Synthesis and in vitro growth inhibitory activity of novel silyl- and trityl-modified nucleosides

Article abstract of DOI:10.1016/j.bmc.2016.04.036

Seventeen silyl- and trityl-modified (5′-O- and 3′,5′-di-O-) nucleosides were synthesized with the aim of investigating the in vitro antiproliferative activities of these nucleoside derivatives. A subset of the compounds was evaluated at a fixed concentra

Full text of DOI:10.1016/j.bmc.2016.04.036

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro growth inhibitory activity of novel silyl- and
trityl-modified nucleosides
Jenny-Lee Panayides a,b, Véronique Mathieu c, Laetitia Moreno Y. Banuls c, Helen Apostolellis d,
Nurit Dahan-Farkas d, Hajierah Davids d,e, Leonie Harmse d, M. E. Christine Rey f, Ivan R. Green g,
Stephen C. Pelly g, Robert Kiss c, Alexander Kornienko h, Willem A. L. van Otterlo a,g,
⇑
a Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, PO Wits, Johannesburg 2050, South Africa
b Pioneering Health Sciences, CSIR Biosciences, PO Box 395, Pretoria 0001, South Africa
c Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium
d Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, PO Wits, Johannesburg 2050, South Africa
e Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa
f School of Molecular and Cellular Biology, University of the Witwatersrand, PO Wits, Johannesburg 2050, South Africa
g Department of Chemistry and Polymer Science, Stellenbosch University, Stellenbosch, Matieland 7602, South Africa
h Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA
a r t i c l e i n f o
a b s t r a c t
Seventeen silyl- and trityl-modified (50-O- and 30,50-di-O-) nucleosides were synthesized with the aim of
Article history:
Received 16 March 2016
Revised 14 April 2016
Accepted 19 April 2016
Available online xxxx
investigating the in vitro antiproliferative activities of these nucleoside derivatives. A subset of the
compounds was evaluated at a fixed concentration of 100 lM against a small panel of tumor cell lines
(HL-60, K-562, Jurkat, Caco-2 and HT-29). The entire set was also tested at varying concentrations against
two human glioma lines (U373 and Hs683) to obtain GI50 values, with the best results being values of
ꢀ25
lM.
Keywords:
Nucleosides
Ó 2016 Elsevier Ltd. All rights reserved.
Silyl protecting groups
Antiproliferative activities
tert-Butyldimethylsilyl
Dimethoxytrityl
1,3-Dihydroxy-1,1,3,3-
tetraisopropyldisiloxane
1. Introduction
(low-grade lymphomas and chronic lymphocytic leukemia13) and
cladribine (low-grade lymphomas,14 chronic lymphocytic
In recent years, nucleoside derivatives and analogues have
found significant application in the field of oncology.1 Of interest
is that ꢀ10 nucleoside base-derived compounds are currently
approved for clinical use2 and there is still much interest in the
development of newer generations of nucleoside compounds with
anticancer properties.3,4 Both purine and pyrimidine nucleoside
analogues are currently in clinical use as antimetabolites, with
examples including the pyrimidine analogues cytarabine5 (myel-
ogenous leukemia, multiple myeloma, Hodgkin’s and non-Hodg-
kin’s lymphomas)6 and gemcitabine7 (pancreatic cancer,8
metastatic bladder cancer,9 non-small cell lung cancer,10 breast,11
ovarian and neck cancers12), the purine analogues fludarabine
leukemia,15 hairy cell leukemia15 and non-Hodgkin’s lymphoma16
)
and the fluoropyrimidines, for example, 50-fluorouracil (gastroin-
testinal, pancreatic, head and neck, renal, skin, prostate, breast
cancers)17 and its prodrug capecitabine17 (metastatic colorectal
cancer,18 metastatic breast cancer19).
In 2006, Herczegh and co-workers noted that silylated leinamy-
cin antibiotic nucleoside analogues, such as compound 1 shown in
Figure 1, had significantly higher in vitro growth inhibitory activity
toward human cancer cells than their ‘free’ non-protected
derivatives.20 These authors postulated that the effect was due to
differences in lipophilic character imparted by the tert-
butyldimethylsilyl (TBDMS) groups used. In a more recent set of
studies, Peterson and colleagues disclosed the synthesis and
antiproliferative activities of
a
number of N-6,50-bis-urei-
⇑
Corresponding author at present address: De Beers Building, Office 1003, Cnr:
Merriman Avenue/De Beers St, Stellenbosch University, Stellenbosch 7600, South
Africa. Tel.: +27 21 808 3344; fax: +27 21 808 3360.
doadenosine derivatives containing one or two TBDMS
groups—see for example, compound 2, as depicted in Figure 1.21
These researchers further developed these scaffolds22–25 and also
E-mail address: wvo@sun.ac.za (W.A.L. van Otterlo).
http://dx.doi.org/10.1016/j.bmc.2016.04.036
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Panayides, J.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.036

2
J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
N
O
N
With this in mind, and based on a recent disclosure from our
Me
research collaborations that these types of compounds show unu-
sual in vitro antiproliferative effects,48 it was decided to report on
the synthesis of a broader set of different nucleosides including pur-
ine and pyrimidines, each in turn modified with a wider range of
silyl-protecting groups at the 50- or 30,50-(di)hydroxyl groups. In
addition, the utilization of another lipophilic protecting group for
the 50-position, namely the trityl group, would also be investigated.
Due to our continued interest in the design and synthesis of novel
anticancer entities,44,49–54 the aim of this study was thus to provide
additional compounds for the study of in vitro growth inhibitory
effects displayed by silyl-derivatized nucleosides.
O
N
HN
N
NHPh
HO
NH
N
N
NH
O
O
MeHN
O
S
S
O
O
O
O
HN
O
O
O
O
O
Si
N
O
TBDMSO
OTBDMS
TBDMSO
OTBDMS
2
1
3
Figure 1. Examples of cytotoxic leinamycin nucleoside analogue 1,20 a bis-sila-
substituted N-6,50-bis-ureidoadenosine derivative 221 and 30-O-silatranylthymidine
3.27
investigated their mode of action and possible targets.23,26 In
another earlier, but related example, Fenlon and co-workers pub-
lished details of nucleoside-derivatives that contained carbon-oxy-
gen-silicon bonds in the form of silatrane functional groups.27 The
researchers tested the in vitro anti-cancer activity of 30-O-sila-
tranylthymidines, such as compound 3 (Fig. 1), against human
breast (MDA-MB-435) and central nervous system (SNB-19 and
2. Results and discussion
2.1. Chemistry
The main set of compounds targeted, comprised of a number of
nucleosides with different silyl substituents on the 50-O position.
All of these compounds were readily synthesized by the reaction
of the purchased nucleosides 5 with the various silyl chlorides, to
afford 6 or 7 as described in the boxed generalized reaction in
Scheme 1. As such, the nucleosides uridine and 5-methyluridine
were converted into their TBDMS- 4d and 8, ThexylDMS- 4e and
9, and TBDPS-derivatives 12 and 13, respectively. In addition, the
di-sila derivatives 10 and 11 were also generated from the same
nucleosides by reaction with the commercially available 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane. Finally, the TBDPS
derivatives of cytidine (14) and adenosine (15) were also prepared,
so that the library would contain representative members of both
the purine and pyrimidine families.
It was also of interest to see whether the masking of any other
functional groups present in the nucleosides would affect their
ability to act as in vitro growth inhibitory agents (for a detailed dis-
cussion on the molecular mechanisms of the nucleoside family, see
the following review55). To this end, the free alcohols of the TBDPS-
uridine derivative 12 were protected as their acetyl derivatives to
give 16, followed by the protection of the base NH as the benzoyl
derivative 17 (Scheme 2). In a similar manner, disila-10 was readily
converted into the benzoyl derivative 18, while treatment of 10
U251) cancer cell lines at a fixed concentration (100 lM) and found
them to exhibit modest activities in the high micromolar ranges
(for a review on silatranes and their applications see Ref. 28). It
is important to note that another class of silyl-containing
tert-butyldimethylsilyl-spiroaminooxathioledioxide (TSAO) com-
pounds have been found to be effective non-nucleoside reverse
transcriptase inhibitors (for examples, see review29 and recent
Refs. 30,31).
In terms of some final nucleoside examples, during a study
involving triorganosilyl derivatives of some biologically active
heterocyclic bases, Lukevits and co-workers32 showed that, in con-
trast to uridine, 50-O-tert-butyldimethylsilyluridine 4d (Fig. 2)
exhibited some anti-tumor activity. These researchers were also
able to show that 50-O-tert-butyldimethylsilyluridine 4d
suppressed the growth of HT-1080 (fibrosarcoma in human
lungs—96% inhibition at 279
lM) and NiH 3T3 cells (fibroblasts
in mice—95% inhibition at 279
lM) in culture. As expected, under
the same conditions, uridine showed a complete absence of any
in vitro activity, as it is a normal product of nucleoside synthesis.
The researchers also commented that, in addition to the lipophilic
properties imparted by the TBDMS groups, the oxygen–silicon
bond in these compounds appeared to be reasonably stable to
hydrolysis.32 Note should also be taken here of research performed
by the Schmalz group, in which they demonstrated that nucleo-
sides with unsaturated carbocyclic portions,33,34 as well as metal
derivatives of the same motif,35–37 benefited from having organosi-
lyl fragments as part of their structure (mainly on the 50 position,
structures not shown).
R'
R'' SiO
R'
Base
Base
Base
1.1 eq R'2R''SiCl
or [(iPr)2SiCl]2O
HO
O
O
O
Si
O
O
or
0.1 eq DMAP,
OH OH
OH OH
O
OH
pyridine, rt, 18 h
Si
6
7
5
As shown by the examples briefly described in the introduction,
observations that silyl derivatives of nucleosides afford compounds
with interesting antiproliferative activities, provided a motivation
for this present study. Other researchers,38–43 and ourselves,44 have
recently also noted that the introduction of bulky silyl groups has
resulted in increased antiproliferative activity of a variety of scaf-
folds – in fact, the introduction of silyl groups into pharmaceutically
relevant molecules has become an active area of research.45–47
O
O
N
O
R
R
R
NH
NH
NH
N
O
O
N
O
Si
O
O
Si
O
O
Si
O
O
O
OH OH
O
OH
OH OH
Si
R = H, 4e (99%)
R = H, 4d (88%)
R = Me, 9 (80%)
R = Me, 8 (93%)
R = H, 10 (85%)
R = Me, 11 (40%)
O
R
NH2
N
NH2
O
NH
N
N
N
NH
4a: R = SiMe3
N
O
N
O
N
N
O
Si
O
Si O
4b: R = SiEt3
4c: R = Si(i-Pr)3
Si
O
RO
O
O
O
O
4d: R = Si(t-Bu)Me2
4e: R = Si(CMe2CHMe2)Me2
OH OH
OH OH
OH OH
OH OH
15 (73%)
R = H, 12 (100%)
R = Me, 13 (100%)
14 (100%)
Figure 2. Trialkylsilyl derivatives of uridine 4a–e, synthesized by Lukevits and co-
workers.32
Scheme 1. Synthesis of the silyl derivatives of a number of nucleosides.
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J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
O
N
Bz
O
NH
N
py, Ac2O, rt, 18 h
Hünig's base,
N
O
12
Si
O
Si O
BzCl, py, rt, 2 h
O
O
OAc OAc
OAc OAc
17 (71%)
16 (89%)
O
O
Bz
O
NH
N
N
O
O
N
O
O
Na2CO3, TBAB, BzCl,
CH2Cl2 : H2O, rt, 2 h
CrO3, py, Ac2O,
CH2Cl2, rt, 2 h
Si
O
O
Si
O
10
O
O
OH
O
Si
Si
19 (80%)
18 (99%)
Scheme 2. Synthesis of additional uridine derivatives.
with chromium(VI)oxide/pyridine/acetic anhydride (1:2:1) also
afforded the keto-uridine derivative 19.
2.2.1. Effect of synthetic compounds on growth of cancer cell
lines and white blood cells
Having synthesized a number of silyl derivatives, it was decided
to also utilize a dimethoxytrityl-protecting group as this would
also result in nucleosides attached to another very bulky non-polar
group on the 50-O position. With this in mind, uridine was treated
with dimethoxytrityl chloride (DMTCl) in the presence of base to
readily afford the uridine-derivative 20. To afford additional
compounds with modulated lipophilicity, this compound was
converted into the diacetyl derivative 21 and subsequently into
the benzoyl-uridine derivative 22 (Scheme 3).
The effect of the synthetic compounds on cell population
growth was evaluated by exposing a cell population to compounds
12, 14, 15, 16, 20 and 21 and then monitoring the mitochondrial
enzymatic reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] to formazan crystals. The cancer
cells were initially exposed to the synthetic compounds (final
concentration of 100 lM) at 37 °C for 24 h. The MTT assay was
then used to quantify cell viability after exposure (Table 1). A com-
pound was considered active if it reduced the growth of the cells
by at least 50% (the GI50 concentration), after exposure of the cells
for 24 or 72 h with the compound of interest, as compared to the
control arbitrarily normalized at 100%.
2.2. Bio-evaluation
An initial screen utilizing a number of cell viability assays was
performed on a small group of synthetic compounds at a single
concentration, in order to obtain information for the activity of a
subset of the compounds synthesized. For this study, five human
cancer cell lines were used: three leukemic cell lines [HL-60
(promyelocytic leukemia), K-562 (myelogenous leukemia) and
Jurkat (T-cell leukemia)] and two colorectal adenocarcinoma cell
lines (HT-29 and Caco-2). Human leucocytes were used as a normal
control and as an indicator for toxicity. In addition, the synthetic
compounds were assayed against two established human glioma
lines (U373 and Hs683) to determine GI50 values for the entire
library of modified nucleosides.
The results from the initial screen were promising, with the
TBDPS-containing compounds 12, 14 and 15 showing activity. Of
the three, 50-O-(tert-butyldiphenylsilyl)uridine 12 was the best,
inhibiting the proliferation of the suspension cell lines (leukemia)
to below 10%, while the adherent colorectal Caco-2 and HT-29 cells
showed between 60% and 75% inhibition (Table 1). Of interest, was
that the 50-O-(4,40-dimethoxytrityl)uridine 20 displayed similar
activity values to the TBDPS-uridine derivative 12 (Table 1, entries
1 and 5). The activity of the compounds in which the 20- and
30-alcohols had been acetylated was lower for TBDPS-protected
compound 16 (versus diol 12), while the acetylated dimethoxytri-
tyl derivative 21 appeared to lose all potency when compared to
the diol 20 (Table 1, entries 4 vs 1 and 6 vs 5).
Freshly isolated leucocytes were also exposed to the same set of
synthetic derivatives (compounds 12, 14, 15, 16, 20 and 21) and
camptothecin, at a final concentration of 100 lM, for a period of
24 h. Cell viability was then determined by the MTT assay (Table 1).
All the synthetic derivatives screened showed reduced in vitro
growth inhibitory effects toward the leucocytes when compared
OMe
O
N
O
N
NH
NH
DMTCl, DMAP,
py, 60oC, 18 h
O
O
Ac2O, py,
rt, 18 h
HO
O
O
O
to 100 lM camptothecin, suggesting that the synthetic derivatives
OH OH
OH OH
prepared for this study may be sparing to non-cancerous cells.
Finally, from Table 1 it could be seen that compounds 12 and 20
show selective activity toward the leukemic cells, which is
promising.
OMe
Uridine
20 (83%)
OMe
OMe
O
O
Bz
N
NH
2.2.2. Effect of synthetic compounds on cancer cell population
growth and determination of GI50 values
N
O
N
O
Hünig's base,
O
O
O
O
BzCl, py, rt, 2 h
Having identified that certain compounds possessed interesting
activity in terms of the inhibition of cancer cell population growth,
it was decided to test an extended library of seventeen synthetic
compounds against two adherent human glioma lines (U373 and
Hs683), across a range of concentrations in order to determine
OAc OAc
OAc OAc
OMe
OMe
21 (90%)
22 (59%)
Scheme 3. Use of trityl-protecting group to generate nucleoside derivatives.
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4
J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
Percentage of cell viability of the cell line panel and normal, un-stimulated leucocytes (white blood cells, WBC), treated with 100
for 24 h
lM of the synthetic compounds or camptothecin
Entry
Compound
Cell viability (% surviving at 100 lM as compared to control (100%)
HL-60
Jurkat
K-562
Caco-2
HT-29
WBC
1
2
3
4
5
6
7
12
14
15
16
20
21
5.6 0.3
26.0 0.6
27.9 0.5
11.2 0.5
6.5 0.5
NA
6.3 0.6
16.0 1.3
18.3 1.2
NA
5.2 1.0
NA
9.4 0.1
35.6 0.9
45.5 0.7
35.5 2.4
8.3 0.3
NA
37.4 1.0
37.2 0.4
NA
NA
26.1 0.2
NA
29.4 0.6
25.6 0.7
NA
NA
27.8 1.2
NA
61.5 4.5
NI
59.3 2.0
NI
82.7 2.1
NI
34.6 2.0
Camptothecin
23.4 0.7
8.9 0.7
39.6 0.9
12.8 0.2
11.4 0.2
NA = not active, NI = no inhibition (cell viability >50% for cell lines, cell viability 100% for WBC).
GI50 values. In terms of a positive control for this study, narci-
clasine was selected, as this particular compound has been shown
by Kiss and co-workers to potently inhibit growth of these cell
lines.56,57 In this series of experiments, GI50 concentrations were
determined after having cultured the glioma cells for 72 h in the
presence of the compound of interest (as compared to the controls,
i.e. untreated cells).
showed increased growth inhibitory activity ranging between 25
and 46 M, irrespective of the uridine 12, 5-methyluridine 13,
l
cytidine 14 or adenosine 15 base utilized. It can be tentatively
concluded that it is the lipophilic TBDPS group that has a positive
effect on the inhibition of cancer cell population growth. Two other
derivatives based on the TBDPS-containing uridine scaffold were
also tested, namely 16 and 17. To this end, it was found that the
Regarding comparison with results available in the literature,
the first compounds to be evaluated were the TBDMS-protected
uridine derivatives 4d (R = H) and 8 (R = Me). Compound 4d was
poorly active in the human glioma lines U373 and Hs683, with
GI50 values against U373 and Hs683 cells were 32
1 and
29 respectively for
1 lM
20,30-O-diacetyl-50-O-(tert-
butyldiphenylsilyl)uridine 16, which were similar to those values
obtained for the unprotected compound 12. In addition, when
the amine on this particular base was protected with a benzoyl
group to afford compound 17, the mean growth inhibitory results
were comparable in potency to compounds 12 and 16.
The next group of compounds investigated in our study were
those containing the 1,3-dihydroxy-1,1,3,3-tetraisopropyldisilox-
ane protecting group, which to our knowledge have not been
tested before in this manner. The simplest of these was the
GI50 values of 71
pound 8 did not fare better with GI50 values of >100
U373 and 77 M for Hs683 (as a reference point, when
cisplatin was tested against the same two cell lines, GI50 values
2
l
M and 84
3
l
M respectively, and com-
l
M for
1
l
of 5 lM and 0.4 lM were obtained for U373 and Hs683, respec-
tively58). In a similar fashion, the thexyl-protected compound 4e
showed poor inhibition of glioma cell population growth (Table 2,
entry 3). Of interest, was that the related 50-O-(dimethylthexylsi-
disilyl-compound 10, for which GI50 values of 81
4 and
lyl)-5-methyluridine 9 showed GI50 values of 68
1
l
M and
83 M were determined for the U373 and Hs683 glioma cells,
2 l
64 M for the U373 and Hs683 cells respectively, indicating a
2
l
respectively. The methyluridine derivative 11 had similar potency,
slight increase in efficacy. In this particular case, the addition of
a methyl group to compound 4e, resulting in 9, does therefore
appear to improve the compound’s in vitro growth inhibitory
activity.
and while the benzoyl-protected derivative 18 had better activity
(29
2 l
M for both cell lines), the 20-keto-compound 19 was again
in the same region of activity (71 2 and 70
4 lM, for U373 and
Hs683 glioma cells respectively).
Next, the set of TBDPS-derivatives were evaluated, and the
results showed that the compounds containing the TBDPS group
Finally, a series of compounds where the tert-butyldiphenylsilyl
protecting group on the 50-oxygen was exchanged for a 4,40-
dimethoxytrityl protecting group was evaluated. The GI50 values
were found to be in the 28–44 l
M range for all the 4,40-dimethox-
Table 2
ytrityl derivatives, including the simple 50-O-(4,40-dimethoxytrityl)
uridine 20, the diacylated compound 21 and the 3-N-benzoyl-20,30-
O-diacetyl-protected uridine derivative 22.
In vitro compound-induced anti-growth effects on various cancer cell lines expressed
as GI50 values
Entry
Compound
GI50
(
lM) [72 h culture
ALogP
Finally, the lipophilicity (ALogP) values were calculated for each
of the nucleoside analogues. Although no obvious trend in the rela-
tionship between the GI50 values and the ALogP values was
observed, it should be noted that the more water-soluble com-
pounds (e.g. compounds 4d/e and 8) tended to be less active, while
those less water-soluble tended to have improved (i.e. decreasing)
GI50 values (e.g. compounds 17 and 18). This observation could
imply that the bulkier non-polar silyl groups could be assisting
in the compounds being able to cross cell membranes or being
responsible for bioactivity in terms of disrupting cellular mem-
branes. It should be noted that in previous papers (for exam-
ple20,29,32) mention has been made of the ability of lipophilic silyl
groups on nucleoside cores to facilitate the crossing of cell mem-
brane barriers. In addition, it has also been noted that the efficacy
of the silyl-containing compounds is related to the rate of hydrol-
ysis,32 explaining why sterically hindered groups like TBDPS and
thexyl-DMS could be considered potentially important structural
elements in the design of novel growth inhibitory compounds, at
least in vitro.
with compounds]
U373 Hs683
71
1
2
3
4
5
6
7
8
4d
8
4e
9
10
11
12
13
14
15
16
17
18
19
20
21
2
84
77
3
1
1.4
1.8
2.1
2.6
6.5
7.0
4.6
5.0
4.6
5.1
5.3
7.0
8.2
8.5
2.6
3.3
5.0
ND
>100
>100
>100
68
81
87
36
25
28
44
32
26
29
71
40
40
39
1
64
83
91
35
27
40
46
29
35
29
70
39
28
44
2
4
2
1
1
1
1
1
1
2
2
3
3
1
2
3
1
1
2
3
1
2
2
4
2
5
2
9
10
11
12
13
14
15
16
17
18
22
Narciclasine
0.029
0.040
Note: ND = not determined.
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J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
CDCl3): d 10.53 (br s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 5.91 (d,
J = 2.1 Hz, 1H), 5.65 (d, J = 8.1 Hz, 1H), 5.52 (br s, 1H), 4.26–4.23
(m, 2H), 4.14 (d, J = 5.3 Hz, 1H), 4.03 and 3.85 (2 ꢁ d, J = 11.3 Hz,
2H), 3.66 (br s, 1H), 0.92 (s, 9H), 0.12 (s, 6H); 13C NMR (50 MHz,
CDCl3): d 164.0, 151.3, 140.5, 102.0, 90.2, 84.8, 75.6, 69.2, 61.8,
25.9, 18.4, ꢂ5.6; HRMS (ESI) m/z calculated for C15H27N2O6Si (M
+H)+ 359.16329, found 359.16336.
3. Conclusions
Seventeen novel nucleoside derivatives were synthesized and
tested against a range of cancer cell lines. The majority of the com-
pounds comprised of 50-O-silyl- and trityl-protected nucleoside
derivatives and a number of previously unknown modifications
of the nucleoside base or ribose portion were also achieved. For
the cell viability assay at 100 lM, compounds 12 and 20 reduced
cell viability by 65–95%, while for the GI50 determinations against
two human glioma lines (U373 and Hs683), compounds 13–18, 20,
21 gave GI50 values in the range of 25–40 lM. From the results it
appeared that in general, the nucleoside derivatives with the more
4.1.2.2. 50-O-(tert-Butyldimethylsilyl)-5-methyluridine (8).
Yield: 93% (white powder); Spectroscopic data correlated well to
that previously reported by Debarge and co-workers.59 1H NMR
(300 MHz, CDCl3/MeOD, signals for NH and OH peaks not
observed): d 7.57 (s, 1H), 5.96 (d, J = 5.0 Hz, 1H), 4.15–4.07 (m,
3H), 3.95 and 3.86 (2 ꢁ dd, J = 2.1, 11.6 Hz, 2H), 1.91 (s, 3H), 0.96
(s, 9H), 0.15 (s, 3H), 0.16 (s, 3H); 13C NMR (50 MHz, DMSO): d
163.6, 150.7, 135.6, 109.7, 87.1, 84.3, 73.1, 70.0, 63.1, 25.8, 18.0,
12.1, ꢂ5.3; HRMS (ESI) m/z calculated for C16H29N2O6Si (M+H)+
373.17894, found 373.17903.
lipophilic silyl/trityl groups had improved GI50 values.
4. Experimental procedures
4.1. Chemistry
4.1.1. General chemistry methods
All solvents and reagents were used as obtained from commercial
sources unless otherwise indicated. All reactions were performed
under an Ar atmosphere unless otherwise stated. The 1H and 13C
NMR spectra were recorded on a Bruker Advance-300 spectrometer
at 300.13 MHz for 1H and 75 MHz for 13C using standard pulse
sequences. Deuterated chloroform or deuterated dimethylsulfoxide
were used as solvents for NMR spectroscopy experiments. 1H chem-
icalshiftvalues(d)arereferencedtotetramethylsilane (d = 0.00 ppm
for CDCl3) or the residual nondeuterated components of the NMR
solvents (d = 2.50 ppm for d6-DMSO). The 13C chemical shifts (d)
are referenced to CDCl3 (central peak, d = 77.0 ppm) or d6-DMSO
(d = 39.5 ppm) as the internal standard. Mass spectra were mea-
sured in positive mode electrospray ionization (ESI) and the data
was obtained on a VG7-SEQ Double Focusing Mass Spectrometer at
4.1.2.3. 50-O-(Dimethylthexylsilyl)uridine (4e).
Yield: 99%
(white foam); 1H NMR (300 MHz, CDCl3): d 10.50 (br s, 1H), 8.02
(d, J = 8.1 Hz, 1H), 5.90 (d, J = 2.6 Hz, 1H), 5.65 (d, J = 8.1 Hz, 1H),
4.26–4.11 (m, 2H), 4.20 (d, J = 5.4 Hz, 1H), 3.88 and 3.71 (2 ꢁ d,
J = 11.4 Hz, 2H), 3.49 (br s, 1H), 2.04 (br s, 1H), 1.63 (sept,
J = 6.6 Hz, 1H), 0.88 (br s, 12H), 0.15 (s, 6H); 13C NMR (50 MHz,
CDCl3): d 164.0, 151.3, 140.4, 102.0, 90.2, 84.9, 75.6, 69.3, 61.7,
34.1, 25.4, 20.2, 18.4, ꢂ3.5; HRMS (ESI) m/z calculated for C17H31
-
N2O6Si (M+H)+ 387.19459, found 387.19505.
4.1.2.4. 50-O-(Dimethylthexylsilyl)-5-methyluridine (9).
Yield:
80% (white solid); 1H NMR (300 MHz, CDCl3/d6-DMSO): d 12.13
(br s, 1H), 8.22 (s, 1H), 6.73 (d, J = 5.2 Hz, 1H), 6.14
(d, J = 5.2 Hz, 1H), 5.83 (d, J = 3.7 Hz, 1H), 4.85–4.84 (m, 2H),
4.79–4.77 (m, 1H), 4.67 and 4.62 (2 ꢁ dd, J = 2.6, 11.5 Hz, 2H),
4.20 (s, 3H), 1.49 (sept, J = 6.7 Hz, 1H), 1.75 (br s, 12H), 1.00
(s, 6H); 13C NMR (50 MHz, DMSO): d 163.6, 150.7, 135.6, 109.5,
87.0, 84.3, 72.9, 69.9, 63.0, 33.6, 24.6, 20.1, 18.4, 12.0, ꢂ3.5; HRMS
(ESI) m/z calculated for C18H33N2O6Si (M+H)+ 401.21024, found
401.20987.
70 eV and 200 lA. All melting points were obtained on a Reichert
hot-stage microscope and are uncorrected. TLC was performed using
aluminum-backed Macherey-Nagel ALUGRAM Sil G/UV254 plates,
pre-coated with 0.25 mm silica gel 60. Column chromatography
was performed using Macherey-Nagel Silica gel 60 (particle size
0.063–0.200 mm). Basified silica gel was used for the column
chromatography of modified nucleosides, where specified in the
text, and was prepared as follows: the silica required for the column
was shaken with a 10% NEt3/hexane (v/v) solution, used in the ratio
of 1 cm3 solution/1 g silica, for 15 min at rt. Thereafter, the silica was
dried by removing the solvent in vacuo.
4.1.3. 1-[(6aR,8R,9R,9aS)-9-Hydroxy-2,2,4,4-tetraisopropyltetrahy-
dro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl]pyrimidine-2,4
(1H,3H)-dione (10)
4.1.2. General procedure for the silylation of the 50-OH of
uridine and 5-methyluridine
A solution of uridine (1.68 g, 6.87 mmol) in distilled pyridine
(20.5 mL) was prepared and cooled to 0 °C in an ice-water bath,
before the addition of the 1,3-dichloro-1,1,3,3-tetraisopropyldis-
iloxane (2.3 mL, 7.2 mmol). The reaction mixture was stirred over-
night and subsequently quenched by the addition of distilled H2O
(21 mL). The solvents were removed under reduced pressure and
the residue obtained was partitioned between EtOAc (70 mL) and
distilled H2O (2 ꢁ mL). The organic fraction was dried over anhy-
drous Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by column chromatography on silica
gel, using a gradient of EtOAc–hexane (10–50%) for elution. Pro-
duct 10 (2.80 g) was obtained as a white foam, yield: 85%. 1H
and 13C NMR spectra obtained correlated well to those reported
by Matsuda and co-workers.60 1H NMR (300 MHz, CDCl3): d 9.64
(br s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 5.75 (d, J = 2.1 Hz, 1H), 5.73–
5.69 (m, 1H), 4.32 (dd, J = 4.7, 8.8 Hz, 1H), 4.25–4.14 (m, 3H),
4.00 (dd, J = 2.5, 13.2 Hz, 1H), 3.62 (br s, 1H), 1.10–1.02 (m, 28H);
13C NMR (50 MHz, CDCl3): d 163.5, 150.2, 139.9, 102.0, 90.9, 81.8,
75.1, 68.7, 60.1, 17.4, 17.33, 17.25, 17.2, 17.0, 16.91, 16.88, 16.8,
13.3, 12.93, 12.88, 12.4; HRMS (ESI) m/z calculated for C21H39N2O7-
Si2 (M+H)+ 487.22903, found 487.22848.
The glassware and attachments were filled with a 5% TMSCl:
hexane solution and were allowed to stand overnight. After this
time, the glassware was oven-dried, placed under high vacuum
and allowed to cool under an Ar atmosphere. To a stirred solution
of uridine/5-methyluridine (1.0 equiv) and DMAP (0.1 equiv) in
distilled pyridine (10 mL:1 g starting material) was added the
corresponding silyl chloride (1.1 equiv). The reaction mixture
was stirred overnight and then quenched by the addition of
distilled H2O (5 mL:1 g starting material) with vigorous stirring.
The mixture was dissolved in excess EtOAc and concentrated under
reduced pressure. The residue obtained was partitioned between
EtOAc (20 mL:1 g starting material) and furthermore washed with
distilled H2O (3 ꢁ 10 mL:1 g starting material). The organic fraction
was dried over anhydrous Na2SO4, filtered and the solvent was
removed in vacuo. No further purification was required.
4.1.2.1. 50-O-(tert-Butyldimethylsilyl)uridine (4d).
Yield:
88% (white foam); Spectroscopic data correlated well to that previ-
ously reported by Debarge and co-workers.59 1H NMR (300 MHz,
Please cite this article in press as: Panayides, J.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.036

6
J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.4. 1-[(6aR,8R,9S,9aR)-Tetrahydro-9-hydroxy-2,2,4,4-tetraiso-
propyl-6H-furo[3,2-f]-[1,3,5,2,4]trioxadisilocin-8-yl](5-methyl-
pyrimidine)-2,4(1H,3H)-dione (11)
4.1.5.4. 50-O-(tert-Butyldiphenylsilyl)adenosine (15).
The
residue was purified by column chromatography, using basified
silica gel and 10% EtOH–CH2Cl2 for elution. Yield: 73% (white
foam). Proton NMR spectroscopic data correlated well to that pre-
viously reported by Beaton and co-workers.63 1H NMR (300 MHz,
Protocol the same as for the preparation of 10, starting from 5-
methyluridine. Product 11 (0.78 g) was obtained as a white foam,
yield: 40%. Proton NMR spectroscopic data correlated well to that
previously reported by Turkman and co-workers.61 1H NMR
(300 MHz, CDCl3): d 9.47 (br s, 1H), 7.46 (s, 1H), 5.72 (s, 1H),
4.37–4.33 (m, 1H), 4.23–4.14 (m, 2H), 4.12–3.98 (m, 2H), 3.68 (d,
J = 1.4 Hz, 1H), 1.91 (s, 3H), 1.10–1.04 (m, 28H); 13C NMR
(50 MHz, CDCl3): d 164.0, 150.2, 135.6, 110.6, 91.1, 81.8, 75.0,
68.9, 60.2, 17.4, 17.3, 17.2, 17.2, 17.0, 17.0, 16.9, 16.8, 12.9, 12.7,
12.6, 12.5; HRMS (ESI) m/z calculated for C22H41N2O7Si2 (M+H)+
501.24468, found 501.24409.
CDCl3/d6-DMSO):
d
8.11 (s, 1H), 8.08 (s, 1H), 7.59 (2 ꢁ d,
J = 7.0 Hz, 4H), 7.38–7.30 (m, 6H), 7.03 (br s, 2H), 5.95 (d,
J = 4.9 Hz, 1H), 5.45 (d, J = 5.6 Hz, 1H), 5.07 (d, J = 5.4 Hz, 1H),
4.61–4.59 (m, 1H), 4.36–4.34 (m, 1H), 4.07–4.03 (m, 1H), 3.93
and 3.78 (2 ꢁ dd, J = 3.9, 11.3 Hz, 2H), 0.99 (s, 9H); 13C NMR
(50 MHz, CDCl3/d6-DMSO): d 155.8, 152.3, 149.2, 138.5, 134.8,
132.4, 129.4, 127.4, 119.1, 87.5, 84.1, 73.5, 69.7, 63.5, 26.4, 18.7;
HRMS (ESI) m/z calculated for C26H32N5O4Si (M+H)+ 506.22181,
found 506.22098.
4.1.5. General procedure for the silylation of the 50-OH of
nucleosides using tert-butydiphenylsilyl chloride
The nucleoside (1.0 equiv) and DMAP (0.1 equiv) were stirred
with distilled pyridine (5 mL:1 g starting material), to this solution
was added tert-butyldiphenylsilyl chloride (1.1 equiv) and the
reaction mixture stirred under an Ar atmosphere overnight. The
reaction mixture was diluted with CH2Cl2 (40 mL:1 g starting
4.1.6. 20,30-O-Diacetyl-50-O-(tert-butyldiphenylsilyl)uridine (16)
The silylated uridine 12 (4.19 g, 8.69 mmol) was stirred with
distilled pyridine (1.41 mL, 17.4 mmol) until a solution had formed
and was then cooled to 0 °C in an ice-water bath. To this cooled
solution was then added drop-wise a mixture of distilled Ac2O
(2.50 mL, 26.1 mmol) and distilled pyridine (2.11 mL, 26.1 mmol).
The reaction mixture was then warmed and stirred overnight
under an Ar atmosphere. EtOAc (400 mL) was added to the reaction
mixture and extracted with brine (3 ꢁ 400 mL), the combined
aqueous layers were back-extracted with CH2Cl2 (3 ꢁ 400 mL)
and the combined organic fractions were then extracted with
saturated NH4Cl that had been basified to pH ꢀ 10 using NH3
(600 mL). The combined organics were dried over anhydrous
MgSO4, filtered and the solvent was removed in vacuo to give
product 16 (4.39 g) as a cream-colored foam, yield: 89%. 1H NMR
(300 MHz, CDCl3): d 9.39 (br s, 1H), 7.76–7.64 (m, 5H), 7.43–7.35
(m, 6H), 6.30 (d, J = 6.5 Hz, 1H), 5.53–5.51 (m, 1H), 5.47–5.40
(m, 2H), 4.18 (br s, 1H), 4.02 and 3.85 (2 ꢁ d, J = 11.8 Hz, 2H),
2.07 (s, 3H), 2.04 (s, 3H), 1.13 (s, 9H); 13C NMR (50 MHz, CDCl3):
d 170.0, 169.8, 163.2, 150.8, 139.6, 135.9, 135.8, 132.8, 130.4,
129.8, 128.1, 127.8, 103.4, 85.4, 83.4, 73.3, 71.2, 63.7, 27.2, 20.9,
20.6, 19.5; HRMS (ESI) m/z calculated for C29H34N2O8SiNa (M+H)+
589.19766, found 589.19699.
material) and extracted with
a saturated NaHCO3 solution
(2 ꢁ 40 mL:1 g starting material) and brine (40 mL:1 g starting
material). The combined organics were then dried over anhydrous
Na2SO4, filtered and the solvent removed in vacuo. The residue was
purified as described below.
4.1.5.1. 50-O-(tert-Butyldiphenylsilyl)uridine (12).
The resi-
due was purified by column chromatography on the basified silica
gel, using 10% EtOH–CH2Cl2 for elution. Yield: 100% (white foam).
Spectroscopic data correlated well to that previously reported by
Sproat et al.62 1H NMR (300 MHz, CDCl3): d 10.38 (br s, 1H), 7.97
(d, J = 8.1 Hz, 1H), 7.65 (2 ꢁ d, each J = 6.1 Hz, 4H), 7.44–7.37(m,
6H), 5.93 (d, J = 2.1 Hz, 1H), 5.47 (br s, 1H), 5.37 (d, J = 8.1 Hz,
1H), 4.37–4.35 (m, 1H), 4.32–4.30 (m, 1H), 4.25–4.08 (m, 2H),
3.89 (d, J = 10.7 Hz, 1H), 3.38 (br d, J = 5.8 Hz, 1H), 1.08 (s, 9H);
13C NMR (50 MHz, CDCl3): d 164.1, 151.4, 140.4, 135.8, 135.6,
132.3, 130.3, 128.22, 128.19, 102.5, 90.5, 84.8, 75.7, 69.4, 62.8,
27.2, 19.5; HRMS (ESI) m/z calculated for C25H31N2O6Si (M+H)+
483.19459, found 483.19407.
4.1.7. 3-N-Benzoyl-20,30-O-diacetyl-50-O-(tert-butyldiphenylsilyl)
uridine (17)
The protected uridine 16 (1.07 g, 1.88 mmol) was stirred with
distilled pyridine (20 mL) and to this pale yellow solution was
added N,N-diisopropylethylamine (1.64 mL, 9.41 mmol) with
stirring. Distilled benzoyl chloride (1.10 mL, 9.41 mmol) was
added, with the liberation of a white gas, and the reaction mixture
was stirred under an Ar atmosphere for 2 h. The dark burgundy-
colored reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with distilled H2O (3 ꢁ 100 mL). The aqueous fractions
were next extracted with CH2Cl2 (100 mL), and the combined
organic layers dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The residue was purified by
column chromatography on silica gel and eluted with a gradient
of EtOAc–hexane (10–50%). Product 17 (0.90 g) was obtained as a
yellow foam, yield: 71%. 1H NMR (300 MHz, CDCl3): d 7.95
(d, J = 7.3 Hz, 2H), 7.86 (d, J = 8.3 Hz, 1H), 7.67–7.63 (m, 4H),
7.48–7.41 (m, 9H), 6.24 (d, J = 6.0 Hz, 1H), 5.55–5.48 (m, 3H),
4.20 (d, J = 2.1 Hz, 1H), 3.87 and 4.05 (2 ꢁ d, J = 11.9 Hz, 2H), 2.10
(s, 3H), 2.05 (s, 3H), 1.15 (s, 9H); 13C NMR (50 MHz, CDCl3): d
170.0, 168.6, 161.9, 149.7, 139.3, 135.9, 135.6, 135.3, 132.7,
131.5, 130.8, 130.6, 130.5, 130.3, 129.3, 128.4, 103.2, 85.9, 83.6,
73.5, 71.2, 63.7, 27.2, 20.8, 20.6, 19.5; HRMS (ESI) m/z calculated
for C36H39N2O9Si (M+H)+ 671.24193, found 671.24173.
4.1.5.2. 50-O-(tert-Butyldiphenylsilyl)-5-methyluridine (13). The
residue was purified by the azeotropic distillation of pyridine,
using toluene as the co-solvent. Yield: 100% (white foam). 1H
NMR (300 MHz, CDCl3/MeOD): d 8.53 (br d, 1H), 7.82 (s, 1H),
7.71–7.68 (m, 4H), 7.43–7.40 (m, 6H), 6.02 (br d, J = 5.7 Hz, 1H),
4.33–4.24 (m, 2H), 4.11–4.03 (m, 2H), 3.90 (dd, J = 2.3, 11.6 Hz,
1H), 1.49 (s, 3H), 1.12 (s, 9H); 13C NMR (50 MHz, DMSO):
d 163.5, 150.6, 134.6, 132.6, 130.0, 127.9, 109.6, 87.3, 84.1,
72.9, 69.8, 64.2, 26.6, 18.6, 11.6; HRMS (ESI) m/z calculated for
C
26H33N2O6Si (M+H)+ 497.21024, found 497.20971.
4.1.5.3. 50-O-(tert-Butyldiphenylsilyl)cytidine(14).
The resi-
due was purified by column chromatography using basified silica
gel, with a gradient of EtOH–CH2Cl2 (10–20%) for elution. Yield:
100% (white foam).1H NMR (300 MHz, d6-DMSO): d 7.66 (d,
J = 7.4 Hz, 1H), 7.65–7.63 (m, 4H), 7.49–7.39 (m, 6H), 7.15 (br s,
2H), 5.83 (d, J = 3.4 Hz, 1H), 5.53 (d, J = 7.4 Hz, 1H), 5.43 (d,
J = 5.1 Hz, 1H), 5.07 (d, J = 6.0 Hz, 1H), 4.11–4.08 (m, 1H), 3.95–
3.90 (m, 3H), 3.79–3.74 (m, 1H), 1.02 (s, 9H); 13C NMR (50 MHz,
d6-DMSO): d 165.5, 155.1, 140.6, 135.0, 132.3, 130.0, 128.0, 93.7,
89.3, 82.9, 74.2, 68.9, 63.2, 26.7, 18.8; HRMS (ESI) m/z calculated
for C25H32N3O5Si (M+H)+ 482.21057, found 482.20978.
Please cite this article in press as: Panayides, J.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.036

J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.8. 3-Benzoyl-1-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraiso-
propyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl]
pyrimidine-2,4(1H,3H)-dione (18)
1-[(6aR,8R,9R,9aS)-9-Hydroxy-2,2,4,4-tetraiso-propyltetrahy-
dro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl]pyrimidine-2,4
(1H,3H)-dione 10 (6.02 g, 12.5 mmol), Na2CO3 (10.10 g,
96.88 mmol) and tetrabutylammonium bromide (0.16 g,
mixture was stirred at 60 °C under an Ar atmosphere overnight and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography on basified silica, using gradi-
ent MeOH–CH2Cl2 (0–10%) for elution. The cream foam isolated
after chromatography was dissolved in MeOH (4.5 mL) and the
solution was added drop-wise to vigorously stirring Et2O
(35 mL). A white precipitate was observed and the mixture was
cooled to 0 °C in an ice-water bath to allow for complete crystal-
lization. The white solid was filtered off through a sintered funnel
and the mother liquor was evaporated and dried in vacuo. The
mother liquor gave product 20 (0.95 g) as a cream-colored foam,
yield: 83%. Spectroscopic data correlated well to that previously
reported by Yang and co-workers.65 1H NMR (300 MHz, CDCl3): d
7.90 (d, J = 8.1 Hz, 1H), 7.22–7.39 (m, 9H), 6.83 (d, J = 8.7 Hz, 4H),
5.95 (d, J = 3.4 Hz, 1H), 5.34 (d, J = 8.1 Hz, 1H), 4.44 (t, J = 5.2 Hz,
1H), 4.34–4.32 (m, 1H), 4.20–4.18 (m, 1H), 3.78 (s, 6H), 3.49–
3.45 (m, 2H), NH and OH peaks not observed; 13C NMR (50 MHz,
DMSO): d 163.1, 158.1, 150.5, 144.6, 140.7, 140.5, 135.4, 135.2,
129.7, 128.9, 127.9, 127.7, 127.6, 126.7, 126.4, 113.2, 101.4, 88.9,
85.8, 82.4, 73.4, 69.6, 63.0, 55.0; HRMS (ESI) m/z calculated for
0.48 mmol) were dissolved in
a biphasic mixture of CH2Cl2
(240 mL) and distilled H2O (480 mL). Benzoyl chloride (1.88 mL,
16.3 mmol) was added and the reaction mixture was vigorously
stirred for 2 h. The reaction mixture was diluted with excess
CH2Cl2 and the aqueous phase was further extracted with CH2Cl2
(3 ꢁ 480 mL). The combined organic fractions were dried over
anhydrous Na2SO4, filtered and evaporated under reduced pres-
sure. The residue was dissolved in 1,2-dichloroethane (120 mL)
with gentle heating and the solution was allowed to stand for
66 h, to allow the O-/N-rearrangement to occur. The solvent was
removed under reduced pressure and the residue was purified by
column chromatography on silica gel, with gradient EtOAc–hexane
(5–50%) used for elution. Compound 18 (7.26 g) was isolated as a
white foam, yield: 99%. 1H and 13C NMR spectra obtained corre-
lated well to those reported previously by Sekine.64 1H NMR
(300 MHz, CDCl3): d 7.93 (d, J = 7.2 Hz, 2H), 7.81 (d, J = 8.2 Hz,
1H), 7.66 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.7 Hz, 2H), 5.80
(d, J = 8.2 Hz, 1H), 5.76 (s, 1H), 4.37 (dd, J = 4.8, 8.6 Hz, 1H), 4.21–
4.11 (m, 3H), 4.01 (dd, J = 2.6, 13.3 Hz, 1H), 2.04 (br s, 1H), 1.10–
1.03 (m, 28H); 13C NMR (50 MHz, CDCl3): d 168.5, 162.1, 148.9,
139.6, 135.1, 131.4, 130.4, 129.1, 101.8, 90.8, 82.1, 75.2, 68.9,
60.2, 17.4, 17.3, 17.22, 17.19, 17.0, 16.9, 16.8, 16.7, 14.1, 13.3,
12.9, 12.5; HRMS (ESI) m/z calculated for C28H43N2O8Si2 (M+H)+
591.25525, found 591.25494.
C
30H31N2O8 (M+Na)+ 569.18944, found 569.18944.
4.1.11. 20,30-O-Diacetyl-50-O-(4,40-dimethoxytrityl)uridine (21)
The protected uridine 20 (2.00 g, 3.65 mmol) was dissolved in
distilled pyridine (1.50 mL, 18.3 mmol), cooled to 0 °C in an ice-
water bath and to the solution was added drop-wise a mixture of
distilled Ac2O (1.05 mL, 11.0 mmol) and distilled pyridine
(0.90 mL, 11 mmol). The reaction mixture was then warmed and
stirred under an Ar atmosphere overnight. The reaction mixture
was diluted with EtOAc (200 mL) while stirring, and the organic
fraction was washed with brine (3 ꢁ 200 mL) and the combined
aqueous fractions were extracted with CH2Cl2 (3 ꢁ 200 mL). All
of the organic fractions were combined and washed with saturated
NH4Cl that had been basified to pH ꢀ 10 with NH3 (300 mL). The
combined organics were dried over anhydrous MgSO4, filtered
and the solvent removed in vacuo to give product 21 (2.06 g) as
a pale orange foam, yield: 90%. 1H NMR (300 MHz, CDCl3): d 9.34
(br s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.29–7.40 (m, 9H), 6.88 (d,
J = 8.1 Hz, 4H), 6.28 (d, J = 6.3 Hz, 1H), 5.65–5.62 (m, 1H), 5.60–
5.58 (m, 1H), 5.36 (d, J = 8.1 Hz, 1H), 4.27–4.25 (m, 1H), 3.82 (s,
6H), 3.53–3.50 (m, 2H), 2.14 (s, 3H), 2.13 (s, 3H); 13C NMR
(50 MHz, CDCl3): d 170.0, 169.8, 163.1, 159.0, 150.8, 144.0, 139.9,
134.9, 130.39, 130.34, 128.4, 128.3, 127.5, 113.6, 103.3, 87.8,
85.5, 82.2, 73.0, 71.5, 62.9, 55.4, 20.9, 20.7; HRMS (ESI) m/z calcu-
4.1.9. 1-[(6aR,8R,9aR)-2,2,4,4-Tetraisopropyl-9-oxotetrahydro-
6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl]pyrimidine-2,4
(1H,3H)-dione (19)
The oxidation complex was pre-formed by preparing 3 equiv. of
the complex relative to the starting material 10.60 Initially, chro-
mium(VI) trioxide (0.69 g, 6.8 mmol) was dissolved in freshly
distilled CH2Cl2 (24 mL). To this solution was added distilled pyri-
dine (1.10 mL, 13.5 mmol) and distilled Ac2O (0.64 mL, 6.8 mmol).
The mixture was stirred for a few minutes to allow for complete
formation of the oxidation complex. A solution was then prepared
of 10 (1.09 g, 2.25 mmol) in distilled CH2Cl2 (3 mL). The starting
material solution was added drop-wise to the oxidation complex
solution with stirring. The reaction mixture was allowed to stir
under an Ar atmosphere for 2 h. After this time, the reaction mix-
ture was poured drop-wise into stirring EtOAc (180 mL). The sus-
pension was then pumped through a short silica gel column,
using excess EtOAc to rinse the column. The filtrate was concen-
trated to dryness to yield a yellow-brown residue. This residue
was then purified by column chromatography, with gradient
EtOAc–hexane solutions (20–50%) for elution. Product 19 (0.87 g)
was obtained pure as a white foam, yield: 80%. NMR spectroscopic
data obtained correlates well to that reported by Matsuda and co-
workers.60 1H NMR (300 MHz, CDCl3): d 8.80 (br s, 1H), 7.16 (d,
J = 8.0 Hz, 1H), 5.76 (d, J = 8.0 Hz, 1H), 5.04 (d, J = 9.0 Hz, 1H),
5.00 (s, 1H), 4.14–4.13 (m, 2H), 3.97–3.91 (m, 1H), 1.14–1.03 (m,
28 H); 13C NMR (50 MHz, CDCl3): d 204.7, 163.0, 149.2, 143.7,
103.2, 85.5, 79.6, 71.8, 62.4, 17.4, 17.2, 16.9, 16.82, 16.79, 16.76,
16.67, 16.6, 13.4, 13.0, 12.5, 12.3; HRMS (ESI) m/z calculated for
lated for C34H35N2O10 (M+H)+ 631.22862, found 631.22851, C34
34N2O10Na (M+Na)+ 653.21057, found 653.21020.
-
H
4.1.12. 3-N-Benzoyl-20,30-O-diacetyl-50-O-(4,40-dimethoxytrityl)
uridine (22)
The protected uridine 21 (0.39 g, 0.63 mmol) was stirred with
distilled pyridine (8 mL). To this solution was then added N,N-
diisopropylethylamine (0.55 mL, 3.15 mmol) and stirring contin-
ued for five min. To the solution was then added distilled benzoyl
chloride (0.37 mL, 3.2 mmol) with the immediate liberation of a
white-colored gas. The reaction mixture was stirred under an Ar
atmosphere for 2 h, after which it was diluted with CH2Cl2
(40 mL) and extracted with distilled H2O (3 ꢁ 40 mL), the com-
bined aqueous layers were then extracted with CH2Cl2 (40 mL)
and the combined organic layers were dried over anhydrous Na2-
SO4, filtered and evaporated. The residue was purified by column
chromatography on silica gel, with gradient elution EtOAc–hexane
(10–50%), to give product 22 (0.27 g) as a yellow foam, yield: 59%.
1H NMR (300 MHz, CDCl3): d 7.96 (d, J = 8.8 Hz, 2H), 7.84 (d,
J = 8.2 Hz, 1H), 7.68–7.27 (m, 12H), 6.87 (d, J = 8.8 Hz, 4H), 6.21
(d, J = 6.2 Hz, 1H), 5.67–5.62 (m, 1H), 5.50–5.58 (m, 1H), 5.45
(d, J = 8.2 Hz, 1H), 4.25 (d, J = 2.6 Hz, 1H), 3.81 (s, 6H), 3.53–3.51
C
21H37N2O7Si2 (M+H)+ 485.21338, found 485.21289.
4.1.10. 50-O-(4,40-Dimethoxytrityl)uridine (20)
Uridine (0.51 g, 2.1 mmol) was dissolved in pyridine (10 mL),
and to this solution was then added 4,40-dimethoxytrityl chloride
(0.85 g, 2.5 mmol) and DMAP (0.0033 g, 0.025 mmol). The reaction
Please cite this article in press as: Panayides, J.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.036

8
J.-L. Panayides et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(m, 2H), 2.10 (s, 3H), 2.07 (s, 3H); 13C NMR (50 MHz, CDCl3): d 170.0,
169.9, 168.7, 162.0, 159.0, 149.7, 144.0, 139.7, 135.3, 135.1, 134.9,
131.6, 130.8, 130.4, 129.3, 128.4, 128.3, 127.6, 113.6, 103.1, 87.9,
86.1, 82.4, 73.4, 71.5, 62.8, 55.5, 20.8, 20.7; HRMS (ESI) m/z calcu-
lated for C34H35N2O10 (M+H)+ 631.22862, found 631.22851, C41H38
N2O11Na (M+Na)+ 757.23678, found 757.23653.
were of human (GI50 range of 5–99 nM) or rodent origin
(GI50 range of 28–35 nM).57
Using the same methodology to assess cell viability, freshly
isolated human peripheral leucocytes (white blood cells) were also
exposed to the test nucleosides and camptothecin for 24 h. This
was done to obtain an indication of relative toxicity to normal
non-cancerous cells. Permission to isolate white blood cells from
human volunteers was obtained from the University of the Witwa-
tersrand Human Ethics Committee clearance no: M070519.
4.2. Bio-evaluation of nucleosides on cancer cell lines
Seven cell lines representing three different types of cancer
(leukemia, colorectal and brain) were used for the bio-evaluation
of the nucleoside derivatives. HL-60, K562 and Jurkat cells repre-
sent leukemic cell lines; HT-29 and Caco-2 cells originate from
colorectal carcinomas, while U373 and Hs683 are glioblastoma cell
lines. HL-60, K562, Jurkat, HT-29, Caco-2 and HS683 cell lines
originated from the American Type Culture Collection (ATCC)
Manassas, VA. The U373 cell line was purchased from the European
Collection of Cell Culture (ECACC), Salisbury, UK.
Acknowledgments
This work was supported by the National Research Foundation
(NRF), Pretoria, the University of the Witwatersrand (University
and Science Faculty Research Councils) and Stellenbosch Univer-
sity (Faculty and Departmental funding), South Africa. The South
African Medical Research Council, (MRC) is acknowledged for on-
going support of the small molecule anti-cancer research projects
in the group at Stellenbosch University under the Self-Initiated
Research Grant initiative. Opinions expressed in this publication
and conclusions arrived at, are those of the authors, and are not
necessarily attributed to the NRF or the MRC. We gratefully
acknowledge Mr R. Mampa for the NMR spectroscopy service
(University of the Witwatersrand). Finally, we acknowledge Ms J.
Schneider and Ms M. Ismail (Mass Spectroscopy Service, University
of Dortmund and Max Planck Institute for Molecular Physiology,
Dortmund, Germany) and Mr B. Moolman, Mr F. Hiten and Dr M.
Stander (Stellenbosch University) for mass spectroscopy services.
We thank D. Lamoral-Theys and T. Gras for their excellent techni-
cal help. R.K. is a director of research with the Fonds National de la
Recherche Scientifique (FRS-FNRS, Belgium). The authors thank
one of the article’s reviewers for making them aware of some of
the Refs. 33–37 concerning silylated nucleoside analogues contain-
ing unsaturated carbohydrate motifs.
4.2.1. Maintenance of cell lines
The HL-60, K562 and Jurkat leukemic cell lines were maintained
in RPMI-1640 medium (Sigma–Aldrich, St. Louis, USA) supple-
mented with 10% (v/v) heat inactivated fetal bovine serum (FBS)
obtained from Gibco-BRL. The colorectal cancer cell lines, HT-29
and Caco-2 as well as HS683 were maintained in Dulbecco’s
Modified Eagles Medium (DMEM) (Sigma–Aldrich, St. Louis, USA)
supplemented with 5–10% (v/v) FBS. U373 cells were maintained
in Earles’s Modified Eagles medium supplemented with 10% FBS
and 1 mM sodium pyruvate.
All cells were grown in 75 cm3 tissue culture flasks at 37 °C in a
humidified incubator with a 5% CO2 atmosphere using standard
aseptic culture techniques. The HL-60, K562 and Jurkat cell lines
were grown in suspension and sub-cultured when the cells
reached a density of 1.5 ꢁ 106/mL. Cell densities were reduced to
0.5 ꢁ 106/mL and the growth media replaced. The adherent cell
lines, HT-29, Caco-2, U373 and Hs683 were sub-cultured when
the cells were confluent using a Trypsin-versene solution and
reseeded into the same culture flasks. Prior to cell viability studies,
cells were harvested and their viability status confirmed by the
trypan blue assay.
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