Synthesis and antitumor activity of ether glycero-phospholipids bearing a carbamate moiety at the sn-2 position: Selective sensitivity against prostate cancer cell lines

Article abstract of DOI:10.1002/cmdc.201000060

Analogues of 1-O-hexadecyl-sn-3-glycerophosphonocholine (compounds 1-4) or sn-3-glycerophosphocholine (compound 5) bearing a carbamate or dicarbamate moiety at the sn-2 position were synthesized and evaluated for their antiproliferative activity against c

Full text of DOI:10.1002/cmdc.201000060

DOI: 10.1002/cmdc.201000060
Synthesis and Antitumor Activity of Ether Glycero-
phospholipids Bearing a Carbamate Moiety at the sn-2
Position: Selective Sensitivity Against Prostate Cancer Cell
Lines
Hoe-Sup Byun,[a] Robert Bittman,*[a] Pranati Samadder,[b] and Gilbert Arthur*[b]
Analogues of 1-O-hexadecyl-sn-3-glycerophosphonocholine
(compounds 1–4) or sn-3-glycerophosphocholine (compound
5) bearing a carbamate or dicarbamate moiety at the sn-2 po-
sition were synthesized and evaluated for their antiproliferative
activity against cancer cells derived from a variety of tissues.
Although all of the compounds are antiproliferative, surprising-
ly the carbamates (1 and 2) are more effective against the hor-
mone-independent cell lines DU145 and PC3 than toward
other cancer cell lines we examined. This selectivity was not
observed with the dicarbamates (3 and 4). Phosphocholine car-
bamate analogue 5 is as effective against the prostate cancer
cell lines as the corresponding phosphonocholine analogue 1.
Cell death induced by 2’-(trimethylammonio)ethyl 4-hexadecyl-
oxy-3(R)-N-methylcarbamoyl-1-butanephosphonate (carbamate
analogue 2) appeared to be mediated by apoptosis, as as-
sessed by caspase activation and loss of mitochondrial mem-
brane potential. The in vivo activity of 2 was evaluated in a
murine prostate cancer xenograft model. Oral and intravenous
administration showed that 2 is effective in inhibiting the
growth of PC3 tumors in Rag2M mice. Our studies show that
the glycerolipid carbamates reported herein represent a class
of prostate-cancer-selective cytotoxic agents.
Introduction
Prostate cancer is the most common male cancer and the
second leading cause of cancer deaths in males.[1] Although
androgen deprivation is initially effective, it does not cure the
disease, and invariably the tumor recurs in an androgen-inde-
pendent form that usually metastasizes primarily to bone.[2,3]
The molecular events underlying the progression of the dis-
ease have yet to be elucidated; however, it is clear that once
the disease progresses, it does not respond to the current
array of chemotherapeutic agents.[4–6] There is, therefore, a
clear need to develop new agents that kill hormone-independ-
ent prostate tumor cells and/or prevent their metastases.[7]
A group of antitumor compounds collectively known as anti-
tumor ether lipids (AELs) act by perturbing intracellular signal-
ing pathways, leading to cell death.[8–14] These compounds are
long-lived analogues of the naturally occurring phospholipid
lysophosphatidylcholine. Insertion of ether bonds into the sn-1
and sn-2 positions of lysophosphatidylcholine in place of the
usual two ester bonds gives an analogue that is highly resist-
ant to metabolism at sites other than in the vicinity of the
phosphodiester linkage. AELs have the potential to deliver an-
titumor activity without any mutagenicity because, unlike
many other anticancer agents, they do not interact directly
with DNA. They possess cell-selective effects by inhibiting the
proliferation and killing of cancer cells at concentrations that
do not affect normal cells,[12,15–17] but the basis for this is un-
clear. The prototype or gold standard AEL is 1-O-octadecyl-2-O-
methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), which in-
hibits a broad panel of tumor cell lines,[14,18–20] but exhibits no
known selectivity against specific cancer cell types. We have
[a] Dr. H.-S. Byun, Prof. R. Bittman
Department of Chemistry and Biochemistry
Queens College of The City University of New York
Flushing, NY 11367-1597 (USA)
Fax: (+1)718-997-3349
E-mail: Robert.Bittman@qc.cuny.edu
[b] Dr. P. Samadder, Prof. G. Arthur
Department of Biochemistry and Medical Genetics
University of Manitoba, 745 Bannatyne Avenue
Winnipeg, Manitoba R3E 0J9 (Canada)
Fax: (+1)204-789-3421
E-mail: arthurg@ms.umanitoba.ca
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000060.
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G. Arthur, R. Bittman, et al.
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shown that replacing the phosphocholine moiety at the sn-3
position of ET-18-OCH3 with a variety of glycosyl residues leads
to differences in efficacy against various cell types and a differ-
ence in the mechanisms responsible for cell death.[21–23] In this
study, we retained a long-chain O-alkyl group at the sn-1 posi-
tion, but replaced the methoxy group at the sn-2 position of
ET-18-OCH3 with another hydrolytically stable group (carba-
mate), and we assessed the efficacy of these novel analogues
against epithelial cancer cell lines. In addition, we replaced the
phosphocholine head group, which is susceptible to hydrolysis
catalyzed by phospholipase C, with a hydrolytically stable iso-
steric analogue bearing a carbon–phosphorus bond (i.e., a
phosphonocholine head group). Herein we report the prefer-
ential effects of carbamate analogues 1–5 against hormone-
independent prostate cancer cell lines. We also show that oral
or intravenous delivery of the lead compound results in inhibi-
tion of the growth of prostate cancer xenografts.
Results and Discussion
Chemistry
The synthesis of target compounds 1 and 3 commenced with
(S)-(À)-1,2,4-butanetriol, which was converted into benzylidene
acetal (S)-6 as described previously[24] (Scheme 1). O-Alkylation
of 6 under phase-transfer conditions afforded ether 7,[24] which
was treated with N-bromosuccinimide and barium carbonate
to afford bromide 8[24] in high yield. Arbuzov reaction of bro-
mide 8 with neat triethyl phosphite at 1508C gave benzoyl
phosphonate 9 in 79% yield. Base-induced hydrolysis of the
benzoyl group of phosphonate 9 provided hydroxyphospho-
nate 10 in 86% yield. During the deprotection of the benzoate
group by methanolysis, the ethyl groups in phosphonate 9
were also converted into methyl groups by transesterification.
Hydroxyphosphonate 10 was reacted with methyl isocyanate,
which was generated in situ by the reaction of methyl iodide
with potassium cyanate in tetrahydrofuran/N,N-dimethylform-
amide (10:1), to furnish a mixture of carbamates 11 and 12 in
11 and 53% yields, respectively. If the amount of DMF was in-
creased, the rate of carbamate formation was faster but the
yield of carbamate 11 decreased. After compounds 11 and 12
were separated by flash chromatography (eluted with a gradi-
ent of chloroform/methanol; see Experimental Section), the
methyl groups in the phosphonate ester were removed by
treatment of 11 or 12 with trimethylsilyl bromide in dichloro-
methane. The resulting phosphonates were then coupled to a
choline group with choline toluenesulfonate promoted by tri-
chloroacetonitrile in pyridine at 508C[25] to afford the target
phosphonocholines 1 and 3 in good yield. Similarly, the target
compounds 2 and 4 (the respective enantiomers of 1 and 3)
were prepared by starting from commercially available 3(R)-
1,3-O-benzylidine-1,3,4-butanetriol (which is prepared from
d-malic acid).[26]
Scheme 1. Synthesis of compounds 1 and 3. Reagents and conditions:
a) NBS, BaCO3, ClCH2CH2Cl, reflux, 4 h; b) NaH, nBu4NBr, THF, RT, 24 h;
c) (EtO)3P, 1508C, overnight; d) MeI, KNCO, nBu4NBr, (iPr)2NEt, THF/DMF
(10:1); e) NaOMe, MeOH, RT; f) 1. Me3SiBr, CH2Cl2, RT, 2. choline toluenesulfo-
nate, Cl3CCN, py, 508C, 48 h.
Scheme 2. Synthesis of compound 5. Reagents and conditions: a) Ph3P, DIAD,
Me3SiN3, CH2Cl2, 08C then RT; b) 2-chloro-2-oxo-1,3,2-dioxaphospholane,
(iPr)2NEt, CH2Cl2, 08C then RT; c) 1. Me3SiBr, CH2Cl2, 2. Me3N(aq), CHCl3/MeCN/
iPrOH (0.9:1.5:1.5), RT, 48 h; d) Pd/C, H2, EtOH, RT, overnight; e) ClCO2Me,
Et3N, CHCl3, 08C then RT, overnight.
As depicted in Scheme 2, the preparation of carbamate
phosphocholine 5 started with 3-O-hexadecyl-sn-glycerol (13),
which was prepared from 1,2-O-isopropylidene-sn-glycerol via
O-alkylation and deketalization, as described previously.[27] Re-
gioselective and stereospecific azidation of glycerol 13 with
azidotrimethylsilane under Mitsunobu conditions[28] gave azido
alcohol 14 in 76% yield. Insertion of the phosphocholine head
group into alcohol 14 provided sn-2-azidoglycerophosphocho-
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Antitumor Activity of Ether Glycerophospholipids
line 16 in 69% overall yield by the following sequence of reac-
tions: 1) phosphorylation of alcohol 14 with 2-chloro-2-oxo-
1,3,2-dioxaphospholane, 2) ring opening of phospholane inter-
mediate 15 with trimethylsilyl bromide, and 3) quaternization
of the resulting ring-opened product with aqueous trimethyl-
amine. Finally, reduction of the azido group of 16 by catalytic
hydrogenation, followed by reaction of amine 17 with methyl
chloroformate, furnished the target carbamate phosphocholine
5 in 85% overall yield.
2. In addition, the apparent preferential effect of 2 against
prostate cancer cell lines relative to the other cells was not ob-
served with 4. To examine the influence of chirality on the ac-
tivity, we synthesized compounds 1 and 3 (the respective
enantiomers of 2 and 4). Compound 1 was active in the same
range as 2 and displayed a similar selectivity for DU145 and
PC3 as compound 2. Compound 3 was found to be less active
than 4 and showed no selectivity for prostate cancer cells.
Compound 5 differs from compounds 1 and 2 in two struc-
tural features: first, it has an isosteric phosphocholine moiety
in place of the phosphonocholine head group; second, the
carbamate connectivity to the glycerol backbone is reversed,
as in this analogue there is an N-carbamoyl group in place of
the O-carbamoyl group. The activity of compound 5 against
the prostate cancer lines DU145 and PC3 was similar to the ac-
tivity observed for compounds 1 and 2, and was less active
against cancer cells derived from other tissues (Table 1;
figure 1, Supporting Information).
Biology
Growth-inhibiting activity
The ability of compounds 1–5 to inhibit growth of a number
of epithelial cancer cell lines was assessed using the CyQuant
assay.[21] The cell lines were derived from breast (MCF-7, BT549,
MDA-MB-231), lung (A549), cervix (HeLa), prostate (DU145,
PC3), kidney (A498), and liver (HepG2). The compounds were
added to exponentially growing cells, and the cells were incu-
bated with the drugs for 48 h. The results are shown in
figure 1 of the Supporting Information, and the IC50 values de-
rived from these data are listed in Table 1. Compound 2 inhib-
Toxicity
We compared the toxicity of the phosphonocholine carba-
mates 1 and 2 with that of phosphonocholine dicarbamate 4
against DU145 and PC3 cells (Figure 1). Compound 2 had LD50
Table 1. Growth inhibitory potency of carbamate and dicarbamate com-
pounds 1–5 against a panel of human cancer cell lines.
IC50 [mm][a]
Cell Line
1
2
3
4
5
DU145
PC3
BT549
MDA-MB-231
MCF-7
HeLa
A549
HepG2
A498
2.1
2.5
6.8
6.1
10.1
7.9
>20
>20
13.0
1.2
8.2
4.8
2.8
4.9
2.4
1.2
4.4
3.7
4
3.3
10.7
4.1
6.6
6.6
1.9
13.4
10.5
9.1
18.3
13.4
8.7
>20
>20
13.5
>20
14.9
>20
>20
16.1
19.3
>20
>20
12.7
>20
9.5
[a] IC50: compound concentration that effects 50% growth inhibition;
values were determined from growth curves (see Supporting Information)
after 48 h incubation with 1–5 (0–20 mm); each sample is the mean of
eight experiments.
ited the proliferation of all of the cell lines with the exception
of HepG2 cells. The most striking observation was an apparent
higher sensitivity of the two hormone-insensitive prostate
cancer cell lines, DU145 and PC3, to compound 2 relative to
the concentrations required to inhibit the growth of most of
the other cell lines. The IC50 value for 2 against DU145 and PC3
cells is 1.2 mm. Whereas compound 2 completely inhibited pro-
liferation at 4 mm, a concentration of at least 12 mm was re-
quired to elicit a similar effect on the other cell lines with the
exception of HeLa cells (Supporting Information figure 1). The
dicarbamate analogue 4 was less active than the correspond-
ing carbamate analogue 2 and, with the exception of BT549
cells, the IC50 value and concentrations needed to completely
inhibit growth were greater than that obtained for compound
~
&
*
Figure 1. Effects of compounds 1 ( ), 2 ( ), and 4 ( ) on the viability of
A) DU145 and B) PC3 prostate cancer cells. Cell viability was assessed with
the ToxiLight assay in 96-well plates. Data represent the mean ÆSD of eight
replicates.
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G. Arthur, R. Bittman, et al.
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values of 3.3 and 2.0 mm for PC3 and DU145 cells, respectively,
compared with LD50 values of 5.4 mm for PC3 and 3.9 mm for
DU145 obtained with compound 1. The values for dicarbamate
4 were 5.4 mm for PC3 and 6.3 mm for DU145. Compound 2
was more active than compound 4 against DU145 cells. Thus,
glycerolipid carbamates 1, 2, and 5 and dicarbamates 3 and 4
are effective antiproliferative and cytotoxic compounds against
epithelial cancer cells and represent novel analogues of AELs.
Unexpectedly, the hormone-independent prostate cancer cell
lines DU145 and PC3 appeared to be more affected by 1, 2,
and 5 than the epithelial cancer cell lines derived from the
other tissues investigated.
These two prostate cancer cell lines are well-known cellular
models of the hormone-insensitive stage of prostate
cancer,[29,30] the most deadly form of the disease that does not
respond to current conventional chemotherapy. Because hor-
mone-insensitive prostate cancer cells are highly sensitive to
compounds 1, 2, and 5, we postulate that it may be possible
to selectively kill prostate cancer cells with low concentrations
of the carbamate analogues of ET-18-OCH3.
Carbamate 2 induces apoptosis in DU145 and PC3 cells
The alkyllysophospholipid class of AELs kill cells by apopto-
sis.[14,17,21] To investigate if compound 2 induces apoptosis, we
examined the levels of activated caspases in the cells using a
general caspase assay kit. Incubation of DU145 or PC3 cells
with compound 2 at 5 mm for 8 h led to a 2.8- to 3.6-fold in-
crease in the levels of activated caspases (Figure 2). We also
Figure 3. Effect of compound 2 on the mitochondrial membrane potential
of DU145 cells. Cells were incubated with or without compound 2 (5 mm) for
8 h. The mitochondrial membrane potential was assayed with the JC-10 re-
agent.
carbamate 2 involves induction of apoptosis. However, further
studies are required to establish why the DU145 and PC3 pros-
tate cancer cells are more susceptible to these compounds rel-
ative to other cell types.
Effect of 2 on PC3 xenograft growth
Figure 2. Effect of compound 2 on caspase activity in prostate cancer cell
lines DU145 and PC3. Cells were incubated with compound 2 at 5 mm for
8 h. Caspase activity was measured by the CaspGlow assay[21, 28] with FITC–
VAD–FMK using a fluorescence plate reader. Data represent the mean ÆSD
of five replicates.
To investigate the activity in vivo, we examined the efficacy of
compound 2 in inhibiting tumor growth in Rag2M mice im-
planted with PC3 cells. Animals were given either 30 or
50 mgkgÀ1 orally or 10 or 15 mgkgÀ1 of compound 2 intrave-
nously. These concentrations, which were established from tol-
erability studies, had no toxic effects on the animals. The ani-
mals were segregated into groups based on the sizes of the
tumors at the start of the treatment. The experimental animals
received compound 2 at the start of the treatment, and once a
week for two weeks for a total of three treatments.
In the animals with a starting tumor size of 30 mm3, oral
treatment with 30 mgkgÀ1 of 2 delayed growth to 300 mm3 by
9 days, while the delay for those receiving 50 mgkgÀ1 of 2
orally was 24 days. Growth to 500 mm3 was delayed by 14 and
28 days, respectively, in animals given 30 and 50 mgkgÀ1 (Fig-
ure 4A). At an initial tumor volume of 80 mm3, a 12 day delay
assessed the effect of compound 2 on the mitochondrial mem-
brane potential in DU145 and PC3 cells by using the JC-10
assay, which determines the loss of mitochondrial membrane
potential as a measure of irreversible apoptosis.[21,31] Incubation
of DU145 cells with compound 2 at 5 mm for 8 h resulted in a
change in the red color of the aggregates in the mitochondria
to the monomeric green color, indicative of the loss of mito-
chondrial membrane potential in these cells (Figure 3). Similar
results were obtained with PC3 cells (see figure 2, Supporting
Information). These observations indicate that cell death by
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Antitumor Activity of Ether Glycerophospholipids
Conclusions
In summary, we discovered a selective toxicity of the carba-
mate and dicarbamate glycerophospholipids and phosphono-
lipids against two hormone-independent prostate cancer cells
lines, DU145 and PC3. In addition, compound 2 inhibited the
growth of PC3 cells in an in vivo study using both oral and in-
travenous administration to Rag2M mice. In contrast to the
present study in which compound 2 was administered only
once per week, a recent study has indicated that frequent oral
administration of ET-18-OCH3 is needed to increase oral bio-
availability.[32] The results reported herein warrant further inves-
tigation to optimize the frequency of dosing to achieve greater
shrinkage and/or inhibition of tumor growth.
Experimental Section
Chemistry
THF was distilled from Na and benzophenone before use. Pyridine,
DMF, ClCH2CH2Cl, (iPr)2NEt, and CH2Cl2 were dried over CaH2. 1H,
13C, and 31P NMR spectra were recorded at 400, 100, and
161.9 MHz, respectively, and were referenced to the residual CHCl3
at d=7.24 (1H) and d=77.00 ppm (13C) and to 85% H3PO4 (31P).
Optical rotations were measured in a cell with a path length of
1 dm on a digital polarimeter. TLC was carried out on Al-backed
silica gel GF plates (250 mm thickness), and the compounds were
visualized by charring with 10% H2SO4 in EtOH and/or short wave-
length UV light. The products were purified by flash chromatogra-
phy on silica gel 60 (230–400 ASTM mesh). HRMS data were
obtained by electrospray ionization.
(3S)-4-O-Hexadecyl-1,3-O-benzylidine-1,3,4-butanetriol
(7).[24]
(3S)-1,3-O-Benzylidine-1,3,4-butanetriol (6)[24] (3.89 g, 20.0 mmol) in
20 mL THF was added to a suspension of NaH (1.60 g, 40.0 mmol;
60% in white oil, washed twice with dry hexane) in 100 mL dry
THF at 08C. After the evolution of H2 gas had stopped, 1-bromo-
hexadecane (6.2 mL, 20.2 mmol) and nBu4NBr (0.67 g, 2.0 mmol)
were added. The mixture was stirred for 24 h at room temperature,
and then the reaction was quenched with 10 mL MeOH. The vola-
tile solvents were removed under reduced pressure, and the resi-
due was diluted with 200 mL Et2O and washed with H2O. The or-
ganic layer was dried over Na2SO4 and concentrated under reduced
pressure to give a residue, which was purified by column chroma-
tography on silica gel (elution with hexane/EtOAc 25:1) to afford 7
Figure 4. Effect of compound 2 on the growth of PC3 tumor xenografts in
Rag2M mice. Mice bearing PC3 tumors were given three treatments of com-
pound 2 orally or by intravenous injection at seven-day intervals. A) oral
3
À1
À1
!
*
*
treatment of a 30-mm tumor: control ( ), 30 mgkg
(
), 50 mgkg
( );
3
À1
À1
*
*
B) oral treatment of a 80-mm tumor: control ( ), 30 mgkg
); c) intravenous treatment of a 60-mm tumor: control ( ), 15 mgkg
(
), 50 mgkg
3
À1
!
*
*
),
(
(
10 mgkgÀ1
(
). Data represent the mean ÆSD of four animals; data points
!
with no error bars represent the means of two animals.
1
was observed in the growth of the tumor to 950 mm3 in ani-
mals given 50 or 30 mgkgÀ1 (Figure 4B). Not surprisingly, the
effectiveness of the compound was dependent on the size of
the tumor at the start of drug treatment, with the effect on
smaller tumors being greater than that on larger tumors. The
delays were shorter with increasing size of the tumor.
In animals receiving intravenous injections of 2, growth of
the tumor from 60 to 800 mm3 was delayed by 14 days with
three treatments of compound 2 at 15 mgkgÀ1 (Figure 4C). The
delay with animals administered compound 2 at 10 mgkgÀ1
was 9 days.
(7.62 g, 91%) as a white solid; mp: 54–558C; H NMR (CDCl3): d=
0.88 (t, 3H, J=6.6 Hz), 1.25 (brs, 26H), 1.54–1.60 (m, 3H), 1.80–1.91
(m, 1H), 3.44–3.52 (m, 3H), 3.62 (dd, 1H, J=5.8, 10.3 Hz), 3.95–4.01
(m, 1H), 4.01–4.09 (m, 1H), 4.29 (dd, 1H, J=4.1, 11.4 Hz), 5.53 (s,
1H), 7.31–7.38 (m, 3H), 7.49 ppm (dd, 2H, J=1.6, 7.9 Hz); 13C NMR
(CDCl3): d=14.1, 22.7, 26.1, 28.3, 29.3, 29.5, 29.6, 29.7, 30.9, 31.9,
66.9, 71.9, 73.7, 76.3, 101.2, 126.1, 128.2, 128.7, 129.0, 129.7,
138.6 ppm; MS (ES) [M+H]+ m/z calcd for C27H47O3: 419.3, found:
419.2.
(3R)-4-O-Hexadecyl-1,3-O-benzylidine-1,3,4-butanetriol. The en-
antiomer of 7 was prepared in 90% yield from (3R)-1,3-O-benzyl-
idine-1,3,4-butanetriol[33] by the procedure described above.
The results of the PC3 xenograft studies (Figure 4) show the
potential utility of carbamate 2 in inhibiting the growth of
prostate tumors. The compound caused a significant decrease
in tumor growth for both oral and intravenous administration.
3(S)-Benzoyl-4-hexadecyloxy-1-bromobutane (8).[24] A mixture of
7 (5.19 g, 12.4 mmol), NBS (2.65 g, 14.9 mmol, and BaCO3 (1.10 g,
5.57 mmol) in 100 mL CCl4 (or ClCH2CH2Cl) was heated at reflux for
4 h. The reaction mixture was passed through a pad of silica gel,
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G. Arthur, R. Bittman, et al.
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which was rinsed with 100 mL hexane/EtOAc (10:1). The filtrate
was concentrated to give a residue, which was purified by column
chromatography on silica gel (elution with hexane/EtOAc 25:1) to
provide bromide 8 (5.36 g, 87%) as a pale-yellow oil; [a]2D5 =À24.9
(c=6.7, CHCl3); 1H NMR (CDCl3): d=0.88 (t, 3H, J=6.6 Hz), 1.26
(brs, 26H), 1.50–1.56 (m, 2H), 2.31–2.41 (m, 2H), 3.42–3.50 (m, 4H),
3.62–3.65 (m, 2H), 5.35–5.41 (m, 1H), 7.42–7.46 (m, 2H), 7.54–7.59
(m, 1H), 8.05 ppm (dd, 2H, J=1.4, 8.4 Hz); 13C NMR (CDCl3): d=
14.1, 22.6, 26.0, 28.7, 29.3, 29.4, 29.5, 29.6, 31.9, 34.5, 71.2, 71.7,
71.7, 128.3, 129.6, 130.0, 133.0, 165.9 ppm; MS (ES) [M+H]+ m/z
calcd for C27H46BrO3: 497.2, found: 497.2.
10 mmol), and hydroxyphosphonate 10 (2.12 g, 5.02 mmol) in
25 mL THF/DMF (10:1). The mixture was stirred until compound 10
was completely consumed. The reaction mixture was diluted with
CHCl3 and washed with brine solution. The organic layer was dried
over Na2SO4 and concentrated. The product was purified by
column chromatography on silica gel (elution with a gradient of
CHCl3, CHCl3/MeOH 100:1, CHCl3/MeOH 50:1, then CHCl3/MeOH
25:1) to give carbamoyl phosphonate 11 (265 mg, 11%) and dicar-
bamoyl phosphonate 12 (1.43 g, 53%) as colorless oils. Data for
compound 11: [a]2D5 =À5.82 (c=5.5, CHCl3); 1H NMR (CDCl3): d=
0.88 (t, 3H, J=6.4 Hz), 1.26 (brs, 26H), 1.48–1.62 (m, 2H), 1.15–2.00
(m, 4H), 2.87 (d, 3H, J=4.8 Hz), 3.25–3.50 (m, 6H), 3.75 (d, 6H, J=
10.8 Hz), 4.90–5.00 (m, 1H), 7.60–7.70 ppm (brs, 1H); 13C NMR
(CDCl3): d=14.1, 20.7 (d, J=151 Hz), 22.6, 24.0 (d, J=5.0 Hz), 26.0,
26.1, 26.4, 29.3, 29.4, 29.5, 29.6, 31.9, 52.4, 67.8, 74.4 ppm (d, J=
6.0 Hz), 153.7; 31P NMR (CDCl3): d=35.5; MS (ES) [M+H]+ m/z calcd
3(R)-Benzoyl-4-hexadecyloxy-1-bromobutane. The enantiomer of
bromide 8 was prepared in 85% yield from (3R)-4-O-hexadecyl-1,3-
O-benzylidine-1,3,4-butanediol by the procedure described above;
[a]2D5 =+20.5 (c=6.9, CHCl3).
for C24H51NO6P: 480.3, found: 480.3. Data for compound 12: [a]D25
=
Diethyl 3(S)-benzoyl-4-hexadecyloxy-1-butanephosphonate (9).
A solution of bromide 8 (4.98 g, 10.0 mmol) in 25 mL (EtO)3P
(150 mmol) was heated at 1508C (oil bath temperature) overnight.
After excess (EtO)3P was removed by using a stream of air, the resi-
due was purified by column chromatography on silica gel (elution
with CHCl3/MeOH 25:1) to give benzoyl phosphonate 9 (4.39 g,
79%) as a colorless oil; [a]2D5 =À6.88 (c=5.8, CHCl3); 1H NMR
(CDCl3): d=0.88 (t, 3H, J=6.7 Hz), 1.26 (brs, 26H), 1.28–1.33 (m,
6H), 1.70–2.00 (m, 2H), 3.43–3.62 (m, 4H), 4.06–4.12 (m, 4H), 5.23–
5.27 (m, 1H), 7.42–7.46 (m, 2H), 7.54–7.59 (m, 1H), 8.04 ppm (d,
2H, J=7.0 Hz); 13C NMR (CDCl3): d=14.1, 16.4 (d, J=5.8 Hz), 22.2
(d, J=143.2 Hz), 22.6, 24.3 (d, J=4.0 Hz), 29.3, 29.4, 29.6, 29.7, 31.9,
61.6, 71.3, 71.7, 72.8 (d, J=18.1 Hz), 127.7, 128.3, 129.6, 130.1,
133.0, 166.0 ppm; MS (ES) [M+H]+ m/z calcd for C31H56O6P: 555.4,
found: 555.3.
À4.70 (c=5.6, CHCl3); 1H NMR (CDCl3): d=0.88 (t, 3H, J=6.0 Hz),
1.25 (brs, 26H), 1.50–1.62 (m, 2H), 1.75–1.86 (m, 2H), 1.90–2.10 (m,
2H), 2.87 (d, 3H, J=4.4 Hz), 3.22 (s, 3H), 3.42–3.52 (m, 4H), 3.51 (t,
2H, J=5.2 Hz), 3.75 (d, 6H, J=10.8 Hz), 4.90–5.00 (m, 1H), 8.40–
8.50 ppm (brs, 1H); 13C NMR (CDCl3): d=14.1, 20.7 (d, J=143 Hz),
22.4, 24.0 (d, J=4.3 Hz), 26.0, 27.1, 29.3, 29.4, 29.5, 29.6, 30.7, 31.9,
52.5, 71.1, 71.7, 75.0 (d, J=17.5 Hz), 155.2, 155.9 ppm; 31P NMR
(CDCl3): d=33.6; ESI-HRMS [M+H]+ m/z calcd for C26H54N2O7P:
537.3663, found: 537.3667.
Dimethyl
phosphonate and dimethyl 4-hexadecyloxy-3(R)-[N-(N’-methyl-
carbamoyl)-N-methylcarbamoyl]-1-butanephosphonate. The
4-hexadecyloxy-3(R)-N-methylcarbamoyl-1-butane-
enantiomers of carbamoyl phosphonate 11 and dicarbamoyl phos-
phonate 12 were prepared in 12 and 50% yields, respectively, from
4-hexadecyloxy-3(R)-hydroxy-1-butanephosphonate by the proce-
dure described above. Data for the enantiomer of 11: [a]2D5 =+5.49
(c=5.6, CHCl3); MS (ES) [M+H]+ m/z calcd for C24H51NO6P: 480.3,
found: 480.3. Data for the enantiomer of 12: [a]2D5 =+4.55 (c=5.7,
CHCl3); ESI-HRMS [M+H]+ m/z calcd for C26H54N2O7P: 537.3663,
found: 537.3659.
Diethyl 3(R)-benzoyl-4-hexadecyloxy-1-butanephosphonate. The
enantiomer of benzoyl phosphonate 9 was prepared in 80% yield
from 3(R)-benzoyl-4-hexadecyloxy-1-bromobutane by the proce-
dure described above; [a]2D5 =+6.71 (c=6.0, CHCl3).
Dimethyl
4-hexadecyloxy-3(S)-hydroxy-1-butanephosphonate
(10).[25] Na0 (0.18 g, 7.83 mmol) was added to 100 mL dry MeOH.
After complete disappearance of Na0, a solution of phosphonate 9
(3.91 g, 7.05 mmol) in 10 mL dry MeOH was added. After the mix-
ture was stirred overnight, the reaction was quenched by the addi-
tion of 500 mL HOAc (8.73 mmol) and then concentrated under re-
duced pressure to give a residue, which was purified by column
chromatography on silica gel (elution with CHCl3/MeOH 10:1) to
provide hydroxyphosphonate 10 (2.56 g, 86%) as a colorless oil;
[a]2D5 =À5.20 (c=5.0, C6H6); 1H NMR (CDCl3): d=0.88 (t, 3H, J=
6.8 Hz), 1.26 (brs, 26H), 1.54–1.58 (m, 2H), 1.70–1.90 (m, 4H), 2.78
(brs, 1H), 3.29 (dd, 1H, J=7.1, 9.4 Hz), 3.40–3.48 (m, 3H), 3.74 (d,
6H, J=10.8 Hz), 3.70–3.85 ppm (m, 1H); 13C NMR (CDCl3): d=14.0,
20.7 (d, J=142.1 Hz), 22.6, 26.0, 26.1 (d, J=4.7 Hz), 29.3, 29.4, 29.5,
29.6, 31.9, 52.3, 69.8 (d, J=14.8 Hz), 71.6, 74.4; ESI-HRMS [M+H]+
m/z calcd for C22H48O5P: 423.3234, found: 423.3242.
2’-(Trimethylammonio)ethyl 4-hexadecyloxy-3(S)-N-methylcarba-
moyl-1-butanephosphonate (1). Me3SiBr (500 mL, 3.79 mmol) was
added to a solution of carbamoyl phosphonate 11 (512 mg,
1.07 mmol) in 25 mL CH2Cl2. After the mixture was allowed to
stand overnight at room temperature, volatile materials were re-
moved under reduced pressure to give a residue. Choline toluene-
sulfonate (1.45 g, 3.01 mmol) was added to the residue, and the
mixture was dried overnight under high vacuum. After the dry mix-
ture was dissolved in pyridine (50 mL), Cl3CCN (1.5 mL, 15.0 mmol)
was added, and the reaction mixture was heated for 48 h at 508C
(oil bath temperature). Upon removal of most of the pyridine by
rotary evaporation, a brown residue was formed, which was dis-
solved in THF/H2O (10 mL, 9:1 v/v) and passed through a column
of TMD-8 resin (previously equilibrated with the same solvent mix-
ture). The product was purified by silica gel chromatography (elu-
tion with CHCl3/MeOH/H2O 65:25:4). The fractions containing the
product were pooled and concentrated under reduced pressure.
The residue was dissolved in CHCl3 (15–25 mL) and passed through
an Osmonics filter three times to remove the suspended silica gel.
The filtrate was concentrated to give a residue, which was lyophi-
lized from benzene to afford phosphonate 1 (395 mg, 69%) as a
white powder; [a]2D5 =À2.53 (c=0.21, CHCl3/MeOH 1:1); 1H NMR
(CDCl3/CD3OD): d=0.89 (t, 3H, J=6.4 Hz), 1.26 (brs, 26H), 1.32–
1.35 (m, 2H), 1.44–1.62 (m, 4H), 1.86–1.95 (m, 2H), 2.86 (d, 3H, J=
4.8 Hz), 3.24 (s, 9H), 3.30–3.70 (m, 8H), 4.90–5.00 ppm (m, 1H);
Dimethyl 4-hexadecyloxy-3(R)-hydroxy-1-butanephosphonate.
The enantiomer of hydroxyphosphonate 10 was prepared in 88%
yield from diethyl 3(R)-benzoyl-4-hexadecyloxy-1-butanephospho-
nate by the procedure described above; [a]2D5 =+5.22 (c=5.1,
C6H6).
Dimethyl
phosphonate (7) and dimethyl 4-hexadecyloxy-3(S)-[N-(N’-meth-
ylcarbamoyl)-N-methylcarbamoyl]-1-butanephosphonate (11).
4-hexadecyloxy-3(S)-N-methylcarbamoyl-1-butane-
MeI (3.2 mL, 51.4 mmol) and (iPr)2NEt (1.8 mL, 10.4 mmol) were
added to a mixture of KNCO (8.11 g, 100 mmol), nBu4NBr (3.22 g,
1050
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Antitumor Activity of Ether Glycerophospholipids
13C NMR (CDCl3): d=14.1, 20.7 (d, J=143 Hz), 22.6, 25.1 (d, J=
4.1 Hz), 26.0, 26.1, 26.4, 29.3, 29.4, 29.5, 29.6, 31.9, 56.5 (d, J=
4.7 Hz), 65.8, 70.6 (d, J=5.8 Hz), 75.4, 75.5, 74.9, 153.7 ppm; ESI-
HRMS [M+H]+ m/z calcd for C27H58N2O6P: 537.4027, found:
537.4037.
azido alcohol 14 was consumed completely to afford intermediate
15, Me3SiBr (1.0 mL, 7.6 mmol) was added at 08C to carry out the
opening of the phospholane ring. After the mixture was stirred for
2 h at room temperature, the volatile material was removed under
reduced pressure to give a residue, which was dissolved in 40 mL
CHCl3/MeCN/iPrOH (0.9:1.5:1.5) and treated with an aqueous solu-
tion of Me3N (40 mL, 40%) for two days at room temperature.
After concentration under reduced pressure, the residue was puri-
fied by column chromatography on silica gel (elution with CHCl3/
MeOH/H2O 65:25:4) to give azido phosphocholine 16 (350 mg,
69%) as a white solid; [a]2D5 =À4.3 (c=0.11, CHCl3), [a]2D5 =À4.5
2’-(Trimethylammonio)ethyl 4-hexadecyloxy-3(R)-N-methylcarba-
moyl-1-butanephosphonate (2). The enantiomer of 1 was pre-
pared in 66% yield from dimethyl 4-hexadecyloxy-3(R)-N-methyl-
carbamoyl-1-butanephosphonate by the procedure described
above; [a]2D5 =+2.44 (c=0.21, CHCl3/MeOH 1:1); ESI-HRMS [M+H]+
m/z calcd for C27H58N2O6P: 537.4027, found: 537.4029.
1
(c=1.0, CHCl3); H NMR (CDCl3/CD3OD): d=0.88 (t, 3H, J=6.4 Hz),
1.26 (s, 26H), 1.50–1.60 (m, 2H), 3.30 (s, 9H), 3–43–3.65 (m, 4H),
3,25–3.50 (m, 3H), 3.90–4.10 (m, 2H), 4.35–4.45 ppm (m, 2H);
13C NMR (CDCl3/CD3OD): d=13.6, 22.3, 25.6, 29.0, 29.1, 29.2, 29.3,
31.6, 44.4, 53.9, 59.5 (d, J=5.0 Hz), 60.7 (d, J=7.0 Hz), 65.7 (d, J=
5.0 Hz), 69.6, 71.7 ppm; 31P NMR (CDCl3/CD3OD): d=À2.03 ppm.
2’-(Trimethylammonio)ethyl 4-hexadecyloxy-3(S)-[N-(N’-methyl-
carbamoyl)-N-methylcarbamoyl]-1-butanephosphonate (3). Com-
pound 3 was prepared in 71% yield from dicarbamoyl phospho-
nate 12 by the procedure described above; [a]2D5 =À2.07 (c=0.23,
CHCl3/MeOH 1:1); 1H NMR (CDCl3/CD3OD): d=0.89 (t, 3H, J=
6.4 Hz), 1.26 (brs, 26H), 1.44–1.62 (m, 4H), 1.86–1.95 (m, 2H), 2.87
(d, 3H, J=4.0 Hz), 3.24 (s, 3H), 3.26 (s, 9H), 3.35–3.70 (m, 8H),
4.95–5.05 ppm (m, 1H); 13C NMR (CDCl3): d=14.1, 20.7 (d, J=
143 Hz), 22.6, 25.1 (d, J=4.1 Hz), 26.0, 26.1, 26.4, 29.3, 29.4, 29.5,
29.6, 31.9, 56.5 (d, J=4.7 Hz), 65.8, 70.6 (d, J=5.8 Hz), 75.4, 75.5,
74.8, 155.4, 156.1 ppm; ESI-HRMS [M+H]+ m/z calcd for
C29H61N3O7P: 594.4242, found: 594.4246.
2’-(Trimethylammonio)ethyl 2(S)-(N-methoxycarbonylamido)-3-
hexadecyloxypropanephosphate (5). A mixture of 16 (102 mg,
0.20 mmol) and Pd/C (30 mg) in EtOH was stirred overnight under
H2 atmosphere. After the catalyst was removed by filtration, the fil-
trate was concentrated under reduced pressure to give crude
amine 17. Et3N (60 mL, 0.44 mmol) and ClCO2Me (30 mL, 0.39 mmol)
were added to a solution of vacuum-dried amine 17 in 10 mL alco-
hol-free CHCl3 at 08C. The mixture was stirred overnight at room
temperature and then concentrated under reduced pressure to
give a residue, which was purified by column chromatography on
silica gel (elution with CHCl3/MeOH/H2O 65:25:4) to give carbamate
phosphocholine 5 (93 mg, 85%) as a white solid after filtration of a
CHCl3 solution of 5 through an Osmonics filter to remove suspend-
2’-(Trimethylammonio)ethyl 4-hexadecyloxy-3(R)-[N-(N’-methyl-
carbamoyl)-N-methylcarbamoyl]-1-butanephosphonate (4). The
enantiomer of 3 was prepared in 68% yield from dimethyl 4-hexa-
decyloxy-3(R)-[N-(N’-methylcarbamoyl)-N-methylcarbamoyl]-1-buta-
nephosphonate by the procedure described above; [a]2D5 =+1.98
(c=0.21, CHCl3/MeOH 1:1); ESI-HRMS [M+H]+ m/z calcd for
C29H61N3O7P: 594.4242, found: 594.4250.
1
ed silica gel: [a]2D5 =À3.9 (c=0.10, CHCl3); H NMR (CDCl3): d=0.88
(t, 3H, J=6.4 Hz), 1.26 (s, 26H), 1.50–1.60 (m, 2H), 3.43 (s, 9H),
3.40–3.75 (m, 4H), 3.67 (s, 3H), 3.90–4.30 (m, 5H), 4.60–4.80 (m,
2H), 6.51 (brs, 1H); 31P NMR (CDCl3): d=À2.61; ESI-HRMS [M+H]+
m/z calcd for C26H56N2O7P: 593.3820, found: 593.3830.
2(S)-Azido-2-deoxy-3-O-hexadecyl-1,3-propanediol (14). Diiso-
propyl azodicarboxylate (DIAD; 3.2 mL, 15 mmol) was added to a
solution of 3-O-hexadecyl-sn-glycerol (13)[33] (3.17 g, 10.0 mmol)
and Ph3P (3.42 g, 13.0 mmol) in 180 mL CH2Cl2 at 08C. After the
mixture was stirred for 3 h under N2 gas, Me3SiN3 was added. The
mixture was stirred at the same temperature for 3 h, and then at
room temperature until glycerol 13 had reacted completely. The
reaction mixture was concentrated to give a yellow residue, which
was dissolved in a minimal volume of CH2Cl2 and passed through a
pad of silica gel in a sintered glass funnel. The pad was rinsed with
hexane/EtOAc (50:1) until the excess yellow DIAD began to elute.
After concentration of the eluted silyloxy azide, the residue was
dissolved in 30 mL THF and treated with a solution of (nBu)4NF
(1m, 25 mL) in THF. The mixture was stirred at room temperature
until all of the silyloxy azides were consumed completely, and then
was diluted with 250 mL Et2O and washed with H2O and brine. The
organic layer was separated, dried over Na2SO4, and concentrated.
The crude product was purified by column chromatography on
silica gel (elution first 150 mL of 50:1 hexane/EtOAc and then with
6:1 hexane/EtOAc) to give azido alcohol 14[34] (2.60 g, 76%) as a
white solid; mp: 37–398C; [a]2D5 =+14.5 (c=1.0, CHCl3), ref. [34]:
[a]2D7 =+14.1 (c=1.0, CHCl3); 1H NMR (CDCl3): d=0.88 (t, 3H, J=
6.4 Hz), 1.26 (s, 26H), 1.52–1.62 (m, 2H), 2.19 (brs, 1H), 3.47 (t, 2H,
J=6.8 Hz), 3.55–3.63 (m, 4H), 3.65–3.80 ppm (m, 1H); 13C NMR
(CDCl3): d=14.1, 22.7, 26.0, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 62.3,
63.0, 70.9, 71.9 ppm; ESI-HRMS [M+Na]+ m/z calcd for
C19H39N3NaO2: 364.2935, found: 364.2941.
Biological methods
Cell culture. Cell lines were obtained from frozen stocks of cell
lines originally obtained from ATTC. The cells were grown in basal
medium supplemented with 10% fetal bovine serum, penicillin,
and streptomycin. PC3 cells were grown in F12K medium; BT549 in
RPMI; MDA-MB-231 in MEM; A549 in F12; and DU145, MCF-7,
HepG2, and HeLa cells were grown in DMEM.
Antiproliferation assay. Cell proliferation was monitored by the
CyQuant assay according to the instructions of the manufacturer
(Invitrogen, Mississauga, ON, Canada) and as previously de-
scribed.[21] Cells were dispersed 96-well plates and incubated until
they were in log phase. The medium was subsequently replaced
with one containing varying concentrations of compounds 1–5 (0–
20 mm dissolved in EtOH) for 48 h. The concentration of EtOH in all
wells was 0.1%. Each drug concentration was replicated eight
times.
Toxicity assay. The toxicity of 1, 2, and 3 was measured by using
the ToxiLight assay kit according to the instructions of the manu-
facturer (Lonza, Rockland, ME, USA) as previously described[21] in
clear-bottom 96-well black plates.
2’-(Trimethylammonio)ethyl 2(S)-azido-3-hexadecyloxypropane-
phosphate (16). 2-Chloro-1,3,2-dioxaphospholane-2-oxide (250 mL,
2.72 mmol) was added to a solution of 14 (342 mg, 1.00 mmol)
and (iPr)2NEt (390 mg, 3.02 mmol) in 20 mL CH2Cl2 at 08C. After
Assay of mitochondrial membrane potential. The effect of com-
pound 2 on the mitochondrial membrane potential was deter-
mined with the JC-10 reagent (ABD Bioquest, Sunnyvale, CA, USA)
by using the assay previously described for JC-1.[21]
ChemMedChem 2010, 5, 1045 – 1052
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G. Arthur, R. Bittman, et al.
MED
[6] A. M. Lin, C. J. Ryan, E. J. Small, Crit. Rev. Oncol. Hematol. 2007, 61, 243–
254.
[7] B. Pandit, A. L. Gartel, Prostate 2010, 70, 825–833.
[8] G. Arthur, R. Bittman, Biochim. Biophys. Acta Lipids Lipid Metab. 1998,
1390, 85–102.
Caspase activation. The activation of caspases in cells was moni-
tored by using the CaspGlow fluorescein caspase staining kit (Bio-
vision Inc., Mountain View, CA, USA) as previously described.[21,35]
Animal studies. Rag2M mice (8–10 weeks old) from the Animal Re-
source Centre of the British Columbia Cancer Research Centre (Van-
couver, BC, Canada) were used for the studies. The methodology
used for the studies was approved by the Institutional Animal Care
Committee at the University of British Columbia prior to conduct-
ing the studies (protocol #A05-1582). The toxicity of the selected
doses of compound 2 to be used in the efficacy studies was estab-
lished as follows: The mice were randomly distributed into groups
of four and individually weighed. Compound 2 was dissolved in
saline solution for intravenous injections or in double-distilled
water for oral gavage. The compound was introduced according to
body weight. The animals were monitored for acute signs of toxici-
ty for the first 2 h; if this was observed, they were sacrificed by
CO2 asphyxiation followed by necropsies. All mice were monitored
daily for 14 days for signs of toxicity, and body weight was moni-
tored.
[9] C. Gajate, A. M. Santos-Beneit, M. Modolell, F. Mollinedo, Mol. Pharma-
col. 1998, 53, 602–612.
[10] P. Samadder, R. Bittman, H.-S. Byun, G. Arthur, J. Med. Chem. 2004, 47,
2710–2713.
[11] T. Nieto-Miguel, C. Gajate, F. Gonzalez-Camacho, F. Mollinedo, Oncogene
2008, 27, 1779–1787.
[12] T. Nieto-Miguel, R. I. Fonteriz, L. Vay, C. Gajate, S. Lopez-Hernandez, F.
Mollinedo, Cancer Res. 2007, 67, 10368–10378.
[13] C. Gajate, F. Mollinedo, Blood 2007, 109, 711–719.
[14] F. Mollinedo, C. Gajate, S. Martin-Santamaria, F. Gago, Curr. Med. Chem.
2004, 11, 3163–3184.
[15] W. E. Berdel, Br. J. Cancer 1991, 64, 208–211.
[16] P. Samadder, G. Arthur, Cancer Res. 1999, 59, 4808–4815.
[17] W. J. van Blitterswijk, M. Verheij, Curr. Pharm. Des. 2008, 14, 2061–2074.
[18] W. J. Houlihan, M. Lohmeyer, P. Workman, S. H. Cheon, Med. Res. Rev.
1995, 15, 157–223.
[19] A. Ashagbley, P. Samadder, R. Bittman, R. V. Erukulla, H.-S. Byun, G.
Arthur, Anticancer Res. 1996, 16, 1813–1818.
Xenograft studies were conducted as follows: Rag2M mice were in-
jected subcutaneously with 10ꢁ106 PC3 cells in a volume of 50 mL.
Tumors were allowed to grow for 29–35 days. Animals were select-
ed and randomized into groups comprising: 1) vehicle-treated con-
trol, 2) oral 2, and 3) intravenous 2. Compound 2 was delivered
once per week by oral gavage (200 mL per 20 g mouse) or intrave-
nously (200 mL per 20 g mouse) for 3 weeks. Tumor growth was
monitored with digital calipers beginning on the day of adminis-
tering the compounds and then every 2–3 days thereafter. Tumor
volume was calculated according to the formula: volume=
(length)ꢁ(width)2/2. The body weight of the animals was deter-
mined at the time of tumor measurement.
[20] W. E. Berdel, R. Andreesen, P. G. Munder in Phospholipids and Cellular
Regulation, Vol. 2 (Ed.: J. F. Kuo), CRC Press, Boca Raton, 1985, pp. 41–
73.
[21] P. Samadder, R. Bittman, H.-S. Byun, G. Arthur, Biochem. Cell Biol. 2009,
87, 401–414.
[22] L. Jahreiss, M. Renna, R. Bittman, G. Arthur, D. C. Rubinsztein, Autophagy
2009, 5, 835–846.
[23] R. K. Erukulla, X. Zhou, P. Samadder, G. Arthur, R. Bittman, J. Med. Chem.
1996, 39, 1545–1548.
[24] G. Yang, R. W. Franck, H.-S. Byun, R. Bittman, P. Samadder, G. Arthur,
Org. Lett. 1999, 1, 2149–2151.
[25] R. Bittman, H.-S. Byun, B. Mercier, H. Salari, J. Med. Chem. 1994, 37, 425–
430.
[26] B. Herradon, Tetrahedron: Asymmetry 1991, 2, 191–194.
[27] K. Yokoyama, D. L. Baker, T. Virag, K. Liliom, H.-S. Byun, G. Tigyi, R. Bitt-
man, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2002, 1582, 295–308.
[28] L. He, M. Wanunu, H.-S. Byun, R. Bittman, J. Org. Chem. 1999, 64, 6049–
6055.
[29] N. M. Navone, C. J. Logothetis, A. C. von Eschenbach, P. Troncoso,
Cancer Metastasis Rev. 1998–1999, 17, 361–371.
[30] R. E. Sobel, M. D. Sadar, J. Urol. 2005, 173, 342–359.
[31] S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith,
G. D. Steele, Jr., L. B. Chen, Proc. Natl. Acad. Sci. USA 1991, 88, 3671–
3675.
Acknowledgements
We thank the Advanced Therapeutics Department at the British
Columbia Cancer Research Centre (Vancouver, BC, Canada) for
performing the animal studies. This study was supported by a
grant from the Elsa U. Pardee Foundation.
Keywords: antitumor
glycerophosphonocarbamates · medicinal chemistry · prostate
cancer
agents
·
cytotoxic
lipids
·
[32] A. E-H. De Mendoza, M. A. Campanero, J. de La Iglesia-Vincente, C.
Gajate, F. Mollinedo, M. J. Blanco-Prieto, Clin. Cancer Res. 2009, 15, 858–
864.
[33] P. N. Guivisdalsky, R. Bittman, J. Org. Chem. 1989, 54, 4637–4642.
[34] M. M. Ponpipom, R. L. Bugianesi, Chem. Phys. Lipids 1984, 35, 29–37.
[35] R. Bittman, Z. Li, P. Samadder, G. Arthur, Cancer Lett. 2007, 251, 53–58.
[1] A. Jemal, T. Murray, A. Samuels, A. Ghafoor, E. Ward, M. J. Thun, CA
Cancer J. Clin. 2003, 53, 5–26.
[2] H. Saitoh, M. Hida, T. Shimbo, K. Nakamura, J. Yamagata, T. Satoh,
Cancer 1984, 54, 3078–3084.
[3] S. C. Jacobs, Urology 1983, 21, 337–344.
Received: February 11, 2010
Revised: April 26, 2010
[4] M. Koutsilieris, G. Tolis, Prostate 1985, 7, 31–39.
[5] S. Chowdhury, S. Burbridge, P. G. Harper, Int. J. Clin. Pract. 2007, 61,
2064–2070.
Published online on June 8, 2010
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ChemMedChem 2010, 5, 1045 – 1052