Design and Synthesis of Novel Anti-metastatic Hypoxic Cytotoxin TX-2137 Targeting AKT Kinase

Materials and Methods

Materials. Nuclear magnetic resonance (NMR) ¹H spectra were obtained using a JNM-EX400 spectrometer (Jeol, Tokyo, Japan) at 400 MHz. Solvents were evaporated under reduced pressure on a rotary evaporator. Thin-layer chromatography was performed on glass-backed silica gels (Merck 60 F254; Merck Japan, Tokyo, Japan) and components were visualized using ultraviolet (UV) light. Column chromatography was performed using a silica gel (60 N, spherical neutral; 40-50 μm; KANTO Chemical, Tokyo Japan). The molecular orbital structure was calculated by WinMOPAC 3.0 (PM3, Fujitsu, Kawasaki, Japan).

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Figure 1.

Design of TX-2137 based on tirapazamine and AKT1/2 inhibitor.

Cell culture. B16-F10 mouse melanoma cells (kindly provided by Dr. Tsuruo, Tokyo University, Tokyo, Japan) and A549 human lung carcinoma cells (supplied by Dr. Kondo, Kyoto University, Kyoto, Japan) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Wako Pure Chemical, Osaka, Japan), while HT-1080 human sarcoma cells (purchased from American type culture collection, Manassas, VA, USA) were cultured in Eagle's minimum essential medium (EMEM) (Wako Pure Chemical). MKN-45 human adenocarcinoma cells (Dr. Suzuki, Fukushima Medical College, Fukushima, Japan) and U87MG human neuronal glioblastoma cells (American Type Culture Collection Manassas, VA, USA) were maintained in RPMI-1640 medium (Wako Pure Chemical). All media were supplemented with 10% fetal bovine serum and cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C. Hypoxic culture was performed in a humidified atmosphere of 0.1% O₂ at 37°C using an AnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan).

Synthesis of 3-chloro-1,2,4-benzotriazine 1-oxide. 2-Nitroaniline (10 g, 72.4 mmol; Sigma-Aldrich Japan, Tokyo, Japan) and cyanamide (6.0 g, 144.8 mmol; Tokyo Chemical, Tokyo, Japan) were melted at 100°C. Thereafter, 36% HCl (40 ml; Wako Pure Chemical) was slowly added and the mixture was stirred at 100°C for 2 h then the solution was cooled to room temperature. To this mixture, 7.5 M NaOH (200 ml; Wako Pure Chemical) was slowly added and stirred at 100°C for 2.5 h. Finally, the solution was cooled to room temperature and water (200 ml) was added and a substance precipitated from the solution was filtered with filter paper to yield compound 1 (3-amino-1,2,4-benzotriazine 1-oxide) as a yellow solid (17). 3-Amino-1,2,4-benzotriazine 1-oxide was dissolved in trifluoroacetic acid (60 ml; Wako Pure Chemical) at 5°C and sodium nitrite (4.8 g, 71.4 mmol; Tokyo Chemical, Tokyo, Japan) was added. The solution was stirred at room temperature for 2 h and was added dropwise to ice/water. The precipitate was filtered, washed with water, and dried. The solid produced (compound 2; 3-hydroxy-1,2,4-benzotriazine 1-oxide) was suspended in phosphoryl chloride (27 ml; Wako Pure Chemical) and N,N-dimethylformamide (5 ml; Wako Pure Chemical) and stirred at 100°C for 1 h. Once cooled, the solution was added dropwise to ice/water, the precipitate was filtered, washed with water, and dried. The precipitates were purified by silica-gel column chromatography with dichloromethane (CH₂Cl₂) to give compound 3 (3-chloro-1,2,4-benzotriazine 1-oxide) (1.9 g, 14.8% yield) (18).

Synthesis of TX-2137. 4-Aminophenol (1.0 g, 9.2 mmol; Tokyo Chemical) and imidazole (1.2 g, 18.3 mmol; Tokyo Chemical) were suspended in CH₂Cl₂ under a nitrogen atmosphere. tert-Butylchlorodimethylsilane (2.1 g, 13.7 mmol; Tokyo Chemical) was added and the solution was stirred at room temperature for 1 h, poured into saturated NaCl solution and extracted with CH₂Cl₂. The organic layer was evaporated to give compound 4 (1.5 g, 73.1% yield). Next, compound 4 (0.25 g, 1.1 mmol) was dissolved in CH₂Cl₂. Triethylamine (156 μl, 1.1 mmol; Wako Pure Chemical) and 3-chloro-1,2,4-benzotriazine 1-oxide (0.1 g, 0.6 mmol) were added and the solution was stirred at room temperature for 2 h, poured into water and extracted with CH₂Cl₂. The organic layer was evaporated and purified by silica-gel column chromatography with CH₂Cl₂ to give compound 5 (0.17 g, 84.1% yield). Next, compound 5 (0.1 g, 0.27 mmol) and NaHCO₃ (0.045 g, 0.54 mmol; Wako Pure Chemical) were dissolved in CH₂Cl₂. m-Chloroperoxybenzoic acid (m-CPBA) (0.093 g, 0.54 mmol; Tokyo Chemical) was added and the mixture stirred at room temperature for 24 h. The solution was collected using a filter paper, the filtrate was evaporated and the residue was purified by silica-gel column chromatography with ethyl acetate (EtOAc) and EtOAc/methanol (MeOH), forming compound 6 (0.062 g, 54.9% yield). Finally, compound 6 (0.61 g, 1.6 mmol) was dissolved in tetrahydrofuran at 5°C and 1.0 M of tetrabutylammonium fluoride solution (3.2 ml, 3.2 mmol; Sigma-Aldrich Japan) was added. The solution was stirred at room temperature for 5 min, solvents were evaporated and the residue was purified by silica-gel column chromatography with 10%-MeOH/EtOAc to obtain compound 7 (TX-2137) (0.37 g, 84.7% yield) in the form of a purple powder; ¹H NMR [(CD₃)₂SO] δ 9.96 (s, 1H, PhOH), 9.41 (s, 1H, PhNH), 8.22 (t, J=8.5 Hz, 2H), 7.97 (td, J=7.8, 1.4 Hz, 1H), 7.61 (td, J=7.8, 1.4 Hz, 1H), 7.38 (d, J=8.7 Hz, 2H), 6.79 (d, J=8.7 Hz, 2H); MS (EI) m/z 270 (M⁺, 13), 254 (68), 238 (56), 210 (100); Anal. calcd for C₁₃H₁₀N₄O₃: C, 57.78; H, 3.73; N 20.73. Found C, 57.56; H, 3.76; N, 20.75.

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Figure 2.

Synthesis of the TX-2137. Reagents: a: Cyanamide, 36% HCl, 7.5 M NaOH; b: Trifluoroacetic acid, sodium nitrite; c: phosphoryl chloride, N,N-dimethylformamide; d: tert-butylchlorodimethylsilane, imidazole, CH₂Cl₂; e: triethylamine, CH₂Cl₂; f: m-chloroperoxybenzoic acid, NaHCO₃, CH₂Cl₂; g: 1.0 M of tetrabutylammonium fluoride solution, tetrahydrofuran.

In vitro WST-8 assay to evaluate the effect of TX-2137 on cell proliferation. In vitro cell proliferation was examined using a colorimetric assay with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, B16-F10, HT-1080 and MKN-45 cells were seeded at a density of 5×10³ cells/well in a 96-well plate and TX-2137, dissolved in dimethyl sulfoxide, was added to the culture medium at concentrations between 0.1-100 μM. After 72 h incubation, the medium was replaced with fresh medium containing the WST-8 reagent. After 3 h, the absorbance in each well was determined at 450 nm (with a reference wavelength of 620 nm) using an ImmunoMini NJ-2300 microplate spectrophotometer (BioTec, Tokyo, Japan, Tokyo, Japan). The percentage of cell growth inhibition was calculated by applying the following formula: % of cell growth inhibition=(1-[T/C]) ×100, where C and T were the mean absorbances of the control group and treated group, respectively. The 50% inhibitory concentration (IC₅₀) value was measured graphically from the dose–response curve with at least three drug concentration points.

In vitro hypoxia-selective cytotoxicity of TX-2137 using WST-1 assay. A549 cells were seeded in two 96-well plates at a density of 3×10³ cells/well and incubation for 24 h. After 24 h, TX-2137 was added at the final concentrations of 0.1 to 30 μM and each plate was incubated either normoxic (21% O₂) or hypoxic (0.1% O₂) conditions for 24 h. After 24 h, each well was washed with 1×PBS and fresh medium containing the WST-1 reagent (Wako Pure Chemical) was added. The absorbance was measured at a wavelength of 450 nm using a Tecan Infinite M200 microplate reader (Tecan, Männedorf, Switzerland).

AKT inhibition by TX-2137 by western blot analysis on U87MG cells. The harvested cells were homogenized in RIPA buffer (Thermo Scientific, IL, USA) supplemented with protease inhibitors (cOmplete™, Mini, EDTA-free®, Roche Applied Science Tokyo, Japan). After 5 min of centrifugation at 13,000 × g, the protein concentration in the supernatant was assayed with BCA reagent (PIERCE, Tokyo, Japan). After reduction in 60 mM Tris-HCl buffer (pH 6.8) containing 10% glycerol, 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol (DTT) and 0.002% bromophenol blue, 50 μg of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (BIO-RAD, Hercules, CA, USA) membranes. The membranes were immersed for 1 h in blocking buffer [5% non-fat dry milk or 5% bovine serum albumin (in tris-buffered saline and then incubated with primary antibodies to AKT1, AKT2, AKT3 (1:1,000; Cell Signaling, Danvers, MA, USA), or phospho-AKT (Ser473) (1:2,000; Cell Signaling in Can Get Signal Solution 1 (TOYOBO, Osaka, Japan). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies in Can Get Signal Solution 2 (dilution 1:3,000; TOYOBO). The protein–antibody complexes were detected with Amersham ECL® plus (GE Healthcare, Little Chalfont, Buckinghamshire, UK) using a Lumino image analyzer (Image Quant LAS4000 mini; GE Healthcare) and NIH ImageJ 1.46 software (http://rsb.info.nih.gov/ij/). Each experiment was repeated three times.

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Figure 3.

AKT inhibition assay of TX-2137 by western blot analysis and the cell viability in U87MG cells. Each experiment was performed four times, data are the mean±SD. A: Western blot analysis to detect AKT protein expression in U87MG cells. Quantitative analysis using Image J (B, C) and the effect of TX-2137 on the cell viability of U87MG (D). Significantly different from the control at *p<0.05.

Assay of MMP9 inhibition by TX-2137 by gelatin zymography. To analyze the effect of TX-2137 on the activation of pro-MMP2 into its activated form which is induced by MT1-MMP and on the expression and secretion of MMP2 and MMP9, HT1080 cells (5×10⁴ cells/ml) were seeded into a 48-well culture plate and cultured in 100 μl of complete media for 24 h. After washing twice with serum-free DMEM, the cells were further cultured in OPTI-MEM (Invitrogen) with various concentrations of TX-2137 for 3 h. Then, an aliquot of conditioned medium was analyzed by gelatin zymography. Zymography was performed with an 10% SDS-polyacrylamide gel containing 0.1% gelatin as described previously (19). After electrophoresis, SDS was replaced by Triton X-100, followed by overnight incubation in Tris-based buffer. Gels were stained with Coomassie Brilliant Blue, and gelatinolytic activity of MMP2 and MMP9 was detected as clear bands in a background of uniform staining.

In vivo anti-metastatic activity of TX-2137 using chick embryo model. Fertilized chicken eggs (Plymouth Rock × White Leghorn) were obtained from the Goto Chicken Farm (Gifu, Japan). The assay was performed as originally described by Endo et al. (20). Briefly, 5×10⁴ cells were injected into the chorioallantoic membrane (CAM) veins of the chicken egg with a 30G needle 11 days after fertilization and eggs were incubated for a further 3 days. TX-2137 (in 0.1 ml) or doxorubicin (Kyowa Hakko Kirin, Tokyo, Japan) was administered into the CAM vein. Doses were as follows: 40 μg/egg for doxorubicin, 62.5 μg/egg and 125 μg/egg for TX-2137. After drug administration, eggs were incubated for another 4 days. Embryo livers were then dissected 7 days after tumor cell injection, and the total DNA was extracted. A fragment of mouse β-globin gene in the oncogene-transformed cells that colonized liver tissue was amplified by 25 cycles of polymerase chain reaction (PCR) using species-specific primers. Each PCR cycle consisted of 1 min of denaturing at 94°C, 1 min of annealing at 50°C, and 1.5 min of extension at 72°C. The amplified fragment (633 base pairs) was separated by electrophoresis in a 1.2% agarose gel. The signal intensity of the band in agarose gel was measured using image processing program Image J. Oligonucleotide primers were as follows: sense primer Mgp1 (5’-GGA TCA GTT GCT CCT ACA TT-3’) and antisense primer Mgp5 (5’-TAT CCG AAC TCT TGT CAA CA-3’).

Statistical analysis. Data are expressed as the mean and standard deviations of at least three independent experiments. The statistical significance of the differences between the results was analyzed using Student's t-test. A p<0.05 was considered statistically significant.