potency against the four tested tumor cell lines. Among them, compounds 7e, 8e, 9b, 9c, 9e, 10c, 10e, 11c and 11e displayed high potency against HL-60, Hep3B, H460 and COLO 205 cells, with
IC50 value less than 1 μM (Table 1). Notably, 11e displayed the most prominent growth inhibitory activities against the above cell lines with IC50 values ranging from 14 to 40 nM. Moreover, none of the active compounds showed cytotoxicity toward Detroit 551 (IC50 > 50 μM) cells. These results suggested that this new series of 4- benzyloxyquinolin-2(1H)-one derivatives could effectively suppress tumor growth
without causing toxicity to normal somatic cells.
Based on the obtained biological data, SAR correlations were determined. Firstly, we evaluated the effects of methoxy-substitution of the C-4 benzyloxy ring (C ring) on the cytotoxic activity. Generally, compounds with 3',5'-dimethoxybenzyloxy side chain (7e–15e) showed the highest potency in their respective series (7–15). Among them, compounds 7e, 8e, 9e, 10e and 11e exhibited significant activity against Hep 3B, H460 and COLO 205 cancer cell lines (IC50 < 1 μM). These results indicated 3',5'- dimethoxy-benzyloxy substitution is preferred relative to other benzyl substitution.
Compounds 9b, 9c, 10b, 10c, 11b, 11c with a 2'- or 3'-methoxybenzyloxy side chain demonstrated moderate activity (IC50 0.2 – 5.0 μM), whereas compounds bearing side chains of benzyloxy or 4'-methoxybenzyloxy were inactive (IC50 > 50 μM) or exhibited
only marginal activity (IC50 4.5 – 10μM).
Next, we explored the SAR of the 2-quinolone A-ring. Compounds with a
substituted benzyloxy moiety at C-4 and various functional groups at C-6, -7 and -8 were studied, and different anticancer effects were found. Regarding the C-6 substitution, compound 8e (6-fluoro), 9e (6-chloro), 10e (6-methyl) and 11e (6- methoxy) were more potent than 7e (no substitution). Moreover, compound 11e (IC50
0.014–0.04 μM) displayed the strongest growth inhibitory activity among the C-6 substituted compounds, suggesting that the C-6 methoxy group might play a pivotal role. Moving the methoxy group from C-6 to C-7 (12e, IC50 2.11–4.9 μM) or C-8 (13e, IC50 2.2–3.8 μM) led to dramatically decreased inhibitory activity. Activity also decreased when the C-8 methoxy of 13e was replaced with chlorine (14e), while activity was retained when the methoxy was replaced with methyl (15e). Thus, in this series of 4-benzyloxy-2-quinolones, optimal anti-proliferative effects were found with
a 6-methoxy group on the 2-quinolone ring.
In the present work, the above findings can be summarized in the following two SAR conclusions:
1. The in vitro anticancer activity of the substituted benzyloxy moiety (C ring) on the 4-position of 2-quinolone derivatives can be ranked in the following order of decreasing activity: 3',5'-dimethoxybenzyloxy (7e–15e) > 3'-methoxybenzyloxy (7c–15c) > 2'-methoxybenzyloxy (7b–15b) > benzyloxy (7a–15a) > 4'-
methoxybenzyloxy (7d–15d).
2. C-6 substituents on the 2-quinolone (A ring) resulted in better activity compared with C-7 and C-8 substituent. The following rank order of in vitro anticancer activity was found relative to the identity of the C-6 substituent: 6-methoxy > 6- chloro > 6-methyl > 6-fluoro > no substitution.
Anticancer drug screen panel of compound 9b, 9c, 9e and 11e against NCI-60 human
cancer cell lines
We selected four potent compounds 9b, 9c, 9e and 11e and submitted them for screening against the NCI-60 HTCL panel assay through the US NCI Developmental Therapeutics Program . The cell lines represent nine tumor subpanels, leukemia, melanoma and cancers of lung, colon, brain (CNS), ovary, kidney, prostate and breast.
Initially, the compounds were added at a single dose (10 μM) and the culture incubated for 48 h. End point determinations were made with a SRB (sulforhodamine B) assay.
Results for each compound are given in Table 2, with a negative value in the cell growth percentage indicating an anti-proliferative effect against that cell line.
Compound 9b displayed positive cytotoxic effects toward 11 out of 60 cell lines, and the positive cytotoxic proportions of 9c, 9e, and 11e were 10/59, 18/60, and 26/57. Our prominent compound 11e exhibited inhibitory effects ranging from –59.50% to –
0.80%. At the primary single high dose 10 μM (10-5 M), 9b, 9c, 9e, and 11e showed greatest effects against colon carcinoma COLO 205 with cell growth percentage of – 55.40, –57.40, –64.41, and –59.50, respectively. The melanoma MDA-MB-435 cell line was also sensitive to these compounds (growth percentages –46.28%, –43.14%, –
43.16%, and –41.44%, respectively).
At the second evaluation stage, the selected compounds were evaluated at five different concentrations (0.01, 0.1, 1, 10, and 100 μM) against same NCI-60 HTCL panel. The outcomes were represented by three calculated response parameters (GI50, TGI, and LC50) for each cell line through growth percent inhibition curves . The GI50
value (growth inhibitory activity) corresponds to the concentration of compound causing 50% decrease in net cell growth, the TGI value (cytostatic activity) is the concentration of compound resulting in total growth inhibition (100% growth inhibition) and LC50 value (cytotoxic activity) is the lethal dose of compound causing net 50% death of initial cells. The calculated results are presented as log concentration (given in supplementary data), as shown in Table 3. The NCI data revealed broad- spectrum sensitivity profiles for 9b, 9c, 9e and 11e toward all nine cancer subpanels with GI50 values less than 1 μM, and less than 0.01 μM (log GI50 < –8.0) against some cell lines for 9e and 11e. The anticancer effects of these compounds were comparable
to those of fluorouracil (5-FU), which is widely used clinically for treating malignancy . These screening results correspond with the one single dose results, showing broad anticancer spectra for 9b, 9c, 9e and 11e. Notably, prominent compound 11e exhibited GI50 values ranging from 0.01 to 8.08 μM in 51 of the 56 cell lines, with GI50 values below 0.01 μM in five cell lines (leukemia K-562 and SR, non- small cell lung cancer NCI-H522, colon cancer COLO 205, melanoma MDA-MB-
435).
To further determine which cancer subtypes were more sensitive to these 4- benzyloxy-2-quinolones, we calculated subpanel-selectivity ratios based upon GI50
values. The calculated results are shown in Table 4 and Fig. 3. Selectivity ratios less than 3 were rated non-selective, ratios ranging from 3 to 6 were termed moderately selective, and ratios greater than 6 were designated highly selective . With all ratios less than 3, compounds 9b and 9c were rated non-selective toward all nine subpanels.
Interestingly, both 9e and 11e, which contain a 3',5'-dimethoxybenzyloxy moiety, were much more selective than 9b and 9c (Fig. 3). As shown in Table 4, the average selectivity ratios of 9e and 11e (ratios = 6.51 and 4.05) were higher than those of 9b and 9c (ratios = 1.12 and 1.28). Compound 9e exhibited selectivity against leukemia, colon cancer, CNS cancer, melanoma, renal cancer and prostate cancer with selectivity
ratios of 7.56, 9.09, 10.28, 10.00, 7.89 and 8.57, respectively. Regarding total MID, the most prominent compound 11e displayed significant activity (0.31 μM) and was moderately selective toward the breast cancer subpanel with a selectivity ratio of 3.83 and highly selective against leukemia, colon cancer and prostate cancer with selectivity ratios of 7.36, 10.42, and 10.42, respectively. Among the subpanels rated highly selective, colon cancer was extremely sensitive to compound 11e (NSC 764592; Fig.
3). This compound showed exceptional potency against the individual cell line COLO 205 (LC50 0.09 μM, TGI 0.03 μM) (Table 3). From the dose-response curves against six colon cancer cell lines (Fig. 4), it is also obvious that 11e exhibited unique
selectivity against COLO 205.
Morphological changes and apoptosis in COLO 205 cells induced by compound 11e Based on the in vitro cytotoxicity data, 11e (NSC 764592), the most potent compound
against COLO 205 cells, was selected for further biological studies. Apoptosis is well known as a process of programmed cell death (cell suicide) . In our previous study, 4- phenyl-2-quinolone analogs (4-PQs) could induce cell cycle arrest and apoptosis in both HL-60 and H460 cells . In order to characterize the cellular basis for the antiproliferative effects of selected derivative 11e, we investigated the compound’s apoptosis inducing ability in COLO 205 cells. Morphological analysis confirmed the
cytotoxic effects of 11e. As shown in Fig. 5A, the apoptotic morphological changes included cell rounding and shrinkage after 24 h incubation with 50 nM of 11e (the black arrowhead indicates an apoptotic nucleus). To confirm the induction of apoptosis by 11e, COLO 205 cells were stained with Hoechst 33258, a fluorescent DNA-staining dye, and cell morphology was investigated using fluorescence microscopy. As shown in Fig. 5B, control cells exhibited uniformly dispersed chromatin, homogeneous blue fluorescence in the nuclei, normal organelles, and intact cell membranes. In cells treated with 50 nM of 11e for 24, 36, and 48 h, the nuclei budded off into several fragments, and nuclear condensation and fragmentation were observed (Fig. 5B), indicating typical characteristics of apoptosis, including condensation of chromatin, shrinkage of nuclei, and appearance of apoptotic bodies (the black arrowhead indicates
an apoptotic nucleus).
Annexin V-FITC/PI double-labeling was used to detect phosphatidylserine (PS) externalization, a hallmark of apoptosis . In Fig. 5C, populations of viable (annexin V–, PI–), early apoptotic (annexin V+, PI–), late apoptotic (annexin V+, PI+) and necrotic (annexin V–, PI+) cells are found in quadrants (Q) 3, 4, 2, and 1, respectively.
Cells incubated in the absence of 11e were undamaged and stained with negative for annexin V-FITC and PI (Q3). After incubation with 50 nM of 11e, the number of early
apoptotic cells stained positive by annexin V-FITC and negative with PI (Q4) increased significantly with incubation time, from 5.5% (control) to 7.7% after 12 h, 12.7% after 24 h, 20.9% after 36 h, and 21.8% after 48 h incubation. The number of late apoptotic cells stained positive by annexin V-FITC and PI (Q2) also increased with incubation time, from 9.9% (control) to 10.2% after 12 h, 10.4% after 24 h, 8.6%
after 36 h, and 16.1% after 48 h incubation. Thus, when COLO 205 cells were stained with Annexin-V/PI and analyzed with flow cytometry, early and late apoptotic (Annexin-V-stained) cells increased in a time-dependent manner (Fig. 5C), which indicates that 11e can induce apoptosis.
Toxicity of COLO 205 cells induced by 11e
Exposure of COLO 205 cells to 11e for 48 h, followed by MTT metabolism assays, confirmed the effects of 11e on cell viability. The IC50 value was 27.2 ± 1.4 nM, and 11e reduced COLO 205 cell viability in a dose-dependent manner. Exposure of COLO
205 cells to 10 nM, 25 nM, 50 nM, 75 nM, and 100 nM 11e reduced the survival to 86.3 ± 0.6%, 49.3 ± 1.4%, 22.4 ± 0.3%, 19.2 ± 0.5%, and 18.7 ± 1.2% of the control (0.1% DMSO), respectively (Fig. 6B). 11e inhibited COLO 205 cell growth in dose- and time-dependent manners (Fig. 6C).
11e induced apoptotic cell death and interfered with the cell-cycle distribution in
G2/M phase arrest
COLO 205 cells were treated with 50 nM of 11e for 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h, followed by flow cytometry analysis to determine the cell cycle distribution of treated cells, investigated the 11e-induced inhibition of COLO 205 cell growth by cell cycle arrest and apoptotic mechanisms. As shown in Fig. 7A, 11e induced a time- dependent accumulation of G2/M cells (11.72% G2/M at 0 h; 33.76% G2/M at 6 h;
58.98% G2/M at 12 h; 90.43% G2/M at 24 h; 82.82% G2/M at 36 h; 59.99% G2/M at 48 h), and apoptotic (sub-G1) cells (0.32% sub-G1 at 0 h; 1.29% sub-G1 at 6 h; 1.91%
sub-G1 at 12 h; 7.18% sub-G1 at 24 h; 26.15% sub-G1 at 36 h; 45.56% sub-G1 at 48 h). The results showed that 11e disturbed the cell-cycle process and induced COLO
205 cell death through an increase in the sub-G1 phase.
11e inhibits microtubule polymerization in COLO 205 cells
COLO 205 cells were treated with 50 nM 11e for 24 h and visualization using confocal microscopy investigated the effects of 11e on microtubule function. As shown in Fig.
7B, treatment with 11e resulted in microtubule changes similar to those induced by colchicine. Both compounds caused cellular microtubule depolymerization with short microtubule fragments scattered throughout the cytoplasm. On the contrary, taxol
induced significantly increased tubulin polymerization.
Molecular modeling and computational studies
Using a molecular docking method and molecular mode of tubulin and N-deacetyl-N- (2-mercaptoacetyl)-colchicine (DAMA-colchicine), we docked 11e into the colchicine- binding domain of tubulin. As shown in Fig. 8A and B, 11e probed deeply into the colchicine-binding pocket of α- and β-tubulin, which is very similar to the binding mode of DAMA-colchicine. Superimposition of compounds in the colchicine-binding site indicated that ring C and ring A are similar pharmacophores between DAMA- colchicine and 11e (Fig. 8C and 8D). The 3',5'-dimethoxy of 11e overlaps with the 1,3- (OMe)2 group in the C ring of DAMA-colchicine. As shown in Fig. 8D, The 1-NH group of 11e overlaps with the acetamide-NH group of DAMA colchicine. Moreover, the 1-NH of 11e forms a hydrogen bond with Thr179α as is observed with the acetamide-NH group of DAMA-colchicine. The -O-CH2- group of 11e occupies a region in space in proximity to the C5 and C6-positions in the B-ring of DAMA colchicine and is involved in hydrophobic interactions with Lys254β, Ala250β, Leu248β and Leu255β. The quinolin-2(1H)-one scaffold of 11e partly overlaps with the A-ring of DAMA-colchicine and forms hydrophobic interactions with Asn258β and Lys352β.
11e changes on expression and phosphorylation status of G2/M regulatory proteins in
COLO 205 cells
Analysis of cell cycle-related protein expression elucidated the mechanisms by which 11e induced G2/M arrest. Cyclin B1 and CDK1 are markers for induction of mitotic
arrest. COLO 205 cells were treated with 50 nM of 11e increased cyclin B1 and CDK1 protein levels (Fig. 9A). Given the importance of the Aurora kinase in cancer cell mitosis and metastasis, its expression was examined. Treatment of COLO 205 cells with 50 nM 11e was used to investigate the effects on aurora kinase function. As shown in Fig. 9B, 11e decreased aurora A, phosphor-aurora A, aurora B, and phosphor-aurora B expression. Histone H3 is one of the substrates of aurora B kinase.
During mitosis, aurora-B is required for phosphorylation of histone H3 on serine 10, and this might be important for chromosome condensation . We therefore examined whether 11e inhibited phosphorylation of histone H3 in COLO 205 cells by western blot. As shown in Fig. 9B, 11e decreased phosphor-H3 expression after a 6 h treatment. This finding suggests that inactivation of aurora kinases A and B is involved
in 11e-induced G2/M arrest.
11e-induced apoptosis associated with caspase-3, caspase-8, caspase-9, and PARP cleavage
To confirm the possibility that 11e-induced apoptosis is related to contributions from the intrinsic or extrinsic signal pathway, COLO 205 cells were treated with 50 nM of 11e for 6 h, 12 h, 24 h, 36 h, and 48 h, then determination of the activities of caspase-3,
caspase-8, and caspase-9 using the western blot assay. As shown in Fig. 10, 11e induced significant caspase-3, caspase-8, and caspase-9 activity. PARP cleavage is an important apoptosis marker, it’s cleavaged by caspas3 between Asp214 and Gly215 to yield the p85 and p25 fragment. Results from the western blot assay indicated that 11e induced PARP cleavage.
Intrinsic apoptotic pathway proteins are modulated during 11e-induced apoptosis The mitochondria are key organelles in the control of apoptosis, we investigated whether 11e was capable of inducing depolarization ot the mitochondrial membrane potential (Δψm) using JC-1, a lipophilic fluorescent cation that incorporates into the mitochondrial membrane. COLO 205 cells were treated with 50 nM of 11e for 6 h, 12 h, 24 h, 36 h, and 48 h, followed by staining with JC-1, confirmed apoptosis as the cause of decreased Δψm. As shown in Fig. 11A, in healthy cells with high mitochondrial Δψm. JC-1 spontaneously forms complexes knows as the JC-1 polymer (P2) with intense red fluorescence (0 h). A significant increase occurs in cells with reduced red fluorescence (P3), indicative of a change in Δψm, in the population in
which apoptosis is induced (6-36 h). Moreover, it is well known that the dissipation of Δψm causes release of cytochrome c, Apaf-1, apoptosis-inducing factor (AIF), and Endo G into the cytosol, with consequence activation of the execution phase of apoptosis. In this study, we demonstrated that mitochondrial cytochrome c, Apaf-1, AIF, and Endo G were released into the cytosol during 11e-induced apoptosis (Fig.
11B).
The Bcl-2 family proteins are key regulators of mitochondrial-related apoptotic pathways . Some of these proteins such as Bcl-2 and Bcl-xL are anti-apoptotic (pro- survival) proteins, whereas others such as Bad, Bax, and Bid are pro-apoptotic proteins. The Bcl-2 and Bcl-xL proteins located in the outer mitochondrial membrane is necessary for maintaining mitochondrial integrity. Furthermore, its phosphorylation is a common characteristic of destabilized mitochondria. The balance of pro- and anti- apoptotic Bcl-2 proteins influences the sensitivity of cells to apoptotic stimuli . Previous research has shown that an increase in the ratio of Bax/Bcl-2 within a cell to predisposes it to certain apoptotic stimuli. Bax and Bak induce the release of cytochrome c and loss of mitochondrial membrane potential, whereas Bcl-2/Bcl-xL inhibits these effects. Because 11e results in caspase-9 activation, which is also a mitochondria-mediated caspase, we sought to determine whether 11e would affect the
protein levels of these Bcl-2 family members. To verify the involvement of Bcl-2 protein activity in 11e-induced apoptosis, COLO 205 cells were treated with 50 nM of 11e for 6 h, 12 h, 24 h, 36 h, and 48 h. As shown in Fig. 11C, results indicated that 11e
reduced anti-apoptotic Bcl-2 and Bcl-xL levels and increased pro-apoptotic Bax and Bad levels, leading to changes in the Bax/Bcl-2 ratio and the release of cytochrome c and pro-caspase 9 cleavage from the mitochondria to the cytosol. Release of cytochrome c leads to activate caspase-9, which cleaves and activates caspase-3. These results demonstrate that 11e-induced cell apoptosis involves the mitochondria- dependent pathway in COLO 205 cells.
Effects of 11e on death receptors and expression of their ligands
Death receptors, on binding to their ligands, trigger apoptosis by stimulating the caspase-8 mediated caspase cascades. In this study, expression of several death receptors (Fas, DR4, and DR5) and their ligands (FasL and TRAIL) were detected in COLO 205 cells (Fig. 12A and B). Compound 11e treatment induced an increase in DR5 but not alter levels of Fas. These results suggest that DR5 up-regulation plays an important role in 11e-mediated apoptosis through extrinsic signaling pathways in COLO 205 cells.
11e-induced apoptosis is mediated via JNK signaling pathway
Mitogen-activated protein kinases (MAPK) respond to extracellular stimuli and regulate cellular activitives, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. COLO 205 cells were treated with 50 nM of 11e for 6 h, 12 h, 24 h, 36h, and 48 h investigated the effects of 11e on extracellular signal-regulated kinases (ERK1/2), JNK and p38 signaling pathway. As shown in Fig. 13, 11e decreased phosphor-ERK1/2, p38, and phospho-p38 expression. 11e induced JNK phosphorylation after 12 h incubation with 50 nM of 11e. These observations suggest that JNK activation is involved in 11e-induced apoptosis.
Discussion and conclusion
In our continuing investigations of 4-phenylquinolin-2(1H)-one (4-PQ), new 4- benzyloxyquinolin-2(1H)-one derivatives (7a–e~15a–e) were designed and synthesized. These novel molecules retain the 2-quinolone central scaffold of 4-PQ, but extend the linkage to the 4-phenyl aromatic ring by adding a CH2O moiety making a more flexible bridge. Nine compounds (7e, 8e, 9b, 9c, 9e, 10c, 10e, 11c and 11e) displayed high potency against HL-60, Hep3B, H460 and COLO 205 cells (IC50 < 1 μM) without affecting normal human Detroit 551 cells (IC50 > 50 μM). Among them,
11e exhibited the most potent activities against the above tumor cell lines with IC50
values of 0.014, 0.035, 0.04 and 0.028 μM, respectively. Notably, compound 11e exhibited improved cytotoxicity in comparison with6,7-methylenedioxy-4-(2,4- dimethoxyphenyl)quinolin-2(1H)-one, the most potent 4-phenyl-2-quinolone analogs (4-PQs) previously reported (IC50 0.4, 1.0, 0.9, and 7.4 μM against the above four tumor cell lines) . SAR study on these new compounds revealed that a 3',5'- dimethoxybenzyloxy moiety, linked at the 4-position of a 6-methoxy-2-quinolone backbone is most favorable for increased antiproliferative activity.
In the NCI-60 assay, compounds 9b, 9c, 9e and 11e showed broad-spectrum antitumor properties at the nanomolar level. Especially, compound 11e not only inhibited the growth of numerous cancer cell lines at the low micromolar range, but also exhibited high selectivity against COLO 205 (colon cancer). Furthermore, the preliminary biological studies indicated that 11e inhibited cell growth and induced apoptosis in COLO 205 cells. Herein, compound 11e has been identified as a
promising hit and candidate for future development.
Investigation of the anticancer activity of a novel 2-quinolone analog, provided data indicates that 11e exerted highly anti-proliferative activity and cytotoxicity against COLO 205 cells in a dose- and time-dependent manner (Fig. 6C), and resulted in
G2/M arrest and apoptosis (Fig. 7A). Microtubules are important cellular targets for anticancer therapy, because of their key role in mitosis . Microtubule-targeting agents bind to different side of tubulin and affect stabilization or destabilization of microtubule dynamics, including the taxanes, vinca alkaloids and colchicine . To clarify the molecular regulation of 11e in G2/M arrest, we first examined its influence of microtubules. Our data showed that 11e results in the depolymerization of microtubules in COLO 205 cells, disrupts intracellular microtubule networks in intact cells, as shown in the immunofluorescence studies (Fig. 7B). Treatment of 11e for 24 h results in microtubule changes similar to those induced by colchicine. The docked conformation of 11e was selected as a working model (Fig. 8), based on its similarity to the crystal structure of the bound conformation of DAMA-colchicine in tubulin. The superimposition of 11e and DAMA-colchicine based on A-ring showed an extensive overlapping of the 2-quinolone core, and C-ring are posed into the similar orientation.
This result supports the hypothesis that the spatial arrangement of the two aromatic A- and C-ring plays a crucial role in the activity and binding of compounds that bind to the main binding site of colchicine domain on α- and β-tubulin. These findings characterize 11e as a anti-mitotic agent.
Previous investigations have reported that cyclin B1/CDK1 complexes are involved
in the regulation of G2/M phase and M-phase transitions . Our data showed increased levels of cyclin B1/CDK1 after 11e treatment within 6 h to 24 h of treatment (Fig. 9A).
These results reveal that treatment with 11e not only directly contributes to disrupting microtubules in COLO 205 cells, but also induces accumulation of cyclin B1/CDK1.
Aurora kinases also play important roles in chromosome alignment, segregation, and cytokinesis during mitosis . Our data showed decreased aurora A, phosphor-aurora A, aurora B, phosphor-aurora B, and phosphor-H3 expression after 11e treatment (Fig.
9B). Therefore, 11e inhibited the growth of COLO 205 cells and arrested cells at the
G2/M phase through the inactivation of aurora kinases.
Apoptosis induced by anti-mitotic agents is widely known to be related to alternations of cellular signaling pathways . Compound 11e not only demonstrates broad-spectrum anticancer abilities but also provokes apoptosis, as shown by the findings of annexin V/PI in COLO 205 cells. Apoptosis regulators are extensively studied and provide the basis for novel therapeutic strategies aimed at promoting tumor cell death . To investigate the involvement of apoptosis pathways in 11e-mediated cytotoxicity, we assessed the caspase cascades. The results showed 11e induced significant caspase-3, caspase-8, and caspase-9 activities (Fig. 10). Moreover, caspase 8 is one of the regular caspases on extrinsic pathway, and caspase-9 is one of the
regular caspases on intrinsic pathway.
The intrinsic pathway is initiated with loss of membrane potential in mitochondria and then the release of cytochrome c, AIF, and Endo G from the mitochondria into the cytosol. Cytochrome c in conjunction with Apaf-1 and procaspase-9 form an apoptosome. This complex promotes the activation of caspase-9, which in turn activates caspase-3, leading to apoptosis . Proteolytic degradation of PRAP, a substrate of caspase-3, indicated that activation of caspases was involved in 11e-induced apoptosis in COLO 205 cells (Fig. 10B). To confirm that mitochondria-mediated intrinsic pathways are involved in 11e-mediated apoptosis, we further monitored the changes of mitochondrial membrane potential. Our data showed a loss of mitochondrial membrane potential in cells treated with 11e (Fig. 11A). Fig. 11B shows that 11e induces a time-dependent effect on cytochrome c, AIF, and Endo G translocation from the mitochondria into the cytosol. The Bcl-2 family proteins largely mediate the mitochondrial apoptotic pathway. These proteins include pro-apoptotic members, such as Bax and Bad, which promote mitochondrial permeability, and anti- apoptotic members, such as Bcl-2 and Bcl-xL, which inhibit the pro-apoptotic proteins effects or inhibit the mitochondrial release of cytochrome c . Over-expression of Bcl-2 increases cell survival by suppressing apoptosis. Bax levels increase in conjunction
with Bax inhibition of Bcl-2, and the cells undergo apoptosis . The present results showed that 11e treatment resulted in a decrease in the level of anti-apoptotic protein Bcl-xL, Bcl-2 and an increase in the level of pro-apoptotic protein Bax, Bad (Fig.
11C).
The extrinsic pathway is initiated by ligation of transmembrane death receptors (Fas, DR4/5, and TNFR1) with their respective ligands (FasL, TRAIL, and TNFα) trigger the formation of a death-inducing complex to active caspase-8, which in turn cleave and activate caspase-3 . Enhanced TRAIL expression and stimulation of DR4- and/or DR5-induced apoptosis had been shown in certain types of cancers, including colon cancer, ovarian cancer, prostate cancer, bladder cancer, and chronic lymphocytic leukemia . In the present study, we found that 11e treatment upregulated the expression of the DR5 protein and influenced the expression of TRAIL (Fig. 12). Caspase-8 is activated by the death receptor. Activated caspase-8 can cleave and activate downstream caspase-3. On the other hand, caspase-8 can induce Bid cleavage. The cleaved Bid causes the cytochrome c efflux from mitochondria, then activation of caspase-9 and caspase-3. We showed that induced the cleavage of full-length Bid producing the truncated Bid (t-Bid, 17 kDa) that translocated to the mitochondria.
These findings together suggest that 11e induced apoptosis by activating both intrinsic
