Synthesis and cellular uptake of 2'-substituted analogues of (e)-5-(2- [125i]iodovinyl)-2'-deoxyuridine in tumor cells transduced with the herpes simplex type-1 thymidine kinase gene. Evaluation as probes for monitoring gene therapy

Article abstract of DOI:10.1021/jm9606406

A useful synthetic methodology was developed to synthesize and radiolabel a series of (E)-5(2-[¹²⁵I)iodovinyl)uracil nucleoside substrates for herpes simplex virus type-1 thymidine kinase (HSV-1 TK). (E)- 5-(2-[¹²⁵I]Iodovinyl)-2'-deoxyuridine ([¹²⁵I]IVDU, 10), (E)-5-(2- [¹²⁵I]iodovinyl)2'-fluoro-2'-deoxyuridine ([¹²⁵I]IVFRU, 11), (E)-5-(2- [¹²⁵I]iodovinyl)-2'-fluoro-2'-deoxyarabinouridine ([¹²⁵I]IVFAU, 12), and (E)-5-(2-[¹²⁵I]iodovinyl)arabinouridine ([¹²⁵I]IVAU, 13) were synthesized in 63-83% radiochemical yield by reaction of the unprotected (E)- 5-(2-(trimethylsilyl)vinyl) precursors (6-9)with [¹²⁵I]ICl. Cellular uptake of these labeled compounds (1018) was evaluated in vitro. All compounds showed minimal uptake in the KBALB cell line. However, increased uptake was observed for all compounds in KBALB-STK cells which are transduced with a replication incompetent Moloney murine leukemia virus vector encoding the HSV-1 TK gene. The results indicate that uptake of these compounds in KBALB-STK cells is variable and highly dependent on the nature of the sugar 2'-substituent. When a fluoro (12) or a hydroxy (13) substituent is present in the arabinofuranosyl (up) configuration at the 2'-position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen (10) or fluorine (11) in the ribofuranosyl (down) configuration at the 2'-position. Our results indicate that radiolabeled IVFRU (11) is most promising for further in vivo studies.

Full text of DOI:10.1021/jm9606406

2184
J . Med. Chem. 1997, 40, 2184-2190
Syn th esis a n d Cellu la r Up ta k e of 2′-Su bstitu ted An a logu es of
(E)-5-(2-[125I]Iod ovin yl)-2′-d eoxyu r id in e in Tu m or Cells Tr a n sd u ced w ith th e
Her p es Sim p lex Typ e-1 Th ym id in e Kin a se Gen e. Eva lu a tion a s P r obes for
Mon itor in g Gen e Th er a p y
Kevin W. Morin, Elena D. Atrazheva, Edward E. Knaus, and Leonard I. Wiebe*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
Received September 10, 1996X
A useful synthetic methodology was developed to synthesize and radiolabel a series of (E)-5-
(2-[125I]iodovinyl)uracil nucleoside substrates for herpes simplex virus type-1 thymidine kinase
(HSV-1 TK). (E)-5-(2-[125I]Iodovinyl)-2′-deoxyuridine ([125I]IVDU, 10), (E)-5-(2-[125I]iodovinyl)-
2′-fluoro-2′-deoxyuridine ([125I]IVFRU, 11), (E)-5-(2-[125I]iodovinyl)-2′-fluoro-2′-deoxyarabino-
uridine ([125I]IVFAU, 12), and (E)-5-(2-[125I]iodovinyl)arabinouridine ([125I]IVAU, 13) were
synthesized in 63-83% radiochemical yield by reaction of the unprotected (E)-5-(2-(trimeth-
ylsilyl)vinyl) precursors (6-9) with [125I]ICl. Cellular uptake of these labeled compounds (10-
13) was evaluated in vitro. All compounds showed minimal uptake in the KBALB cell line.
However, increased uptake was observed for all compounds in KBALB-STK cells which are
transduced with a replication incompetent Moloney murine leukemia virus vector encoding
the HSV-1 TK gene. The results indicate that uptake of these compounds in KBALB-STK
cells is variable and highly dependent on the nature of the sugar 2′-substituent. When a fluoro
(12) or a hydroxy (13) substituent is present in the arabinofuranosyl (up) configuration at the
2′-position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen (10)
or fluorine (11) in the ribofuranosyl (down) configuration at the 2′-position. Our results indicate
that radiolabeled IVFRU (11) is most promising for further in vivo studies.
The potential of gene therapy as a clinical treatment
modality for a variety of disorders has attracted con-
siderable attention in recent years. Clinical trials inves-
tigating the benefits of gene transfer have proliferated
rapidly. In particular, gene therapy protocols for the
treatment of cancer have occupied a central role in this
rapidly evolving area of research. A gene therapy
strategy for cancer that draws heavily on advances in
medicinal chemistry is directed enzyme prodrug therapy
(DEPT), where expression of metabolic suicide genes
convert relatively nontoxic prodrugs into highly cyto-
toxic agents.1 The suicide gene that has been studied
most extensively in the laboratory and clinic for treat-
ment of cancer is the herpes simplex type-1 thymidine
kinase (HSV-1 TK) gene.2,3 This virus-specific kinase
has been exploited successfully as a target for activation
of antiviral nucleoside prodrugs.4 The high degree of
selectivity observed in HSV-1-infected cells is attributed
to selective phosphorylation of nucleoside substrates by
HSV-1 TK.5
Transfer of the HSV-1 TK gene into proliferating
tissue and expression of the gene product can result in
sensitization to the cytotoxic effects of various purine
and uracil nucleoside analogs.6 Selective phosphoryla-
tion of these analogs by HSV-1 TK and further phos-
phorylation of these prodrugs by cellular kinases results
in generation of highly toxic triphosphate derivatives.
The acyclic guanosine analog ganciclovir has been
shown to be particularly effective as a cytotoxic agent
in gene therapy protocols involving HSV-1 TK gene
transfer.7 However, several 5-substituted uracil nucleo-
side derivatives have been shown to have more potent
and selective cytotoxic activity in cells transfected with
the HSV-1 TK gene.8,9 (E)-5-(2-Bromovinyl)-2′-deoxy-
uridine (BVDU) and related compounds are particularly
potent cytotoxic agents against FM3A murine mam-
mary carcinoma expressing HSV-1 TK.10,11 The obser-
vation that (E)-5-(2-[125I]iodovinyl)-2′-deoxyuridine ac-
cumulates by metabolic trapping in tumor cells ex-
pressing HSV-1 TK implicates phosphorylation as a
requisite for cytotoxic activity.12
Previous studies with radiolabeled IVDU have dem-
onstrated that, despite rapid accumulation in cells
expressing HSV-1 TK in vitro, it is an unsuitable agent
for in vivo detection of HSV-1 TK expressing tissue due
to rapid phosphorylase-mediated deglycosylation.13 Con-
sequently, the catabolic instability of IVDU precluded
its development as an imaging agent for herpes simplex
encephalitis. However, several structural analogs of
IVDU have enhanced stability toward phosphorylases.
Incorporation of a fluorine (ribo or arabino configura-
tion) or hydroxyl (arabino configuration) at the 2′-
position results in resistance to phosphorylase-mediated
degradation, retention of antiviral activity, and selective
metabolic trapping in herpes-infected (TK+) cells.14-16
These are desirable properties for a putative radio-
pharmaceutical for imaging HSV-1 TK expression dur-
ing gene therapy.
Noninvasive imaging of gene expression during gene
therapy could provide potentially useful information.
The extent of gene delivery and expression may be
assessed with a radiopharmaceutical selectively local-
izing in HSV-1 TK expressing tissue. This is particu-
larly important since treatment failures of gene therapy
with HSV-1 TK in animal models is associated with
inefficient gene delivery and expression prior to ganci-
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(96)00640-1 CCC: $14.00 © 1997 American Chemical Society

Probes for Monitoring Gene Therapy
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2185
Sch em e 1a
clovir administration.17 Recently, the feasibility of such
an approach has been demonstrated using quantitative
autoradiography and single photon emission computed
tomography (SPECT) with radiolabeled 5-iodo-2′-fluoro-
2′-deoxyarabinouridine (FIAU) in tumors transduced
with the HSV-1 TK gene.18,19 In this report, we describe
new radiosyntheses of (E)-5-(2-[125I]iodovinyl)-2′-de-
oxyuridine ([125I]IVDU), (E)-5-(2-[125I]iodovinyl)-2′-fluoro-
2′-deoxyuridine ([125I]IVFRU), (E)-5-(2-[125I]iodovinyl)-
2′-fluoro-2′-deoxyarabinouridine ([125I]IVFAU), and (E)-
5-(2-[125I]iodovinyl)arabinouridine ([125I]IVAU) as poten-
tial noninvasive radiopharmaceuticals for imaging HSV-1
TK expression. In order to select a radiopharmaceutical
with high sensitivity for scintigraphic imaging of HSV-1
TK expression, we have compared the uptake of each
compound in cells expressing HSV-1 TK.
Ch em istr y
Several strategies have been reported for the synthe-
sis of radiolabeled IVDU. Radiohalogen exchange has
been shown to be effective for the synthesis of lower
specific activity product.20 Similarly, carrier-added and
high specific activity products have been isolated in
respectable radiochemical yields using modified Huns-
diecker reactions.21 Recent reports for the highly ef-
ficient radiosyntheses of labeled IVAU from vinylstan-
nane and vinylsilane precursors under mild conditions
are particularly appealing.22,23 However, radioiodina-
tion of these precursors has only been attempted using
protected nucleosides. Deprotection after radiohaloge-
nation requires additional undesired laboratory han-
dling of radioactive materials, which results in some loss
of radiolabeled product. A general radiosynthetic strat-
egy where the final radiohalogenation is carried out
using an unprotected (E)-5-(2-(trimethylsilyl)vinyl) pre-
cursor is now reported.
The introduction of an (E)-5-(2-(trimethylsilyl)vinyl)
functionality at the 5-position of 5-iodoarabinouridine
has been reported using a reaction sequence which
involved palladium-catalyzed coupling of (trimethylsi-
lyl)acetylene followed by reduction over a Lindlar
catalyst.23 However, this procedure produces mixtures
of reduced compounds that require HPLC separation.
Direct coupling of (E)-2-(tributylstannyl)-1-(trimethyl-
silyl)ethene with protected 5-iodo-2′-deoxyuridine in the
presence of a palladium catalyst gives the desired
protected (E)-5-(2-(trimethylsilyl)vinyl)-2′-deoxyuridine
in good yield.24 It was therefore of interest to investi-
gate whether a similar coupling reaction could be
performed using unprotected nucleosides to simplify the
subsequent radioiodination procedure.
The palladium-catalyst coupling reaction proceeded
smoothly for 5-iodo-2′-deoxyuridine (3) and the 2′-
fluorinated derivatives (4, 5). Although the nucleoside
precursors had a low solubility in acetonitrile or THF,
the reactions proceeded to completion, providing yields
ranging from 64 to 80%. The purification of the (E)-5-
(2-(trimethylsilyl)vinyl) products (6-8) was troublesome
since trace impurities tended to co-elute with the desired
products during silica gel column chromatography, and
these impurities impeded subsequent crystallization
attempts. Therefore, it was necessary to perform ad-
ditional column chromatography purification of fractions
containing small amounts of impurities prior to crystal-
lization. The choice of recrystallization solvent was
a
Reagents: (i) Bu3SnCHdCHSiMe3, (Ph3P)2Pd(II)Cl2; (ii) LiOH,
benzene; (iii) 125ICl, acetonitrile.
important since isomerization of vinylsilane nucleosides
from the E to Z stereochemistry has been observed in
polar solvents.23 Recrystallization from ethyl acetate
did not appear to induce this isomerization.
Although the coupling reaction could be performed
using the unprotected 5-iodo-2′-deoxy nucleosides (3-
5), the extremely low solubility of 5-iodoarabinouridine
in the reaction solvents necessitated the introduction
of protecting groups. The reaction of (E)-2-(tributyl-
stannyl)-1-(trimethylsilyl)ethene with 2′,3′,5′-tri-O-
acetyl-5-iodoarabinouridine (1) in the presence of the
palladium catalyst in dry acetonitrile gave the coupled
product (2) in 64% yield (Scheme 1). Several methods
to remove the acetyl protecting groups, while retaining
the reactive (E)-5-(2-(trimethylsilyl)vinyl) moiety, were
investigated. Selective deprotection of 2 was achieved
after 80 h at 25 °C by treatment with a suspension of
2% lithium hydroxide in dry benzene to give (E)-5-(2-
(trimethylsilyl)vinyl)arabinouridine (9, 39%).
Unlabeled iodination and carrier-added radioiodina-
tion reactions were performed using the unprotected (E)-
5-(2-(trimethylsilyl)vinyl) precursors (6-9). Thus, treat-
ment with 1 equiv of iodine monochloride in dry
acetonitrile resulted in rapid formation of the desired
(E)-5-(2-iodovinyl) products (10-13). It was noted that
when the vinylsilane compounds were added to the
iodine monochloride solution immediately after dissolu-
tion, only formation of the E isomer was observed.
Carrier-added radiolabeling was carried out by simply
adding Na125I to small-scale reactions using iodine
monochloride. However, radiochemical yields were
improved when acetic acid (20%) was included in the
reaction mixture. Radiochemical yields, after reverse-
phase HPLC purification, were about 85% for all reac-
tions except for [125I]IVAU (13), which was obtained in
63% radiochemical yield. The lower solubility of (E)-5-
(2-(trimethylsilyl)vinyl)arabinouridine (9) in acetonitrile
may account for the lower radiochemical yield obtained
in this reaction. The specific activity of products
obtained in carrier-added reactions, which ranged be-
tween 30 and 41 GBq/mmol (0.81-1.1 Ci/mmol), was
acceptable for cellular uptake experiments.

2186 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Morin et al.
F igu r e 1. In vitro uptake of [125I]IVDU (a), [125I]IVFRU (b), [125I]IVFAU (c), and [125I]IVAU (d) in KBALB-STK (9) and KBALB
(2) cells.
Resu lts a n d Discu ssion
four compounds (10-13) occurred in the KBALB cell
line since less than 0.3 pmol compound/105 cells was
present at all time periods in KBALB cells.
Cellular uptake of each radiolabeled compound (10-
13) was determined in two cell lines of murine origin.
The KBALB cell line is a Kirsten virus-transformed
sarcoma tumor cell line.25 KBALB-STK cells are de-
rived from the KBALB cell line, express HSV-1 TK, and
are sensitive to the cytotoxic effects of ganciclovir in
vitro and in vivo.26 The KBALB-STK cells have been
transduced with the STK vector which is a replication-
incompetent Moloney murine leukemia retrovirus vector
containing the HSV-1 TK gene promoted by the SV40
early promoter-enhancer and the neomycin resistance
gene driven by the viral long terminal repeat.27 Con-
tinuous in vitro selection by culturing in the presence
of the neomycin analogue G-418 ensures that resistant
cells are propagated. In order to accurately compare
cellular uptake, each radiolabeled compound was as-
sayed under identical molar concentration (0.076 µM)
and cellular density (1 × 105 cells) in cell cultures of
KBALB and KBALB-STK cells. The cellular uptake of
each compound (10-13) in both cell lines over a period
of 8 h is illustrated in Figure 1. Minimal uptake of all
In contrast to the KBALB cells which lack HSV-1 TK
expression, the KBALB-STK cell line was capable of
accumulating the nucleoside analogues to varying de-
grees.
[
125I]IVDU was metabolically trapped to the
greatest extent, with cellular radioactivity increasing
up to 8 h after exposure (see Figure 1). Rapid influx of
compound was evident at 15 min, when approximately
11% of the total radioactivity or 4.1 pmol/105 cells was
present. At 8 h after exposure, 66% of the radioactivity
(25 pmol/105 cells) was intracellularly trapped. The
kinetic profile of the uptake process is qualitatively
similar to that observed previously with [131I]IVDU in
TK+ HSV-1-infected rabbit kidney cells.28 125I]IVFRU
[
also accumulated rapidly in KBALB-STK cells, with
increasing cellular radioactivity over time (see Figure
1b). However, the presence of a 2′-fluorine substituent
in the ribofuranosyl configuration appears to affect
cellular nucleoside uptake, since there was substantially
lower cellular uptake of [125I]IVFRU relative to [125I]-

Probes for Monitoring Gene Therapy
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2187
IVDU. Influx of [125I]IVFRU after 15 min was 2.2 pmol/
105 cells or 6% of the total radioactivity. After 8 h
exposure to [125I]IVFRU, KBALB-STK cells accumulated
8.2 pmol/105 cells or 21.6% of the total activity added
to the culture.
cytoplasmic membrane integrity due to an absence of
cytopathic viral infection imply that facilitated nucleo-
side transport mechanisms may be of increased impor-
tance relative to HSV-1 cell infection. It is known that
NBMPR-sensitive, equilibrative nucleoside transporters
in both human and murine cells are capable of accepting
a variety of nucleoside substrates which are relatively
insensitive to modifications at the 2′-position of uracil
nucleosides.31,32 Thus, it is likely that other biochemical
processes are affecting metabolic trapping of [125I]-
IVFAU and [125I]IVAU. IVDU, IVFRU, and IVFAU are
effective substrates for HSV-1 TK, display comparable
cytostatic activity against HSV-1 TK gene-transfected
murine mammary carcinoma, and appear to evoke this
cytostatic effect primarily through inhibition of thymidy-
Unexpected uptake kinetics were observed for both
[
125I]IVFAU (see Figure 1c) and [125I]IVAU (see Figure
1d) which possess 2′-fluoro and 2′-hydroxy substituents
in the arabinofuranosyl configuration, respectively. In
both cases, rapid uptake of radioactivity was evident
at the earliest time intervals. However, the influx of
labeled compounds was minimal after 30 min exposure.
In the case of [125I]IVFAU, initial uptake at 15 min was
2.4 pmol/105 cells, which is comparable to [125I]IVFRU
at this early time point. However, after 8 h exposure
to [125I]IVFAU, only 2.7 pmol/105 cells had accumulated
in the KBALB-STK cells. This represents a 3-fold
decrease in uptake relative to [125I]IVFRU, which differs
structurally from [125I]IVFAU only in that [125I]IVFRU
has a 2′-fluoro substituent in the ribofuranosyl config-
uration. A similar time-response profile with substan-
tially lower uptake of activity was observed for [125I]-
IVAU. Preferential uptake in HSV-1 TK expressing
cells was evident at 15 min in KBALB-STK cells rela-
tive to KBALB cells lacking HSV-1 TK. However, less
than 1 pmol/105 cells had accumulated at the earliest
time point in the KBALB-STK cells. Exposure to [125I]-
IVAU for up to 8 h did not substantially increase its
influx into KBALB-STK cells. After 8 h, cellular uptake
of [125I]IVAU was only 0.9 pmol/105 cells, representing
a 3-fold decrease in uptake relative to [125I]IVFAU
where the 2′-substituent in the arabino configuration
is fluorine.
late synthase.33
However, the observation that the
structurally related (E)-5-(2-bromovinyl)-2′-arabinouri-
dine completely lacks cytostatic and thymidylate syn-
thase inhibitory activity in HSV-1 TK expressing tumor
cells whereas (E)-5-(2-bromovinyl)-2′-deoxyuridine is one
of the most potent cytostatic agents in these cells
demonstrates that a small modification in structure can
have dramatic biochemical consequences even though
both compounds exhibit potent antiviral activity against
HSV-1.34
In conclusion, the objective of this study was to
identify a suitable radiolabeled probe which could prove
useful as a marker for cells transduced or transfected
with the HSV-1 TK gene during in vivo gene therapy.
All of the compounds evaluated (10-13) displayed very
low uptake in KBALB cells that lack the HSV-1 TK
gene. In HSV-1 TK gene-transduced tumor cells
(KBALB-STK), the order of cellular uptake is [125I]IVDU
> [125I]IVFRU > [125I]IVFAU > [125I]IVAU. Although
Structure-activity correlations for a variety of 2′-
fluoro-2′-deoxyuridine analogs have indicated that the
sugar 2′-configuration results in differential antiviral
effects.15 Moreover, 5-substituted 2′-fluoro-2′-deoxyuri-
dine analogs possessing the arabinofuranosyl configu-
ration are generally more potent against HSV-1 than
similar compounds possessing the ribofuranosyl config-
uration.29 Similarly, we observed that there was a
correlation between nucleoside uptake in rabbit kidney
cells infected with HSV-1 (TK+) and antiviral effect,
since radiolabeled 5-iodo-2′-fluoro-2′-deoxyarabinouri-
dine (FIAU) accumulated to a greater extent than
5-iodo-2′-fluoro-2′-deoxyuridine (FIRU) in HSV-1-in-
fected cells over a 24 h period.30 However, a compara-
tive uptake investigation revealed that [125I]IVDU and
[
125I]IVDU displayed the highest uptake in KBALB-STK
cells at all time periods examined, it is considered
unsuitable as an imaging agent due to its susceptibility
to rapid, phosphorylase-mediated deglycosylation.13 The
structure-uptake relationship observed in this study
indicates that for these 2′-substituted 2′-deoxyuridine
derivatives possessing an (E)-5-(2-iodovinyl) substituent,
the presence of a 2′-fluoro substituent in the ribo
configuration confers a superior ability to accumulate
in tumor cells expressing HSV-1 TK. The combination
of avid cellular accumulation of radiolabeled IVFRU in
HSV-1 TK expressing cells and resistance to phospho-
rylase-mediated deglycosylation after in vivo adminis-
tration to rodents are desirable characteristics for a
putative HSV-1 TK imaging agent.14 However, it should
be noted that cellular accumulation of IVFRU in human
cells expressing HSV-1 TK may differ from that ob-
served in murine cells. A potential concern regarding
the predictive value of the murine models should be
addressed by performing uptake studies in human cells.
Therefore, IVFRU radiolabeled with radionuclides that
emit tissue-penetrating photons (131I, 123I, or 124I) war-
rant further investigation as a noninvasive in vivo probe
to detect HSV-1 TK gene expression during gene
therapy.
[
125I]IVFRU had significantly higher cellular uptake
than [125I]IVFRU in HSV-1 (TK+)-infected rabbit kidney
cells despite the comparable antiviral activity of IV-
FAU.14 Interestingly, this latter study indicated that
the change in cellular uptake of [125I]IVFAU between 4
and 24 h was quite low compared to that of [125I]IVDU
and [125I]IVFRU. The present results for HSV-1 TK
expressing tumor cells is in agreement with the previous
observations since [125I]IVDU and [125I]IVFRU accumu-
lated to a greater degree than [125I]IVFAU and [125I]-
IVAU, with the latter two compounds showing very little
change in uptake between 15 min and 8 h. In addition,
the extent of [125I]IVAU uptake in KBALB-STK cells is
similar to that previously observed in HSV-1 (TK+)-
infected rabbit kidney cells.23
Exp er im en ta l Section
Melting points were determined on a Buchi capillary ap-
paratus and are uncorrected. NMR spectra were acquired on
a Bruker AM-300 spectrometer. The assignment of exchange-
able protons (NH, OH) were confirmed by addition of D2O. 13
C
The reasons for the different time-response profiles
are not directly apparent in this study. Uncompromised
NMR spectra were acquired using the J -modulated spin echo

2188 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Morin et al.
technique where methyl and methine carbon resonances
appear as positive peaks and methylene and quaternary
carbon resonances appear as negative peaks. High-resolution
mass spectrometry was performed on a AEI MS-50 mass
spectrometer. Solvents and reagents used were of reagent
quality. Solvents were dried using standard methods and were
freshly distilled prior to use. TLC analysis was performed
using Whatman MK6F silica gel microslides. Column chro-
matography was performed on Merck 7734 silica gel (60-200
µm particle size). HPLC separations of radiolabeled com-
pounds were performed on a preparative Whatman Partisil
M9 10/25 ODS reverse phase column using a Waters HPLC
system consisting of Model 510 solvent pumps, Model 860
gradient controller, Model U6K injector unit, and a Hewlett-
Packard Model 1040A diode array ultraviolet detector. Ra-
dioactivity was determined by gamma counting using a
Beckman Gamma 8000 scintillation counter. 5-Iodo-2′-deox-
yuridine was purchased from the Aldrich Chemical Co. 5-Iodo-
2′-fluoro-2′-deoxyuridine (4)35 and 2′,3′,5′-tri-O-acetyl-5-iodo-
arabinouridine (1)23 were prepared using literature pro-
cedures. 5-Iodo-2′-fluoro-2′-deoxyarabinouridine (5) was ob-
tained as a generous gift from Bristol Meyers Squibb. (E)-2-
(Tributylstannyl)-1-(trimethylsilyl)ethene was prepared as
previously described.36 KBALB cells were obtained from the
American Type Culture Collection (ATCC), and KBALB-STK
cells were a kind gift from Dr. Scott Freeman.
SnCHdCHSiMe3 (412 mg, 1.06 mmol) were added with
stirring to a solution of 4 (197 mg, 0.53 mmol) in dry THF (5
mL) at 50 °C, and the reaction mixture was stirred for 16 h at
50 °C under an atmosphere of argon at which time TLC
analysis indicated the reaction was completed. Removal of the
solvent in vacuo and purification of the residue by silica gel
column chromatography with methanol-dichloromethane (1:
25, v/v) as eluent afforded 7 as a white solid (146 mg, 80%
yield) after recrystallization from ethyl acetate: mp 164-165
1
°C; H NMR (DMSO-d6) δ 0.08 (s, 9H, SiMe3), 3.62-3.65 (m,
1H, J gem ) 9.9 Hz, H-5′), 3.84-3.88 (m, 1H, H-5′′), 3.89 (d, 1H,
J 3′,4′ ) 8.8 Hz, H-4′), 4.14-4.25 (m, 1H, J 3′,F ) 24.2, J 2′,3′ ) 3.8
Hz, H-3′), 5.04 (dd, 1H, J 2′,F ) 53.3, J 2′,3′ ) 3.8 Hz, H-2′), 5.43
(t, 1H, J OH,5′ ) 4.8 Hz, C-5′ OH), 5.63 (d, 1H, J OH,3′ ) 6.6 Hz,
C-3′ OH), 5.92 (d, 1H, J 1′,F ) 17.0 Hz, H-1′), 6.52 and 6.59 (two
d, 1H each, J trans ) 19.8 Hz, CHdCHSiMe3), 8.33 (s, 1H, uracil
H-6), 11.51 (s, 1H, NH); exact mass calculated for C14H21FN2O5-
Si 344.1204, found (HRMS) 344.1208 (M+, 2.2). Anal. (C14H21
FN2O5Si) C, H, N.
-
(E)-5-(2-(Tr im et h ylsilyl)vin yl)-2′-flu or o-2′-d eoxya r a -
bin ou r id in e (8). (E)-Bu3SnCHdCHSiMe3 (220 mg, 0.56
mmol) was added to a solution of 5 (100 mg, 0.27 mmol) and
(Ph3P)2Pd(II)Cl2 (20 mg, 0.028 mmol) in dry acetonitrile (10
mL), and the reaction was allowed to proceed at 60 °C for 16
h under an argon atmosphere with stirring. Removal of the
solvent in vacuo, purification of the residue by silica gel column
chromatography with methanol-chloroform (1:10, v/v) as
eluent, and recrystallization of the product from EtOAc gave
8 as a white solid (59 mg, 64% yield): mp 182-183 °C; 1H
NMR (DMSO-d6) δ 0.09 (s, 9H, SiMe3), 3.60-3.70 (m, 2H,
2′,3′,5′-Tr i-O-a cet yl-(E)-5-(2-(t r im et h ylsilyl)vin yl)a r a -
bin ou r id in e (2). (E)-Bu3SnCHdCHSiMe3 (10.3 g, 26.2 mmol)
was added to a stirred solution of 1 (5.2 g, 9.9 mmol) and
(Ph3P)2Pd(II)Cl2 (0.99 g, 1.41 mmol) in dry acetonitrile (300
mL) under an argon atmosphere, and the reaction was allowed
to proceed for 16 h at 50 °C with stirring. Removal of the
solvent in vacuo gave a residue that was purified by silica gel
column chromatography using hexane-ethyl acetate (1.5:1,
v/v) as eluent to yield 2 (3.32 g, 64%) as a white crystalline
H-5′), 3.78-3.80 (m, 1H, H-4′), 4.24-4.31 (m, 1H, J 3′,F
)
20.9 Hz, H-3′), 5.11 (dt, 1H, J 2′,F ) 52.8, J 1′,2′ ) 4.4, J 2′,3′
) 4.4 Hz, H-2′), 5.27 (t, 1H, J OH,5′ ) 5.5 Hz, C-5′ OH), 5.90
(d, 1H, J OH,3′ ) 5.0 Hz, C-3′ OH), 6.15 (dd, 1H, J 1′,F ) 13.2,
J 1′,2′ ) 4.4 Hz, H-1′), 6.59 (two d, 1H each, J trans ) 19.2
Hz, CHdCHSiMe3), 8.04 (s, 1H, uracil H-6), 11.58 (s, 1H,
NH); exact mass calculated for C14H21FN2O5Si 344.1204,
found (HRMS) 344.1202 (M+, 3.53). Anal. (C14H21FN2O5Si)
C, H, N.
1
product, mp 159-160 °C. The H and 13C NMR spectra were
identical to those reported previously.23
(E)-5-(2-(Tr im eth ylsilyl)vin yl)ar abin ou r idin e (9). Com-
pound 2 (0.18 g, 0.34 mmol) was added to a 2% suspension of
LiOH in dry benzene (200 mL), and the reaction was allowed
to proceed for 80 h at 25 °C with stirring. After filteration to
remove solid LiOH, and removal of the solvent in vacuo, the
residue obtained was purified by silica gel column chroma-
tography using methanol-chloroform (12:88, v/v) as eluent.
Recrystallization of the product 9 from methanol gave white
crystals (51 mg, 39%): mp 103-105 °C; UV (CH3OH) λmax 246,
Gen er a l P r oced u r e for Iod in a t ion of (E)-5-(2-(Tr i-
m eth ylsilyl)vin yl) P r ecu r sor s (6-9) (Meth od A). After
dissolution of 6-9 (0.116 mmol) in dry acetonitrile (2 mL),
iodine monochloride (19 mg, 0.116 mmol) was added im-
mediately, and the reaction was allowed to proceed at 25 °C
for 30 min with stirring. The solvent was removed in vacuo,
and the residue was purified by silica gel column chromatog-
raphy using methanol (7-10%) in chloroform as eluent. The
solvent from pooled fractions was removed in vacuo, and the
respective product (10, 11, 12, or 13) was recrystallized from
methanol.
Gen er a l P r oced u r e for Ca r r ier -Ad d ed Ra d ioiod in a -
tion of (E)-5-(2-(Tr im eth ylsilyl)vin yl) P r ecu r sor s (6-9)
(Meth od B). Na125I (6.5 MBq) in 0.1 N NaOH solution (5 µL)
was placed in a Wheaton vial, and a solution of ICl (25 µg,
0.154 µmol) in 20% acetic acid in acetonitrile (10 µL) was
added. A solution of 6, 7, 8, or 9 (0.73 µmol) in 20% acetic
acid in acetonitrile (10 µL) was added to the contents in the
Wheaton vial, and the reaction was allowed to proceed for 15
min at 25 °C. The desired product (10, 11, 12, or 13) was
purified by preparative reverse phase HPLC on a Whatman
Partisil M9 10/25 C8 column using isocratic elution with
acetonitrile-H2O (3:7, v/v) at a flow rate of 1.5 mL/min and
UV detection at 254 nm. Radiolabeled compounds (10-13)
displayed identical HPLC chromatographic retention times as
observed for authentic unlabeled reference samples under a
variety of chromatographic conditions.
1
296 nm; H NMR (CD3OD) δ 0.10 (s, 9H, SiMe3), 3.79-3.92
(m, 3H, H-5′, H-4), 4.10 (dd, 1H, J 3′,4′ ) 4.2, J 2′,3′ ) 3.9 Hz,
H-3′), 4.19 (dd, 1H, J 1′,2′ ) 4.5, J 2′,3′ ) 3.9 Hz, H-2′), 6.16 (d,
1H, J 1′,2′ ) 4.5 Hz, H-1′), 6.69 and 6.53 (two d, 1H each, J trans
) 19.4 Hz, CHdCHSiMe3), 8.07 (s, 1H, uracil H-6); 13C NMR
(CD3OD) δ 164.70 (C-4, CdO), 151.60 (C-2, CdO), 140.56 (C-
6), 135.71 and 130.19 (vinyl, C-1 and C-2), 112.78 (C-5), 87.12
(C-1′), 85.82 (C-4′), 77.19 (C-2′), 76.93 (C-3′), 61.99 (C-5′), -1.20
(SiMe3); exact mass calculated for C14H22N2O6Si 342.1247,
found (HRMS), 342.1243 (M+, 1.74).
(E)-5-(2-(Tr im eth ylsilyl)vin yl)-2′-d eoxyu r id in e (6). (E)-
Bu3SnCHdCHSiMe3 (220 mg, 0.56 mmol) and (Ph3P)2Pd(II)-
Cl2 (20 mg, 0.028 mmol) were added to a solution of 3 (100
mg, 0.28 mmol) in dry acetonitrile (10 mL), and the reaction
was allowed to proceed at 60 °C for 16 h under an argon
atmosphere with stirring. Removal of the solvent in vacuo
gave a residue that was purified by silica gel column chroma-
tography. Elution with methanol-chloroform (3:20, v/v) af-
forded 6, which was recrystallized from ethyl acetate as a
1
colorless solid (67 mg, 73% yield): mp 108-110 °C; H NMR
(DMSO-d6) δ 0.09 (s, 9H, SiMe3), 2.08-2.22 (m, 2H, H-2′),
3.56-3.64 (m, 2H, H-5′), 3.78-3.79 (m, 1H, H-4′), 4.22-4.30
(m, 1H, H-3′), 5.17 (t, 1H, J OH,5′ ) 5.0 Hz, C-5′ OH), 5.27 (d,
1H, J OH,3′ ) 4.4 Hz, C-3′ OH), 6.16 (t, 1H, J 1′,2′ ) 6.1 Hz, H-1′),
6.59 (s (A2), 2H, CHdCHSiMe3), 8.18 (s, 1H, H-6), 11.42 (s,
1H, NH); exact mass calculated for C14H22N2O5Si 326.1298,
found (HRMS) 326.1301 (M+, 2.39). Anal. (C14H22N2O5Si) C,
H, N.
(E)-5-(2-Iod ovin yl)-2′-d eoxyu r id in e (10, Meth od A) a n d
(E)-5-(2-[125I]Iod ovin yl)-2′-d eoxyu r id in e (10, Meth od B).
(E)-5-(2-Iodovinyl)-2′-deoxyuridine (10) was prepared in 77%
yield (method A) after recrystallization from methanol: mp
1
167-169 °C; H NMR (DMSO-d6) δ 2.10-2.16 (m, 2H, H-2′),
3.52-3.68 (m, 2H, H-5′), 3.76-3.80 (m, 1H, H-4′), 4.20-4.26
(m, 1H, H-3′), 5.10 (t, 1H, J OH,5′ ) 4.0 Hz, C-5′ OH), 5.27 (d,
1H, J OH,3′ ) 3.0 Hz, C-3′ OH), 6.13 (t, 1H, J 1′,2′ ) 4.0 Hz, H-1′),
7.12 (d, J trans ) 14.3 Hz, 1H, CHdCHI), 7.20 (d, J trans ) 14.3
Hz, 1H, CHdCHI), 8.07 (s, 1H, H-6), 11.55 (s, 1H, NH).
(E)-5-(2-(Tr im et h ylsilyl)vin yl)-2′-flu or o-2′-d eoxyu r i-
d in e (7). (Ph3P)2PdCl2 (37 mg, 0.05 mmol) and (E)-Bu3-

Probes for Monitoring Gene Therapy
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2189
(E)-5-(2-[125I]Iodovinyl)-2′-deoxyuridine ([125I]IVDU) (10, 5.6
MBq, 86% radiochemical yield, >98% radiochemical purity,
specific activity 38 GBq/mmol), prepared according to method
B, had a HPLC retention time of 11.49 min whereas the
unreacted (trimethylsilyl)vinyl precursor (6) had a retention
time of 22.51 min.
Heritage Foundation for Medical Research for a stu-
dentship to one of us (K.W.M.). We also thank Dr. Scott
Freeman for providing the KBALB-STK cell line.
Refer en ces
(E)-5-(2-Iodovin yl)-2′-flu or o-2′-deoxyu r idin e (11, Meth od
A) a n d (E)-5-(2-[125I]Iod ovin yl)-2′-flu or o-2′-d eoxyu r id in e
(11, Meth od B). (E)-5-(2-Iodovinyl)-2′-fluoro-2′-deoxyuridine
(IVFRU) was prepared in 78% yield (method A) after recrys-
tallization from methanol as a white solid: mp 107-109 °C
(lit.14 mp 108-110 °C); 1H NMR (DMSO-d6) δ 3.65 (d, 1H, J gem
) 12 Hz, H-5′), 3.82-3.96 (m, 2H, H-4′, H-5′′), 4.20 (dd, 1H,
J 3′,F ) 23, J 2′,3′ ) 6 Hz, H-3′), 5.06 (dd, 1H, J 2′,F ) 54, J 2′,3′ ) 6
Hz, H-2′), 5.47 (br s, 1H, C-5′ OH), 5.72 (br s, 1H, C-3′ OH),
5.92 (d, 1H, J 1′,F ) 18 Hz, H-1′), 7.09 (d, 1H, J trans ) 16 Hz,
CHdCHI), 7.20 (d, 1H, J trans ) 16 Hz, CHdCHI), 8.23 (s, 1H,
uracil H-6), 11.60 (br s, 1H, NH).
(E)-5-(2-[125I]Iodovinyl)-2′-fluoro-2′-deoxyuridine ([125I]IVF-
RU) (11, 5.5 MBq, 85% radiochemical yield, >98% radiochemi-
cal purity, specific activity 41 GBq/mmol), prepared according
to method B, had a HPLC retention time of 11.76 min, whereas
the unreacted (trimethylsilyl)vinyl precursor (7) had a reten-
tion time of 24.19 min.
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(E)-5-(2-Iodovin yl)-2′-flu or o-2′-deoxyar abin ou r idin e (12,
Meth od A) a n d (E)-5-(2-[125I]Iod ovin yl)-2′-flu or o-2′-d eoxy-
a r a b in ou r id in e (12, Met h od B). (E)-5-(2-Iodovinyl)-2′-
fluoro-2′-deoxyarabinouridine (IVFAU) was prepared in 64%
yield (method A) after recrystallization from methanol: mp
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2′-deoxyuridine against murine mammary carcinoma (FM3A)
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1
178 °C (lit.37 mp 178-179 °C); H NMR (DMSO-d6) δ 3.61-
3.71 (m, 2H, H-5′), 3.79-3.80 (m, 1H, H-4′), 4.17-4.27 (m, 1H,
J 3′,F ) 20 Hz, H-3′), 5.06 (dt, 1H, J 2′,F ) 54, J 1′,2′ ) 3.0 Hz,
H-2′), 5.20 (t, 1H, J OH,5′ ) 6.0 Hz, C-5′ OH), 5.94 (d, 1H, J OH,3′
) 4.9 Hz, C-3′ OH), 6.10 (dd, 1H, J 1′,F ) 10, J 1′,2′ ) 4.9 Hz,
H-1′), 7.15 (d, 1H, J trans ) 14.8 Hz, CHdCHI), 7.23 (d, 1H, J trans
) 14.8 Hz, CHdCHI), 7.98 (s, 1H, uracil H-6), 11.73 (s, 1H,
NH).
(E)-5-(2-[125I]Iodovinyl)-2′-fluoro-2′-deoxyarabinouridine([125I]-
IVFAU) (12, 5.6 MBq, 86% radiochemical yield, >98% radio-
chemical purity, specific activity 38 GBq/mmol), prepared
according to method B, had a HPLC retention time of 13.21
min whereas the unreacted (trimethylsilyl)vinyl precursor (8)
had a retention time of 27.59 min.
(E)-5-(2-Iod ovin yl)a r a bin ou r id in e (13, Meth od A) a n d
(E)-5-(2-[125I]Iod ovin yl)a r a b in ou r id in e (13, Met h od B).
(E)-5-(2-Iodovinyl)arabinouridine was isolated in 75% yield
(method A) after recrystallization from methanol: mp 171-
1
175 °C (lit.23 mp 170-175 °C); H NMR (DMSO-d6) δ 3.62-
3.65 (m, 2H, H-5′), 3.72-3.76 (m, 1H, H-4′), 3.90-3.93 (m, 1H,
H-3′), 4.00-4.05 (m, 1H, H-2′), 5.13 (t, 1H, J OH,5′ ) 5.5 Hz,
C-5′ OH), 5.46 (d, 1H, J OH,3′ ) 4.4 Hz, C-3′ OH), 5.56 (d, 1H,
J OH,2′ ) 5.3 Hz, C-2′ OH), 5.98 (d, 1H, J 1′,2′ ) 4.7 Hz, H-1′),
7.13 (d, 1H, J trans ) 14.6 Hz, CHdCHI), 7.19 (d, 1H, J trans
)
14.6 Hz, CHdCHI), 7.88 (s, 1H, uracil H-6), 11.54 (s, 1H, NH).
(E)-5-(2-[125I]Iodovinyl)arabinouridine ([125I]IVAU) (13, 4.1
MBq, 63% radiochemical yield, >98% radiochemical purity,
specific activity 30 GBq/mmol), prepared using Method B had
a HPLC retention time of 10.24 min, whereas the unreacted
trimethylsilylvinyl precursor (9) had a retention time of 17.69
min.
In Vitr o Up ta k e Stu d ies. Cells (1 × 105) of each cell line
(KBALB, KBALB-STK) were grown in 24-well culture plates.
Radiolabeled compound (10, 11, 12, or 13; 38 pmol; specific
activity ) 30-41 GB1/mmol) was added to each well and
incubated at 37 °C in Dulbecco’s Modified Eagles Medium (0.5
mL). At varying times after exposure to the radiolabeled
compounds, the supernatants were removed, the cells were
rinsed with phosphate-buffered saline, and the adherant cells
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radioactivity was determined by gamma counting in a Beck-
mann 8000 gamma counter.
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J M9606406