Prenylation inhibitors have gained increasing interest as potential therapeutics for cancer. mixture was heated to reflux at 70 and (CH3)3In (Perez et al. 2001 (15 ml 1.5 mmol in THF) was added dropwise. After 4 h 2 ml of MeOH was added and the reaction mixture was concentrated. The reaction mixture was next taken up in 30 ml of ether washed with 10% HCl (10 ml) aqueous NaHCO3 (15 ml) and brine dried Toceranib (PHA 291639, SU 11654) over MgSO4 and concentrated. Purification by flash chromatography (hexanes/ethyl acetate 95:5) gave 313 mg (59%) of compound 3 as a colorless oil. Note that this procedure using trimethylindium (Perez et al. 2001 affords superior results to the Stille coupling procedure with tetramethyltin used in previous work (Zahn et al. 2001 on 2Z-GGPP (compound 9). 1H NMR (300 MHz CDCl3): δ 1.55 (t = 6.9 Hz 3 1.87 (m 9 1.94 (s 3 2.15 (s 3 2.27 2.4 (m 10 2.9 (t = 7.8 Hz 2 4.41 (q = 6.9 Hz 2 5.44 (m 3 5.92 (s 1 Compound 4. To the solution of ester compound 3 (313 mg 0.94 mmol) in 7 ml of toluene was added diisobutyl aluminum hydride Toceranib (PHA 291639, SU 11654) (1.0 M solution in toluene 2.82 ml 2.82 mmol) less than argon at ?78°C. The response was stirred at ?78°C for 1 h. The response was quenched with the addition of 30 ml of ethyl Rabbit Polyclonal to SFRS17A. acetate and permitted to warm to space temp. Thirty milliliters of drinking water was added as well as the aqueous remedy was extracted with ethyl acetate (2 × 20 ml). The mixed organic layers had been cleaned with brine (2 × 20 ml) and dried out over MgSO4. Focus followed by adobe flash chromatography (hexanes/ethyl acetate 4:1) afforded alcoholic beverages substance 4 (210 mg 76 like a colorless essential oil. 1H NMR (300 MHz CDCl3): δ 1.64 (m 15 1.95 (m 12 4.03 (d = 7.2 Hz 2 5.03 (s 3 5.34 (t = 7.25 1 Substance 5. A remedy of 4-chloro-= 6Hz 2 4.46 (m 2 4.96 (d = 8.5 2 5.08 (m 3 5.34 (t = 7.5 1 6.6 (d = 4Hz 1 7.2 (d = 3.5Hz 1 13 NMR (CDCl3 125 Hz): 16.27 17.94 23.83 25.38 25.95 26.86 26.99 29.7 32.39 33.45 39.95 44.91 48.48 59.44 63.28 (d P-C = 5.1 Hz) 112.3 112.97 120.04 (d P-C = 6.9Hz) 123.48 124.32 124.59 131.55 135.32 136.25 143.04 153.55 31 NMR (CDCl3 121 MHz): ?14 ppm. MS: ESI 621/623 +Na (Fig. 1). In Vitro GGTase I Inhibition Assay 2 [substance 8; synthesized from 2and counted having a hemacytometer. MTT Assay Cells had been plated in a denseness of 2500 cells per well including 200 μl of development press with inhibitors or automobile in 96-well plates and cultured for 72 h. Twenty microliters of 3-(4 5 5 bromide) (MTT) (Invitrogen) share remedy (5 mg/ml in phosphate-buffered saline) was after that added as well as the plates had been incubated for 4 h. The medium was removed and the formazan precipitate formed was dissolved in 150 μl of DMSO. Absorbance values were measured with a plate reader (SpectraFluor Plus; Tecan Salzburg Australia) at 485-nm wavelength. After normalizing the absorbance values for media and vehicle controls the data were analyzed with GraphPad Prism version 4.0c by nonlinear regression (curve fit) and plotting sigmoidal dose-response to obtain GI50 (concentration of drug for 50% inhibition of growth) values which were further plotted on an isobologram for synergy analysis. Flow Cytometric Analysis STS-26T cells were treated and collected for DNA analysis as described previously (Mattingly et al. 2006 DNA content was analyzed with a FACScalibur instrument (BD Biosciences). A minimum of 104 cells per sample was analyzed to determine the percentage Toceranib (PHA 291639, SU 11654) of apoptotic cells and cells in G1 S and G2/M phases (Modfit; Variety Software Topsham ME). DEVDase Activity Assay Lysates of STS-26T cells were prepared and used for DEVDase assays as described previously (Wojtkowiak et al. 2008 Changes in fluorescence over time were converted to picomoles of product by comparison with a standard curve made with 7-amino-4-methylcoumarin. DEVDase-specific activities are reported as nanomoles of product made per minute Toceranib (PHA 291639, SU 11654) per milligram of protein. The bicinchoninic acid assay using bovine serum albumin as a standard was used to estimate protein concentrations. Results Inhibition of Geranylgeranylation of GTPases by GGTI-2Z and Lovastatin Combination. Recently we demonstrated that the monophosphate derivatives of certain farnesyl pyrophosphate analogs are potent FTIs and that prodrugs derived from these analogs block protein farnesylation (Clark et al. 2007 Wojtkowiak et al. 2008 We have also synthesized and evaluated novel GGPP analogs and found several.