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Mol Cancer Ther. 2004;3:1365-1374
© 2004 American Association for Cancer Research
Discovery and mechanism of action of a novel series of apoptosis inducers with potential vascular targeting activity
Shailaja Kasibhatla1,
Henriette Gourdeau2,
Karen Meerovitch2,
John Drewe1,
Sanjeeva Reddy1,
Ling Qiu1,
Hong Zhang1,
Frederick Bergeron2,
David Bouffard2,
Quan Yang2,
John Herich1,
Serge Lamothe2,
Sui Xiong Cai1 and
Ben Tseng1
1 Maxim Pharmaceuticals, Inc., San Diego, California and 2 Shire BioChem, Inc., Laval, Quebec, Canada
Requests for reprints: Shailaja Kasibhatla, Maxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, CA 92121. Phone: 858-202-4042; Fax: 858-202-4000. E-mail: skasibhatla{at}maxim.com
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Abstract
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A novel series of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes was identified as apoptosis-inducing agents through our cell-based apoptosis screening assay. Several analogues from this series, MX-58151, MX-58276, MX-76747, MX-116214, MX-126303, and MX-116407, were synthesized and further characterized. MX-116407, a lead compound from this series, induced apoptosis with an EC50 of 50 nmol/L and inhibited cell growth with a GI50 of 37 nmol/L in T47D breast cancer cells. Treatment of cells with these analogues led to G2-M arrest, cleavage of essential proapoptotic caspase substrates, and induction of nuclear fragmentation. We identified these compounds as tubulin destabilizers with binding site at or close to the colchicine binding site. Compounds in this series were also active in drug-resistant cancer cell lines with a GI50 value for one of the analogues (MX-58151) of 2.5 nmol/L in paclitaxel-resistant, multidrug-resistant MES-SA/DX5 tumor cells. This series of compounds displayed high selectivity against proliferating versus resting cells. Interestingly, these compounds were shown to disrupt preformed endothelial cell capillary tubules in vitro and affect functional vasculature to induce tumor necrosis in vivo and are thus likely to work as tumor vasculature targeting agents. Among these compounds, MX-116407 showed capillary tubule disruption activity in vitro at concentrations well below the cytotoxic dose. In a separate study, we further characterized the antitumor efficacy and pharmacokinetic profile of this series of compounds and identified MX-116407 as a potent apoptosis-inducing agent with apparent activity as tumor vasculature targeting agent.
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Introduction
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Most chemotherapeutic agents presently used for cancer treatment were developed by screening in a growth inhibition assay for chemical substances, which inhibit the growth of neoplastic cells. Inherent to the growth inhibition assay, agents active in this screen fall into two general classes, cytostatic and cytotoxic, the latter falling into two modes of action, necrotic and apoptotic. Necrosis is typically described as a "nonspecific" form of cell death (1) and apoptotic cell death is the consequence of a series of precisely regulated events that are frequently altered in tumor cells (2). Subsequently, it was determined that the resultant clinically useful cytotoxic agents primarily act by inducing apoptosis in cancer cells (3–5). The principal proapoptotic chemotherapeutic agents used for both childhood and adult cancers target tubulin (taxanes consisting of Taxol, Taxotere, and Vinca alkaloids consisting of vincristine, vinblastine, and vinorelbine). The limitations for their widespread use are the emergence of drug-resistant tumor cells as well as dose-limiting levels of neurologic and bone marrow toxicity. Recently, a subclass of tubulin inhibitors was shown to preferentially target tumor endothelial cells while sparing the normal vasculature. These compounds, called vascular targeting agents, act by disrupting the tumor vasculature by targeting endothelial cells (6, 7). Two such agents, combretastatin A-4 phosphate prodrug (CA-4P) and ZD6126, are presently undergoing clinical trials (8, 9). Novel and synthetic compounds that induce apoptosis in cancer cells targeting the clinically validated tubulin/microtubule system as well as lacking neurotoxicity and retaining activity in multidrug-resistant tumors remain compelling for drug discovery in oncology (10–12).
The mechanism of apoptosis involves a cascade of initiator and effector caspases that are activated sequentially (4, 13). Within the caspase family, caspase-3, caspase-6, and caspase-7 have been identified as key effector caspases that cleave multiple protein substrates in cells leading to irreversible cell death (13–15). To discover novel apoptosis-inducing anticancer agents, we have developed a cell-based high throughput screening system for inducers of apoptosis using our novel pro-fluorescent caspase substrate (16). In the current study, we report the discovery of a novel series of 2-amino-4-aryl-3-cyano-4H-chromenes as potent inducers of apoptosis. From the initial hit compound, several analogues in the 4-aryl-4H-chromene series were synthesized in-house and identified as small molecule tubulin destabilizers with a binding site close to or overlapping the colchicine binding site on tubulin. These compounds were also active in vitro in multidrug-resistant cell lines and showed antivascular activity in vitro (this study) and antitumor activity in vivo in mouse tumor models (17). The lead candidate from this series, MX-116407, showed potential antivascular properties as characterized in the current and adjoining article. These 4-aryl-4H-chromenes are therefore considered as novel vascular targeting agents with MX-116407 exhibiting antitumor efficacy at well-tolerated doses.
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Materials and Methods
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Chemical Synthesis and Characterization of the Compounds
The substituted 4H-chromenes were prepared by reaction of a mixture of 3-bromo-4,5-dimethoxybenzaldehyde (0.5 mmol), malononitrile (0.5 mmol), and the desired substituted phenol (0.5 mmol) in ethanol (2 mL) and piperidine (1 mmol) at room temperature.3 The reaction mixture was stirred overnight. The precipitate formed was collected by filtration, washed with ethanol, and dried to provide the product. As an example, MX-58151 was synthesized from a mixture of 3-bromo-4,5-dimethoxybenzaldehyde, malononitrile, and 3-dimethylaminophenol and was isolated as a white solid. Melting point 200-202°C; 1H nuclear magnetic resonance (CDCl3): 6.89 (d, J = 1.8 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.72 (d, J = 1.8 Hz, 1H), 6.46-6.43 (m, 1H), 6.28 (d, J = 2.7 Hz, 1H), 4.58 (s, 1H), 4.57 (s, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 2.94 (s, 6H). Elemental analysis calculated for (C20H20BrN3O3): C, 55.83; H, 4.68; N, 9.77. Found C, 55.87; H, 4.85; N, 9.63. The compounds were also characterized by mass spectrometry and high performance liquid chromatography, with purity >95% as determined by high performance liquid chromatography.
Cell Lines and Tissue Culture
Human breast tumor cell lines (T47D, MDA-MB-435, and MCF-7), human lung and colon cancer cell lines (H1299 and DLD-1), and human leukemia cell lines (Jurkat and HL-60) were obtained from the American Type Culture Collection (Manassas, VA). Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Walkersville, MD) and peripheral blood lymphocytes were from healthy volunteers. Cells were grown either in RPMI 1640 (T47D, MCF-7, MDA-MB-435, H1299, and DLD-1,) supplemented with 10% heat-inactivated fetal bovine serum (FBS) or EGM-2 BulletKit (HUVEC, Cambrex) and maintained at 37°C with 5% CO2. The human uterine sarcoma cell line MES-SA and its doxorubicin-resistant, P-glycoprotein-overexpressing variant, MES-SA/DX5, were purchased from American Type Culture Collection and cultured in McCoy plus 10% FBS (the DX5 cells were kept in 5 µg/mL doxorubicin to maintain their resistance phenotype). Vinblastine, doxorubicin, and paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO). Combretastatin A4 active drug (CA-4) and ZD6126 were provided (see adjoining article) by Shire Biochem (Laval, Quebec, Canada). All other tissue culture–grade reagents were from Sigma-Aldrich. Cell lines were started from frozen stocks and maintained in culture for only 10 to 15 passages to avoid any spontaneous changes. All cancer cell lines used in this study are reported to be cancerous in origin (18–20).
Caspase Activation Assay
The initial compound hit from the 4-aryl-4H-chromene series was identified as an inducer of apoptosis from our cell-based screening assay (21). Our screening library is composed of commercially available diversity-based selected compounds with various scaffolds that were used in our cell-based high throughput screening assay for apoptosis induction in HL-60 cells and confirmed in T47D cells. Analogues of the initial hit were designed and synthesized to understand the structure-activity relationship as well as to improve chemical and pharmacologic properties. The analogues were then tested in the caspase assay using T47D cells. Briefly, cells were incubated with the test compounds in 384-well plates for 24 hours. Caspase-3 fluorogenic substrate N-(Ac-DEVD)-N'-ethoxycarbonyl-R110 (16) was then added to cells and the samples were mixed by agitation and incubated at room temperature for 3 hours. The fluorescent signal was measured using a fluorescent plate reader (Tecan Model Spectraflour Plus). Compounds that are considered to activate apoptosis yield a fluorescent signal (signal-to-background ratio) that is at least three times background. Compounds confirmed to be active were then tested at several concentrations to provide a dose response and EC50 calculation. The EC50 was determined by a sigmoidal dose-response calculation (XLFit3, IDBS) and represents the concentration of compound that produces 50% of maximum response.
Cell Viability Assay
Exponentially growing cells were seeded in 96-well plates at a density of 3 x 104 cells per well (cell numbers were determined using a hemocytometer) and allowed to attach overnight. Test compounds diluted in DMSO were then added at a 1:10 serial dilutions in the appropriate medium at final concentrations ranging from 10–5 to 10–10 mol/L (200 µL volume, 0.01% DMSO). Cells were exposed to drugs for 48 hours and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 2 mg/mL, 50 µL) solution in PBS was added for 4 hours. Cell viability was assessed by measuring the NADH-dependent reduction of MTT to form a formazan product as described by Plumb et al. (22). Each point represents the mean value of three measurements. The GI50 was calculated and represents the concentration of drug causing 50% inhibition of the increase in absorbance compared with control cells. For peripheral blood lymphocytes, human lymphocytes from whole blood obtained from healthy volunteers were isolated using a Ficoll-Paque Plus solution (Amersham Biosciences, Baie d'Urfé, Quebec, Canada). After separation, the lymphocytes were washed three times in PBS and then counted using a hemocytometer. Cells were then seeded at 2 x 105 per well in a 96-well round-bottomed tissue culture plates in RPMI 1640 plus 10% FBS. The effect of drugs on resting peripheral blood lymphocytes was measured using the Live/Dead Cell-Mediated Cytotoxicity kit according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Concanavalin A (0.1 µg/well, Calbiochem, San Diego, CA) was used as a mitogen. Before stimulation, appropriate dilutions of drugs were added to their respective wells. Cytotoxicity of the drugs was measured 72 hours later by adding 3H-thymidine during the last 16 hours of incubation. The cells were harvested (Tomtec, Orange, CT) and radioactivity was measured using a Wallac 1450 MicroBeta Trilux (Perkin-Elmer, Wallac, Turku, Finland). Each point represents the mean value of triplicate. The IC50 values were calculated and represent the concentration of drug causing 50% inhibition in cell proliferation.
Cell Cycle Analysis
A total of 2 x 104 MCF-7 cells were seeded into a six-well plate in MEM/10% FBS/100 mmol/L sodium pyruvate (Wisent, St-Bruno, Quebec, Canada) and incubated for 72 hours at 37°C in a 5% CO2 atmosphere. Cells were then serum starved for 48 hours before adding the drugs. At the end of drug exposure, cells were collected, washed twice in PBS, and resuspended in staining buffer (500 µL; 0.1% sodium citrate, 0.37% NP40, 20 µg/mL RNase A, 50 µg/mL propidium iodide) and incubated for 30 minutes at 4°C. The samples were read on a flow cytometer EPICS XL-MCL (Beckman Coulter, Mississauga, Ontario, Canada) and data were analyzed with the Expo 32 software (Applied Cytometry Systems, Sheffield, United Kingdom).
Jurkat cells were maintained and harvested as described above and used to determine the effect of caspase inhibitors. Cells (1 x 106) were pretreated with MX-1013 (10 µmol/L), our proprietary pan-caspase inhibitor (23), for 1 hour before treatment with MX-58151 (1 µmol/L) for 18 hours at 37°C. As a control, cells were also incubated with an equivalent amount of solvent (DMSO). Cells were harvested at 200 x g and washed twice with EDTA/PBS (5 mmol/L). Cells were then resuspended in EDTA/PBS (300 µL) and 100% ethanol (700 µL), vortexed, and incubated at room temperature for 1 hour. Samples were centrifuged at 200 x g for 5 minutes and the supernatant was removed. A solution containing propidium iodide (100 µg/mL) and RNase A (1 mg/mL) was added to the samples and incubated for 1 hour at room temperature. Samples were then transferred to 12 x 75 mm polystyrene tubes and analyzed on a flow cytometer. Flow cytometry analyses were done on FACSCalibur (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest analysis software.
In vitro Tubulin Binding Assays
Microtubule-associated protein–rich lyophilized tubulin (Cytoskeleton ML113, Cytoskeleton, Denver, CO) was assayed for the effect of the test compound on tubulin polymerization according to the manufacturer's recommended procedure. Briefly, each experimental compound (1 µL, from a 100x stock) was incubated with GTP-supplemented tubulin supernatant (99 µL) in a 96-well plate. Incubation was done in a Molecular Devices (Sunnyvale, CA) plate reader at 37°C, and absorbance readings at 340 nm were recorded every minute for 1 hour. The IC50 for tubulin inhibition was the concentration found to decrease the initial rate of tubulin polymerization by 50%.
Colchicine Binding Assay
A radioactive 96-well plate tubulin binding assay was used to measure the ability of drugs to compete with 3H-colchicine (American Radiolabeled Chemicals, St. Louis, MO) on tubulin. Briefly, 5.5 µg/well of biotin-labeled tubulin (Cytoskeleton), diluted in PIPES (80 mmol/L, pH 6.8) containing EGTA (1 mmol/L), MgCl2 (1 mmol/L, tubulin binding buffer), and GTP (1 mmol/L), was coated onto a FlashPlate streptavidin-coated 96-well plates (Perkin-Elmer, Woodbridge, Ontario, Canada) and incubated for 30 minutes at 37°C. The wells were then washed with tubulin binding buffer. A serial dilution of the test compounds (10–4 to 10–9 mol/L) were added in the tubulin binding buffer with GTP (1 mmol/L) and 3H-colchicine (0.36 µmol/L) to a final volume of 100 µL. The reaction was incubated at 37°C for 30 minutes and the wells were then washed in tubulin binding buffer. The wells were allowed to dry and ScintiSafe Econo 1 (50 µL, Fisher Scientific, St-Laurent, Quebec, Canada) was added to each well and counted for radioactivity in a Wallac 1450 MicroBeta Trilux. Triplicates at each concentration of drug were measured and IC50 values were determined.
HUVEC Tube Disruption Assay
Aliquots of Matrigel basement membrane matrix (300 µL, BD Biosciences, Mississauga, Ontario, Canada) were added to each well of 24-well plates (Fisher Scientific Ltd., Nepean, Ontario, Canada) and allowed to incubate for 1 hour at 37°C. HUVEC cells (3 x 104) were added per well in EGM-2 and incubated for 4 hours at 37°C in a 5% CO2 atmosphere to allow the cells to form tube-like structures. Compounds diluted in DMSO to their respective concentrations were added to cells and allowed to incubate for 1 hour at 37°C in a 5% CO2 atmosphere. Following the incubation, medium was gently aspirated and fresh EGM-2 was added and cells were further incubated for 24 hours. Pictures were taken with a Zeiss LSM 510 confocal microscope (Zeiss Canada Ltd., Toronto, Ontario, Canada). The effect of compounds on capillary tube disruption was evaluated by light microscopy (x40 magnification) and on growth inhibition by MTT assay.
Western Blotting
Cells were harvested by centrifugation, washed with PBS, and lysed in a buffer containing Tris-HCl (50 mmol/L, pH 7.4), NaCl (150 mmol/L), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors (Complete Protease Inhibitor Tablets, Roche Molecular Systems, Pleasanton, CA). The amount of protein in each sample was measured by the method of Bradford using a Bio-Rad Protein Assay Kit (Hercules, CA). Samples of lysate containing equivalent amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Bcl-2 phosphorylation: MDA-MB-435 cells in exponential growth were exposed to drugs at 1 and 10 nmol/L for 24 hours. Cells were then harvested by trypsinization and recovered by centrifugation (500 x g, 10 minutes) and the resulting cell pellets were lysed directly in SDS protein loading buffer (200 µL) separated on SDS-PAGE under reducing conditions. Western blot analysis was done using a mouse monoclonal anti-human Bcl-2 antibody (MAB827, R&D Systems, Inc., Minneapolis, MN) followed by enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ). Actin antibody (C-11, Santa Cruz Biotechnology, Santa Cruz, CA) was used as protein loading control. For reprobing, membranes were stripped by treating with Tris-HCl (62.6 mmol/L, pH 6.8), 2% SDS, and ß-mercaptoethanol (100 mmol/L) for 20 minutes at 50°C and then washed 5 times in TBS at room temperature.
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Results
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Caspase Activation and Growth Inhibition
A novel series of 4-aryl-4H-chromenes were identified as potent apoptosis inducers with potential antitumor activity (Fig. 1). The initial hit compound was identified by high throughput screening by measuring the induction of apoptosis in HL-60 promyelocytic leukemia cells using a proprietary pro-fluorescent substrate (20) as briefly described in Materials and Methods. MX-58151, MX-58276, MX-76747, MX-116214, MX-126303, and MX-116407 (Fig. 1) were analogues synthesized as part of the structure-activity relationship studies and were highly potent with activities in the low nanomolar range in our caspase activation assay (Table 1). These compounds were further profiled for growth inhibitory activity on a panel of normal and tumor cell lines (Table 2). A direct correlation was observed across cell lines between caspase activity and growth inhibitory activity (comparing Tables 1 and 2). Indeed, MX-126303 was the most potent compound in activating caspases, as well as growth inhibition, with potency in the subnanomolar range. These compounds were cytotoxic to all proliferating cells tested but had no effect on nonproliferating cells, similar to that observed with vinblastine and paclitaxel (Table 2). Contrary to the standard chemotherapeutics, paclitaxel and vinblastine, the 4-aryl-4H-chromenes were also effective in the MES-SA/DX5 cell line (Table 2), a P-glycoprotein-overexpressing, multidrug-resistant cell line that is highly resistant toward vinblastine and paclitaxel.
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Figure 1. A, structure of MX-58151. B, structure of other 4-aryl-4H-chromene compounds. R1 and R2 separately are hydrogen, amino, or dimethylamino or are fused five-membered ring heterocycle.
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Table 2. In vitro growth inhibitory activity of compounds on a selected panel of tumor and normal cell lines (GI50 nmol/L)
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Effect on Cell Cycle
To further understand the mechanism of action, these novel apoptosis-inducing compounds were evaluated for their effect on cell cycle by measuring DNA content. Compounds specifically arrested cells in the G2-M phase of the cell cycle leading to significant apoptosis as shown by an increase in sub-G1 content (Table 3). To confirm that the caspase activation resulted in apoptosis, we used MX-1013, a pan-caspase inhibitor previously described by us, to inhibit caspase activation (23) and then assessed apoptosis. In the presence of both MX-58151 and MX-1013, Jurkat cells were protected from apoptosis and remained growth arrested in the G2-M phase of the cell cycle (Table 3). This confirmed that the cells treated with these agents undergo a G2-M arrest prior to caspase activation and cell death.
Compounds Identified as Microtubule Destabilizers
Because G2-M block leading to apoptosis is a hallmark of antimitotic drugs, the effect of the compounds on tubulin polymerization was evaluated. MX-58151 was identified as a tubulin destabilizer with an IC50 of 0.4 µmol/L comparable with the Vinca alkaloid, vinblastine (Fig. 2). The binding site on tubulin for the 4-aryl-4H-chromenes was further characterized using a 3H-colchicine binding displacement assay and the results are summarized in Table 4. The 4-aryl-4H-chromenes displaced labeled 3H-colchicine with an IC50 range of 0.3 to 8 µmol/L, suggesting the binding occurs at or close to the colchicine site on ß-tubulin. These compounds were about as potent as colchicine in the displacement binding assay (IC50, 4.8 µmol/L). These data suggest that the 4-aryl-4H-chromene series of compounds are likely to induce apoptosis through the inhibition of tubulin polymerization.
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Figure 2. Inhibition of tubulin polymerization. Inhibition of polymerization of bovine tubulin was assayed in vitro. MX-58151 and vinblastine (used as control) were tested at different concentrations (0.01-4 µmol/L) as described in Materials and Methods.
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Microtubule inhibitors are reported to be apoptosis-inducing agents (24); observed mechanisms are phosphorylation of Bcl-2 leading to protein degradation and induction of apoptosis (25). We observed that Bcl-2 was phosphorylated in MDA-MB-435 cells treated with the 4-aryl-4H-chromenes at their apoptosis-inducing concentrations comparable with known microtubule inhibitors such as paclitaxel, vinblastine, and CA-4 (Fig. 3). As predicted, in compound-treated cells, cleavage of a caspase-3 natural substrate, poly(ADP-ribose) polymerase, was also observed (data not shown).
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Figure 3. Effect of selected 4-aryl-4H-chromenes on Bcl-2 phosphorylation. MDA-MB-435 cells were treated with 1 or 10 nmol/L of MX-126303, MX-58151, and MX-76747 for 24 hours. Vinblastine, CA-4, and paclitaxel were used as controls. At the end of the incubation period, cells were lysed and equal amount of proteins (20 µg) were electrophoresed on a 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Top, Bcl-2 was detected using an anti-human Bcl-2 antibody; bottom, ß-actin was used to verify loading of similar amounts of cell lysates.
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Effect on Endothelial Cell Organization
The ability for endothelial cells to form tubular structures when plated on a Matrigel matrix measures the three-dimensional organization of endothelial cells and offers an in vitro model of angiogenesis (26). Within 4 hours of being seeded on Matrigel, endothelial cells flattened into a reticular network on the surface of the gel; within 24 hours, the cells form a mesh of capillary-like vessels (Fig. 4A). To evaluate the potential for 4-aryl-4H-chromenes as antivascular agents, increasing concentrations of the compounds were added to endothelial cells preplated for 4 hours on Matrigel, incubated for 1 hour, and then washed and further incubated for additional 20 hours. The ability of the compounds to disrupt endothelial tubular structures was qualitatively measured and representative results are shown in Fig. 4B. Of all the compounds tested, MX-126303 was the most potent, causing tube disruption at 1 nmol/L, with a complete inhibition at 3 nmol/L (Table 5). MX-76747 was also quite potent causing tube disruption at 3 nmol/L, with complete inhibition at 10 nmol/L. CA-4 also completely disrupted tube formation at 10 nmol/L, whereas MX-58151, MX-116214, and MX-116407 were effective at 30 nmol/L. MX-58276 was less active, disrupting tube formation at 100 nmol/L.
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Figure 4. Effect of selected 4-aryl-4H-chromenes on in vitro endothelial tubule formation. Spontaneous formation of capillary-like structures by HUVECs on Matrigel was used to assess vascular disruption potential. A, photomicrographs (x40 magnification) of HUVECs seeded on plastic or Matrigel-coated plates after 4 and 24 hours. B, effect of MX-116407 and MX-126303 on preformed endothelial tubes. CA-4 and ZD6126 were used as positive controls.
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It may be important for tube disruption to occur at a concentration that is not cytotoxic to endothelial cells during the 1-hour exposure (27). The ability of the compounds to inhibit endothelial cell proliferation after a 1-hour exposure was therefore evaluated in a 48-hour cell proliferation assay. Table 5 summarizes the concentration of the compounds necessary to completely disrupt tube formation and the concentration required for growth inhibition following a 1-hour exposure. A short exposure of the cells to the compounds indicates that quite different concentration was effective in inhibiting growth, with some compounds active in the nanomolar range, whereas ZD6126, MX-58276, and MX-116407 required micromolar levels (Table 5). The ratio of the concentrations required for endothelial cell cytotoxicity versus the concentration required for endothelial tube disruption indicated that MX-116407 has the largest difference in concentration between tube disruption and endothelial cell cytotoxicity.
The exposure times required for MX-58151, MX-58276, MX-116407, and MX-126303 to induce G2-M arrest and apoptosis were further characterized. It was observed that a 3-hour exposure of cells to the compounds and subsequent washout still resulted in apoptosis by MX-58151 and MX-126303 but not by MX-58276 (data not shown) and MX-116407 (Fig. 5). Thus, MX-58276 and MX-116407, with reversible effect on cells, also showed endothelial tube disruption activity at a concentration that is several logs from the cytotoxic doses observed for these cells. This property may also be a desirable characteristic for a vascular targeting agent.
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Figure 5. Functional reversibility of selected 4-aryl-4H-chromenes. Human breast T47D tumor cells were incubated in the presence of graded concentrations of MX-58151, MX-116407, and MX-126303 for 3 or 24 hours. After the 3-hour incubation period, cells were washed and resuspended in cell culture medium. At the end of the 24-hour incubation period, cells were harvested and analyzed for DNA content by flow cytometry as described in Materials and Methods.
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Discussion
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The current study shows that the substituted 4-aryl-4H-chromenes belong to a novel class of microtubule inhibitors with potential antivascular activity in vitro (current data) and in vivo together with potent antitumor activity seen in experimental tumor models (17). This series of compounds were also active in multidrug-resistant, P-glycoprotein-positive cancer cell lines. This distinguishes the 4-aryl-4H-chromenes from paclitaxel, vinblastine, and colchicine (24, 28). This novel series of compounds is therefore part of an emerging group of vascular targeting agents, of which CA-4P and ZD6126 are the most clinically advanced (29).
Data presented in this study show that the 4-aryl-4H-chromenes bind at or near the colchicine binding site on tubulin, causing disruption of tubulin polymerization. Six compounds from this series that were synthesized as part of the structure-activity relationship, MX-58151, MX-58276, MX-76747, MX-116214, MX-116407, and MX-126303, were further characterized. These compounds seem to have similar modes of action. Consistent with a mechanism of tubulin disruption, analysis of DNA content showed a time-dependent block in the G2-M phase followed by a decrease in mitotic cells and an increase in apoptotic cells, suggesting a mitotic catastrophe as the cause for apoptosis. The accumulation of cells in G2-M with no increase in the apoptotic cells in the presence of a pan-caspase inhibitor further supports this data. Although the process by which previously described microtubule inhibitors induce apoptosis is not completely understood, it could likely be through modulation of genes that regulate apoptosis such as p53, Bcl-2, and Bcl-x (24, 30, 31). We have tested a subset of compounds on Bcl-2 phosphorylation and observed a close correlation between caspase activity and Bcl-2 phosphorylation.
Following drug treatments and subsequent washout of the free compound, we observed that the G2-M arrest and apoptosis were reversible with MX-58276 and MX-116407 and not with MX-58151 or MX-126303. This may suggest that, although the compounds are structurally similar, they may have different tubulin binding kinetics, leading to differences in the observed vascular disruption activity. The improved therapeutic index observed with CA-4P compared with colchicine has been correlated to their differences in the on/off rate of binding to tubulin (32–34), leading to the hypothesis that a successful vascular targeting agent would have reversible binding kinetics and relatively rapid clearance in vivo (27). It has been suggested that the reversible activity of CA-4P and ZD6126 distinguishes them from colchicine, the narrow therapeutic window of which has been ascribed to its pseudo-irreversible binding to tubulin (34, 35). Furthermore, CA-4, ZD6126, MX-58276, and MX-116407 showed a 40- to 100-fold difference between growth inhibition and tube disruption, thereby affecting endothelial cell morphology at noncytotoxic concentrations. This observation is characteristic of antiangiogenic and vascular targeting agents (34). Indeed, CA-4P and ZD6126 are thought to produce antitumor activity by selective destruction of tumor vasculature at noncytotoxic doses (36). These observations, together with our in vitro data, would suggest that MX-116407, although less potent than MX-126303, would have better antitumor activity. Consistent with this, we have observed in vivo antitumor activity with MX-116407 at doses that are significantly lower than the toxic dose (17).
In summary, the 4-aryl-4H-chromenes are a new class of antimitotic and potential antivascular agents with in vitro properties that may overcome some of the limitations of colchicine, vinblastine, and paclitaxel. The lead candidate, MX-116407, is characterized as a potent activator of caspases that induces apoptosis in all cell lines tested (11-42 nmol/L) with potential vascular targeting activity. MX-116407 showed promising antitumor activity in several in vivo mouse tumor models with evidence of antivascular activity (17). MX-116407 and analogues that showed antivascular activity may prove to be useful for the treatment of a wide range of neoplastic diseases.
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Acknowledgments
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We thank Candace Crogan-Grundy for technical assistance.
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Footnotes
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Grant support: NIH grant 1R43 CA90120-01 (Maxim Pharmaceuticals).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: S. Kasibhatla and H. Gourdeau contributed equally to this work.
3 W. Kemnitzer et al. Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay; structure-activity relationships of the 4-aryl group. J Med Chem. In Press 2004. 
Received 5/ 7/04;
revised 7/16/04;
accepted 9/15/04.
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Abstract
Introduction
