Synthesis and evaluation of sulfonylethyl-containing phosphotriesters of 3′-azido-3′-deoxythymidine as anticancer prodrugs

Article abstract of DOI:10.1016/j.bmc.2014.09.046

A series of bis(sulfonylethyl) and mono(sulfonylethyl) phenyl phosphotriesters of zidovudine (3′-azido-3′-deoxythymidine, AZT) were synthesized as potential anticancer prodrugs that liberate AZT monophosphate via nonenzymatic β-elimination mechanism. Stab

Full text of DOI:10.1016/j.bmc.2014.09.046

Accepted Manuscript
Synthesis and evaluation of sulfonylethyl-containing phosphotriesters of 3′-azi-
do-3′-deoxythymidine as anticancer prodrugs
Jiang Wang, Yi-Jun Wang, Zhe-Sheng Chen, Chul-Hoon Kwon
PII:
S0968-0896(14)00700-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.046
BMC 11833
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2014
13 September 2014
22 September 2014
Please cite this article as: Wang, J., Wang, Y-J., Chen, Z-S., Kwon, C-H., Synthesis and evaluation of sulfonylethyl-
containing phosphotriesters of 3′-azido-3′-deoxythymidine as anticancer prodrugs, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.046
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Synthesis and evaluation of sulfonylethyl-containing phosphotriesters of
3'-azido-3'-deoxythymidine as anticancer prodrugs
Jiang Wang, Yi-Jun Wang, Zhe-Sheng Chen and Chul-Hoon Kwon*
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s
University, Jamaica, New York 11439
*Corresponding author. Tel: (718)-990-5214. Fax: (718)-990-6551. E-mail: kwonc@stjohns.edu.
Abstract
A series of bis(sulfonylethyl) and mono(sulfonylethyl) phenyl phosphotriesters of
zidovudine (3'-azido-3'-deoxythymidine, AZT) were synthesized as potential anticancer prodrugs
that liberate AZT monophosphate via nonenzymatic β-elimination mechanism. Stability studies
demonstrated that all the synthesized prodrugs spontaneously liberate AZT monophosphate with
half-lives in the range of 0.07-278.8 h under model physiological conditions in 0.1 M phosphate
o
buffer at pH 7.4 and 37 C. Analogous to aldophosphamide, the elimination rates were
accelerated in the presence of reconstituted human plasma under the same conditions. Among the
compounds, 3, 4, 8, and 10 were comparable or superior to AZT against five established human
cancerous cell lines in vitro. Moreover, the selected compounds were equally sensitive to both
the wild-type osteosarcoma 143B and the thymidine kinase-deficient 143B/TK- cell lines. The
findings are consistent with that these compounds deliver AZT monophosphate intracellularly.
Key words:
AZT; AZT monophosphate; sulfonylethyl; phosphotriester; pronucleotide; β-elimination;
prodrugs.
1 / 44

1. Introduction
Zidovudine (3'-azido-3'-deoxythymidine, AZT), a nucleoside reverse transcriptase inhibitor,
was the first drug approved by FDA for the treatment of HIV/AIDS infections. However, interest
in repositioning AZT as an anticancer agent has emerged since 1989.1 It was reported that AZT
could potentiate the anticancer effects of other chemotherapeutic agents such as paclitaxel, 5-
fluorouracil, emodin, and cisplatin.2-5 When used alone, AZT exhibited potent inhibitory activity
against human breast cancer, melanoma, and multiple myeloma cell growth.6-8 The anticancer
activity of AZT may be associated with its intracellular conversion to 5'-triphosphate, which
functions as a competitive inhibitor of DNA polymerase, and it incorporates itself into the
growing DNA strand.9 It terminates DNA chain replication, which induces apoptosis of the
target cells. AZT triphosphate was also found to inhibit human telomerase activity in many
cancer cell lines in vitro.10-12 Approximately 90% of all human cancers exhibit enhanced
telomerase activity, and it is proposed that telomerase may be involved in cell immortalization,
tumorigenesis and inhibition of apoptosis.11
Cellular drug resistance, however, can develop due to prolonged exposure to AZT, which
enhances the cellular expression of P-glycoprotein (P-gp), multidrug resistance proteins (MRPs),
and breast cancer resistance protein (BCRP), which in turn cause efflux of AZT and its
metabolites from the cells.13-16 In AZT-resistant cells the activity of thymidine kinase is reduced,
and prolonged exposure of cells to AZT results in decreased levels of thymidine kinase,17 the
enzyme responsible for the intracellular transformation of AZT into AZT monophosphate. This
phosphorylation is essential to the activation of the drug to AZT triphosphate. Moreover, AZT
possesses such undesirable pharmacokinetic properties as short half-life and small volume of
distribution.18
2 / 44

In order to improve the pharmacokinetic profile of AZT and overcome the drawbacks of
AZT-based therapy, many pronucleotides of AZT have been developed, and their anticancer
activities have been investigated. Iyer et al. evaluated the anticancer activity of a series of AZT
phosphoramidate monoesters containing amino acid methyl ester and N-alkylamide moieties and
found that the synthesized prodrugs were less potent than AZT against MCF-7 and CEM cell
lines.18 When the cytotoxicity of AZT 5'-chloromethylphosphonates against the human cancer
cell lines KB and MCF-7 were examined by Celewicz et al., the phosphonates exhibited less
anticancer activity than did AZT, and the hydrolysis studies showed that these pronucleotides act
as depot forms of AZT rather than prodrugs of AZT 5'-phosphate.9 More recently, Liu et al.
studied a series of AZT phosphorothioamidates and proposed that these pronucleotides were able
to release AZT monothiophosphate inside the cell.19 These prodrugs presented selective toxicity
toward cancer cells, as they were more potent than AZT against RKO colon carcinoma, but less
cytotoxic than AZT towards 293T embryonic kidney cells.19
Substituted sulfonylethyl groups have successfully been employed in our laboratory for the
intracellular delivery of phosphate-containing drug molecules in the design of phosphoramide
mustard prodrugs20 and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) prodrugs.21 Now
we applied the same approach to AZT, synthesizing and evaluating a series of bis(sulfonylethyl)
phosphotriesters of AZT (Figure 1A). The proposed activation mechanism for the these prodrugs
involves two successive β-elimination steps to release AZT monophosphate (Scheme 1A).
Moreover, phosphodiesterases may have great influence on the activity of the pronucleotides.22
The phenyl phosphodiester of AZT proved to be a good substrate of type I phosphodiesterase
and was hydrolyzed rapidly in the presence of the enzyme.23 Therefore, mono(sulfonylethyl)
phenyl phosphotriesters of AZT (Figure 1B) were also prepared and investigated. The
3 / 44

decomposition of the mono(sulfonylethyl) phenyl prodrugs is expected to follow β-elimination
and subsequent phosphodiesterase-hydrolyzing steps (Scheme 1B).
2. Results and Discussion
2.1. Chemistry
All the sulfonylethyl-containing phosphotiesters were synthesized using P(III)
(phosphoramidite) chemistry. 2-(phenylthio)ethanol (1c) is commercially available. Reaction of
2-chloroethanol with corresponding p-substituted thiophenols yielded compounds 2c-5c (Scheme
2). Thiophenol and allyl alcohol were used to prepare 2-(phenylthio)propan-1-ol (6c) with sulfur
as catalyst (Scheme 3). Other α- and β-substituted thioethanols, compounds 7c-10c, were
prepared by adding suitable epoxides to sodium thiophenolate in methanol (Scheme 4).
Phosphoramidite intermediates 1b-15b were prepared via two synthetic steps (Scheme 5). In the
first step, crystalline bis(diisopropylamino)chlorophosphine was added to the mixture of dry
triethylamine and thioethanol derivatives, yielding the bis(diisopropylamino)alkoxyphosphine
intermediates. In the second step, the crude products were treated with either thioethanol or
phenol derivatives in the presence of the acid catalyst 4,5-dicyanoimidazole (DCI). Intermediates
1b-15b were coupled with AZT using 1H-tetrazole in anhydrous acetonitrile, followed by in situ
oxidation using tert-butyl hydroperoxide (TBHP), resulting in the formation of the
phosphotriester derivatives 1a-15a. The thioethyl groups were then oxidized to form
sulfonylethyl groups using an excess of meta-chloroperoxybenzoic acid (mCPBA) to obtain the
target compounds 1-15 (Scheme 6).
Some compounds were obtained as mixtures of diastereoisomers due to the asymmetrical
center formed at the phosphorus atom in the reactions. Moreover, more than two signals were
present in 31P NMR spectra of compounds 7, 8, and 10 due to the chiral α- and β-carbons of their
4 / 44

sulfonylethyl moieties. Unfortunately, these compounds could not be isolated by preparative
TLC or column chromatography and further purification was not attempted. All the synthesized
analogues presumably permeate the cell membrane by passive diffusion and release the identical
mononucleotide in the cell.
2.2. Stability studies
Stability studies were conducted to determine the half-lives of the synthesized
o
pronucleotides in 0.1 M phosphate buffer at pH 7.4 and 37 C and in the presence of human
plasma under the same conditions. The results are presented in Table 1.
As anticipated, the phosphotriesters bearing various substituted sulfonylethyl moieties
possess different kinetic properties. Compound 2 with a p-methyl group was observed to have a
slightly longer half-life than compound 1. The half-life of compound 3 with a p-methoxy
substitution was twice of that of compound 1. The electron-donating methyl and methoxy groups
make the α-proton of the sulfonylethyl moiety less acidic, resulting in decreased β-elimination
rates. Since methoxy is more electron-donating than methyl, compound 3 had a longer half-life
than compound 2. Conversely, the electron-withdrawing p-chloro group in 4 and p-nitro group in
5 enhance the acidity of α-proton; as a result, compounds 4 and 5 showed higher β-elimination
rates than compound 1. The p-substituents confer the β-elimination reactivity in the following
order: -NO2 > -Cl > -Me > -OMe. All the α- and β- substituted analogues provided longer half-
lives than the nonsubstituted compound 1. The α-methyl-substituted prodrug 6 exhibited a much
slower elimination rate compared to its β-methyl-substituted counterpart, prodrug 7.
Combination of the steric hindrance, the reduced acidity of α-proton due to the presence of
methyl group and the availability of only one α-proton in compound 6 could explain the retarded
elimination rate. Compound 8, with a β-ethyl substitution, showed more chemical stability than
5 / 44

compound 7 because the steric hindrance provided by the ethyl group was more prominent than
it was in the methyl group. Both α- and β-methyl substitutions made compound 9 extremely
stable: the half-life was approximately 12 days in the phosphate buffer. Unexpectedly, the α-
phenyl-substituted prodrug 10 had a much shorter half-life than compound 6, which has a much
smaller substitution at the α position. We had assumed that the electron-inductive effect of the α-
phenyl group might increase the acidity of α-proton.
All the mono(sulfonylethyl)phenyl phosphotriesters 11-15 exhibited longer half-lives than
compound 1. The half-lives varied slightly, depending on the p-substituents on the phenoxy
group. The overall trend was that the electron-donating p-substitutions decelerated the β-
elimination rates. Conversely, electron-withdrawing p-substitutions accelerated the elimination
rates. Since the half-lives of the analogues were determined by the rate of disappearance of the
remaining prodrugs, not the generation of nucleoside monophosphate, it might be argued that the
half-lives of these mono(sulfonylethyl) phenyl phosphotriesters could only give the information
for the β-elimination rates of the sulfonylethyl group, but the rates of actual release of
monophosphate was still not established. Mono(thioethyl) phenyl phosphotriester 12a which
lacks a sulfonyl group displayed much greater stability. It had a half-life 12 h in phosphate buffer,
and was stable for 20 h in human plasma, suggesting that the cleavage of the phenoxy group
from the phosphate constituted a rate-limiting step for the liberation of AZT monophosphate.
All the synthesized phosphotriesters of AZT displayed a similarly accelerated β-elimination
profile in plasma. The extent of enhancement of β-elimination reaction was 2.2-4.2 times in
plasma what it was in the phosphate buffer. The results were consistent with the previous
findings: plasma proteins, specifically, human serum albumin (HSA), contribute to the faster
6 / 44

elimination of cyclophosphamide, sulfonylethyl analogues of phosphoramide mustard and
FdUMP.20, 21, 24
2.3. Anticancer activity
The sulfonylethyl prodrugs were investigated by employing MTT assay for their cytotoxic
activity against five human cancer cell lines: MCF-7 (breast adenocarcinoma), H460 (lung
carcinoma), KB-3-1 (epidermoid carcinoma), SW620 (colorectal adenocarcinoma), and 143B
(osteosarcoma). AZT was used as a positive control. The IC50 values are presented in Table 2.
Cytotoxicity of AZT and all the synthesized pronucleotides is directly associated with the
intracellular level of AZT monophosphate. As all the phosphotriesters were designed to release
active metabolite AZT monophosphate inside the cell, their ability to penetrate the cell
membrane and rate of intracellular conversion are of great importance in determining their
cytotoxic effects.
AZT and all the synthesized compounds displayed the same trend of cytotoxic effects on the
tested cell lines. They were most sensitive to H460 cells but displayed highest IC50 values in
SW620 cells. Compounds 1 and 2 were slightly less toxic than AZT to all the cell lines.
Compounds 3 and 4 exhibited higher potency compared to AZT against all the cell lines.
Compound 5, with p-nitro substitution, was approximately half as active as AZT. No direct
relationship between their stability and cytotoxicity can be deduced based on these results, but a
compound with an extremely short half-life may not be a good drug candidate as active AZT
monophosphate might be released more in the extracellular medium than in the intracellular
compartment. The α-methyl-substituted analogue 6 showed higher IC50 value than the β-methyl-
substituted analogue 7, but both were less cytotoxic than AZT. However, analogue 8, which
displayed higher stability than 7, exhibited slightly better activity than AZT. Compound 9, which
7 / 44

had an extremely long half-life, was 1/3 to 1/2 as potent against all the cell lines as was AZT.
The α-phenyl-substituted analogue 10 was as potent as AZT. Since the intracellular conversion
rate might be slow in the prodrugs with extremely high stability, as in the cases of compounds 6
and 9, the acute toxicity of these prodrugs was reduced due to there being less AZT
monophosphate present in the target cells. Moreover, compounds 8 and 10 possess higher
lipophilicity due to the presence of β-ethyl and α-phenyl groups, respectively, which may allow
for the increased permeation through the cell membrane. Thus lower IC50 values were displayed
by compounds 8 and 10 than by the other α- and β-substituted sulfonylethyl analogues.
The mono(sulfonylethyl) phenyl phosphotriesters 11-15 presented very similar potency,
perhaps because of the slow intracellular liberation of AZT monophosphate. Unlike
bis(sulfonylethyl) prodrugs which require two consecutive β-elimination steps to liberate active
AZT monophosphate, the activation of this series of compounds is a phosphodiesterase-involved
process. The level of phosphodiesterases in the target cells might be a determining factor in the
intracellular generation of AZT monophosphate.
In order to further probe the mode of action of the phosphotriester prodrugs, we evaluated
compounds 3, 4, 8, 10, 13, and AZT against both a parental 143B cell line and thymidine kinase-
deficient 143B/TK- cell line. The data are shown in Table 3. The IC50 value of AZT for
143B/TK- cells is more than 12 times as high as that for wild-type 143B cells. However, the IC50
values of all the selected analogues were not significantly different for 143B and 143B/TK- cells.
In other words, these compounds could reverse the cellular resistance to AZT. This confirmed
that the mechanism of action of these phosphotriester prodrugs involved the intracellular
liberation of AZT monophosphate.
3. Conclusion
8 / 44

The phosphotriesters bearing sulfonylethyl pro-moieties were successfully applied to AZT
as anticancer prodrugs. Efficient preparation of these compounds were achieved by employing
bis(diisopropylamino)chlorophosphine as a phosphitylating agent via P(III) chemistry. Stability
studies showed that the half-lives of these compounds vary in a substituent-dependent manner.
Higher β-elimination rates were observed for all compounds in model physiological phosphate
o
buffer solution as well as in the presence of human plasma at pH 7.4 and 37 C. The in vitro
anticancer activities of these compounds were evaluated against MCF-7, H460, KB-3-1, SW620,
and 143B cancer cell lines. Compounds 3, 4, and 8 were more cytotoxic to five human cancer
cell lines than AZT. Compound 3 was most potent among all the synthesized prodrugs. AZT was
less than 1/12 as effective against thymidine kinase-deficient 143B/TK- cells as it was against
wild-type 143B cells, while all the selected prodrugs were equally potent against both cell lines.
These results were consistent with an activation mechanism that involves intracellular delivery of
AZT monophosphate, which circumvents the first phosphorylation step.
4. Experimental section
4.1. Chemistry
All water-sensitive experiments were conducted under scrupulously anhydrous conditions
using oven-dried glassware. Moisture-sensitive reactions were performed under nitrogen
atmosphere. Triethylamine was dried by refluxing over calcium hydride and stored over
activated 4Å molecular sieves. Evaporation of solvents was carried out on a rotary evaporator
under reduced pressure. Melting points were determined on a Thomas-Hoover capillary melting
1
point apparatus and were uncorrected. H, 13C, and 31P NMR spectra were recorded on a
Bruker spectrometer (1H, 400 MHz; 13C, 100 MHz). All 13C and 31P NMR spectra were recorded
1
proton-decoupled. All NMR spectra were run at ambient temperature. Chemical shifts of H
9 / 44

NMR spectra are given in δ values relative to tetramethylsilane (TMS) peak used as the internal
reference. Chemical shifts of 31P NMR spectra are reported relative to external reference
phosphoric acid. Abbreviations for the signal patterns are described as singlet (s), doublet (d),
triplet (t), quartet (q), quintet (quin), sextet (sex), septet (sep), multiplet (m), broad (br), tertiary
1
carbons (C tert), quaternary carbons (C quat). Many H NMR signals were split due to the
presence of phosphate diastereoisomers in the samples. Elemental analyses were carried out by
Atlantic Microlab Inc., Norcross, GA and the observed values were within 0.4% of the
calculated values. The column chromatography refers to flash chromatography carried out using
Merck silica gel 60 (40-60 µm) as stationary phase. Preparative thin-layer chromatography was
performed on Merck pre-coated glass plates (20 × 20 cm, silica gel GF, 1000 µm thick)
purchased from Analtech Inc., Newark, DE, and visualized under 254 nm UV light. AZT was
purchased from AK Scientific, Inc., Union City, CA.
4.1.1. Standard procedure 1: preparation of p-substituted phenylthioethanol (2c-5c)
Compounds 2c-5c were prepared according to the published procedure.21
4.1.1.1. 2-(p-Tolylthio)ethanol (2c). Starting with 2-chloroethanol (2.42 g, 30 mmol) and p-
thiocresol (2.48 g, 20 mmol), product 2c was obtained as a pale yellow oil (2.68 g, 80%). Rf 0.36
(EtOAc/n-hexane = 1:4); 1H NMR (DMSO) δ 7.26 (2H, m), 7.12 (2H, d, J = 8.4Hz), 3.56 (2H, t,
J = 7.2 Hz), 2.99 (2H, t, J = 6.8Hz), 2.26 (3H, s).
4.1.1.2. 2-(p-Methoxyphenylthio)ethanol (3c). Starting with 2-chloroethanol (2.42 g, 30 mmol)
and 4-methoxythiophenol (2.80 g, 20 mmol), product 3c was obtained as a colorless solid (2.76 g,
1
75%). mp 40-41 ºC; Rf 0.28 (EtOAc/n-hexane = 1:4); H NMR (DMSO) δ 7.35 (2H, m), 6.91
(2H, m), 3.74 (3H, s), 3.50 (2H, m), 2.91(2H, t, J = 7.0 Hz).
10 / 44

4.1.1.3. 2-(p-Chlorophenylthio)ethanol (4c). Starting with 2-chloroethanol (2.42 g, 30 mmol)
and 4-chlorothiophenol (2.89 g, 20 mmol), product 4c was obtained as a pale yellow oil (2.64 g,
1
70%). Rf 0.24 (EtOAc/n-hexane = 1:4); H NMR (DMSO) δ 7.36 (4H, m), 3.58 (2H, q, J = 6.4
Hz), 3.06 (2H, t, J = 6.7 Hz).
4.1.1.4. 2-(p-Nitrophenylthio)ethanol (5c). Starting with 2-chloroethanol (2.42 g, 30 mmol) and
4-nitrothiophenol (3.10 g, 20 mmol), product 5c was obtained as a yellow solid (2.03 g, 51%).
mp 61-62 ºC; Rf 0.30 (EtOAc/n-hexane = 1:1); 1H NMR (DMSO) δ 8.12 (2H, m), 7.51 (2H, m),
3.66 (2H, m), 3.21 (2H, t, J = 6.4 Hz).
4.1.2. Preparation of 2-(phenylthio)propan-1-ol (6c)
Compound 6c was synthesized according to the procedure described previously.25 Starting
with thiophenol (11 g, 0.1 mol) and allyl alcohol (6.96 g, 0.12 mol), product 6c was obtained as a
pale yellow liquid (2.01 g, 12%). Rf 0.50 (EtOAc/n-hexane = 1:3); 1H NMR (DMSO) δ 7.38 (2H,
d, J = 7.2 Hz), 7.32 (2H, t, J = 7.6 Hz), 7.23 (1H, t, J = 7.2 Hz), 3.52 (1H, m), 3.31 (2H, m), 1.23
(3H, d, J = 6.1 Hz); 13C NMR (DMSO) δ 134.9 (C quat), 130.4 (C tert), 128.9 (C tert), 126.4 (C
tert), 65.1 (C1), 43.9 (C2), 17.6 (CH3).
4.1.3. Standard procedure 2: preparation of 2-(phenylthio)ethanol derivatives (7c-10c)
Compounds 7c-10c were synthesized according to the published procedure.26
4.1.3.1. 1-(Phenylthio)propan-2-ol (7c). Starting with sodium thiophenolate (1.58 g, 12 mmol)
and propylene oxide (0.58 g, 10 mmol), product 7c was obtained as a pale yellow oil (1.49 g,
1
89%). Rf 0.50 (EtOAc/n-hexane = 1:3); H NMR (DMSO) δ 7.32 (4H, m), 7.16 (1H, t, J = 7.0
13
Hz), 3.78 (1H, sep, J = 5.9 Hz), 2.96 (2H, m), 1.16 (3H, d, J = 6.3 Hz); C NMR (DMSO) δ
136.9 (C quat), 128.9 (C tert), 127.8 (C tert), 125.3 (C tert), 65.2 (C1), 41.0 (C2), 22.5 (CH3).
11 / 44

4.1.3.2. 1-(Phenylthio)butan-2-ol (8c). Starting with sodium thiophenolate (1.58 g, 12 mmol)
and 1,2-butylene oxide (0.72 g, 10 mmol), product 8c was obtained as a pale yellow oil (1.26 g,
1
69%). Rf 0.44 (EtOAc/n-hexane = 1:4); H NMR (DMSO) δ 7.31 (4H, m), 7.16 (1H, t, J = 7.0
Hz), 3.52 (1H, sex, J = 6.0 Hz), 2.97 (2H, d, J = 6.1 Hz), 1.58 (1H, m), 1.38 (1H, m), 0.88 (3H, t,
J = 7.3 Hz).
4.1.3.3. 3-(Phenylthio)butan-2-ol (9c). Starting with sodium thiophenolate (1.58 g, 12 mmol)
and 2,3-epoxybutane (0.72 g, 10 mmol), product 9c was obtained as a pale yellow oil (1.33 g,
73%). Rf 0.42 (EtOAc/n-hexane = 1:4); 1H NMR (DMSO) δ 7.37 (2H, d, J = 7.2 Hz), 7.32 (2H, t,
J = 7.6 Hz), 7.21 (1H, t, J = 7.2 Hz), 3.76 (1H, sex, J = 5.5 Hz), 3.35 (1H, m), 1.21 (3H, d, J =
6.8 Hz), 1.12 (3H, d, J = 6.3 Hz).
4.1.3.4. 2-Phenyl-2-(phenylthio)ethanol (10c). Starting with sodium thiophenolate (1.58 g, 12
mmol) and styrene oxide (1.20 g, 10 mmol), product 10c was obtained as a pale yellow oil (0.92
1
g, 40%). Rf 0.36 (EtOAc/n-hexane = 1:4); H NMR (DMSO) δ 7.30 (10H, m), 4.45 (1H, t, J =
6.6 Hz), 3.77 (2H, m).
4.1.4. Standard procedure 3: preparation of phosphoramidites (1b-15b)
The phosphoramidite intermediates 1b-15b were prepared by a modified procedure
previously described.27 To a stirred solution of thioethanol (4.0 mmol) in anhydrous THF (20 mL)
containing triethylamine (6.06 g, 6 mmol) was added bis(diisopropylamino)chlorophosphine
(1.28 g, 4.8 mmol) at room temperature under N2. After 2h, TLC analysis indicated complete
conversion of the alcohol into a product with a higher Rf value. After removal of the (Et3)N·HCl
salt by filtration and concentration of the filtrate under reduced pressure afforded the product as
viscous liquid.
12 / 44

The residue was dissolved in anhydrous CH2Cl2 (20 mL) and the corresponding alcohol (3.2
mmol) together with 4,5-dicyanoimidazole (1.89 g, 1.6 mmol) was added at ambient temperature
and stirred for 30 min. The resulting mixture was washed with cold saturated NaHCO3 solution
(2 × 20 mL) and cold saturated NaCl solution (20 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by
preparative TLC with an appropriate solvent containing 1% N,N-dimethylethylamine to obtain
the products.
4.1.4.1. Bis[2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (1b). Product 1b was
obtained as a yellow viscous liquid (0.81 g, 58%). Rf 0.86 (EtOAc/n-hexane = 1:4 containing 1%
N,N-dimethylethylamine); 1H NMR (CDCl3) δ 7.36 (4H, d, J = 7.1 Hz), 7.26 (4H, t, J= 7.6 Hz),
7.17 (2H, t, J = 7.3 Hz), 3.85-3.67 (4H, m), 3.61-3.50 (2H, m), 3.18-3.08 (4H, m), 1.12 (12H, d, J
= 6.8 Hz); 31P NMR (CDCl3) δ 147.74.
4.1.4.2. Bis[2-(p-tolylthio)ethyl] N,N-diisopropylphosphoramidite (2b). Product 2b was
obtained as a yellow viscous liquid (0.86 g, 58%). Rf 0.88 (EtOAc/n-hexane = 1:4 containing 1%
N,N-dimethylethylamine); 1H NMR (CDCl3) δ 7.31 (4H, d, J = 7.2 Hz), 7.11 (4H, d, J = 7.3 Hz),
3.81-3.73 (4H, m), 3.58-3.48 (2H, m), 3.13-3.09 (4H, m), 2.34 (6H, s), 1.16 (12H, d, J = 6.8 Hz);
31P NMR (CDCl3) δ 147.90.
4.1.4.3. Bis[2-(p-methoxyphenylthio)ethyl] N,N-diisopropylphosphoramidite (3b). Product
3b was obtained as a yellow viscous liquid (1.04 g, 65%). Rf 0.81 (EtOAc/n-hexane = 1:4
containing 1% N,N-dimethylethylamine); 1H NMR (CDCl3) δ 7.36 (4H, d, J = 6.7 Hz), 6.82 (4H,
d, J = 6.9 Hz), 3.77 (6H, s), 3.76-3.61 (4H, m), 3.58-3.46 (2H, m), 3.07-2.92 (4H, m), 1.10 (12H,
d, J = 6.1 Hz); 31P NMR (CDCl3) δ 147.72.
13 / 44

4.1.4.4. Bis[2-(p-chlorophenylthio)ethyl] N,N-diisopropylphosphoramidite (4b). Product 4b
was obtained as a yellow viscous liquid (0.81 g, 50%). Rf 0.72 (EtOAc/n-hexane = 1:4
containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.39-7.32 (8H, m), 3.80-3.63 (4H,
m), 3.55-3.43 (2H, m), 3.17 (4H, t, J = 6.5 Hz), 1.06 (12H, d, J = 6.6 Hz); 31P NMR (DMSO) δ
148.56.
4.1.4.5. Bis[2-(p-nitrophenylthio)ethyl] N,N-diisopropylphosphoramidite (5b). Product 5b
was obtained as a yellow viscous liquid (1.33 g, 79%). Rf 0.72 (EtOAc/n-hexane = 1:1
containing 1% N,N-dimethylethylamine); 1H NMR (CDCl3) δ 8.11 (4H, d, J = 8.6 Hz), 7.37 (4H,
d, J = 8.9 Hz), 3.96-3.79 (4H, m), 3.64-3.54 (2H, m), 3.39 (4H, t, J = 6.8 Hz), 1.16 (12H, d, J =
6.8 Hz); 31P NMR (CDCl3) δ 147.90.
4.1.4.6. Bis[2-(phenylthio)propyl] N,N-diisopropylphosphoramidite (6b). Product 6b was
obtained as a yellow viscous liquid (0.81 g, 54%). Rf 0.88 (EtOAc/n-hexane = 1:4 containing 1%
N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.40 (4H, d, J = 7.7 Hz), 7.32 (4H, t, J = 7.4 Hz),
7.25 (2H, t, J = 7.3 Hz), 3.68-3.54 (2H, m), 3.53-3.48 (2H, m), 3.47-3.38 (4H, m), 1.27-1.22 (6H,
m), 1.05 (12H, t, J = 6.8 Hz); 31P NMR (DMSO) δ 147.65, 147.40.
4.1.4.7. Bis[1-(phenylthio)propan-2-yl] N,N-diisopropylphosphoramidite (7b). Product 7b
was obtained as a yellow viscous liquid (1.16 g, 78%). Rf 0.73 (EtOAc/n-hexane = 1:6
containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.37-7.26 (8H, m), 7.18 (2H, t, J=
7.1 Hz), 3.99-3.86 (2H, m), 3.51 (2H, m), 3.06 (4H, m), 1.25 (6H, m), 1.16 (12H, d, J = 6.2 Hz);
31P NMR (DMSO) δ 146.45, 145.99, 145.48.
4.1.4.8. Bis[1-(phenylthio)butan-2-yl] N,N-diisopropylphosphoramidite (8b). Product 8b was
obtained as a yellow viscous liquid (1.04 g, 66%). Rf 0.74 (EtOAc/n-hexane = 1:6 containing 1%
N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.36-7.25 (8H, m), 7.18 (2H, t, J = 7.3 Hz), 3.95-
14 / 44

3.75 (2H, m), 3.61-3.46 (2H, m), 3.16-3.01 (4H, m), 1.76-1.49 (4H, m), 1.09 (12H, d, J = 6.9 Hz),
0.88-0.81 (6H, m); 31P NMR (DMSO) δ 146.78, 146.21, 145.81.
4.1.4.9. Bis[3-(phenylthio)butan-2-yl] N,N-diisopropylphosphoramidite (9b). Product 9b was
obtained as a yellow viscous liquid (0.92 g, 58%). Rf 0.86 (EtOAc/n-hexane = 1:6 containing 1%
N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.39 (4H, d, J = 7.2 Hz), 7.32 (4H, t, J = 7.4 Hz),
7.24 (2H, t, J = 7.2 Hz), 3.95-3.81 (2H, m), 3.55-3.44 (2H, m), 3.43-3.34 (2H, m), 1.23-1.16
(12H, m), 1.04 (12H, d, J = 6.6 Hz); 31P NMR (DMSO) δ 146.65, 146.16, 145.66.
4.1.4.10. Bis[2-phenyl-2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (10b). Product
11b was obtained as a yellow viscous liquid (1.39 g, 74%). Rf 0.87 (EtOAc/n-hexane = 1:6
1
containing 1% N,N-dimethylethylamine); H NMR (DMSO) δ 7.37-7.20 (20H, m), 4.59-3.50
31
(2H, m), 3.92-3.62 (4H, m), 3.36-3.25 (2H, m), 0.91 (12H, d, J = 6.9 Hz); P NMR (DMSO) δ
147.21, 146.78.
4.1.4.11. Phenyl [2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (11b). Product 11b
was obtained as a yellow viscous liquid (0.64 g, 53%). Rf 0.79 (EtOAc/n-hexane = 1:5
containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.42-7.24 (6H, m), 7.19 (1H, t, J =
7.2 Hz), 7.06-6.90 (3H, m), 3.90-3.74 (2H, m), 3.72-3.58 (2H, m), 3.21 (2H, t, J = 6.4 Hz), 1.11
(12H, d, J = 7.1 Hz); 31P NMR (DMSO) δ 146.69.
4.1.4.12. 2-(Phenylthio)ethyl p-tolyl N,N-diisopropylphosphoramidite (12b). Product 12b
was obtained as a yellow viscous liquid (1.12 g, 90%). Rf 0.80 (EtOAc/n-hexane = 1:5
containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.41-7.23 (4H, m), 7.18 (1H, t, J =
7.1 Hz), 7.06-6.92 (4H, m), 3.88-3.72 (2H, m), 3.70-3.55 (2H, m), 3.20 (2H, t, J = 6.3 Hz), 2.55
(3H, s), 1.11 (12H, d, J = 6.9 Hz); 31P NMR (DMSO) δ 146.52.
15 / 44

4.1.4.13. p-Methoxyphenyl [2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (13b).
Product 13b was obtained as a yellow viscous liquid (1.05 g, 81%). Rf 0.79 (EtOAc/n-hexane=
1:5 containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.42-7.25 (4H, m), 7.20 (1H, t,
J = 7.4 Hz), 6.94-6.78 (4H, m), 3.89-3.74 (2H, m), 3.69 (3H, s), 3.67-3.49 (2H, m), 3.20 (2H, t, J
= 6.5 Hz), 1.11 (12H, d, J = 6.8 Hz); 31P NMR (DMSO) δ 146.48.
4.1.4.14. p-Fluorophenyl [2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (14b).
Product 14b was obtained as a yellow viscous liquid (1.10g, 87%). Rf 0.79 (EtOAc/n-hexane =
1:5 containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.37 (2H, d, J = 7.5 Hz), 7.31
(2H, t, J = 7.4 Hz), 7.20 (1H, t, J = 7.2 Hz), 7.11 (2H, t, J = 7.6 Hz), 7.01-6.92 (2H, m), 3.89-3.73
(2H, m), 3.72-3.56 (2H, m), 3.19 (2H, t, J = 6.8 Hz), 1.12 (12H, d, J = 6.8 Hz); 31P NMR (DMSO)
δ 147.91.
4.1.4.15. p-Chlorophenyl [2-(phenylthio)ethyl] N,N-diisopropylphosphoramidite (15b).
Product 15b was obtained as a yellow viscous liquid (0.92 g, 70%). Rf 0.80 (EtOAc/n-hexane =
1:5 containing 1% N,N-dimethylethylamine); 1H NMR (DMSO) δ 7.42-7.24 (6H, m), 7.20 (1H, t,
J = 7.2 Hz), 6.99 (2H, d, J = 8.0 Hz), 3.91-3.72 (2H, m), 3.71-3.58 (2H, m), 3.21 (2H, t, J = 6.5
Hz), 1.11 (12H, d, J = 7.0 Hz); 31P NMR (DMSO) δ 147.72.
4.1.5. Standard procedure 4: preparation of phosphotriester derivatives of AZT (1a-15a)
AZT phosphotriesters 1a-15a were prepared according to the published procedure of with
modifications.28 1H-tetrazole (8.8 mL, 4 mmol, 0.45 M in acetonitrile, 4 equiv.) was added to a
stirred solution of 3'-azido-3'-deoxythymidine (267 mg, 1 mmol) and phosphoramidite (1.2 mmol)
in anhydrous acetonitrile (8 mL) at room temperature. After 3 h, the reaction mixture was cooled
to 0 ºC, and a solution of TBHP (0.44 mL, 2.4 mmol, 5.5 M in nonane, 2.4 equiv.) was added;
the reaction mixture was then allowed to warm to room temperature for over 1 h. The solvent
16 / 44

was removed under reduced pressure. The residue was diluted in CH2Cl2 (50 mL) and washed
with 10% Na2S2O3 solution (50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL)
and the combined organic phase was washed with water (100 mL). The organic layer was dried
over anhydrous Na2SO4, and evaporated to dryness under reduced pressure followed by
preparative TLC purification to get the desired compounds.
4.1.5.1. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(phenylthio)ethyl] phosphate (1a). Product 1a
1
was obtained as a colorless viscous liquid (0.26 g, 42%). Rf 0.58 (CH2Cl2/MeOH = 9:1); H
NMR (DMSO) δ 11.39 (1H, br s, NH), 7.45 (1H, s), 7.40-7.27 (8H, m), 7.21 (2H, t, J = 7.0 Hz),
6.12 (1H, t, J = 6.5 Hz), 4.43 (1H, q, J = 5.9 Hz), 4.24-4.15 (2H, m), 4.09 (4H, q, J = 6.9 Hz),
4.02-3.93 (1H, m), 3.24 (4H, t, J = 6.5 Hz), 2.43-2.25 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ
-0.39.
4.1.5.2. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-tolylthio)ethyl] phosphate (2a). Product 2a
1
was obtained as a colorless viscous liquid (0.29 g, 45%). Rf 0.59 (CH2Cl2/MeOH = 9:1); H
NMR (DMSO) δ 11.39 (1H, br s, NH), 7.44 (1H, s), 7.30-7.22 (4H, m), 7.12 (4H, d, J = 7.2 Hz),
6.12 (1H, t, J = 6.8 Hz), 4.42 (1H, q, J = 5.8 Hz), 4.21-4.14 (2H, m), 4.05 (4H, q, J = 6.8 Hz),
31
3.99-3.92 (1H, m), 3.22-3.11 (4H, m), 2.38-2.32 (2H, m), 2.26 (6H, s), 1.74 (3H, s); P NMR
(DMSO) δ -0.38.
4.1.5.3. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-methoxyphenylthio)ethyl] phosphate (3a).
Product 3a was obtained as a pale yellow viscous liquid (0.27 g, 40%). Rf 0.53 (CH2Cl2/MeOH =
9:1); 1H NMR (CDCl3) δ 9.03 (1H, br s, NH), 7.42-7.33 (5H, m), 6.88-6.80 (4H, m), 6.21 (1H, t,
J = 6.5 Hz), 4.34-4.26 (2H, m), 4.25-4.21 (1H, m), 4.19-4.12 (4H, m), 4.03-3.96 (1H, m), 3.79
(6H, s), 3.08-2.99 (4H, m), 2.46-2.24 (2H, m), 1.90 (3H, s); 31P NMR (CDCl3) δ -0.45.
17 / 44

4.1.5.4. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-chlorophenylthio)ethyl] phosphate (4a).
Product 4a was obtained as a pale yellow viscous liquid (0.26 g, 38%). Rf 0.55 (CH2Cl2/MeOH =
9:1); 1H NMR (DMSO) δ 11.39 (1H, br s, NH), 7.45 (1H, s), 7.41-7.33 (8H, m), 6.13 (1H, t, J =
6.7 Hz), 4.43 (1H, q, J = 6.1 Hz), 4.23-4.15 (2H, m), 4.10 (4H, q, J = 6.9 Hz), 3.99-3.93 (1H, m),
3.29-3.22 (4H, m), 2.42-2.26 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ -0.44.
4.1.5.5. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-nitrophenylthio)ethyl] phosphate (5a).
Product 5a was obtained as a pale yellow viscous liquid (191 mg, 27%). Rf 0.48 (CH2Cl2/MeOH
= 95:5); 1H NMR (DMSO) δ 11.32 (1H, br s, NH), 8.10 (4H, d, J = 8.1 Hz), 7.52 (4H, t, J = 6.3
Hz), 7.43 (1H, s), 6.10 (1H, t, J = 6.3 Hz), 4.42 (1H, q, J = 6.2 Hz), 4.26-4.12 (6H, m), 3.98-3.92
(1H, m), 3.48-3.40 (4H, m), 2.42-2.26 (2H, m), 1.74 (3H, s); 31P NMR (DMSO) δ -0.48.
4.1.5.6. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(phenylthio)propyl] phosphate (6a). Product
6a was obtained as a pale yellow viscous liquid (0.2 g, 31%). Rf 0.62 (CH2Cl2/MeOH = 9:1); 1H
NMR (DMSO) δ 11.37 (1H, br s, NH), 7.45 (1H, s), 7.43-7.37 (4H, m), 7.36-7.30 (4H, m), 7.29-
7.23 (2H, m), 6.12 (1H, t, J = 6.4 Hz), 4.42 (1H, q, J = 5.7 Hz), 4.23-4.13 (2H, m), 4.05-3.86 (5H,
m), 3.57-3.49 (2H, m), 2.41-2.28 (2H, m), 1.75 (3H, s), 1.23 (6H, t, J = 5.6 Hz); 31P NMR
(DMSO) δ -0.50.
4.1.5.7. 3'-Azido-3'-deoxythymidin-5'-yl bis[1-(phenylthio)propan-2-yl] phosphate (7a).
Product 7a was obtained as a pale yellow viscous liquid (0.25 g, 39%). Rf 0.61 (CH2Cl2/MeOH =
9:1); 1H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.47 (1H, s), 7.38-7.26 (8H, m), 7.19 (2H, t, J =
7.2 Hz), 6.15-6.08 (1H, m), 4.55-4.45 (2H, m), 4.43-4.36 (1H, m), 4.24-4.08 (2H, m), 3.98-3.91
(1H, m), 3.24-3.13 (4H, m), 2.41-2.26 (2H, m), 1.75 (3H, s), 1.38-1.29 (6H, m); 31P NMR
(DMSO) δ -1.80, -1.93, -1.96, -2.17.
18 / 44

4.1.5.8. 3'-Azido-3'-deoxythymidin-5'-yl bis[1-(phenylthio)butan-2-yl] phosphate (8a).
Product 8a was obtained as a pale yellow viscous liquid (0.17 g, 25%). Rf 0.61 (CH2Cl2/MeOH =
9:1); 1H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.48 (1H, s), 7.39-7.25 (8H, m), 7.24-7.15 (2H,
m), 6.15-6.07 (1H, m), 4.45-4.37 (2H, m), 4.36-4.30 (1H, m), 4.22-4.10 (2H, m), 3.99-3.91 (1H,
m), 3.25-3.16 (4H, m), 2.40-2.25 (2H, m), 1.76 (3H, s), 1.74-1.56 (4H, m), 0.87-0.79 (6H, m);
31P NMR (DMSO) δ -1.44, -1.56, -1.59, -1.81.
4.1.5.9. 3'-Azido-3'-deoxythymidin-5'-yl bis[3-(phenylthio)butan-2-yl] phosphate (9a).
Product 9a was obtained as a pale yellow viscous liquid (0.17 g, 25%). Rf 0.61 (CH2Cl2/MeOH =
9:1); 1H NMR (DMSO) δ 11.38 (1H, br s, NH), 7.47 (1H, s), 7.43-7.36 (4H, m), 7.34-7.28 (4H,
m), 7.27-7.22 (2H, m), 6.16-6.06 (1H, m), 4.52-4.45 (2H, m), 4.43-4.38 (1H, m), 4.20-4.11 (2H,
m), 3.98-3.90 (1H, m), 3.59-3.49 (2H, m), 2.36-2.28 (2H, m), 1.76 (3H, s), 1.36-1.26 (6H, m),
1.24-1.18 (6H, m); 31P NMR (DMSO) δ -1.42, -1.48.
4.1.5.10. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-phenyl-2-(phenylthio)ethyl] phosphate (10a).
Product 10a was obtained as a pale yellow viscous liquid (0.28 g, 38%). Rf 0.60 (CH2Cl2/MeOH
= 9:1); 1H NMR (DMSO) δ 11.39 (1H, br s, NH), 7.39-7.29 (21H, m), 6.15-6.05 (1H, m), 4.70-
4.56 (2H, m), 4.33-4.20 (3H, m), 4.18-4.06 (2H, m), 3.98-3.86 (2H, m), 3.85-3.78 (1H, m), 2.33-
2.18 (2H, m), 1.69 (3H, s); 31P NMR (DMSO) δ -0.83, -0.86, -0.88.
4.1.5.11. 3'-Azido-3'-deoxythymidin-5'-yl phenyl [2-(phenylthio)ethyl] phosphate (11a).
Product 11a was obtained as a pale yellow viscous liquid (0.24 g, 43%). Rf 0.65 (CH2Cl2/MeOH
= 9:1); 1H NMR (DMSO) δ 11.38 (1H, br s, NH), 7.45 (1H, s), 7.42-7.28 (6H, m), 7.26-7.15 (4H,
m), 6.13 (1H, t, J = 6.6 Hz), 4.45 (1H, q, J = 5.8 Hz), 4.40-4.27 (2H, m), 4.25-4.17 (2H, q, J = 6.6
Hz), 4.03-4.01 (1H, m), 3.31-3.24 (2H, m), 2.42-2.26 (2H, m), 1.71 (3H, s); 31P NMR (DMSO) δ
-5.28, -5.38.
19 / 44

4.1.5.12. 3'-Azido-3'-deoxythymidin-5'-yl 2-(phenylthio)ethyl p-tolyl phosphate (12a).
Product 12a was obtained as a pale yellow viscous liquid (0.20 g, 35%). Rf 0.65 (CH2Cl2/MeOH
= 9:1); 1H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.44 (1H, s), 7.40-7.28 (4H, m), 7.22 (1H, t, J
= 6.8 Hz), 7.16 (2H, t, J = 7.0 Hz), 7.06 (2H, d, J = 8.1 Hz), 6.14 (1H, t, J = 6.7 Hz), 4.44 (1H, q,
J = 5.9 Hz), 4.39-4.25 (2H, m), 4.23-4.16 (2H, m), 4.03-3.96 (1H, m), 3.32-3.20 (2H, m), 2.44-
2.30 (2H, m), 2.27 (3H, s), 1.71 (3H, s); 31P NMR (DMSO) δ -5.23, -5.32.
4.1.5.13. 3'-Azido-3'-deoxythymidin-5'-yl p-methoxyphenyl [2-(phenylthio)ethyl] phosphate
(13a). Product 13a was obtained as a pale yellow viscous liquid (0.29 g, 49%). Rf 0.65
1
(CH2Cl2/MeOH = 9:1); H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.45 (1H, d, J = 10.1 Hz),
7.39-7.29 (4H, m), 7.25-7.19 (1H, m), 7.11 (2H, d, J = 9.1 Hz), 6.95-6.86 (2H, m), 6.13 (1H, t, J
= 6.8 Hz), 4.44 (1H, q, J = 5.6 Hz), 4.36-4.26 (2H, m), 4.25-4.16 (2H, m), 4.01-3.97 (1H, m),
31
3.73(3H, s), 3.30-3.21 (2H, m), 2.43-2.28 (2H, m), 1.71 (3H, s); P NMR (DMSO) δ -4.92, -
5.03.
4.1.5.14. 3'-Azido-3'-deoxythymidin-5'-yl p-fluorophenyl [2-(phenylthio)ethyl] phosphate
(14a). Product 14a was obtained as a pale yellow viscous liquid (0.28 g, 49%). Rf 0.49
1
(CH2Cl2/MeOH = 95:5); H NMR (DMSO) δ 11.36 (1H, br s, NH), 7.44 (1H, d, J = 9.2 Hz),
7.39-7.29 (4H, m), 7.25-7.18 (4H, m),6.13 (1H, t, J = 6.7 Hz), 4.45 (1H, q, J = 5.9 Hz), 4.40-4.27
(2H, m), 4.26-4.19 (2H, m), 4.02-3.97 (1H, m), 3.31-3.24 (2H, m), 2.42-2.28 (2H, m), 1.71 (3H,
s); 31P NMR (DMSO) δ -5.27, -5.38.
4.1.5.15. 3'-Azido-3'-deoxythymidin-5'-yl p-chlorophenyl [2-(phenylthio)ethyl] phosphate
(15a). Product 15a was obtained as a pale yellow viscous liquid (0.22 g, 37%). Rf 0.50
1
(CH2Cl2/MeOH = 95:5); H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.43 (3H, t, J = 7.2 Hz),
7.38-7.28 (4H, m), 7.22 (3H, t, J = 8.2 Hz),6.14 (1H, t, J = 5.8 Hz), 4.45 (1H, q, J = 5.9 Hz),
20 / 44

4.41-4.29 (2H, m), 4.24 (2H, q, J = 6.3 Hz), 4.08-3.98 (1H, m), 3.32-3.22 (2H, m), 2.46-2.28 (2H,
m), 1.72 (3H, s); 31P NMR (DMSO) δ -5.53, -5.63.
4.1.6. Standard procedure 5: preparation of sulfone products (1-15)
Oxidation of sulfide to sulfone was achieved according to the procedure described
previously with some modifications.29 To a stirring solution of a sulfide-containing phosphate
(0.5 mmol) in CH2Cl2 (10 mL) was added an excess amount of mCPBA (0.56 g, 2.5 mmol, 5
equiv.). The reaction mixture was stirred at ambient temperature for 3 h and then concentrated at
reduced pressure. The residue was diluted with CH2Cl2 (30 mL) and washed with water (30 mL),
and the water layer was extracted with CH2Cl2 (3 × 30 mL). The combined extract was washed
with saturated NaCl solution (30 mL), dried over anhydrous Na2SO4, and filtered. The filtrate
was evaporated under reduced pressure and the crude product was purified by preparative TLC
to afford the sulfone product.
4.1.6.1. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(phenylsulfonyl)ethyl] phosphate (1). Product
1
1 was obtained as a white foam (198 mg, 58%). Rf 0.51 (CH2Cl2/MeOH = 9:1); H NMR
(DMSO) δ 11.39 (1H, br s, NH), 7.94-7.86 (4H, m), 7.75 (2H, t, J = 7.2 Hz), 7.71-7.61 (4H, m),
7.42 (1H, s), 6.12 (1H, t, J = 6.6 Hz), 4.39 (1H, q, J = 5.7 Hz), 4.24-4.12 (4H, m), 4.06-3.96 (2H,
m), 3.93-3.87 (1H, m), 3.76-3.66 (4H, m), 2.44-2.26 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ
-1.44. Anal. Calcd for C26H30N5O11PS2: C, 45.68; H, 4.42; N, 10.24; S, 9.38. Found: C, 45.50; H,
4.54; N, 9.98; S, 9.15.
4.1.6.2. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-tolylsulfonyl)ethyl] phosphate (2). Product
1
2 was obtained as a white foam (135 mg, 38%). Rf 0.52 (CH2Cl2/MeOH = 9:1); H NMR
(DMSO) δ 11.40 (1H, br s, NH), 7.84-7.72 (4H, m), 7.49-7.43 (4H, m), 7.41 (1H, s), 6.13 (1H, t,
J = 6.5 Hz), 4.39 (1H, q, J = 5.7 Hz), 4.22-4.12 (4H, m), 4.06-3.93 (2H, m), 3.92-3.86 (1H, m),
21 / 44

31
3.72-3.63 (4H, m), 2.40 (6H, s), 2.38-2.29 (2H, m), 1.74 (3H, s); P NMR (DMSO) δ -1.38.
Anal. Calcd for C28H34N5O11PS2·1/4H2O: C, 46.96; H, 4.86; N, 9.78; S, 8.95. Found: C, 46.94;
H, 4.84; N, 9.41; S, 8.80.
4.1.6.3. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-methoxyphenylsulfonyl)ethyl] phosphate
(3). Product 3 was obtained as a white foam (141 mg, 38 %). Rf 0.47 (CH2Cl2/MeOH = 9:1); 1H
NMR (DMSO) δ 11.37 (1H, br s, NH), 7.85-7.77 (4H, m), 7.40 (1H, s), 7.22-7.10 (4H, m), 6.13
(1H, t, J = 6.6 Hz), 4.39 (1H, q, J = 5.7 Hz), 4.25-4.13 (4H, m), 4.07-3.99 (2H, m), 3.94-3.89 (1H,
m), 3.85 (6H, s), 3.74-3.60 (4H, m), 2.41-2.26 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ -1.30.
Anal. Calcd for C28H34N5O13PS2·1/5CH3COOCH2CH3: C, 45.44; H,4.71; N,9.20; S, 8.42.
Found: C, 45.37; H, 4.69; N, 8.98; S, 8.10.
4.1.6.4. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-chlorophenylsulfonyl)ethyl] phosphate (4).
Product 4 was obtained as a white foam (120 mg, 32%). Rf 0.49 (CH2Cl2/MeOH = 9:1); 1H NMR
(DMSO) δ 11.39 (1H, br s, NH), 7.95-7.88 (4H, m), 7.78-7.70 (4H, m), 7.41 (1H, s), 6.13 (1H, t,
J = 6.7 Hz), 4.42-4.38 (1H, m), 4.25-4.17 (4H, m), 4.05-3.99 (2H, m), 3.92-3.89 (1H, m), 3.82-
3.75 (4H, m), 2.42-2.25 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ -1.43. Anal. Calcd for
C26H28Cl2N5O11PS2·1/2CH3COOCH2CH3: C, 42.22; H, 4.05; N, 8.79; S, 8.05. Found: C, 42.43;
H, 4.03; N, 8.62; S, 7.92.
4.1.6.5. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(p-nitrophenylsulfonyl)ethyl] phosphate (5).
1
Product 5 was obtained as a white foam (162 mg, 42%). Rf 0.48 (CH2Cl2/MeOH = 19:1); H
NMR (DMSO) 11.35 (1H, br s, NH), 8.51-8.40 (4H, m), 8.24-8.12 (4H, m), 7.37 (1H, s), 6.09
(1H, t, J = 6.5 Hz), 4.36 (1H, q, J = 5.8 Hz), 4.29-4.17 (4H, m), 4.01-3.94 (2H, m), 3.93-3.79 (5H,
m), 2.42-2.26 (2H, m), 1.75 (3H, s); 31P NMR (DMSO) δ -1.57. Anal. Calcd for
22 / 44

C26H28N7O15PS2·1/4CH3COOCH2CH3: C, 40.76; H, 3.80; N, 12.32; S, 8.06. Found: C, 40.80; H,
3.87; N, 12.07; S, 8.00.
4.1.6.6. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-(phenylsulfonyl)propyl] phosphate (6).
Product 6 was obtained as a white foam (160 mg, 45%). Rf 0.54 (CH2Cl2/MeOH = 9:1); 1H NMR
(DMSO) δ 11.37 (1H, br s, NH), 7.87 (4H, t, J = 7.7 Hz), 7.77 (2H, t, J = 7.6 Hz), 7.66 (4H, t, J =
7.4 Hz), 7.43 (1H, s), 6.13 (1H, t, J = 6.8 Hz), 4.47-4.36 (1H, m), 4.22-4.10 (4H, m), 4.09-4.01
(2H, m), 3.98-3.89 (1H, m), 3.78-3.66 (2H, m), 2.42-2.28 (2H, m), 1.76 (3H, s), 1.20 (6H, m);
31P NMR (DMSO) δ -1.16. Anal. Calcd for C28H34N5O11PS2·3/5H2O: C, 46.55; H, 4.91; N, 9.69;
S, 8.88. Found: C, 46.81; H, 4.89; N, 9.31; S, 8.69.
4.1.6.7. 3'-Azido-3'-deoxythymidin-5'-yl bis[1-(phenylsulfonyl)propan-2-yl] phosphate (7).
Product 7 was obtained as a white foam (174 mg, 49%). Rf 0.50 (CH2Cl2/MeOH = 9:1); 1H NMR
(DMSO) δ 11.37 (1H, br s, NH), 7.97-7.88 (4H, m), 7.76 (2H, t, J = 7.5 Hz), 7.69-7.62 (4H, m),
7.45 (1H, s), 6.13 (1H, t, J = 7.0 Hz), 4.82-4.62 (2H, m), 4.48-4.34 (1H, m), 4.19-4.06 (2H, m),
31
3.97-3.89 (1H, m), 3.75-3.63 (4H, m), 2.41-2.27 (2H, m), 1.75 (3H, s), 1.40-1.27 (6H, m); P
NMR (DMSO) δ -2.98, -3.03, -3.77. Anal. Calcd for C28H34N5O11PS2·1/4H2O: C, 46.96; H, 4.86;
N, 9.78; S, 8.95. Found: C, 47.35; H, 5.01; N, 9.43; S, 8.58.
4.1.6.8. 3'-Azido-3'-deoxythymidin-5'-yl bis[1-(phenylsulfonyl)butan-2-yl] phosphate (8).
Product 8 was obtained as a white foam (236 mg, 64%). Rf 0.53 (CH2Cl2/MeOH = 9:1); 1H NMR
(DMSO) δ 11.38 (1H, br s, NH), 7.98-7.86 (4H, m), 7.79-7.72 (2H, m), 7.70-7.62 (4H, m), 7.45
(1H, s), 6.13 (1H, t, J = 6.8 Hz), 4.67-4.53 (2H, m), 4.45-4.35 (1H, m), 4.25-4.08 (2H, m), 3.99-
3.87 (1H, m), 3.78-3.58 (4H, m), 2.43-2.28 (2H, m), 1.75 (3H, s), 1.73-1.56 (4H, m), 0.80 (6H, t,
J = 7.3 Hz); 31P NMR (DMSO) δ -2.32, -2.67, -2.73, -3.37. Anal. Calcd for C30H38N5O11PS2: C,
48.71; H, 5.18; N, 9.47; S, 8.67. Found: C, 48.50; H, 5.13; N, 9.19; S, 8.39.
23 / 44

4.1.6.9. 3'-Azido-3'-deoxythymidin-5'-yl bis[3-(phenylsulfonyl)butan-2-yl] phosphate (9).
Product 9 was obtained as a white foam (207 mg, 56%). Rf 0.52 (CH2Cl2/MeOH = 9:1); 1H NMR
(DMSO) δ 11.37 (1H, br s, NH), 7.91-7.83 (4H, m), 7.81-7.74 (2H, m), 7.72-7.63 (4H, m), 7.45
(1H, s), 6.13 (1H, t, J = 6.8 Hz), 4.83-4.66 (2H, m), 4.45-4.32 (1H, m), 4.19-4.02 (2H, m), 3.95-
3.83 (1H, m), 3.73-3.58 (2H, m), 2.42-2.31 (2H, m), 1.76 (3H, s), 1.37-1.27 (6H, m), 1.15 (6H, t,
31
J = 7.7 Hz); P NMR (DMSO) δ -2.31, -2.46. Anal. Calcd for C30H38N5O11PS2: C, 48.71; H,
5.18; N, 9.47. Found: C, 49.04; H, 5.21; N, 9.23.
4.1.6.10. 3'-Azido-3'-deoxythymidin-5'-yl bis[2-phenyl-2-(phenylsulfonyl)ethyl] phosphate
(10). Product 10 was obtained as a white foam (175 mg, 42%). Rf 0.52 (CH2Cl2/MeOH = 9:1);
1H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.69 (2H, t, J = 7.7 Hz), 7.65-7.58 (4H, m), 7.57-7.50
(4H, m), 7.38-7.24 (7H, m), 7.22-7.13 (4H, m), 6.09 (1H, t, J = 6.8 Hz), 5.03-4.91 (2H, m), 4.59-
31
4.33 (4H, m), 4.32-4.23 (1H, m), 3.99-3.77 (3H, m), 2.34-2.22 (2H, m), 1.71 (3H, s); P NMR
(DMSO) δ -1.17, -1.22, -1.28. Anal. Calcd for C38H38N5O11PS2: C, 54.60; H, 4.58; N, 8.38; S,
7.67. Found: C, 54.64; H, 4.82; N, 8.12; S, 7.43.
4.1.6.11. 3'-Azido-3'-deoxythymidin-5'-yl phenyl [2-(phenylsulfonyl)ethyl] phosphate (11).
Product 11 was obtained as a pale yellow foam (154 mg, 52%). Rf 0.50 (CH2Cl2/MeOH = 9:1);
1H NMR (DMSO) δ 11.36 (1H, br s, NH), 7.95-7.86 (2H, m), 7.75 (1H, t, J = 7.2 Hz), 7.65 (2H,
t, J = 7.3 Hz), 7.46-7.31 (3H, m), 7.28-7.18 (1H, m), 7.13 (2H, d, J = 7.7 Hz), 6.13 (1H, t, J = 6.5
Hz), 4.48-4.32 (3H, m), 4.30-4.16 (2H, m), 4.02-3.92 (1H, m), 3.86-3.72 (2H, m),2.43-2.28 (2H,
m), 1.74 (3H, s); 31P NMR (DMSO)
δ
-5.90, -6.05. Anal. Calcd for
C24H26N5O9PS·1/3CH3COOCH2CH3: C, 49.00; H, 4.65; N, 11.28. Found: C, 49.29; H, 4.66; N,
11.25.
24 / 44

4.1.6.12. 3'-Azido-3'-deoxythymidin-5'-yl 2-(phenylsulfonyl)ethyl p-tolyl phosphate (12).
1
Product 12 was obtained as a white foam (175 mg, 58%). Rf 0.51 (CH2Cl2/MeOH = 9:1); H
NMR (DMSO) δ 11.38 (1H, br s, NH), 7.94-7.85 (2H, m), 7.75 (1H, t, J = 7.4 Hz), 7.65 (2H, t, J
= 7.4 Hz), 7.41 (1H, d, J = 11.9 Hz), 7.15 (2H, t, J = 8.0 Hz), 7.01 (2H, d, J = 8.2 Hz), 6.13 (1H, t,
J = 6.7 Hz), 4.43-4.30 (3H, m), 4.27-4.15 (2H, m), 4.01-3.92 (1H, m), 3.83-3.71 (2H, m), 2.44-
2.30 (2H, m), 2.27 (3H, s), 1.70 (3H, s); 31P NMR (DMSO) δ -5.71, -5.85. Anal. Calcd for
C25H28N5O9PS: C, 49.59; H, 4.66; N, 11.57; S, 5.30. Found: C, 49.64; H, 4.77; N, 11.29; S, 5.13.
4.1.6.13. 3'-Azido-3'-deoxythymidin-5'-yl p-methoxyphenyl [2-(phenylsulfonyl)ethyl]
phosphate (13). Product 13 was obtained as a white foam (180 mg, 58%). Rf 0.54
(CH2Cl2/MeOH = 9:1); 1H NMR (DMSO) δ 11.36 (1H, br s, NH), 7.94-7.87 (2H, m), 7.75 (1H, t,
J = 7.5 Hz), 7.65 (2H, t, J = 7.5 Hz), 7.42 (1H, d, J = 12.1 Hz), 7.04 (2H, d, J = 8.1 Hz), 6.89 (2H,
t, J = 7.8 Hz), 6.13 (1H, t, J = 6.8 Hz), 4.48-4.41 (1H, m), 4.39-4.29 (2H, m), 4.27-4.13 (2H, m),
31
3.99-3.92 (1H, m), 3.84-3.75 (2H, m), 3.74-3.66 (3H, s), 2.43-2.28 (2H, m), 1.71 (3H, s); P
NMR (DMSO) δ -5.40, -5.54. Anal. Calcd for C25H28N5O10PS: C, 48.31; H, 4.54; N, 11.27; S,
5.16. Found: C, 48.09; H, 4.64; N, 11.23; S, 5.14.
4.1.6.14.
3'-Azido-3'-deoxythymidin-5'-yl
p-fluorophenyl
[2-(phenylsulfonyl)ethyl]
phosphate (14). Product 14 was obtained as a white foam (140 mg, 46%). Rf 0.49
(CH2Cl2/MeOH = 9:1); 1H NMR (DMSO) δ 11.37 (1H, br s, NH), 7.94-7.87 (2H, m), 7.75 (1H, t,
J = 7.4 Hz), 7.65 (2H, t, J = 7.3 Hz), 7.42 (1H, d, J = 11.8 Hz), 7.25-7.13 (4H, m), 6.13 (1H, t, J
= 6.8 Hz), 4.47-4.32 (3H, m), 4.31-4.17 (2H, m), 4.01-3.93 (1H, m), 3.84-3.73 (2H, m), 2.44-
2.28 (2H, m), 1.71 (3H, s); 31P NMR (DMSO) δ -5.77, -5.92. Anal. Calcd for C24H25FN5O9PS: C,
47.29; H, 4.13; N, 11.49; S, 5.26. Found: C, 47.56; H, 4.35; N, 11.18; S, 5.06.
25 / 44

4.1.6.15.
3'-Azido-3'-deoxythymidin-5'-yl
p-chlorophenyl
[2-(phenylsulfonyl)ethyl]
phosphate (15). Product 15 was obtained as a white foam (200 mg, 64%). Rf 0.49
(CH2Cl2/MeOH = 9:1); 1H NMR (DMSO) δ 11.39 (1H, br s, NH), 7.93-7.87 (2H, m), 7.75 (1H, t,
J = 7.6 Hz), 7.65 (2H, t, J = 7.7 Hz), 7.43 (3H, t, J = 8.2 Hz), 7.18 (2H, d, J = 8.5 Hz), 6.13 (1H, t,
J = 6.5 Hz), 4.48-4.33 (3H, m), 4.31-4.18 (2H, m), 4.01-3.93 (1H, m), 3.85-3.72 (2H, m),2.46-
2.29 (2H, m), 1.72 (3H, s); 31P NMR (DMSO) δ -6.05, -6.18. Anal. Calcd for
C24H25ClN5O9PS·1/2H2O: C, 45.40; H, 4.13; N, 11.03; S, 5.05. Found: C, 45.79; H, 4.19; N,
10.79; S, 5.04.
4.2. Stability studies
4.2.1. Stability tests of sulfonylethyl nucleotide analogues in 0.1 M phosphate buffer at pH
7.4
Each incubation mixture contained 0.4 mL of pre-incubated 0.1 M phosphate buffer at pH
7.4 and 0.1 mL of the drug solution (5 mg/mL) prepared in acetonitrile. The vials were incubated
at 37 °C in a shaking water bath (Dubnoff Metabolic Shaking Incubator, Precision Scientific) for
varying time intervals. The incubation was initiated by the addition of the compound and
terminated by the addition of 0.1 mL 2.0 M acetate buffer (pH 5) and 0.2 mL ethanol (0.4 mL
ethanol for compound 5) and immediately cooled in crushed ice. An aliquot of the resulting
mixture (15 µL) were spotted on 10 × 20 mm silica gel TLC plates and eluted with appropriate
solvent system (CH2Cl2/MeOH = 95:5). The starting materials from all the samples were
visualized as a dark spot under UV light and quantitatively analyzed by using the Uniscan Video
Densitometer. The results were expressed as the percentage of starting material remaining after
each incubation period relative to the zero time incubation control sample. These results were
26 / 44

plotted against the respective incubation times and the half-lives were calculated from rate
constant k of the decay equation (t1/2 = ln2/k).
4.2.2. Stability tests of sulfonylethyl nucleotide analogues in the presence of human plasma
Lyophilized human plasma (Lee Biosolutions, Inc., St. Louis, MO) was used for this study
and it was reconstituted immediately prior to the experiment. The protocol used for the study was
essentially the same as in 4.2.1 except the following: Each incubation mixture contained 0.4 mL
of reconstituted solution of human plasma in 0.1 M phosphate buffer at pH 7.4 and 0.1 mL drug
solution. After the termination of the incubation, the vials were cooled in crushed ice and
centrifuged at high speed (3000 × g) for 30 min to precipitate the proteins. Subsequently, an
aliquot of the resulting supernatant (15 µL) was spotted on TLC plates, developed and analyzed
as described above in procedure 4.2.1.
4.3. In vitro cytotoxicity assessment
4.3.1. Chemicals and equipment
Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium
(EMEM), fetal bovine serum (FBS), penicillin/streptomycin and trypsin 0.25% were products of
Hyclone, Thermo Scientific, Logan, UT. Phosphate buffered saline (PBS) 20× concentrate (pH
7.5) was purchased from AMRESCO, Solon, OH. 5-Bromo-2'-deoxyuridine (5-BrdU) was
purchased from Alfa Aesar, Ward Hill, MA. Dimethyl sulfoxide (DMSO), 3-(4,5-
dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were obtained
from Sigma Chemical Co., St. Louis, MO. OPSYS microplate reader was purchased from Dynex
Technologies, Chantilly, VA.
4.3.2. Cell lines and cell culture
27 / 44

The cell lines MCF-7, H460, KB-3-1, and SW620 were cultured at 37 °C, 5% CO2 with
DMEM containing 10% FBS and 1% penicillin/streptomycin. The 143B cell line (ATCC No.
CRL-1543) was cultured at 37 °C, 5% CO2 with EMEM containing 10% FBS and 1%
penicillin/streptomycin. The cell line 143B/TK- (ATCC No. CRL-8303) was maintained in the
EMEM with 0.015 mg/mL 5-BrdU. All cells were grown as adherent monolayer in drug-free
culture media for more than 2 weeks before the assay.
4.3.3. Cytotoxicity determination by MTT assay
A modified MTT colorimetric assay30 was utilized to detect the sensitivity of cells to AZT
and anticancer prodrugs in vitro. In the cytotoxicity assay, stocking solutions of the compounds
were prepared at 10 mM using diemthyl sulfoxide (DMSO). Then 0.5 mM PBS (PH 7.4) was
used to dilute the stocking solutions of compounds into different concentrations (0.03 µM to 300
µM) right before the MTT assay. Briefly, cells were seeded in 180 µL medium in 96-well plates
in triplicate at 5000~6000 cells/well. After incubation at 37 °C, 5% CO2 for 24 h to allow the
cells to attach to the wells, different concentrations of AZT and the synthesized compounds (20
µL/well) were added into designated wells. After 72 h of incubation at 37 °C, 20 µL of MTT
solution (4 mg/mL) was added to each well. The plates were further incubated at 37 °C for 4 h,
allowing viable cells to change the yellow-colored MTT into dark-blue formazan crystals.
Subsequently, the MTT medium was removed from each well without disturbing the cells, and
100 µL of DMSO was added into each well. Plates were placed on a shaking table to thoroughly
mix the formazan into the solvent. Finally, the absorbance was determined at 570 nm by OPSYS
microplate reader. The percentage of cell survival was expressed as the absorbance of the cells
treated with certain drug concentration relative to that of the untreated control cells. IC50 values
were obtained from the slopes of cell survival rate versus log drug concentration curves.
28 / 44

Acknowledgement
This research is supported by the Department of Pharmaceutical Sciences of St. John's
University.
References and notes
1. Chow, W. A.; Jiang, C.; Guan, M. Lancet Oncol. 2009, 10, 61.
2. Johnston, J. S.; Johnson, A.; Gan, Y.; Wientjes, M.G.; Au, J. L. Pharm. Res. 2003, 20, 957.
3. Brunetti, I.; Falcone, A.; Calabresi, P.; Goulette, F. A.; Darnowski, J. W. Cancer Res. 1990,
50, 4026.
4. Chen, P.; Liu, Y.; Sun, Y.; Chen, C.; Qi, Y.; Zhang, Y. Pharm. Biol. 2013, 51, 1586.
5. Mattson, D. M.; Ahmad, I. M.; Dayal, D.; Parsons, A. D.; Aykin-Burns, N.; Li, L.; Orcutt, K.
P.; Spitz, D. R.; Dornfeld, K. J.; Simons, A. L. Free Radic. Biol. Med. 2009, 46, 232.
6. Wagner, C. R.; Ballato, G.; Akanni, A. O.; McIntee, E. J.; Larson, R. S.; Chang, S. L.;
Abulhajj, Y. J. Cancer Res. 1997, 57, 2341.
7. Humer, J.; Ferko, B.; Waltenberger, A.; Rapberger, R.; Pehamberger, H.; Muster, T.
Melanoma Res. 2008, 18, 314.
8. Pereira, J.; Levy, D.; Ruiz, J. L.; Brocardo, G. A.; Ferreira, K. A.; Costa, R. O.; Queiroz, R.
G.; Maria, D. A.; Neto, A. E.; Chamone, D. A.; Bydlowski, S. P. Anticancer Agents Med.
Chem. 2013, 13, 186.
9. Celewicz, L.; Jóźwiak, A.; Ruszkowski, P.; Laskowska, H.; Olejnik, A.; Czarnecka,
A.; Hoffmann, M.; Hładoń, B. Bioorg. Med. Chem. 2011, 19, 6375.
10. Jeng, K. S.; Sheen, I. S.; Jeng, W. J. Hepatogastroenterology. 2011, 58, 2091.
11. Falchetti, A.; Franchi, A.; Bordi, C.; Mavilia, C.; Masi, L.; Cioppi, F.; Recenti, R.; Picariello,
L.; Marini, F.; Del Monte, F.; Ghinoi, V.; Martineti, V.; Tanini, A.; Brandi, M. L. J. Bone Miner
29 / 44