2′-Deoxy-2′-azidonucleoside analogs: synthesis and evaluation of antitumor and antimicrobial activity

Article abstract of DOI:10.1007/s11696-017-0339-9

Abstract: A series of ten pyrimidine nucleosides modified in 2′ position with azide or amine group was tested for the antibacterial, antifungal and cytotoxic activity. The cytotoxic effect was determined on three cancer (CCRF-CEM, MCF7, HeLa) and one norm

Full text of DOI:10.1007/s11696-017-0339-9

Chem. Pap.
DOI 10.1007/s11696-017-0339-9
ORIGINAL PAPER
2
′‑Deoxy‑2′‑azidonucleoside analogs: synthesis and evaluation
of antitumor and antimicrobial activity
1
2
1,2
Adam Mieczkowski · Patrycja Wińska · Marta Kaczmarek
·
1
,3
1
1
Magdalena Mroczkowska · Damian Garbicz · Tomasz Pilżys ·
1
1
1
Michał Marcinkowski · Jan Piwowarski · Elżbieta Grzesiuk
Received: 9 August 2017 / Accepted: 5 November 2017
©
Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract A series of ten pyrimidine nucleosides modiꢁed
in 2′ position with azide or amine group was tested for the
antibacterial, antifungal and cytotoxic activity. The cyto-
toxic eꢂect was determined on three cancer (CCRF-CEM,
MCF7, HeLa) and one normal (HEK293) cell lines, while
antibacterial activity was evaluated on ꢁve bacterial strains.
Among others, 2′-azido-2′deoxycytidine and 2′-amino-
Graphical Abstract
2
′-deoxycytidine exhibited the strongest antiproliferative
activity at 200 μM concentration, decreasing the viability
of CCRF-CEM cells to 33 ± 1 and 36 ± 2%, respectively.
Newly synthesized 2′-amino-2′-deoxythymidine exhibited
cytotoxic eꢂect exclusively toward HeLa cancer cell line,
but not toward the normal HEK293 cells. Also, investigated
compounds did not exhibit any antibacterial or antifungal
activity at a concentration of 40 mM. The obtained results
suggest that the presence of cytosine base is desirable for the
appearance of cytotoxic eꢂect, while the structural varia-
tions of the sugar ring play a minor role. Future modiꢁcation
of 2′-amino-2′-deoxythymidine could be a promising way to
obtain more active anticancer substances.
Keywords Azidonucleosides · Cytotoxicity · Antifungal ·
Antibacterial · CCRF-CEM · MCF7 · HeLa HEK293 cell
lines
Introduction
Nucleoside derivatives which possess the azide group
(
Pathak 2002) have attracted the great attention of medicinal
chemists after the discovery that 3′-azido-3′-deoxythymidine
(
zidovudine, 1, Scheme 1) could act as an eꢀcient HIV
reverse transcriptase inhibitor and could be applied to the
treatment of acquired immune deꢁciency syndrome (AIDS)
(
Mitsuya et al. 1985) (Fig. 1). The utility and high poten-
tial of azide-modiꢁed nucleoside analogs were further sup-
ported by the discovery of 4′-azidocytidine (2, R1479), a
relatively potent (IC = 320 nM) inhibitor of RNA synthesis
*
Adam Mieczkowski
amiecz@ibb.waw.pl
50
mediated by NS5B, the RNA polymerase encoded by HCV
(Smith et al. 2007). Earlier reports have also revealed the
great potential of 2-azido-2′-deoxy-3-(β-d-arabinofuranosyl)
cytosine (3, Cytarazid) against various human tumor cell
lines (Bobek et al. 1978; Cheng et al. 1981; Matsuda et al.
1991) and HSV virus strains (Cheng et al. 1981). It was
1
2
3
Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland
Faculty of Chemistry, Warsaw University of Technology,
Noakowskiego 3, 00-664 Warsaw, Poland
Department of Chemistry, University of Warsaw, Pasteura 1,
0
2-093 Warsaw, Poland
Vol.:(0
1
123456789)
3

Chem. Pap.
ꢁ
rst designed as cytidine deaminase-resistant analog of the
did not exhibit any signiꢁcant binding activity, 2′-azido-
2′-deoxyuridine (4) exhibited strong binding eꢂect to this
enzyme, with inhibitory potency of 82.1 ± 28.5 μM (3.42
times greater potency than the reference compound—β-
d-uridine). Among the tested compounds, 2′-azido-
2′-deoxycytidine (5) was also the most active one
(IC = 0.05 ± 0.01 μM, SI = > 2030 ± 1347) against
anticancer compound—3-(β-d-arabinofuranosyl)cytosine
(
AraC).Scheme 1 Synthesis of 5-halogeno-2′-deoxy-2′-azidouridine
derivatives 8a–c
In the pyrimidine 2′-azido-2′-deoxyribonucleoside
series, only the unmodiꢁed 2′-azido-2′-deoxyuridine (4)
and 2′-azido-2′-deoxycytidine (5) are vastly described in
the literature, ignoring the remaining representatives of
the class. Reported data are greatly focused on interac-
tions of compounds 4 and 5 with diꢂerent types of cellu-
lar enzymes. Because of the lack of the 2′-hydroxy group,
pyrimidine 2′-azido-2′-deoxynucleosides are recognized
and phosphorylated to the appropriate monophosphates
by human deoxyribonucleoside salvage enzymes, such
as deoxycytidine kinase (dCK) (Iwata et al. 1979; Wanf
et al. 1998; Kierdaszuk et al. 1998, 1999). The diphos-
phate of 2′-azido-2′-deoxycytidine (5) exhibits strong
inhibition eꢂect on ribonucleotide reductase, a pivotal
enzyme involved in the DNA synthesis, which trans-
forms ribonucleosides into their 2′-deoxyribonucleoside
analogs (Akerblom 1985; Wnuk et al. 2002; Roy et al.
5
0
a highly pathogenic avian H5N1 inꢃuenza A virus strain
(Kumaki et al. 2011), Vietnam/1203/2004. Also, it inhib-
ited polyoma virus DNA replication (Bjursel 1977, 1978;
Eliasson et al. 1981). In another report, this compound
exhibited signiꢁcant inhibitory eꢂect against leukemic
L1210 cell line, rather weak antiviral activity against vac-
cinia virus, and lack of signiꢁcant activity against HSV
(Herpes Simples Virus) and VSV (vesicular Stomatitis
Virus) virus strains (de Clercq et al. 1980).
Although 2′-azido-2′-deoxycytidine (5) does not exhibit
any signiꢁcant antiretroviral activity, because of its ribo-
nucleotide reductase-inhibition activity, it can be used as a
potentiator of anti-HIV drugs, such as AZT (Giacca et al.
1994, 1996). Cytidine deaminase, an enzyme responsible for
deactivation of cytidine-derived anticancer drugs, e.g., cyta-
rabine, by limiting their bioavailability and half-life time, is
also strongly inhibited by azidonucleoside 5 (Cacciamani
et al. 1991). Consequently, due to its cytidine deaminase-
inhibition activity, compound 5 could extend the bioavail-
ability and half-life time and decrease therapeutic doses and
side eꢂects of several anticancer drugs. Polynucleotides pos-
sessing 2′-azido-2′-deoxyuridine (4) were also investigated
as possible interferon inducers (Torrence et al. 1973a, b).
In our ongoing quest for the design and synthesis of
new medicinally relevant nucleoside analogs and hetero-
cyclic derivatives this reference should be: Mieczkowski
et al. 2015, 2016a, b), we envisioned the possibility of
synthesizing novel, 2′-azido modiꢁed pyrimidine nucleo-
sides. Because of the lack of synthetic and biological
data corresponding to the modiꢁed pyrimidine 2′-azido-
2′-deoxyribonucleosides in literature, we decided to syn-
thesize novel analogs of 2′-azido-2′-deoxyuridine (4) and
2′-azido-2′-deoxycytidine (5) and compared their biological
2
004). Additionally, in its 5′-triphosphate form, it also
inhibited the action of primase in a reconstructed Escheri-
chia coli enzyme system (Reichard et al. 1978). During
investigation of structural requirements and determinants
for nucleoside aꢀnity to the proteins responsible for the
nucleoside transport through cell membranes, 2′-azido-2′-
deoxyuridine (4) exhibited rather high aꢀnity to the human
concentrative nucleoside transporters (hCNTs) (hCNT1 K
i
[
μM] = 11.5 ± 0.5, hCNT3 K [μM] = 33.3 ± 2.0, hCNT2
i
K [μM] > 3000) (Zhang et al. 2003, 2005) and human
i
equilibrative nucleoside transporters (hENTs) (hENT1 K
i
[
μM] = 13 ± 1, hENT1 K [μM] = 169 ± 14) (Vickers
i
et al. 2004), investigated in the yeast expression system.
2
′-Azido-2′-deoxyuridine (4) and 2′-azido-2′-deoxycytidine
(
5) were both tested in studies determining the eꢂects of
modiꢁcations in the pentose moiety and conformational
changes on the binding of nucleoside ligands to uridine
phosphorylase (UrdPase) from Toxoplasma gondii (el
Kouni et al. 1996). While 2′-azido-2′-deoxycytidine (5)
O
NH2
NH2
O
NH2
H C
3
N
NH
NH
N
N
N
O
N
O
N
O
N
O
N
O
O
O
O
O
O
HO
HO
HO
HO
HO
N3
HO
HO
N3
HO
N3
N3
N3
HO
N3
1
2
3
4
5
Fig. 1 Representative, medicinally relevant pyrimidine azidonucleosides: 1 zidovudine (anti-HIV), 2 R1479 (anti-HCV), 3 Cytarazid (antican-
cer, anti-HSV), 4 2′-azido-2′-deoxyuridine, 5 2′-azido-2′-deoxycytidine (antiviral, anticancer)
1
3

Chem. Pap.
activities against cancer lines, bacteria and fungi with parent
compounds. For comparative studies and investigation of
structure–activity relationships, we also used three 2′-amino-
brine. The organic phase was dried over magnesium sulfate,
and the volatiles were evaporated under reduced pressure.
The solid residue was puriꢁed by column chromatography
on silica gel using toluene:ethyl acetate, 9:1–8:2. Yield:
2
′-deoxy pyrimidine nucleoside analogs.
0
.32 g (28%). Compound 7a was obtained as white crystals,
1
m.p.: 78.4–78.9 °C. H NMR (500 MHz, CDCl ): 8.96 (bs,
3
3
Experimental
1H, NH), 7.99 (s, 1H, H ), 6.24 (d, 1H, J = 7.0 Hz, 1H, H ),
6
1′
3
3
4
.35 (dd, J = 2.0, 5.0 Hz, 1H, H ), 4.10 (q, J = 2.0 Hz,
3
′
3
2
Commercially available chemicals were of reagent grade
purity and used as received. 2,2′-Anhydrouridine, 2,2′-anhy-
drothymidine (12), 2′-azido-2′-deoxycytidine (3), 2′-amino-
1H, H ), 3.96 (dd, J = 2.0 Hz, J = 12.0 Hz, 1H, H ), 3.77
4′
5′b
3
3
3
(dd, J = 2.0 Hz, J = 12.0 Hz, 1H, H ), 3.51 (dd, J = 5.0,
5
′a
7.0 Hz, 1H, H ), 0.95 (s, 18H, 2 × t-Bu ), 0.19 (s, 3H,
2
′
Si
2
′-deoxyuridine (15) and 2′-amino-2′-deoxycytidine (16)
Me ), 0.17 (s, 3H, Me ), 0.16 (s, 3H, Me ), 0.14 (s, 3H,
Si Si Si
1
3
were purchased from Carbosynth (UK). The reactions were
monitored by thin layer chromatography (TLC) analysis
using silica gel plates (Kieselgel 60F , E. Merck). Col-
Me ); C NMR (125 MHz, CDCl ): 158.6, 149.5, 138.5,
Si 3
97.7, 86.9, 85.8, 73.5, 65.6, 62.6, 26.1, 25.7, 18.5, 18.1,
+
− 4.7, − 5.0, − 5.2, − 5.3. HRMS (ESI): m/z [M+H] cal-
2
54
umn chromatography was performed on silica gel 60 M
culated for C H BrN O Si : 576.16676, found: 576.16672.
21
39
5
5
2
(
0.040–0.063 mm, E. Merck). Melting points were uncor-
1
13
rected and measured on a Koꢃer apparatus. The H and
C
2′‑azido‑2′‑deoxy‑5‑iodo‑3′,5′‑O‑di(t‑butyldimethylsilyl)
NMR spectra were recorded at the Department of Chemis-
try, Warsaw University, using Varian Unity Plus spectrom-
eter (500 MHz) and Bruker AVANCE III HD (300 MHz)
spectrometer, in CDCl and DMSO-d , with shift values in
uridine (7b)
1.00 g (2.0 mmol, 1 eq.) of 2′-azido-2′-deoxy-3′,5′-O-di(t-
butyldimethylsilyl)uridine (6) was dissolved in 50 ml of
dichloromethane and 0.88 g (4.0 mmol, 2 eq.) of silver trif-
luoroacetate, followed by the addition of 1.52 g (6.0 mmol,
3 eq.) of iodine, and reaction mixture was stirred at RT for
18 h. The inorganic silver salts were separated by ꢁltration
through Celite, and the organic phase was washed twice with
a saturated solution of sodium thiosulfate and once with
brine. The organic phase was dried over magnesium sulfate,
and the volatiles were evaporated under reduced pressure.
The solid residue was puriꢁed by column chromatography
on silica gel using toluene:ethyl acetate, 9:1 then 8:2. Yield:
3
6
parts per million relative to SiMe as internal reference. The
4
resonance assignments are based on peak integration, peak
multiplicity and 2D correlation experiments. Multiplets were
assigned as s (singlet), d (doublet), t (triplet), q (quartet),
dd (doublet of doublet), dt (doublet of triplet), ddd (doublet
of doublet of doublet), m (multiplet) and bs (broad singlet).
High-resolution mass spectra were performed by the Labo-
ratory of Mass Spectrometry, Institute of Biochemistry and
Biophysics PAS, on LTQ Orbitrap Velos, Thermo Scientiꢁc.
The antibacterial and antifungal activity of compounds was
assayed using standardized disc diꢂusion agar test. The disc
diꢂusion method was carried out according to the standard
method (Bauer et al. 1966). Clinically antimicrobial drugs,
kanamycin and amphotericin B, were used as reference drugs
and DMSO was used as a negative control. The results were
recorded as the average diameter of growth inhibition zones,
and for the reference drugs a minimum 20 mm of bacterial or
fungal growth around the discs was inhibited. For the tested
compounds, there were no growth inhibition zones observed.
0.96 g (72%). Compound 7b was obtained as white crystals,
1
m.p.: 150.0–150.3 °C. H NMR (500 MHz, CDCl ): 9.15 (s,
3
3
1H, NH), 8.00 (s, 1H, H ), 6.24 (d, 1H, J = 7.5 Hz, 1H, H ),
6
1′
3
3
4.36 (dd, J = 2.0, 5.0 Hz, 1H, H ), 4.10 (q, J = 2.0 Hz,
3
′
3
2
1H, H ), 3.93 (dd, J = 2.0 Hz, J = 11.5 Hz, 1H, H ), 3.76
4′
5′b
3
2
3
(dd, J = 2.0 Hz, J = 11.5 Hz, 1H, H ), 3.49 (dd, J = 5.0,
5
′a
7.5 Hz, 1H, H ), 0.96 (s, 9H, t-Bu ), 0.95 (s, 9H, t-Bu ),
2
′
Si
Si
0.19 (s, 3H, Me ), 0.18 (s, 3H, Me ), 0.17 (s, 3H, Me ),
Si
Si
Si
1
3
0.14 (s, 3H, Me ); C NMR (125 MHz, CDCl ): 159.8,
Si
3
1
50.0, 143.5, 87.0, 85.6, 73.7, 69.5, 65.4, 62.7, 26.3, 25.8,
2
′‑Azido‑5‑bromo‑2′‑deoxy‑3′,5′‑O‑di(t‑butyldimethylsi
18.6, 18.1, − 4.7, − 4.9, − 5.1, − 5.2. HRMS (ESI): m/z
+
lyl)uridine (7a)
[M+H] calculated for C H IN O Si : 624.15289, found:
2
1
39
5
5
2
6
24.15229.
1
.00 g (2.0 mmol, 1 eq.) of 2′-azido-2′-deoxy-3′,5′-O-di(t-
4
butyldimethylsilyl)uridine (6) was dissolved in 50 ml of
dichloromethane and 0.88 g (4.0 mmol, 2 eq.) of silver tri-
ꢃuoroacetate followed by addition of 0.31 ml (6.0 mmol,
2′‑Azido‑2′‑deoxy‑O ‑(2,4,6‑triisopropylbenzenesulfonyl)
‑3′,5′‑O‑di(t‑butyldimethylsilyl)uridine (9)
3
eq.) of bromine, and the reaction mixture was stirred at RT
To the solution of 0.50 g (1.0 mmol, 1 eq.) of 2′-azido-2′-
deoxy-3′,5′-O-di(t-butyldimethylsilyl)uridine (6) dissolved
in 30 ml of dry dichloromethane, 0.91 g (3.0 mmol, 3 eq.)
of TPSCl, 25 mg (0.2 mmol, 0.2 eq.) of DMAP and 0.41 ml
for 18 h. The inorganic silver salts were separated by ꢁltra-
tion through Celite, and the organic phase was washed twice
with a saturated solution of sodium thiosulfate and once with
1
3

Chem. Pap.
of TEA were added, and the reaction mixture was stirred
at RT for 18 h. The obtained solution was then evaporated
with silica gel and chromatographed using 100% toluene
2′-Azido-5-bromo-2′-deoxyuridine (8a) yield: 93%,
1
m.p.: 116.9–117.3 °C, white crystals. H NMR (300 MHz,
DMSO-d ): 11.89 (s, 1H, NH), 8.44 (s, 1H, H ), 5.93 (d,
6
6
3
3
and then toluene:ethyl acetate 9:1. Yield: 0.56 g (73%) of
J = 5.7 Hz, 1H, OH ), 5.77 (d, J = 4.5 Hz, 1H, H ),
3 1′
′
1
3
3
colorless oil. H NMR (500 MHz, CDCl ): 8.48 (d, 1H,
5.36 (t, J = 4.8 Hz, 1H, OH ), 4.33 (q, J = 5.7 Hz, 1H,
5′
3
3
3
3
J = 7.5 Hz, H ), 7.26 (s, 1H, H ), 7.21 (s, 1H, H ), 6.02
H ), 4.17 (dd, J = 4.5, 5.7 Hz, 1H, H ), 3.89 (dt, J = 2.7,
3′ 2′
6
Ar
Ar
3
3
3
2
(
d, 1H, J = 7.5 Hz, H ), 5.76 (d, 1H, J = 1.5 Hz, 1H,
5.7 Hz, 1H, H ), 3.73 (ddd, J = 2.7, 4.8 Hz, J = 12.3 Hz,
4′
5
3
3
2
H ), 4.31 (dd, J = 5.0, 7.0 Hz, 1H, H ), 4.25 (septet, 2H,
1H, H ), 3.59 (ddd, J = 2.7, 4.8 Hz, J = 12.3 Hz, 1H,
1
′
3′
5′b
3
3
13
J = 7.0 Hz, 2 × i-Pr), 4.06 (m, 2H, H + H ), 3.93 (dd,
H ); C NMR (125 MHz, DMSO-d ): 159.2, 149.7, 139.8,
4
′
5′b
5′a
6
+
J = 1.5, 5.0 Hz, 1H, H ), 3.76 (m, 1H, H ), 2.91 (septet,
96.0, 86.4, 84.9, 69.6, 65.2, 59.4. HRMS (ESI): m/z [M+H]
2
′
5′a
3
3
1
H, J = 7.0 Hz, i-Pr), 1.33 (d, 6H, J = 7.0 Hz, i-Pr), 1.27
calculated for C H BrN O : 347.99381, found: 347.99561.
9 11 5 5
3
3
(
d, 6H, J = 7.0 Hz, i-Pr), 1.26 (d, 6H, J = 7.0 Hz, i-Pr),
2′-Azido-5-iodo-2′-deoxyuridine (8b) yield: 64%, m.p.:
1
0
0
.94 (s, 9H, t-Bu ), 0.91 (s, 9H, t-Bu ), 0.13 (s, 3H, Me ),
146.0–146.3 °C, white crystals. H NMR (500 MHz, DMSO-
si
si
Si
3
.12 (s, 3H, Me ), 0.11 (s, 3H, Me ), 0.10 (s, 3H, Me );
d ): 11.76 (s, 1H, NH), 8.45 (s, 1H, H ), 5.93 (d, J = 5.5 Hz,
Si
Si
Si
6
6
1
3
3
3
C NMR (125 MHz, CDCl ): 167.3, 154.6, 153.8, 151.2,
1H, OH ), 5.77 (d, J = 4.5 Hz, 1H, H ), 5.34 (t, J = 5.0 Hz,
3
3′ 1′
3
3
1
2
45.6, 130.6, 124.1, 95.0, 89.2, 83.9, 69.1, 66.6, 60.2, 34.3,
1H, OH ), 4.33 (q, J = 5.5 Hz, 1H, H ), 4.16 (dd, J = 4.5,
5 3′
′
3
9.7, 26.0, 25.6, 24.7, 24.4, 23.5, 23.5, 18.4, 18.0, − 4.4,
5.5 Hz, 1H, H ), 3.90 (ddd, J = 2.5, 3.0, 5.5 Hz, 1H, H ),
2
′
4
′
+
3
2
−
5.0, − 5.3, − 5.6. HRMS (ESI): m/z [M+H] calculated
3.72 (ddd, J = 3.0, 4.5 Hz, J = 12.0 Hz, 1H, H ), 3.59
5
′b
3
2
13
for C H N O SSi : 764.39030, found: 764.38988.
(ddd, J = 2.5, 4.5 Hz, J = 12.0 Hz, 1H, H ); C NMR
3
6
62
5
7
2
5′a
(
125 MHz, DMSO-d ): 160.5, 150.1, 144.5, 86.3, 84.9, 79.2,
6
+
2
′‑Azido‑2′‑deoxy‑4‑oxime‑3′,5′‑O‑di(t‑butyldimethylsi
69.7, 69.6, 65.2, 59.5. HRMS (ESI): m/z [M+H] calculated
lyl)uridine (10)
for C H IN O : 395.97994, found: 395.98029.
9
11
5
5
2
′-Azido-2′-deoxy-4-oxime-uridine (11) yield: 55%,
1
A solution of 0.80 g of 9 (1.05 mmol, 1 eq.) dissolved in
m.p.: 192.2–192.4 °C, white crystals. H NMR (500 MHz,
4
1
0 ml of THF was treated with 0.62 ml of 50% hydrazine
DMSO-d ): 10.05 (s, 1H, OH), 9.66 (d, J(NH-H ) = 2.0 Hz,
6
5
3
solution in water (10.50 mmol, 10 eq.) and the reaction mix-
ture was stirred at RT for 1 h. The volatiles were evaporated
under reduced pressure, followed by co-evaporation with
1H, NH), 7.03 (d, J = 8.5 Hz, 1H, H ), 5.93 (m, 1H,
6
4
3
OH + H ), 5.61 (dd, J(NH-H ) = 2.0 Hz, J = 8.5 Hz,
3
′
1′
5
3
1H, H ), 5.09 (t, J = 5.0 Hz, 1H, OH ), 4.25 (dt, J = 3.5,
5
5′
3
2
0 ml of toluene (three times). The oily residue was chro-
5.0 Hz, 1H, H ), 3.88 (dd, J = 5.0, 7.0 Hz, 1H, H ), 3.85
3
′
2
′
3
13
matographed on silica gel using toluene:ethyl acetate 8:2.
(q, J = 3.5 1H, H ), 3.55 (m, 2H, H + H ); C NMR
4 5′a 5′b
′
1
Yield: 0.43 g (80%) of colorless oil. H NMR (500 MHz,
(125 MHz, DMSO-d ): 149.3, 143.1, 129.2, 99.1, 85.3, 84.3,
6
3
+
CDCl ): 8.83 (bs, 1H, NH), 7.10 (d, J = 8.5 Hz, 1H, H ),
71.2, 63.2, 60.9. HRMS (ESI): m/z [M+H] calculated for
3
6
3
3
6
.11 (d, 1H, J = 5.5 Hz, 1H, H ), 5.65 (d, J = 8.5 Hz, 1H,
C H N O : 285.09419, found: 285.09299.
1
′
9
13
6
5
3
H ), 4.35 (dd, J = 3.5, 5.5 Hz, 1H, H ), 4.02 (m, 1H, H ),
5
3′
4′
3
2
3
.90 (dd, J = 2.5 Hz, J = 11.5 Hz, 1H, H ), 3.73 (dd,
The synthesis of 2′‑azido‑5‑chloro‑2′‑deoxyuridine (8c)
5
′b
3
2
3
J = 1.5 Hz, J = 11.5 Hz, 1H, H ), 3.59 (t, J = 5.5 Hz,
5
′a
1
3
H, H ), 0.94 (s, 9H, t-Bu ), 0.92 (s, 9H, t-Bu ), 0.17 (s,
The solution of 0.38 g of 4 (0.68 mmol, 1 eq.) dissolved in
5 ml of dry THF was treated with 0.1 ml of sulfuryl chloride
and the reaction mixture was stirred overnight at RT. The
volatiles were evaporated under reduced pressure, and the
oily residue was chromatographed on the silica gel using 5%
2
′
Si
Si
1
3
H, Me ), 0.13 (s, 3H, Me ), 0.10 (s, 6H, 2xMe );
C
Si
Si
Si
NMR (125 MHz, CDCl ): 149.4, 145.1, 129.9, 98.7, 85.6,
3
8
5.4, 72.7, 65.0, 62.3, 25.9, 25.7, 18.4, 18.1, − 4.7, − 5.0,
+
−
5.5, − 5.6. HRMS (ESI): m/z [M+H] calculated for
C H N O Si : 513.26715, found: 513.26632.
methanol in chloroform. Yield: 85 mg (41%) of colorless
2
1
41
6
5
2
1
oil. H NMR (300 MHz, DMSO-d ): 11.92 (s, 1H, NH),
6
3
General procedure for silyl group removal
′,5′-O-di(t-butyldimethylsilyl)nucleosides 7a–b and 10 (1 eq)
8.36 (s, 1H, H ), 5.93 (d, J = 5.4 Hz, 1H, OH ), 5.77 (d,
6
3′
3
3
J = 4.5 Hz, 1H, H ), 5.36 (t, J = 4.5 Hz, 1H, OH ), 4.33
1
′
5′
3
3
3
(q, J = 5.4 Hz, 1H, H ), 4.15 (dd, J = 4.5, 5.4 Hz, 1H,
3
′
3
3
were dissolved in THF (10 ml per 1 mmol of nucleoside).
Then, the TBAF hydrate (3 eq) was added and the reaction
mixture was stirred at RT for 4 h. After evaporation of the
solvent and co-evaporation with toluene (20 ml, three times),
the residue was dissolved in methanol, evaporated with silica
gel and chromatographed using 5% methanol in chloroform.
H ), 3.89 (dt, J = 2.7, 5.4 Hz, 1H, H ), 3.73 (ddd, J = 2.7,
2
′
4
′
2
3
4.5 Hz, J = 12.3 Hz, 1H, H ), 3.60 (ddd, J = 2.7, 4.2 Hz,
5
′b
2
13
J = 12.3 Hz, 1H, H ); C NMR (125 MHz, DMSO-d ):
5
′a
6
159.1, 149.6, 137.4, 107.4, 86.5, 85.0, 69.6, 65.2, 59.5.
+
HRMS (ESI): m/z [M+H] calculated for C H ClN O :
9
11
5
5
304.04432, found: 304.04433.
1
3

Chem. Pap.
2
′‑Azido‑2′‑deoxy‑5‑methyluridine (13)
blood T lymphoblast cell line) were cultured in RPMI
medium (Gibco) supplemented with 10% fetal bovine
serum and antibiotics (100 U/mL penicillin, 100 µg/mL
The solution of 3.00 g of 2,2′-anhydrothymidine (12,
2
1
2.49 mmol, 1 eq) dissolved in 100 ml of dry DMF
streptomycin). Cells were grown in 75 cm cell culture
was treated with 0.58 g (22.48 mmol, 1.8 eq) of lithium
ꢃuoride, 2.98 ml of azidotrimethylsilane (22.48 mmol,
ꢃasks (Sarstedt) in a humidiꢁed atmosphere of CO /air
2
(5/95%) at 37 °C. Cervical cancer cells HeLa and human
embryonic kidney cells HEK293 were cultured in DMEM
medium (Life Technology) supplemented with 10% fetal
bovine serum (Life Technology) and 0.1% antibiotics (pen-
icillin, streptomycin, Life Technology). Cells were grown
1
.8 eq) and 3.37 ml of TMEDA (22.48 mmol, 1.8 eq).
The reaction mixture was stirred for 48 h at 110–115 °C.
The volatiles were evaporated under reduced pressure and
the oily residue was chromatographed on the silica gel
using 10% methanol in chloroform. Yield: 1.59 g (45%)
in a humidiꢁed atmosphere of CO /air (5/95%) at 37 °C.
2
1
of colorless oil. H NMR (500 MHz, DMSO-d ): 11.40
6
4
®
(
(
s, 1H, NH), 7.72 (q, J(Me-H ) = 1.0 Hz, 1H, H ), 5.95
MTT‑based cytotoxicity and alamarBlue cell viability
6
6
3
3
d, J = 5.5 Hz, 1H, OH ), 5.90 (d, J = 5.5 Hz, 1H,
assay
3
′
3
H ), 5.20 (t, J = 5.0 Hz, 1H, OH ), 4.31 (m, 1H, H ),
1
′
5′
3′
3
4
.04 (t, J = 5.5 Hz, 1H, H ), 3.89 (m, 1H, H ), 3.68
Before the treatment, MCF7 cells were trypsinized in 0.25%
trypsin–EDTA solution (Sigma-Aldrich) and seeded into
2
′
4′
3
2
(
ddd, J = 3.5, 5.0 Hz, J = 12.0 Hz, 1H, H ), 3.58 (ddd,
5′b
3
2
4
4
J = 3.5, 5.0 Hz, J = 12.0 Hz, 1H, H ), 1.78 (d, J (Me-
96-well microplates at a density of 1.5 × 10 cells/well.
5
′a
1
3
H ) = 1.0 Hz, 1H, Me). C NMR (125 MHz, DMSO-
Cells were treated with speciꢁc compounds dissolved in
DMSO or DMSO (0.5%) at the corresponding concentra-
tions, 18 h after plating (at 70% of conꢃuency). CCRF-CEM
6
d ): 163.7, 150.5, 135.7, 109.7, 85.3, 85.2, 70.6, 64.3,
6
+
6
0.4, 12.3. HRMS (ESI): m/z [M+H] calculated for
4
C H N O : 284.09895, found: 284.09914.
were seeded at 10 cells/well and treated with the tested
1
0
14
5
5
compounds. MTT stock solution (Sigma-Aldrich) was added
to each well to a ꢁnal concentration of 0.3 mg/mL. After 2 h
of incubation at 37 °C, water-insoluble dark blue formazan
crystals were dissolved in DMSO (200 µL) (37 °C/10 min
incubation), and Sorensen’s glycine buꢂer (0.1 M glycine,
0.1 M NaCl, pH 10.5) was added (25 μL per well). Opti-
cal absorbance was measured at 570 nm using Synergy H4,
BioTek microplate reader. All measurements were carried
out in triplicate and the results are expressed in percentage
of cell viability relative to control (cells without inhibitor
in 0.5% DMSO). HeLa and HEK293 cells were seeded in
2
′‑Amino‑2′‑deoxy‑5‑methyluridine (14)
1
.00 g (3.53 mmol) of 2′-azido-2′-deoxy-5-methyluridine
(
13) was dissolved in 40 ml of isopropyl alcohol, and
1
00 mg of 10% palladium on charcoal was added. The
reaction vessel was connected to a balloon ꢁlled with
hydrogen and the reaction mixture was stirred at room
temperature for 20 h. The resultant solution was ꢁltered
through Celite and evaporated giving 772.0 mg (85%) of
1
3
1
4 as a colorless oil. H NMR (500 MHz, DMSO-d ): 7.67
96-well microplates at a density of 7 × 10 cells/well. After
6
4
3
(
q, J(Me-H ) = 0.5 Hz, 1H, H ), 5.66 (d, J = 8.0 Hz,
18 h, cells were treated with test compounds dissolved in
DMSO at the corresponding concentrations for 24 h. Then,
10% Alamar Blue (Invitrogen) was added according to the
manufacturer’s protocol. After 4 h incubation, emission at
585 nm was measured with excitation at 570 nm, using a
scanning multi-well spectrophotometer, DTX 880.
6
6
3
1
H, H ), 3.90 (dd, J = 1.5, 5.5 Hz, 1H, H ), 3.89 (dd,
1
′
3
′
3
J = 1.5, 3.5 Hz, 1H, H ), 3.56 (m, 2H, H ), 3.30 (dd,
4
′
5′
3
4
J = 5.5, 8.0 Hz, 1H, H ), 1.78 (d, J (Me-H ) = 0.5 Hz,
2
′
6
1
1
1
H, Me). Acidic protons: NH, OH, NH not present in
2
1
3
H-NMR spectra. C NMR (125 MHz, DMSO-d ): 163.8,
6
52.2, 136.5, 109.4, 87.7, 85.9, 71.3, 61.7, 57.2, 12.3.
+
HRMS (ESI): m/z [M+H] calculated for C H N O :
1
0
16
3
5
2
58.10845, found: 258.10874.
Results and discussion
The starting point of our synthesis was the 2′-azido-2′-
deoxy-3′,5′-O-di(t-butyldimethylsilyl)uridine (6) prepared
according to the reported procedure (Gai et al. 2010). Silyl
protected nucleoside 6 was then brominated in the C5 posi-
tion using the Br /CF COOAg system, which resulted in the
Cell culture and treatment
MCF7 adherent cells (human breast cancer cell line) were
cultured in DMEM medium (Lonza) supplemented with
2
3
1
0% fetal bovine serum (Biowest), 2 mM l-glutamine,
formation of the bromo derivative 7a with 28% yield. In a
similar manner, derivative 6 was iodinated in the C5 posi-
tion with I /CF COOAg (Kobayasji et al. 1980), giving the
antibiotics (100 U/ml penicillin, 100 µg/mL streptomy-
cin) and 10 µg/mL of human recombinant insulin (Sigma-
Aldrich). CCRF-CEM suspension cells (human peripheral
2
3
iodo derivative 7b with 72% yield (Scheme 1). Alternatively,
1
3

Chem. Pap.
iodine monochloride ICl could be used for the synthesis of
b (Gold et al. 1995). Silyl intermediates 7a–b were then
2005). 2′-Azidothymidine (13) was then dissolved in iso-
propyl alcohol and hydrogenated in the presence of cata-
lytic amount of 5% palladium on activated charcoal, giving
2′-aminothymidine (14) with 85% yield. The reduction of
azide to the amine group could also be done with triph-
enylphosphine in ammonium hydroxide (Pokrovskii et al.
2005).
7
deprotected with the TBAF hydrate in THF to the ꢁnal
nucleosides 8a and 8b with 93 and 64% yield, respectively.
The treatment of protected nucleoside 6 with sulfuryl chlo-
ride in DCM resulted in the formation of 5-chloro deriva-
tive with concurrent cleavage of the silyl groups. The depro-
tected 3-chloro nucleoside 8c was isolated as a sole product
with 41% yield.
In the initial experiments, the synthesized compounds
4, 8a–c, 11, 13, 14 and commercially available reference
substances 5, 15 and 16 were tested for their antiprolifera-
tive activity on MCF7 (human breast adenocarcinoma) and
CCRF-CEM (T cell lymphoblastic leukemia) cell lines
(Table 1). The investigated compounds possessed sub-
stituent variations on sugar (N versus NH in C posi-
Silyl protected nucleoside 6 was also transformed into
4
the O -TPS derivative 9 with 73% yield, using TPSCl, in the
presence of triethylamine excess and catalytic amounts of
4
-(dimethylamino)pyridine (Scheme 2) (Hari et al. 2011).
The obtained intermediate 9 was treated then with hydroxy-
lamine solution in THF, which resulted in the formation of
oxime derivative 10 with 80% yield, and deprotected to the
3
2
2′
tion) and/or pyrimidine ring (H, Cl, Br, I in C position,
5
C=O and C=N–OH in C position), giving an attractive
4
ꢁ
nal product 11 with the TBAF hydrate in THF (55% yield).
pool for the investigation of structure–activity relation-
ships. The most active compound in the 2′-azido-2′-
deoxy series, the 2′-azido-2′-deoxycytidine (5), decreased
cell viability at 200 μM concentration to 33 ± 1% for the
CCRF-CEM cell line. The structural modifications of
2
′-Azido-2′-deoxyuridine (4) was synthesized accord-
ing to a literature procedure (Kirschenheuterer al. 1994)
and the reported conditions were then applied to the syn-
thesis of 2′-azido-2′-deoxy-5-methyluridine (2′-azidothy-
midine, 13). The treatment of 2,2′-anhydrothymidine (12)
with lithium ꢃuoride and azidotrimethylsilane in DMF, in
the presence of TMEDA at 110–115 °C, led to the opening
of the anhydro bridge and formation of 2′-azido derivative
4
2′-azido-2′-deoxycytidine (5) led to the N -hydroxy-2′-
azido-2′-deoxycytidine (11) (introduction of the –OH
4
group in the N position of the cytidine ring) and 2′-azido-
2′-deoxyuridine (4) (exchange of cytosine into the uracil
ring)—compounds deprived of antiproliferative activity.
5-Chloro- (8c) 5-bromo- (8a), 5-iodo (8b) and 5-methyl
derivative (13) also did not exhibit any significant
1
3 with 45% yield (Scheme 3). The opening of anhydro-
nucleoside 12 could be also performed with lithium azide/
HMPA instead of azidotrimethylsilane (Pokrovskii et al.
O
N
O
N
O
O
Cl
X
X
NH
NH
I or Br ,
NH
NH
2
2
SO Cl
CF COOAg
2
2
3
TBAF
O
O
N
O
N
O
O
O
O
O
DCM, rt,
8 hrs
18 hrs, rt
DCM, rt
HO
TBDMSO
TBDMSO
TBDMSO
TBDMSO
HO
1
1
8 hrs
HO
N3
N3
N3
HO
N3
8
c
6
7a X = Br
8a X = Br
8b X = I
7
b X = I
Scheme 1 Synthesis of 5-halogeno-2′-deoxy-2′-azidouridine derivatives 8a–c
OH
OH
NH
O
N
OTPS
N
N
N
NH
N
N
TPSCl, DMAP
TEA
NH
NH OH
2
TBAF
O
O
O
N
O
O
O
O
DCM, rt,
THF, rt, 1h
O
18 hrs, rt
TBDMSO
TBDMSO
TBDMSO
TBDMSO
TBDMSO
TBDMSO
HO
18 hrs
N3
N3
N3
HO
N3
6
9
10
11
Scheme 2 Synthesis of 4-oxime-uridine derivative 11
1
3

Chem. Pap.
O
N
O
N
O
N
H C
H C
CH3
3
3
NH
NH
N
O
LiF, TMEDA,
Me SiN
3
3
Pd/C/H2
O
O
O
O
O
O
i-PrOH, 20 hrs
DMF, 110 C
HO
HO
HO
HO
HO
N3
HO
NH2
1
2
13
14
Scheme 3 Synthesis of 2′-azidothymidine (13) and 2′-aminothymidine (14)
Table 1 Antiproliferative activity against MCF7 and CCRF-CEM
cell lines of 2′-amino and 2′-azidonucleoside analogs
demonstrate any signiꢁcant eꢂect on cell growth. The above
results suggest that in the case of CCRF-CEM and MCF-
7
cell lines, only the cytidine derivatives 5 and 16 exhibit
Compound
Concentration 200 µM (cell viability
± SD, after 24 h incubation)
%
noticeable cytotoxic eꢂect, and apparently the presence of
cytosine ring is required for the antiproliferative eꢂect. The
modiꢁcations of the sugar ring did not alter cell viability, as
both 2′-azide- and 2′-amino derivatives showed similar anti-
proliferative eꢂect. As for the cell lines, CCRF-CEM cells
were more susceptible to cytidine derivatives, while MCF7
cells did not exhibit signiꢁcant susceptibility. In comparison
to CCRF-CEM and MCF7, HeLa and HEK293 cell lines
were less susceptible to the tested compounds. Because of
the minor eꢂect on HeLa and HEK293 cell survival, most
of the examined 2′-modiꢁed nucleosides seem to be unsuit-
able scaꢂolds for novel anticancer drugs. However, the ꢁnd-
ing that newly synthesized compound 14 showed cytotoxic
eꢂect exclusively on cancer cell line HeLa and not on nor-
mal HEK293 is extremely interesting. Future modiꢁcation of
this particular compound could be a promising way to obtain
more powerful anticancer substances.
MCF7
CCRF-CEM
4
5
101 ± 2
82 ± 5
98 ± 3
100 ± 2
97 ± 4
95 ± 3
96 ± 3
94 ± 2
94 ± 2
89 ± 4
102 ± 5
33 ± 1
101 ± 1
103 ± 5
99 ± 6
92 ± 7
95 ± 7
101 ± 6
70 ± 6
36 ± 2
8
8
8
1
1
1
1
1
a
b
c
1
3
4
5
6
antiproliferative activity against both lines of cancer cells.
In the 2-amino-2′-deoxy- series, 2′-amino-2′-deoxycytidine
(
16) showed the highest cytotoxic activity, decreasing cell
2′-Azido-2′-deoxy- and 2′-amino-2′-deoxy- nucleoside
derivatives were evaluated for their antibacterial and anti-
fungal (against Candida albicans ATCC 10231) activities.
Antibacterial studies were carried out against two Gram-
positive bacterial strains (Staphylococcus aureus ATCC
6538 and Bacillus subtilis ATCC 6633) and three Gram-
negative bacterial strains (E. coli ATCC 8739, Salmonella
typhimurium ATCC 14028 and Pseudomonas aeruginosa
ATCC 9027). The results showed that all the synthesized
compounds did not exhibit antibacterial or antifungal activ-
ity at 40 mM concentration.
viability at 200 μM concentration to 36 ± 2% for CCRF-
CEM, with an eꢂect similar to the one observed for 2′-azido-
2
′-deoxycytidine (5). The exchange of the azido group into
the amino group in compound (5) had almost no inꢃuence on
the cytotoxic eꢂect on the investigated cell lines. In contrast,
uridine analogs of compound 16: 2′-amino-2′-deoxuridine
(
15) and its 5-methyl derivative (14) did not decrease cell
viability at 200 μM concentration.
In further experiments, the investigated compounds 4, 5,
8
a–c, 13, 14, 15 and 16 were tested for their antiproliferative
activity with the use of HeLa (cervical cancer) and Hek293
(
human embryonic kidney) cell lines (Fig. 2).
The obtained results showed that compounds 8a and 14
Conclusions
exhibited the strongest antiproliferative eꢂect on HeLa and
Hek293 cells. Compound 8a showed similar cytotoxic eꢂect
on both HEK293 and HeLa cell lines (IC approximately
A series of ten pyrimidine nucleosides modified at the
2′ position with azide or amine group was subjected
to the investigation of biological activity. The tested
compounds exhibited a rather weak cytotoxic effect.
5
0
3
.5 mM), whereas molecule 14 exhibited toxic eꢂect only on
HeLa cells (IC about 1.7 mM). Other compounds did not
5
0
1
3

Chem. Pap.
Fig. 2 Cytotoxic properties of 2′-amino and 2′-azidonucleosides against HeLa and HEK293 cell lines
2
′-Azido-2′-deoxycytidine and 2′-amino-2′-deoxycytidine
thymidine kinases TK1 and TK2, we are currently working
on the synthesis of appropriate monophosphate pro-drugs of
the obtained 2′-azido-2′-deoxynucleoside analogs, followed
by investigation and comparison of the cytotoxic eꢂects of
nucleosides and their 5′-monophosphate esters. These results
will be published in due course.
showed antiproliferative activity against CCRF-CEM cells,
while newly synthesized 2′-amino-2′-deoxythymidine exhib-
ited cytotoxic eꢂect exclusively toward HeLa cancer cell
line, but not toward the normal HEK293 cells. The lack of
inhibition of cell growth by 2′-azido-2′-deoxyuridine ana-
logs could be attributed to the poor phosphorylation by TK1
and TK2 enzymes to appropriate monophosphates, a key
determinant responsible for exhibiting the cytotoxic eꢂect.
Although in rare cases the nucleoside derivatives did not
require transformation to appropriate monophosphates,
the observed biological eꢂect is related to the non-nucleo-
sidic mode of action (McGuigan et al. 2004). Therefore, to
increase the bioavailability of synthesized compounds and to
overcome the problem with the ꢁrst phosphorylation step, by
Acknowledgements The equipment used was sponsored in part by
the Centre for Preclinical Research and Technology (CePT), a project
co-sponsored by European Regional Development Fund and Innovative
Economy, The National Cohesion Strategy of Poland. We thank Jacek
Olędzki (IBB PAS) for recording ES-MS spectra.
Compliance with ethical standards
Conꢀict of interest The authors declare that they have no conꢃict
of interest.
1
3

Chem. Pap.
Kierdaszuk B, Krawiec K, Kazimierczuk Z, Jacobsson U, Johansson
NG, Munch-Petersen B, Eriksson S, Shugar D (1998) Substrate/
inhibitor speciꢁcities of human deoxycytidine kinase (dCK) and
thymidine kinases (TK1 and TK2). In: Griesmacher A et al (eds)
Purine and pyrimidine metabolism in man IX. Plenum, New York,
pp 623–627
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