All chemicals, solvents, reagents and enzymes were purchased from Sigma-Aldrich (St. Louis, MO.) and used without further purification, with the exceptions of laromustine which was provided by Vion Pharmaceuticals Inc., (New Haven, CT.), the fluorescent dye Hoechst 33258 which was purchased from Molecular Probes, Inc., (Eugene, OR.) and purified recombinant human AGT which was a kind gift from Dr. Joann Sweasy (Yale Medical School, New Haven, CT).
Melting points were determined on a Thomas-Hoover Unimelt melting point apparatus and are uncorrected. NMR spectra were recorded on a Varian EM-390 (90 MHz) or Bruker Avance DRX-400 (400 MHz) NMR spectrometer with tetramethylsilane as an internal standard. Mass spectra were recorded on a VG-ZAB-SE mass spectrometer in the fast atom bombardment mode (glycerol matrix). Column chromatography was conducted with Merck silica gel 60, 230–400 mesh. Thin layer chromatography was performed on EM pre-coated silica gel sheets containing a fluorescent indicator. High resolution mass spectra (HRMS) were obtained at the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. The purity of the tested compounds was determined by analytical HPLC, using a Beckman System Gold HPLC system, comprising a 168 UV/Vis detector, a 502 autosampler, and a 127P solvent module monitoring at 280 nm with a 40 nm bandwidth. The purity of the tested compounds was >95% in each and every case.
(6-(Benzyloxy)-2-((((4-nitrobenzyl)oxy)carbonyl)amino)-9H-purin-9-yl)methyl pivalate (6)
To a stirred solution of (2-amino-6-(benzyloxy)-9H-purin-9-yl)methyl pivalate (4) (1.06 g, 3 mmol) and dry pyridine (3 mL) in 70 mL of anhydrous dichloromethane was added a 20% solution of phosgene in toluene (1.5 mL, 3.4 mmol) dropwise at 0–5 °C. The mixture was stirred overnight while the ice bath was allowed to warm to room temperature. A solution of 4-nitrobenzyl alcohol (0.46 g, 3 mmol) in anhydrous dichloromethane (10 mL) was added, and the reaction mixture was stirred at room temperature for 2 h. It was then evaporated with 5 g of silica gel to dryness in vacuo and the residue was purified by column chromatography (silica gel, dichloromethane-ethanol 30:1) to give 1.42 g (89%) of the title compound as a white solid; m.p. 146–148 °C; 1H NMR (CDCl3, 90 MHz) δ 10.20 (s, D2O exchangeable,1H), 8.24 (d, J = 8.5 Hz, 2H), 8.03 (s, 1H), 7.33–7.61 (m, 7H), 6.08 (s, 2H), 5.62 (s, 2H), 5.37 (s, 2H), 1.16 (s, 9H); MS m/z 535 [M+H]+.
Compounds 7 and 8 were synthesized using procedures analogous to the one described above except that for compound 7, after the addition of 1-(4-nitrophenyl)ethanol, the reaction mixture was stirred at room temperature for 6 h, and for compound 8, the reaction mixture was stirred at room temperature for 40 h after the addition of 2-(4-nitrophenyl)propan-2-ol.
(6-(Benzyloxy)-2-(((1-(4-nitrophenyl)ethoxy)carbonyl)amino)-9H-purin-9-yl)methyl pivalate (7)
Compound 7 was obtained in an 81% yield as a white solid; m.p. 140–142 °C; 1H NMR (CDCl3, 90 MHz) δ 10.34 (s, D2O exchangeable, 1H), 8.26 (d, J = 9 Hz, 2H), 8.04 (s, 1H), 7.54 (d, J = 9 Hz, 2H), 7.34–7.41 (m, 5H), 6.07 (s, 2 H), 6.06 (q, J = 6.6 Hz, 1H), 5.61 (s, 2H), 1.65 (d, J = 6.6 Hz, 3H), 1.15 (s, 9H); MS m/z 549 [M+H]+.
(6-(Benzyloxy)-2-((((2-(4-nitrophenyl)propan-2-yl)oxy)carbonyl)amino)-9H-purin-9-yl)methyl pivalate (8)
Compound 8 was obtained in a 68% yield as a white solid; m.p. 92–94 °C; 1H NMR (CDCl3, 90 MHz) δ 10.20 (s, D2O exchangeable, 1H), 8.19 (d, J = 9 Hz, 2H), 7.97 (s, 1H), 7.45 (d, J = 9 Hz, 2H), 7.29–7.45 (m, 5H), 6.02 (s, 2 H), 5.58 (s, 2H), 1.87 (s, 6H), 1.13 (s, 9H ); MS m/z 563 [M+H]+.
4-Nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1)
A suspension of 6 (0.53 g, 1 mmol) in 0.1 M ammonia in methanol (40 mL) was stirred at room temperature until TLC showed a complete disappearance of the starting material (approximately 40 h). The white solid that was formed was collected by filtration after cooling the reaction mixture in an ice-water bath for 1 h, washed with methanol and ether and dried to give 0.36 g (85%) of the target molecule; m.p. 233–235 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 10.51 (s, 1H), 8.29-8.21 (m, 3H), 7.74 (d, J = 8.7 Hz, 2H), 7.61-7.49 (m, 2H), 7.46-7.26 (m, 3H), 5.59 (s, 2H), 5.34 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 151.8, 151.7, 147.0, 144.7, 136.3, 128.9, 128.5, 128.3, 123.6, 67.5, 64.5. HRMS, calculated for C20H16N6O5, m/z: 421.1255 [(M+H)+], found, 421.1257.
1-(4-Nitrophenyl)ethyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (2)
Method 1: To an ice-cooled solution of 7 (1.2 g, 2.2 mmol) in ethanol (60 mL) was added an ice-cooled solution of 1 M sodium hydroxide (10 mL) and the mixture was stirred at 0 °C for 30 min. The reaction mixture was neutralized with 10% acetic acid and was evaporated with 5.8 g of silica gel to dryness in vacuo. The residue was chromatographed on a silica gel column (60 Å, 70–230 mesh) and eluted with CH2Cl2/EtOH, 20:1 (v/v) to give 0.57 g (60%) of the title compound as a white solid. Method 2: Compound 2 was also synthesized using a procedure analogous to the one described for 1 except that 1 M ammonia in methanol was used as a base in lieu of 0.1 M ammonia and was obtained as a white solid; yield, 79%; m.p. 134–135 °C (decomp.); 1H NMR (400 MHz, DMSO-d6) δ 13.22 (s, 1H), 10.44 (s, 1H), 8.42-8.12 (m, 3H), 7.76 (d, J = 8.7 Hz, 2H), 7.56 (dd, J = 7.9, 1.5 Hz, 2H), 7.46-7.26 (m, 3H), 5.96 (q, J = 6.6 Hz, 1H), 5.59 (s, 2H), 1.56 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ151.7, 151.3, 146.9, 136.3, 128.9, 128.5, 128.3, 126.9, 123.7, 71.2, 67.5, 22.5. HRMS, calculated for C21H18N6O5, m/z: 435.1411 [(M+H)+], found, 435.1414.
2-(4-Nitrophenyl)propan-2-yl (6-(benzyloxy)-9H-purin-2-yl)carbamate (3)
Compound 3 was synthesized using procedures analogous to the ones described for 2 and was obtained as a white solid; yield: 63% (method 1); 82% (method 2); m.p. 246 °C (decomp.); 1H NMR (400 MHz, DMSO-d6) δ 13.21 (s, 1H), 10.36 (s, 1H), 8.34-8.19 (m, 3H), 7.79 (dd, J = 9.2, 2.1 Hz, 2H), 7.57 (dd, J = 7.8, 1.4 Hz, 2H), 7.46-7.27 (m, 3H), 5.61 (s, 2H), 1.84 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ154.1, 151.8, 150.5, 146.3, 136.3, 128.9, 128.4, 128.3, 125.8, 123.5, 80.2, 67.5, 28.4. HRMS, calculated for C22H20N6O5, m/z: 449.1568 [(M+H)+], found, 449.1571.
AGT inactivation assay
AGT substrate DNA was prepared by treating L1210 DNA (175 μg/mL) in 10 mM Tris-HCl buffer (pH ~7.4) with 0.2 mM 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine for 3 min at 37 °C. This substrate DNA was then stored at 0 °C until used. A 10 μL aliquot of substrate DNA containing O6
-(2-chloroethyl)guanine and N1,O6
-ethanoguanine lesions (~50 fmol) was incubated for 30 min at 37 °C with 40 fmol of purified recombinant human AGT which was pretreated (or not) with various inhibitors for 30 min at 37 °C. This assay gives a moderately linear decrease in these DNA lesions in proportion to the level of AGT activity until the lesions have been largely titrated. The level of remaining cross-link precursors in this mixture was then determined and the level of inhibition of AGT calculated from this value. The procedure is as follows: ~40 fmol of AGT in AGT stabilization buffer of the following composition: 20 mM Tris-HCl, 1 mM EDTA, 1 mg/mL of bovine serum albumin, 0.1 mM dithiothreitol, and 2 μg/mL of L1210 DNA, pH 7.4 (the L1210 DNA greatly contributes to the AGT stability but does not add significantly to the total DNA in the final assay) was mixed with an equal volume of 2x concentration of inhibitor in an equivalent amount of buffer, to give a mixture of the desired inhibitor and AGT concentration. This mixture was then incubated for 30 min at 37 °C, then 10 μL were added to 10 μL of substrate DNA, and the mixture incubated for a further 30 min at 37 °C to allow the residual AGT activity to repair the DNA. This mixture was then diluted 10-fold with 5 mM Tris-HCl, 1 mM EDTA, and 1 mM NaN3
, pH 8.0 buffer, then incubated at 50 °C for 2–3 h to allow the unrepaired cross-link precursors to progress to cross-links and the level of cross-linking was then measured using previously described fluorescence methodology.6
This involved dilution of the sample to a volume of 1.5 mL with 5 mM Tris-HCl, 1.0 mM EDTA, and 1.0 mM NaN3
buffer, pH 8.0, containing 0.1 μg/mL of Hoechst H33258 fluorescent dye, and measuring the fluorescence using a Hoefer Scientific Instruments TKO 100 fluorometer. The mixture was then heated in a 100 °C hot-block for 5 min then plunged into a water bath at room temperature for 5 min and the fluorescence measured again. The percentage of DNA molecules that were cross-linked (i.e., containing at least one cross-link per molecule) was then calculated from the change in fluorescence.
Polarographic determination of half-wave reduction potentials
Differential pulse polarography (DPP) voltagrams of the synthesized agents were obtained using a pH 7.0 buffer composed of 100 mM potassium chloride and 50 mM potassium phosphate as the supporting electrolyte containing 10% DMSO by volume. Compounds were added as 0.5% of a 10 mM solution in DMSO to give final concentrations of 50 μM. The samples were purged with nitrogen to remove dissolved oxygen, and DPP voltagrams were obtained using a Princeton Applied Research electrochemical trace analyzer model 394, linked to a model 303A static mercury drop electrode (Princeton Applied Research, Oak Ridge, TN, USA). Scans were performed from 0 to −900 mV (2 mV/sec) using a platinum counter electrode versus an Ag/AgCl reference electrode (saturated KCl/AgCl electolyte). A pulse amplitude of 50 mV was used and the polarographic half-wave reduction potential E1/2
was calculated from the peak current potential (EP
) according to the following equation: E1/2
− pulse amplitude/2.27
Determination by HPLC
HPLC measurements of the concentrations of nitrobenzene, N-phenylhydroxylamine, aniline, 1, 2 and 3 were performed using a Beckman 127P solvent module and a Beckman 168 UV/vis detector (Beckman, Fullerton, CA, USA). A standard curve for estimation of total agent was established and a linear relationship between concentration and the area under the curve was found in all cases. Samples in 50% v/v acetonitrile were separated on a 5 micron 220 × 4.6 mm Applied Biosystems RP-18 C-18 reverse phase column (Applied Biosystems, Carlsbad, California, USA) by elution with 34% acetonitrile in buffer (0.03 M KH2PO4, 1.0 mM NaN3, pH 5.4) for 5 min followed by a 34–70% acetonitrile linear gradient in buffer, at a flow rate of 0.6 ml/min from 5 to 35 min. After this point the concentration of acetonitrile was maintained at this level for 5 min then returned to the starting concentration over an additional 5 min. Absorbance was monitored at 280 nm using a Beckman 168 UV/vis detector.
Reduction of Nitrobenzene with Zn/EDTA
A solution of nitrobenzene at a concentration of 1 mM in 50 mM Tris-HCl, 10 mM EDTA, pH 7.0, was quickly shaken with approximately 4 mg/ml of Zn dust and allowed to settle at room temperature, then centrifuged at 10,000g for 4 min. The supernatant was then diluted 10-fold with 30 mM potassium phosphate buffer, pH 5.4, and analyzed by HPLC.
Reduction of Nitrobenzene with Xanthine/Xanthine Oxidase
A solution of nitrobenzene at a concentration of 100 μM in 100 mM potassium phosphate buffer, pH 7.4, containing 2.5 mM xanthine added as 1% of a 250 mM solution in 1 M NaOH was purged with nitrogen and 0.16 units/ml of xanthine oxidase (Sigma, bovine milk, X4500-5UN) added and the mixture incubated at 37 °C. Samples were taken at 2 h and analyzed by HPLC for aniline, nitrobenzene, and N-phenylhydroxylamine. Nitrobenzene, N-phenylhydroxylamine, and aniline eluted at approximately 20, 10 and 8 min, respectively.
Reduction of Nitrobenzene with NADPH:Cytochrome P450 Reductase
A solution of nitrobenzene at a concentration of 100 μM in 100 mM potassium phosphate buffer, pH 7.85, the pH optimum for NADPH:cytochrome P450 reductase (Sigma, human recombinant C8113), containing 10 mM NADPH, 10 mM glucose, 2 units/mL of glucose oxidase, 120 units/mL of catalase and 4 units/mL of NADPH:cytochrome P450 reductase, added after 5 min to allow the oxygen to be depleted, was incubated for 2 h in a sealed tube at 37 °C and then the mixture was analyzed by HPLC for aniline, nitrobenzene, and N-phenylhydroxylamine.
Reduction of 1, 2 and 3 with Zn/EDTA
A solution of each agent at a concentration of 50 μM in 50 mM Tris-HCl, 10 mM EDTA, pH 7.0, was quickly shaken with approximately 4 mg/mL of Zn dust and allowed to settle at room temperature, then centrifuged at 10,000g for 4 min. The supernatant was then analyzed by HPLC.
Reduction of 1, 2 and 3 by NADPH:Cytochrome P450 Reductase and Xanthine Oxidase
In these experiments, oxygen deficiency was enzymatically generated as previously described.28
Briefly, glucose/glucose oxidase was used to rapidly consume the available free oxygen and catalase was employed to remove the generated hydrogen peroxide. In the absence of other components, this system itself does not measurably reduce 1
in the time frames employed in these experiments. The reaction mixtures in a total volume of 0.5 mL were as follows: 100 mM potassium phosphate buffer, pH 7.4, containing 10 mM glucose, 5 μL of a solution of 200 units/mL of glucose oxidase and 12,000 units/mL of catalase, 20 μM test agent added as a 100x solution in DMSO plus either 5 μL of 104 units/mL of NADPH:cytochrome P450 reductase plus 1 mM NADPH or 0.16 units/mL of xanthine oxidase plus 1 mM xanthine. The aerobic reaction was identical except that the glucose needed for the oxygen deficiency system to operate was omitted. Furthermore, the aerobic reaction volumes were scaled up 10-fold and the mixture incubated as a shallow layer with shaking in sealed 25 cm2
plastic culture flasks to ensure that full air saturation was maintained and evaporation prevented. The reaction mixtures were incubated at 37 °C and samples were removed at 0 and 1 h after initiation, mixed with an equal volume of CH3
CN and centrifuged at 10,000g
for 10 min to sediment any precipitated protein. The supernatant was then analyzed as described above for the remaining agent and reduction products using HPLC.
Cellular activation of 1, 2 and 3 under Normoxic and Oxygen Deficient Conditions
Experiments, following the time course of prodrug loss and O6-BG generation when ~20 μM of 1, 2, or 3 was incubated with either EMT6 or DU145 cells, were conducted as follows. Cells at a concentration of 107/ml in Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (EMT6) or in α-Minimum Essential Medium with 10% fetal bovine serum (DU145) containing 10 mM glucose were shaken as cell suspensions as shallow layers (5 ml) in 25 cm2 plastic culture flasks for the normoxic incubations. For the oxygen deficient condition incubations, the cells were incubated in sealed microfuge tubes with an additional 10 mM glucose and 2 units/mL of glucose oxidase plus 120 units/mL of catalase as additional components. Samples were removed at 0, 1, 2, and 3 h after the start of the incubations and mixed with an equal volume of acetonitrile to lyze the cells, precipitate the macromolecules, and extract the remaining prodrug and generated O6-BG. The mixture was allowed to stand for 15 min at room temperature, centrifuged at 10,000g for 10 min and the supernatant analyzed by HPLC for parental prodrug and O6-BG.
Cell survival (clonogenic) assays were performed using a previously described method.3
plastic tissue culture flasks were seeded with 2.5 × 105
cells each and 3 days later cells were pretreated for 6 h in the presence of 3
prior to the addition of onrigin dissolved in 10 mL of medium for 24 h at 37 °C. All agents were initially dissolved in DMSO and then diluted to the required concentration. For oxygen-deficient conditions, cells were incubated with laromustine in the presence of 2 units/mL of glucose oxidase (Sigma G6641), 120 units/mL of catalase (Sigma, C1345) in high glucose DMEM (Invitrogen).28,29
Flasks were flushed with nitrogen for 10 sec and the caps screwed on tightly. This action facilitated oxygen depletion of the medium by glucose oxidase through removal of residual oxygen containing air and denial of the entry of additional air. After treatment, monolayers were rinsed with phosphate buffered saline, and cells were detached by trypsinization, suspended in culture medium, counted and sequential cell dilutions were plated in duplicate into 6-well plates at a density of 1 × 102
, 1 × 103
, and 1 × 104
cells per well. Ten to fourteen days later, colonies were fixed, stained with 0.25% crystal violet in 80% methanol and quantified. DMSO concentrations were ≤ 0.05%, and non-toxic. Cells under aerobic conditions were treated under similar conditions and cytotoxic agent concentrations, but in unsealed flasks without glucose oxidase and catalase. Cells were then washed, harvested by trypsinization, and assayed for survival using a clonogenic assay described previously.3,30,31
In the absence of cells, no measurable direct metabolism of 3
or of laromustine was detected in the presence of the glucose oxidase and catalase enzyme components of the oxygen deficiency generating system.28