Anti-leukemic activity of ubiquinone-based compounds targeting trans-plasma membrane electron transport

Article abstract of DOI:10.1021/jm301585z

Trans-plasma membrane electron transport (tPMET) is a ubiquinone-dependent cell survival pathway for maintaining intracellular redox homeostasis in rapidly dividing cells. To target this pathway, fifteen ubiquinone-based compounds were designed and synthesized to position at the plasma membrane and disrupt tPMET. We established that quaternary ammonium salt moieties carrying highly hindered, positive electronic charges located to the plasma membrane. A ten-carbon chain linked to these moieties was effective at positioning the redox-active ubiquinone-like function within the lipid bilayer to disrupt tPMET in human leukemic cells (IC₅₀ 9 ± 1 μM). TPMET inhibition alone was not sufficient to induce significant cell death, but positively charged compounds could also enter the cell and disrupt intracellular redox balance, distinct from their effects on mitochondrial electron transport. The synergistic effect of tPMET inhibition plus intracellular redox disruption gave strong antiproliferative activity (IC₅₀ 2 ± 0.2 μM). Positively charged ubiquinone-based compounds inhibit human leukemic cell growth.

Full text of DOI:10.1021/jm301585z

Article
pubs.acs.org/jmc
Anti-Leukemic Activity of Ubiquinone-Based Compounds Targeting
Trans-plasma Membrane Electron Transport
Carole Grasso,† Lesley Larsen,§ Melanie McConnell,† Robin A. J. Smith,§ and Michael V. Berridge*,†
†Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, New Zealand
§Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand
S
* Supporting Information
ABSTRACT: Trans-plasma membrane electron transport
(tPMET) is a ubiquinone-dependent cell survival pathway for
maintaining intracellular redox homeostasis in rapidly dividing
cells. To target this pathway, fifteen ubiquinone-based compounds
were designed and synthesized to position at the plasma
membrane and disrupt tPMET. We established that quaternary
ammonium salt moieties carrying highly hindered, positive
electronic charges located to the plasma membrane. A ten-carbon
chain linked to these moieties was effective at positioning the
redox-active ubiquinone-like function within the lipid bilayer to
disrupt tPMET in human leukemic cells (IC50
9
1 μM).
TPMET inhibition alone was not sufficient to induce significant
cell death, but positively charged compounds could also enter the
cell and disrupt intracellular redox balance, distinct from their effects on mitochondrial electron transport. The synergistic effect of
tPMET inhibition plus intracellular redox disruption gave strong antiproliferative activity (IC50
ubiquinone-based compounds inhibit human leukemic cell growth.
2
0.2 μM). Positively charged
active compound at the outer or inner leaflet of the plasma
membrane, allowing the quinone component to locate within the
lipid bilayer and disrupt redox cycling (Figure 1). Similar membrane
positioning studies have been carried out with a series of MitoQ
compounds where a 10-carbon (C10) chain was found to be most
effective for antioxidant activity.24
We tested each compound for inhibition of tPMET activity
and for antiproliferative activity in HL60 cells which have active
mitochondrial and plasma membrane electron transport function,
and HL60ρo mitochondrial gene-knockout cells that are defective in
mitochondrial electron transport25 but have elevated tPMET.15
This allowed the effect of tPMET inhibition to be distinguished
from inhibition of mitochondrial electron transport. We show that
compounds in which a redox-active quinone is linked by a C10 chain
to a quaternary ammonium salt with a highly hindered positive
charge had the highest tPMET inhibitory activity and antiprolifer-
ative activity, independent of mitochondrial electron transport.
INTRODUCTION
■
The trans-plasma membrane electron transport (tPMET) complex
is a simple redox-active ubiquinone system spanning the plasma
membrane of all living cells. It is linked to a diverse range of biological
functions including cell metabolism, defense, growth, and survival.1−5
Electrons from intracellular reductants flow through enzyme-
mediated tPMET pathways to reduce extracellular molecules,2,6,7
allowing cells to respond to changes in their redox microenvironment
and maintain intracellular NAD+/NADH homeostasis.8−10
Cancer cells that use glycolytic metabolism to accommodate
their metabolic demands even in the presence of oxygen11−13
employ tPMET pathways in addition to lactate dehydrogenase
to replenish NAD+ pools.14−17 A significant imbalance of the
NAD+/NADH ratio can create intracellular reductive stress
resulting in cell death.18−20 Due to the dependence of glycolytic
cancer cells on tPMET for their survival, the pathway is a potential
target for drug development.21
We have previously shown that the redox-active, anticancer
drug, phenoxodiol, inhibits tPMET and is linked to antiprolifer-
ative activity in human leukemic HL60 cells.22,23 Here, we report
on the synthesis and structure−activity profile of a series of fifteen
redox-active ubiquinone-based compounds designed to inhibit
tPMET, and compare tPMET inhibition with antiproliferative
activity against human leukemic HL60 cells. The compounds are
composed of a redox-active dimethoxymethylquinone compo-
nent, functionally related to ubiquinone, coupled by a 3, 5, or 10-
carbon chain to a terminal moiety of varying polarity and/or
electronic charge. These moieties are designed to position the
SYNTHESIS
■
Alcohols. The C3 (1) and C5 (2) alcohols were prepared as
described by Hiroshi Morimoto26 and our group, Asin-Cayuela
et al.27 The C10 compound idebenone (3) was obtained from
commercial sources.
Sulfonic Acids. The sulfonic acids were prepared as outlined
in Scheme 1. The key step in the synthesis was the use of the isobutyl
Received: July 12, 2012
Published: February 22, 2013
© 2013 American Chemical Society
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a
Scheme 1. Structures of 1−3 and 14
a
Synthetic routes to 9, 10, and 13.
Phloroglucinols. Alkylation of phloroglucinol with (11)
using potassium carbonate in acetone gave the O-alkyated product
(12) which was debenzylated by hydrogenation and oxidized in air to
give 13. The preparation of the corresponding C-alkylated compound
(14) has been described by us previously.29
Amines and Quaternary Ammonium Salts. Nitrogen was
introduced at the end of the alkyl chain by nucleophilic sub-
stitution of idebenone mesylate (15) with various amines in tert-
butanol (Scheme 2). Reaction with imidazole, morpholine, or
dibenzylamine gave the tertiary amines 16, 17, and 18, respectively.
Similar reaction of 15 with N-methylimidazole or N-methyl-
morpholine gave the quaternary salts 19, 20. Other quaternary
salts, which could not be formed directly in this way, were
obtained via alkylation of the appropriate amine; thus, benzyl
bromide in dichloroethane was reacted with 16 to give 21 and
reaction of 18 with excess methyl iodide led to the dibenzylmethyl-
ammonium salt 22. The tribenzylammonium salt 23 was formed in
low yield by the prolonged reaction of excess benzyl bromide and
sodium iodide with 18 at 50 °C.
Figure 1. Model of enzyme-mediated tPMET pathway showing the
positioning of ubiquinone-based compounds within the lipid bilayer to
·
disrupt ubiquinone (CoQ) redox cycling via CoQ during oxidation of
intracellular NADH to NAD+.
protecting group for aromatic sulfonic acids.28 Reduction and
O-benzylation29 of 1 and 3 gave the alcohols (4,5), the anions of which
were then reacted with mesylate 6 to produce the sulfonic esters
(7, 8). The sulfonate ester 6 was obtained from 3-(chlorosulfonyl)-
benzoyl chloride by reaction with isobutanol, followed by reduc-
tion with RedAl then mesylation. Deprotection of 7 and 8 by
hydrogenation, followed by oxidation in air and removal of the
isobutyl group with sodium iodide, gave the required quinone
sulfonic acids 9 and 10.
1
The synthetic yields, HPLC, H NMR, and ESI-MS of un-
reported compounds are described in the Experimental Section
and 13C NMR data are presented in Table 2.
RESULTS AND DISCUSSION
■
Previous studies have shown that small, amphipathic, redox-
active molecules have tPMET inhibitory activity (TIA) as well as
antiproliferative activity (APA).22,23 We therefore synthesized a
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Scheme 2. Synthetic Routes to 16−23
Table 1. In Vitro tPMET Inhibitory Activity and
Antiproliferative Activity of Ubiquinone-Based Compounds
electron transport (Table 1). The ability of key active com-
pounds to induce apoptosis was also determined through cell
cycle analysis at 24, 48, and 72 h.
tPMET inhibitory activity
antiproliferative activity
(APA) IC50 (μM)
a
Alcohols − 1, 2, and 3. Initial efforts focused on readily
accessible, uncharged, polar C3, C5, and C10 quinone alcohols.
Compounds 1 and 2 did not have TIA in HL60 cells but had the
same moderate APA (13 1 μM), suggesting these compounds
did not position at the plasma membrane and disrupt tPMET,
but entered the cell and were active intracellularly. There was,
however, weak TIA in HL60ρo cells for 1 and 2 (92 22 and
123 53 μM, respectively), potentially due to HL60ρo cells
having upregulated tPMET as a consequence of dysfunctional
oxidative phosphorylation and increased cellular NADH.15 APA
(TIA) IC50 (μM)
compound
HL60
HL60ρo
HL60
HL60ρo
b
1
Inactive
Inactive
92 22
13
1
1
3
15
8
5
2
123 53
13
36
3
3
77 19
Inactive
70
4
Inactive
Inactive
9
Inactive
Inactive
10
13
14
16
17
18
19
20
21
22
23
81
6
6
70
13
9
5
2
143
16
3
120
5
1
5
63 35
Inactive
15 13
15 13
Inactive
44 10
Inactive
21
14
1
1
1
Inactive
33
46
9
8
3
3
2
2
activity of 1 and 2 with HL60ρo was similar to HL60 cells (15
and 8 3 μM) confirming that these compounds were not
5
33 11
Inactive
Inactive
78
Inactive
33 10
35 17
working primarily at the level of mitochondrial electron transport.
Compound 3 had weak TIA with HL60 and HL60ρo (77 19
and 70 4 μM), with moderate APA in HL60 cells (36 μM 3 μM)
but no APA in HL60ρo, suggesting that, in addition to effects on
tPMET, 3 was entering the cell and disrupting redox activity at
the level of mitochondrial electron transport. These results
support the view that inhibition of tPMET in HL60ρo cells is
independent of mitochondrial electron transport. Cell cycle data
for 3 showed cell cycle arrest in G1 in the surviving HL60 cells
within 24 h with a 30 μM dose, but minimal cell cycle changes in
HL60ρo cells (Figure 2).
Compounds with carbon chains longer than 10 were not con-
sidered given the synthetic chemical demands and our prior
experience,27 which indicated that incorporation of longer aliphatic
chains resulted in very lipophilic compounds that did not interact
effectively with cellular membranes. Well-known lipophilic bio-
molecules such as Coenzyme Q10 do not easily cross cell mem-
branes but are synthesized and function within the hydrophobic
core of membranes.
Overall, these results led to our designing compounds with a
stronger negative charge and a restricted range of carbon-linker
chains.
Sulfonic Acids − 9 and 10. Compounds 9 and 10 contain an
arylsulfonic acid function which, at biological pH, would be
completely deprotonated and thus embody a net negative charge.
The short chain compound (9) resulted in inactive TIA and APA
in both HL60 and HL60ρo cells, indicating the strong negative
23
122
9
1
8
8
1
2
3
2
0.3
0.2
0.1
0.1
1
68
47
6
4
5
3
3
2
10
14
1
3
0.4
1
54 12
a
Values are means of dose−response experiments SEM (n = 2/3).
b
IC50 = half maximal inhibitory concentration. Inactive = IC50 > 200 μM.
series of 15 compounds composed of a redox-active quinone
moiety, functionally related to ubiquinone, linked with either a 3,
5, or 10-carbon chain to a terminal moiety with varying polarity
and/or electronic charge and investigated their TIA and APA.
The compounds were designed to determine the optimum
carbon chain length and charge required to position the quinone
within the plasma membrane lipid bilayer and thereby disrupt
tPMET activity (see Figure 1).
Compounds were tested directly for TIA by their ability to
block reduction of the cell-impermeable tetrazolium salt WST-130,31
in real time following a 30 min preincubation. APA was measured
by reduction of cell-permeable MTT after 48 h incubation with
the compound. The IC50 for each compound was determined for
TIA and APA. Data were compared between the human leukemic
cell line HL60, which has active mitochondrial and plasma mem-
brane electron transport function, and HL60ρo mitochondrial gene-
knockout cells that are defective in mitochondrial electron
transport25 but have elevated tPMET.15 This allowed the effect of
tPMET inhibition to be distinguished from effects on mitochondrial
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Table 2. 13C NMR Data for Compounds
compound
a
a
b
b
b
b
b
b
b
b
b
position
9
10
13
16
17
18
19
20
21
22
23
1
186.3
146.1
146.1
185.8
144.1
140.7
61.9
186.3
146.1
146.1
185.9
144.4
140.3
61.9
184.8
144.3
144.2
184.4
143.2
138.9
61.2
61.2
11.9
25.8
26.4
29.0
29.0
29.1
29.1
29.2
29.7
28.6
68.0
184.7
144.3
144.3
184.2
143.0
138.7
61.1
61.1
11.9
26.4
26.5
28.7
29.0
29.3
29.3
29.3
29.7
31.1
47.0
184.7
144.3
144.2
184.1
143.0
138.6
61.1
61.1
11.9
26.3
26.5
27.5
28.7
29.3
29.4
29.5
29.5
29.8
59.8
184.6
143.9
143.1
184.1
140.1
138.6
61.1
184.6
144.2
144.2
184.1
143.0
138.7
61.1
61.1
11.9
26.1
26.3
28.6
28.8
29.1
29.2
29.7
30.2
36.5
50.0
184.6
144.2
144.2
184.1
143.0
138.7
61.1
61.1
11.9
21.8
26.0
26.3
28.5
29.0
29.1
29.1
29.1
29.6
59.9
184.6
144.2
144.2
184.1
142.9
138.6
61.1
184.5
144.2
144.1
184.0
142.8
138.6
61.0
184.6
144.3
144.2
184.1
142.9
138.7
61.1
2
3
4
5
6
OMe
OMe
ArMe
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
1″
Ar1
Ar2
Ar3
Ar4
Ar5
Ar6
Im2
Im4
Im5
NMe
Morph2
Morph3
61.9
61.9
61.1
61.1
61.0
61.1
12.2
12.2
11.9
11.8
11.8
11.9
c
c
c
c
c
c
c
c
c
c
24.6
27.4
26.4
26.3
23.0
24.1
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
29.9
27.6
27.0
28.6
26.2
26.3
c
c
c
c
c
c
71.3
30.0
27.2
28.8
28.4
26.8
c
30.7
28.7
29.1
28.9
28.6
c
30.8
29.3
29.1
29.1
28.8
c
30.8
29.5
29.1
29.1
29.1
c
30.9
29.6
29.6
29.1
29.1
c
31.1
29.9
26.1
29.2
29.1
c
31.1
29.8
30.1
29.6
29.6
72.0
58.2
50.1
59.57
64.6
60.1
c
73.5
140.5
126.4
146.1
126.4
129.7
130.9
73.6
53.4
53.2
64.3
140.9
126.4
146.1
126.4
129.7
131.0
161.3
94.8
126.6
128.1
128.7
128.2
128.7
128.1
133.0
129.0
129.3
129.4
129.3
129.0
137.1
121.8
121.8
127.0
129.2
133.1
130.6
133.1
129.2
128.0
129.3
133.2
130.6
133.2
129.3
157.6
95.6
157.6
94.8
137.1
129.4
118.7
138.3
123.2
121.5
39.6
c
c
c
c
c
c
c
c
c
c
47.6
60.7
65.7
46.4
53.8
67.0
a
b
c
CD3OD solvent. CDCl3 solvent. Denotes signals not formally assigned. Numbering systems as shown below.
charge was potentially repelling the quinone function from the
outer plasma membrane. This idea was supported by results with
10, which showed weak TIA in HL60 and HL60ρo (81 6 and
70 5 μM) and weak APA (143 3 and 120 5 μM) indicating
the longer alkyl chain was allowing the quinone to position within the
lipid bilayer.
These results indicated the need for a moiety with reduced
negative charge to prevent repulsion from the plasma membrane,
and a sufficiently long carbon chain to allow the quinone to posi-
tion within the lipid bilayer. Therefore, all further work concentrated
on the longer C10 chain length exclusively.
Phloroglucinols − 13 and 14. Compound 13 is a C10
quinone linked to a phloroglucinol through oxygen with two
residual free phenolic groups. Therefore at biological pH, 13
would be expected to have a small residual negative charge.
Compound 13 had strong TIA in HL60 cells (6 1 μM) and
moderate APA (16 5 μM), with cell death induced within 24 h
with a 30 μM dose (data not shown). HL60ρo cells had moder-
ately strong TIA (13 2 μM), but weak APA (63 35 μM), with
cell death at 72 h. In contrast, 14, a quinone linked through a C10
carbon to a phloroglucinol with three phenolic groups, had
moderately strong TIA in HL60 (21
1 μM) and HL60ρo
(9 1 μM) but no APA activity in either cell line. This suggests
that 14 partitions at the plasma membrane and does not enter the
cell, but tPMET inhibition alone is insufficient to induce cell
death. Since 13 had similar TIA to 14 but also had APA, we
suggest that 13 enters the cell where it has an intracellular target.
Thus, a compound with synergistic effects on tPMET and on intra-
cellular targets may have optimum activity against leukemic cells.
Amines −16, 17, and 18. Based on estimated pKa values for
16, 17 and 18, 16 could be relatively protonated and therefore
positively charged at biological pH compared to 17 and especially
18. Results for 16 showed moderate TIA for HL60 (14 1 μM)
and HL60ρo (33 9μM), with strong APA in HL60 cells (3 2μM)
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Figure 2. Effects of 3 and 22 on HL60 and HL60ρo on cell cycle arrest at 24 h time point. HL60 and HL60ρo cells were incubated with and without
30 μM 3 or 22 for 24 h, stained with propidium iodide (PI), and analyzed by flow cytometry to measure apoptosis by cell cycle arrest in G1 and
accumulation of cells in <G1 in surviving cell populations. A. HL60. B. HL60 + 30 μM 3. C. HL60 + 30 μM 22. D. HL60ρo. E. HL60ρo + 30 μM 3.
F. HL60ρo + 30 μM 22. Representative of 2 experiments.
and moderately strong activity in HL60ρo cells (15 13 μM).
Compound 17 showed a similar trend except that TIA for HL60
21, with a hindered and delocalized positive charge, gave
moderately strong TIA in HL60 cells (9 1 μM) and weak TIA
in HL60ρo cells (68 6 μM), but very strong APA in both cell
lines (2 and 5 2 μM). These promising results led to the
synthesis of 22 and 23, highly hindered, positively charged
compounds that could potentially inhibit tPMET and disrupt
intracellular redox systems thereby maximizing inhibition of cell
proliferation.
(33
11 μM) was increased. In contrast, the most basic
compound, 18, had no TIA on HL60 or HL60ρo cells and only
moderate APA effects on HL60 cells (33 10 μM). Compound
18 appeared not to partition at the membrane, but was entering
the cell and affecting the cell at the level of mitochondrial elec-
tron transport.
The dibenzylmethylammonium salt, 22, and tribenzylammonium
salt, 23, both gave very strong APA in both cell lines (2−3 μM), with
cell death induced within 24 h with a 30 μM dose (see Figure 2).
However, TIA was moderate in HL60 cells (10 1 and 14 3 μM)
and weak in HL60ρo cells (47 4 and 54 12 μM). These results
again strongly suggest that these compounds interact intracellularly,
but not at the level of mitochondrial electron transport.
Results from 16 and 17 highlighted the potential synergy of
positively charged compounds. Compounds with varying levels
of delocalized positive charge were therefore synthesized and
evaluated.
Quaternary Ammonium Salts − 19, 20, 21, 22, and 23.
Compound 19 containing a dispersed, mildly hindered delocalized
charge, had the least APA activity in this group of compounds,
with only moderate (HL60) and weak (HL60ρo) TIA. The
N-methylmorpholine salt 20, whose positive charge is less
distributed and moderately hindered had weak TIA for HL60
(122 8 μM), but very strong APA (1 + 0.3 μM). Examination of
20 with HL60ρo cells showed no TIA or APA. These results give a
strong indication that 20 is disrupting redox-sensitive mechanisms at
the mitochondrial level. In contrast, the benzylimidazolium compound,
CONCLUSION
■
Ubiquinone-like functions linked by a C10 carbon chain to
quaternary ammonium salts with a highly hindered positive
electronic charge had both the greatest inhibition of tPMET and
highest antiproliferative activity in vitro, independent of mito-
chondrial electron transport. Our data suggest that these compounds
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partition at the plasma membrane to inhibit tPMET activity but
this inhibition alone is not sufficient to induce cell death. These
positively charged compounds also enter the cell to disrupt non-
mitochondrial redox-sensitive mechanisms and it is this synergistic
effect that creates strong antiproliferative effects in human leukemic
cells.
We conclude that C10 ubiquinone-based compounds with
highly hindered, positively charged moieties are promising candidates
for further development as anticancer drugs.
Isobutyl 3-[10-(2,5-Bis(benzyloxy)-3,4-dimethoxy-6-meth-
ylphenyl)decyloxymethylene]benzenesulfonate (8). Similarly,
treatment of 3,4-dimethoxy-6-methyl-2,5-bis(phenylmethoxy)-
benzenedecanol (5)29 (0.10 g, 0.31 mmol) with isobutyl 3-(mesylo-
xymethyl)benzenesulfonate (7) (0.16 g, 0.31 mmol) gave 8 as a
colorless oil (70 mg, 30%). 1H NMR (CDCl3) δ: 7.88 (1H, bs), 7.81
(1H, bd, J = 8 Hz), 7.62 (1H, bd, J = 8 Hz), 7.52 (1H, t, J = 8 Hz),
7.49 (4H, bd), 7.39 (4H, bt), 7.34 (2H, bt), 5.00 (2H, s), 4.95 (2H,
s), 4.56 (2H, s), 3.94 (3H, s), 3.93 (3H, s), 3.81 (2H, d, J = 7 Hz),
3.50 (2H, t, J = 7 Hz), 2.57 (2H, bt, J = 7 Hz), 2.15 (3H, s), 1.95
(1H, m), 1.63 (2H, m), 1.24−1.44 (14H, m), and 0.89 (6H, d,
J = 7 Hz). HRMS (+ve ESI) calc for C44H58NaO8S 769.3745 m/z
[MNa+], found 769.3791 m/z.
3-[3-(2,5-Dioxo-3,4-dimethoxy-6-methylphenyl)-
propyloxymethylene]benzenesulfonic Acid (9). A solution of
isobutyl 3-[3-(2,5-bis(benzyloxy)-3,4-dimethoxy-6-methyl-
phenyl)propyloxymethylene]benzenesulfonate (7) (0.20 g,
0.31 mmol) in methanol (20 mL) in the presence of Pd/C
(5 mg, 5%) was stirred under hydrogen for 60 min when TLC
showed that all the starting material had been consumed. The
mixture was then stirred in air overnight, filtered through Celite,
and the solvents removed in vacuo to give a yellow gum. Puri-
fication by column chromatography over silica gel eluting with
DCM/ethyl acetate gave isobutyl 3-[3-(2,5-dioxo-3,4-dimethoxy-6-
methylphenyl)propyloxymethylene]benzenesulfonate as a yellow
gum (90 mg, 62%). HPLC: 13.50 min (method A), 14.32 min
(method B). 1H NMR (CDCl3) δ: 7.97 (1H, bs), 7.82 (1H, bd, J = 8
Hz), 7.61 (1H, bd, J = 8 Hz), 7.53 (1H, t, J = 8 Hz), 4.55 (2H, s) 3.97
(6H, s), 3.82 (2H, d, J = 7 Hz), 3.53 (2H, t, J = 7 Hz), 2.59 (2H, t, J =
7 Hz), 2.04 (3H, s), 1.95 (1H, m), 1.76 (2H, m), and 0.89 (6H, d, J =
7 Hz). HRMS (+ve ESI) calc for C23H30NaO8S 489.1554 m/z
[MNa+], found 489.1567 m/z.
EXPERIMENTAL SECTION
■
General Chemistry Methods. 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 visualized
with a UV lamp, and then by dipping in a vanillin solution (1%
vanillin, 1% H2SO4 in EtOH) and heating. High resolution mass
spectrometry (HRMS) was recorded using a Bruker microTOF
electrospray mass spectrometer. UV spectra were recorded in
methanol using a Jasco V-550. IR spectra were recorded on a
Bruker Optics Alpha FT-IR spectrometer (with a diamond
Attenuated Total Reflectance (ATR) top plate). NMR spectra, at
1
25 °C, were recorded at 500 or 300 MHz for H 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 7.26 and CDCl3 at 77.0; or (CH3)2CO at
2.15 and (CD3)2CO at 30.5; or CH3OD at 3.3 and CD3OD at 49.0;
or (CH3)2SO at 2.62 and (CD3)2SO at 39.6. 13C NMR data are
presented in Table 2. All tested compounds had a purity of ≥95%
shown by HPLC.
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 × 3 mm) with a
2 × 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 μL.
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 × 4.6 mm) with a 2 × 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 μL.
A stirred solution of isobutyl 3-[3-(2,5-dioxo-3,4-dimethoxy-
6-methylphenyl)propyloxymethyl]benzenesulfonate (90 mg) in
dry acetonitrile (10 mL) with sodium iodide (90 mg) was heated
to reflux for 3 h when TLC analysis showed all the starting
material had been consumed. Evaporation of the solvent in vacuo
and purification by column chromatography over silica gel eluting
with chloroform/methanol 0−20% gave quinone 9 as an orange
solid (40 mg, 51%). This was dissolved in water and freeze−dried
1
to give a fluffy orange solid. HPLC: 9.66 min (method A). H
NMR (CD3OD) δ: 7.84 (1H, s), 7.78 (1H, d, J = 7 Hz), 7.44
(2H, m), 4.52 (2H, s) 3.93 (6H, s), 3.55 (2H, t, J = 7 Hz), 2.61
(2H, t, J = 7 Hz), 2.03 (3H, s), and 1.76 (2H, m). HRMS (−ve
ESI) calc for C19H21O8S 409.0963 m/z [M-H−], found 409.0987 m/z.
3-[10-(2,5-Dioxo-3,4-dimethoxy-6-methylphenyl)-
decyloxymethylene]benzenesulfonic Acid (10). Similarly,
treatment of 8 (0.20 g, 0.27 mmol) gave isobutyl 3-[10-(2,5-
dioxo-3,4-dimethoxy-6-methylphenyl)decyloxymethylene]-
benzenesulfonate (0.11 g, 73%) as an orange solid. HPLC: 16.40 min
(Method A). 1H NMR (CDCl3) δ: 7.87 (1H, bs), 7.81 (1H, bd,
J = 8 Hz), 7.63 (1H, bd, J = 8 Hz), 7.53 (1H, t, J = 8 Hz), 4.56
(2H, s), 3.99 (3H, s), 3.98 (3H, s), 3.81 (2H, d, J = 7 Hz), 3.50
(2H, t, J = 7 Hz), 2.44 (2H, bt, J = 7 Hz), 2.01 (3H, s), 1.95 (1H,
m), 1.63 (2H, m), 1.24−1.44 (14H, m), and 0.89 (6H, d, J =
7 Hz). HRMS (+ve ESI) calc for C30H44NaO8S 587.2649 m/z
[MNa+], found 587.2665 m/z.
Procedures for the Synthesis of Compounds. Isobutyl 3-
[3-(2,5-Bis(benzyloxy)-3,4-dimethoxy-6-methylphenyl)-
propyloxymethylene]benzenesulfonate (7). To a stirred
solution of 3,4-dimethoxy-6-methyl-2,5-bis(phenylmethoxy)-
benzenepropanol (4) (0.13 g, 0.31 mmol) in dry dimethylfor-
mamide (0.5 mL) at RT was added sodium hydride (20 mg,
60% w/w, 0.47 mmol) and the mixture stirred for 0.5 h. Isobutyl
3-(mesyloxymethyl)benzenesulfonate (7) (0.10 g, 0.31 mmol)
was added and stirring continued for 12 h. The mixture was
added to ice/water then extracted into dichloromethane (DCM).
Purification by column chromatography over silica gel eluting with
1
DCM/ethyl acetate gave 7 (115 mg, 57%) as a colorless oil. H
Similarly, isobutyl 3-[10-(2,5-dioxo-3,4-dimethoxy-6-methyl-
phenyl)decyloxymethylene]benzenesulfonate (25 mg, 0.044
mmol) gave sulfonic acid 10 as an orange solid (11 mg, 47%).
HPLC: 13.56 min (Method A). 1H NMR (CD3OD) δ: 7.83 (1H,
bs), 7.75 (1H, bd, J = 8 Hz), 7.43 (1H, bd, J = 8 Hz), 7.40 (1H, t,
J = 8 Hz), 4.52 (2H, s), 3.94 (6H, s), 3.48 (2H, t, J = 6 Hz),
NMR (CDCl3) δ: 7.85 (1H, s), 7.81 (1H, d, J = 8 Hz), 7.59 (1H, d,
J = 8 Hz), 7.29−7.53 (11H, m), 5.00 (2H, s), 4.96 (2H, s), 4.52 (2H,
s), 3.95 (3H, s), 3.94 (3H, s), 3.80 (2H, d, J = 7 Hz), 3.50 (2H, t, J =
7 Hz), 2.70 (2H, m), 2.16 (3H, s), 1.94 (1H, m), 1.75 (2H, m), and
0.88 (6H, d, J = 7 Hz); HRMS (+ve ESI) calc for C37H44NaO8S
671.2649 m/z [MNa+], found 671.2644 m/z.
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Journal of Medicinal Chemistry
Article
2.45 (2H, m), 1.98 (3H, s), 1.59 (2H, m), and 1.28−1.40
(14H, m). HRMS (−ve ESI) calc for C26H35O8S 507.2058 m/z
[M-H−], found 507.2077 m/z.
3439, 2929, 2853, 1647, 1610, 1461, 1272 cm−1. UV λmax (log ε)
421 (2.11) and 281 (3.68) nm.
2-[10-(Dibenzylamino)decyl]-5,6-dimethoxy-3-methyl-2,5-
cyclohexadiene-1,4-dione (18). Similarly quinine mesylate 15
(100 mg, 0.24 mmol) and dibenzylamine (200 mg, 0.96 mmol)
gave 18 as a yellow gum (82 mg, 66%). HPLC: 11.07 min
(Method A), 13.92 min (Method B). Found: C, 75.15; H, 8.68;
N, 2.54. C33H43NO4. 1/2H2O requires: C, 75.25; H, 8.42; N,
5-[10-(2,5-Bis(benzyloxy)-3,4-dimethoxy-6-methylphenyl)-
decyloxy]benzene-1,3-diol (12). A solution of phloroglucinol
(0.30 g, 2.4 mmol) and 1-(10-iododecyl)-3,4-dimethoxy-6-
methyl-2,5-bis(phenylmethoxy)benzene (11)28 (0.20 g, 0.32 mmol)
in acetone (5 mL) containing potassium carbonate (0.30 g, 2.2 mmol)
was refluxed for 6 h. Treatment with 3 M HCl until acidic,
extraction into ethyl acetate, drying, and evaporation in vacuo
gave a brown gum. Purification by column chromatography over
silica gel eluting with methanol in chloroform gave 12 (65 mg,
33%) as a pale yellow gum. HPLC 16.70 min (Method A), 16.82
1
2.66. H NMR (CDCl3) δ: 7.18−7.38 (10H, m), 3.99 (6H, s),
3.54 (4H, s), 2.43 (2H, m), 2.38 (2H, t, J = 8 Hz), 2.01 (3H, s),
1.49 (2H, m), and 1.10−1.40 (14H, m). HRMS (+ve ESI) calc
for C33H44NO4 518.3265 m/z [MH+], found 518.3268 m/z. IR
υmax 2927, 2854, 1650, 1611, 1494, 1453, and 1266 cm−1. UV λmax
(log ε) 387 (2.55) and 279 (4.01) nm.
1
min (Method B). H NMR (CDCl3) δ: 7.48 (4H, m), 7.38
(4H, m), 7.34 (2H, m), 5.96 (2H, s), 5.89 (1H, bs), 5.00 (2H, s),
4.95 (2H, s), 3.94 (3H, s), 3.93 (3H, s), 3.84 (2H, t, J = 7 Hz),
2.56 (2H, bt, J = 7 Hz), 2.14 (3H, s), 1.71 (2H, quin, J = 7 Hz),
1.39 (4H, m), and 1.24−1.31 (10H, m). 13C NMR (CDCl3) δ:
161.2, 157.5 (2C), 146.7, 146.6, 144.8, 144.8, 138.0, 137.7, 130.8,
128.4 (2C), 128.2 (2C), 127.9 (2C), 127.9 (2C), 127.8 (2C),
125.5, 95.5, 94.8 (2C), 75.4, 75.1, 69.0, 61.3, 61.3, 30.1, 30.0,
29.4, 29.3 (5), 29.3, 29.2, 29.1, 27.2, 25.9, and 12.0. HRMS
(+ve ESI) calc for C39H48NaO7 651.3292 m/z [MNa+], found
651.3278 m/z.
3-[10(4,5-Dimethoxy-2-methyl-3,6-dioxo-cyclohexa-1,4-
dienyl)decyl]-1-methyl-3H-imidazol-1-ium Mesylate (19).
Similarly, quinone mesylate 15 (110 mg, 0.26 mmol) and
N-methylimidazole (200 mg, 24 mmol) in tert-butanol (10 mL)
gave 19 as an orange gum (56 mg, 54%). HPLC 9.47 min
(Method A), 12.54 min (Method B). 1H NMR (CDCl3) δ: 9.95
(1H, bs), 7.36 (1H, bs), 7.26 (1H, s), 4.24 (2H, t, J = 8 Hz), 4.04
(3H, s), 3.96 (6H, s), 2.75 (3H, s), 2.41 (2H, m), 1.98 (3H, s),
1.86 (2H, m), and 1.20−1.35 (14H, m). HRMS (+ve ESI) calc
for C23H35N2O4 403.2591 m/z [M+], found 403.2584 m/z. IR
υmax 3440, 2929, 2853, 1647, 1610, 1575, 1460, 1273, 1193 cm−1.
UV λmax (log ε) 420 (2.06) and 278 (3.86) nm.
5-[10-(2,5-Dioxo-3,4-dimethoxy-6-methylphenyl)-
decyloxy]benzene-1,3-diol (13). A solution of compound 12
(56 mg, 0.09 mmol) in methanol (10 mL) containing Pd/C
(10%, 4 mg) was stirred under an atmosphere of hydrogen for 3 h
when TLC showed that all the starting material had been con-
sumed. The mixture was then stirred in air overnight, filtered
through Celite, and the solvents removed in vacuo to give 13
(38 mg, 96%) as a yellow gum. HPLC: 13.34 min (Method A),
15.00 min (Method B). 1H NMR (CDCl3) δ: 5.99 (2H, s), 5.97
(1H, bs), 5.53 (2H, bs), 3.98 (3H, s), 3.97 (3H, s), 3.86 (2H, t,
J = 7 Hz), 2.44 (2H, dd, J = 6 Hz, 7 Hz), 2.00 (3H, s), 1.71 (2H,
quin, J = 7 Hz), 1.38 (4H, m), and 1.24−1.31 (10H, m). HRMS
(+ve ESI) calc for C25H34NaO7 469.2197 m/z [MNa+], found
469.2198 m/z. IR: υmax 3405, 2928, 2855, 1644, 1610, 1468, and
1266 cm−1. UV: λmax (log ε) 411 (2.52) and 277 (4.15) nm.
2-(10-Imidaz-1-yl)decyl-5,6-dimethoxy-3-methylcyclo-
hexa-2,5-diene-1,4-dione (16). A solution of the quinone
mesylate 15 (100 mg, 0.24 mmol) and imidazole (200 mg, 0.73
mmol) in tert-butanol (10 mL) was refluxed for 2 h. The solvent
was removed in vacuo, then the crude product purified by
column chromatography over silica gel when 5% methanol in
chloroform eluted 16 as a yellow solid (67 mg, 72%). HPLC:
9.38 min (Method A), 12.61 min (Method B). Found: C, 67.84;
H, 8.43; and N, 7.09. C22H32N2O4 requires: C, 68.01; H, 8.30;
and N, 7.21. 1H NMR (CDCl3) δ: 7.44 (1H, bs), 7.04 (1H, bs),
6.89 (1H, bs), 3.97 (6H, s), 3.90 (2H, t, J = 7 Hz), 2.43 (2H, t, J =
7 Hz), 1.99 (3H, s), 1.75 (4H, m), and 1.26 (16H, m). HRMS
(+ve ESI) calc for C22H32N2O4 389.2435 m/z [MH+], found
389.2417 m/z. IR: υmax 2928, 2855, 1651, 1610, 1456, 1267 cm−1.
UV: λmax (log ε) 411 (2.49) and 278 (4.14) nm.
4-[10(4,5-Dimethoxy-2-methyl-3,6-dioxo-cyclohexa-1,4-
dienyl)decyl]-4-methylmorpholin-4-ium Mesylate (20). Sim-
ilarly, quinone mesylate 15 (110 mg, 0.24 mmol) and N-methyl-
morpholine (100 mg, 0.99 mmol) in tert-butanol (10 mL) gave
20 as a yellow gum (42 mg, 42%). HPLC: 8.96 min (Method A),
1
12.15 min (Method B). H NMR (CDCl3) δ: 3.95−4.20 (4H,
m), 3.96 (6H, s), 3.55−3.90 (6H, m), 3.52 (3H, s), 2.41 (2H, m),
1.98 (3H, s), 1.79 (2H, m), and 1.20−1.40 (14H, m). HRMS
(+ve ESI) calc for C24H40NO5 422.2901 m/z [M+], found
422.2897 m/z. IR υmax 3439, 2926, 2854, 1647, 1610, 1456,
1267 cm−1. UV λmax (log ε) 387 (2.67) and 278 (4.06) nm.
3-[10(4,5-Dimethoxy-2-methyl-3,6-dioxo-cyclohexa-1,4-
dienyl)decyl]-1-benzyl-3H-imidazol-1-ium Mesylate (21). A
solution of the imidazole quinone 16 (70 mg, 0.18 mmol) and
benzyl bromide (100 mg, 0.88 mmol) in dichloroethane (7 mL)
was refluxed for 2 h. The solvent was removed in vacuo, then the
crude product purified by column chromatography over silica gel
when 5% methanol in chloroform eluted 21 as a yellow gum (45
mg, 52%). HPLC: 10.54 min (Method A), 13.29 min (Method
B). 1H NMR (CDCl3) δ: 10.60 (1H, s), 7.46 (2H, m), 7.33 (3H,
m), 7.31 (2H, s), 5.58 (2H, s), 4.25 (2H, t, J = 7.5 Hz), 3.94 (6H,
s), 2.39 (2H, t, J = 7 Hz), 1.96 (3H, s), 1.86 (2H, m), 1.32 (2H,
m), 1.28 (2H, m), and 1.25 (10H, m). HRMS (+ve ESI) calc for
C29H39N2O4 479.2904 m/z [M+], found 479.2892 m/z. IR υmax
3418, 2927, 2855, 1650, 1610, 1562, 1456, and 1266 cm−1. UV
λmax (log ε) 416 (2.39) and 278 (4.05) nm.
Dibenzyl[10(4,5-dimethoxy-2-methyl-3,6-dioxo-cyclo-
hexa-1,4-dienyl)decyl]methylammonium Mesylate (22). A
solution of the dibenzylaminoquinone 18 (100 mg, 0.19
mmol) in methyl iodide (5 mL) was stood for 18 h. The solvent
was removed in vacuo, then the crude product purified by
column chromatography over silica gel when 5% methanol in
chloroform eluted 22 as a yellow gum (82 mg, 64%). HPLC:
11.15 min (Method A), 13.71 min (Method B). 1H NMR (CDCl3)
δ: 7.61 (4H, d, J = 8 Hz), 7.40 (6H, m), 4.99 (4H, s), 3.94 (6H, s),
3.22 (2H, m), 3.07 (3H, s), 2.40 (2H, t, J = 7 Hz), 1.97 (3H, s),
1.94 (2H, m), and 1.10−1.40 (14H, m). HRMS (+ve ESI) calc
2,3-Dimethoxy-5-methyl-6-[10(4-morpholinyl)decyl]-2,5-
cyclohexadiene-1,4-dione (17).25 Similarly, quinone mesylate
15 (110 mg, 0.264 mmol) and morpholine (100 mg, 1.1 mmol)
in tert-butanol (10 mL) gave 17 as a yellow gum (62 mg, 59%).
HPLC: 9.03 min (Method A), 12.43 min (Method B). 1H NMR
(CDCl3) δ: 3.96 (6H, s), 3.68 (4H, t, J = 8 Hz), 2.42 (6H, m),
2.28 (2H, dd, J = 7,8 Hz), 1.98 (3H, s), 1.45 (2H, m), 1.35 (2H,
m), and 1.24−1.30 (12H, m). HRMS (+ve ESI) calc for
C23H38NO5 408.2744 m/z [MH+], found 408.2761m/z. IR υmax
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for C34H46NO4 532.3421 m/z [M+], found 532.3415 m/z. IR
υmax 2927, 2854, 1651, 1611, 1456, and 1267 cm−1. UV λmax (log
ε) 387 (2.31) and 278 (3.95) nm.
at 2000 rpm for 2 min, washed and resuspended in RPMI-1640
medium at a concentration of 0.4 × 106 cells/mL. 50 μL of cell
suspension was added to a well containing 50 μL of compound,
resulting in a final concentration of 0.2 × 106 cells/mL. Following
48 h incubation at 37 °C in a humidified incubator maintained at
5% CO2, dye reduction was initiated by adding 10 μL of 5 mg/mL
MTT to each well. After 2 h, 100 μL of lysing buffer was added and
incubated overnight to allow formazan crystals to dissolve. Micro-
titer plates were then measured at A570 in a FLUOstar OPTIMA
plate reader (BMG Laboratories, Offenberg, Germany).
Tribenzyl[10-(4,5-dimethoxy-2-methyl-3,6-dioxo-cyclo-
hexa-1,4-dienyl)decyl]ammonium Mesylate (23). A solution
of the quinone 18 (50 mg, 0.096 mmol) and benzyl bromide
(70 mg, 0.40 mmol) with sodium iodide (70 mg) was heated at
50 °C for 96 h. The solvent was removed in vacuo, then the crude
product purified by column chromatography over silica gel when
10% methanol in chloroform eluted 23 as an orange gum (12 mg,
18%). HPLC: 12.01 min (Method A), 14.32 min (Method B).
1H NMR (CDCl3) δ: 7.65 (6H, d, J = 8 Hz), 7.44 (9H, m), 5.11
(6H, s), 3.96 (6H, s), 3.25 (2H, m), 2.42 (2H, t, J = 8 Hz), 1.98
(3H, s), and 1.10−1.40 (16H, m). HRMS (+ve ESI) calc for
C40H50NO4 608.3734 m/z [M+], found 608.3736 m/z. IR υmax
2927, 2855, 1651, 1610, 1456, and 1267 cm−1. UV λmax (log ε)
387 (2.11), 278 (3.85) nm.
Cells and Cell Culture. Unless otherwise noted, tissue culture
plasticware was from Becton Dickinson (Auckland, New Zealand),
and cell culture reagents were from Invitrogen (Auckland, NZ).
HL60 cells, originally from ATCC, were obtained from Dr. Graeme
Findley (University of Auckland, NZ). Cells were grown in RPMI-
1640 medium, supplemented with 5% fetal bovine serum (ICP
Biotechnology, Auckland, NZ), GlutaMAX-1 (2 mM), penicillin
(10 000 U/mL), streptomycin (10 000 μg/mL). The mitochon-
drial DNA-knockout cell line, HL60ρo, was derived from its
parental cell line, HL60, by culturing in the presence of ethidium
bromide (50 ng/mL)29 for 6−8 weeks and lack of mitochondrial
DNA verified by PCR and stable phenotype. Cells were cultured
in RPMI-1640 medium, supplemented with 5% (v/v) heat-
inactivated fetal bovine serum, 25 μg/mL penicillin, 25 μg/mL
streptomycin, 50 μg/mL uridine, and 1 mM pyruvate. Both
HL60 and HL60ρo were grown to densities of (0.5−0.8) × 106
cells/mL (exponential stage) at 37 °C in a humidified incubator
maintained at 5% CO2.
Cell Cycle Arrest. Cells were seeded at 0.2 × 106 cells per mL
in media (as described above). 30 μM of compound was added
and cells incubated for 24 h at 37 °C in a humidified incubator
maintained at 5% CO2. After 24 h, cells were removed, washed in
HBSS, and fixed with 70% cold ethanol. Fixed cells were spun at
3000 rpm for 5 min, resuspended in 1 mL of propidium iodide
staining solution per million cells and incubated at 37 °C in a
humidified incubator maintained at 5% CO2 for 30 min. Cells
were then analyzed for cell cycle arrest as a measure of apoptosis
using flow cytometry (BD FACSort, Becton Dickson, San Jose, CA).
ASSOCIATED CONTENT
* Supporting Information
NMR spectra, HPLC, and characterization checklist. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +644 499 6914; fax: +644 499 6915; e-mail: mberridge@
malaghan.org.nz.
■
Author Contributions
Manuscript equally written by Carole Grasso and Lesley Larsen.
Notes
The authors declare no competing financial interest.
Biological Materials. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1)
and 1-methoxyphenazine methylsulfate (mPMS) were purchased
from Dojindo Laboratories (Kumamoto, Japan). 2-(4,5-Dimethyl-
2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) was pur-
chased from Sigma-Aldrich (St Louis, USA).
ACKNOWLEDGMENTS
The authors would like to thank Genesis Oncology Trust for
financial support for this work.
■
ABBREVIATIONS USED
■
tPMET Activity Measured by WST-1/mPMS Reduction.
TPMET activity was measured as previously described.30,31
Briefly, each compound dissolved in dimethyl sulfoxide (DMSO)
was diluted 10-fold in HBSS from a starting concentration of
200 μM. 50 μL of each dilution was added to a 96-well, flat-bottomed
microtiter plate (in triplicate). Exponentially growing cells were
centrifuged at 2000 rpm for 2 min, washed and resuspended in
HBSS buffer at a concentration of 2.0 × 106 cells/mL. 50 μL of
cell suspension was added to a well containing 50 μL of com-
pound, resulting in a final concentration of 1.0 × 106 cells/mL.
Cells were incubated for 30 min at 37 °C in a humidified
incubator maintained at 5% CO2. Dye reduction was initiated by
adding 10 μL of a 10× stock solution of WST-1/mPMS in Milli-
Q water (final concentrations of 500 μM WST-1 and 20 μM
mPMS). WST-1 reduction was measured in real time at 450 nm
over 40 min in a FLUOstar OPTIMA plate reader (BMG
Laboratories, Offenberg, Germany).
APA, antiproliferative activity; DCM, dichloromethane; HRMS,
high resolution mass spectrometry; PI, propidium iodide; TIA,
tPMET inhibitory activity; tPMET, trans-plasma membrane
electron transport
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