Prekinamycin and an Isosteric-Isoelectronic Analogue Exhibit Comparable Cytotoxicity Towards K 562 Human Leukemia Cells

* corresponding author a Department of Chemistry, 200 University Ave. West, Waterloo, Canada, N2L 3G1. Fax: 1-519-746-0435; Tel: 1519-888-4567 ext. 84642; E-mail: dmitrien@scimail.uwaterloo.ca b Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Ave., Winnipeg, Canada, R3E 0T5. Fax: 1-204-474-7617; Tel: 1-204-474-8325; E-mail: B_Hasinoff@Umanitoba.ca c Department of Chemistry, Washington University, 750 Campus Box 1134, One Brookings Drive, St Louis, U.S.A., 63130. E-mail: birman@wustl.edu


Theoretical calculations
All computations were performed at the DFT level of theory employing the hybrid density functional B3LYP 1, 2 (Becke three-parameter Lee-Yang-Parr exchange-correlation functional) and the 6-31G(d) basis set 3 (a valence double-zeta polarized basis set) using the suite of programs accessible in the commercially available software package Gaussian 03 by Gaussian Inc. (Copyright  1994-2003, Gaussian, Inc) 4 installed on a desktop computer running the Linux operating system (Redhat Enterprise Linux 4).Unless otherwise stated, all calculations were carried out in the gas phase at T = 298.15K and P = 1 atm.

Cell culture and growth inhibition assays
Human leukemia K562 cells, obtained from the American Type Culture Collection, were maintained as suspension cultures in Dulbecco's modified Eagle's medium (Invitrogen, Burlington, Canada) containing 4 mM L-glutamine and supplemented with 20 mM Hepes, 10% fetal calf serum (Invitrogen), 100 units/ml penicillin G, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 and 95% air at 37 °C (pH 7.4).For the measurement of growth inhibition, K562 cells in exponential growth were harvested and seeded at 6000 cells/well in 96-well plates (100 μl/well).At 24 h later cells were treated with vehicle or various concentrations of kinamycin F and allowed to grow an additional 72 h.Kinamycin F was dissolved in DMSO and the final concentration of DMSO did not exceed 0.5% (v/v), which was an amount that was shown not to affect cytotoxicity.After treatment cells were assayed with the MTS CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega, Madison, WI).The spectrophotometric 96-well plate cell growth inhibition assay measures the ability of the cells to enzymatically reduce MTS.Three replicates were measured at each drug concentration and the IC 50 -values for growth inhibition were measured by fitting the absorbancedrug concentration data to a three-or four-parameter logistic equation as described. 5The IC 50 data reported are the results of three such experiments carried out on three separate days.

General procedures
Materials and Methods.All reactions were carried out under an atmosphere of nitrogen and/or argon in flame-dried and/or oven-dried glassware with magnetic stirring unless otherwise stated.Where required, the purification of solvents and reagents was accomplished according to standard procedures. 6All solvents were reagent grade unless otherwise stated.Dichloromethane and triethylamine were distilled from calcium hydride.DMSO was distilled from calcium hydride under reduced pressure.Toluene was distilled from sodium benzophenone ketyl.Dimethylformamide was pre-dried with calcium hydride and distilled under reduced pressure from 3Å molecular sieves and stored over 3Å molecular sieves.Alternatively, solvents were purified using the M. Braun Solvent Purification System.All commercial reagents used were purchased from the Aldrich Chemical Co. or VWR Canada (EMD, BDH, Alfa Aesar and J.T. Baker) and were used as received unless otherwise stated.Reactions were monitored by analytical thin layer chromatography (TLC) with silica coated aluminum sheets (EMD TLC Silica gel 60 F 254 ).Visualization was accomplished using UV light (254 nm) or basic KMnO 4 stain.Unless otherwise stated, purification of crude reaction products was carried out using flash silica gel chromatography (Silicycle SiliaFlash ® P60 40-63 μm, 230-400 mesh) according to established procedures. 7All reported yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated.
Characterization Methods.In some instances, compounds previously reported in the literature in which the characterization data was insufficient are reported here with a more complete characterization data set.Melting points were obtained on a MEL-TEMP ® apparatus (Laboratory Devices Inc., Holliston MA, USA) and are uncorrected. 1H NMR NMR spectra were acquired on a Brüker AC300 (300 MHz), Brüker AVANCE300 (300 MHz) or Brüker AVANCE500 (500 MHz) spectrometer and are reported in parts per million (ppm) in either CD 2 Cl 2 or CDCl 3 using the solvent residual peak as the internal standard.For CDCl 3 this was 7.24 and 77.0 ppm for 1 H NMR and 13 C NMR, respectively.Data are reported as chemical shift in ppm, integrated intensity; peak multiplicities; coupling constants J (Hz) and assignment.The following abbreviations are used for reporting peak multiplicities: br = broad, w = weak, s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet.

13
C NMR spectra were broadband decoupled and acquired on Brüker AC300 (75.5 MHz), Brüker AVANCE300 (75.5 MHz) or Brüker AVANCE500 (125.8MHz) spectrometers and are reported in ppm in CD 2 Cl 2 or CDCl 3 , using the carbon signal of the deuterated solvent as the internal standard.Infrared spectra (IR) were recorded on a Perkin Elmer Spectrum RX I FT-IR System spectrometer either as thin films on a NaCl plate, or as a KBr pellet or as a solution in CDCl 3 using an IR solution cell with 0.1 mm path length and CaF 2 windows.Low and high resolution mass spectra were recorded in electron impact (EI) mode, chemical ionization (CI) mode and/or electrospray ionization (ESI) mode obtained at the WATSPEC mass spectrometry facilities, University of Waterloo, Waterloo, Ontario, Canada.

1,4,8-Trimethoxynaphthalen-2-amine (18)
. Following a known procedure, 13 a mixture of the imine (0.251 g, 0.63 mmol) obtained from the previous reaction, 5% palladium on carbon (0.162 g, 12 mol%) and ammonium formate (0.681 g, 10.9 mmol) in methanol (5 mL) was heated at reflux for 1 h 40 min.The mixture was cooled to room temperature and diluted ten-fold with dichloromethane.The mixture was filtered through a bed of Celite 545, washed once with 0.1 M NaOH, dried over sodium sulfate, filtered and concentrated in vacuo to give 0.232 g of a light brown solid that was further purified by flash silica gel chromatography (1:12 hexanes:ether) to afford 0.123 g (84%) of the title compound as a light tan solid: mp 118-119 °C (lit.value 14

6,7,11-Trimethoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[b]carbazole-5(2H)-carbonitrile.
A solution of the carbazole 21 (0.017 g, 0.051 mmol), phenyl cyanate 16 (0.040mL, 0.37 mmol) and triethylamine (0.030 mL, 0.21 mmol) in dry DMSO (0.8 mL) was stirred at room temperature for 4 h after which another 2 drops of triethylamine (~0.012 g, 0.12 mmol) was added and stirring was continued overnight.The solution was then diluted with water (10 mL) and extracted with ethyl acetate three times and the organic extracts were pooled.The pooled extracts were washed three times with water, brine once and dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to yield the title compound as a light tan solid (0.019 g, 99%): mp 183-184 °C; IR (NaCl, film, cm  The cyanamide (0.012 g, 0.033 mmol) obtained from the previous reaction was dissolved in acetonitrile (1 mL) and the solution cooled to 0 °C.To this solution was added ceric ammonium nitrate (0.048 g, 0.087 mmol) dissolved in distilled water (0.3 mL), in 0.04 mL aliquots over a 5 min period.The solution was then allowed to come to room temperature over a period of 10 minutes and water (20 mL) was added.This mixture was extracted with CH 2 Cl 2 four times and the extracts were pooled, washed with water, a saturated solution of NaHCO 3 , water, brine and dried over Na 2 SO 4 .The solution was then filtered and concentrated in vacuo to give the crude product as an orange solid which was purified chromatographically (silica, 2:5 then 1:5 hexanes:ethyl acetate) to afford the title compound as an orange solid (0.011 g, 95%): mp  240 °C decomposition; IR (CDCl 3 , cm -1 ) ν max 3693, 2259, 1701, 1662, 1586, 1528, 1472,  1454, 1440, 1408, 1298, 1272, 1240, 1168, 1041, 1005,   17 N-cyanocarbazoloquinone 22 (0.216 g, 0.65 mmol) was dissolved in dichloromethane (12 mL) and the solution was cooled to 0 C.To this was added TBSOTf (0.24 mL, 1.05 mmol) and the mixture was stirred for 2 min.Triethylamine (0.25 mL, 1.79 mmol) was added and was stirring continued at room temperature for 1 h.Dichloromethane was then added and the reaction was quenched with an excess of an ice cold solution of saturated sodium bicarbonate.The organic phase was dried over sodium sulfate, evaporated in vacuo and purified by flash silica column chromatography (2:3 hexanes:ether) to afford 0.103 g (35%) of the title compound as an orange solid: IR (CDCl