Autofluorescent fused-pyrimidine nucleosides: Synthesis and evaluation as permeants and inhibitors of human nucleoside transporters

Article abstract of DOI:10.1135/cccc2011061

Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4-C5 of pyrimidin-2-one were prepared by ring closures with 5-(alkyn-1-yl)pyrimidin-2-one intermediates, heterocyclic atom replacements, and ring closure with a 5-aminocyti

Full text of DOI:10.1135/cccc2011061

Autofluorescent Nucleosides/Human Nucleoside Transporters
1395
AUTOFLUORESCENT FUSED-PYRIMIDINE NUCLEOSIDES: SYNTHESIS
AND EVALUATION AS PERMEANTS AND INHIBITORS OF HUMAN
NUCLEOSIDE TRANSPORTERS
c
Ireneusz NOWAKa1, Vijaya L. DAMARAJUb1, Carol E. CASSb2, James D. YOUNG
a2,
and Morris J. ROBINS
*
a
Department of Chemistry and Biochemistry, Brigham Young University,
1
2
Provo 84602-5700, Utah, U.S.A.; e-mail: i_nowak@hotmail.com, morris_robins@byu.edu
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2;
b
c
1
2
e-mail: vijaya.damaraju@albertahealthservices.ca, carol.cass@albertahealthservices.ca
Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7;
e-mail: james.young@ualberta.ca
Received March 9, 2011
Accepted August 23, 2011
Published online November 30, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in appreciation of a long
and rewarding friendship and in recognition of his outstanding contributions to nucleic acid chemistry,
biochemistry, and medicinal applications.
Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4–C5 of
pyrimidin-2-one were prepared by ring closures with 5-(alkyn-1-yl)pyrimidin-2-one interme-
diates, heterocyclic atom replacements, and ring closure with a 5-aminocytidine derivative.
Ultraviolet absorption and emission properties of the autofluorescent products enabled stud-
ies on permeation and inhibition of the trans-cellular trafficking effected by human equi-
librative nucleoside transporters (hENTs). Some of the autofluorescent nucleosides were
shown to be potent and selective inhibitors of human concentrative nucleoside transporters
(hCNTs) in a companion study reported elsewhere.
Keywords: Alkynylpyrimidine cyclizations; Autofluoresent nucleoside analogues; Nucleoside
transport studies; Ring-fused pyrimidines; Sonogashira coupling.
Pyrimidine nucleoside analogues and derivatives have been applied
extensively in biochemistry, pharmacology, and medicine. We reported
Sonogashira coupling of terminal alkynes with 5-iodouracil compounds
in 1981 and demonstrated that copper(I)-catalyzed cyclization of the
5-(alkyn-1-yl) products gave fluorescent furo[2,3-d]pyrimidin-2-one ana-
logues1. Considerable interest has been focused on such nucleosides with
furan, pyrrole, and thiophene rings fused at C4–C5 of the pyrimidine ring
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011061

1396
Nowak, Damaraju, Cass, Young, Robins:
(Fig. 1). Bicyclic hybrids of this type have been found to target bacterial
enzymes, and oligonucleotides containing pyrrolopyrimidine nucleobase
analogues in a CpG motif were recognized by toll-like receptor 9 (TLR9)
and stimulated the immune system2. The autofluorescence properties we
noted1 have been employed in probes for analysis of DNA and RNA hybrid-
ization3 and for detection of single nucleotide polymorphisms4. Potent and
selective antiviral activities were identified by extensive structure activity
relationship studies with longer-chain bicyclic derivatives5, and the se-
lectivities and potencies were found to depend on the chain length and na-
ture of alkyl/aryl substituents at C6 of the fused five-membered rings5.
Because such appendages affect hydrophobic properties that can increase
interactions with cellular membrane components and/or simple diffusion,
we chose the parent bicyclic ring analogues (and those with a minimal
6-methyl substituent) for studies with human nucleoside transporters. We
recently reported real-time analysis of nucleoside transport into living cells
with confocal microscopy using furo[2,3-d]pyrimidin-2-one 2′-deoxy- and
ribonucleoside fluorophores6, and other applications of fluorescent probes
for measurement of uptake of nucleoside drugs into cancer cells7,8
.
FIG. 1
Structures, abbreviations, ring numbering, and compound numbers of fused-pyrimidine nu-
cleosides
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1397
The role of human equilibrative and concentrative nucleoside transport-
ers (hENTs and hCNTs) as biomarkers for prediction of response to
nucleoside drugs is gaining increased recognition. The presence of hENT1
in membranes has been shown to be of major importance for entry of
nucleoside analogue drugs into cells and is a useful predictor of drug activi-
ties9. Studies on patients with pancreatic cancer have demonstrated positive
correlations between clinical responses to gemcitabine (2′-deoxy-2′,2′-di-
fluorocytidine) and abundances of hENT1 protein in the cancer tissues10.
Immunohistochemistry (IHC) has been used to estimate hENT1-mediated
transport of gemcitabine into cancer cells, and while that methodology is
applicable to paraffin-embedded or frozen tissue samples, it is time con-
suming and less useful for routine monitoring of nucleoside transport ca-
pacity of freshly isolated cells. The abundance of hENT1 on cell membranes
can be enumerated by equilibrium binding with tritium-labeled 6-S-
(4-nitrobenzyl)thioinosine ([3H]NBMPR) but procedures that involve mea-
surement of radioactivity are less attractive. High-affinity autofluorescent
analogues that identify cell-surface abundances of hENT1 enable rapid
quantification with some clinical samples and provide initial predictions of
potential treatment outcomes with nucleoside drugs9c. We recently de-
scribed a new approach for real-time measurement of nucleoside traffick-
ing6 with a novel fluorescent cell-surface hENT1 probe11. Autofluorescent
permeants and inhibitors of other nucleoside transporters will provide tools
for new investigations in transporter research. We now report preparation
of the autofluorescent analogues shown in Fig. 1 and further evaluation of
their activities as permeants and/or inhibitors of human nucleoside trans-
porters. Five were shown to be permeants for hENT1 in the present study.
In a related study using a Xenopus oocyte expression system, all the com-
pounds inhibited hCNT1-mediated uptake of uridine, two were shown to
be potent inhibitors of hCNT1 with negligible permeant activity, and some
were shown to be permeants for hCNT3 12. (Note that the bicyclic rings
were numbered incorrectly in ref.12, in which N3 was erroneously num-
bered N1 as in the pyrimidine nucleoside precursors.)
RESULTS AND DISCUSSION
Chemistry
Heating a mixture of 2′,3′,5′-tri-O-benzoyl-5-iodouridine13 (1; Scheme 1),
propyne, triethylamine, tetrakis(triphenylphosphine)palladium, and cop-
per(I) iodide in dichloromethane (50 °C, sealed flask) for 3.5 h resulted
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1398
Nowak, Damaraju, Cass, Young, Robins:
in copious precipitation of furopyrimidine derivative 2a (the usual1 5-
(propyn-1-yl) Sonogashira coupling product was not observed). The highly
fluorescent 2a also was produced by similar treatment of 1 for 75 min, and
no intermediate was detected by TLC. Apparently, cuprous-ion catalyzed
cyclization of the 5-alkynyl intermediate occurred rapidly under these reac-
tion conditions. We previously observed14 that the rates of such cycli-
zations were much slower in DMF/Et3N mixtures. The limited solubility of
2a in CH2Cl2/Et3N might also shift the equilibrium from a cuprous-alkyne
complex toward 2a.
The fused furan ring in 2a is sensitive to opening with bases. De-
protective cleavage of O-acetyl groups had been effected by brief exposure
to cold methanolic ammonia6,15, but removal of the more stable O-benzoyl
groups from 2a was incomplete under conditions that resulted in attack on
the ring system. As expected16, heating 2a in NH3/MeOH produced
6-methyl-3-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2(3H,7H)-one (3).
Our weakly basic procedure with lithium trifluoroethoxide in trifluoro-
ethanol17 was the only one of several attempted deprotection methods that
gave 6-methyl-3-(β-D-ribofuranosyl)furo[2,3-d]pyrimidin-2(3H)-one (2b)
without significant byproducts.
The thiophene analogue 7b was then targeted. Subjection of 1 to the
Divakar–Reese procedure18 gave the 4-(triazol-1-yl) intermediate 4. How-
ever, attempted Sonogashira coupling of 4 and propyne resulted in
reductive deiodination with nearly quantitative conversion to 1-(2,3,5-tri-
O-benzoyl-β-D-ribofuranosyl)-4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (4a).
The π-deficient triazole ring was displaced from 4 using methanol super-
heated at 110 °C, which gave 5 with an electron-donating 4-methoxyl
group. Intermediate 5 was a good substrate for Sonogashira coupling with
propyne, and the product was subjected to acidic hydrolysis to give the
5-(propyn-1-yl)uracil derivative 6. Conversion of 6 into its 4-triazolyl deriv-
ative followed by treatment with thioacetic acid19 gave the benzoyl-
protected thiophene compound 7a, which was much more stable under ba-
sic conditions than its furan analogue 2a. The benzoyl esters were cleaved
with dilute methanolic potassium hydroxide to give 6-methyl-3-(β-D-ribo-
furanosyl)thieno[2,3-d]pyrimidin-2(3H)-one (7b). A similar sequence was
used for preparation of the 2′-deoxy analogue 10. The propynyl derivative
8 20 underwent smooth conversion into its 5-(propyn-1-yl)-4-(1,2,4-triazol-
1-yl) intermediate, which was treated directly with potassium thioacetate in
DMF. Nucleophilic displacement of the triazolyl group by sulfur, loss of the
acetyl group, and ring closure occurred in situ. Direct treatment of the re-
sulting thiophene derivative 9 with methanolic potassium hydroxide
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1399
cleaved the 4-methylbenzoyl esters to give 3-(2-deoxy-β-D-erythro-pento-
furanosyl)-6-methylthieno[2,3-d]pyrimidin-2(3H)-one (10).
SCHEME 1
Reagents and conditions: (a) (Ph3P)4Pd/CuI/Et3N/propyne/CH2Cl2/50 °C; (b) CF3CH2OLi/
TFE/80 °C; (c) NH3/MeOH/80 °C; (d) POCl3/1,2,4-triazole/Et3N/CH3CN; (e) MeOH/110 °C;
(f) TFA/H2O; (g) AcSH/CH3CN; (h) (i) KOH/MeOH, (ii) AcOH; (i) AcSK/DMF
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1400
Nowak, Damaraju, Cass, Young, Robins:
Analogues without a 6-methyl substituent were significantly less stable.
Sonogashira coupling of trimethylsilylacetylene and 1 followed by fluoride-
promoted removal of the TMS group and copper(I) iodide catalyzed
cyclization produced the furopyrimidine derivative 11a (Scheme 2). Mild
solvolysis of 11a with trifluoroethanol17 gave 3-(β-D-ribofuranosyl)furo-
[2,3-d]pyrimidin-2(3H)-one (11b), and methanolic ammonia at 80 °C con-
verted 11a into 3-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2(3H,7H)-one
(12). The unsubstituted thieno analogue 14 was not isolated in pure form.
The Sonogashira coupling intermediate from 1 was converted into the
4-(1,2,4-triazol-1-yl)-5-TMSethynyl derivative 13 and subjected to tandem
in situ substitution of triazole with thioacetate, fluoride-promoted desilyl-
ation, and cyclization. That sequence was the most successful of several at-
tempts, but gave a mixture containing two major products (TLC). Partial
separation followed by mild solvolysis gave 14 as the presumed major (un-
1
stable) product with H NMR signals similar to those of the other bicyclic
analogues. The geminal H5′/H5′′ signals in spectra of 2b, 3, 7b, 11b, 12,
and 14 are separated by ∆δ 0.16–0.19 ppm, whereas those signals in spectra
of their 5-(alkyn-1-yl)uracil nucleoside precursors have narrow separations.
SCHEME 2
Reagents and conditions: (a) (Ph3P)4Pd/CuI/Et3N/TMS-acetylene/CH2Cl2/50 °C; (b) NH4F/
THF/MeOH/H2O/50 °C; (c) CuI/Et3N/DMF/80 °C; (d) CF3CH2OLi/TFE/90 °C; (e) NH3/MeOH/
80 °C; (f) POCl3/1,2,4-triazole/Et3N/CH3CN; (g) AcSK/NH4F/H2O/pyridine; (h) Dowex 1 X2
(–OCH2CF3)/TFE/90 °C
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1401
Vicinal proton signals for the –CH=CH–X system in spectra of [14] (X = S),
3
3JHH = 5.9 Hz (0.26 ppm signal separation), 12 (X = N), JHH = 3.9 Hz
3
(0.75 ppm signal separation), and 11b (X = O), JHH = 2.9 Hz (0.95 ppm
signal separation), show effects of the relative S, N, and O electronegati-
vities on values of the coupling constants (decreasing) and signal separa-
tions (increasing).
The imidazo-pyrimidine analogue 17 21 was prepared by a slight modifi-
cation of a published procedure (Scheme 3). Heating 5-aminocytidine22
(15) in diethoxymethyl acetate was reported21a to give 17. However, the
solubility of 15 is very limited in diethoxymethyl acetate, and mixtures
were obtained. Protection of 15 with tert-butyldimethylsilyl chloride in
pyridine gave the TBDMS ether 16. Heating 16 in diethoxymethyl acetate
at 145 °C and successive trifluoroacetic acid-catalyzed desilylation, neutral-
ization, and chromatography on silica gel and then on Dowex 1 X2 (OH–)
resin gave 3-(β-D-ribofuranosyl)imidazo[4,5-d]pyrimidin-2(3H,5/7H)-one (17).
SCHEME 3
Reagents and conditions: (a) TBDMSCl/pyridine; (b) (EtO)2CHOAc/145 °C; (c) TFA/H2O/0 °C;
(d) NH3/MeOH
Nucleoside Transport
Concentration-dependent inhibition of transporter-mediated uptake of
[3H]uridine by the compounds in Fig. 1 was examined using recombinant
hENTs produced in yeast as described in Experimental. Values for 50% in-
hibitory concentrations (IC50) with the seven analogues for hENT1 and
hENT2 are listed in Table I along with previously reported12 IC50 values for
hCNT1, hCNT2, and hCNT3 (which are included for completeness). All of
the compounds were potent inhibitors of hCNT1 with IC50 values less than
1 µM, whereas only one had an IC50 value less than 1 µM for hCNT3 and
none were effective inhibitors of hCNT2 or of the equilibrative transporters
hENT1 and hENT2 12
.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1402
Nowak, Damaraju, Cass, Young, Robins:
Live-cell imaging of uptake of the analogues into BeWo and CEM cells
was evaluated using confocal microscopy at an excitation wavelength of
351 nm. The extracellular medium emitted green fluorescence instantly
upon addition of an analogue to the well, and the fluorescence intensity of
the medium changed but slightly during the observation time. Figure 2a il-
lustrates that cells had increased fluorescence intensities after exposure to
the analogues. Significant intracellular fluorescence enhancement was ob-
served within 1 min, and the fluorescence intensities continued to increase
over a period of 10 min (data not shown). Data presented are fluorescence
intensities after subtracting values in the presence of NBMPR for each of
the analogues. Longer time-courses exhibited two-phase accumulation of
intracellular fluorescence with rapid initial rates of uptake during the first
phase (data not shown). The almost complete inhibition of uptake of the
analogues by NBMPR (10 µM) indicated that transport was mediated pri-
marily by hENT1. Similar results with CEM/hENT1 cells were observed with
compounds 10 (Fig. 2b) and 7b (Fig. 2c).
Because 7b and 10 were shown12 to be inhibitors of hCNT1 with negligi-
ble transport activity, we analyzed uptake of 7b (50 µM) using confocal
microscopy with a panel of CEM cell lines (which exhibited some auto-
fluorescence). Uptake was measured with CEM/hENT1 cells (in the absence
or presence of NBMPR), with nucleoside-transport deficient CEM/ARAC
cells, and with CEM/hCNT1 cells (Fig. 3a–3d). CEM/hENT1 cells exhibited
uptake of 7b (Fig. 3a) as shown by the enhanced green fluorescence inside
the cells, and NBMPR blocked that uptake (Fig. 3b). CEM/ARAC cells
(Fig. 3c) and CEM/hCNT1 cells (Fig. 3d) showed no uptake, which demon-
strated that hENT1 was essential for the transport of 7b and that 7b did not
enter cells via hCNT1.
In summary, our current and recently reported12 studies with yeast pro-
ducing recombinant hNTs revealed that all seven compounds in Fig. 1 were
high-affinity inhibitors of hCNT1, moderate-affinity inhibitors of hCNT3,
and low-affinity inhibitors of hENT1, hENT2, and hCNT2. Laser scanning
confocal microscopy allowed direct visualization of autofluorescence of the
analogues in living BeWo and CEM/hENT1 cells (including blockage of up-
take by the potent hENT1 inhibitor NBMPR). The results indicated that
transport of 7b was mediated predominantly by hENT1 (Fig. 3a and 3b).
Live-cell imaging was undertaken with a panel of CEM cell lines that are
deficient in nucleoside transporter activity or that have either hENT1 or
hCNT1 as the sole activity. The absence of uptake of the autofluorescent
nucleoside analogues in transport-deficient cells compared with their trans-
portability in hENT1-containing cells suggests that they could be used for
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1403
FIG. 2
Time course of uptake of autofluorescent analogues by cultured cells: Compounds 11b, 2b, 10,
7b, 12, and 3 were added individually to BeWo cells and representative time courses of uptake
of the autofluorescent analogues are shown (A). The whole-cell fluorescence intensities of the
analogues and the extracellular fluorescence intensities at different time points were measured
with six to eight cells and one extracellular region (similar in size to a cell). The extracellular
fluorescence values were subtracted, and the resulting mean values SE after correcting for
values in the presence of NBMPR were plotted against time (compound numbers are shown
beside the curves). Analogous experiments were performed with 10 (B) and 7b (C) in CEM/hENT1
cells. Time-dependent increases in fluorescence were observed with both compounds
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1404
Nowak, Damaraju, Cass, Young, Robins:
TABLE I
Effects of autofluorescent nucleoside analogues on the uptake of uridine into S. cerevisiae
producing recombinant hENT1, hENT2, hCNT1, hCNT2, or hCNT3
hENT1a
hENT2a
hCNT1b
hCNT2b
hCNT3b
Compound
IC50 SE, µM
FuPmR (11b)
MeFuPmR (2b)
dMeThPmR (10)
MeThPmR (7b)
PrPmR (12)
161 42
424 31
1757 280
714 18
1036 53
526 25
>3000
0.12 0.01
0.37 0.08
0.33 0.08
0.18 0.03
0.39 0.03
0.26 0.05
0.19 0.07
393 42
1419 246
133 11
360 24
239 29
175 43
270 22
5.3 1.2
25.0 1.2
52.0 7.6
7.5 0.1
1.2 0.6
0.7 0.2
1.3 0.1
33
2
79 12
101 14
MePrPmR (3)
ImPmR (17)
313 24
1235 42
201 21
1450 270
a
IC50 values ( SE) for inhibition of initial rates of uptake (i.e., transport) of [3H]uridine
(1 µM) were determined by computer-generated concentration-effect curves. Each experiment
b
was conducted with six replicates. We recently reported these data12 for hCNT1, hCNT2,
and hCNT3 (obtained by methods similar to those presently described for the hENTs).
FIG. 3
Live-cell imaging to measure uptake of 7b by cultured cells with different transport capabili-
ties: Uptake of compound 7b into CEM/hENT1 cells occurred in the absence of NBMPR (a).
Uptake of 7b into CEM/hENT1 cells was blocked in the presence of NBMPR (10 µM) (b). Uptake
of 7b into CEM/ARAC (c) and CEM/hCNT1 (d) cells was not observed. Images were taken 300 s
after the addition of 7b (500 µM) to cells as described in Experimental
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1405
rapid assessment of hENT1 abundance in cell samples. The lack of uptake in
hCNT1-viable cells showed that 7b was not transported by hCNT1.
Our results clearly demonstrate that selected fused-pyrimidine nucleo-
sides have a number of potential applications. The autofluorescence of such
analogues allows measurement of uptake in live cells as shown earlier6, and
presently with six of the seven analogues. Compounds 7b and 10 also are
inhibitors of hCNT1 as well as permeants of hCNT3 12. Therefore, they have
potential for analysis of nucleoside uptake by hCNT3.
EXPERIMENTAL
Chemistry
All chemicals and solvents were of reagent grade and were used as received from suppliers
unless specified. Solutions in CDCl3 were used for 1H (500 MHz) and 13C (125 MHz) NMR
spectra unless noted. Chemical shifts are given in ppm (δ-scale), coupling constants (J) in
Hz. 13C NMR peaks with the same chemical shift for more than one carbon are specified,
and a shift range (ovlp) is given for overlapping signals from multiple carbon atoms. Solu-
tions in methanol were used for ultraviolet (UV) absorption and emission spectra. Purities
(>95%) of final products (except 14) conformed to our reported criteria23 for evaluation of
impurities in nucleoside samples.
3-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-6-methylfuro[2,3-d]pyrimidin-2(3H)-one (2a)
A 30-ml pressure flask equipped with a Teflon valve was charged with 2′,3′,5′-tri-O-benzoyl-
5-iodouridine13 (1; 2.80 g, 4.11 mmol) and CH2Cl2 (10 ml) and Et3N (5 ml) were added. N2
was bubbled through the solution for 15 min, the solution was cooled to –20 °C, and
propyne gas (~2 l balloon) was bubbled through. (Ph3P)4Pd (160 mg, 0.14 mmol) and CuI
(60 mg, 0.32 mmol) were added and the mixture was sealed and stirred at 50 °C for 1.5 h
during which time copious precipitation of crystalline material occurred. The mixture was
filtered and the filter cake was washed with CH2Cl2 to dissolve the precipitated product.
Volatiles were evaporated from the filtrate and the residue was chromatographed (CH2Cl2).
Crystallization of the purified residue (CH2Cl2/MeOH) gave 2a (2.17 g, 89%). 1H NMR: 2.31
(s, 3 H), 4.69 (dd, J = 2.9, 12.2, 1 H), 4.81–4.84 (m, 1 H), 4.95 (dd, J = 2.4, 12.2, 1 H), 5.69
(s, 1 H), 5.83 (t, J = 4.9, 1 H), 5.92 (t, J = 5.9, 1 H), 6.61 (d, J = 4.4, 1 H), 7.35–7.66 (m, 9 H),
7.92–8.13 (m, 6 H), 8.06 (s, 1 H). 13C NMR: 14.1, 63.3, 70.5, 75.1, 80.5, 90.0, 99.3, 108.8,
128.4–129.9 (ovlp), 133.6, 133.65, 133.69, 133.8, 154.5, 156.6, 165.1, 165.3, 166.0, 172.3.
FAB-MS m/z: 617 ([M + Na+] 10%), 445 (100%). HRMS (C33H26N2NaO9): calculated 617.1531,
found 617.1527.
6-Methyl-3-(β-D-ribofuranosyl)furo[2,3-d]pyrimidin-2(3H)-one (2b)
A 250-ml pressure flask equipped with a Teflon valve was charged with 2a (4.0 g,
6.73 mmol) and a solution of LiOCH2CF3/CF3CH2OH prepared by addition of BuLi (1.6 M,
8.0 ml, 12.8 mmol) to TFE (40 ml). The solution was stirred at 80 °C for 19 h, neutralized
with AcOH, and concentrated. Chromatography (EtOAc → EtOAc/MeOH, 3:1) gave purified
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1406
Nowak, Damaraju, Cass, Young, Robins:
2b (1.82 g, 96%). A sample for analysis was obtained by PTLC [EtOAc/MeOH (4:1) followed
by CH2Cl2/MeOH (3:1)]. 1H NMR (CD3OD): 2.37 (q, J = 1.5, 3 H), 3.84, 4.01 (2 × dd, J = 2.4,
12.2, 2 × 1 H), 4.11–4.20 (m, 3 H), 5.96 (d, J = 1.5, 1 H), 6.36 (d, J = 1.5, 1 H), 8.77 (s, 1 H).
13C NMR (CD3OD): 13.9, 61.1, 69.7, 77.0, 85.9, 94.1, 101.3, 109.9, 138.5, 157.2, 157.7,
173.3. FAB-MS m/z: 283 ([M + H+] 100%). HRMS (C12H15N2O6): calculated 283.0925, found
283.0924.
6-Methyl-3-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2(3H,7H)-one (3)
A solution of 2b (300 mg, 1.06 mmol) in NH3/MeOH (~14%, 15 ml) was heated in a sealed
flask at 80 °C for 24 h. Volatiles were evaporated and the residue was chromatographed
(EtOAc/MeOH, 3:1) to give 3 (280 mg, 94%). A sample for analysis was obtained by PTLC
[EtOAc/MeOH (3:1) followed by CH2Cl2/MeOH (3:1)]. 1H NMR (CD3OD): 2.29 (s, 3 H), 3.83,
4.00 (2 × dd, J = 2.4, 12.2, 2 × 1 H), 4.10–4.20 (m, 3 H), 5.98 (d, J = 0.9, 1 H), 6.02 (d, J =
2.4, 1 H), 8.77 (s, 1 H). 13C NMR (CD3OD): 13.7, 61.5, 70.0, 77.1, 85.9, 93.9, 99.0, 122.3,
136.4, 140.6, 157.3, 160.3. FAB-MS m/z: 282 ([M
+
H+] 50%), 301 (100%). HRMS
(C12H16N3O5): calculated 282.1085, found 282.1091.
1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-5-iodo-4-methoxypyrimidin-2(1H)-one (5)
Et3N (2.0 ml, 1.45 g, 14.4 mmol) was added dropwise to a stirred, cooled (~0 °C) mixture of
1,2,4-triazole (1.01 g, 14.7 mmol), POCl3 (0.31 ml, 510 mg, 3.32 mmol), and MeCN (9 ml).
A solution of 1 (1.00 g, 1.47 mmol) in CH2Cl2 (10 ml) was added and stirring was continued
at ambient temperature for 30 min. Volatiles were evaporated and the residue was
chromatographed (EtOAc → EtOAc/MeOH, 10:1) to give 1-(2,3,5-tri-O-benzoyl-β-D-ribo-
furanosyl)-5-iodo-4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (4; 820 mg, 76%). Sonogashira
treatment of 4 with propyne resulted in deiodination to give 1-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (4a; 89%). 1H NMR: 4.73–4.92
(m, 3 H), 5.84 (t, J = 5.1, 1 H), 5.92 (t, J = 5.4, 1 H), 6.50 (d, J = 4.9, 1 H), 6.97 (d, J = 7.3,
1 H), 7.37–7.69 (m, 9 H), 7.98 (d, J = 7.3, 2 H), 8.10, 8.19 (2 × d, J = 7.3, 2 × 1 H), 8.12, 9.25
(2 × s, 2 × 1 H). 13C NMR: 63.4, 70.8, 74.7, 80.8, 90.2, 95.2, 128.2–129.7 (ovlp), 131.75,
131.81, 131.9, 133.5–133.6 (ovlp), 143.2, 146.5, 153.86, 153.95, 159.4, 165.0, 165.1, 165.8.
A solution of 4 (1.6 g, 2.18 mmol) in MeOH (15 ml) was heated in a pressure flask at 110 °C
for 4 h. Volatiles were evaporated and the residue was chromatographed (EtOAc/hexanes,
1:2 → 1:1) to give 1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-iodo-4-methoxypyrimidin-
2(1H)-one (5; 1.23 g, 81%). 1H NMR: 4.00 (s, 3 H), 4.71–4.88 (m, 3 H), 5.72 (t, J = 5.9, 1 H),
5.91 (dd, J = 3.9, 5.9, 1 H), 6.52 (t, J = 3.9, 1 H), 7.35–7.63 (m, 9 H), 7.96–7.98 (m, 4 H), 7.99
(s, 1 H), 4.12–8.14 (m, 2 H). 13C NMR: 56.0, 57.6, 63.8, 71.3, 74.7, 81.0, 88.4, 128.3–129.9
(ovlp), 133.5–133.6 (ovlp), 147.5, 154.4, 165.2, 165.3, 166.0, 168.5. FAB-MS m/z: 719 ([M +
Na+] 20%), 445 (100%). HRMS (C31H24IN2NaO9): calculated 719.0497, found 719.0501.
2′,3′,5′-Tri-O-benzoyl-5-(propyn-1-yl)uridine (6)
Sonogashira treatment of 5 (500 mg, 0.72 mmol) with propyne as described for 1 → 2a,
evaporation of volatiles, and chromatography of the residue (EtOAc/hexanes, 1:2 → 1:1)
gave 1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-4-methoxy-5-(propyn-1-yl)pyrimidin-2(1H)-one
(400 mg, 92%). This material was treated directly with TFA/H2O (2:1, 3 ml), the mixture was
stirred at ambient temperature for 4 h, and volatiles were evaporated. The residue was
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1407
chromatographed (EtOAc/hexanes, 1:2 → 1:1) to give 6 (350 mg, 90%). 1H NMR: 1.85 (s,
3 H), 4.70–4.81 (m, 3 H), 5.75 (t, J = 6.1, 1 H), 5.88 (dd, J = 3.9, 5.9, 1 H), 6.37 (d, J = 5.9,
1 H), 7.35–7.64 (m, 9 H), 7.65 (s, 1 H), 7.92–8.14 (m, 6 H), 8.26 (br s, 1 H). 13C NMR: 4.4,
63.9, 69.6, 71.2, 73.8, 80.6, 87.6, 91.1, 102.0, 128.1–129.8 (ovlp), 133.3, 133.6, 133.7, 141.0,
149.2, 161.6, 165.1, 165.2, 166.0. FAB-MS m/z: 595 ([M + H+] 10%), 445 (100%). HRMS
(C33H27N2O9): calculated 595.1711, found 595.1710.
3-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-6-methylthieno[2,3-d]pyrimidin-2(3H)-one (7a)
Et3N (3.0 ml, 2.18 g, 21.6 mmol) was added dropwise to a stirred, cooled (~0 °C) mixture of
1,2,4-triazole (1.62 g, 23.48 mmol), POCl3 (0.50 ml, 820 mg, 5.36 mmol) and MeCN (14 ml).
A solution of 6 (1.40 g, 2.35 mmol) in MeCN (14 ml) was added to this mixture, and stirring
was continued at ambient temperature for 2 h. Volatiles were evaporated and the residue
was chromatographed (EtOAc/hexanes, 1:1
→ EtOAc) to give 1-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-5-(propyn-1-yl)-4-(1,2,4-triazol-1-yl)pyrimidin-2(3H)-one (1.48 g, 97%). A
solution of this material (1.4 g, 2.16 mmol) and thioacetic acid (1.0 ml, 1.07 g, 14.0 mmol)
was stirred at ambient temperature for 3 h. Volatiles were evaporated and the residue was
chromatographed (CH2Cl2 → EtOAc) to give 7a (1.1 g, 83%). 1H NMR: 2.35 (s, 3 H), 4.68
(dd, J = 3.4, 12.7, 1 H), 4.83–4.86 (m, 1 H), 5.00 (dd, J = 2.9, 12.7, 1 H), 5.86 (dd, J = 4.4,
5.4, 1 H), 5.91 (d, J = 1.5, 1 H), 5.93 (t, J = 5.9, 1 H), 6.60 (d, J = 4.4, 1 H), 7.35–7.66 (m,
9 H), 7.91–8.14 (m, 6 H), 8.19 (s, 1 H). FAB-MS m/z: 633 ([M + Na+] 10%), 445 (100%).
HRMS (C33H26N2NaO8S): calculated 633.1302, found 633.1291.
6-Methyl-3-(β-D-ribofuranosyl)thieno[2,3-d]pyrimidin-2(3H)-one (7b)
A suspension of 7a (600 mg, 0.98 mmol) in a solution of KOH (250 mg, 4.46 mmol) in
MeOH (50 ml) was stirred at 60 °C for 1 h. The clear solution was then neutralized (AcOH),
volatiles were evaporated, and the residue was chromatographed (EtOAc → EtOAc/MeOH,
3:1) to give 7b (200 mg, 68%) that was recrystallized (MeOH) to give purified 7b. 1H NMR
(DMSO-d6): 2.41 (d, J = 1.0, 3 H), 3.65, 3.84 (2 × ddd, J = 2.0, 4.9, 12.2, 2 × 1 H), 3.94–4.02
(m, 3 H), 5.03 (d, J = 5.9, 1 H), 5.31 (t, J = 5.1, 1 H), 5.64 (d, J = 4.9, 1 H), 5.80 (d, J = 2.0,
1 H), 6.74 (q, J = 1.5, 1 H), 8.98 (s, 1 H). 13C NMR (DMSO-d6): 16.1, 59.3, 67.8, 74.7, 84.1,
91.6, 117.0, 118.4, 136.4, 138.4, 151.9, 178.7. FAB-MS m/z: 299 ([M + H+] 30%), 141 (100%).
HRMS (C12H15N2O5S): calculated 299.0696, found 299.0709.
3-(2-Deoxy-β-D-erythro-pentofuranosyl)-6-methylthieno[2,3-d]pyrimidin-2(3H)-one (10)
The procedure described above with 6 was applied to 1-[2-deoxy-3,5-di-O-(4-methyl -
benzoyl)-β-D-erythro-pentofuranosyl]-5-(propyn-1-yl)uracil20 (8; 500 mg, 1.0 mmol) to give
1-[2-deoxy-3,5-di-O-(4-methylbenzoyl)-β-D-erythro-pentofuranosyl]-5-(propyn-1-yl)-4-(1,2,4-
triazol-1-yl)pyrimidin-2(1H)-one, which was stirred with potassium thioacetate (180 mg,
1.58 mmol) in DMF (15 ml) at ambient temperature for 1 h. Volatiles were evaporated and
the residue was chromatographed (CH2Cl2) to give 3-[2-deoxy-di-O-(4-methylbenzoyl)-
β-D-erythro-pentofuranosyl]-6-methylthieno[2,3-d]pyrimidin-2(3H)-one (9; 470 mg, 91% for
two steps), a suspension of which in a solution of KOH (130 mg, 2.32 mmol) in MeOH
(15 ml) was stirred for 2 h. The solution was neutralized (AcOH), volatiles were evaporated,
and the residue was chromatographed (EtOAc → EtOAc/MeOH, 3:1) to give 10 (210 mg,
82%). 1H NMR (DMSO-d6): 2.07 (td, J = 5.9, 13.2, 1 H), 2.41 (d, J = 1.0, 3 H), 2.42 (ddd, J =
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1408
Nowak, Damaraju, Cass, Young, Robins:
4.4, 6.3, 13.2, 1 H), 3.59–3.71 (m, 2 H), 3.92–3.94 (m, 1 H), 4.20–4.24 (m, 1 H), 5.14 (t, J =
5.1, 1 H), 5.30 (d, J = 3.9, 1 H), 6.11 (t, J = 5.9, 1 H), 6.78 (q, J = 1.5, 1 H), 8.83 (s, 1 H).
13C NMR (DMSO-d6): 16.1, 41.2, 60.6, 69.5, 87.6, 88.3, 117.1, 118.4, 136.1, 138.0, 151.7,
178.5. FAB-MS m/z: 305 ([M + Na+] 30%), 587 ([2 M + Na+] 100%). HRMS (C12H14N2NaO4S):
calculated 305.0567, found 305.0570. UV (absorption) max 274 (ε 5200), 358 (ε 2800),
367 nm (ε 2700); min 303 nm (ε 200). UV/VIS (emission) max 390, 400, 461, 496 nm.
3-(β-D-Ribofuranosyl)furo[2,3-d]pyrimidin-2(3H)-one (11b)
(Ph3P)4Pd (120 mg, 0.10 mmol) and CuI (45 mg, 0.24 mmol) were added to a solution of 1
(2.1 g, 3.1 mmol) and TMS–acetylene (0.6 ml) in Et3N (3.0 ml) and CH2Cl2 (10 ml), and the
mixture was stirred at 50 °C for 2 h, during which time copious quantities of crystalline
material precipitated. The mixture was filtered and the filter cake was washed with CH2Cl2
to dissolve the precipitated product. Volatiles were evaporated from the filtrate and the resi-
due was chromatographed (CH2Cl2) to give 2′,3′,5′-tri-O-benzoyl-5-(trimethylsilylethynyl)-
uridine (2.0 g, 99%). A solution of that material (5.2 g, 8.0 mmol) and NH4F (300 mg,
8.1 mmol) in THF (100 ml), MeOH (820 ml), and H2O (10 ml) was stirred at 50 °C for
30 min. Volatiles were evaporated and the residue was chromatographed (CH2Cl2/hexanes,
1:2 → CH2Cl2). Crystallization of the purified residue (EtOAc/hexanes) gave 2′,3′,5′-tri-
O-benzoyl-5-ethynyluridine (1.59 g, 34%), which was stirred with CuI (520 mg, 2.74 mmol)
in Et3N (7 ml) and DMF (16 ml) at 80 °C for 12 h. Volatiles were evaporated, CH2Cl2 was
added, and the mixture was filtered using a short column of silica. Volatiles were evapo-
rated, EtOAc was added, and the solution was filtered through silica. Evaporation of volatiles
gave clean (TLC) 11a 17 (1.0 g, 63%).
A 250-ml pressure flask equipped with a Teflon valve was charged with 11a (1.0 g,
1.72 mmol) and a LiOCH2CF3/CF3CH2OH solution prepared by addition of BuLi (1.6 M,
2.0 ml, 3.2 mmol) to TFE (10 ml). The solution was stirred at 90 °C overnight, neutralized
(AcOH), concentrated, and chromatographed (EtOAc → EtOAc/MeOH, 4:1) to give 11b
(340 mg, 74%). Successive PTLC with EtOAc/MeOH (4:1) and CH2Cl2/MeOH (3:1) gave TLC-
homogeneous 11b. 1H NMR (DMSO-d6): 3.66, 3.82 (2 × d, J = 12.4, 2 × 1 H), 3.94–4.02 (m,
3 H), 5.00–5.80 (br s, 3 H), 5.85 (d, J = 2.0, 1 H), 6.81 (d, J = 2.9, 1 H), 7.76 (d, J = 2.9, 1 H),
9.01 (s, 1 H). 13C NMR (DMSO-d6): 69.0, 77.4, 84.6, 93.8, 101.3, 114.4, 114.9, 149.1, 154.6,
163.7, 181.0. EI-MS m/z: 268 ([M+] 5%), 136 (100%). HRMS (C11H12N2O6): calculated
268.0695, found 268.0701. UV (absorption) max 328 nm (ε 2200); min 267 nm (ε 400).
UV/VIS (emission) max 400 nm.
3-(β-D-Ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2(3H,7H)-one (12)
A solution of 11a (200 mg, 0.35 mmol) in NH3/MeOH (~14%, 10 ml) was heated in a sealed
pressure flask at 80 °C for 3 h. Volatiles were evaporated and the residue was chrom-
atographed (EtOAc/MeOH, 3:1) to give 12 (64 mg, 69%). PTLC (EtOAc/MeOH, 3:1) provided
a sample of 12 for analysis. 1H NMR (CD3OD): 3.85, 4.02 (2 × dd, J = 2.4, 12.7, 2 × 1 H),
4.12–4.21 (m, 3 H), 6.02 (d, J = 2.0, 1 H), 6.35 (d, J = 3.9, 1 H), 7.10 (d, J = 3.9, 1 H), 9.04 (s,
1 H). 13C NMR (CD3OD): 61.4, 69.9, 77.2, 85.9, 94.1, 102.5, 111.1, 128.8, 139.1, 157.2,
159.5. EI-MS m/z: 267 ([M+] 5%), 135 (100%). HRMS (C11H13N3O5): calculated 267.0855,
found 267.0847. UV (absorption) max 273 (ε 3600), 340 nm (ε 2800); min 248 (ε 2000),
292 nm (ε 800). UV/VIS (emission) max 424 nm.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1409
3-(β-D-Ribofuranosyl)thieno[2,3-d]pyrimidin-2(3H)-one (14)
Coupling of 1 and TMS–acetylene (described in the first step of 1 to 11a) was followed by
conversion of 2′,3′,5′-tri-O-benzoyl-5-(trimethylsilylethynyl)uridine (3.0 g, 4.6 mmol) into
1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-(trimethylsilylethynyl)-4-(1,2,4-triazol-1-yl)pyri-
midin-2(3H)-one (13) (as described for the first step in the conversion of 6 to 7a). The crude
material was chromatographed (EtOAc/hexanes, 1:2 → EtOAc) and recrystallized from
EtOAc/hexanes to give 13 (3.98 g, 92%). A solution of 13 (900 mg, 1.28 mmol), NH4F
(300 mg, 8.11 mmol), and KSAc (600 mg, 5.26 mmol) in pyridine (14 ml) and H2O (1.5 ml)
was stirred at ambient temperature for 40 min. Volatiles were evaporated and the residue
was chromatographed (EtOAc/hexanes, 1:1 → 3:1) to give a mixture (3:2, 465 mg) of the
presumed fused-thieno intermediate and an unidentified product. Solvolysis of the benzoyl
esters with Dowex 1 X2 (OH–) in trifluoroethanol at 80 °C for 2 h and PTLC (EtOAc →
EtOAc/MeOH, 4:1) gave 14 as an unstable material. 1H NMR (DMSO-d6): 3.66, 3.84 (2 × d,
J = 12, 2 × 1 H), 3.92–4.04 (m, 3 H), 5.20–5.60 (br s, 3 H), 5.81 (d, J = 2.0, 1 H), 7.06 (d, J =
5.9, 1 H), 7.32 (d, J = 5.9, 1 H), 9.19 (s, 1 H).
3-(β-D-Ribofuranosyl)imidazo[4,5-d]pyrimidin-2(3H,5/7H)-one
[1-(β-D-Ribofuranosyl)purin-2(1H,7/9H)-one] (17)
A solution of 5-aminocytidine22 15 (200 mg, 0.78 mmol) and TBDMSCl (140 mg, 0.93 mmol)
in dried pyridine (2 ml) was stirred at ambient temperature overnight. Volatiles were evapo-
rated and the residue was chromatographed (EtOAc → EtOAc/MeOH, 3:1) to give 5′-O-
TBDMS-5-aminocytidine 16 (260 mg, 90%). A solution of 16 (200 mg, 0.70 mmol) in
(EtO)2CHOAc (5 ml) was heated at 145 °C for 1.5 h and volatiles were evaporated. The resi-
due was chromatographed (EtOAc → EtOAc/MeOH, 10:1) and the resulting blue fluorescent
residue (100 mg) was stirred at 0 °C for 1 h with TFA/H2O (9:1, 1 ml). Volatiles were evapo-
rated, the residue was dissolved in NH3/MeOH (~14%, 10 ml), and the solution was stirred
at ambient temperature for 1 h. Volatiles were evaporated and the residue was chrom-
atographed (MeOH → H2O). The UV-active fractions were pooled, concentrated, and applied
to a column of Dowex 1 X2 (OH–) resin in H2O. Elution (H2O → MeOH → MeOH/AcOH,
3:1) and evaporation of volatiles from the UV-active fractions gave 17 (46 mg, 32%).
1H NMR (DMSO-d6): 3.66, 3.83 (2 × d, J = 11.7, 2 × 1 H), 3.94–4.06 (m, 3 H), 5.01, 5.38, 5.55
(3 × br s, 3 × 1 H), 5.87, 8.19, 9.12 (3 × s, 3 × 1 H), 12.40 (br s, 1 H). 13C NMR (DMSO-d6):
59.3, 67.9, 75.0, 83.8, 91.4, 123.0, 135.9, 146.8, 154.2, 160.3. FAB-MS m/z: 291 ([M + Na+]
20%), 131 (100%). HRMS (C10H12N4NaO5): calculated 291.0700, found 291.0701. UV (ab-
sorption) max 322 nm (ε 4100); min 270 nm (ε 2100). UV/VIS (emission) max 390 nm.
Nucleoside Transport
Materials: [5,6-3H]Uridine (21.3 Ci/mmol) was purchased from Moravek Biochemicals
(Brea, CA). Tissue culture (96) plates, Roswell Park Memorial Institute (RPMI) 1640 cell cul-
ture media, and fetal bovine serum (FBS) were from Invitrogen (Burlington, Ontario). Ecolite
was from ICN Pharmaceuticals (Montreal, Quebec). Yeast nitrogen base, amino acids, and
glucose were from Difco (Detroit, MI). Filter mats for yeast studies were from Molecular
Devices (Sunnyvale, CA). Unless otherwise noted, NBMPR, (p-nitrobenzylmercaptopurine
riboside) [6-S-(4-nitrobenzyl)-6-thioinosine] and other chemicals were from Sigma–Aldrich
(Oakville, Ontario).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1410
Nowak, Damaraju, Cass, Young, Robins:
The hENT1-containing human CCRF-CEM leukemia cell line (referred to as CEM/hENT1)
was obtained from W. T. Beck (University of Illinois at Chicago). CEM/ARAC-8C, a nucleo-
side transport-deficient derivative of CCRF-CEM 24 (referred to as CEM/ARAC) was a gift
from Dr. B. Ullman (Oregon Health and Science University, Portland). The origin and char-
acterization of the hCNT1-containing cell line (referred to as CEM/hCNT1), which was pro-
duced by stable transfection of CEM/ARAC with a vector containing a cDNA encoding
hCNT1, are described elsewhere25. BeWo human choriocarcinoma cells were obtained from
the American Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640
medium supplemented with 10% FBS as suspension (CEM) or adherent (BeWo) cultures.
Cells were determined to be free of mycoplasma, maintained in the absence of antibiotics,
incubated at 37 °C in a humidified atmosphere (5% CO2), and subcultured at two- to four-
day intervals to maintain active proliferation.
Yeast strains were maintained in complete minimal medium (CMM) containing 0.67%
yeast nitrogen base (Difco, Detroit, MI), amino acids (as required to maintain auxotrophic
selection), and 2% glucose (CMM/GLU). Agar plates contained CMM with various supple-
ments and 2% agar (Difco). Plasmids were propagated in E. coli strain TOP10F (Invitrogen,
Carlsbad, CA) and maintained in Luria broth with 100 µg/ml ampicillin as described26
.
Methods: Measurement of [3H]uridine uptake mediated by recombinant hENTs produced
in Saccharomyces cerevisiae: Construction of the yeast expression systems for hENT1 and
hENT2 has been described earlier27a. Yeast producing individual recombinant hENTs were
maintained in logarithmic growth phase CMM/GLU.
Transport experiments were conducted with a high-throughput assay described previ-
ously26a with 96-well plates and a semi-automated cell harvester (Micro96 Harvester; Skatron
Instruments, Lier, Norway). Relative affinities of the transporters for the auto-fluorescent
nucleoside analogues were assessed by measuring the concentration dependence of their in-
hibition of uptake of [3H]uridine as follows. Yeast producing recombinant hENT1 or hENT2
were incubated with graded concentrations of the analogues in the presence of [3H]uridine
(1 µM) for 10 min. Each experiment was repeated at least three times. Nonspecifically associ-
ated radioactivity was determined in the presence of non-radioactive uridine (10 mM), and
the resulting values were subtracted from the total uptake values. Data were subjected to
nonlinear regression analysis using GraphPad Prism Software version 3.0 (GraphPad
Software Inc., San Diego, CA) to obtain IC50 values (inhibitor concentrations that produced
50% inhibition of uridine transport).
Live-cell imaging of the uptake of autofluorescent nucleoside analogues into BeWo and
CEM cells with confocal microscopy: Time-lapse microscopy was performed with a Zeiss
LSM510 Confocal Laser Scanning microscope (Carl Zeiss, Jena) mounted on an Axiovert
100M inverted microscope as described6. Coverslips with adhered BeWo cells (attached dur-
ing growth) or CEM cells (attached with poly-L-lysine and air dried) were washed three times
with phosphate-buffered saline (PBS, pH 7.4) and glued onto the edge of an open hole lo-
cated in the middle of a 2-µm thick metal slide to form a well. PBS alone or PBS containing
NBMPR (10 µM) was added to the well, which was maintained at ambient temperature for
5 min before the confocal measurements. The solution in the well was gently removed by
suction, and time-lapse image capturing was begun a few seconds before adding PBS (200 µl)
containing the individual analogue into the well. The live-cell imaging was continued for
10 min. Fluorescence intensities were measured by placing a circle around the stored digital
image of a targeted cell or intracellular part. Integrated intensities were exported to a linked
Microsoft Excel worksheet. Background fluorescence (defined as the fluorescence intensity of
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

Autofluorescent Nucleosides/Human Nucleoside Transporters
1411
a comparable measurement circle in a nearby extracellular region) and fluorescence in the
presence of NBMPR were determined and subtracted from each cellular measurement to cal-
culate specific uptake values.
We thank Brigham Young University and unrestricted gift funds from pharmaceutical companies
for financial support of the chemistry; the nucleoside transport work was funded by grants from the
Canadian Cancer Society Research Institute and the Canadian Institute of Health Research. We thank
the Cell Imaging Facility at the Cross Cancer Institute for help with the confocal microscopy studies,
and Gerry Barron, Delores Mowles, Tracey Tackaberry, and Marnie Wilson for expert technical
assistance.
REFERENCES
1. a) Robins M. J., Barr P. J.: Tetrahedron Lett. 1981, 22, 421; b) Robins M. J., Barr P. J.:
J. Org. Chem. 1983, 48, 1854.
2. Yu D., Putta M. R., Bhagat L., Li Y., Zhu F., Wang D., Tang J. X., Kandimalla E. R.,
Agrawal S.: J. Med. Chem. 2007, 50, 6411.
3. a) Liu C., Martin C. T.: J. Mol. Biol. 2001, 308, 465; b) Liu C., Martin C. T.: J. Biol. Chem.
2002, 277, 2725.
4. a) Srivatsan S. G., Weizman H., Tor Y.: Org. Biomol. Chem. 2008, 6, 1334; b) Sinkeldam
R. W., Greco N. J., Tor Y.: Chem. Rev. 2010, 110, 2579.
5. a) McGuigan C., Brancale A., Barucki H., Srinivasan S., Jones G., Pathirana R., Blewett S.,
Alvarez R., Yarnold C. J., Carangio A., Velazquez S., Andrei G., Snoeck R., De Clercq E.,
Balzarini J.: Drugs Future 2000, 25, 1151; b) De Clercq E.: Med. Res. Rev. 2003, 23, 253;
c) De Clercq E.: Annu. Rev. Pharmacol. Toxicol. 2011, 51, 1.
6. Zhang J., Sun X., Smith K. M., Visser F., Carpenter P., Barron G., Peng Y., Robins M. J.,
Baldwin S. A., Young J. D., Cass C. E.: Biochemistry 2006, 45, 1087.
7. Cai J., Damaraju V. L., Groulx N., Mowles D., Peng Y., Robins M. J., Cass C. E., Gros P.:
Cancer Res. 2008, 68, 2349.
8. Paproski R. J., Ng A. M. L., Yao S. Y. M., Graham K., Peng Y., Robins M. J., Young J. D.,
Cass C. E.: Mol. Pharmacol. 2008, 74, 1372.
9. a) Wiley J. S., Jones S. P., Sawyer W. H., Paterson A. R.: J. Clin. Invest. 1982, 69, 479;
b) Cass C. E., King K. M., Montano J. T., Janowska-Wieczorek A.: Cancer Res. 1992, 52,
5879; c) Gati W. P., Paterson A. R., Belch A. R., Chlumecky V., Larratt L. M., Mant M. J.,
Turner A. R.: Leuk. Lymphoma 1998, 32, 45; d) Mackey J. R., Baldwin S. A., Young J. D.,
Cass C. E.: Drug Resist. Updat. 1998, 1, 310; e) Gati W. P., Larratt L. M., Turner A. R.:
Proc. West. Pharmacol. Soc. 2001, 44, 93.
10. a) Spratlin J., Sangha R., Glubrecht D., Dabbagh L., Young J. D., Dumontet C., Cass C.,
Lai R., Mackey J. R.: Clin. Cancer Res. 2004, 10, 6956; b) Farrell J. J., Elsaleh H.,
Garcia M., Lai R., Ammar A., Regine W. F., Abrams R., Benson A. B., Macdonald J., Cass
C. E., Dicker A. P., Mackey J. R.: Gastroenterology 2009, 136, 187; c) Marechal R., Mackey
J. R., Lai R., Demetter P., Peeters M., Polus M., Cass C. E., Young J., Salmon I., Deviere J.,
Van Laethem J. L.: Clin. Cancer Res. 2009, 15, 2913.
11. Robins M. J., Peng Y., Damaraju V. L., Mowles D., Barron G., Tackaberry T., Young J. D.,
Cass C. E.: J. Med. Chem. 2010, 53, 6040.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412

1412
Nowak, Damaraju, Cass, Young, Robins:
12. Damaraju V. L., Smith K. M., Mowles D., Nowak I., Karpinski E., Young J. D., Robins
M. J., Cass C. E.: Biochem. Pharmacol. 2011, 81, 82.
13. Asakura J., Robins M. J.: Tetrahedron Lett. 1988, 29, 2855.
14. Robins M. J., Vinayak R. S., Wood S. G.: Tetrahedron Lett. 1990, 31, 3731.
15. Robins M. J., Miranda K., Rajwanshi V. K., Peterson M. A., Andrei G., Snoeck R.,
De Clercq E., Balzarini J.: J. Med. Chem. 2006, 49, 391.
16. a) Woo J., Meyer R. B., Jr., Gamper H. B.: Nucleic Acids Res. 1996, 24, 2470; b) Berry
D. A., Jung K.-Y., Wise D. S., Sercel A. D., Pearson W. H., Mackie H., Randolph J. B.,
Somers R. L.: Tetrahedron Lett. 2004, 45, 2457.
17. Nowak I., Jones C. T., Robins M. J.: J. Org. Chem. 2006, 71, 3077.
18. Divakar K. J., Reese C. B.: J. Chem. Soc., Perkin Trans. 1 1982, 1171.
19. Brancale A., McGuigan C., Algain B., Savy P., Benhida R., Fourrey J.-L., Andrei G.,
Snoeck R., De Clercq E., Balzarini J.: Biorg. Med. Chem. Lett. 2001, 11, 2507.
20. De Clercq E., Descamps J., Balzarini J., Giziewicz J., Barr P. J., Robins M. J.: J. Med. Chem.
1983, 26, 661.
21. a) Fox J. J., Van Praag D.: J. Org. Chem. 1961, 26, 526; b) Holý A.: Collect. Czech. Chem.
Commun. 1979, 44, 2846.
22. Fukuhara T. K., Visser D. W.: J. Am. Chem. Soc. 1955, 77, 2393.
23. Robins M. J., Sarker S., Wnuk S. F.: Nucleosides Nucleotides 1998, 17, 785.
24. Ullman B., Coons T., Rockwell S., McCartan K.: J. Biol. Chem. 1988, 263, 12391.
25. Lang T. T., Young J. D., Cass C. E.: Mol. Pharmacol. 2004, 65, 925.
26. a) Zhang J., Visser F., Vickers M. F., Lang T., Robins M. J., Nielsen L. P. C., Nowak I.,
Baldwin S. A., Young J. D., Cass C. E.: Mol. Pharmacol. 2003, 64, 1512; b) Zhang J.,
Smith K. M., Tackaberry T., Visser F., Robins M. J., Nielsen L. P. C., Nowak I.,
Karpinski E., Baldwin S. A., Young J. D., Cass C. E.: Mol. Pharmacol. 2005, 68, 830.
27. a) Vickers M. F., Kumar R., Visser F., Zhang J., Charania J., Raborn R. T., Baldwin S. A.,
Young J. D., Cass C. E.: Biochem. Cell Biol. 2002, 80, 639; b) Vickers M. F., Zhang J.,
Visser F., Tackaberry T., Robins M. J., Nielsen L. P. C., Nowak I., Baldwin S. A., Young
J. D., Cass C. E.: Nucleosides Nucleotides Nucleic Acids 2004, 23, 361.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1395–1412