Cyclopropane-Containing Polyamine Analogues Are Efficient Growth Inhibitors of a Human Prostate Tumor Xenograft in Nude Mice

Polyamine analogues 7, 10, 18, 27, and 32 containing cyclopropane rings were obtained by chemical synthesis. Their antineoplastic activities were assessed against the cultured human prostate tumor cell lines DU-145, DuPro, and PC-3. Decamines 32 and 27 exhibited variable levels of cytotoxicity against all three cell lines, while 7, 10, and 18 were efficacious against DU-145 and DuPro. Maximum tolerated doses (MTD) for all five compounds in a NCr-nu mouse model were determined at dosing schedules of q1d × 5 (ip) in two cycles with a break of 10 days between cycles. Their antitumor efficacies were then tested against DU-145 tumor xenografts in mice treated with all five agents at their respective MTDs. In addition, the efficacies of 7 and 10 against the same tumor xenograft were assessed at doses below their respective MTDs. In all experiments, administration began two weeks after tumor implantation. All compounds efficiently inhibited tumor growth for up to 50 days postimplantation, with negligible animal body weight loss. Tetramine 10 and hexamine 18 were the most efficient among the five analogues in arresting tumor growth. Tetramine 10 containing two cyclopropane rings had the lowest systemic toxicity as reflected in animal body weight loss. It was further assessed at a weekly administration regimen of (q1w × 4) in two cycles with a four-week break between the cycles. At this dosing schedule, 10 again efficiently arrested tumor growth with negligible effect on animal body weight. Tetramine 10 also arrested the growth of large tumors (ca. 2000 mm3) treated 66 days postimplantation. Studies on the metabolism of 10 showed that it accumulates in tumor within 6 h after the end of administration and reached a maximum level 72 h after cessation of dosing. Intracellular concentrations of 10 in liver and kidney were much smaller when compared to those in the tumor when measured 72 h after cessation of dosing. In liver and kidney, the deethyl metabolites of 10 accumulated over a 96 h period after cessation of dosing

Additional file 1: of Pharmacological inhibition of ABCC3 slows tumour progression in animal models of pancreatic cancer

Figure S1. MCI-715- a specific inhibitor of ABCC3. Figure S2. Pharmacological inhibition of ABCC3 with MCI-715 reduces the activity of pSTAT3 Y705 and HIF1Îą. Figure S3. MCI-715 treatment in the animal models of PDAC. Figure S4. MCI-715 treatment induces apoptosis in the KPC primary cell line. Figure S5. Modulation of ABCC3 activity influences the viability of epithelial and stromal PDAC cells. (ZIP 4873 kb

Supplemental Figure 4A from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

NMR of ADT 061.</p

Supplemental Figure 3C from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

HPLC chromatogram of compound 5.</p

Supplemental Figure 4C from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

HPLC chromatogram of ADT 061</p

Supplemental Figure Legends from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

Supplemental Figure Legends</p

Supplemental Figure 2C from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

HPLC chromatogram of compound 4.</p

Supplemental Figure 6 from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

ADT 061 pharmacokinetics and tissue distribution. ADT 061 was detected in plasma at a concentration of 2.8 µM 0.5 hr after a single oral administration of a dose of 100 mg/kg in 0.5% CMC/0.25% T80 in water to female C57BL/6 mice and reached plasma Cmax of 4.9 µM 4 hr after administration, which is >10 times higher than human colon cancer cell growth inhibition IC50 values. ADT 061 reached a Cmax in colon mucosa within 2 hr after oral administration (62.6 nmol/g) and was still present at concentrations exceeding IC50 values 8 hr after administration (28.9 nmol/g). ADT 061 concentrations in lungs, ovaries, and uterus (1-2 nmol/g) were appreciably lower than colon mucosa levels, while the ADT 061 concentration in the brain was nearly undetectable 2 and 8 hr post treatment (0.1 and 0.2 nmol/g, respectively) (n=4, mean {plus minus} SD).</p

Supplemental Figure 1 from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

Reaction steps for synthesis of ADT 061 (a) 3-(4-methoxyphenyl)-2-methylacrylic acid (compound 1): 4-Methoxybenzaldehyde (219 g, 1.61 mole), propionic anhydride (315g, 2.42 mole), and sodium propionate (155g, 1.61 mole) were stirred at 140 ºC until a clear solution was achieved (~48 h). The solution was cooled to room temperature and poured into 8L of ice water. The precipitation formed was collected by filtration, transferred into a 2L round-bottom flask, and refluxed in 1.5 L of ethanol for 3h. The flask was stored at -20 ºC overnight. Compound 1 was obtained as a colorless crystal (213g) after filtration. Compound 1 is also commercially available from AstraTech (# W18287,95% purity). (b) 3-(4-Methoxyphenyl)-2-methylpropanoic acid (compound 2): p-Methoxy-α-methylcinnamic acid (213g) and palladium on active charcoal (Pd-C, 10%, 2g) were suspended in 1.5 L of 95% ethanol and warmed to 60 ºC in a water bath. The warm suspension was immediately put on a catalytic hydrogenator, treated with hydrogen (40 psi). The reaction was completed within 45 min, as indicated by the complete dissolving of the solid starting material. The catalyst was removed by filtration, and the filtrate was concentrated to give compound 2 as a colorless oil (215g), which was used for the next reaction step without further purification and characterization. Compound 2 is also commercially available from PharmaBlocks (# PBTQ6955, 97% purity). (c) 6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one (compound 3): Phosphoryl acid (98%, 500g) and polyphosphoryl acid (PPA, 450g) were pre-warmed separately to 70{degree sign}C in a water bath before being transferred to a 2L, three-necked flask equipped with a thermometer, a mechanical stirrer, and a dropping funnel. The flask was kept in an oil bath at 55-60 {degree sign}C for 1 h until smooth stirring was achieved. Compound 2 (95 g) was added dropwise over a period of 5 min. The temperature was carefully raised to 70-75{degree sign}C for 15 min. The reaction solution was immediately transferred to 6L of ice water and stirred until the PPA was completely dissolved. The mixture was extracted with ethyl ether (1L Ã- 3). The organic layer was dried with sodium sulfate and concentrated. The residue was purified by a silica gel column and eluted with hexane and acetone. Purity was monitored by TLC. Compound 3 was obtained as a clear, colorless oil (76g) that was used for the next reaction step without further purification and characterization. Compound 3 is also commercially available from AstraTech (# 96721, 95% purity). (d) 2-(5-Methoxy-2-methyl-1H-inden-3-yl) acetic acid (compound 4): A mixture of compound 3 (76g, 0.39 mole), cyanoacetic acid (36.6g, 0.43 mole), acetic acid (40 mL) and ammonium acetate (9 g) in 500 mL of toluene was refluxed with stirring for 48h. The liberated water was collected by a Dean-Stark trap. The reaction mixture was cooled and filtered, and the filtrate was concentrated. The residue was dissolved in 250 mL of ethanol. A solution of potassium hydroxide (90 g) in 360 mL of water was added to the solution. The mixture was refluxed overnight under argon. The organic solvents were removed under vacuum, and the remaining aqueous solution was diluted with 500 mL of water, extracted with ethyl ether, boiled with active charcoal for 1h, then filtered. The filtrate was acidified with 6N HCl and sonicated for 2h. The precipitate was collected by filtration, washed with water, refluxed in 180 mL of acetone for 2h, and stored at -20 ºC overnight. The precipitate was collected by filtration and dried under vacuum to give compound 4 as a colorless solid (42g). The mother liquor was concentrated, and a second crop (3.5g) was obtained after repeating the recrystallization procedure. The structure was confirmed by mass spectrometry (M+H: 219.04) and 1H-NMR (DMSO-d6) Î'(ppm): 7.240(d, 1H); 6.804(d, 1H); 6.658(m, 1H); 3.781 (s, 3H); 3.741(s, 2H); 3.250(s, 2H); 2.050(s, 3H). Purity was determined by HPLC (98.7%) (Suppl. Fig. 2a-c). (e) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid (compound 5): Compound 4 (32.7g, 0.150 mole), 3, 4, 5-trimethoxybenzaldehyde (35.3g, 0.180 mole) and sodium methoxide (21g) in 300 mL of anhydrous methanol were refluxed in a 500 mL round-bottomed flask overnight. After cooling, the reaction mixture was diluted with 150 mL of acetone and sonicated for 30 min. The precipitate was collected by filtration and washed twice with acetone, then dissolved in 150 mL of water. The aqueous solution was acidified with 6N HCl and sonicated for 30 min. The precipitate was collected by filtration, washed with water, recrystallized from methanol, and dried under vacuum to afford compound 5 as a yellow crystal (51.7g). The structure was confirmed by mass spectrometry (M+H: 397.11) and 1H-NMR (DMSO-d6) Î'(ppm): 12.377 (s, 1H); 7.429 (d, 1H); 7.139 (s, 1H); 6.877(s, 2H); 6.669 (d, 1H); 6.546(dd, 1H); 3.780 (s, 3H); 3.780(s, 3H); 3.737(s, 6H); 3.545(s, 2H); 21.121(s, 3H). Purity was determined by HPLC (99.64%) (Suppl. Fig. 3a-c). (f) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N-(pyridin-3-yl) acetamide (ADT 061): To a 500 mL round-bottomed flask containing compound 5 (39.6g, 0.100 mole) and 300 mL of anhydrous dichloromethane, 1,1'-carbonyldiimidazole (CDI, 20.0g, 0.115 mole) was added in portions and stirred for 15 min at room temperature. 3-Amino-pyridine (10.6g, 0.115 mole) was added, followed by anhydrous pyridine (80 mL). The solution was stirred at 40{degree sign}C overnight, treated with 5g of sodium hydroxide in 20 mL of water for 10 min, then diluted with 300 mL of dichloromethane, and washed with water (250 mL x 3). The solution was concentrated, and the residue was purified with a silica gel column. Recrystallization from methanol and then from ethyl acetate afforded ADT 061 as a yellow crystal (32.7g). The structure of ADT 061 was confirmed by mass spectrometry (M+H: 473.2) and 1H-NMR (DMSO-d6) Î'(ppm): 10.453(sb, 1H); 8.765(d, 1H); 8.276(dd, 1H); 8.044(m, 1H); 7.431(d, 1H); 7.351(dd, 1H); 7.142(s,1H); 6.914(s, 1H); 6.880(s, 2H); 6.542(m, 1H); 3.781 (s, 6H); 3.731(s, 3H); 3.726(s, 3H); 3.696(s, 2H); 2.194(s, 3H). Purity was determined by HPLC (>99.7%) (Suppl. Fig. 4a-c).</p

Supplemental Figure 5 from Suppression of Colon Tumorigenesis in Mutant <i>Apc</i> Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

Eadie-Hofstee plot of data from Fig. 2C. Error bars = SEM</p