Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-nitrobenzyl)adenosine; Inhibition of nucleoside transport and proliferation of cancer cells

Article abstract of DOI:10.1002/cmdc.201402047

Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by S_NAr displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2′,3′,5′-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1.

Full text of DOI:10.1002/cmdc.201402047

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DOI: 10.1002/cmdc.201402047
Synthesis of Purine and 7-Deazapurine Nucleoside
Analogues of 6-N-(4-Nitrobenzyl)adenosine; Inhibition of
Nucleoside Transport and Proliferation of Cancer Cells
Ramanjaneyulu Rayala,[a] Patricia Theard,[a] Heysell Ortiz,[a] Sylvia Yao,[b] James D. Young,[b]
Jan Balzarini,[c] Morris J. Robins,[d] and Stanislaw F. Wnuk*[a]
Human equilibrative nucleoside transporter 1 (hENT1) is a pro-
totypical nucleoside transporter protein ubiquitously expressed
on the cell surface of almost all human tissue. Given the role
of hENT1 in the transport of nucleoside drugs, an important
class of therapeutics in the treatment of various cancers and
viral infections, efforts have been made to better understand
the mechanisms by which hENT1 modulates nucleoside trans-
port. To that end, we report here the design and synthesis of
novel tool compounds for the further study of hENT1. The 7-
deazapurine nucleoside antibiotic tubercidin was converted
into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkyla-
tion at N3 followed by a Dimroth rearrangement to the 4-N-
isomer or by fluoro-diazotization followed by SNAr displace-
ment of the 4-fluoro group by a benzylamine. The 4-N-(4-nitro-
benzyl) derivatives of sangivamycin and toyocamycin antibiot-
ics were prepared by the alkylation approach. Cross-membrane
transport of labeled uridine by hENT1 was inhibited to
a weaker extent by the 4-nitrobenzylated tubercidin and sangi-
vamycin analogues than was observed with 6-N-(4-nitrobenzy-
l)adenosine. Type-specific inhibition of cancer cell proliferation
was observed at micromolar concentrations with the 4-N-(4-ni-
trobenzyl) derivatives of sangivamycin and toyocamycin, and
also with 4-N-benzyltubercidin. Treatment of 2’,3’,5’-O-acetyla-
denosine with aryl isocyanates gave the 6-ureido derivatives
but none of them exhibited inhibitory activity against cancer
cell proliferation or hENT1.
Introduction
Analogues and derivatives of 7-deazapurine nucleoside antibi-
otics have been synthesized and subjected to extensive biolog-
ical testing.[1] A sizeable number of active nucleoside com-
pounds with such a pyrrolo[2,3-d]pyrimidine base has been
prepared by chemical modifications of the parent antibiotics
as well as by coupling base derivatives with protected
sugars.[2] Noteworthy examples of such molecules include: 5-
iodotubercidin,[3] an up-field activator of the p53 pathway; san-
givamycin analogues, such as 6-hydrazinosangivamycin[4] and
xylocidine,[5] in vitro down-field inhibitors of protein kinase C
and cyclin-dependent kinases in cancer cell lines; a methyl-
substituted tubercidin,[6] which acts against the replication of
polio and dengue viruses; the anti-herpes simplex virus agent
xylotubercidin;[7] substituted toyocamycin analogues[8] and 2’-
b-C-methyl derivative of toyocamycin,[9] sangivamycin,[9] and
tubercidin[10] that have activity against the hepatitis C virus; 2’-
deoxy-2’-fluoroarabinotubercidin[11] and 2-amino-2’-deoxy-2’-
fluoroarabinotubercidin,[12] which exhibit antiviral activity; 4N,5-
diaryltubercidin derivatives, which act as adenosine kinase in-
hibitors;[2l,13] and tubercidin derivatives, such as 4-(het)aryl,[14]
5-(het)aryl[15] and some 4-substituted-5-(het)aryl[16] compounds
with nanomolar cytostatic activity against several cancer cell
lines.
Various methods to access 4-N-substituted-7-deazapurine
derivatives have been reported;[8,16–18] however, direct alkyla-
tion of exocyclic amino groups on 7-deazapurine nucleosides
has thus far received much less attention.[2b] Thus, treatment of
tubercidin with methyl iodide followed by sodium hydroxide
produced 4-N-methyltubercidin[2b] (6-N-methyl-7-deazaadeno-
sine) via a Dimroth rearrangement. Several other 4-N-substitut-
ed tubercidin derivatives have previously been prepared by
SNAr displacement of chloride from the 4-chloro analogue[2b,16]
of tubercidin or by displacement of 1,2,4-triazole from 4-N-
(1,2,4-triazol-4-yl) intermediates.[17] Such 4-N-substituted toyo-
camycin derivatives also were prepared from the correspond-
ing 4-chloro compounds.[8] The 7-chloro derivative of the C-nu-
cleoside antibiotic formycin (3-b-d-ribofuranosylpyrazolo[4,3-
d]pyrimidine) was prepared and then converted into 7-N-ben-
zylformycin.[18]
[a] R. Rayala, P. Theard, H. Ortiz, Prof. S. F. Wnuk
Department of Chemistry & Biochemistry, Florida International University
11200 SW 8th Street, Miami, Florida 33199 (USA)
E-mail: wnuk@fiu.edu
[b] Dr. S. Yao, Prof. J. D. Young
Department of Physiology, University of Alberta
7-25 Medical Sciences Building, Edmonton, Alberta, T6G 2H7 (Canada)
[c] Prof. J. Balzarini
Purine and pyrimidine nucleosides and their derivatives play
crucial roles in human physiology and pharmacology.[19] These
“salvage” metabolites can be converted into nucleotides,
which in turn serve as the energy-rich currency of intermediary
metabolism and as precursors of nucleic acid biosynthesis. Ad-
Rega Institute for Medical Research, KU Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
[d] Prof. M. J. Robins
Department of Chemistry & Biochemistry, Brigham Young University
C100 Benson Science Building, Provo, Utah 84602 (USA)
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enosine functions as a key signaling molecule and modulates
diverse physiological processes through interactions with cell
surface P1 purinergic receptors.
lution to give the N6-alkylated product.[27] Alkylation of tuberci-
din (1a) with benzyl bromide in DMF occurred at N3 (same as
ring nitrogen N1 in adenosine), and treatment of the 3-benzyl
intermediate with dimethylamine (2m in THF) in methanol re-
sulted in rearrangement to give 4-N-benzyltubercidin (2a;
67%).
Many types of cancer and viral infections are treated with
nucleoside drugs,[20] but drug availabilities within cells and in
the extracellular environment are determined primarily by spe-
cific nucleoside transporter (NT) proteins that facilitate their
movement across plasma membranes and some organellar
membranes. Because of the hydrophilic nature of nucleosides,
NTs play key roles in the physiology, pathophysiology, and
therapeutic actions of many nucleoside drugs.[19] The human
equilibrative nucleoside transporter 1 (hENT1) is the prototypi-
cal NT present in the outer plasma membrane and some intra-
cellular membranes of most, if not all, human cells.[21]
Because the 6-N-(4-nitrobenzyl) derivatives of adenosine and
other adenine nucleosides are more potent inhibitors of nu-
cleoside transport,[22] we also synthesized 4-nitrobenzyl deriva-
tives of the 7-deaza antibiotics. Alkylation of 1a with 4-nitro-
benzyl bromide followed by treatment with dimethylamine
(2m in THF) in methanol produced 4-N-(4-nitrobenzyl)tuberci-
din (2b; 56%). Analogous treatment of sangivamycin (1b)
with 4-nitrobenzyl bromide and then dimethylamine (2m in
THF) in methanol gave 4-N-(4-nitrobenzyl)sangivamycin (2c;
45%).
Nitrobenzylmercaptopurine ribonucleoside (NBMPR) and
structurally related hENT1 probes such as 6-N-(4-nitrobenzyl)a-
denosine[22] and 5’-S-{2-(6-[3-(fluorescein-5-yl)thioureido-1-yl]-
hexanamido)}ethyl-6-N-(4-nitrobenzyl)-5’-thioadenosine (FITC-
SAHENTA)[23] bind with nanomolar affinity within, or closely as-
sociated with, the outward-facing permeant-binding site of
hENT1. Development of such probes has greatly aided investi-
gations on the structure, function, and cell-surface abundances
of hENT1.[19,23,24] We now report the synthesis of analogues of
these transport inhibitors with purine and 7-deazapurine bases
that exhibit pronounced cytotoxicity in several cancer cell lines
as well as moderate inhibition of hENT1 transport activity.
Treatment of toyocamycin (1c) with 4-nitrobenzyl bromide
unexpectedly gave 4-N-(4-nitrobenzyl)toyocamycin (2d) direct-
ly. Formation of an intermediate alkylated at N3 apparently did
not occur; TLC analysis of the reaction mixture showed only
a more rapidly migrating material corresponding to 2d with-
out indication of a more polar (cationic) product. Therefore,
successive treatment with dimethylamine was not required. Al-
though direct alkylation on the exocyclic amino group of ade-
nine and guanine with quinone methides is known,[28] the ob-
served direct alkylation on the exocyclic amine group of toyo-
camycin with 4-nitrobenzyl bromide has not been previously
reported. This might have resulted from alteration of the nu-
cleophilicity of the endocyclic N3 of toyocamycin relative to
that of the exocyclic 4-amino nitrogen by the electron-with-
drawing pull of the cyano group at C5. A possible accompany-
ing stereoelectronic effect might result from interference of
the cylindrical cyano group with a synperiplanar orientation of
the exocyclic amino group relative to the planar heterocyclic
ring that would diminish lone-pair donation from the amino ni-
trogen toward N3 of that amidine system.[29]
Results and Discussion
Chemistry
The N-benzylated 7-deazaadenosine analogues (2a–d)
(Scheme 1) were prepared by two procedures. One involved al-
kylation of the 7-deazaadenosine antibiotics (1a–c) at N3 with
a benzyl bromide followed by a base-promoted Dimroth rear-
rangement.[18,25] The second route employed diazotization–flu-
orodediazoniation followed by SNAr displacement of fluoride
with a benzyl amine.[26] Alkylation of adenosine occurs at N1,
and the resulting cation undergoes rearrangement in basic so-
Alternatively, tubercidin (1a) was acetylated and then sub-
jected to diazotive-fluorodeamination[23,30] followed by SNAr
displacement of fluoride with benzylamines (Scheme 2).[23,26]
Thus, 2’,3’,5’-tri-O-acetyltubercidin (3) was treated with sodium
nitrate in freshly prepared hydrogen fluoride pyridine (~55%)
at ꢀ108C[31] for 15 minutes to yield protected 4-fluorotuberci-
din 4 (82%). The concentration of hydrogen fluoride pyridine
as well as the temperature are crucial.[31b] Treatment of 4 with
4-nitrobenzylamine hydrochloride in methanol and in the pres-
ence of triethylamine[23] gave 2’,3’,5’-tri-O-acetyl-4-N-(4-nitro-
benzyl)tubercidin (5; 47%), which was deacetylated (ammonia
in methanol) to give 2b (85%).
Some 2’,3’-bis-O-silylated adenosine derivatives with ureido
substituents at the 5’- and 6-positions exhibit antiproliferative
activity.[32] We prepared the 6-N-[N-arylcarbamoyl]adenosine
derivatives (9a–c; Scheme 3) with phenyl, 4-methoxyphenyl,
and 4-nitrophenyl moieties to probe for binding to hENT1 and
inhibition of nucleoside transport as observed with 6-N-(4-ni-
trobenzyl)adenosine.[22] The 2’,3’,5’-tri-O-acetyl derivative (7) of
adenosine (6) was treated with phenyl isocyanate to give 8a.
Deacetylation gave 6-N-[N-phenylcarbamoyl]adenosine (9a),
Scheme 1. Synthesis of 4-N-benzylated nucleosides by alkylation. Reagents
and conditions: a) ArCH2Br, DMF, 40–808C, 24–63h; b) Me2NH (2m in THF),
MeOH, reflux, 20–32 h. Yield: 45–67%.
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Inhibition of cell proliferation
Inhibition of the proliferation of
murine leukemia (L1210) cells
and human T-lymphocyte (CEM),
cervix carcinoma (HeLa), prostate
adenocarcinoma (PC-3), and
kidney carcinoma (Caki-1) cells
by the adenine and 7-deazaade-
nine nucleoside analogues was
evaluated. No pronounced inhibi-
tory effect was observed with
the 6-ureido compounds 9a, 9b,
and 9c in any of the tumor cell
lines (Table 1). Marked inhibition
Scheme 2. SNAr-based synthesis of 4-N-(4-nitrobenzyl)tubercidin (2b). Reagents and conditions: a) Ac2O, Py, 08C!
RT, 21 h, 86%; b) Py·HF (55% HF), NaNO2, ꢀ108C, 0.25 h, 82%; c) Et3N, MeOH, RT, 5 h, 47%; d) saturated NH3 in
MeOH, RT, 20 h, 85%.
of the proliferation of PC-3 cells (IC50: 0.92 mm) and HeLa cells
(IC50: 7.4 mm) by 4-N-benzyltubercidin (2a) was observed but
no significant activity was found with its nitrobenzyl analogue
Table 1. Inhibitory effects on the proliferation of cells in culture.
Compd
IC50 [mm][a]
L1210
CEM
HeLa
PC-3
Caki-1
2a
2b
2c
2d
9a
9b
9c
172ꢁ47
125ꢁ28
182ꢁ20
7.4ꢁ2.2
143ꢁ4
1.8ꢁ0.2
9.4ꢁ3.0
201ꢁ44
161ꢁ10
>250
0.92ꢁ0.67
76ꢁ10
116ꢁ23
132ꢁ30
116ꢁ23
98ꢁ10
ꢂ250
0.92ꢁ0.04
115ꢁ28
109ꢁ16
221ꢁ0
3.4ꢁ0.9
5.6ꢁ3.3
115ꢁ20
110ꢁ31
45ꢁ26
5.5ꢁ1.5
>250
>250
>250
Scheme 3. Synthesis of 6-ureidoadenosine analogues. Reagents and condi-
tions: a) Ac2O, Py, 08C!RT, 24 h, 90%; b) CH2Cl2, RT, 24–68 h, 80–96%; c) sa-
turated NH3 in MeOH, RT, 2 h, 65–85%.
150ꢁ33
>250
>250
ꢂ250
>250
[a] Half-maximal inhibitory concentration (IC50); data represent the meanꢁ
SD of at least n=2–3 independent experiments. Cell lines: murine leukemia
(L1210); human CD4+ T-lymphocytes (CEM); human cervix carcinoma
(HeLa); human prostate adenocarcinoma (PC-3); human kidney carcinoma
(Caki-1).
and the analogous sequential treatment of 7 with the respec-
tive isocyanates produced the 4-methoxyphenyl and 4-nitro-
phenyl analogues 9b and 9c.
2b (IC50: >50 mm). The 4-N-(4-nitrobenzyl)sangivamycin (2c)
and 4-N-(4-nitrobenzyl)toyocamycin (2d) analogues inhibited
proliferation of L1210, HeLa, and PC-3 cells, with IC50 values
ranging from approximately 0.9–9.4 mm with 2c showing more
potent effects (IC50: 0.92–3.4 mm). It is intriguing that such strik-
ing differences in cytostatic activity were dependent on the
nature of the tumor cell lines. HeLa and PC-3 tumor cells were
highly susceptible to the cytostatic activity of 2a, 2c, and 2d,
whereas the L1210 cells were sensitive only to 2c and 2d, and
the CEM and Caki-1 cells were weakly sensitive to the antiproli-
ferative effects of any of compounds 2a–2d.
Nucleoside transport
Inhibition of the initial rate of [3H]-uridine (20 mm) uptake by
recombinant hENT1 produced in the Xenopus oocyte heterolo-
gous expression system was determined as described previ-
ously.[21] Minimal inhibition (<25%) of labelled uridine uptake
was observed at a concentration of 100 mm for each of the 6-
ureidoadenosine compounds 9a, 9b, and 9c. The 4-N-benzyl-
tubercidin (2a) and 4-N-(4-nitrobenzyl)toyocamycin (2d) ana-
logues also exhibited weak inhibition (<45%) but both 4-N-(4-
nitrobenzyl)tubercidin (2b) and 4-N-(4-nitrobenzyl)sangivamy-
cin (2c) fully inhibited labelled uridine uptake at that concen-
tration. Dose–response evaluations indicated that 2c (IC50 =
123ꢁ31 nm) was a better inhibitor of uridine transport by
hENT1 than 2b (IC50 >1000 nm). However, the 7-deaza ana-
logue 2c was a weaker inhibitor of hENT1-mediated transport
than 6-N-(4-nitrobenzyl)adenosine, NBMPR, and other deriva-
tives[22] containing a nitrogen atom at the 7-position in the ad-
enine ring.
Conclusions
Alkylation at N3 of tubercidin with 4-nitrobenzyl bromide and
a Dimroth rearrangement gave 4-N-(4-nitrobenzyl)tubercidin,
which also was prepared by fluoro-diazotization of tubercidin
and SNAr displacement of the 4-fluoro group by 4-nitrobenzyla-
mine. The alkylation–rearrangement sequence was employed
to convert sangivamycin (5-carboxamidotubercidin antibiotic)
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(Ph), 126.6, 122.3 (C6), 103.4, 99.2 (C5), 87.6 (C1’), 85.1 (C4’), 73.7
(C2’), 70.7 (C3’), 61.8 (C5’), 43.1 ppm (CH2); MS (ESI): m/z 357 [M+
H]+ (100%); HRMS (ESI): m/z [M+H]+ calcd for C18H29N4O4:
357.1563; found: 357.1581.
into its 4-N-(4-nitrobenzyl) derivative. However, no evidence of
formation of an initial N3-alkylated intermediate was observed
upon treatment of toyocamycin (5-cyanotubercidin antibiotic)
with 4-nitrobenzyl bromide. Alkylation occurred directly on the
amino group to give 4-N-(4-nitrobenzyl)toyocamycin. The 4-N-
(4-nitrobenzyl) derivatives of tubercidin and sangivamycin in-
hibited cross-membrane transport of labelled uridine by hENT1
at IC50 values of >1 mm and ~120 nm, respectively. Inhibition
of the proliferation of L1210, HeLa, and PC-3 tumor cells in cul-
ture was observed with 4-N-benzyltubercidin and the 4-N-(4-ni-
trobenzyl) derivatives of sangivamycin and toyocamycin at IC50
values of 0.92–9.4 mm.
4-(4-Nitrobenzylamino)-7-(b-d-ribofuranosyl)pyrrolo[2,3-d]pyri-
midine (2b)
Method A. Tubercidin (1a; 266 mg, 1.0 mmol) was treated with 4-
nitrobenzyl bromide (648 mg, 3.0 mmol) at 808C for 24 h as de-
scribed above for 2a. The resultant solid was redissolved in MeOH
(15 mL), and the solution was treated with Me2NH in THF (2m,
8 mL). The reaction mixture was heated at reflux (658C, oil bath)
with stirring for 20 h. TLC showed ~85% conversion to a less polar
product, which was isolated and purified as described for 2a to
give 2b as a yellow oil (244 mg, 56%): UV (MeOH): lmax =278 nm,
1
lmin =240 nm; H NMR ([D6]DMSO): d=8.30 (t, 1H, J=6.1 Hz, NH),
Experimental Section
8.19 (“d”, 2H, J=8.8 Hz, Ph), 8.12 (s, 1H, H2), 7.58 (d, 2H, J=8.8 Hz,
Ph), 7.43 (d, 1H, J=3.7 Hz, H6), 6.69 (d, 1H, J=3.5 Hz, H5), 6.04 (d,
1H, J=6.3 Hz, H1’), 5.28–5.30 (m, 2H, 2ꢁOH), 5.14 (s, 1H, OH),
4.80–4.91 (m, 2H, CH2), 4.45 (“d”, 1H, J=4.4 Hz, H2’), 4.11–4.13 (m,
1H, H3’), 3.92 (q, 1H, J=3.5 Hz, H4’), 3.64 (dt, 1H, J=3.9, 11.8 Hz,
H5’), 3.52–3.57 ppm (m, 1H, H5’’); 13C NMR ([D6]DMSO/D2O): d=
155.8, 151.0, 148.8, 147.9, 146.3, 127.9 (Ph), 123.4 (Ph), 122.8 (C6),
103.6, 99.3 (C5), 87.5 (C1’), 84.9 (C4’), 73.5 (C2’), 70.4 (C3’), 61.6
(C5’), 42.7 ppm (CH2); MS (ESI): m/z 402 [M+H]+ (100%); Anal.
calcd for C18H19N5O6·1.25H2O (423.89): C, 51.00; H, 5.11; N, 16.52;
found: C, 51.05; H, 5.06; N, 16.03.
General: Reagent-grade chemicals were used, and solvents were
dried by heating at reflux over CaH2 under N2 unless otherwise
specified. Reactions were performed under an atmosphere of N2.
Reaction progress was monitored by thin-layer chromatography
(TLC) on Merck Kieselgel 60-F254 sheets with product detection by
254 nm light. Products were purified by column chromatography
using Merck Kiselgel 60 (230–400 mesh) or by automated flash
chromatography using a CombiFlash system. Purity of the synthe-
sized compounds was determined to be ꢂ95% by elemental anal-
ysis (C, H, N) and/or high-performance liquid chromatography
(HPLC).
Method B
1H (400 MHz), 13C (100.6 MHz), and 19F (376 MHz) NMR spectra were
recorded at RT in solutions of CDCl3 or [D6]DMSO. Chemical shifts
(d) are reported in parts per million (ppm) referenced to the residu-
al solvent peak, and coupling constants (J) are given in Hertz (Hz).
Multiplicity is reported using standard abbreviations: singlet (s);
doublet (d); triplet (t); multiplet (m); broad (br); inverted commas
indicate observed multiplicity. UV spectra were recorded with
a Varian Cary 100 Bio UV-visible spectrophotometer.
Step a. Ac2O (377 mL, 408 mg, 4 mmol) was added to a stirred sus-
pension of tubercidin (1a; 266 mg, 1 mmol) in dry pyridine (5 mL)
at 08C (ice bath), and the solution was stirred at 08C for 12 h and
then at RT for 9 h (total reaction time: 21 h). MeOH was added,
and the reaction mixture was stirred at RT for a further 30 min. The
volatiles were removed in vacuo (<258C) with coevaporation
using MeOH. The resulting gum was partitioned between CHCl3
(50 mL) and 2% aq AcOH (50 mL), and the aqueous layer was ex-
tracted with CHCl3 (2ꢁ10 mL). The combined organic phase was
washed sequentially with saturated aq NaHCO3 (50 mL) and brine
(50 mL), dried (MgSO4), filtered and concentrated in vacuo. Purifica-
tion of the residue using column chromatography (100%, EtOAc)
gave 2’,3’,5’-tri-O-acetyltubercidin (3) as a colorless foam (337 mg,
86%); characterization data were in agreement with those previ-
ously reported.[33]
4-Benzylamino-7-(b-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine
(2a): BnBr (345 mL, 496 mg, 2.9 mmol) was added to a stirred solu-
tion of tubercidin (1a; 266 mg, 1 mmol) in dried DMF (5 mL). After
stirring for 48 h at 408C, TLC showed almost complete conversion
to a more polar product. The volatiles were evaporated under
vacuum (<408C) to ~1 mL, and the resulting syrup was added
dropwise to dried acetone (30 mL) with vigorous stirring. Et2O
(60 mL) was added to the resultant suspension, which was then
chilled for 20 min at 08C. The precipitate was isolated by vacuum
filtration. The hygroscopic solid was quickly redissolved in MeOH
(10 mL), and Me2NH in THF (2m, 8 mL) was added. The resulting
solution was heated at reflux (658C, oil bath) with stirring for 20 h.
TLC showed ~80% conversion to a less polar product, and volatiles
were removed in vacuo with coevaporation using MeOH (ꢁ2). The
residue was redissolved in warm MeOH (10 mL), H2O (60 mL) was
added, and the solution was extracted with EtOAc (5ꢁ20 mL). The
combined organic phase was dried (Na2SO4), filtered, and concen-
trated in vacuo, with coevaporation using EtOH (ꢁ2). Purification
by flash chromatography (100%, EtOAc) gave 2a as a colorless oil
(238 mg, 67%): UV (MeOH): lmax =276 nm, lmin =244 nm; 1H NMR
([D6]DMSO): d=8.09–8.13 (m, 2H, NH, H2), 7.38 (d, 1H, J=3.7 Hz,
H6), 7.29–7.35 (m, 4H, Ph), 7.22–7.25 (m, 1H, Ph), 6.67 (d, 1H, J=
3.4 Hz, H5), 6.01 (d, 1H, J=5.9 Hz, H1’), 5.27–5.32 (m, 2H, 2’-OH, 5’-
OH), 5.12 (d, 1H, J=4.3 Hz, 3’-OH), 4.70–4.78 (m, 2H, CH2), 4.44 (q,
1H, J=5.7 Hz, H2’), 4.09 (“q”, 1H, J=4.1 Hz, H3’), 3.90 (q, 1H, J=
3.4 Hz, H4’), 3.60–3.65 (m, 1H, H5’), 3.50–3.56 ppm (m, 1H, H5’’);
13C NMR ([D6]DMSO): d=156.1, 151.4, 149.5, 140.1, 128.2 (Ph), 127.1
Step b. NaNO2 (66 mg, 0.95 mmol) was added to a solution of 3
(150 mg, 0.38 mmol) in freshly prepared ~55% HF in pyridine[31b]
(1.9 mL) at ꢀ108C in a sealed polypropylene vessel. The mixture
was stirred at ꢀ108C for 15 min, and TLC showed almost complete
conversion to a less polar product. Ice/H2O (50 mL) was added, and
the mixture was extracted with CH2Cl2 (4ꢁ25 mL). The combined
organic phase was washed sequentially with saturated aq NaHCO3
(50 mL) and brine (50 mL), and dried (Na2SO4), filtered and concen-
trated in vacuo. Purification of the resulting brown oil using
column chromatography (30%, EtOAc/hexane) gave 7-(2,3,5-tri-O-
acetyl-b-d-ribofuranosyl)-4-fluoropyrrolo[2,3-d]pyrimidine (4) as
a colorless oil (124 mg, 82%): UV (MeOH): lmax =258 nm, lmin
=
1
4
233 nm; H NMR (CDCl3): d=8.53 (d, 1H, JH-F =0.5 Hz, H2), 7.37 (d,
1H, J=3.8 Hz, H8), 6.66 (d, 1H, J=3.8 Hz, H7), 6.45 (d, 1H, J=
6.0 Hz, H1’), 5.74 (t, 1H, J=5.8 Hz, H2’), 5.55 (dd or m, 1H, J=4.1,
5.6 Hz, H3’), 4.32–4.42 (m, 3H, H4’, H5’, H5’’), 2.12 (s, 3H, CH3), 2.13
(s, 3H, CH3), 2.02 ppm (s, 3H, CH3); 19F NMR (CDCl3): d=
ꢀ64.81 ppm (s); 13C NMR (CDCl3): d=170.2, 169.6, 169.4 (3ꢁC=O),
1
3
162.3 (d, JC-F =253.8 Hz, C4), 155.1 (d, JC-F =11.9 Hz, C7a), 151.1 (d,
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2
3JC-F =14.5 Hz, C2), 125.7 (d, 4JC-F =2.6 Hz, C6), 105.4 (d, JC-F
=
precipitate was collected by vacuum filtration, and recrystallization
from MeOH gave 2d as colorless crystals (99 mg, 46%): mp: 214–
3
33.4 Hz, C4a), 99.6 (d, JC-F =4.9 Hz, C5), 86.1 (C1’), 79.9 (C4’), 73.1
(C2’), 70.1 (C3’), 63.3 (C5’), 20.7 (CH3), 20.5 (CH3), 20.3 ppm (CH3);
MS (ESI): m/z 396 [M+H]+ (100%).
2168C; UV (MeOH): lmax =274, 236 nm (e=22100, 18900), lmin
=
250, 228 nm (e=12800, 18000); 1H NMR ([D6]DMSO): d=8.35 (s,
1H, H2), 8.19–8.23 (m, 3H, H6, Ph), 7.59 (d, 2H, J=8.6 Hz, Ph), 6.87
(s, 1H, NH), 5.93 (d, 1H, J=5.6 Hz, H1’), 5.47 (d, 1H, J=6.0 Hz, 2’-
OH), 5.35 (“s”, 2H, CH2), 5.21 (d, 1H, J=5.0 Hz, 3’-OH), 5.09 (t, 1H,
J=5.4 Hz, 5’-OH), 4.31 (q, 1H, J=5.5 Hz, H2’), 4.08 (q, 1H, J=
4.6 Hz, H3’), 3.92 (q, 1H, J=3.7 Hz, H4’), 3.62–3.68 (m, 1H, H5’),
3.53–3.58 ppm (m, 1H, H5’’); 13C NMR ([D6]DMSO/D2O): d=152.6,
149.4, 146.7, 144.9, 142.7, 129.4, 128.5 (Ph), 123.5 (Ph), 115.2, 105.0,
87.7 (C1’), 85.8 (C4’), 85.5, 74.6 (C2’), 70.1 (C3’), 61.1 (C5’), 48.8 ppm
(CH2); MS (ESI): m/z 427 [M+H]+ (100%); Anal. calcd for
C19H18N6O6·0.5H2O (435.39): C, 52.41; H, 4.40; N, 19.30; found: C,
52.17; H, 4.29; N, 19.30.
Step c. Freshly distilled Et3N (113 mL, 82 mg, 0.81 mmol) was added
to a stirred suspension of 4 (93 mg, 0.23 mmol) and 4-nitrobenzyla-
mine hydrochloride (67 mg, 0.35 mmol) in MeOH (3 mL), and the
mixture was stirred for 5 h at RT. The volatiles were evaporated in
vacuo, and the residue was purified by column chromatography
(50%, EtOAc/hexane) to give 2’,3’,5’-tri-O-acetyl-4-N-(4-nitrobenzyl)-
tubercidin (5) as a colorless oil (58 mg, 47%): UV (MeOH): lmax
=
1
277 nm, lmin =238 nm; H NMR (CDCl3): d=8.36 (s, 1H, H2), 8.17 (d,
2H, J=8.7 Hz, Ph), 7.51 (d, 2H, J=8.7 Hz, Ph), 7.12 (d, 1H, J=
3.77 Hz, H6), 6.42–6.46 (dd, 2H, J=3.8, 6.0 Hz, H1’, H5), 5.73 (t, 1H,
J=5.8 Hz, H2’), 5.54–5.59 (m, 2H, H3’, NH), 4.95 (d, 2H, J=6.1 Hz,
CH2), 4.31–4.40 (m, 3H, H4’, H5’, H5’’), 2.14 (s, 6H, 2ꢁCH3),
2.04 ppm (s, 3H, CH3); 13C NMR (CDCl3): d=170.4, 169.7, 169.5 (3ꢁ
C=O), 156.0 (C4), 152.3 (C2), 150.8 (C7a), 147.3 (Ph), 146.7 (Ph),
128.0 (Ph), 123.9 (Ph), 121.5 (C6), 103.8 (C4a), 99.4 (C5), 85.4 (C1’),
79.5 (C4’), 73.1 (C2’), 70.8 (C3’), 63.5 (C5’), 44.2 (CH2), 20.8 (CH3),
20.6 (CH3), 20.4 ppm (CH3); MS (ESI): m/z 528 [M+H]+ (100%).
6-N-[N-phenylcarbamoyl]adenosine (9a)
Step a. A solution of phenyl isocyanate (35 mL, 39 mg, 0.32 mmol)
and 2’,3’,5’-tri-O-acetyladenosine (7; 106 mg, 0.27 mmol) in CH2Cl2
(5 mL) was stirred at RT under N2 for 48 h. An additional portion of
phenyl isocyanate (35 mL, 39 mg, 0.324 mmol) was then added,
and stirring was continued for 20 h (total reaction time: 68 h). The
reaction mixture was partitioned between saturated aq NaHCO3
(25 mL) and CHCl3 (25 mL), and the organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo. Purification by
column chromatography (70%, EtOAc/hexane) gave 2’,3’,5’-tri-O-
Step d. A saturated (at 08C) solution of NH3 in MeOH (5 mL) was
added to a stirred solution of 5 (54 mg, 0.1 mmol) in MeOH (1 mL),
and the mixture was stirred at RT for 20 h. The volatiles were
evaporated in vacuo, and the residue was purified by column chro-
matography (2!4% gradient, EtOAc/iPrOH/H2O (4:1:2) in EtOAc)
to give 2b as a yellow oil (35 mg, 85%): see method A for charac-
terization data.
acetyl-6-N-[N-phenylcarbamoyl]adenosine (8a) as
a pale-yellow
1
solid (111 mg, 80%): H NMR (CDCl3): d=11.89 (s, 1H, NH), 9.40 (s,
1H, NH), 8.62 (s, 1H, H2), 8.56 (s, 1H, H8), 7.63 (“d”, 2H, J=7.6 Hz,
Ph), 7.35 (“t”, 2H, J=7.9 Hz, Ph), 7.11 (“t”, 1H, J=7.4 Hz, Ph), 6.25
(d, 1H, J=5.4 Hz, H1’), 6.03 (t, 1H, J=5.5 Hz, H2’), 5.69 (“dd”, 1H,
J=4.4, 5.4 Hz, H3’), 4.37–4.48 (m, 3H, H4’, H5’, H5’’), 2.15 (s, 3H,
CH3), 2.08 (s, 3H, CH3), 2.07 (s, 3H, CH3).
5-Carboxamido-4-(4-nitrobenzylamino)-7-(b-d-ribofuranosyl)-
pyrrolo[2,3-d]pyrimidine (2c): Sangivamycin (1b; 155 mg,
0.5 mmol) was treated with 4-nitrobenzyl bromide (162 mg,
1.5 mmol) for 26 h at 408C as described for the conversion of
1a!2a. The alkylated intermediate was then dissolved in MeOH
(12 mL), treated with Me2NH in THF (2m, 6 mL), and stirred for 45 h
at RT. The reaction was then treated with additional Me2NH in THF
(2m, 2 mL), and the mixture was heated at reflux while stirring for
32 h (total reaction time: 77 h). TLC showed ~80% conversion to
the less polar product (2c). Volatiles were evaporated in vacuo to
give a yellow solid. Recrystallization from EtOH gave 2c as off-
white crystals (101 mg, 45%): mp: 154–1588C (dec.); UV (MeOH):
lmax =286 nm (e=23100), lmin =229 nm (e=8700); 1H NMR
([D6]DMSO): d=10.29 (t, 1H, J=6.0 Hz, NH), 8.17–8.21 (m, 4H, Ph,
H2, H6), 8.11 (s, 1H, CONH2), 7.59 (br d, 2H, J=8.8 Hz, Ph), 7.47 (s,
1H, CONH2), 6.06 (d, 1H, J=6.0 Hz, H1’), 5.43 (d, 1H, 2’-OH, J=
6.3 Hz), 5.20 (d, 1H, J=5.0 Hz, 3’-OH), 5.09 (t, 1H, J=5.8 Hz, 5’-OH),
4.90 (“d”, 2H, J=6.2 Hz, CH2), 4.37 (q, 1H, J=5.8 Hz, H2’), 4.10 (“q”,
1H, J=4.5 Hz, H3’), 3.93 (q, 1H, J=4.0 Hz, H4’), 3.61–3.67 (m, 1H,
H5’), 3.53–3.58 ppm (m, 1H, H5’’); 13C NMR ([D6]DMSO): d=166.5,
156.5, 152.6, 150.4, 148.0, 146.4, 128.0 (Ph), 125.7, 123.6, 110.9,
101.7, 87.2 (C1’), 85.3 (C4’), 73.9 (C2’), 70.5 (C3’), 61.9 (C5’),
42.9 ppm (CH2); MS (ESI): m/z 445 [M+H]+ (100%); Anal. calcd for
C19H20N6O7·1.5 H2O (471.42): C, 48.41; H, 4.92; N, 17.83; found: C,
48.59; H, 4.66; N, 17.65.
Step b. A saturated (at 08C) solution of NH3 in MeOH (6 mL) was
added to a solution of 8a (103 mg, 0.2 mmol) in MeOH (3 mL), and
the mixture was stirred at RT for 2 h. The volatiles were evaporat-
ed, and the residual solid was recrystallized from MeOH to give 9a
as a white powder (66 mg, 85%): mp: 193–1958C; UV (MeOH):
lmax =279 nm (e=28250), lmin =241 nm (e=9200); 1H NMR
([D6]DMSO): d=11.76 (s, 1H, NH), 10.17 (s, 1H, NH), 8.72 (s, 1H,
H2), 8.69 (s, 1H, H8), 7.63 (d, 2H, J=7.7 Hz, Ph), 7.36 (t, 2H, J=
7.9 Hz, Ph), 7.10 (t, 1H, J=7.4 Hz, Ph), 6.01 (d, 1H, J=5.6 Hz, H1’),
5.54 (d, 1H, J=6.0 Hz, 2’-OH), 5.24 (d, 1H, J=5.0 Hz, 3’-OH), 5.14 (t,
1H, J=5.5 Hz, 5’-OH), 4.61 (q, 1H, J=5.6 Hz, H2’), 4.19 (q, 1H, J=
4.7 Hz, H3’), 3.99 (q, 1H, J=3.8 Hz, H4’), 3.67–3.73 (m, 1H, H5’),
3.55–3.61 ppm (m, 1H, H5’’); 13C NMR ([D6]DMSO): d=150.83 (C=
O), 150.81 (C2), 150.6, 150.0, 142.4 (C8), 138.4, 128.9 (Ph), 123.2
(Ph), 120.6, 119.4 (Ph), 87.7 (C1’), 85.7 (C4’), 73.8 (C2’), 70.3 (C3’),
61.3 ppm (C5’); MS (ESI): m/z 387 [M+H]+ (100%); Anal. calcd for
C17H18N6O5·0.5H2O (395.37): C, 51.64; H, 4.84; N, 21.26; found: C,
51.38; H, 4.77; N, 21.02.
6-N-[N-(4-Methoxyphenyl)carbamoyl]adenosine (9b)
Step a. A solution of 7 (197 mg, 0.5 mmol) and 4-methoxyphenyl
isocyanate (78 mL, 90 mg, 0.6 mmol) in CH2Cl2 (5 mL) was stirred at
RT under N2 for 12 h. Additional 4-methoxyphenyl isocyanate
(78 mL, 90 mg, 0.6 mmol) was then added, and stirring was contin-
ued for 12 h (total reaction time: 24 h). The reaction mixture was
partitioned between saturated aq NaHCO3 (25 mL) and CHCl3
(25 mL), and the organic layer was dried (Na2SO4), filtered, and con-
centrated in vacuo to give 2’,3’,5’-tri-O-acetyl-6-N-[N-(4-methoxy-
phenyl)carbamoyl]adenosine (8b) as a pale-yellow solid of suffi-
cient purity for NMR characterization and use in the next step
5-Cyano-4-(4-nitrobenzylamino)-7-(b-d-ribofuranosyl)pyrrolo[2,3-
d]pyrimidine (2d): Toyocamycin (1c; 146 mg, 0.5 mmol) was
heated with 4-nitrobenzyl bromide (364 mg, 1.5 mmol) in dried
DMF (3 mL) at 408C for 63 h (TLC showed ~85% conversion to
a less polar product). The volatiles were evaporated under vacuum
(<408C) to ~1 mL, and this material was added dropwise to vigo-
rously stirred dry acetone (20 mL). Et2O (40 mL) was added to the
stirred suspension, which was then chilled at 08C for 20 min. The
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(260 mg, 96%): H NMR (CDCl3): d=11.45 (s, 1H, NH), 8.62 (s, 1H,
(449.37): C, 45.44; H, 4.26; N, 21.82; found: C, 45.10; H, 3.96; N,
21.59.
H2), 8.59 (s, 1H, H8), 8.12 (s, 1H, NH), 7.53 (“d”, 2H, J=8.9 Hz, Ph),
6.91 (“d”, 2H, J=9.0 Hz, Ph), 6.21 (d, 1H, J=5.3 Hz, H1’), 5.97 (t,
1H, J=5.4 Hz, H2’), 5.66 (“t”, 1H, J=4.8 Hz, H3’), 4.38–4.48 (m, 3H,
H4’, H5’, H5’’), 2.16 (s, 3H, CH3), 2.14 (s, 3H, CH3), 2.10 ppm (s, 3H,
CH3).
Cytostatic activity assays
All assays were performed in 96-well microtiter plates. Each well
containing 5–7.5ꢁ104 tumor cells (200 mL) was treated with
a given amount of test compound in DMSO in cell culture medium
at fivefold dilutions starting at 250 mm as the highest compound
concentration. Cells were allowed to proliferate for 48 h (murine
leukemia; L1210), 72 h (human T-lymphocytes; CEM), 96 h (human
cervix carcinoma; HeLa), 144 h (human prostate adenocarcinoma;
PC-3), or 168 h (human kidney; Caki-1) at 378C in a humidified 5%
CO2 controlled atmosphere. At the end of the incubation period,
cells were counted in a Coulter counter. The IC50 value was defined
as the concentration of the compound that inhibited cell prolifera-
tion by 50%. Data are the meanꢁSD of at least 2–3 independent
experiments.
Step b. A saturated (at 08C) solution of NH3 in MeOH (6 mL) was
added to a solution of crude 8b (260 mg, 0.5 mmol) in MeOH
(3 mL), and the mixture was stirred at RT for 2 h. The volatiles were
evaporated, and the residue was recrystallized from MeOH to give
9b as a white powder (170 mg, 80%): mp: 181–1828C; UV (MeOH):
lmax =280 nm (e=21300), lmin =247 nm (e=11100); 1H NMR
([D6]DMSO): d=11.59 (s, 1H, NH), 10.07 (s, 1H, NH), 8.70 (s, 1H,
H2), 8.67 (s, 1H, H8), 7.53 (“d”, 2H, J=4.5 Hz, Ph), 6.94 (“d”, 2H, J=
4.5 Hz, Ph), 6.01 (d, 1H, J=5.7 Hz, H1’), 5.54 (d, 1H, J=6.0 Hz, 2’-
OH), 5.24 (d, 1H, J=5.0 Hz, 3’-OH), 5.14 (t, 1H, J=5.6 Hz, 5’-OH),
4.61 (q, 1H, J=5.5 Hz, H2’), 4.18 (q, 1H, J=4.8 Hz, H3’), 3.98 (q, 1H,
J=3.8 Hz, H4’), 3.75 (s, 3H, OCH3), 3.67–3.72 (m, 1H, H5’), 3.55–
3.61 ppm (m, 1H, H5’’); 13C NMR ([D6]DMSO): d=155.4 (C=O), 150.9
(C2), 150.8, 150.4, 149.8, 142.3 (C8), 131.1, 121.2 (Ph), 120.3, 114.1
(Ph), 87.7 (C1’), 85.6 (C4’), 73.7 (C2’), 70.1 (C3’), 61.1 (C5’), 55.2 ppm
(OCH3); MS (ESI): m/z 417 [M+H]+ (100%); Anal. calcd for
C18H20N6O6·1.5H2O (443.41): C, 48.76; H, 5.23; N, 18.95; found: C,
48.54; H, 4.97; N, 18.85.
Acknowledgements
This work was partially supported by the US National Institute of
General Medical Sciences (NIGMS)/US National Cancer Institute
(NCI) (1SC1A138176; S.F.W.) and the Katholieke Universiteit
Leuven (KU Leuven), Belgium (GOA 10/014; J.B.). The authors
thank the US National Institutes of Health (NIH) MARC U*STAR
program (GM083688-02) for the fellowships supporting H.O. and
P.T., and Mrs. Lizette van Berckelaer (Rega Institute) for technical
assistance with the cytostatic evaluations.
6-N-[N-(4-Nitrophenyl)carbamoyl]adenosine (9c)
Step a. A solution of 4-nitrophenyl isocyanate (54 mg, 0.33 mmol)
and 7 (117 mg, 0.30 mmol) in CH2Cl2 (20 mL) was stirred at RT
under N2 for 17 h (TLC showed ~85% conversion to a less polar
product), and then heated at reflux for 24 h (total reaction time:
41 h). The reaction mixture was partitioned between saturated aq
NaHCO3 (25 mL) and CHCl3 (25 mL), and the organic layer was
dried (Na2SO4), filtered and concentrated to give 2’,3’,5’-tri-O-
acetyl-6-N-[N-(4-nitrophenyl)carbamoyl]adenosine (8c) as a pale-
yellow solid of sufficient purity for NMR characterization and use in
Keywords: 7-deazapurine nucleosides
agents nitrogen heterocycles nucleoside transporter
inhibition · nucleosides
·
antiproliferative
·
·
1
the next step (160 mg, 96%): H NMR ([D6]DMSO): d=12.13 (s, 1H,
NH), 10.62 (s, 1H, NH), 8.75 (s, 1H, H2), 8.71 (s, 1H, H8), 8.27 (d, 2H,
J=9.2 Hz, Ph), 7.90 (d, 2H, J=9.2 Hz, Ph), 6.33 (d, 1H, J=5.3 Hz,
H1’), 6.05 (d, 1H, J=5.7 Hz, H2’), 5.65 (t, 1H, J=5.4 Hz, H3’), 4.42–
4.47 (m, 1H, H4’, H5’), 4.25–4.3 (m, 1H, H5’’), 2.13 (s, 3H, CH3), 2.05
(s, 3H, CH3), 2.03 ppm (s, 3H, CH3).
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Step b. A saturated (at 08C) solution of NH3 in MeOH (6 mL) was
added to a solution of crude 8c (160 mg, 0.29 mmol) in MeOH
(3 mL), and the mixture was stirred at RT for 2 h. The volatiles were
evaporated, and the residual solid was recrystallized from MeOH to
give 9c as an off-white powder (80 mg, 65%): mp: 202–2088C; UV
(MeOH): lmax =317, 278 nm (e=16200, 11300), lmin =286, 241 nm
1
(e=10300, 5600); H NMR ([D6]DMSO): d=12.27 (s, 1H, NH), 10.61
(s, 1H, NH), 8.73 (s, 1H, H2), 8.72 (s, 1H, H8), 8.27 (“d”, 2H, J=
9.2 Hz, Ph), 8.00 (“d”, 2H, J=9.2 Hz, Ph), 6.01 (d, 1H, J=5.7 Hz,
H1’), 5.52 (“s”, 1H, 2’-OH), 5.24 (d, 1H, J=14.1 Hz, 3’-OH), 5.14 (“s”,
1H, 5’-OH), 4.61 (“s”, 1H, H2’), 4.18 (“s”, 1H, H3’), 3.98 (q, 1H, J=
3.8 Hz, H4’), 3.68–3.71 (m, 1H, H5’), 3.55–3.61 ppm (m, 1H, H5’’);
1H NMR ([D6]DMSO/D2O): d=8.71 (s, 1H, H2), 8.68 (s, 1H, H8), 8.24
(“d”, 2H, J=9.2 Hz, Ph), 7.88 (“d”, 2H, J=9.2 Hz, Ph), 6.00 (d, 1H,
J=5.7 Hz, H1’), 4.58 (t, 1H, J=5.4 Hz, H2’), 4.15 (“t”, 1H, J=4.3 Hz,
H3’), 3.99 (q, 1H, J=3.7 Hz, H4’), 3.68 (dd, 1H, J=3.8, 12.1 Hz, H5’),
3.57 ppm (dd, 1H, J=3.9, 12.2 Hz, H5’’); 13C NMR ([D6]DMSO): d=
150.9, 150.72, 150.66, 149.4, 144.5, 142.6 (C8), 142.2, 125.0, 120.5,
119.1, 87.7 (C1’), 85.6 (C4’), 73.7 (C2’), 70.1 (C3’), 61.1 ppm (C5’); MS
(ESI): m/z 432 [M+H]+ (100%); Anal. calcd for C17H17N7O7·H2O
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Received: February 26, 2014
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Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 8
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7
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These are not the final page numbers!

FULL PAPERS
R. Rayala, P. Theard, H. Ortiz, S. Yao,
J. D. Young, J. Balzarini, M. J. Robins,
S. F. Wnuk*
Transporters! The 4-N-(4-nitrobenzyl)
derivatives of tubercidin, toyocamycin
and sangivamycin inhibited cross-mem-
brane transport of labelled uridine by
nucleoside transporter hENT1 and prolif-
eration of L1210, HeLa, and PC-3 tumor
cells at micromolar levels. These com-
pounds represent tool compounds for
the continued study of the structure
and function of nucleoside transporter
proteins.
&& – &&
Synthesis of Purine and 7-
Deazapurine Nucleoside Analogues of
6-N-(4-Nitrobenzyl)adenosine;
Inhibition of Nucleoside Transport and
Proliferation of Cancer Cells
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 8
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8
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These are not the final page numbers!