Amino acids and peptide coupling reagents were obtained from Merck (Novabiochem). All other chemical reagents were obtained from SigmaAldrich or Fisher Scientific and used without further purification. Thin-layer chromatography (TLC) was carried out on Merck silica gel plates with a fluorescence indicator (254 nm). Visualization of TLC plates was carried out by UV light, then by either a cerium sulphate/ammonium molybdate or potassium permanganate stain. Flash chromatography was preformed with 35–70 μm silica gel (Fisher Davisil). Infrared spectra were recorded using a FTIR spectrometer fitted with an ATR accessory. 1H NMR and 13C NMR were recorded using a Bruker DPX400 (400 and 100 MHz). Chemical shifts δ are given in ppm. All enzymatic assays were performed using a Varian Cary 100 Spectrometer. All peptides were purified on a Waters HPLC, using a Waters Alantis Prep T3 OBD 5μm 19 × 100 mm column using a water/acetonitrile at a flow rate of 17 mL min−1. For cell culture experiments, all reagents were obtained from Invitrogen. MCF-7 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Autogen Bioclear), penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (292 μg/ml).
Cyclic Peptide synthesis
The alanine-scanning analogues of CRYFNV were synthesized and purified as previously described for CRYFNV.[8a]
Synthesis of capped dipeptides
The dipeptides in were synthesized as outlined for compound 8 in on a 500 mg scale. Further experimental details and analytical information for compounds 8–13 and 15–17 are provided as supplemental data
. The synthesis of compound 14 is detailed below:
Synthesis of Boc-Phe(4-NO2)-diethylamide
Dry CH2Cl2 (8 mL), and Boc-4-nitro-L-phenylalanine (500 mg, 1.61 mmol) were added to a three- neck flask under an inert atmosphere, with vigorous stirring. To this mixture, EDC hydrochloride (309 mg, 1.61 mmol) and HOBt (218 mg, 1.61 mmol) were added, and the solution was stirred for 30min at room temperature. Diethylamine (0.34 mL, 3.38 mmol) was added and the resulting yellow solution was left to stir for 4h. The progress of this reaction was checked by TLC, and was shown to be complete in 4 hours. The reaction was stopped, diluted with CH2Cl2 (30mL) and washed with citric acid (10 mL), NaHCO3 saturated solution (10 mL) and brine (10 mL). The organic phase was dried with anhydrous Na2SO4, filtered and evaporated in vacuo to give a pale yellow oil that solidified upon standing. The crude material was dried on argon stream and re-precipitated using cold hexane to give a white solid that was collected by Buchner funnel (483mg, 82% yield). Rf = 0.82 (EtOAc/Hex 2:1). m.p. 90–90.5 °C. 1H-NMR (300MHz, DMSO-d6): δ = 8.17 (d, 2H, J = 8.8Hz, Ar), 7.57 (d, 2H, J = 8.8Hz, Ar), 7.31 (d, 1H, J = 8.4Hz, NH), 4.57 (q, 1H, J = 7.7Hz, CH2); 2.97–3.41 (m, 6H, CH), 1.30 (s, 9H, tertBut), 1.08 (t, 3H, J = 6.9Hz, CH3), 1.01 (t, 3H, J = 6.9Hz, CH3). 13C-NMR (75MHz, DMSO-d6): 169.7, 154.9, 146.3, 130.8, 123.7, 78.1, 51.1, 41.1, 37.5, 27.9, 14.2, 12.7. HRMS/ES+ (m/z): calculated for C18H27N3O5Na: 388.1843 [M+Na]+ found: 388.1854 [M+Na]+.
Synthesis of fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-L-Diethylamide
Dry CH2Cl2 (6 mL), Boc-4-nitro-L-phenyldiethylamide (350 mg, 1.32 mmol) were added to a flask under an inert atmosphere with vigorous stirring. Trifluoracetic acid (6 mL) was added to this mixture and stirred for 1h. The reaction was monitored by TLC. After this time, the solvent was removed in vacuo and the orange oil co-evaporated with ethylacetate (3 × 10 mL) to give a yellow oil (350 mg, 100% yield) whose identity was confirmed by mass spectrometry. The deprotected amine was added to stirred dry CH2Cl2 (15 mL) in a flask under an inert atmosphere. fmoc-Arg(Pbf)-OH (1.88 mg, 3.0mmol) was added to this solution and the reaction was cooled to −20°C. HOBt (535 mg, 3.96 mmol) and EDC (380 mg, 1.98, mmol) were added, and the reaction was left to stir overnight and allowed equilibrate to room temperature. The reaction was diluted with CH2Cl2 (100 mL), washed with citric acid (20 mL), saturated NaHCO3 solution (20 mL) and brine (20 mL). The yellow solution was evaporated in vacuo to leave a yellow solid, which was purified by column chromatography using CH2Cl2/MeOH 95:5 to give the product (Rf = 0.45) as an off-white solid (520mg, 44% yield). m.p. 110°C decomp. 1H-NMR (300MHz, DMSO-d6): 8.25δ (d, J = 8.4Hz, 1H), 8.03 (d, J = 8.4Hz, 2H), 7.84 (d, J = 7.3Hz, 2H), 7.65 (d, J = 8.8Hz, 2H), 7.46–7.34 (m, 7H), 4.96–4.89 (m, 1H), 4.34–4.22 (m, 3H), 4.04–4.00 (m, 1H), 3.35–2.98 (m, 12H), 2.47 (s, 3H), 2.04 (s, 3H), 1.43 (s br, 10H), 1.07–0.97 (m, 6H). 13C-NMR (75MHz, DMSO-d6): 172.3, 170.1, 158.4, 157.0, 156.7, 147.2, 146.6, 144.8, 144.6, 141.7, 138.2, 132.4, 131.7, 131.6, 128.6, 128.0, 126.1, 125.2, 124.0, 123.9, 121.0, 117.2, 87.2, 66.5, 60.7, 55.1, 50.3, 47.6, 43.4, 42.1, 38.6, 29.2, 19.9, 18.5, 15.2, 13.7, 13.2. LRMS/ES+ (m/z): 896 [M+H]+ 5%, 918 [M+Na]+100%, 1813 [2M+Na]+9.6%. HRMS/ES+ (m/z): calculated for C47H58N7O9S: 896.4011 [M+H]+ found: 896.4010 [M+H]+.
Synthesis of Amino-Arg(Pbf)-CONH-Phe(4-NO2)-L-Diethylamide
Dry ethylacetate(14 mL) was added to fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-diethylamide (520 mg, 0.58 mmol) in a flask under an inert atmosphere and stirred vigorously. To this mixture, DBU (93 mg, 0.1 mL, 0.61 mmoL) was added; the solution was stirred for 1h and the reaction was monitoring by TLC. The solvent was removed in vacuo to give a brown solid, which was purified by column chromatography using CH2Cl2/MeOH 9:1 to give the product (Rf = 0.35) as a white solid (363 mg, 93% yield). m.p. 121°C decomp. 1H-NMR (300MHz, DMSO-d6): 8.31δ (d, J = 8.4Hz, 1H), 8.15 (d, J = 8.4Hz, 2H), 7.51 (d, J = 7.3Hz, 2H), 4.96–4.91 (m, 1H), 3.43–2.94 (m, 13H), 2.54–2.46 (m, 8H overlap DMSO d6), 2.03 (s br, 5H), 1.44 (s br, 10H), 1.07–0.97 (m, 6H). 13C-NMR (75MHz, DMSO d6): 174.3, 169.3, 157.3, 156.0, 146.2, 145.7, 137.2, 134.2, 131.3, 130.7, 124.2, 123.0, 116.2, 86.2, 54.1, 48.7, 42.4, 41.1, 37.9, 32.1, 28.2, 25.3, 18.9, 17.5, 14.3, 12.7, 12.2. LRMS/ES+ (m/z): 674 [M+H]+ 39%, 696 [M+Na]+100%, 1869 [2M+Na]+11%. HRMS/ES+ (m/z): calculated for C32H47N7O7S: 674.3330 [M+H]+ Found: 674.3319 [M+H]+.
Synthesis of Acetyl-Arg(Pbf)-CONH-Phe(4-NO2)-L-Diethylamide
Dry ethylacetate (7 mL) and fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-diethylamide (363 mg, 0.54 mmol) were added to flask under an inert atmosphere and stirred vigorously. Triethylamine (58 mg, 0.08 mL, 0.57 mmoL), and acetic anhydride (58 mg, 0.05 mL, 0.57 mmol) were to theis mixture and stirred overnight. The solvent was removed in vacuo giving a sticky yellow oil that was purified by column chromatography (short path) using CH2Cl2/MeOH 9:1 to give a white solid (Rf = 0.52) in quantitative yield (386mg, 100% yield). m.p. 113°C decomp. 1H-NMR (300MHz, DMSO-d6): 8.36δ (d, J = 8.4Hz, 1H), 8.14 (d, J = 8.8Hz, 2H), 7.89 (d, J = 8.2Hz, 2H), 7.56 (d, 8.8Hz, 2H), 4.92–4.84 (m, 1H), 4.28–4.24 (m, 1H), 3.36–2.99 (m, 16H), 2.54–2.51 (s br, 8H overlap DMSO-d6), 2.45 (s, 3H), 1.94 (s, 3H), 1.84 (s, 3H), 1.55–1.28 (s br, 11H), 1.05–0.96 (m, 6H). 13C-NMR (75MHz, DMSO-d6): 172.1, 170.1, 168.9, 157.3, 155.9, 147.1, 146.7, 138.1, 135.1, 135.1, 133.3, 131.7, 125.2, 124.0, 117.1, 87.2, 50.3, 43.4, 42.0, 38.5, 30.4, 29.1, 26.3, 23.3, 22.0, 18.5, 15.2, 13.6, 13.2. LRMS/ES+ (m/z): 716 [M+H]+ 100%, 1431 [2M+Na]+26%. HRMS/ES+ (m/z): Calcd. for C34H50N7O8S: 716.3436 [M+H]+ Found: 716.3434 [M+H]+.
Synthesis of Acetyl-Arg-CONH-Phe(4-NO2)-L-Diethylamide
To a flask, a cocktail of TFA/TIS/H2O 9.5/0.25/0.25 (22 mL) and acetyl-Arg(Pbf)-CONH-Phe(4-NO2)-diethylamide (386 mg, 0.54 mmol) were added, and stirred at r.t. for 2h. The reaction was monitored by TLC, and the solvent evaporated in vacuo to leave a viscous yellow oil. This was co-evaporated with CH2Cl2 (3 × 5 mL) and MeOH (3 × 5 mL) and the residue dried with an argon stream for ~20min. Diethylether (20 mL) was then added and the white precipitate collected by filtration (263 mg, 100% crude yield). The crude product was purified by reverse phase HPLC (retention time 7.63 minutes) and lyophilized to obtain the product as a white solid (125 mg, 50% yield). m.p. 113°C decomp. 1H-NMR (300MHz, DMSO-d6): 8.31δ (d, J = 8.7Hz, 1H), 8.10 (d, J = 8.6Hz, 2H), 7.91 (d, J =8.1Hz, 2H), 7.65 (t, J = 5.6Hz, 1H), 7.50 (d, 8.6Hz, 2H), 7.17 (s br, 3H), 4.90–4.82 (m, 1H), 4.32–4.23 (m, 1H), 3.37–3.00 (m, 7H), 2.95 (dd, 1H), 1.82 (s, 3H), 1.61–1.50 (m, 3H), 1.00 (t, 7.12Hz, 3H), 0.95 (t, 7.13 Hz, 6H). 13C-NMR (75MHz, DMSO-d6): 171.1,169.1, 169.1, 156.8, 146.3, 145.8, 130.8, 123.1, 51.8, 49.4, 41.4, 40.4, 39.8, 37.6, 29.2, 25.0, 22.4, 14.2, 12.7. LRMS/ES+ (m/z): 464 [M+H]+ 100%. HRMS/ES+ (m/z): Calcd. for C21H33N7O5: 464.2616 [M+H]+ Found: 464.2610 [M+H]+.
Protein expression and enzyme kinetics
Avian and human ATIC fused to an N-terminal 6X histidine tag were expressed from pET28 vectors in BL21(DE3) strains of E. coli, and purified as previously detailed [8a, 19]
. AICAR Tfase assays were conducted with avian ATIC and carried out in 1 cm path length quartz cuvettes at 25 °C as previously described [8a, 19]
. Each inhibitor was dissolved in DMSO to give a 100 mM stock solution that was further diluted for use in each assay. The data was analyzed using KaleidaGraph (Synergy Software) and Microsoft Excel, by assuming competitive inhibition with 10-f-THF as previously detailed [8a]
. Errors in the Ki were calculated by linear regretion. Compounds 1, 8, and 14 were also assayed with human ATIC, with the same Ki (within error) being observed for each compound as for the avian enzyme.
Size exclusion chromatography
Three 50 μM solutions of human ATIC were prepared as above, in 1 mL of buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 50 mM KCl, 5 mM EDTA, 5 mM dithiothreitol). 10 μM of compound 14 or 9 (negative control) was added to two of these solutions. Each solution was filtered through a Millex GP 0.22 μm filter (Millipore), and loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) pre-equilibrated with the above buffer. The flow rate was 1 mL/min and the flow monitored by UV on an AKTAprime FPLC (GE Healthcare). The column was calibrated using a HMW gel filtration calibration kit (GE Healthcare).
Cell proliferation assays
Cells were plated at 2000 cells per well in 96-well plates 24 h prior to dosing with appropriate drug in 100 μl fresh medium, final concentration of solvent DMSO in each well was 0.5%. MTS-based cell proliferation assays were performed 48 h post-dosing according to manufacturer’s instructions (CellTitre Aqueous One Cell Proliferation assay, Promega).
Cells were plated at 10,000 cells per well in 24-well plates. Time-lapse microscopy 
was initiated on dosing, with images captured every 45 min for 72 hours. Analysis was carried out using Image J software. All conditions were assayed in triplicate, and expressed as means ± S.E.M. Statistical significance was calculated using the one-way ANOVA parametric test and Tukey’s post hoc test using Prism software (GraphPad).