Synthesis of novel 2′,3′-Didehydro-2′,3′- dideoxyinosine phosphoramidate prodrugs and evaluation of their anticancer activity

Article abstract of DOI:10.1080/15257770.2014.898070

An efficient synthesis of 4-chlorophenyl N-alkyl phosphoramidates of 2 ′,3 ′-didehydro-2 ′,3 ′-dideoxyinosine employing 4-chlorophenyl phosphoroditetrazolide as a phosphorylating agent is reported. Improved method for the synthesis of 2 ′,3 ′-didehydro-2

Full text of DOI:10.1080/15257770.2014.898070

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Synthesis of Novel 2′,3′-
Didehydro-2′,3′-dideoxyinosine
Phosphoramidate Prodrugs and
Evaluation of their Anticancer Activity
Weronika Boreka, Marta Lewandowskaa, Natalia Kleczewskaa, Piotr
Ruszkowskib, Karol Kacprzaka & Lech Celewicza
a Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka St
6, 60-780 Poznań, Poland
b Department of Pharmacology, Poznań University of Medical
Sciences, Rokietnicka St 5a, 60-806 Poznań, Poland
Published online: 10 Jul 2014.
To cite this article: Weronika Borek, Marta Lewandowska, Natalia Kleczewska, Piotr Ruszkowski,
Karol Kacprzak & Lech Celewicz (2014) Synthesis of Novel 2′,3′-Didehydro-2′,3′-dideoxyinosine
Phosphoramidate Prodrugs and Evaluation of their Anticancer Activity, Nucleosides, Nucleotides and
Nucleic Acids, 33:8, 507-518, DOI: 10.1080/15257770.2014.898070
To link to this article: http://dx.doi.org/10.1080/15257770.2014.898070
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Nucleosides, Nucleotides and Nucleic Acids, 33:507–518, 2014
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.898070
SYNTHESIS OF NOVEL 2ꢀ,3ꢀ-DIDEHYDRO-2ꢀ,3ꢀ-DIDEOXYINOSINE
PHOSPHORAMIDATE PRODRUGS AND EVALUATION OF THEIR
ANTICANCER ACTIVITY
Weronika Borek,1 Marta Lewandowska,1 Natalia Kleczewska,1
Piotr Ruszkowski,2 Karol Kacprzak,1 and Lech Celewicz1
1Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka St 6, 60-780 Poznan´,
Poland
2Department of Pharmacology, Poznan´ University of Medical Sciences, Rokietnicka St 5a,
60-806 Poznan´, Poland
An efficient synthesis of 4-chlorophenyl N-alkyl phosphoramidates of 2 ꢁ,3 ꢁ-didehydro-2 ꢁ,3 ꢁ-
2
dideoxyinosine employing 4-chlorophenyl phosphoroditetrazolide as a phosphorylating agent is re-
ported. Improved method for the synthesis of 2 ꢁ,3 ꢁ-didehydro-2 ꢁ,3 ꢁ-dideoxyinosine starting from
inosine is also described. The synthesized phosphoramidates 11–18 were examined for their cytotoxic
activity in three human cancer cell lines: cervical (HeLa), oral (KB), and breast (MCF-7) employing
sulforhodamine B assay. The highest activity in all investigated cancer cell lines was displayed by
phosphoramidate 13 with N-n-propyl substituent.
Keywords Phosphorylation; phosphoramidate; 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine;
prodrug; anticancer activity
INTRODUCTION
Many nucleoside analogs have found important use as antiviral[1] and
anticancer[2] therapeutics. Particularly, 2ꢁ,3ꢁ-dideoxyinosine (ddI, didano-
sine) has been developed as an anti-HIV drug for the treatment of ac-
quired immunodeficiency syndrome.[3] Significant interest also concerned
synthesis of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (d4I) due to its potential
biological activity.[4,5] Mechanism of antiviral or anticancer action of 2ꢁ,3ꢁ-
dideoxynucleosides (ddN) primarily involves their intracellular conver-
sion to 2ꢁ,3ꢁ-dideoxynucleoside 5ꢁ-triphosphates (ddNTPs) via 5ꢁ-mono- and
Received 12 December 2013; accepted 21 February 2014.
Research support from National Science Centre (Grant No. N N204 343037) is gratefully acknowl-
edged.
Address correspondence to Lech Celewicz, Faculty of Chemistry, Adam Mickiewicz University,
Grunwaldzka St 6, 60-780 Poznan´, Poland. E-mail: celewicz@amu.edu.pl
507

508
W. Borek et al.
5ꢁ-diphosphates. The ddNTPs act as competitive inhibitors of DNA poly-
merases and chain terminators of a growing DNA strand due to the lack
of a 3ꢁ-hydroxyl group.[6] Since, the first step of phosphorylation is usually
the slowest one, attempts are made to synthesize prodrugs (pronucleotides)
of 2ꢁ,3ꢁ-dideoxynucleoside 5ꢁ-monophosphates (ddNMPs) with a protected
5ꢁ-phosphate group.[7] These prodrugs are designed to easily cross the cell
membrane and release ddNMPs inside the cell as a result of chemical or enzy-
matic hydrolysis.[6,8] Thus, ddNMPs liberated inside the cell require only the
second and third phosphorylation steps for conversion to the ddNTPs. Of
the current pronucleotide strategies, the phosphoramidate diester approach
appears to be the most viable.[9]
The aim of our study was to synthesize novel phosphoramidate pro-
drugs of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine with potential antiviral or an-
ticancer properties. In this paper, we report a method for the synthesis
of 4-chlorophenyl N -alkyl phosphoramidate diesters of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-
dideoxyinosine (11–18) and evaluation of their anticancer activity. Improved
method for the synthesis of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine starting from
inosine is also presented.
RESULTS AND DISCUSSION
Chemistry
2ꢁ,3ꢁ-Didehydro-2ꢁ,3ꢁ-dideoxyinosine was synthesized from inosine ac-
cording to the procedure shown in Scheme 1 adopting the methods de-
scribed by Bhat[10] and Robins[5,11] with some modifications. In the first
step of the synthesis, inosine (1) was reacted with 2-acetoxyisobutyryl bro-
mide (2) in acetonitrile at room temperature to give the mixture of two
isomeric inosine bromoacetates 3 and 4 in 72% yield. The amount of the 2-
acetoxyisobutyryl bromide could be reduced to 2.5 equivalents. It was found
that precooling of the reaction mixture to 5◦C was not necessary as indicated
in the original procedure.[10] Moreover, it was established that the addition of
water to the acetonitrile was not essential.[5,11] Attempts to use nitromethane
instead of acetonitrile did not improve the reaction yield.[4] Treatment of
the mixture of bromoacetates 3 and 4 with activated zinc in THF effected
reductive elimination to provide 5ꢁ-O-(2,5,5-trimethyl-1,3-dioxalan-4-on-2yl)-
2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (5) in 65% yield. The use of zinc-copper
couple instead of the zinc did not increase the reaction yield appreciably.
When reductive elimination of the bromo acetates 3 and 4 was carried out in
DMF[5,11] or pyridine[4] the product 5 was obtained with a lower yield. In the
last step of the synthesis, 5ꢁ-O-(2,5,5-trimethyl-1,3-dioxalan-4-on-2yl) group
was removed from compound 5 by treatment with methanolic ammonia to
furnish 6 in 89% yield.

Synthesis of Novel 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine
509
RO
Hyp
RO
Hyp
HO
Hyp
RO
Hyp
O
O
O
Br
O
Br
O
i
ii
O
Br
O
OAc
HO OH
AcO
3
1
2
4
5
iii
O
N
O
O
O
N
NH
Hyp =
R =
HO
Hyp
CH3
N
CH3
O
CH3
6
SCHEME 1 Synthesis of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (6) from inosine (1). Reagents and condi-
tions: (i) CH3CN, r.t., 3 hours; (ii) Zn, AcOH, THF, r.t., 5 hours; and (iii) NH3/CH3OH, r.t., overnight.
A series of novel 4-chlorophenyl N -alkyl phosphoramidate diesters
of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (11–18) were synthesized by phos-
phorylation of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (6) with 4-chlorophenyl
phosphoroditetrazolide (9) according to the synthetic route outlined in
Scheme 2.
O
P
O
P
N
N
N
N
N
i
Cl
Cl
O
Cl
+
O
N
N
N
H
N
Cl
N
7
8
N
N
9
O
P
N
O
ii
iii
HO
Hyp
Cl
O
Cl
Hyp
O
Hyp
O
P
O
O
O
O
NH
R1
N
N
N
6
10
11 - 18
11: R1 = CH3
O
N
12: R1 = CH2CH3
13: R1 = CH2CH2CH3
14: R1 = CH2CH=CH2
N
NH
Hyp =
15: R1 = CH2C
N
CH
16: R1 = CH2-(2-furyl)
17: R1 = (S)-CH(CH3)CO2CH3
18: R1 = (S)-CH(CH2Ph)CO2CH3
SCHEME 2 Synthesis of 4-chlorophenyl N -alkyl phosphoramidate diesters of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-
dideoxyinosine (11–18). Reagents and conditions: (i) NEt3, CH3CN, r.t., 30 minutes; (ii) 9, pyridine, r.t.,
1 hour; and (iii) R-NH2, r.t., 1 hour.
4-Chlorophenyl phosphoroditetrazolide (9) was obtained by reaction
of 4-chlorophenyl phosphorodichloridate (7) with 1H-tetrazole (8) in the
presence of triethylamine in acetonitrile. Reaction of compound 9 with
2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (6) in the presence of pyridine afforded
reactive intermediate 10, which was treated in situ with the appropriate

510
W. Borek et al.
TABLE 1 Preparation of 4-chlorophenyl N -alkyl phosphoramidates of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-
dideoxyinosine (11–18) by phosphorylation of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (6) with
4-chlorophenyl phosphoroditetrazolide (9)
Product
R1
Yielda (%)
11
12
13
14
15
16
CH3
CH2CH3
CH2CH2CH3
CH2CH CH2
CH2C≡CH
68
70
87
85
83
86
17
18
(S)-CH(CH3CO2CH3)
(S)-CH(CH2Ph2)CO2CH3)
67
66
aYield of isolated product.
amine (or amine hydrochloride in the presence of triethylamine) to give
the desired products 11–18 in 66–87% yield (Table 1).
It should be noted that the use of 4-chlorophenyl phosphorodichloride
(7), rather than its tetrazolide counterpart 9, resulted in the formation of
a considerable amounts of the symmetrical (5ꢁ-5ꢁ)dinucleoside phosphate.
We also tried to employ 4-chlorophenyl phosphoroditriazolide[12] (obtained
from 7 and 1H-1,2,4-triazole) as the phosphorylating reagent of 6 but the
reaction yield was lower (Table 1, Table 2 entry 1). The addition of 1-
methylimidazole as the nucleophilic catalyst[13] only slightly increased the
yield of phosphorylation reaction (Table 2, entry 2). The application of 2-
and 4-chlorophenyl phosphoroditriazolides[14–16] for the phosphorylation
of 5ꢁ-protected nucleosides has been reported in the phosphotriester syn-
thesis of oligonucleotides and provided the stimulus for the development
of our method. We anticipate that developed by us method of the syn-
thesis of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine phosphoramidates will also ap-
ply to the other 2ꢁ,3ꢁ-dideoxynucleosides, e.g., 2ꢁ,3ꢁ-dideoxyinosine (ddI),
2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxythymidine (d4T), or 3ꢁ-azido-3ꢁ-deoxythymidine
(AZT).
31P NMR spectra of products 11–18 revealed the presence of two di-
astereoisomers due to a chiral center being formed at the phosphorus atom.
There were two close signals, in the ratio of approximately 1:1, in each 31P
NMR spectrum. Thin-layer chromatography (TLC) of compounds 11–18 was
also consistent with the presence of two diastereoisomers showing two over-
lapping spots but we were unable to resolve them by silica gel column chro-
matography. However, it was possible to resolve the two diastereoisomers by
HPLC on a reversed-phase column (see experimental data for compound
13).

Synthesis of Novel 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine
511
TABLE 2 Optimization of the reaction conditions for the preparation of 4-chlorophenyl N -n-propyl
phosphoramidate of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (13)
Entry
1
Phosphorylating reagent
Extra nucleophilic catalyst
Not added
Yielda (%) of 13
74
2
3
4
1-Methylimidazole
Not added
76
87
26
Not added
aYield of isolated product.
Anticancer Activity
The synthesized phosphoramidates 11–18 were evaluated for their cyto-
toxic activity in three human cancer cell lines: cervical (HeLa), oral (KB),
and breast (MCF-7) employing sulforhodamine B (SRB) assay.[17] The result-
ing cytotoxic activity data of the obtained phosphoramidates and reference
TABLE 3 In vitro cytotoxic activity of the phosphoramidates 11–18 and the parent nucleoside 6 in
three human cancer cell lines: cervical (HeLa), oral (KB), and breast (MCF-7)
Compound
Cytotoxicity (IC50, μM)a SDb
log Pc
HeLa
KB
MCF-7
11
12
13
14
15
16
72.5 0.8
55.1 0.5
46.7 0.3
71.2 0.8
60.1 0.7
>100
71.1 0.7
54.6 0.6
45.1 0.2
73.4 0.9
62.5 0.6
>100
78.4 0.9
64.3 0.7
53.4 0.4
81.2 0.8
71.3 0.7
>100
1.09
1.47
1.97
1.74
1.25
1.44
17
18
96.7 0.6
>100
95.9 0.7
>100
99.1 0.9
>100
1.29
2.75
6
>100
3.5 0.1
>100
4.1 0.1
>100
3.8 0.1
−0.97
Cytarabine (standard)
−2.32 [18]
aIC50 is the compound concentration required to inhibit cell growth by 50%.
bSD (standard deviation) of three independent experiments.
clog P (logarithm of partition coefficient) was calculated using “log PKnowwin” method.[19]

512
W. Borek et al.
compounds are presented in Table 3. The phosphoramidates 11–15 and
17 showed moderate cytotoxic activity. Among them the highest activity, in
all investigated cancer cell lines, was displayed by phosphoramidate 13 with
N -n-propyl substituent. Phosphoramidate 12 and 15 exhibited also relatively
high activity whereas phosphoramidates 11, 14, and 17 were somewhat less
potent. On the other hand, the phosphoramidates 16 and 18 as well as the
parent nucleoside 6 proved inactive in three the cell lines.
Partition coefficient (log P) values of the compounds 11–18 were calcu-
lated[19] to determine a possible correlation between the cytotoxicity data
and lipophilicity (Table 3). All of the d4I phosphoramidates were more
lipophilic than d4I itself (log P = −0.97), with log P values ranging from
1.09 to 2.75. The most active compounds 12 and 13 showed moderate values
of log P, 1.47 and 1.97, respectively. However, linear regression analysis did
not reveal any correlation between log P values and the cytotoxicity data.
CONCLUSIONS
In summary, we have developed an efficient method for the synthe-
sis of 4-chlorophenyl N -alkyl phosphoramidates of 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-
dideoxyinosine employing 4-chlorophenyl phosphoroditetrazolide as a
phosphorylating agent. 4-Chlorophenyl phosphoroditetrazolide was more
effective than its di(1,2,4-triazolo) counterpart. The obtained phosphorami-
dates 11–18 were examined for their cytotoxic activity in three human cancer
cell line: cervical (HeLa), oral (KB), and breast (MCF-7). The highest activity
in all the investigated cancer cell lines was displayed by phosphoramidate
13 with N -n-propyl substituent. Improved method for the synthesis of 2ꢁ,3ꢁ-
didehydro-2ꢁ,3ꢁ-dideoxyinosine starting from inosine was also elaborated.
EXPERIMENTAL SECTION
Chemistry (General Methods)
1H, 13C, and 31P NMR spectra were recorded on a Varian-Gemini
400 MHz spectrometer. Chemical shifts (δ) are reported in ppm relative to
the tetramethylsilane (TMS) peak. For 31P NMR spectra 85% phosphoric(V)
acid in D2O was used as an external standard (coaxial inner tube). Mass
spectra were measured on a Waters Micromass ZQ electrospray (ES) mass
spectrometer. Elemental analyses were performed on EL III elemental an-
alyzer (Elementar Analysensysteme GmbH, Germany). TLC was performed
on silica gel 60 F254 precoated (0.2 mm) plates and vacuum flash column
chromatography on silica gel 60 H (5–40 μm) purchased from Merck. High
performance liquid chromatography (HPLC) was performed on a Waters
chromatograph equipped with a Waters 996 UV-Vis photodiode array detec-
tor. Analytical HPLC was carried out on Waters XBridge C18 reversed-phase

Synthesis of Novel 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine
513
column (4.6 × 150 mm, 5 μm) using as an eluting system phosphate buffer
(20 mM Na2HPO4, pH was adjusted to 7.1 with H3PO4)–methanol (40:60).
The flow rate was 1 mL/min and detection at 266 nm. Chemical reagents
were purchased from Sigma-Aldrich.
2ꢀ,3ꢀ-Didehydro-2ꢀ,3ꢀ-dideoxyinosine (6)
To a stirred suspension of inosine (2.00 g, 7.46 mmol) in dry ace-
tonitrile (15 mL) was added dropwise 2-acetoxyisobutyryl bromide (3.89 g,
18.61 mmol, 2.5 equiv) and stirring was continued at room temperature for
3 hours. Then the mixture was poured into saturated aqueous solution of
sodium bicarbonate (50 mL) and extracted with ethyl acetate (4 × 15 mL).
The combined ethyl acetate extracts were washed with water (10 mL), dried
over anhydrous magnesium sulfate, filtered and evaporated under vacuum to
dryness; yield of 3 and 4: 2.69 g (72%). Next, the mixture of 3 and 4 (2.69 g,
5.37 mmol) was dissolved in THF (55 mL) and zinc dust (2.69 g) was added
followed by acetic acid (0.25 mL). The reaction mixture was stirred at room
temperature for 5 hours. After that, the mixture was filtered and the residue
was washed with THF (15 mL). The combined filtrates were concentrated to
about 20 mL and poured into 5% aqueous solution of EDTA trisodium
salt (100 mL). The resulting solution was extracted with ethyl acetate
(6 × 20 mL). The combined ethyl acetate extracts were washed 5% aqueous
solution of sodium bicarbonate (20 mL) and with water (10 mL), dried over
anhydrous magnesium sulfate, filtered and evaporated under vacuum to dry-
ness; yield of 5: 1.26 g (65%). Afterward, compound 5 (1.26 g, 3.48 mmol)
was suspended in methanol saturated with ammonia (50 mL) and the mix-
ture was stirred at room temperature for 12 hours. Then the mixture was
evaporated under vacuum to dryness and the residue was crystallized from
methanol; yield of 6: 0.73 g (89%).
1H NMR (400 MHz, DMSO-d6): δ 3.62–3.58 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 4.89
(m, 1H, H-4ꢁ), 4.92 (s, 1H, OH), 5.88–6.15 (m, 1H, H-3ꢁ), 6.36–6.49 (m, 1H,
H-2ꢁ), 6.91 (dd, J = 6.2 Hz, J = 3.8 Hz, 1H, H-1ꢁ), 8.09 (s, 1H, H-8), 8.12 (s,
1H, H-2), 12.33 (s, 1H, H-1); 13C NMR (100 MHz, DMSO-d6): δ 62.63, 87.99,
88.26, 124.00, 125.23, 134.68, 138.51, 145.97, 147.98, 156.65; MS (ESI+):
m/z 235 [M + H]+, 257 [M + Na]+; Anal. Calcd for C10H10N4O3: C, 51.28;
H, 4.30; N, 23.92. Found: C, 51.33; H, 4.31; N, 23.93.
Synthesis of 2ꢀ,3ꢀ-didehydro-2ꢀ,3ꢀ-dideoxyinosine
phosphoramidates (11–18)
General Procedure
To a solution of 4-chlorophenyl phosphorodichloridate (7) (260 mg,
1.06 mmol) in acetonitrile (2 mL) was added 1H-tetrazole (8) (193 mg,

514
W. Borek et al.
2.75 mmol) followed by triethylamine (220 mg, 2.17 mmol) and the reac-
tants were stirred for 30 min at room temperature. Alternatively, instead
of 1H-tetrazole other additives were used, as demonstrated for the synthe-
sis of compound 13 (see Table 2), they include: 1H-1,2,4-triazole (190 mg,
2.75 mmol) (entry 1), mixture of 1H-1,2,4-triazole (190 mg, 2.75 mmol)
and 1-methylimidazole (35 mg, 0.43 mmol) (entry 2). Then to the mixture
2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine (6) (100 mg, 0.43 mmol) and pyridine
(2.7 mL) were added. The reaction mixture was stirred at room temper-
ature for a further 1 hour and the appropriate amine (2.15 mmol) was
added. In the case of synthesis of compounds 11 and 12 amine hydrochlo-
ride (2.15 mmol) and triethylamine (327 mg, 3.23 mmol) were added. When
compounds 17 and 18 were synthesized L-alanine methyl ester hydrochlo-
ride or L-phenylalanine methyl ester hydrochloride (2.15 mmol) and triethy-
lamine (327 mg, 3.23 mmol) were added. After 1 h, the reaction mixture
was evaporated under reduced pressure. To the residue was added saturated
aqueous sodium bicarbonate (15 mL) and the mixture was extracted with
chloroform (5 × 20 mL). The combined chloroform extracts were washed
with water (15 mL), dried over anhydrous magnesium sulfate, filtered and
evaporated to dryness. The residue was purified by silica gel column chro-
matography using as an eluent the mixture chloroform – methanol (from
100 : 1 to 40 : 1, v/v) to afford products 11–18 (yield 66–87%).
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-(4-chlorophenyl
N-methylphosphate) (11)
1H NMR (400 MHz, DMSO-d6): δ 2.60–2.86 (m, 3H, CH3), 4.15–4.31
(m, 2H, H-5ꢁ, H-5ꢁꢁ), 5.08 (m, 1H, H-4ꢁ), 5.46 (m, 1H, P-NH), 6.08 (m, 1H,
H-3ꢁ), 6.54 (m, 1H, H-1ꢁ), 7.08–7.20 (m, 1H, H-2ꢁ), 7.28, 7.30 (d, J = 9.0 Hz,
2H, 4-ClPh), 7.46, 7.49 (d, J = 9.0 Hz, 2H, 4-ClPh), 8.09 (s, 1H, H-8), 8.30
(s, 1H, H-2), 12.24 (s, 1H, NH-1); 13C NMR (100 MHz, DMSO-d6): δ 25.74,
64.06, 86.11, 88.24, 121.21, 124.58, 126.06, 129.51, 130.10, 133.83, 138.57,
146.01, 148.95, 149.31, 169.61; 31P NMR (160 MHz, DMSO-d6): δ 5.11, 5.38;
MS (ESI+): m/z 438, 440 [M + H]+, 460, 462 [M + Na]+; Anal. Calcd. for
C17H17ClN5O5P: C, 46.64; H, 3.91; N, 16.00. Found: C, 46.67; H, 3.92; N,
16.01.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-(4-chlorophenyl N-ethylphosphate)
(12)
1H NMR (400 MHz, DMSO-d6): δ 1.09 (t, J = 7.2 Hz, 3H, CH3), 2.61–2.86
(m, 2H, N-CH2), 4.15–4.37 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 5.22 (m, 1H, H-4ꢁ), 5.68 (m,
1H, P-NH), 6.12 (m, 1H, H-3ꢁ), 6.72 (m, 1H, H-1ꢁ), 7.09–7.18 (m, 1H, H-2ꢁ),
7.29, 7.34 (d, J = 8.3 Hz, 2H, 4-ClPh), 7.51, 7.56 (d, J = 8.3 Hz, 2H, 4-ClPh),
8.12 (s, 1H, H-8), 8.31 (s, 1H, H-2), 12.94 (s, 1H, NH-1); 13C NMR (100 MHz,
DMSO-d6): δ 15.28, 35.60, 68.24, 85.49, 88.74, 122.38, 124.81, 126.18, 129.03,

Synthesis of Novel 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine
515
130.90, 133.88, 138.94, 145.17, 147.89, 149.31, 167.84; 31P NMR (160 MHz,
DMSO-d6): δ 5.42, 5.61; MS (ESI+): m/z 452, 454 [M + H]+, 474, 476 [M +
Na]+; Anal. Calcd. for C18H19ClN5O5P: C, 47.85; H, 4.24; N, 15.50. Found:
C, 47.88; H, 4.25; N, 15.51.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-(4-chlorophenyl
N-n-propylphosphate) (13)
1H NMR (400 MHz, DMSO-d6): δ 0.82, 0.90 (t, J = 10.2 Hz, 3H, CH3),
1.39, 1.48 (s, J = 9.8 Hz, 2H, N C CH2), 2.88–2.96 (m, 2H, N-CH2),
4.10–4.28 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 5.01 (m, 1H, H-4ꢁ), 5.43–5.58 (m, 1H, P-NH),
6.01 (m, 1H, H-3ꢁ), 6.49 (m, 1H, H-1ꢁ), 7.12–7.14 (m, 1H, H-2ꢁ), 7.26, 7.28
(d, J = 8.8 Hz, 2H, 4-ClPh), 7.44, 7.48 (d, J = 8.8 Hz, 2H, 4-ClPh), 8.07 (s,
1H, H-8), 8.24 (s, 1H, H-2), 13.04 (s, 1H, NH-1); 13C NMR (100 MHz, DMSO-
d6): δ 10.98, 24.82, 43.32, 66.26, 85.81, 88.31, 121.35, 124.40, 126.05, 129.54,
129.90, 133.46, 138.55, 145.68, 148.65, 149.19, 168.50; 31P NMR (160 MHz,
DMSO-d6): δ 4.89, 5.18; MS (ESI+): m/z 466, 468 [M + H]+, 488, 490 [M +
Na]+; Anal. Calcd. for C19H21ClN5O5P: C, 48.99; H, 4.54; N, 15.03. Found:
C, 49.05; H, 4.55; N, 15.04; HPLC: retention time (tR) of 5.17 and 5.66 min
in the ratio 1: 1.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-(4-chlorophenyl N-allylphosphate)
(14)
1H NMR (400 MHz, DMSO-d6): δ 3.23–3.37 (m, 2H, N-CH2), 4.02–4.24
(m, 3H, H-4ꢁ, H-5ꢁ, H-5ꢁꢁ), 5.01–5.19 (m, 2H, N C C CH2), 5.70–5.82 (m,
2H, P-NH, N-C-CH C), 6.18–6.27 (m, 1H, H-3ꢁ), 6.45–6.54 (m, 1H, H-1ꢁ),
6.78–6.89 (m, 1H, H-2ꢁ), 7.17–7.26 (m, 2H, 4-ClPh), 7.44–7.56 (m, 2H, 4-
ClPh), 7.98 (s, 1H, H-8), 8.11 (s, 1H, H-2), 12.42 (s, 1H, NH-1); 13C NMR
(100 MHz, DMSO-d6): δ 38.06, 67.39, 85.39, 92.60, 115.07, 121.95, 122.04,
126.09, 129.48, 131.60, 133.22, 136.43, 138.22, 146.04, 148.06, 149.62, 166.99;
31P NMR (160 MHz, DMSO-d6): δ 5.55, 5.59; MS (ESI+): m/z 464, 466 [M
+ H]+, 486, 488 [M + Na]+; Anal. Calcd. for C19H19ClN5O5P: C, 49.20; H,
4.13; N, 15.10. Found: C, 49.24; H, 4.14; N, 15.12.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-(4-chlorophenyl
N-propargylphosphate) (15)
1H NMR (400 MHz, DMSO-d6): δ 2.09 (s, 1H, N-C-C-CH), 3.62–3.78 (m,
2H, N-CH2-C-C), 4.35 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 5.17 (m, 1H, H-4ꢁ), 6.13–6.22
(m, 1H, H-3ꢁ), 6.42–6.50 (m, 1H, H-2ꢁ), 6.78–6.89 (m, 1H, H-1ꢁ), 7.02–7.26
(m, 2H, 4-ClPh), 7.48–7.62 (m, 2H, 4-ClPh), 8.07 (s, 1H, H-8), 8.22 (s, 1H,
H-2), 12.83 (s, 1H, NH-1); 13C NMR (DMSO-d6) δ 29.67, 71.82, 71.89, 80.60,
85.56, 88.46, 121.37, 121.44, 126.16, 129.54, 129.68, 133.37, 138.75, 145.70,
148.59, 148.94, 158.45; 31P NMR (DMSO-d6) δ 4.24, 4.58; MS (ESI+): m/z

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W. Borek et al.
462, 464 [M + H]+, 484, 486 [M + Na]+; Anal. Calcd. for C19H17ClN5O5P:
C, 49.42; H, 3.71; N, 15.17. Found: C, 49.46; H, 3.72; N, 15.18.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-[4-chlorophenyl
N-methyl(2-furyl)phosphate] (16)
1H NMR (400 MHz, DMSO-d6) δ 3.64 (m, 2H, N-CH2-2-furyl), 3.82–3.96
(m, 2H, H-5ꢁ, H-5ꢁꢁ), 4.36 (m, 1H, H-4ꢁ), 6.11–6.34 (m, 2H, H-3ꢁ, H-2ꢁ),
6.43–6.58 (m, 1H, furyl), 6.81 (m, 1H, furyl) 7.02 (m, 1H, H-1ꢁ), 7.25–7.40
(m, 2H, 4-ClPh) 7.50–7.68 (m, 2H, 4-ClPh), 7.96 (s, 1H, H-8), 8.18 (s, 1H,
H-2), 12.79 (s, 1H, NH-1); 13C NMR (DMSO-d6) δ 29.70, 66.03, 85.70, 88.41,
107.22, 110.30, 116.80, 121.36, 126.05, 129.51, 130.08, 133.62, 138.78, 142.06,
145.70, 148.66, 148.96, 149.12, 158.48; 31P NMR (DMSO-d6) δ 4.19, 4.64; MS
(ESI+): m/z 504, 506 [M + H]+, 526, 528 [M + Na]+; Anal. Calcd. for
C21H19ClN5O6P: C, 50.06; H, 3.80; N, 13.90. Found: C, 50.11; H, 3.81; N,
13.91.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-[4-chlorophenyl
N-(methoxy-(S)-alaninyl) phosphate] (17)
1H NMR (400 MHz, DMSO-d6) δ 1.25 (m, 3H, CCH3), 3.78 (s, 3H,
OCH3), 3.94 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 4.18–4.28 (m, 1H, N-CH), 4.36–4.54 (m,
1H, H-4ꢁ), 6.11–6.28 (m, 1H, H-3ꢁ), 6.84 (m, 1H, H-2ꢁ) 7.08 (m, 1H, H-1ꢁ),
7.25–7.31 (m, 2H, 4-ClPh), 7.62–7.81 (m, 2H, 4-ClPh), 7.98 (s, 1H, H-8),
8.21 (s, 1H, H-2), 12.81 (s, 1H, NH-1); 13C NMR (DMSO-d6) δ 45.80, 52.59,
68.12, 85.61, 85.62, 88.57, 121.20, 121.51, 126.20, 129.65, 130.87, 133.38,
138.50, 145.31, 146.96, 148.95, 158.12, 167.76; 31P NMR (DMSO-d6) δ 3.18,
3.12; MS (ESI+): m/z 510, 512 [M + H]+, 532, 534 [M + Na]+; Anal. Calcd.
for C20H21ClN5O7P: C, 47.12; H, 4.15; N, 13.74. Found: C, 47.17; H, 4.16; N,
13.75.
2 ꢀ,3 ꢀ-Didehydro-2 ꢀ,3 ꢀ-dideoxyinosine 5 ꢀ-O-[4-chlorophenyl
N-(methoxy-(S)-phenylalaninyl) phosphate] (18)
1H NMR (400 MHz, DMSO-d6) δ 2.98–3.08 (m, 2H, CH2Ph), 3.69 (s,
3H, OCH3), 3.89 (m, 2H, H-5ꢁ, H-5ꢁꢁ), 4.31–4.40 (m, 1H, H-4ꢁ), 4.68 (m,
1H, N-CH) 6.18–6.38 (m, 1H, H-3ꢁ), 6.42–6.54 (m, 1H, H-2ꢁ), 6.89 (m, 1H,
H-1ꢁ), 7.05–7.50 (m, 9H, 4-ClPh, C-Ph), 8.02 (s, 1H, H-8), 8.24 (s, 1H, H-2),
12.89 (s, 1H, NH-1); 13C NMR (DMSO-d6) δ 38.17, 52.17, 53.28, 70.15, 84.98,
89.45, 116.18, 122.32, 127.15, 128.85, 129.05, 129.39, 130.07, 131.87, 133.81,
135.05, 137.77, 147.81, 149.11, 151.28, 158.65, 172.18; 31P NMR (DMSO-d6)
δ 5.21, 5.38; MS (ESI+): m/z 586, 588 [M + H]+, 608, 610 [M + Na]+; Anal.
Calcd. for C26H25ClN5O7P: C, 53.30; H, 4.30; N, 11.95. Found: C, 53.35; H,
4.31; N, 11.96.

Synthesis of Novel 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxyinosine
517
BIOLOGICAL EVALUATION
Cell Cultures
Human cancer cells HeLa (cervical cancer cell line) and KB (carcinoma
nasopharynx) were cultured in RPMI 1640 medium and human cancer cells
MCF-7 (breast cancer cell line) were cultured in DMEM medium. Each
medium was supplemented with 10% fetal bovine serum, 1% L-glutamine,
and 1% penicillin/streptomycin solution. The cell lines were kept in the
incubator at 37◦C. The optimal plating density of cell lines was determined
to be 5 × 104. All the cell lines were obtained from The European Collection
of Cell Cultures (ECACC) supplied by Sigma-Aldrich.
In vitro Cytotoxicity Assay
The protein-staining SRB (Sigma-Aldrich) microculture colorimetric as-
say, developed by the National Cancer Institute (USA) for in vitro antitumor
screening was used in this study, to estimate the cell number by providing
a sensitive index of total cellular protein content, being linear to cell den-
sity.[17] The monolayer cell culture was trypsinized and the cell count was
adjusted to 5 × 104 cells. To each well of the 96 well microtiter plate, 0.1 mL
of the diluted cell suspension (approximately 10,000 cells) was added. After
24 hours, when a partial monolayer was formed, the supernatant was washed
out and 100 μL of six different compound concentrations (0.1, 0.2, 1, 2, 10,
and 20 μM) were added to the cells in microtitre plates. The tested com-
pounds were dissolved in DMSO (containing 10% of water) (100 μL) and
the content of DMSO did not exceed 0.1%; this concentration was found
to be nontoxic to the cell lines. The cells were exposed to compounds for
72 hours. After that, 25 μL of 50% trichloroacetic acid was added to the wells
and the plates were incubated for 1 hour at 4◦C. The plates were then washed
out with the distilled water to remove traces of medium and next dried by
the air. The air-dried plates were stained with 100 μL SRB and kept for 30
minutes at room temperature. The unbound dye was removed by rapidly
washing with 1% acetic acid and then air dried overnight. The optical den-
sity was read at 490 nm. All cytotoxicity experiments were performed three
times. Cell survival was measured as the percentage absorbance compared to
the control (nontreated cells). Cytarabine (Sigma-Aldrich) was used as the
internal standard.
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