Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6215
analogue of a GSAO metabolite that enters cells faster and is
exported slower. The increased residence time of PENAO in
the cytosol correlates with its increased mitochondrial toxi-
city. PENAO is one of the most potent antitumor agents in
mice that has been reported and is well tolerated at efficacious
doses.
cultured in RPMI medium. HMEC-1 cells were cultured in
MCDB131 medium (Gibco, Gaithersburg, MD) supplemented
with 10 ng per mL EGF and 1 μg per mL hydrocortisone. The
cells were supplemented with 10% fetal calf serum (FBS), 2 mM
L-glutamine, and 10 units per mL penicillin and 10 μg per mL
streptomycin. Cell culture plasticware was from Techno Plastic
Products (Trasadingen, Switzerland). All other cell culture
reagents were from Gibco.
Accumulation of the Arsenicals in BAE Cells. BAE cells were
seeded in 6-well plates and allowed to attach overnight. The
medium was replaced, and the cells were incubated for 30 min in
the absence or presence of transport inhibitors and then with
50 μM GSAO, GCAO, CAO, or PENAO for up to 4 h.
Cytosolic arsenic measured by inductively coupled plasma
spectrometry.4
Drug Transporter Transfectants. Canine kidney epithe-
lial MDCKII cells stably overexpressing MRP1, MRP2, or
MRP3,11,12 human embryonic kidney HEK293 cells stably
overexpressing MRP4 and MRP5,13 and murine embryo fibro-
blast MEF3.8 cells stably overexpressing MRP6, MDR1, or
BCRP3 were used as described. Cells were grown and main-
tained as adherent monolayers in DMEM containing 10% calf
serum (Cosmic, Hyclone, Tauranga, New Zealand), 10 units per
mL penicillin, and 10 μg per mL streptomycin. Cytotoxicity
assays were performed as described previously.17
Cell Proliferation Assays. BAE, NB4, K562, MDCKII,
HT1080, LLC, HCT116, PANC-1, MCF-7, and BxPC-3 cells
were seeded at a density of 1.5 ꢀ 103, 3 ꢀ 103, 4 ꢀ 103, 5 ꢀ 102,
2 ꢀ 103, 5 ꢀ 102, 5 ꢀ 102, 6 ꢀ 103, 6 ꢀ 103, and 1 ꢀ 104 cells per
well, respectively, into 96-well plates. Cells were allowed to
adhere overnight at 37 °C in a 5% CO2, 95% air atmosphere.
Cells were untreated or pretreated with transporter inhibitors
and then cultured for 24, 48, or 72 h in medium containing 10%
fetal calf serum and PENAO. See figure legends for reagent
concentrations and times of incubation. Viable cells were de-
termined by incubating cells with the tetrazolium salt MTT
(Sigma, St. Louis, MO), which is metabolized by viable cells to
form insoluble purple formazan crystals. DMSO was added to
lyse cells, the contents of the wells were homogenized, and the
absorbance at 550 nm measured. Cell number in the untreated
control was normalized as 100%, and viable cell number for all
treatments was expressed as percentage of control.
Experimental Section
Synthesis and Purification of PENAO. An aqueous solution
of sodium carbonate (36.6 g, 345 mmol, 250 mL) was added to
a suspension of p-arsanilic acid (50.0 g, 230 mmol) in water
(250 mL) and cooled to 0 °C. Bromoacetyl bromide (92.6 g,
460 mmol) was added over a period of 10 min to the cooled
solution. The reaction was maintained for 1 h below 20 °C. The
precipitated product, BRAA, was collected by filtration and
washed with water. The wet cake was added to a mixture of
methanol (375 mL), 47% aqueous hydrobromic acid (375 mL),
and sodium iodide (5.14 g, 34.5 mmol), followed by dropwise
addition of a solution of sodium sulphite (86.9 g, 690 mmol) in
water (325 mL) over 15 min. The heterogeneous mixture was
maintained at room temperature for 2 h and filtered. The filter
cake was slurried in a mixture of methanol, 47% aqueous
hydrobromic acid, and water (375:150:225 mL), followed by
water (375 mL) and 5% aqueous sodium bicarbonate solution
(375 mL). The product was filtered and dried under vacuum at
1
45 °C for 12 h giving 54 g of BRAO (72.8% yield). H NMR
(CF3COOD): δ 4.14 (s, 2H), 7.80 (d, J = 7.8 Hz, 2H), 7.95 (d,
J = 7.6 Hz, 2H), 11.5 (s, 1H). 13C NMR (CF3COOD): δ 28.47,
123.69, 133.26, 134.07, 171.4.
To a freshly prepared solution of sodium (10.7 g 465 mmol)
in methanol (500 mL) was added D-penicillamine (25.53 g,
171-mmol) under argon atmosphere. The reaction mixture
stirred for 5 min. BRAO (50 g, 155 mmol) was added, stirred
for 10 min, and pH was adjusted to 3-5 using 5% sulfuric acid in
methanol and filtered. The clear filtrate was taken and dry
distilled under vacuum to give crude PENAO (190 g, 125%
yield). The crude PENAO was further purified by C18 silica gel
chromatography using water as eluent. The pure fractions were
collected and distilled completely to give 30 g of purified
material. The obtained solid was dissolved in methanol
(90 mL) and added to a solution of acetone and water mixture
(600 mL:45 mL) and stirred for 14 h. The precipitated white
product was filtered and dried at 40 °C for 4 h to give 18.1 g
(30%) of PENAO. 1H NMR (D2O): δ1.40 (s, 3H), 1.61 (s, 3H),
3.61 (d, J = 6.34 Hz, 2H), 3.72 (s, 1H), 7.58 (d, J = 7.81 Hz, 2H),
7.74 (d, j = 7.81 Hz, 2H). 13C NMR (D2O): δ 25.93, 29.68, 35.91,
49.58, 64.10, 124.02, 132.77, 141.55, 146.86, 173.44 ppm.
PENAO purity was characterized by HPLC (1200 Series;
Agilent Technologies, Santa Clara, CA) on a Zorbax Eclipse
XDB-C18 column (4.6 mm ꢀ 150 mm, 5 μm; Agilent Tech-
nologies) using a mobile phase of acetonitrile-water (25:75 vol/
vol), flow rate of 0.5 mL per min, and detection by absorbance at
256 nm. Purity of PENAO by peak area was 97%.
Mitochondrial Swelling Assay. Mitochondrial permeability
transition induction was assessed by light scattering using
isolated rat liver mitochondria.4
Primary Tumor Growth Assays. Female 7-9 week old Balb C
nude were used (UNSW Biological Resource Centre). Mice were
held in groups of 3-5 at a 12 h day and night cycle and were
given animal chow and water ad libitum. A suspension of 3 ꢀ 106
BxPC-3 cells in 0.1 mL of PBS was injected subcutaneously in
the proximal midline. Tumors were allowed to establish and
grow to a size of ∼50 mm3, after which they were randomized
into groups of 2-10. Animals were implanted with 28 day alzet
model 1004 micro-osmotic pumps (ALZA Corporation, Palo
Alto, CA) subcutaneously in the flank. The pumps delivered
vehicle, 0.025, 0.25, or 0.5 mg/kg/day PENAO or 0.25 or 5 mg/
kg/day GSAO. Tumor volume and animal weight was measured
every 2 or 3 days. Tumor volume was calculated using the
relationship length ꢀ height ꢀ width ꢀ 0.523. Tumor doubling
time (TD) was calculated from the tumor growth rate curve
during exponential growth using the formula TD = 0.693/
ln(VF/VI)/ID, where VF is final tumor volume, VI is initial tumor
volume, and ID the interval in days.18
GSAO, GCAO, and CAO were prepared as previously
described.2,4 All arsenical compounds were dissolved in phos-
phate-buffered saline containing 100 mM glycine, and concen-
trations were determined by titrating with dimercaptopropanol
and calculating the remaining free thiols with 5,50-dithiobis-
(2-nitrobenzoic acid). The solutions were stored at 4 °C in the
dark until use. There was no significant loss in the active
concentration of stock solutions of the arsenicals for at least a
week when stored under these conditions.
Cell Culture. Bovine aortic endothelial (BAE) cells were from
Cell Applications, San Diego, CA, and BxPC-3, HT1080, LLC,
PANC-1, MCF-7, HCT116, bEnd.3, HMEC-1, and K562 cells
were from ATCC, Bethesda, MD. MDCKII cells were from
P. Borst (The Netherlands Cancer Institute, Amsterdam). BAE,
HT1080, PANC-1, MCF-7, HCT116, MDCKII, bEnd.3, and
LLC cells were cultured in DMEM. K562 and BxPC-3 cells were
Immunohistochemistry. Blood vessel density was estimated by
immunostaining sectioned tumors for CD31 (rat antimouse
CD31; BD Pharmingen, Australia). Tumor cell proliferation
was estimated by immunostaining for PCNA (mouse antihu-
man PC10; Dako, Carpinteria, CA). Excised tumors were fixed
in formalin, embedded in paraffin, and 4 μm sections were cut
and mounted on Superfrost slides. Sections were deparaffinized