Synthesis and antiproliferative properties of a photoactivatable analogue of ET-18-OCH3

Article abstract of DOI:10.1016/S0040-4020(01)00901-2

A photoreactive analogue of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH₃, 1) was synthesized for the first time. The 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) moiety was used as the photolabel in ether lipid 2a. The TID group was tethered through an O-undecyloxy linkage to the sn-1 position of the ether lipid. Compound 2a was found to qualitatively mimic the antiproliferative effects of 1 in the dark (i.e. in the absence of carbene generation), suggesting that this photoactivatable probe may be suitable for identification of the proteins that mediate the biological activities of 1. Analogue 2a was converted by Stille reaction to stannane intermediate 2b, which was subjected to ipso [¹²⁵I]-iododestannylation to afford ¹²⁵I-labeled ether lipid 2c. Photolysis (long wavelength UV light, 30 min) of 2c in the presence of cytosolic proteins obtained from a breast cancer cell line (MCF-7), followed by NaDodSO₄/PAGE and autoradiography, showed radioiodination of primarily two polypeptides (with molecular masses of about 47- and 170-kDa), both of which were subject to competition by prior addition of excess 2a. This study indicates that analogue 2c may have utility in the characterization of the putative proteins that interact specifically with antitumor ether lipid 1.

Full text of DOI:10.1016/S0040-4020(01)00901-2

TETRAHEDRON
Pergamon
Tetrahedron 57 &2001) 8925±8932
Synthesis and antiproliferative properties of a photoactivatable
analogue of ET-18-OCH3
a
Guoqing Li, Pranati Samadder, Gilbert Arthur and Robert Bittman
b
b
a,p
a
Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing, NY 11367-1597 USA
b
Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
Received 2 November 2000; accepted 31August 2001
AbstractÐA photoreactive analogue of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine &ET-18-OCH , 1)
3
was synthesized for the ®rst time. The 3-&tri¯uoromethyl)-3-&m-iodophenyl)diazirine &TID) moiety was used as the photolabel in ether lipid
2
a. The TID group was tethered through an O-undecyloxy linkage to the sn-1position of the ether lipid. Compound 2a was found to
qualitatively mimic the antiproliferative effects of 1 in the dark &i.e. in the absence of carbene generation), suggesting that this photo-
activatable probe may be suitable for identi®cation of the proteins that mediate the biological activities of 1. Analogue 2a was converted by
1
25
125
Stille reaction to stannane intermediate 2b, which was subjected to ipso [ I]-iododestannylation to afford I-labeled ether lipid 2c.
Photolysis &long wavelength UV light, 30 min) of 2c in the presence of cytosolic proteins obtained from a breast cancer cell line &MCF-
7
), followed by NaDodSO /PAGE and autoradiography, showed radioiodination of primarily two polypeptides &with molecular masses of
4
about 47- and 170-kDa), both of which were subject to competition by prior addition of excess 2a. This study indicates that analogue 2c may
have utility in the characterization of the putative proteins that interact speci®cally with antitumor ether lipid 1. q 2001Elsevier Science Ltd.
All rights reserved.
1.Introduction
ET-18-OCH interactions and thereby enhance our under-
3
standing of the drug's mechanism&s) of action, we seek to
identify the intracellular signaling proteins that are inhibited
by ET-18-OCH .
3
The antitumor ether lipid &AEL) 1-O-octadecyl-2-O-
methyl-glycero-3-phosphocholine &1), also known as ET-
1
multitude of processes.
8-OCH and edelfosine, inhibits tumor cell growth by a
3
Like other alkyl-lysophospho-
lipids and unlike many other anticancer agents, 1 does not
1
±3
Photoactivatable analogues of biologically active
compounds are an attractive means for determining the
macromolecular targets of the parent compounds. Photo-
8
directly target cellular DNA. Although the principal
mechanism of action of ET-18-OCH has not yet been
reactive analogues generate highly reactive intermediates
on activation with UV light. Carbene precursors have
been used extensively to identify ligand-binding domains.
The 3-tri¯uoromethyl-3-m-iodophenyl-3H-diazirine &TID)
moiety is readily incorporated into the hydrophobic region
3
fully established, it is clear that the drug perturbs intra-
cellular signaling transduction events after it is taken up
4
spontaneously into cells. We showed that ET-18-OCH
3
inhibits the translocation of Raf-1to the plasma membrane
of the mammary epithelial cancer cell line MCF-7, thereby
directly blocking Ras-dependent activation of Raf-1and
subsequently blocking mitogen-activated protein &MAP)
9
of membranes, and is thus a useful probe for the study of
8
,10
lipid±protein interactions.
®rst synthesis of a photoactivatable probe of ET-18-OCH
In this paper, we report the
3
5
,6
11
kinase activity and the Raf-1/MEK/ERK cascade. ET-
8-OCH is also implicated in the c-Jun kinase &JNK)
signaling pathway, since it activated JNK in leukemic
&2a), which bears a tri¯uoromethyl iodophenoxydiazirine
&TID) group at the terminus of the sn-1 O-alkyl moiety. To
test whether the TID photolabel in 2a perturbs its biological
activities as an antitumor agent, we compared the anti-
proliferative activity of 2a with that of the parent compound
&1). Thus, we incubated a variety of epithelial cancer cell
lines with 2a in the dark, i.e. under conditions in which
photoactivation of 2a to produce the reactive carbene func-
tionality is avoided. Our ®ndings indicate that probe 2a
mimics the antiproliferative properties of 1 in the epithelial
cancer cells we tested. To illustrate the utility of the photo-
activatable probe, 2a was converted in two steps into 2c, the
radioiodinated form of 2a. 2c was activated with UV light in
the presence of cytosolic proteins derived from MCF-7
1
3
7
cells. These ®ndings raise the possibility that the anti-
proliferative effects of ET-18-OCH may arise, at least in
3
part, from its ability to interfere with the activities of key
proteins in various mitogenic signaling pathways of cells
that are sensitive to ET-18-OCH . To examine protein-
3
Keywords: antitumor compounds; diaziridenes/diazirines; lipids; phospho-
lipids.
p
Corresponding author. Tel.: 11-718-997-3279; fax: 11-718-997-3349;
e-mail: robert_bittman@qc.edu
0040±4020/01/$ - see front matter q 2001Elsevier Science Ltd. All rights reserved.
PII: S0040-4020&01)00901-2

8926
G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
Scheme 1. Synthesis of photoaf®nity labeled ET-18-OCH
Bu NBr, THF; c. &i) 9-BBN, &ii) NaOH, H ; d. 17 &see Scheme 2), Ph
Et N, Et O; f. NH OH´HCl, Py-EtOH &1:6), 50±608C, 30 h; g. p-TsCl, Et
k. &i) POCl , Et N, 2108C; &ii) choline tosylate, Py, CHCl , rt; l. Bu Sn
3
analogue 2. Reagents and conditions: a. CH
P, DIAD, THF, rt, 18 h; e. &i) CAN, CH
N, DMAP, CH Cl ; h. liq. NH , CH Cl ; i. I
2 4
, cat. Pd&MeCN) Cl , HMPA, 18 h, rt; m. Na I, chloramine-T, NaH PO , EtOH.
2
vCH&CH
CN±H
, Et
2
)
9
OH, n-BuLi, THF, 08C; b. NaH, CH
O &2:1), 08C±rt, &ii) Ac O, DMAP,
N, MeOH, rt; j. NaOMe, MeOH, rt;
3
I,
4
2
O
2
3
3
2
2
3
2
2
3
2
2
3
2
2
2
3
25
1
3
3
3
6
2
2
2
cells. Two proteins were prominently photolabeled; more-
over, the extent of labeling was reduced markedly in com-
petition experiments carried out with an excess of 2a,
indicative of a speci®c interaction with cellular proteins.
recently shown to provide a convenient entry to protected
The reaction of lithium 11-un-
1
2,13
glycerol derivatives.
decenoxide &2 equiv.) with cyclic sulfate 3 proceeded
smoothly in THF at 08C to give the p-methoxyphenyl
&PMP)-protected glycerol derivative 4 in 94% yield. After
the unreacted 11-undecenol was removed by converting it
into 11-undecenyl methyl ether, alcohol 4 was O-methyl-
ated under phase-transfer conditions &excess NaH, MeI, cat.
Bu NBr). Hydroboration of methoxy derivative 5 using
4
9-borabicyclo[3.3.1]nonane &9-BBN) afforded v-alcohol 6.
Mitsunobu reaction &DIAD/Ph P) of phenol derivative 15
14
3
&prepared as outlined in Scheme 2) with primary alcohol 6
afforded the coupling product 7 in 88% yield. The PMP
ether group in 7 was removed with CAN in aqueous aceto-
nitrile to give the free C-3 alcohol, which was acetylated to
give 8 in 86% overall yield. The tri¯uoroacetyl group of
acetate 8 was converted into the tri¯uoromethyldiazirinyl
group in 12 by using conventional procedures. Base-cata-
lyzed hydrolysis of the acetate afforded alcohol 13 in
virtually quantitative yield. Insertion of the phosphocholine
2
.Results and discussion
2
.1. Syntheses of probes 2a and 2c
1
5
0
,3-cyclic sulfate &3) with long-chain nucleophiles were
Ring-opening reactions of &S)-1-&4 -methoxyphenyl)glycerol
2
1
1

G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
8927
Scheme 2. Synthesis of compound 17.
moiety by using phosphorus oxychloride followed by
choline tosylate16 gave product 2a. Aryl iodide 2a was
subjected to Stille coupling with hexa-n-butylditin &bis&tri-
DMF at re¯ux provided precursor 17 in high yield. Phenol
17 was coupled to alcohol 6 via Mitsunobu reaction
&Scheme 1); it can also be coupled to primary alkyl halides
via its phenolate group.
1
7
butyltin)) and catalytic Pd&MeCN) Cl at room temperature,
2
2
1
8
affording aryltin intermediate 2b in 51% yield. The latter
is stable for months at 2208C. To illustrate the utility of 2a
as a photoaf®nity label, radioiodine was incorporated by
2.3. Review of the synthetic procedure
1
25
ipso destannylation of 2b using Na I with chloramine-T
in buffered aqueous ethanol solution. Fractionation on solid-
phase extraction cartridges &aminopropyl and reversed
phase silica) provided product 2c.
We chose an ether linkage as the means of coupling the aryl
tri¯uoromethyldiazirine moiety to the sn-1 O-alkyl chain of
the probe. An advantage of using an ether linkage to tether
the carbene precursor to the lipid is that this linkage affords
resistance to chemical and enzymatic hydrolysis. However,
this linkage has the disadvantage of introducing a polar
atom into the hydrophobic region of the molecule, which
is accommodated in the interior region of the membrane
bilayer.
2
.2. Synthesis of aryl iodide 17
As shown in Scheme 2, tri¯uoroacetylation of the Grignard
reagent of 14 with N-&tri¯uoroacetyl)piperidine provided
tri¯uoroacetophenone analogue 15 in high yield. Electro-
philic aromatic iodination of 15 took place with very high
regioselectivity; the methoxy and tri¯uoroacetyl groups
both direct the iodo group to the desired position.
O-Demethylation of anisole 16 with lithium chloride in
Our synthesis of the new photoactivatable analogue of 1
features several noteworthy steps: &a) regioselective intro-
duction of an iodine atom into the aromatic ring of
compound 15 &Scheme 2) in the presence of excess HgO,
1
9
affording aryl iodide 16 in high yield; &b) O-demethylation
of 16, followed by Mitsunobu coupling of phenol 17 with
v-alcohol 6 &Scheme 1); &c) Stille coupling to replace the
nonradiolabeled iodide in 2a with a tin-containing group,
affording the stable intermediate 2b; and &d) chloramine-T
mediated ipso iododestannylation of 2b, giving radio-
iodinated 2c.
The synthetic sequence used to prepare 2a can be readily
modi®ed to prepare a range of photoactivatable compounds
carrying linkers with different chain lengths at the sn-1
position coupled to the aryldiazirine moiety.
2.4. Biological
Incubation of epithelial cancer cells derived from breast
&
&
MCF-7, MDA-MB-231, MDA-MB-468, BT549), lung
A549), and prostate &DU145) with 2a for 48 h resulted in
Figure 1. Effect of 2a on the proliferation of epithelial cancer cell lines.
Proliferating MCF-7 &X), MDA-MB-468 &B), MDA-MB-231& O), BT-549
a concentration-dependent decrease in the proliferation of
the cells; at 50 mM of 2a, the decrease ranged from 40 to
90% &data not shown). The cytotoxic properties of 2a were
manifested by incubating the cells with 30±50 mM of the
compound for 96 h &Fig. 1). The order of decreasing
susceptibility to the cytotoxic effects was: MDA-MB-
&
P), A549 &W), and DU 145 &A) cells were incubated in 24-well plates with
medium containing 2a &0±50 mM). Cells in representative wells were
counted on day 0 prior to the addition of 2a. After 48 h, the medium was
replaced with freshly prepared drug-containing media for another 48 h. The
cell numbers were determined in each well, and the increase over day 0 for
each concentration was expressed as a percentage of that in control wells
with no drug. The results are the means of two different experiments with
quadruplicate wells per experiment.
2
31.MCF-7.DU145ˆMDA-MB-468. Under the same
experimental conditions, 2a was not cytotoxic toward

8928
G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
Table 1. Growth inhibitory properties of ET-18-OCH
3
&1) and 2a on epithelial tumor cells
IC50 &mM)
Cell line
ET-18-OCH
incubation period, 48 h)
3
2a
&incubation period, 48 h)
2a
&incubation period, 96 h)
&
MCF-7
2
17.5
7
MDA-MB-2313
BT549
A549
MDA-MB-468
DU145
9
6
3.5
8
19
15
37
37
48
.50
15
23
18
9
Cells were incubated with 1 or 2a for 48 or 96 h, as indicated. For details of the in vitro assay, see Section 4 and Ref. 23.
BT549 and A549, but the proliferation of the cells was
reduced to approximately 20% of the controls. One can
infer from these results that incubating BT549 or A549
cells for periods longer than 96 h would ultimately result
in the death of the cells. Table 1presents the IC 50 values
3.Conclusion
The synthesis of 2a, the ®rst photoactivatable probe of ET-
18-OCH &1), in 30% overall yield has been described here.
3
This model photoactivatable probe was found to qualita-
tively mimic the antiproliferative properties of the parent
compound &1) against a variety of epithelial cancer cell
lines in the dark. The ability of 2c to label cytosolic proteins
obtained from MCF-7 cells was also demonstrated. This
study suggests that intracellular proteins that interact with
AELs can be identi®ed by photoactivatable labeling.
&drug concentrations required to inhibit growth by 50%)
found for incubation of the cells with 2a for 48 and 96 h.
Also shown are the IC values obtained when the cells were
5
0
incubated with ET-18-OCH &1) for 48 h. An incubation
3
period of 96 h with 2a was needed to obtain IC50 values
approaching those of the parent compound &1). While the
reason for this is unclear, we speculate that the presence of
polar atoms in the hydrophobic region of 2a and/or the
shorter sn-1chain length in 2a than in 1 may reduce the
rate of uptake of the compound and its accumulation in
the cells. Nevertheless, the above results indicate that 2a
is an effective probe since it retains the antiproliferative
4.Experimental
4.1. General methods
2
0
1
13
H NMR and C NMR spectra were recorded in CDCl
and cytotoxic activity of the parent compound &1).
3
at
00 and 100 MHz on a Bruker spectrometer, respectively.
4
1
Fig. 2 shows the results of radioiodination of MCF-7 cyto-
solic proteins in the presence of 2c &lane 1). Analysis by
sodium dodecylsulfate-polyacrylamide gel electrophoresis
The reference chemical shifts for CDCl were d 7.24 & H)
3
1
3
and 77.0 ppm & C). Melting points are uncorrected. Flash
chromatography was carried out with Merck 60 &230±400
mesh) silica gel. TLC was carried out on EM 0.25-mm thick
silica gel 60 F254 aluminum sheets. Visualization was with
10% sulfuric acid solution in ethanol. Solid-phase `Bond-
Elut' extraction silica gel cartridges &aminopropyl, 500 mg,
and C-18, 2 g) were purchased from Alltech Associates
&Deer®eld, IL). The solvents were dried by distillation
from the following reagents before use: THF &sodium/
&
SDS-PAGE) showed a prominent band of labeled protein
with a molecular mass &Mr) of about 47 kDa. Another
protein with a Mr of about 170 kDa was also present.
Both of these bands were markedly reduced in intensity
when an excess of 2a was present &lanes 2 and 3), indicating
that the interaction of 2c with these proteins is speci®c. A
band having Mr of about 62-kDa is also apparent in the
presence of excess 2a.
benzophenone); chloroform &P
O
); CH
Cl
&CaH
HMPA &redistilled). Na I &17 Ci/mg) in 10 M NaOH
pH 8±11) was purchased from New England Nuclear
);
2
5
2
2
2
1
25
25
&
Life Sciences. High-resolution MS data were recorded at
the University of California, Riverside, Mass Spectrometry
Facility. Aluminum foil was used to exclude light from the
reaction ¯asks containing the TID probe. Exponentially
growing MCF-7 cells were harvested, lysed by sonication,
5
and the cytosol was obtained by centrifugation. The cyto-
solic proteins were irradiated for 30 min at room tempera-
ture in plastic trays &Accutran purchased from Schleicher
and Schuell) with long-wavelength &365 nm) UV light
&Mineralight lamp model UVGL-58, UVP, Inc.) at a
distance of 1cm from the lamp.
0
3). The titled compound was synthesized from &R)-1-&4 -
4
.1.1. ꢀS)-1-ꢀ4 -Methoxyphenyl)glycerol 2,3-cyclic sulfate
0
ꢀ
methoxyphenyl)glycerol as previously reported.
Figure 2. Radioiodination of MCF-7 cytosolic proteins by 2c. Cytosolic
proteins were photolyzed with 2c &lane 1) and with 2c in the presence of a
12
2
00-fold excess &lane 2) and a 100-fold excess &lane 3) of 2a as described in
0
glycerol ꢀ4). To an ice-cooled solution of 11-undecenol
0
4
.1.2. 1-O-ꢀ11 -Undecenyl)-3-O-ꢀ4 -methoxyphenyl)-sn-
Section 4.1. Proteins were separated on 10% SDS gels and subjected to
autoradiography.

G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
8929
&10.2 g, 60.0 mmol) in 250 mL of THF, 24 mL &60 mmol)
of n-butyllithium &a 2.5 M solution in hexane) was added by
dried &MgSO ), and concentrated. Product 6 &1.84 g, 91%)
4
was obtained by chromatography &elution with hexane/
EtOAc 2:1) as a clear oil; R 0.10 &hexane/EtOAc 3:1). H
1
a syringe under argon. The mixture was stirred for about
3
f
0 min until no more bubbles formed. A solution of 7.81g
30 mmol) of cyclic sulfate 3 in 50 mL of THF was added
NMR: d 6.90±6.83 &m, 4H), 4.07±4.17 &m, 2H), 3.79 &s,
3H), 3.79±3.61&m, 5H), 3.54 &s, 3H), 3.50±3.46 &m, 2H),
&
dropwise at 08C. The mixture was stirred overnight at room
1
3
1.61±1.55 &m, 4H), 1.47 &br s, 1H), 1.35±1.28 &m, 12H). C
NMR: d 153.9, 153.0, 115.5, 114.6, 78.8, 71.8, 69.9, 68.3,
63.0, 58.2, 55.7, 32.8, 29.6, 29.5, 29.40, 29.38, 26.1, 25.7.
temperature. To facilitate the workup of the product
&
product 4 and 11-undecenol have very similar R values),
f
1
1
methyl iodide &8.5 g, 60 mmol) was added to convert the
unreacted 11-undecenol into the corresponding methyl
ether. After the reaction mixture was stirred for 10 h, a
solution of 80 mL of concentrated H SO in 400 mL of
DEI-MS 382 &M) ; HRMS calcd for &C H O ) 382.2719,
22 38 5
found 382.2707.
4.1.5. 4-Methoxytri¯uoroacetophenone ꢀ15). The titled
compound &Scheme 2) was synthesized as previously
2
4
water was added. After the solution was stirred overnight,
the product was extracted with Et O. The ether layer was
19b
reported in 91% yield.
2
washed with water, saturated aqueous sodium bicarbonate
solution, saturated aqueous sodium thiosulfate solution,
brine, and then dried &MgSO ). The residue obtained after
4.1.6. 3-Iodo-4-methoxy-tri¯uoroacetophenone ꢀ16). A
mixture of 1.03 g &50 mmol) of 15, 2.16 g &100 mmol) of
HgO, 0.5 mL of concentrated H SO , and 1.27 g &50 mmol)
4
concentration was puri®ed by chromatography &elution with
hexane/EtOAc 6:1) to provide 9.8 g &94%) of 4 as a clear
2
4
of iodine in 25 mL of CCl was heated to re¯ux with
4
1
oil; R 0.35 &hexane/EtOAc 6:1). H NMR: d 6.85±6.79 &m,
vigorous stirring for 5 h. The reaction mixture was cooled
to room temperature, and then ®ltered through a pad of
Celite. The ®ltrate was washed with 2N aqueous sodium
thiosulfate solution and brine, and then dried &Na SO ).
f
4
1
2
1
1
2
H), 5.82±5.76 &m, 1H), 5.00±5.89 &m, 2H), 4.13±4.10 &m,
H), 3.96±3.94 &m, 2H), 3.75 &s, 3H), 3.57±3.44 &m, 4H),
.04±1.99 &m, 2H), 1.61±1.54 &m, 2H), 1.35±1.25 &m,
2
4
1
3
2H). C NMR: d 154.0, 152.7, 139.2, 115.5, 114.6,
14.1, 71.7, 71.4, 69.7, 69.1, 55.7, 33.8, 29.6, 29.5, 29.4,
The residue obtained after evaporation was puri®ed by
column chromatography &elution with hexane/EtOAc 9:1),
affording 1.32 g &80%) of the product as a white solid; mp
1
9.1, 28.9, 26.1. DEI-MS 350 &M) ; HRMS calcd for
1
1
&
C H O ) 350.2457, found 350.2461.
2
52±548C; R 0.22 &hexane/EtOAc 9:1). H NMR: d 8.47 &s,
f
1
34
4
1
H), 8.04 &d, Jˆ8.8 Hz, 1H), 6.89 &d, Jˆ8.8 Hz, 1H), 3.98
0
phenyl)-sn-glycerol ꢀ5). To an ice-cooled solution of 7.65 g
0
13
4
.1.3. 1-O-ꢀ11 -Undecenyl)-2-O-methyl-3-O-ꢀ4 -methoxy-
&s, 1H). C NMR: d 177.9 &q, J ˆ35 Hz), 163.6, 141.7,
C±F
132.7 &d, Jˆ2.1 Hz), 124.3, 116.6 &q, Jˆ145 Hz), 110.3,
&21.8 mmol) of alcohol 4 in 200 mL of THF, 1.05 g
&43.8 mmol) of sodium hydride was added. After about
86.5, 56.9.
4
9
0
0 min &when no bubbles were still formed), a solution of
.29 g &66 mmol) of methyl iodide in 10 mL of THF and
.5 g &1.6 mmol) of tetra-n-butylammonium bromide were
4.1.7. 4-Hydroxy-3-iodo-tri¯uoroacetophenone ꢀ17).
O-Demethylation of 16 was carried out as follows. A
mixture of 3.35 g &10.0 mmol) of 16 and 1.3 g &31 mmol)
of LiCl in 25 mL of DMF was heated at re¯ux under argon
for 2 h. The reaction mixture was cooled to room tempera-
ture, poured into water &100 mL), and acidi®ed with 10%
added. The mixture was stirred for 20 h at room tempera-
ture, and then concentrated. After Et O &200 mL) and water
&100 mL) were added, the aqueous layer was extracted with
Et O &150 mL). The combined ether layer was washed with
2
HCl. The product was extracted with Et O &2£75 mL). The
2
2
water and brine, and dried &MgSO ). The product &5.92 g,
4
ethereal layer was washed with brine twice, dried &MgSO ),
4
7
EtOAc 20:1, then 10:1); R 0.65 &hexane/EtOAc 3:1). H
4%) was puri®ed by chromatography &elution with hexane/
and concentrated. The residue was dried under high
vacuum, affording 2.8 g &87%) of 17 &R 0.45, CH Cl ),
1
f
f
2
2
NMR: d 6.86±6.79 &m, 4H), 5.82±5.75 &m, 1H), 4.99±
4
&
which was used directly in the next step &see Scheme 1,
step d). A small amount of the product was puri®ed by
chromatography &elution with CH Cl ) to give a white
.89 &m, 2H), 4.03±3.07 &m, 2H), 3.74 &s, 3H), 3.74±3.67
m, 1H), 3.60±3.56 &m, 2H), 3.49 &s, 3H), 3.45±3.42 &m,
2
2
1
2
1
7
2
H), 2.04±1.98 &m, 2H), 1.56±1.51 &m, 2H), 1.36±1.24 &m,
solid; mp 87±888C. H NMR: d 8.40 &d, Jˆ0.4 Hz, 1H),
1
2H). C NMR: d 153.8, 153.0, 139.2, 115.5, 114.5, 114.1,
3
7.98 &d, Jˆ8.6 Hz, 1H), 7.09 &d, Jˆ8.8 Hz, 1H), 6.13 &s,
1
3
8.8, 71.8, 69.9, 68.2, 58.2, 55.7, 33.8, 29.6, 29.5, 29.4,
9.1, 28.9, 26.1. DEI-MS 364 &M) ; HRMS calcd for
1H). C NMR: d 178.4 &q, Jˆ35 Hz), 160.8, 141.2 &d,
1
Jˆ1.6 Hz), 132.9 &d, Jˆ1.0 Hz), 125.9, 124.6, 118.0 &q,
1
1
&
C H O ) 364.2614, found 364.2612.
4
Jˆ290 Hz), 115.3, 86.3. DEI-MS 316 &M) ; HRMS calcd
2
2
36
1
for &C H F IO ) 315.9208, found 315.9208.
8
4
3
2
0 0
1-O-ꢀ11 -Hydroxyundecyl)-2-O-methyl-3-O-ꢀ4 -
methoxyphenyl)-sn-glycerol ꢀ6). To a solution of 1.82 g
4
.1.4.
0
decyl)]-2-O-methyl-3-O-ꢀ4 -methoxyphenyl)-sn-glycerol
4.1.8. 1-O-[11 -ꢀ2-ꢀIodo-4-ꢀtri¯uoroacetyl)phenoxy)un-
0
&5.0 mmol) of alkene 5 in 40 mL of THF, 15 mL
&7.5 mmol) of a 0.5 M solution of 9-BBN in THF was
ꢀ7). To a mixture of 627 mg &1.64 mmol) of 6 was added a
solution of 588 mg &1.84 mmol) of 4-hydroxy-3-iodo-
tri¯uoroacetophenone &17) and 504 mg &1.92 mmol) of
added dropwise under argon. After the solution was stirred
for 8 h at room temperature, the reaction was quenched with
1
mL of MeOH. A solution of 0.45 g of NaOH in 4 mL of
Ph P in 12 mL of THF, followed by a solution of 400 mg
3
water was added, and the mixture was cooled in an ice bath;
then 2 mL of 30% H O &18 mmol) was added dropwise.
&1.88 mmol, 95% purity) of diisopropyl azodicarboxylate
&DIAD) in 3 mL of THF. The mixture was stirred for 18 h
at room temperature. After concentration, the residue was
puri®ed by chromatography &elution with hexane/EtOAc
2
2
After the mixture was stirred overnight, THF was removed
under reduced pressure, and the residue was extracted with
Et O twice. The ether layer was washed with brine twice,
6:1) to afford 0.99 g &88%) of 7 as an oil; R 0.40 &hexane/
f
2

8930
G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
1
EtOAc 3:1). H NMR: d 8.49 &d, Jˆ1.7 Hz, 1H), 8.03 &dd,
CH Cl , p-TsCl &434 mg, 2.3 mmol) was added slowly. The
2 2
Jˆ7.6 Hz, 0.8 Hz, 1H), 6.88±6.80 &m, 5H), 4.12 &t, Jˆ
mixture was stirred at room temperature for about 1h. After
CH Cl was added, the mixture was washed with saturated
aqueous sodium bicarbonate solution, brine, and dried
6
.4 Hz, 2H), 4.05±4.00 &m, 2H), 3.76 &s, 3H), 3.69±3.59
m, 3H), 3.51 &s, 3H), 3.48±3.44 &m, 2H), 1.89±1.86 &m,
2
2
&
1
3
2
d
1
6
2
H), 1.61±1.51 &m, 4H), 1.38±1.26 &br m, 12H). C NMR:
177.9 &q, Jˆ35 Hz), 163.2, 153.9, 153.0, 141.7,
32.7,124.0, 115.5, 114.6, 111.0, 87.0, 78.8, 71.8, 69.91,
9.89, 68.2, 58.2, 55.7, 29.7, 29.6, 29.53, 29.45, 29.43,
&MgSO ). After concentration, 1.61 g &99%) of O-tosyl
4
1
oxime 10 was obtained; R 0.80 &CH Cl /MeOH 10:1). H
f
2
2
NMR &CDCl ) &two stereoisomers): d 7.98±7.83 &m, 2H),
3
7.77±7.74 &m, 1H), 7.41±7.29 &m, 3H), 6.81±6.55 &2d, 1H),
4.24±4.20 &m, 1H), 4.10±4.00 &m, 3H), 3.51±3.38 &m, 8H),
2.44±2.43 &2s, 3H), 2.04 &s, 3H), 1.81±1.79 &m, 2H), 1.52±
1
9.2, 28.7, 26.1, 25.9. FAB-MS 680 &M) ; HRMS calcd
1
for &C H F IO ) 680.1822, found 680.1808.
3
0
40
3
6
1
1.48 &m, 4H), 1.36±1.21 &m, 12H). FAB-MS 786 &MH) ;
HRMS calcd for &C H F INO S1H) 786.1786, found
786.1738.
0
1
4
.1.9. 1-O-[11 -ꢀ2-Iodo-4-ꢀtri¯uoroacetyl)phenoxy)un-
3
2
43
3
8
decyl)]-2-O-methyl-3-O-acetyl-sn-glycerol ꢀ8). To an
ice-cooled mixture of 1.89 g &2.76 mmol) of PMP ether 7
in 40 mL of MeCN and 20 mL of water was added 4.76 g
8.69 mmol) of ammonium ceric&IV) nitrate &CAN). The
mixture was stirred for 2.5 h at room temperature, and
then stirred with aqueous 1N Na S O for 30 min. The
0
0
0
00
4.1.12. 1-O-[11 -ꢀ2 -Iodo-4 -ꢀ3 -tri¯uoromethyldiaziri-
dinyl)phenoxy)undecyl]-2-O-methyl-3-O-acetyl-sn-
glycerol ꢀ11). To a solution of 1.50 g &1.91 mmol) of 10 in
35 mL of CH Cl in a pressure tube was added liquid ammo-
&
2
2
3
2
2
mixture was extracted with Et O &2£150 mL), washed
nia &30 mL) at 2788C. After the mixture was stirred at room
temperature for 2.5 h, the excess ammonia was evaporated,
and the product was extracted with CH Cl . The organic
2
with brine, and dried &MgSO ). After concentration, the
4
residue was dissolved in 50 mL of anhydrous Et O. To the
2
2
2
solution of the crude alcohol were added 1.5 g &14.9 mmol)
of Et N, 25 mg &0.20 mmol) of DMAP, and 1.0 g
layer was washed with water and brine, and dried
&MgSO ). The residue was puri®ed by chromatography
3
4
&
overnight and then acidi®ed with 2N HCl. After the mixture
was extracted with Et O &twice), the combined organic
2
9.8 mmol) of acetic anhydride. The mixture was stirred
&elution with CH Cl /MeOH 20:1), providing 1.13 g
2
2
&93%) of 11 as a viscous oil; R 0.60 &CH Cl /MeOH
f 2 2
1
10:1). H NMR: d 7.97 &d, Jˆ2.0 Hz, 1H), 7.51 &dd, Jˆ
8.5 Hz, 1.8 Hz, 1H), 6.76 &d, Jˆ8.6 Hz, 1H), 4.25±4.21
&m, 1H), 4.10±4.06 &m, 1H), 4.00 &t, Jˆ6.3 Hz, 2H),
3.53±3.51&m, H1 ), 3.48±3.46 &m, 2H), 3.43 &s, 3H),
3.42±3.39 &m, 2H), 2.73 &d, Jˆ8.7 Hz, 1H), 2.16 &d, Jˆ
8.7 Hz, 1H), 2.05 &s, 3H), 1.82±1.79 &m, 2H), 1.55±1.46
layers were washed with saturated aqueous sodium bicarb-
onate solution and brine, and dried &MgSO ). The product
4
&
1.46 g, 86% for two steps) was obtained as an oil by chro-
matography &elution with hexane/EtOAc 6:1, then 2:1); Rf
1
0
1
4
3
3
1
1
.30 &hexane/EtOAc 3:1). H NMR: d 8.49 &d, Jˆ1.6 Hz,
1
3
H), 8.03 &d, Jˆ8.6 Hz, 1H), 6.86 &d, Jˆ8.9 Hz, 1H),
&m, 4H), 1.25 &br m, 12H). C NMR: d 171.0, 158.9,
.25 &dd, Jˆ1 1 .7, 3.8 Hz, 1 H), 4.1 4±4.09 &m, 3H )1, 38.9, 129.5, 125.2, 123.4 &q, Jˆ277 Hz), 111.4, 86.5,
.53±3.50 &m, 1H), 3.48±3.46 &m, 2H), 3.43 &s, 3H),
.43±3.39 &m, 2H), 2.05 &s, 3H), 1.87±1.82 &m, 2H),
78.0, 71.8, 69.7, 69.4, 63.7, 58.0, 56.9 &q, Jˆ145 Hz),
29.53, 29.51, 29.47, 29.46, 29.42, 29.19, 28.9, 26.02,
25.97, 20.9.
1
3
.55±1.48 &m, 4H), 1.36±1.23 &m, 12H). C NMR: d
77.9 &q, Jˆ35 Hz), 170.9, 163.2, 141.6, 132.6, 124.0, 116.6
0
0
0
00
&
5
2
q, Jˆ290 Hz), 111.0, 87.0, 78.0, 71.8, 69.9, 69.7, 63.7,
4.1.13. 1-O-[11 -ꢀ2 -Iodo-4 -ꢀ3 -tri¯uoromethyldiazirinyl)-
phenoxy)undecyl]-2-O-methyl-3-O-acetyl-sn-glycerol ꢀ12).
A mixture of 1.03 g &1.63 mmol) of 11, 457 mg &1.79 mmol)
8.0, 29.52, 29.49, 29.43, 29.41, 29.22, 29.14, 28.7, 26.0,
5.9, 20.9.
of iodine, and 412 mg &4.08 mmol) of Et N in 1 5 mL of
3
0
4
.1.10. 1-O-[11 -ꢀ2-Iodo-4-ꢀtri¯uoroacetyloxime)phen-
MeOH was stirred at room temperature for 0.5 h. After
150 mL of Et O was added, the mixture was poured into
oxy)undecyl)]-2-O-methyl-3-O-acetyl-sn-glycerol ꢀ9). A
mixture of 240 mg &0.39 mmol) of and 32 mg
0.47 mmol) of hydroxylamine hydrochloride in 3 mL of
EtOH and 0.5 mL of pyridine was heated at 50±608C for
0 h. The mixture was concentrated and partitioned between
2
8
1 N NaS O solution. The organic layer was washed with
2
2
3
&
brine and dried &MgSO ). The product &1.0 g, 98%) was
4
obtained by chromatography &elution with CH Cl ); R
f
2
2
1
3
0.70 &CH Cl /MeOH 50:1). H NMR: d 7.55 &d,
2 2
Et O and water. The ether layer was washed with 2N HCl,
2
Jˆ2.2 Hz, 1H), 7.16 &dd, Jˆ2.0, 8.6 Hz, 1H), 6.74 &d,
Jˆ8.7 Hz, 1H), 4.25±4.21 &m, 1H), 4.10±4.07 &m, 1H),
3.98 &t, Jˆ6.4 Hz, 2H), 3.52±3.40 &m, 8H), 2.06 &s, 3H),
1.81±1.76 &m, 2H), 1.55±1.46 &m, 4H), 1.30±1.26 &m,
saturated aqueous sodium bicarbonate solution, brine, and
dried &MgSO ). On concentration and drying under vacuum,
4
2
39 mg &97%) of 9 was obtained as an oil, which was pure
1
13
enough to be used in the next step. H NMR: &as a mixture of
the anti and syn stereoisomers) d 8.96±8.67 &two broad
peaks, 1H), 7.95±7.89 &2d, 1H), 7.47±7.40 &2dd, 1H),
12H). C NMR: d 170.9, 158.8, 137.6, 128.2, 122.4, 22.0
&q, Jˆ273 Hz), 111.5, 86.9, 78.1, 71.8, 69.8, 69.4, 63.7,
58.0, 29.55, 29.52, 29.46, 29.43, 29.2, 28.9, 27.4 &q,
Jˆ40.5 Hz), 26.03, 25.96, 20.90.
6
8
1
.83±6.76 &2d, 1H), 4.22±4.01 &m, 4H), 3.71±3.40 &m,
H), 2.06 &s, 3H), 1.81±1.80 &m, 2H), 1.59±1.49 &m, 4H),
.33±1.17 &m, 12H).
0
0
0
00
4.1.14. 1-O-[11 -ꢀ2 -Iodo-4 -ꢀ3 -tri¯uoromethyldiazirinyl)-
phenoxy)undecyl]-2-O-methyl-sn-glycerol ꢀ13). To a
solution of 1.0 g &1.59 mmol) of 12 in 25 mL of MeOH
was added ,8 mg &0.33 mmol) of NaH. The mixture was
stirred at room temperature for 2 h. After three drops of 10%
HCl were added, the mixture was concentrated and the resi-
0
4
.1.11.
1-O-[11 -ꢀ2-Iodo-4-ꢀtri¯uoroacetyl)phenoxy)-
undecyl)]-2-O-methyl-3-O-acetyl-sn-glycerol
oxime ꢀ10). To an ice-cooled solution of 1.31 g
2.07 mmol) of syn and anti oximes 9, 530 mg &5.3 mmol)
of Et N, and 20 mg &0.16 mmol) of DMAP in 40 mL of dry
O-tosyl
&
due was extracted with Et O &2£75 mL). The ether layer
3
2

G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
8931
was washed with brine and dried &MgSO ). The product
4
6.73 &d, Jˆ8.6 Hz, 1H), 4.28 &m, 2H), 3.89±3.77 &m, 6H),
&
with CH Cl /MeOH 100:2.5); R 0.40 &CH Cl /MeOH
0.92 g, 99%) was puri®ed by chromatography &elution
3.50±3.34 &m, 17H), 1.73 &m, 2H), 1.51±1.25 &m, 28H),
1
3
1.03±0.87 &m, 6H), 0.85 &t, Jˆ7.3 Hz, 3H). C NMR: d
164.3, 135.0, 131.5, 128.5, 122.4 &q, Jˆ275 Hz), 120.8,
109.3, 79.7, 71.8, 70.4, 67.9, 66.4, 64.9, 59.3, 57.8, 54.4,
29.7, 29.63, 29.56, 29.46, 29.3, 29.2, 29.1, 29.0, 27.3, 26.14,
26.10, 13.6, 9.9.
2
2
f
2
2
1
5
2
0:1). H NMR: d 7.54 &d, Jˆ2.3 Hz, 1H), 7.16 &dd, Jˆ
.2 Hz, Jˆ8.8 Hz, 1H), 6.74 &d, Jˆ8.6 Hz, 1H), 3.98 &t,
Jˆ6.4 Hz, 2H), 3.73±3.63 &m, 2H), 3.53±3.50 &m, 2H),
3
1
1
8
2
2
.44 &s, 3H), 3.437±3.39 &m, 3H), 1.83±1.76 &m, 2H),
1
3
.56±1.44 &m, 4H), 1.44±1.23 &m, 12H). C NMR: d
58.8, 137.6, 128.3, 122.4, 122.0 &q, Jˆ273 Hz), 111.5,
6.9, 79.8, 71.9, 70.6, 69.9, 69.4, 62.7, 57.7, 29.56, 29.51,
9.47, 29.46, 29.42, 29.2, 28.9, 27.5 &q, Jˆ40.5 Hz), 26.1,
6.0.
0
0
125
0
00
4.1.17. 1-O-[11 -ꢀ2 -[ I]-4 -ꢀ3 tri¯uoromethyldiazirinyl)-
phenoxy)undecyl]-2-O-methyl-sn-glycero-3-phospho-
choline ꢀ2c). The following iododestannylation procedure
was used to incorporate radioiodine. To a 1.5-mL cone-
shaped vial were added 100 mL &0.093 mmol) of a solution
of arylstannane 2b &1.7 mg of 2b in 2 mL of absolute
EtOH), 50 mL &1 0m mol) of a solution of NaH PO
0 0 0 00
.1.15. 1-O-[11 -ꢀ2 -Iodo-4 -ꢀ3 -tri¯uoromethyldiazirinyl)-
4
phenoxy)undecyl]-2-O-methyl-sn-glycero-3-phospho-
choline ꢀ2a). A 50-mL ¯ask was ¯ame-dried and ¯ushed
with argon. A solution of 117 mg &0.76 mmol) of POCl and
2
4
&276 mg of NaH PO ´H O in 1 0 mL of HO), 40 mL
2
4
2
2
1
25
&1.3 mmol) of unlabeled NaI, and 10 mL of Na
I
3
7
7 mg &0.76 mmol) of Et N in 0.5 mL of alcohol-free
3
&1mCi). Then 50 mL &0.23 mmol) of chloramine-T &from
a freshly prepared stock solution of 13 mg &0.046 mmol)
of chloramine-T trihydrate in 10 mL of absolute EtOH)
was added with stirring. The reaction mixture, which
became pale yellow, was stirred for 30 min. The reaction
CHCl3 was cooled to 2108C. A solution of 360 mg
0.61mmol) of 13 in 3 mL of CHCl was added. After the
&
3
mixture was stirred for 1h, choline tosylate &250 mg,
.91mmol) and 0.5 mL of pyridine were added. The reac-
0
tion mixture, which became orange, was stirred overnight at
room temperature. After water &0.2 mL) was added to
quench the reaction, the mixture was stirred for 30 min.
The residue obtained after concentration was passed
was quenched with 50 mL of 30% aqueous NaHSO solu-
3
tion. After 200 mL of chloroform was added, the mixture
was shaken and the bottom layer was collected and concen-
trated to dryness by using a stream of nitrogen. The residue
through a TMD-8 resin &elution with THF/H O 9:1). The
2
was dissolved in 100 mL of CHCl and loaded onto an
3
fractions containing the product were combined, concen-
trated, and puri®ed by chromatography &elution with
CHCl /MeOH 4:1, then with CHCl /MeOH/H O 65:25:4).
aminopropyl solid-phase extraction cartridge &500 mg).
The vial was washed with an additional 100 mL of CHCl3.
The latter solution was also loaded onto the cartridge.
Elution with 4 mL of CHCl3 removed the byproducts
&presumably tributyltin iodide and p-toluenesulfonamide);
3
3
2
The product &330 mg, 72%) was obtained after lyophili-
zation from benzene and ®ltration of a chloroform solution
of the product through a 0.2-mm Millipore ®lter to remove
suspended silica gel; R 0.18 &CHCl /MeOH/H O 65:25:4).
then elution with 4 mL of 1:1 CHCl /MeOH afforded
3
product 2c in a single fraction.
f
3
2
1
H NMR: d 7.53 &d, Jˆ2.1 Hz, 1H), 7.16 &dd, Jˆ8.5 Hz,
2
3
4
1
7
2
2
.2 Hz, 1H), 6.72 &d, Jˆ8.6 Hz, 1H), 4.23±3.74 &m, 9H),
.45±3.30 &m, 16H), 1.78±1.73 &m, 2H), 1.46±1.40 &m,
4.2. Effects of compounds 1 and 2a on cell proliferation
1
3
H), 1.27±1.20 &m, 12H). C NMR: d 158.8, 137.5,
28.2, 122.4, 122.0 &q, Jˆ273 Hz), 111.5, 86.8, 79.6,
1.7, 70.2, 69.4, 66.3, 64.9, 59.2, 57.7, 54.3, 29.7, 29.6,
9.5, 29.2, 29.0, 28.9, 28.7, 27.5 &q, Jˆ40.5 Hz), 26.1,
A 50 mM stock concentration of 2a was prepared in abso-
lute EtOH and stored at 2808C in the dark. The cells were
maintained under subdued light to avoid carbene generation.
The effects of compounds 1 and 2a on the increase in cell
numbers were assessed on the following panel of epithelial
cancer cell lines: breast &MCF-7, MDA-MB-468, MDA-
MB-231, BT549), prostate &DU145), and lung &A549).
1
1
6.0. FAB-MS 752 &MH) , 724 &MH2N ) ; HRMS calcd
2
1
for &C H F IN PO 1H) 752.2150, found 752.2189; UV
7
3
2
46
3
3
2
MeOH) lmax &e, M cm ) 295 nm &1975), 375 nm &329).
1
21
&
0
0
0
00
21±24
4
.1.16. 1-O-[11 -ꢀ2 -ꢀTri-n-butylstannyl)-4 -ꢀ3 -tri¯uoro-
The in vitro assays have been described previously.
methyldiazirinyl)phenoxy)undecyl]-2-O-methyl-sn-
glycero-3-phosphocholine ꢀ2b). A mixture of 48 mg
Brie¯y, cells were subcultured into 24-well plates and
allowed to grow to the exponential phase. The medium
was changed and replaced with one containing 2a &0±
50 mM) or 1 &0±20 mM) in 10% fetal bovine serum supple-
mented medium. The drug-containing media were prepared
shortly before use. The cell numbers in representative wells
were determined at the time of addition of the compounds.
&
ditin, and 8.0 mg &0.031mmol) of Pd&CH CN) Cl in 3 mL
0.064 mmol) of 2a, 371mg &0.64 mmol) of hexa- n-butyl-
3
2
2
of HMPA was stirred for 18 h at room temperature under
argon. The reaction mixture was diluted with 5 mL of
CHCl , and then passed through an aminopropyl silica gel
3
solid-phase extraction cartridge &500 mg) &elution with
CHCl to remove the solvent and unreacted hexabutylditin,
then with CHCl /MeOH 4:1). The ®ltrate was passed
3
The cells were incubated in an atmosphere of 5% CO , and
2
the plates were shielded from light with aluminum foil. At
the end of the incubation period &48 or 96 h), the cells were
detached with trypsin and the cell numbers were determined
with a Coulter counter. The increase in cell numbers was
expressed as a percentage relative to those in wells without
any drug. When the cells were incubated with 2a for 96 h,
the medium in all wells was replaced after 48 h with fresh
medium. All manipulations were carried out in a partially
darkened room.
3
through another aminopropyl cartridge &elution with
CHCl /MeOH 4:1). After concentration, the residue was
3
dissolved in a small volume of MeOH and loaded onto a
C-18 cartridge &2 g) &elution with MeOH). The ®rst part of
the eluant consisted of starting material 2a &9 mg), whereas
the last part afforded 24.2 mg &51%) of the desired product.
1
H NMR: d 7.14 &s, 1H), 7.07 &dd, Jˆ2.0 Hz, 8.7 Hz, 1H),

8932
G. Li et al. / Tetrahedron 57 ꢀ2001) 8925±8932
4
.3. Gel electrophoresis and autoradiography
9. P e rochon, E.; Leray, C.; Cr e mel, G.; Hubert, P. Anal.
Biochem. 1997, 254, 109±118.
For radioiodination of MCF-7 cytosolic proteins, 2 mL of a
10. Brunner, J. Methods Enzymol. 1989, 172, 628±687.
11. The tri¯uoromethyl group suppresses photo-rearrangement to
a linear diazo compound. Tri¯uoromethylaryldiazirines offer
many advantages, e.g. high chemical stability in subdued
ambient light toward various mild reducing agents, oxidizing
agents, acidic and basic conditions, and elevated tempera-
6
solution of 2c in EtOH &10 nmol, 2.2£10 cpm) was added
to 500 mL of cytosolic proteins &1.2 mg of protein) in a 1.5-
mL microfuge tube. Competition experiments were carried
out in the presence of a 100- and a 200-fold excess of 2a in
4
mL of EtOH. The tubes were rotated in the dark at room
8
temperature prior to photolysis. Photolysis was carried out
as described under Section 4.1. After photolysis, the
samples were boiled at 1008C for 5 min in 300 mL of
tures. They are activated in the near UV ꢀl . 360 nm†;
avoiding protein-damaging wavelengths.
12. He, L.; Byun, H.-S.; Bittman, R. J. Org. Chem. 1998, 63,
5696±5699.
5
Laemmli SDS sample buffer. The proteins were separated
by SDS-PAGE on 10% gels, which were dried and exposed
to ®lm &Kodak X-OMAT AR) for 3 days.
13. For a recent review of cyclic sulfate chemistry, see: Byun,
H.-S.; He, L.; Bittman, R. Tetrahedron 2000, 56, 7051±7091.
1
4. For a review of the Mitsunobu reaction, see: Hughes, D. L.
Org. React. 1992, 42, 335±656.
1
5. &a) Harter, C.; B aÈ chi, T.; Semenza, G.; Brunner, J. Bio-
chemistry 1988, 27, 1856±1864. &b) Weber, T.; Brunner, J.
J. Am. Chem. Soc. 1995, 117, 3084±3095.
Acknowledgements
The research at CUNY-Queens College was supported in
part by a PSC-CUNY Faculty Research Award to R. B. The
work at the University of Manitoba was supported by a grant
from the National Cancer Institute of Canada with funds
from the Canadian Cancer Society to G. A.
1
1
1
6. Deroo, P. W.; Rosenthal, A. F.; Isaacson, Y. A.; Vargas, L. A.;
Bittman, R. Chem. Phys. Lipids 1976, 16, 60±70.
7. For a review of the Stille coupling reaction, see: Farina, V.;
Krishnamurthy, V.; Scott, W. J. Org. React. 1997, 50, 1±652.
8. The bromo analogue of 17, which was prepared by reaction of
1
5 with bromine and HgO, followed by O-demethylation,
failed to undergo the Stille reaction with hexaalkylditin &step
l of Scheme 1) even at 608C.
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