Synthesis of novel nucleoside analogue phosphorothioamidate prodrugs and in vitro anticancer evaluation against RKO human colon carcinoma cells

Article abstract of DOI:10.1080/15257770.2013.770523

Novel phosphorothioamidates of pyrimidine nucleoside analogues have been prepared and evaluated in vitro against RKO human colon cancer cell by the MTT cytotoxicity assay. The parent nucleoside analogues were inactive in this assay, while the phosphorothioamidate prodrugs were active at low uM levels in some cases. The O-isopropyl phosphorothioamidate of 2 ′,3 ′-O- isopropylidene-uridine containing the L-phenylalanine ethyl ester 6f was the most active at 148 uM, a 10-fold enhancement in anticancer activity compared with the parent nucleoside 2 with no increase in cytotoxicity.

Full text of DOI:10.1080/15257770.2013.770523

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Synthesis of Novel Nucleoside Analogue
Phosphorothioamidate Prodrugs and in
vitro Anticancer Evaluation Against RKO
Human Colon Carcinoma Cells
Wei Liu a b , Lin Zhang b , Honggang Zhou b c , Cheng Yang b c
Zhiwei Miao c & Yufen Zhao b
,
a College of Sciences, Tianjin University of Science and Technology,
Tianjin, China
b High Throughput Molecular Drug Discovery Center, Tianjin
International Joint Academy of Biotechnology and Medicine, Tianjin,
China
c College of Pharmacy, NanKai University, Tianjin, China
To cite this article: Wei Liu , Lin Zhang , Honggang Zhou , Cheng Yang , Zhiwei Miao & Yufen
Zhao (2013): Synthesis of Novel Nucleoside Analogue Phosphorothioamidate Prodrugs and in vitro
Anticancer Evaluation Against RKO Human Colon Carcinoma Cells, Nucleosides, Nucleotides and
Nucleic Acids, 32:4, 161-173
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Nucleosides, Nucleotides and Nucleic Acids, 32:161–173, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.770523
SYNTHESIS OF NOVEL NUCLEOSIDE ANALOGUE
PHOSPHOROTHIOAMIDATE PRODRUGS AND IN VITRO
ANTICANCER EVALUATION AGAINST RKO HUMAN COLON
CARCINOMA CELLS
Wei Liu,1,2 Lin Zhang,2 Honggang Zhou,2,3 Cheng Yang,2,3 Zhiwei Miao,3
and Yufen Zhao2
1College of Sciences, Tianjin University of Science and Technology, Tianjin, China
2High Throughput Molecular Drug Discovery Center, Tianjin International Joint Academy
of Biotechnology and Medicine, Tianjin, China
3College of Pharmacy, NanKai University, Tianjin, China
Novel phosphorothioamidates of pyrimidine nucleoside analogues have been prepared and eval-
2
uated in vitro against RKO human colon cancer cell by the MTT cytotoxicity assay. The parent nu-
cleoside analogues were inactive in this assay, while the phosphorothioamidateꢁ prodrugs were active
ꢁ
at low uM levels in some cases. The O-isopropyl phosphorothioamidate of 2 ,3 -O-isopropylidene-
uridine containing the L-phenylalanine ethyl ester 6f was the most active at 148 uM, a 10-fold
enhancement in anticancer activity compared with the parent nucleoside 2 with no increase in
cytotoxicity.
Keywords Phosphorothioamidate; prodrug; nucleoside; anticancer activity; colon
cancer
INTRODUCTION
Many nucleoside derivatives and analogues have been synthesized as im-
portant agents for anticancer and antiviral therapy. Preliminary metabolic
studies showed that the nucleosides are phosphorylated to triphosphates
and inhibit RNA synthesis. Phosphorylation to nucleoside monophosphates
by the host cell kinase may be the crucial step in their activation.[1] In many
cases, however, nucleoside analogues are poor substrates for the kinases and
unable to cross the cell membrane. In order to overcome these problems,
their phosphate and phosphoramidate derivatives have been widely used as
Received 9 October 2012; accepted 22 January 2013.
This work was supported by the Tianjin SME Technology Innovation Fund (10ZCKFSY08300,
11ZXCXSY03500) and the National Biomedical Special Project of International Innovation Park (11ZCK-
FSY06800).
Address correspondence to Wei Liu, College of Sciences, Tianjin University of Science and Tech-
nology, The 13th Avenue, TEDA, Tianjin, China. E-mail: liuwei2006@tust.edu.cn
161

162
W. Liu et al.
FIGURE 1 Structures of AZT (1) and 2ꢁ,3ꢁ-O-isopropylidene uridine (2).
prodrug moieties to enhance biological activity.[2–7] Among all reported pro-
drugs approaches, the class of aryl phosphoramidate nucleoside prodrugs
(ProTide) developed by McGuigan has been proven to enhance, in vitro, the
activity of parent nucleosides by increasing the formation rate of NuTP by im-
proved intracellular transport and/or bypassing the rate-limiting first phos-
phorylation step.[8–17] In the past, our group reported the synthesis of several
series of nucleoside 5ꢁ-phosphorothioamidate prodrugs.[18] Except for her-
bicidal activity,[19] few investigations on the medicinal activity of phospho-
rothioamidate derivatives have been reported. Previously, Venkatachalam
reported a comparative study of aryl phosphoramidate and aryl thiophospho-
ramidate derivatives of d4T.[20] By determining the rate of hydrolysis in vitro,
they believed that aryl phosphoramidate derivatives of d4T were more active
than the corresponding aryl phosphorothioamidate derivatives. However,
Bell reported in 2006 that phosphorothioamidates had antimalarial activity
and might be a target for new antimalarial drugs.[21] Recently, further studies
on antimalarial activity revealed that phosphorothioamidates were more ac-
tive than their oxo congeners.[22] These findings prompted us to undertake
further research on phosphorothioamidate derivatives. Herein, the ProTide
strategy has been applied to parent nucleosides (see Figure 1). We report
the synthesis of eight novel pyrimidine nucleoside analogue phosphoroth-
ioamidate prodrugs 6 (see Table 1) bearing amino acid ester moieties with
the aim to improve and/or broaden their biological activity. Both protected
2 ꢁ,3 ꢁ-O-isopropylidene-uridine 2 and AZT 1, as nucleoside analogs having a
defect in the 2ꢁ,3ꢁ positions of ribose ring, have antiviral or anticancer activity
by inhibiting the RNA replication in these cells. The anticancer activity of
compounds 6 was evaluated in RKO human colon cancer cells.
RESULTS AND DISCUSSION
Chemistry
Using the previously described method[18] (see Scheme 1), we synthe-
sized the glycine, L-alanine, L-phenylalanine, and L-valine phosphoroth-
ioamidates 6 of parent nucleosides 1 and 2, each bearing an ethyl ester.

Phosphorothioamidate Prodrugs and Anticancer Evaluation
163
SCHEME 1 General synthetic pathway for the synthesis of phosphorothioamidate prodrugs 6. Reagents
and conditions: (i) Et3N, THF, −5◦C, 2 h; (ii) Et3N, rt, 24 h.
In brief, thiophosphoryl chloride 3a was used as starting material, which
was condensed with 2-propanol to yield O-isopropyl phosphorodichloridoth-
ioate 3b. Two compounds (3a and 3b) were further condensed with L-amino
acid ethyl ester hydrochlorides 4 to obtain the dichloridate and monochlori-
date 5, respectively. Treatment of chloridates 5 in dry tetrahydrofuran (THF)
and triethylamine with 1 or 2 furnished the desired alkyl phosphorothioami-
date derivatives 6. All the phosphorothioamidate prodrugs 6 displayed two
TABLE 1 Structures and yields of phosphorothioamidates 6 synthesized in this work
S
R1
B
P
O
C2H5OOCHCHN
R2
O
X
Y
Compound
R1
R2 Amino acid ethyl ester
Nucleoside (B/X/Y)
Yield (%)
6a
6b
6c
6d
6e
6f
OH
OH
OCH(CH3)2
OCH(CH3)2 Me L-alaninyl
OCH(CH3)2 i-Pr L-valinyl
OCH(CH3)2 Bn L-phenylalaninyl
Me L-alaninyl
i-Pr L-valinyl
2ꢁ,3ꢁ-O-isopropylidene uridine
2ꢁ,3ꢁ-O-isopropylidene uridine
2ꢁ,3ꢁ-O-isopropylidene uridine
2ꢁ,3ꢁ-O-isopropylidene uridine
2ꢁ,3ꢁ-O-isopropylidene uridine
2ꢁ,3ꢁ-O-isopropylidene uridine
Zidovudine (AZT)
71
74
62
65
58
53
62
66
H
Glycinyl
6g
6h
OCH(CH3)2
OCH(CH3)2 i-Pr L-valinyl
H
Glycinyl
Zidovudine (AZT)

164
W. Liu et al.
closely spaced signals in phosphorus-31 nuclear magnetic resonance (31P
NMR) because of the presence of diastereoisomers at the phosphorus stere-
ocenter. Unfortunately, the diastereoisomeric mixtures could not be sep-
arated by silica gel column chromatography. Their structures have been
demonstrated by mass spectrometry (MS), infrared (IR), and a series of one-
and 2-dimensional NMR analyses.
Because of the stereochemistry at the phosphorus center, the final com-
pounds were isolated as mixtures of two diastereoisomers. The confirmation
of the presence of two diastereoisomers was shown by 31P NMR (two peaks)
1
and H and 13C NMR (splitting of many of the nucleoside signals). For ex-
ample, a pair of peaks at 31P NMR 71.96 and 71.63 ppm was derived from
the diastereoisomers 6g. The 1H NMR signal of five CH3 groups appeared at
1.98–1.30 ppm as three groups of signals, which could confirm the structure
of 6g. The 13C NMR spectrum of 6g displayed 17 carbons. The DEPT-135
(135◦ distortionless enhancement by polarization transfer) indicated the
presence of four methylenes (δC 65.5, 61.7, 43.4, 37.6), five methines (δC
135.3, 85.0, 82.5, 73.0, 60.6), one quaternary carbon (δC 111.4), three car-
bonyl carbon (δC 170.6, 163.7, 150.3), and four methyl groups (δC 23.7, 23.4,
1
14.1, 12.5). These carbon signals and the H NMR data also supported the
fact that 6g was composed of an isopropoxy group, a nucleoside moiety, and
1
an amino acid moiety, and was further confirmed by the H–1H COSY and
1H–13C HMQC experiments.
The IR spectra of compounds 6 were recorded in KBr and are given
in the Experimental section. Characteristic frequencies of the azide group
in the spectrogram of the compound 6g were observed at 2111 cm−1. Ab-
sorptions at 1692 cm−1 were due to the presence of the carbonyl group.
Electrospray ionization (ESI) mass spectra showed that the quasimolecular
ion peaks of compound 6g were observed in both the positive mode and
the negative mode. The high-resolution mass spectrometry (HRMS) gave
a quasimolecular ion at m/z 491.1485 [M+H]+ (calculated 491.1478 for
C17H27N6O7PS).
Anticancer Activity
The anticancer activity of phosphorothioamidate prodrugs 6 against hu-
man colon carcinoma cells (RKO cells) was compared with that of the parent
nucleosides 1–2. Meanwhile, cytotoxicity was also evaluated in normal hu-
man embryonic kidney cells (293T cells). The activity values expressed as
IC50 values are shown in Table 2. Phosphorothioamidate prodrugs 6 showed
only very modest activity through more potent activity against the cover cell
line than the parent nucleosides 1 and 2, with IC50 values >1000 μM. This
result could suggest that the phosphorothioamidate approach allows the
efficient conversion to its corresponding monothiophosphate in RKO cells,
whereas the parent nucleosides were poor substrates for the nucleoside

Phosphorothioamidate Prodrugs and Anticancer Evaluation
165
TABLE 2 In vitro cytotoxicity of parent nucleosides 1 and 2 and phosphorothioamidate prodrugs 6
against RKO human colon cancer cells and 293T human embryonic kidney cells
IC50 (uM)
Compound
RKO
293T
6a
6b
6c
>1000
>1000
724
>1000
>1000
178
6d
276
132
6e
561
677
6f
6g
148
187
>1000
578
6h
1 (AZT)
2
160.00
>1000
>1000
147
53
203
Note: The IC50 values were determined from eight different concentrations of compounds and were
the means of three separate experiments.
kinases in these cells. We consider that the mechanisms of phosphoroth-
ioamidate prodrugs in vitro are likely similar to phosphoramidate prodrugs
(see Scheme 2), though the modest activity makes it difficult to draw firm
conclusions.
Structure–activity relationships could be observed with regard to the
nature of the alkoxy moiety and the amino acid moiety in terms of even-
tual anticancer activity of the prodrug derivatives. Although prodrugs 6a
and 6b did not show appreciable activity in RKO cells, prodrugs containing
isopropoxy groups (6c–h) were more potent than those bearing hydroxyl
groups (6a–b), with IC50 values in the 724–148 μM range. Instead of hy-
droxyl, prodrugs containing isopropoxy groups (6c--h) were more active.
According to the metabolism of nucleoside aryl phosphoramidates,[13] alkyl
phosphorothioamidates also need four steps to eventually release the nu-
cleoside analogue monothiophosphate (see Scheme 2). Cyclization as the
second step involves the oxygen of the carboxylate attacking the phosphorus
SCHEME 2 Postulated activation pathway of phosphorothioamidate prodrugs.

166
W. Liu et al.
atom and displacing the isopropyl ester to form a five-membered intermedi-
ate, which eventually is converted to a monothiophosphate. In this second
step, the leaving group ability of the isopropoxy group is expected to be
greater than that of the hydroxy group. As a result, prodrugs 6a–b contain-
ing hydroxyl groups should slow down conversion to monothiophosphate.
With regard to the amino acid modifications, we found that the introduc-
tion of bulky amino acid side chains significantly enhanced potency in the
Et-ester derivatives. The L-phenylalanine derivative 6f and L-valine derivative
6h showed a bit more anticancer activity than other amino acid derivatives in
the uridine and AZT series, respectively. For example, the inhibitory activity
of 6f was fourfold higher than glycine derivative 6c. We utilized the 293T cells
to test the cytotoxicity of the compounds described herein. The results of the
cytotoxicity in vitro evaluations showed that all phosphorothioamidate pro-
drugs 6 were less toxic than the corresponding parent nucleosides 1–2. For
example, prodrugs 6a, 6b, and 6f exhibited an IC50 of >1000 μM. Among
all investigated compounds, prodrug 6f showed the most pronounced in-
hibitory activity against the RKO human tumor cell line and demonstrated
some selectivity relative to its effect on the growth of human normal 293T
cells.
CONCLUSIONS
A series of phosphorothioamidate prodrugs of pyrimidine nucleoside
analogues were prepared and evaluated as inhibitors of RKO tumor cells
in vitro. The phosphorothioamidate approach provided novel compounds
with modest potency in the anticancer assay when compared with the inactive
parent compounds. All phosphorothioamidates tested were only moderately
cytotoxic (IC50 >100 μM). The most active compound prepared in the series
was the O-isopropyl L-phenylalanine ethyl ester phosphorothioamidate 6f
with an IC50 of 148 μM in the anticancer assay and a selectivity index of >6.
This report suggested that phosphorothioamidate prodrugs may bypass the
rate-limiting initial phosphorylation of a ribonucleoside analogue and thus
confer anticancer activity on an inactive parent nucleoside.
EXPERIMENTAL
Chemistry (General Procedures)
All experiments involving water-sensitive compounds were conducted
under scrupulously dry conditions. Triethylamine was dried by refluxing
over calcium hydride. THF was distilled from sodium. Nucleosides were
dried by storage at elevated temperature over P2O5 in vacuo. Proton, car-
bon, and phosphorus nuclear magnetic resonance (1H, 13C, 31P NMR) spec-
tra were recorded on 400 or 600 MHz Bruker Avance DPX spectrometers.

Phosphorothioamidate Prodrugs and Anticancer Evaluation
167
All 13C and 31P spectra were recorded proton decoupled. All NMR spectra
were recorded in CDCl3 or D2O at room temperature (20◦C). Chemical
1
shifts for H and 13C spectra are quoted in ppm downfield using tetram-
ethylsilane (TMS). Coupling constants are referred to as J values. Chemical
shifts for 31P spectra are quoted in ppm relative to an external phosphoric
acid standard. Many proton and carbon NMR signals were split due to the
presence of (phosphate) diastereoisomers in the samples. ESI mass spectra
were acquired using a Bruker ESQUIRELCTM ESI ion trap spectrometer.
HR-ESI mass spectra were obtained with an Agilent Technologies ESI-TOF
spectrometer. FTIR spectra were recorded at room temperature in the re-
gion of 4000–400 cm−1 with a PerkinElmer spectrum 65 FTIR spectrometer
using KBr pellets.
Preparation of O-isopropyl Phosphorodichloridothioate 3b[23]
2-Propanol (50 mmol) was added dropwise to a stirred solution of 3a
(100 mmol) at room temperature over 10 minutes. Following the addition,
pyridine (50 mmol) was added dropwise to the reaction mixture. The reac-
tion mixture was stirred at room temperature for 2 hours. The crude product
was purified by reduced pressure distillation to give the title compound as
an oil in 84% yield.
Standard Procedure: Preparation of Amino Acid Ethyl
Ester Hydrochloride 4
L-amino acid (5.0 mol equiv) in 50 ml dry ethanol was stirred at room
temperature; the mixture was saturated by HCl gas and stirred overnight.
The solvent was removed under reduced pressure to give the crude product
as amino acid ethyl ester hydrochlorides. The product was washed with dry
ethanol, concentrated to remove HCl gas, and recrystallized from EtOH-
Et2O to give the white title compound.
Standard Procedure A: Preparation of Phosphorothioamidate Derivatives
6a–b of Nucleosides
Triethylamine (0.5 g, 5.0 mmol) was added dropwise to a stirred solu-
tion of 3a (0.42 g, 2.5 mmol) and amino acid ethyl ester hydrochloride 4
(2.5 mmol) in dry THF (15.0 ml) under nitrogen. The reaction flask was
externally cooled by ice bath below 0◦C. After completion of the addition of
the base, the reaction mixture was allowed to warm to room temperature and
stirred for additional 2 hours. On completion of the reaction (as determined
by 31P NMR), a solution of 2 (0.71 g, 2.5 mmol) and dry THF (10 ml) was
added dropwise to the above reaction mixture over a period of 20 minutes.
Additional triethylamine (0.25 g, 2.5 mmol) was then added and the reac-
tion mixture was stirred overnight, filtered, and concentrated in vacuo. After
hydrolysis in NH4OH, the residue was purified by column chromatography

168
W. Liu et al.
on silica (200–300 mesh) with elution by propanol-NH4OH-H2O (30:1:1) to
give a white solid 6a–b.
Standard Procedure B: Preparation of Phosphorothioamidate Derivatives
6c–h of Nucleosides
Triethylamine (0.5 g, 5.0 mmol) was added dropwise with vigorous stir-
ring to a solution of amino acid ethyl ester hydrochloride 4 (2.5 mmol)
and 3b (0.5 g, 2.5 mmol) in dry THF (15 ml) at −5◦C over a period of 15
minutes under nitrogen. The reaction mixture was slowly warmed to room
temperature with stirring over 2 hours. Upon complete reaction of 3b (as
determined by 31P NMR), a solution of nucleoside 1 or 2 (2.5 mmol) dis-
solved in dry pyridine (10 ml) and triethylamine (0.25 g, 2.5 mmol) was
added with vigorous stirring. The solvent was removed under vacuum after
24 hours. The residue was dissolved in chloroform (20 ml) and washed with
1 M hydrochloric acid solution (2 × 15 ml), saturated sodium bicarbonate
solution (2 × 10 ml), and then water (3 × 15 ml). The organic phase was
dried (MgSO4) and evaporated under vacuum, and the residue was purified
by column chromatography on silica (200–300 mesh) with elution with 5%
methanol in chloroform to give a white solid 6c–h.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[hydroxy(ethyloxy-L-alaninyl)]thiophosp
hate (Diastereoisomers, 6a). The compound was prepared according to
Standard Procedure A by using uridine 2, L-alanine ethyl ester hydrochlo-
ride, and 3a. Product 6a was obtained as a white solid (0.85 g, 71%). 31P
1
NMR (162 MHz, D2O): δ 57.19, 56.77; H NMR (400 MHz, D2O): δ 7.90,
7.88 (1H, dd, J = 8.0 Hz, H-6), 5.90 (2H, m, H-1ꢁ, 5), 4.98 (2H, m, H-2ꢁ,
3ꢁ), 4.58 (1H, s, H-4ꢁ), 4.20–4.11 (2H, m, H-5ꢁ), 4.06 (2H, m, OCH2CH3),
3.91–3.82 (1H, m, NHCH), 1.60 (3H, s, CH3), 1.41 (3H, s, CH3), 1.31 (3H,
d, J = 7.2 Hz, CHCH3), 1.25 (3H, t, J = 7.6 Hz, OCH2CH3); 13C NMR
(100 MHz, D2O): δ 177.20 (COOEt), 166.46 (C-4), 151.50 (C-2), 142.98,
142.88 (C-6), 114.60, 114.57 (>CMe2), 102.00, 101.91(C-5), 93.23, 92.92
(C-1ꢁ), 85.61, 85.42 (d, JC-P = 10.0 Hz, C-4ꢁ), 84.76, 84.62 (C-2ꢁ), 81.13, 81.01
(C-3ꢁ), 64.80 (C-5ꢁ), 62.38 (OCH2CH3), 50.94, 50.53, (d, JC-P = 8.0 Hz,
Ala-C-α), 26.21 (CH3), 24.49 (CH3), 20.17, 19.99 (d, JC-P = 3.0 Hz, Ala-C-β),
13.50, 13.47 (COOCH2CH3); IR (KBr, cm−1) 3459, 1677, 1401, 1277, 1220,
1116, 978, 938, 905; ESI-MS (pos.): m/z 480 (M+H)+; ESI-MS (neg.):
m/z 478 (M−H)−; HRMS-ESI (m/z) calcd for C17H26N3O9PS [M+H]+
480.1206, found 480.1211.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[hydroxy(ethyloxy-L-valinyl)]thiophosph
ate (Diastereoisomers, 6b). The compound was prepared according to
Standard Procedure A by using uridine 2, L-valine ethyl ester hydrochloride,
and 3a. Product 6b was obtained as a white solid (0.94 g, 74%). 31P NMR
1
(162 MHz, D2O): δ 58.22, 57.30; H NMR (400 MHz, D2O): δ 7.88, 7.86
(1H, dd, J = 8.0 Hz, H-6), 5.94–5.89 (2H, m, H-1ꢁ, 5), 4.97 (2H, m, H-2ꢁ,
3ꢁ), 4.60 (1H, s, H-4ꢁ), 4.22–4.16 (2H, m, OCH2CH3), 4.08–4.04 (2H,

Phosphorothioamidate Prodrugs and Anticancer Evaluation
169
m, H-5ꢁ), 3.58 (1H, dm, NHCH), 1.94 (1H, m, CH(CH3)2), 1.61 (3H, s,
CH3), 1.42 (3H, s, CH3), 1.28 (3H, t, J = 7.2 Hz, OCH2CH3), 0.90 (6H,
m, CH(CH3)2); 13C NMR (100 MHz, D2O): δ 176.40 (COOEt), 166.46
(C-4), 151.43 (C-2), 142.82, 142.78 (C-6), 114.48 (>CMe2), 101.88, 101.79
(C-5), 93.32, 93.15 (C-1ꢁ), 85.70, 85.49 (d, JC-P = 10.0 Hz, C-4ꢁ), 84.81, 84.77
(C-2ꢁ), 81.08, 81.00 (C-3ꢁ), 64.78, 64.73 (C-5ꢁ), 62.11 (OCH2CH3), 60.81,
60.67 (Val-C-α), 31.99 (Val-C-β), 26.13, 26.10 (CH3), 24.36 (CH3), 18.34,
17.65 (Val-CH(CH3)2), 13.51 (COOCH2CH3); IR (KBr, cm−1) 3459, 1644,
1384, 1271, 1214, 1116, 962; ESI-MS (pos.): m/z 508 (M+H)+; ESI-MS
(neg.): m/z 506 (M−H)−; HRMS-ESI (m/z) calcd for C19H30N3O9PS
[M+H]+ 508.1519, found 508.1524.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[isopropyl(ethyloxy-glycinyl)]thiophosph
ate (Diastereoisomers, 6c). The compound was prepared according to
Standard Procedure B by using uridine 2, glycine ethyl ester hydrochloride,
and 3b. Product 6c was obtained as a white solid (0.79 g, 62%). 31P NMR
(162 MHz, CDCl3): δ 71.69, 71.19; 1H NMR (400 MHz, CDCl3): δ 9.10 (1H,
br, H-3), 7.50, 7.45 (1H, dd, J = 8.0 Hz, H-6), 5.85 (1H, dd, J = 2.4, 22.4 Hz,
H-1ꢁ), 5.74 (1H, dd, J = 3.2, 8.0 Hz, H-5), 4.87 (2H, m, H-2ꢁ, 3ꢁ), 4.74 (1H,
m, OCH(CH3)2), 4.39 (1H, m, H-4ꢁ), 4.23–4.19 (4H, m, H-5ꢁ, OCH2CH3),
3.76–3.72 (3H, m, P-NH, NHCH2), 1.58 (3H, s, CH3), 1.36–1.26 (12H,
m, CH3); 13C NMR (100 MHz, CDCl3): δ 170.69, 170.61 (COOEt), 163.13
(C-4), 150.05 (C-2), 141.65, 141.40 (C-6), 114.47 (>CMe2), 102.54, 102.49
(C-5), 93.54, 93.02 (C-1ꢁ), 85.31, 84.97 (d, JC-P = 10.0 Hz, C-4ꢁ), 84.57
(C-2ꢁ), 80.74, 80.68 (C-3ꢁ), 72.73 (OCH(CH3)2), 66.13, 65.83 (d, JC-P
=
5.0 Hz, C-5ꢁ), 61.64, 61.62 (OCH2CH3), 43.40, 43.37 (Gly-C-α), 27.18, 27.15
(CH3), 25.33, 25.31 (CH3), 23.65, 23.60 (d, JC-P = 3.0 Hz, OCH(CH3)2),
14.14 (COOCH2CH3); IR (KBr, cm−1) 3452, 2988, 1637, 1457, 1384, 1271,
1214, 1108, 987; ESI-MS (pos.): m/z 508 (M+H)+; ESI-MS (neg.): m/z 506
(M−H)−; HRMS-ESI (m/z) calcd for C19H30N3O9PS [M+H]+ 508.1519,
found 508.1524.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[isopropyl(ethyloxy-L-alaninyl)]thiophos
phate (Diastereoisomers, 6d). The compound was prepared according to
Standard Procedure B by using uridine 2, L-alanine ethyl ester hydrochlo-
ride, and 3b. Product 6d was obtained as a white solid (0.85 g, 65%). 31P
1
NMR (162 MHz, CDCl3): δ 70.66, 70.10; H NMR (400 MHz, CDCl3): δ
8.23 (1H, br, H-3), 7.51, 7.47 (1H, dd, J = 8.0 Hz, H-6), 5.89–5.75 (2H,
m, H-1ꢁ, 5), 4.86 (2H, m, H-2ꢁ, 3ꢁ), 4.73 (1H, m, OCH(CH3)2), 4.39 (1H,
s, H-4ꢁ), 4.20 (4H, m, H-5ꢁ, OCH2CH3), 4.05 (1H, m, P-NH), 3.70 (1H, m,
NHCH), 1.60 (3H, s, CH3), 1.44–1.27 (15H, m, CH3); 13C NMR (100 MHz,
CDCl3): δ 173.57 (COOEt), 163.23 (C-4), 149.83 (C-2), 141.52, 141.39
(C-6), 114.52 (>CMe2), 102.44 (C-5), 93.38, 92.97 (C-1ꢁ), 84.47, 84.24 (d,
JC-P = 10.0 Hz, C-4ꢁ), 84.55 (C-2ꢁ), 80.74, 80.64 (C-3ꢁ), 72.75 (OCH(CH3)2),
65.54 (C-5ꢁ), 61.58, 61.53 (OCH2CH3), 46.79 (Ala-C-α), 27.16 (CH3), 25.33

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W. Liu et al.
(CH3), 23.61, 23.59 (d, JC-P = 3.0 Hz, OCH(CH3)2), 21.08 (Ala-C-β), 14.13
(COOCH2CH3); IR (KBr, cm−1) 3459, 2985, 1692, 1459, 1384, 1273, 1216,
1108, 990; ESI-MS (pos.): m/z 522 (M+H)+; ESI-MS (neg.): m/z 520
(M−H)−; HRMS-ESI (m/z) calcd for C20H32N3O9PS [M+H]+ 522.1676,
found 522.1670.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[isopropyl(ethyloxy-L-valinyl)]thiophosph
ate (Diastereoisomers, 6e). The compound was prepared according to
Standard Procedure B by using uridine 2, L-valine ethyl ester hydrochloride,
and 3b. Product 6e was obtained as a white solid (0.80 g, 58%). 31P NMR
(162 MHz, CDCl3): δ 72.36, 72.16; 1H NMR (400 MHz, CDCl3): δ 8.83 (1H,
br, H-3), 7.52, 7.46 (1H, dd, J = 8.0 Hz, H-6), 5.90–5.76 (2H, m, H-1ꢁ,
5), 4.86–4.82 (2H, m, H-2ꢁ, 3ꢁ), 4.75 (1H, m, OCH(CH3)2), 4.38 (1H, s,
H-4ꢁ), 4.23–4.15 (4H, m, H-5ꢁ, OCH2CH3), 3.87 (1H, m, P-NH), 3.69 (1H,
m, NHCH), 2.07 (1H, m, CHCH(CH3)2), 1.59 (3H, s, CH3), 1.37 (3H,
s, CH3), 1.32–1.27 (9H, m, COOCH2CH3, OCH(CH3)2), 0.93 (6H, dd,
J = 6.8, 26.0 Hz, CHCH(CH3)2); 13C NMR (100 MHz, CDCl3): δ 172.97,
172.94 (COOEt), 162.95, 162.91 (C-4), 150.00 (C-2), 141.43, 141.21 (C-6),
114.55, 114.51 (>CMe2), 102.66, 102.55 (C-5), 93.30, 92.66 (C-1ꢁ), 85.12
(C-2ꢁ), 84.74, 84.58 (d, JC-P = 10.0 Hz, C-4ꢁ), 80.62 (C-3ꢁ), 72.86, 72.78 (d,
JC-P = 6.0 Hz, OCH(CH3)2), 66.07, 65.87 (d, JC-P = 4.5 Hz, C-5ꢁ), 61.28
(OCH2CH3), 60.46, 60.34 (Val-C-α), 32.19 (Val-C-β), 27.20, 27.16 (CH3),
25.35, 25.30 (CH3), 23.62, 23.49 (d, JC-P = 4.0 Hz, OCH(CH3)2), 18.97,
18.84, 17.73, 17.63 (Val-CH(CH3)2), 14.25(COOCH2CH3); IR (KBr, cm−1)
3450, 2979, 1693, 1460, 1384, 1271, 1215, 1107, 994; ESI-MS (pos.): m/z
550 (M+H)+; ESI-MS (neg.): m/z 548 (M−H)−; HRMS-ESI (m/z) calcd for
C22H36N3O9PS [M+H]+ 550.1989, found 550.1981.
2ꢁ,3ꢁ-O-isopropylidene-uridine-5ꢁ-O-[isopropyl(ethyloxy-L-phenylalaninyl)]thi
ophosphate (Diastereoisomers, 6f). The compound was prepared according
to Standard Procedure B by using uridine 2, L-phenylalanine ethyl ester
hydrochloride, and 3b. Product 6f was obtained as a white solid (0.79 g,
1
53%). 31P NMR (162 MHz, CDCl3): δ 70.72, 70.63; H NMR (400 MHz,
CDCl3): δ 8.75 (1H, br, H-3), 7.47, 7.41 (1H, dd, J = 8.0 Hz, H-6), 7.31–7.17
(5H, m, PhH), 5.80–5.61 (2H, m, H-1ꢁ, 5), 4.71–4.67 (2H, m, H-2ꢁ, 3ꢁ),
4.68–4.51 (1H, m, OCH(CH3)2), 4.30 (2H, m, H-5ꢁ), 4.15 (3H, m, H-4ꢁ,
OCH2CH3), 3.92–3.85 (1H, m, P-NH), 3.68 (1H, m, NHCH), 3.02 (2H,
m, PhCH2), 1.58 (3H, s, CH3), 1.36 (3H, s, CH3), 1.30–1.19 (9H, m,
COOCH2CH3, OCH(CH3)2); 13C NMR (100 MHz, CDCl3): δ 172.72, 172.56
(d, JC-P = 9.0 Hz, COOEt), 163.05 (C-4), 150.05, 149.98 (C-2), 141.44,
141.22 (C-6), 136.05, 135.92 (PhC), 129.53, 129.49 (PhC-meta), 128.60,
128.55 (PhC-ortho), 127.12(PhC-para), 114.51, 114.45 (>CMe2), 102.59,
102.55 (C-5), 93.21, 92.72 (C-1ꢁ), 85.71, 85.61 (d, JC-P = 10.0 Hz, C-4ꢁ), 85.33
(C-2ꢁ), 80.59 (C-3ꢁ), 72.84, 72.77 (d, JC-P = 4.5 Hz, OCH(CH3)2), 65.91,
65.68 (C-5ꢁ), 56.43, 56.24 (OCH2CH3), 61.54, 61.50 (Phe-C-α), 40.58, 40.45
(d, JC-P = 6.0 Hz, Phe-C-β), 27.20, 27.18 (CH3), 25.35 (CH3), 23.60, 23.49
(d, JC-P = 4.0 Hz, OCH(CH3)2), 14.17 (COOCH2CH3); IR (KBr, cm−1)

Phosphorothioamidate Prodrugs and Anticancer Evaluation
171
3467, 2983, 1691, 1456, 1384, 1273, 1216, 1107, 990; ESI-MS (pos.): m/z
599 (M+H)+; ESI-MS (neg.): m/z 597 (M−H)−; HRMS-ESI (m/z) calcd for
C26H36N3O9PS [M+H]+ 598.1989, found 598.1985.
Zidovudine-5ꢁ-O-[isopropyl(ethyloxy-glycinyl)]thiophosphate (Diastereoiso
mers, 6g). The compound was prepared according to Standard Procedure B
by using 1, glycine ethyl ester hydrochloride, and 3b. Product 6g was obtained
as a white solid (0.76 g, 62%). 31P NMR (243 MHz, CDCl3): δ 71.96, 71.63;
1H NMR (600 MHz, CDCl3): δ 8.96 (1H, s, H-3), 7.47, 7.42 (1H, 2s, H-6),
6.25 (1H, d, J = 30 Hz, H-1ꢁ), 4.78 (1H, m, OCH(CH3)2), 4.38 (1H, s, H-3ꢁ),
4.24 (4H, m, H-5ꢁ, OCH2CH3), 4.08 (1H, s, H-4ꢁ), 3.76–3.69 (3H, m, P-NH,
NHCH2), 2.45 (1H, s, H-2ꢁ), 2.31 (1H, s, H-2ꢁ), 1.98 (3H, d, J = 4.8 Hz, 5-
CH3), 1.35–1.30 (9H, m, OCH(CH3)2, COOCH2CH3); 13C NMR (150 MHz,
CDCl3): δ 170.66, 170.60 (d, JC-P = 9.0 Hz, COOEt), 163.68 (C-4), 150.25
(C-2), 135.33, 135.28 (C-6), 111.47, 111.36 (C-5), 85.01, 84.74 (C-1ꢁ), 82.52,
82.46 (C-4ꢁ), 73.02, 72.99 (OCH(CH3)2), 65.52, 65.25 (d, JC-P = 6.0 Hz, C-5ꢁ),
61.76, 61.74 (OCH2CH3), 60.67, 60.62 (C-3ꢁ), 43.45, 43.35 (Gly-C-α), 37.55
(C-2ꢁ), 23.65, 23.44 (d, JC-P = 4.5 Hz, OCH(CH3)2), 14.14 (COOCH2CH3),
12.54 (5-CH3); IR (KBr, cm−1) 3467, 2981, 2111, 1692, 1469, 1374, 1274, 984;
ESI-MS (pos.): m/z 491 (M+H)+; ESI-MS (neg.): m/z 489 (M−H)−; HRMS-
ESI (m/z) calcd for C17H27N6O7PS [M+H]+ 491.1478, found 491.1485.
Zidovudine-5ꢁ-O-[isopropyl(ethyloxy-L-valinyl)]thiophosphate (Diastereoiso
mers, 6h). The compound was prepared according to Standard Procedure
B by using 1, L-valine ethyl ester hydrochloride, and 3b. Product 6h was
obtained as a white solid (0.88 g, 66%). 31P NMR (243 MHz, CDCl3): δ
1
72.94, 72.28; H NMR (600 MHz, CDCl3): δ 8.75 (1H, s, H-3), 7.48, 7.40
(1H, 2s, H-6), 6.26, 6.20 (1H, 2s, H-1ꢁ), 4.77 (1H, m, OCH(CH3)2), 4.37
(1H, s, H-3ꢁ), 4.22–4.16 (4H, m, H-5ꢁ, OCH2CH3), 4.08 (1H, s, H-4ꢁ), 3.90
(1H, s, P-NH), 3.66 (1H, t, J = 9.0 Hz, NHCH), 2.45 (1H, m, H-2ꢁ), 2.27
(1H, m, H-2ꢁ), 2.09 (1H, m, CHCH(CH3)2), 2.00 (3H, s, 5-CH3), 1.34–1.27
(9H, m, COOCH2CH3), 0.99, 0.90 (6H, d, J = 6.6 Hz CHCH(CH3)2);
13C NMR(150 MHz, CDCl3): δ 172.86 (COOEt), 163.53 (C-4), 150.10 (C-
2), 135.09 (C-6), 111.40 (C-5), 84.93, 84.72 (C-1ꢁ), 82.45, 82.32 (d, JC-P
= 10.0 Hz, C-4ꢁ), 73.05, 73.00 (OCH(CH3)2), 65.40, 65.35 (C-5ꢁ), 61.31
(OCH2CH3), 60.76 (C-3ꢁ), 60.64, 60.42 (Val-C-α), 37.48 (C-2ꢁ), 32.20, 32.04
(Val-C-β), 23.62, 23.39 (d, JC-P = 5.0 Hz, OCH(CH3)2), 18.88, 17.63 (Val-
CH(CH3)2), 14.21 (COOCH2CH3), 12.48 (5-CH3); IR (KBr, cm−1) 3454,
2975, 2111, 1692, 1469, 1385, 1273, 996; ESI-MS (pos.): m/z 533 (M+H)+;
ESI-MS (neg.): m/z 531 (M-H)−; HRMS-ESI (m/z) calcd for C20H33N6O7PS
[M+H]+ 533.1948, found 533.1955.
MTT Cytotoxicity Assay
Exponentially growing RKO cell or 293T cell were plated at a density
of 8000 or 15,000 cells, respectively, per well into 96-well plates. They were
incubated for 24 hours before the addition of drugs. Stock solutions of the

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W. Liu et al.
drugs (1, 2, 6) were initially in dimethyl sulfoxide (DMSO) and further
diluted with fresh complete medium.
Cytotoxic activity of phosphorothioamidate prodrugs 6 was determined
in RKO and 293T cells using standard tetrazolium MTT assay[24]; 20 μl each
of the drug dilutions were added per well and cells were exposed to drugs
at a final concentration range of 0–1000 μM. After incubation with the
compounds for 48 hours at 37◦C in a humidified atmosphere containing 5%
CO2, 20 μl of the medium containing MTT (5 mg/ml) were added to each
well. After 4 hours of exposure, the medium was removed and 180 μl of
DMSO were added to each well. The metabolized MTT product dissolved in
DMSO was quantified by reading the absorbance at 490 nm on a micro plate
reader. IC50 value, 50% inhibition of cell growth, was calculated from the
equation of the logarithmic line determined by fitting the best line (origin
7.0) to the curve formed from the data. All experiments were done at least
3 times, with 6 wells for each of the concentrations of the drugs.
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