All compounds were prepared as previously reported78
and were tested as their per-HCl salts. Drugs readily dissolved in H2
O to at least 10 mM concentrations. Compounds were characterized by TLC (using 4:1:1:2 isopropanol/pyridine/glacial acetic acid/H2
H and 13
C NMR, LC/MS and elemental analysis and all data were consistent with structures assigned and supported >97% purities. Characterization details are given below for several representative compounds.
Representative acyl products
L-Lys-ε-(palmitoyl)-N1-spermine (10) - TLC analysis: Rf = 0.19. 1H NMR (D2O, δ): 3.93 (1H), 3.45 (1H), 3.03 (13H), 2.12 (2H), 2.02 (2H), 1.85 (4H), 1.75 (s, 4H), 1.43 (4H), 1.32 (2H), 1.11 (24H), 0.72 (t, 3H). 13C NMR (D2O, ppm): 175.7, 169.8, 53.4, 46.7 (m), 45.2, 44.6, 38.8, 36.5, 36.1, 31.9, 30.4, 30.0 (m), 29.9 (m), 29.6 (m), 29.4, 28.2, 25.7, 25.5, 23.6, 22.8, 22.7, 22.3, 21.7, 13.8. LC/MS (ret time, 7.2 min, 99% purity), calcd for C32H68N6O2 m/z 568, obsd 569 (MH+). Anal. Calcd for C32H72Cl4N6O2: C, 53.77; H, 10.15; N, 11.76. Found: C, 53.51; H, 10.09; N, 11.51.
D-Lys-ε-(palmitoyl)-N1-spermine (11) - TLC analysis: Rf = 0.19. 1H NMR (D2O, δ): 3.94 (1H), 3.47 (1H), 3.06 (13H), 2.13 (2H), 2.04 (2H), 1.87 (4H), 1.75 (4H), 1.47 (4H), 1.36 (2H), 1.16 (24H), 0.78 (3H). 13C NMR (D2O, ppm): 175.7, 169.8, 53.4, 47.2 (m), 45.6, 44.8, 39.0, 36.6 (m), 36.1, 31.9, 29.8 (m), 29.6, 29.3, 28.4, 25.9, 25.7, 23.8, 22.8, 23.1, 22.8, 22.1, 14.0. LC/MS (ret time, 7.2 min; 98% purity), calcd for C32H68N6O2 m/z 568, obsd 569 (MH+). Anal. Calcd for C32H72Cl4N6O2: C, 53.77; H, 10.15; N, 11.76. Found: C, 53.38; H, 10.20; N, 11.52.
Representative alkyl product
L-Lys-ε-(n-hexadecyl)-N1-spermine (27) - TLC analysis: Rf = 0.11. 1H NMR (D2O, δ): 3.97 (1H), 3.48 (1H), 3.04 (15H), 2.04 (2H), 1.91 (4H), 1.75 (8H), 1.48 (2H), 1.22 (26H), 0.91 (3H). 13C NMR (D2O, ppm): 168.8, 53.4, 48.0, 47.3, 47.1 (m), 45.4, 44.7, 36.7, 32.0, 30.6, 29.9 (m), 29.8, 29.5, 29.4, 29.1, 26.5, 25.9, 25.6, 25.5, 23.8, 22.9, 22.8, 21.7, 13.9. LC/MS (ret time, 7.2 min; 97% purity), calcd for C32H70N6O m/z 555, obsd 556 (MH+). Anal. Calcd for C32H75Cl5N6O·3/2H2O: C, 50.29; H, 10.29; N, 11.00. Found: C, 50.30; H, 10.05; N, 10.67.
Cell Growth Assay
All cell lines were obtained from ATCC (Manassas, VA) and cultured in the recommended media, serum, and CO2 concentration. Medias were obtained from Mediatech, Inc. (Herndon, VA) and serums from Gibco BRL (Gaithersburg, MD). 50 U/ml penicillin, 50 µg/ml streptomycin and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD) were included in all cultures. When cells were cultured with inhibitors, 1.0 mM aminoguanidine was included to inhibit serum amine oxidase activity. Cells were plated in 96-well plates such that they would be in log growth for the duration of the assay. The day after plating, inhibitors were added to the cells, and growth, if any, permitted to continue for six days in the presence 0.5 µM spermidine to insure that any growth inhibition was not the result of depletion of external polyamines in the media. At the end of the six days, cell growth was measured by MTS/PMS dye assay (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI). EC50 represents the concentration of inhibitor that resulted in 50% of maximum growth inhibition achievable in the presence of both DFMO (5.0 mM in all cell lines) and inhibitor (at different concentrations depending in part on the cell line used) compared to controls. IC50 represents the concentration of inhibitor that resulted in 50% maximum growth inhibition when used alone. EC50 values are shown in and .
Polyamine Transport and Ki Assays
[2,9-3H]spermidine from DuPont NEN (Boston, MA) was added alone or simultaneously with inhibitors to 24-well plates containing MDA-MB-231 cells in log growth. The cells were incubated at 37°C for 15 min to determine initial rate polyamine uptake. The cells were then washed three times with cold PBS, lysed with 0.1% SDS, and the amount of polyamine incorporation into the cells was determined by scintillation counting of the cell lysates. To determine a Ki, four radioactive substrate concentrations (0.3–3 µM) and five inhibitor concentrations (0.01–1.0 µM) and a control were tested. The Ki values were determined using double reciprocal Lineweaver-Burke plot analyses. Ki values were determined from linear equations derived from graphing the slopes of Lineweaver-Burke plot vs. inhibitor concentration, with Ki = y-intercept / slope.
HPLC Analysis of Dansylated Derivatives
Polyamine levels and analogs were determined by prederivatization by dansyl chloride followed by reverse-phase HPLC by fluorescent detection.79
Mouse Squamous Cell Carcinoma Model
The K6/ODC mouse model on the FVB/N strain background instead of the original C57BL/6J background12
was used in all experiments. All experiments with mice were approved by the Institutional Animal Care and Use Committee of Lankenau Institute for Medical Research. To induce SCCs, newborn (1 day old) pups were treated once topically with 100 nmol of DMBA dissolved in 50 µl acetone. Both male and female mice were used. SCCs typically developed on the treated area (dorsal skin) beginning 5 weeks later. SCC-bearing mice were used in experiments between 8 and 18 weeks of age. Based on pilot data indicating a general lack of responsiveness of SCCs of the face and head, these tumors were excluded from analyses.
There was substantial variability in tumor growth rates, even among multiple SCCs in the same mouse. In an attempt to control for this variability, tumor-bearing mice were randomized based on tumor size distribution to one of four treatment groups: (1) control (phosphate-buffered saline (vehicle for PTI); (2) DFMO (either 0.25% or 0.5% in drinking water); (3) PTI analog and (4) DFMO plus PTI analog. Thus, each treatment group consisted of mice bearing SCCs with approximately equal tumor size distributions (small→large). DFMO solutions were changed every 4 days or less. Compound 11 was dissolved in phosphate-buffered saline at a concentration of 9.75 mM (5 mg/ml) and injected i.p at a dose of 0.7 mg/kg twice daily in early morning and late afternoon, except on weekends when mice were injected once. Animals were observed daily for signs of distress due to tumor burden and/or drug treatment. Tumor volume was calculated according to the equation V = l × w2/2 where l = length (longest dimension) and w = width. Responses of individual tumors to treatment were expressed as “ratio of tumor volume,” which is defined as the tumor volume after treatment divided by the initial (pretreatment) volume.