Glycolytic cancer cells can be sensitised to the novel phloroglucinol PMT7 by induction
of energetic stress.
RUNNING TITLE: Death of glycolytic cancer cells under stress
Kate Broadley1, Lesley Larsen2, Patries M. Herst1, Robin A.J. Smith3, Michael V.
Berridge1 and Melanie J. McConnell1,4
SUPPORTING INFORMATION
SYNTHETIC CHEMISTRY:
General Experimental Procedures
All solvents were removed by rotary evaporation at temperatures up to 35°C. Merck silica
gel 60, 200-400 mesh, 40-63 μm, was used for silica gel chromatography. TLC was
carried out using Merck Silica gel 60 F254, first visualised with a UV lamp, and then by
dipping in a solution of 1% vanillin and 1% H2SO4 in ethanol followed by heating. High
resolution mass spectrometry was recorded using a VG70-250S double focussing
magnetic sector mass spectrometer. UV spectra were recorded in methanol using a Jasco
V-550. NMR spectra, at 25°C, were recorded at 500 or 300 MHz for 1H and 125 or 75
MHz for 13C on Varian INOVA-500 or VXR-300 spectrometers. Chemical shifts are
given in ppm on the δ scale referenced to the solvent peaks CHCl3 at 7.25 and CDCl3 at
77.0.
HPLC methods:
Method A: HPLC analysis was carried out using an Agilent HP1100 at 25 °C on a C18
column (Phenomenex Luna ODS(3) 5 μm 100 A 150 x 3 mm) with a 2 x 4 mm C18
guard column. Peaks were detected at 210 and 254 nm and UV spectra recorded from 190
to 600 nm. The mobile phase was acetonitrile in water, both with 0.1% formic acid:
t0=10%, t12.5=100%, t15=100%, t16=10%, t20=10%. The flow rate was 0.5 mL/min, with
an injection volume of 5 L.
Method B: HPLC analysis was carried out using an Agilent HP1100 at 25 °C on a C8
column (Agilent Zorbax Eclipse XDB-C8 5 μm 100 A 150 x 4.6 mm) with a 2 x 4 mm
C8 guard column. Peaks were detected at 210 and 254 nm and UV spectra recorded from
190 to 600 nm. The mobile phase was methanol in water, both with 0.1% trifluoroacetic
acid: t0=10%, t12.5=100%, t15=100%, t16=10%, t20=10%. The flow rate was 0.5 mL/min,
with an injection volume of 5 L.
1,4-bis(benzyloxy)-2-(10-hydroxydecyl)-5,6-dimethoxy-3-methylbenzene (1)
Yield 9.3g, 83%. Anal. calcd. for C33H44O5 : C, 76.1; H, 8.5; Found: C, 76.2; H, 8.4%.
1
H NMR (CDCl3)  7.30-7.55 (10H, m, PhH), 5.00 (2H, s, CH2Ph), 4.96 (2H, s, CH2Ph),
3.95 (3H, s, OCH3), 3.94 (3H, s, OCH3), 3.63 (2H, t, J=6.5Hz, CH2-OH), 2.57 (2H, t, J =
8.5 Hz, CH2Ar), 2.15 (3H, s, ArCH3), 1.40-1.20 (16H, m) ppm.
1,4-bis(benzyloxy)-2-(10-iododecyl)-5,6-dimethoxy-3-methylbenzene (2).
Yield 1.43 g, 79%.
MS found 653.2111; C33H43IO4.Na requires 653.2098.
1
H NMR (CDCl3) 7.48 (4H, bd, PhH), 7.39 (4H, bt, PhH ), 7.34 (2H, m PhH, ), 4.99
(2H, s, CH2Ph), 4.94 (2H, s, CH2Ph), 3.94 (3H, s, OCH3), 3.93 (3H, s, OCH3), 3.18 (2H,
t, J= 8Hz, CH2I), 2.58 (2H, m, ArCH2), 2.13 (3H, s, ArCH3), 1.80 (2H, m, CH2 CH2I) and
1.24 – 1.43 (14H, m) ppm.
2-(10-(2,5-bis(benzyloxy)-3,4-dimethoxy-6-methylphenyl)decyl)benzene-1,3,5-triol (3).
Yield 125 mg, 31%.
HPLC 16.01 minutes (Method A), 16.47 minutes (Method B).
MS found 651.3292; C39H48O7.Na requires 651.3292.
1
H NMR (CDCl3) 7.48 (4H, bd, PhH), 7.39 (4H, bt, PhH ), 7.34 (2H, m, PhH ), 5.85
(2H, s, ArH), 4.99 (2H, s, CH2Ph ), 4.95 (2H, s, CH2Ph), 3.94 (3H, s, O CH3), 3.93 (3H,
s, O CH3), 2.55 (2H, m), 2.49 (2H, t, J 8Hz, CH2Ar), 2.13 (3H, s, ArCH3), 1.48 (2H, m),
1.39 (2H, m) and 1.24 – 1.31 (12H, m) ppm.
13
C NMR (CDCl3) 155.21 (2C), 154.47, 146.74, 146.61, 144.86, 144.79, 138.00,
137.79, 130.86, 128.42 (4C), 128.27 (2C), 127.94 (3C), 127.85, 125.52, 107.74, 95.63
(2C), 75.39, 75.11, 61.31, 61.29, 30.06, 29.86, 29.39, 29.35 (2C), 29.33, 29.29, 29.22,
27.22, 22.61 and 12.03 ppm.
I.R. max 3391, 2926, 2854, 1620, 1465, 1370 and 1105 cm-1.
UV  max log 280 (3.50) nm.
2-(10-(2,4,6-trihydroxyphenyl)decyl)-5,6-dimethoxy-3-methyl-2,5-cyclohexadiene-1,4dione (PMT7)
Yield 32 mg, 90%.
HPLC 12.40 minutes (Method A), 14.38 (Method B).
MS found 469.22201; C25H34O7.Na requires 469.21967.
1
H NMR (CDCl3) 6.04 (1H, 2xOH), 5.95 (2H, s, ArH), 5.78 (2H, OH), 3.94 (6H, s,
2xOCH3), 2.48 (2H, bs, CH2Ar), 2.40 (2H, m, CH2Ar), 1.97 (3H, s, quinoneCH3), 1.45
(2H, bs) and 1.24 – 1.31 (14H, m) ppm.
13
C NMR (CDCl3) 184.93, 184.56, 155.38 (2C), 154.32 , 144.22, 143.24, 138.94,
108.24, 95.72 (2C), 61.18 (2C), 29.67, 29.58, 29.38, 29.26 (3C), 29.14, 29.08, 28.55,
26.35, 22.65 and 11.92 ppm.
IR  max 3412, 2926, 2854, 1643, 1620, 1456, 1267 cm-1.
UV  max log 277 (4.02) nm.
OBn
MeO
MeO
OH
OBn
1
a
OBn
OH
MeO
+
MeO
I
OBn
HO
2
OH
b
OBn
MeO
OH
MeO
OBn
3
HO
OH
c
O
MeO
OH
MeO
O
Scheme 1.
PMT7
HO
OH
Synthesis of PMT7. a, i, MsCl, Et3N; ii, NaI, CH3CN. b, KOH, MeOH. c,
H2, 5% Pd/C.
Supplemental Figure 1: Reliance on mitochondrial electron transport is measured by the
effect of mitochondrial uncoupling on NADH flux, as determined by WTS1/PMS
reduction after FCCP treatment (Herst PM, Berridge MV. Cell surface oxygen
consumption: a major contributor to cellular oxygen consumption in glycolytic cancer
cell lines. Biochim Biophys Acta 2007 Feb; 1767 (2): 170-177). Cells were incubated
with 2 M FCCP and WST1/PMS reduction was measured as described in the main text.