Synthesis and in vitro cytostatic activity of 1,2- and 1,3- diacylglycerophosphates of clofarabine

Article abstract of DOI:10.1016/j.bmc.2013.06.019

The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2- fluoro-β-d-arabinofuranosyl)adenine] with 1,2- and 1,3- diacylglycerophosphates have been prepared by the phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against HL-60, A-549, MCF-7, and HeLa tumor cell lines.

Full text of DOI:10.1016/j.bmc.2013.06.019

Accepted Manuscript
Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophos‐
phates of clofarabine
Ilona Tsybulskaya, Tamara Kulak, Alexander Baranovsky, Marina Golubeva,
Boleslav Kuzmitsky, Elena Kalinichenko
PII:
S0968-0896(13)00550-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.019
BMC 10912
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 February 2013
31 May 2013
6 June 2013
Please cite this article as: Tsybulskaya, I., Kulak, T., Baranovsky, A., Golubeva, M., Kuzmitsky, B., Kalinichenko,
E., Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of clofarabine, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.019
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Synthesis and in vitro cytostatic activity of
1,2- and 1,3-diacylglycerophosphates of
clofarabine
Leave this area blank for abstract info.
Ilona Tsybulskayaa,*, Tamara Kulaka, Alexander Baranovskya, Marina Golubevaa, Boleslav Kuzmitskya, Elena
Kalinichenkoa
a Institute of Bioorganic Chemisrty, National Academy of Sciences of Belarus, BY-220141 Minsk, Belarus

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of
clofarabine
∗
Ilona Tsybulskaya a, , Tamara Kulak a, Alexander Baranovskya, Marina Golubevaa, Boleslav Kuzmitskya,
and Elena Kalinichenkoa
a Institute of Bioorganic Chemisrty, National Academy of Sciences of Belarus, BY-220141 Minsk, Belarus
ARTICLE INFO
ABSTRACT
Article history:
Received
The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2-fluoro-ꢀ-D-
arabinofuranosyl)adenine] with 1,2- and 1,3-diacylglycerophosphates have been prepared by the
Received in revised form
Accepted
Available online
phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl
protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the
phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against
HL-60, A-549, MCF-7, and HeLa tumor cell lines.
Keywords:
Clofarabine
2009 Elsevier Ltd. All rights reserved.
Prodrugs
Phosphoramidite approach
1,2-, 1,3-Diacylglycerophospholipids
Antitumor activity
———
∗Corresponding author. Tel.: +375-29-590-69-63; fax: +375-17-267-87-61; e-mail: ilonaolenikova@tut.by

1. Introduction
isomeric 1,2- and 1,3-diacylglycerophosphocholines.17 Thus, 1,3-
diacylglyceromonophosphate derivatives of biologically active
nucleosides may exhibit different pharmacokinetic properties and
increased stability in gastrointestinal tract in comparison with
their 1,2-counterparts. We believe that such compounds deserve
further investigation as prodrug forms of biologically active
nucleosides.
Nucleoside and nucleotide analogues play an important role as
anticancer agents.1 It is known, however, that such compounds
often have a number of drawbacks including low bioavailability,
poor pharmacokinetic properties, and toxic effects connected
with insufficiently selective delivery of the agent to the tumor
tissue. It is also shown that the conjugation of these nucleosides
with lipid derivatives can give the prodrugs characterized by
improved pharmacokinetics, different pharmacological profiles,
and fewer side effects as compared with the parent compounds.2
Clofarabine
[2-chloro-9-(2-deoxy-2-fluoro-ꢀ-D-
arabinofuranosyl)adenine, C1-F-ara-A] is a purine nucleoside
used in the therapy of pediatric acute myeloid leukemia.1 An
evaluation of this compound against various tumor systems in
vitro and in mice has demonstrated that C1-F-ara-A exhibits
significant cytotoxicity against human tumor cell lines including
leukemias and several types of solid tumors.18
This approach was firstly employed for preparing the prodrugs
of 1-ꢀ-D-aravinofuranosylcytosine (ara-C). In the 1970s, several
groups of researchers started the investigations aimed at the
preparation of the conjugates of ara-C with natural or synthetic
phospholipids and evaluation of their cytotoxicity.3, A number
4
The usage of C1-F-ara-A is also limited by some
disadvantages such as narrow therapeutic ratio, short biological
half-life, bone marrow toxicity.19 There are few data on the
preparation and properties of lipid derivatives of clofarabine in
the literature. Several dialkylglycerophosphate derivatives of C1-
F-ara-A described to date exhibited higher plasma exposure,
increased terminal half-life, and high efficacy in a variety of solid
tumor xenograft models.20
of
1-ꢀ-D-arabinofuranosylcytosine
5’-diphosphate-1,2-
diacylglycerols demonstrated promising antiproliferative
properties, especially in the in vivo experiments.5-7 1-ꢀ-D-
arabinofuranosylcytosine
5’-monophosphate-L-1,2-dipalmitin
exhibited in vivo and in vitro cytotoxicity against L1210
lymphoid leukemia.5 A series of oxy- and thioether phospholipid
derivatives of ara-C also demonstrated significant antitumor
activity against various leukemias in animals.8,
The lipid
9
The present work focuses on the synthesis of clofarabine
conjugates with natural 1,2-diacylglycerophosphates and their
synthetic 1,3-counterparts as well as on early evaluation of their
in vitro activity in promyelocytic leukemia HL-60, lung
carcinoma A-549, breast carcinoma MCF-7, and cervix
carcinoma HeLa cell lines.
prodrugs of other antitumor nucleoside analogs, such as 5’-
fluoro-2’-deoxyuridine, fludarabine, gemcitabine, have been
synthesized.10-12 The results of the above mentioned studies
indicate that the in vitro activity of the lipid derivatives of
antitumor nucleosides is often lower than that of nucleoside
alone. Contrary to this, when tested in vivo, the same conjugates
tend to be equally or more active than the parent drug. It was
shown that the ability of these compounds to form micelles is
important for improving their activity13. The incorporation of the
conjugates into liposomes often leads to the enhanced antitumor
effect14.
2. Results and Discussion
2.1. Synthesis
The phosphoramidite approach21 widely used in
oligonucleotide chemistry was chosen for the preparation of
diacylglycerophosphates of clofarabine. The method included the
formation of P-O bond via condensation of 3’-O-protected
nucleoside with 1,2- or 1,3-diacylglyceryl phosphoramidites to
obtain phosphite intermediates which were further oxidized with
iodine.
The
metabolic
pathways
of
nucleoside
1,2-
diacylglyceromonophosphates were clarified by the example of
corresponding AZT (3’-azidothymidine) derivatives. It was
shown that, in the cells, these compounds undergo deacylation
catalyzed by phospholipases A and lysophospholipases. The
produced AZT glycerophosphates are further hydrolyzed by
phosphodiesterases to liberate the nucleoside/5’-nucleotide
moiety which, after phosphorylation by kinases, give AZT 5’-
triphosphate, an active metabolite.15 Recently we have
synthesized the conjugates of antiviral nucleoside ribavirin [1-(ꢀ-
D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide] with 1,2- and
1,3-diacylglycerophosphates and demonstrated that 1,3-isomers,
in principle, are also able to enter the first stage of the above
described metabolic pathway catalyzed by phospholipases.16
However, the hydrolysis of 1,3-diacyl derivatives by porcine
pancreatic phospholipase A2 was essentially decelerated as
compared with 1,2-isomers containing natural phosphatidyl
residues, similar to the phenomenon previously observed for
To provide C1-F-ara-A derivative bearing free 5’-OH group
appropriated for the condensation with 1,2- and 1,3-
diacylglyceryl phosphoramidites, we employed 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl (TIPDS) protecting group
previously used for temporarily masking the hydroxyl functions
of various nucleosides.22
Scheme 1. Synthesis of 3’-ꢁ-TIPDS derivative 5. Reagents and
condition: (i): TIPDSiCl2/Py, (ii): TFA/H2O/THF

4 coincides with those of 5’-O-TIPDS derivative 3; however, in
the spectrum of 4 we do not observe a peak from OH group of
siloxane fragment characteristic for nucleoside 3. Moreover, a
comparison of integral intensity of the signals from nucleoside
and isopropyl protons suggests that in the molecule of compound
4 there are two nucleoside residues connected to TIPDS
fragment. The data of mass-spectrometry ([MH]+: 849) confirm
the dimeric structure of compound 4.
The 3’-O-silylated nucleoside 5 having free 5’-OH group
suitable for further coupling with lipid phosphoramidites was
prepared in 90% yield by the selective deblocking of 5’-OH
function of cyclic 3’,5’-ꢁ-TIPDS derivative
2 under the
treatment with a mixture of trifluoroacetic acid/water/THF (1:1:4,
v/v)23 for 3 h at 0ꢂ ꢃ.
Phosphoramidite derivatives of 1,2-diacyl glycerols (6, 7) and
their 1,3-diacyl regioisomers 8, 9 were prepared as described
previously16.
The condensation of 3’-ꢁ-TIPDS-protected nucleoside 5 with
phosphoramidites 6-9 in CH2Cl2 in the presence of tetrazole
followed by I2/H2O oxidation gave the phosphotriesters 10-13
isolated in 82-88% yields.
Compounds 10-13 were treated with a mixture of Py/NEt3
(1:1, v/v) for removing ꢀ-cyanoethyl protecting group from the
phosphate and then with 1M tetrabutylammonium fluoride in
THF to remove 3’-O-TIPDS protecting group of the nucleoside
fragment. The deprotected lipid clofarabine derivatives were
purified by silica gel column chromatography and isolated as
sodium salts 14-17 after the sequential treatment with HCl and
NaOH solutions in 60-97% overall yields.
The structure of the synthesized clofarabine conjugates are
confirmed by the data of NMR spectroscopy including 2D NMR
methods. The data of elemental analysis and mass-spectrometry
are in good accordance with the composition of the lipid-
clofarabine derivatives.
The treatment of clofarabine (1) with 1,3-dichloro-1,1,3,3-
tetraisopropyldisiloxane (TIPDSiCl2) (1.2 molar equiv.) in
pyridine resulted in the formation of cyclic 3’,5’-ꢁ-TIPDS
2.2. In vitro cytostatic activity of diacylglycerophosphates of
clofarabine
clofarabine derivative
compounds, 5’-ꢁ-silylated nucleoside
bis(nucleoside)siloxane 4 (3%).
2 (72%), along with two minor
3
(22%) and
In vitro cytostatic activity of clofarabine derivatives 14-17
against hematologic and solid tumor cell lines (promyelocytic
leukemia HL-60, lung carcinoma A-549, breast carcinoma MCF-
7, cervix carcinoma HeLa) was evaluated in comparison with
parent nucleoside clofarabine (1). The obtained results are given
in Table 1.
The structure and purity of clofarabine derivatives 2-4 have
been confirmed by means of 1H, 13C, and 19F NMR spectroscopy,
mass spectrometry, and elemental analysis. The peaks at 6.02 and
1
5.15 ppm observed in H NMR spectrum of clofarabine (1) in
DMSO-d6, corresponding to the protons of 3’-OH and 5’-OH
groups, disappeared in the spectrum of cyclic 3’,5’-ꢁ-TIPDS
derivative 2; instead, a group of signals appears at 0.96-1.32 ppm
due to the presence of 28 isopropyl protons of TIPDS fragment.
Compound 14 inhibited HL-60 cell line at micromolar
concentrations (IC50 0.8 µM). Compounds 15 and 17 were
weaker inhibitors of HL-60 cells, while conjugate 16 did not
exhibit noticeable cytotoxicity. The antiproliferative activity of
compounds 14, 16, 17 against breast carcinoma MCF-7 and the
cytotoxicity of conjugate 14 in HeLa cell line were of the same
order of magnitude as that of parent nucleoside 1. The tested
conjugates were only weak inhibitors of lung carcinoma A-549.
1
A similar group of signals is observed at 0.78-1.03 ppm in H
NMR spectrum of nucleoside 3; however, in this case there are
also a dublet at 6.06 ppm and a singlet at 6.14 ppm attributed,
correspondingly, to the proton of 3’-OH group of sugar moiety
and hydroxyl group of siloxane fragment.
The introduction of TIPDS group into the 3’,5’-diol fragment
of C1-F-ara-A molecule causes a significant low-field shift of H-
3’ and H-5’,H-5” resonance. In the spectrum of clofarabine (1),
the signals from these protons are observed at 4.43 and 3.62-3.72
ppm, whereas in the case of nucleoside 2 the corresponding peaks
As mentioned above, the in vivo activity of lipid nucleoside
derivatives can be substantially higher in comparison with in
vitro experiments. Hence, further in vivo investigation of
antiproliferative properties of the synthesized clofarabine
derivatives would be helpful for estimating their prospects as
nucleoside prodrugs.
1
are located at 5.05 and 3.96-4.17 ppm. In H NMR spectrum of
5’-O-silylated derivative 3, a shift to the low field is observed
only for H-5’,H-5” resonance (multiplet centered at 3.98 ppm).
The location of the peaks from nucleoside protons of compound

O
C
C
O
O
C
H2n+1Cn
H2n+1Cn
O
O
H2n+1Cn
H2n+1Cn
O
O
P
N
5
+
5
+
O
P
N
C
O
O
O
O
NC
NC
8: n=13
9: n=15
6: n=13
7: n=15
i, ii
i, ii
NH2
N
NH2
N
O
N
N
N
O
C
H2n+1Cn
H2n+1Cn
C
C
O
O
O
H2n+1Cn
H2n+1Cn
O
O
O
P
O
N
N
Cl
N
Cl
O
O
O
P
O
F
F
O
O
C
O
O
O
NC
OH
Si
O
OH
O
NC
O
Si
Si
O
Si
10: n=13
11: n=15
12: n=13
13: n=15
NH2
NH2
N
iii, iv, v
iii, iv, v
O
N
N
N
N
H2n+1Cn
H2n+1Cn
C
C
O
O
O
O
O
H
2n+1Cn
C
O
O
N
N
Cl
N
Cl
O
P O
O
P
O
F
F
O
O
ONa
H2n+1Cn
C
O
O
ONa
OH
OH
14: n=13
15: n=15
16: n=13
17: n=15
Scheme 2. Synthesis of clofarabine conjugates 14 - 17. Reagents and conditions: (i): CH2Cl2, tetrazole; (ii): I2/Py/CH2Cl2/H2O; (iii): Py/(C2H5)3N = 1/1; (iv): 1M
TBAF/THF; (v): HCl/CHCl3, NaOH/MeOH
Table 1. In vitro cytostatic activity of clofarabine conjugates,
4. Experimental
IC50, µM
Clofarabine was kindly provided by Dr. G.G. Sivets24. TLC
was performed on silica gel sheets Merck 60 F 254. Lipid
derivatives were visualized on TLC by spraying with 30 %
H2SO4 and charring. Preparative column chromatography was
performed on silica gel Merck 60 (70-230 µm). Elemental
analysis was done with the use of CHNS-O Analyser EA-3000
(EuroVector). UV spectra were recorded using UV-VIS
spectrometer Cary 100 (Varian). NMR spectra were registered on
Compound
Cell lines
1
14
15
16
17
HL-60
A-549
MCF-7
HeLa
0.1
0.8
10.0
>100
>100
>100
>100
>100
40.0
>100
55.0
90.0
30.0
100.0
8.0
>100
40.0
50.0
50.0
50.0
1
Avance 500 (Bruker). In H- and 13C NMR spectra, chemical
shifts (ꢄ) are given in ppm related to internal SiMe4, coupling
constants (J) in Hz. In 31P NMR spectra, chemical shifts are
given in ppm related to external H3PO4. Assignment of the
3. Conclusions
1
signals in H and 13C NMR spectra was carried out with the use
The method proposed in this work for the synthesis of lipid
clofarabine derivatives is based on a combination of TIPDS
protecting group for the carbohydrate moiety of nucleoside and
cyanoethyl group for the phosphate fragment of lipid-nucleoside
conjugates. It allows obtaining of the desired phospholipid
clofarabine derivatives in reasonable yields through a limited
number of synthetic steps.
of 1H-1H and 1H-13C correlation spectroscopy methods.
4.1. Cytostatic activity
The samples of compounds 14-17 for in vitro testing were
prepared by sonication of drug suspensions in 0.9% NaCl
solution (3 × 1 min) using an ultrasonic dispergator UZDN-A
with working frequency 22 kHz and power up to 100 W/cm2.
Diacylglycerophosphate clofarabine derivatives 14-17 deserve
consideration as clofarabine prodrugs for further in vivo
investigation. The study on the biophysical and biochemical
properties of the synthesized conjugates is now in progress.
Human cell lines were obtained from the Institute of
ꢃytology, Russian Academy of Sciences. The cell lines studied
were maintained in RPMI 1640 medium (AppliChem, Darmstadt,
Germany) supplemented with 20% (HL-60) fetal calf serum
(HyClone, Cramlington, UK), in DMEM (A 549) and Eagle's

MEM (AppliChem) (MCF7, HeLa) with 10% fetal calf serum
(HyClone). Cells were cultivated at 37°C in a humidified
atmosphere of 5% CO2. Cellular sensitivities to clofarabine and
its lipid derivatives were measured with the MTT assay. Cells
were plated in triplicate in 96-well plates (105 cells/mL for
suspended cell cultures and 5×103 cells/ mL for monolayer
cultures); at the same day (suspended cell cultures) or on the
following day (monolayer cultures), compounds were added at
the appropriate dilutions. Plates were incubated under standard
conditions for 48 h. Thereafter, 10 ꢅL MTT (Sigma) in phosphate
buffered saline (5 mg/mL) were added. The plates were
incubated for an additional 4 h and 150 ꢅL dimethylsulphoxide
(DMSO) were added to dissolve the formazan crystals. The
optical density was read on Infinite M 200 plate reader (Tecan,
Switzerland) at 540 nm. The antiproliferative effects were
expressed as IC50.
4.2.3. 1,1,3,3-Tetraisopropyldisiloxane-1,3-di{[2-chloro-9-(2-
deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)adenine]-5'-yl}, 4
1H NMR (DMSO-d6): ꢄ 8.12 (d, 2ꢇ, J 1.5 Hz, ꢇ-8), 7.89 (br s,
4ꢇ, NH2), 6.34 (dd, 2ꢇ, J1’,2’ 5.0 Hz, J1’,F 13.0 Hz, ꢇ-1’), 6.06 (d,
2ꢇ, Jꢁꢇ, 3’ 5.0 Hz, 3’-OH), 5.27 (m, 2ꢇ, J2’,F 52.5 Hz, ꢇ-2’), 4.50
(m, 2ꢇ, J3’,F 19.5 Hz, ꢇ-3’), 3.99 (m, 4ꢇ, ꢇ-5’, ꢇ-5”), 3.91 (m,
2ꢇ, ꢇ-4’), 1.03 - 0.78 (m, 28ꢇ, 4[i-Pr]). 13C NMR (DMSO-d6): ꢄ
156.81, 153.31, 150.14, 139.69, 117.45 (Ade), 95.35 (d, JC,F
193.0 Hz, C(2’)), 82.66 (d, JC,F 6.0 Hz, C(4’)), 81.31 (d, JC,F 17.0
Hz, C(1’)), 72.62 (d, JC,F 23.0 Hz, C(3’)), 61.91 (C(5’)), 17.04,
12.28, 12.25 (i-Pr). 19F NMR (DMSO-d6): ꢄ -198.53. Anal. calcd
for C32H48Cl2F2N10O7Si2 (849.860), %: ꢃ 45.22, ꢇ 5.69, N 16.48;
found, %: ꢃ 44.95, H 5.63, N 16.32. ESI-MS: [MH]+: 849. UV
(EtOH), ꢈmax , nm (lg ꢉ): 212 (4.59), 263 (4.38).
4.2.4. 2-Chloro-9-[2-deoxy-2-fluoro-3-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]-
adenine, 5
4.2. Chemistry
To a solution of nucleoside 2 (352 mg, 0.64 mmol) in THF (2
mL), a mixture of trifluoroacetic acid/water/THF (1:1:4, v:v; 1
mL) was added at 0ꢂ ꢃ. After stirring for 3 h, the reaction mixture
was evaporated to dryness. The residue was evaporated with
toluene (1 mL) and purified on silica gel column (50 cm3) using a
gradient of CH3OH (0ꢆ2%) in CHCl3. The appropriate fractions
were combined and evaporated to give a solid foam of compound
4.2.1.
2-Chloro-9-[2-deoxy-2-fluoro-3,5-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-ꢀ-D-
arabinofuranosyl]adenine, 2
To a solution of 1 (225 mg, 0.74 mmol) in pyridine (1 mL),
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.28 mL, 0.89
mmol) was added, and the mixture was stirred at room
temperature for 24 h. After that the mixture was evaporated and
the residue partitioned between chloroform (100 mL) and water
(30 mL). The chloroform layer was dried over anhydrous sodium
sulfate, evaporated, and co-evaporated with toluene. The residue
was applied on silica gel column (60 cm3), and the products were
eluted using a gradient of CH3OH (0ꢆ20%) in CHCl3. The
appropriate fractions were combined and evaporated to give
compounds 2 (352 mg, 87%), 3 (36 mg, 9%), and 4 (17 mg, 2%)
as solid foams.
1
5 (327 mg, 90%); H NMR (DMSO-d6): ꢄ 8.25 (d, 1ꢇ, J 1.5 Hz,
ꢇ-8), 7.90 (br s, 2ꢇ, NH2), 6.34 (dd, 1ꢇ, J1’,2’ 4.5 Hz, J1’,F 15.0
Hz, ꢇ-1’), 6.14 (s, 1ꢇ, SiOH), 5.30 (m, 1ꢇ, J2’,F 50.0 Hz, ꢇ-2’),
5.25 (m, 1ꢇ, 5’-OH), 4.78 (m, 1ꢇ, J3’,F 17.0 Hz, ꢇ-3’), 3.95 (m,
1ꢇ, ꢇ-4’), 3.72 (m, 1ꢇ, J5’,4’ 4.5 Hz, J5’,5” 12.0 Hz, ꢇ-5’), 3.66
(m, 1ꢇ, J5”,4’ 5.5 Hz, J5”,5’ 12.0 Hz, ꢇ-5”), 0.81-1.04 (m, 28ꢇ, 4[i-
Pr]). 13C NMR (DMSO-d6): ꢄ 156.78, 153.30, 150.16, 139.86,
117.37 (Ade), 95.01 (d, JC,F 194.0 Hz, C(2’)), 84.23 (C(4’)),
81.75 (d, JC,F 17.0 Hz, C(1’)), 73.56 (d, JC,F 25.0 Hz, C(3’)),
60.09 (C(5’)), 17.26, 17.23, 17.00, 16.96, 13.01, 12.47, 12.39 (i-
Pr). 19F NMR (DMSO-d6):
ꢄ -197.81. Anal. calcd for
1
2: H NMR (DMSO-d6): ꢄ 8.17 (s, 1ꢇ, ꢇ-8), 7.91 (br s, 2ꢇ,
C22H39ClFN5O5Si2, (564.198), %: ꢃ 46.83, ꢇ 6.97, N 12.41;
found, %: ꢃ 46.38, H 6.85, N 12.12. ESI-MS: 564 [M+H]+. UV
(EtOH), ꢈmax , nm (lg ꢉ): 212 (4.35), 263 (4.13).
NH2), 6.36 (dd, 1ꢇ, J1’,2’ 6.5 Hz, J1’,F 4.5 Hz, ꢇ-1’), 5.57 (m, 1ꢇ,
J2’,F 54.0 Hz, ꢇ-2’), 5.05 (m, 1ꢇ, J3’,F 21.5 Hz, ꢇ-3’), 4.17 (dd,
1ꢇ, J5’,4’ 5.5 Hz, J5’,5” 12.5 Hz, ꢇ-5’), 3.96 (dd, 1ꢇ, J5”,4’ 2.5 Hz,
J5”,5’ 12.5 Hz, ꢇ-5”), 3.89 (m, 1ꢇ, ꢇ-4’), 1.32-0.96 (m, 28ꢇ, 4[i-
Pr]). 13C NMR (DMSO-d6): ꢄ 156.83, 153.22, 150.13, 139.89,
117.93 (Ade), 95.00 (d, JC,F 196.0 Hz, C(2’)), 79.71 (d, JC,F 17.0
Hz, C(1’)), 78.55 (d, JC,F 4.0 Hz, C(4’)), 74.69 (d, JC,F 21.0 Hz,
C(3’),), 62.00 (C(5’)), 17.28, 17.11, 16.80, 16.71, 12.52, 12.39,
12.07, 11.98 (i-Pr). 19F NMR (DMSO-d6): ꢄ -200.33. Anal. calcd
for C22H37ClFN5O4Si2 (546.183), %: ꢃ 48.38, ꢇ 6.83, N 12.82;
found, %: ꢃ 48.81, H 6.88, N 12.58. ESI-MS: 546 [M+H]+. UV
(EtOH), ꢈmax , nm (lg ꢉ): 212 (4.39), 263 (4.16).
4.2.5. General procedure for the preparation of conjugates
10-13
A solution of lipid phosphoramidite 6-9 (0.39 mmol) in
CH2Cl2 (2 mL) and 0.45 M solution of tetrazole (1.70 mmol) in
CH3CN (3.8 mL) were added to the solution of nucleoside 5
(0.25 mmol) in CH2Cl2 (2 mL) under argon. After stirring for 24
h, a solution of I2 (0.39 mmol) in Py/H2O/CH2Cl2 (3:1:1, v/v/v)
was added. The mixture was diluted with CHCl3 (200 mL) and
extracted with 2% solution of sodium thiosulfate in brine (60
mL), the organic layer was dried (Na2SO4) and evaporated. The
residue was purified by column chromatography (silica gel, 100
cm3) using a gradient of CH3OH (0ꢆ6.25%) in CHCl3. The
compounds 10-13 were isolated in yields ranging from 82% to
88%.
4.2.2. 2-Chloro-9-[2-deoxy-2-fluoro-5-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]adenine,
3
1H NMR (DMSO-d6): ꢄ 8.14 (s, 1ꢇ, ꢇ-8), 7.90 (br s, 2ꢇ,
NH2), 6.35 (dd, 1ꢇ, J1’,2’ 4.5 Hz, J1’,F 13.0 Hz, ꢇ-1’), 6.14 (s, 1ꢇ,
SiOH), 6.06 (d, 1ꢇ, Jꢁꢇ, 3’ 5.5 Hz, 3’-ꢁꢇ), 5.27 (m, 1ꢇ, J2’,F 52.5
Hz, ꢇ-2’), 4.49 (m, 1ꢇ, J3’,F 19.5 Hz, ꢇ-3’), 3.98 (m, 2ꢇ, ꢇ-5’,
ꢇ-5”), 3.92 (m, 1ꢇ, ꢇ-4’), 1.03-0.78 (m, 28ꢇ, 4[i-Pr]). 13C NMR
(DMSO-d6): ꢄ 156.77, 153.30, 150.12, 139.62, 117.35 (Ade),
95.29 (d, JC,F 193.0 Hz, C(2’)), 82.61 (d, J 6.0 Hz, C(4’)), 81.34
(d, JC,F 17.0 Hz, C(1’)), 72.60 (d, JC,F 23.0 Hz, C(3’)), 61.66
(C(5’)), 17.19, 17.18, 17.08, 17.05, 17.02, 12.95, 12.37, 12.33 (i-
4.2.6. {2-Chloro-9-[2-deoxy-2-fluoro-3-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]-
adenine}-5’-[(1,2-di-ꢀ-myristoyl-sn-glycer-3-yl)-(2-
cyanoethyl)phosphate], 10, a mixture of diastereomers
1H NMR (CDCl3): ꢄ 8.14 (d, J 3.0 Hz, ꢇ-8), 8.11 (d, J 3.0
Hz, ꢇ-8;), 6.58 (dd, J1’,2’ 3.0 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.52 (dd,
J1’,2’ 3.0 Hz, J1’,F 22.0 Hz, ꢇ-1’), 6.15 (br s, NH2), 5.24 (m, ꢇ-2
Gly), 5.11 (m, J2’,F 51.0 Hz, ꢇ-2’), 4.81 (m, J3’,F 18.5 Hz, ꢇ-3’),
4.69 (br s, Si-OH), 4.42-4.15 (m, ꢇ-1 Gly, ꢇ-3 Gly, ꢇ-4’, H-5’,
H-5”, ꢃH2ꢃH2CN), 2.77 (m, ꢃꢇ2ꢃꢁ2ꢃN), 2.33-2.31 (m,
Pr). 19F NMR (DMSO-d6):
ꢄ -198.28. Anal. calcd for
C22H39ClFN5O5Si2 (564.198), %: ꢃ 46.83, ꢇ 6.97, N 12.41;
found, %: ꢃ 47.14, H 6.96, N 11.88. ESI-MS: 564 [M+H]+. UV
(EtOH), ꢈmax , nm (lg ꢉ): 212 (4.35), 263 (4.13).

ꢃꢇ3(CH2)11CH2CO), 1.59 (m, ꢃꢇ3(CH2)10CH2CH2CO), 1.30-
1.25 (m, ꢃꢇ3(CH2)10C2H4CO), 1.09-0.95 (m, i-Pr), 0.88 (t,
ꢃꢁ3(CH2)12CO). 13C NMR (CDCl3): ꢄ 173.33 (CH2OC(O)),
172.93 (CHOC(O)), 156.11, 154.40, 150.70, 140.53 (Ade: C(6),
C(2), C(4), C(8)), 117.99, 116.16 (C(5), ꢃꢇ2ꢃꢇ2ꢂN), 94.47 (d,
JC,F 193.0 Hz, C(2’)), 84.50 (m, C(4’)), 83.76 (d, JC,F 16.5 Hz,
C(1’)), 83.70 (d, JC,F 16.0 Hz, C(1’)), 75.08 (d, JC,F 26.0 Hz,
C(3’)), 69.23 (d, J 7.0 Hz, C-2 Gly), 66.85 (br s, C(5’)), 66.33 (d,
ꢂꢇ2ꢃꢇ2ꢃN), 66.27 (d, ꢂꢇ2ꢃꢇ2ꢃN), 62.42, 61.50 (C-1 Gly, C-3
Gly), 34.13, 34.00 (CH2CO), 31.92, 29.70, 29.49, 29.37, 29.29,
29.13, 29.09, 24.93, 24.82, 22.66 (CH3(CH2)11CH2CO), 19.64 (d,
ꢃꢇ2ꢂꢇ2ꢃN), 17.29, 17.24, 17.16, 17.10, 14.10, 13.41, 12.99,
12.80 (i-Pr, ꢂꢇ3(CH2)12CO). 31P NMR (CDCl3): ꢄ -0.41 (m).
ESI-MS: [MH]+: 1192.
adenine}-5’-[(1,3-di-ꢀ-palmitoyl-sn-glycer-2-yl)-(2-
cyanoethyl)phosphate], 13, a mixture of diastereomers
1H NMR (CDCl3): ꢄ 8.14 (d, J 3.0 Hz, ꢇ-8), 8.13 (d, J 3.0 Hz,
ꢇ-8), 6.52 (dd, J1’,2’ 3.0 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.51 (dd, J1’,2’
3.0 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.11 (br s, NH2), 5.11 (m, J2’,F 51.0
Hz, ꢇ-2’), 4.83-4.68 (m, ꢇ-3’, SiOH), 4.45-4.15 (m, CH2CH2CN,
ꢇ-1 Gly, ꢇ-2 Gly, ꢇ-3 Gly, ꢇ-4’, H-5’, H-5”), 2.77 (m,
ꢃꢇ2ꢃꢁ2ꢃN), 2.33 (m, ꢃꢇ3(CH2)13CH2CO), 1.60 (m,
ꢃꢇ3(CH2)12CH2CH2CO), 1.25 (m, ꢃꢇ3(CH2)12C2H4CO), 1.09-
0.95 (m, i-Pr), 0.88 (t, ꢃꢁ3(CH2)14CO). 13C NMR (CDCl3): ꢄ
173.25 (CH2OC(O)), 156.08, 154.39, 150.68, 140.54 (Ade: C(6),
C(2), C(4), C(8)), 118.01, 116.10 (C(5), ꢃꢇ2ꢃꢇ2ꢂN), 94.45 (d,
JC,F 193.0 Hz, C(2’)), 94.40 (d, JC,F 191.5 Hz, C(2’)), 84.53 (m,
C(4’)), 83.83 (d, JC,F 15.5 Hz, C(1’)), 75.18, 74.97 (C(3’), C-2
Gly), 66.83 (m, C(5’)), 62.43 (m, C-1 Gly, C-3 Gly), 33.98,
33.95 (CH2CO), 31.94, 29.72, 29.50, 29.38, 29.30, 29.15, 24.80,
22.71 (CH3(CH2)13CH2CO), 19.59 (d, J 7.5 Hz, ꢃꢇ2ꢂꢇ2ꢃN),
17.30, 17.24, 17.17, 17.11, 14.10, 13.42, 13.00, 12.81 (i-Pr,
ꢂꢇ3(CH2)14CO). 31P NMR (CDCl3): ꢄ -0.40 (m). ESI-MS:
[MH]+: 1248.
4.2.7. {2-Chloro-9-[2-deoxy-2-fluoro-3-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]-
adenine}-5’-[(1,2-di-ꢀ-palmitoyl-sn-glycer-3-yl)-(2-
cyanoethyl)phosphate], 11, a mixture of diastereomers
1H NMR (CDCl3): ꢄ 8.14 (d, J 3.0 Hz, ꢇ-8), 8.11 (d, J 3.0 Hz,
ꢇ-8), 6.55 (dd, J1’,2’ 3.0 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.52 (dd, J1’,2’
3.0 Hz, J1’,F 22.0 Hz, ꢇ-1’), 6.03 (br s, NH2), 5.25 (m, ꢇ-2 Gly),
5.10 (m, J2’,F 51.0 Hz, ꢇ-2’), 4.80 (m, J3’,F 18.0 Hz, ꢇ-3’), 4.68
(br s, Si-OH), 4.42-4.14 (m, ꢇ-1 Gly, ꢇ-3 Gly, ꢇ-4’, H-5’, H-5”,
ꢃH2ꢃH2CN), 2.76 (m, ꢃꢇ2ꢃꢁ2ꢃN), 2.34-2.28 (m,
ꢃꢇ3(CH2)13CH2CO), 1.59 (m, ꢃꢇ3(CH2)12CH2CH2CO), 1.30-
1.25 (m, ꢃꢇ3(CH2)12C2H4CO), 1.09-0.95 (m, i-Pr), 0.88 (t,
ꢃꢁ3(CH2)14CO). 13C NMR (CDCl3): ꢄ 173.31 (CH2OC(O)),
172.91 (CHOC(O)), 156.09, 154.37, 150.68, 140.56 (Ade: C(6),
C(2), C(5), C(8)), 117.97, 116.13 (C(4), ꢃꢇ2ꢃꢇ2ꢂN), 94.44 (d,
JC,F 193.0 Hz, C(2’)), 85.56 (m, C(4’)), 83.78 (d, JC,F 16.0 Hz,
C(1’)), 83.71 (d, JC,F 16.0 Hz, C(1’)), 75.06 (d, JC,F 26.0 Hz,
C(3’)), 69.21 (d, J 7.0 Hz, C-2 Gly), 66.83 (br s, C(5’)), 66.32 (d,
ꢂꢇ2ꢃꢇ2ꢃN), 66.27 (d, ꢂꢇ2ꢃꢇ2ꢃN), 62.42, 61.50 (C-1 Gly, C-3
Gly), 34.13, 33.98 (CH2CO), 31.92, 29.70, 29.50, 29.36, 29.29,
29.13, 29.09, 24.93, 24.82, 22.68 (CH3(CH2)13CH2CO), 19.61 (d,
J 7.0 Hz, ꢃꢇ2ꢂꢇ2ꢃN), 17.32, 17.26, 17.19, 17.13, 14.13, 13.43,
13.02, 12.83 (i-Pr, ꢂꢇ3(CH2)14CO). 31P NMR (CDCl3): ꢄ -0.41
(m). ESI-MS: [MH]+: 1248.
4.2.10. General procedure for the preparation of conjugates
14-17
A solution of diacylglycerophosphates of clofarabine 10-13
(0.30 mmol) in Py/Et3N mixture (1:1, v:v; 6 mL) was stirred for
24 h and evaporated. The residue was evaporated with toluene (2
mL), dissolved in THF (4 mL), and 1M TBAF in THF (0.75 mL)
was added to the obtained solution. After stirring for 4 h, the
reaction mixture was evaporated to dryness. The residue was
evaporated with toluene (2 mL) and purified on silica gel column
(50 cm3) using a gradient of CH3OH (0ꢆ20% + 1.5% (C2H5)3N)
in CHCl3. The residues after evaporation of the appropriate
fractions were dissolved in CHCl3/MeOH (30 mL), H2O (6 mL)
was added, mixed well, and pH of the aqueous layer was adjusted
to 1. The organic layer was separated and methanolic NaOH (0.1
M, 1.0 molar equiv.) was added, the mixture was evaporated
under reduced pressure to yield the sodium salts 14, 15, 16, and
17 (60, 97, 76, and 73%, correspondingly).
4.2.8. {2-Chloro-9-[2-deoxy-2-fluoro-3-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]-
adenine}-5’-[(1,3-di-ꢀ-myristoyl-sn-glycer-2-yl)-(2-
cyanoethyl)phosphate], 12, a mixture of diastereomers
4.2.11. [2-Chloro-9-(2-deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-
adenine]-5’-(1,2-di-ꢀ-myristoyl-sn-glycer-3-yl)phosphate,
sodium salt, 14
1H NMR (DMSO-d6): ꢄ 8.26 (s, 1ꢇ, ꢇ-8), 7.89 (br s, 2ꢇ,
NH2), 6.36 (br s, 1ꢇ, 3’-ꢁꢇ), 6.31 (m, 1ꢇ, J1’,2’ 4.0 Hz, J1’,F 13.0
Hz, ꢇ-1’), 5.22 (m, 1ꢇ, J2’,F 52.5 Hz, ꢇ-2’), 5.05 (m, 1ꢇ, ꢇ-2
Gly), 4.44 (m, 1ꢇ, J3’,F 19.0 Hz, ꢇ-3’), 4.27 (dd, 1ꢇ, J1,2 12.0 Hz,
ꢇ-1 Gly), 4.05 (dd, 1ꢇ, J1,2 6.5 Hz, J1,1 12.0 Hz, ꢇ-1 Gly), 3.94
(m, 1ꢇ, ꢇ-4’), 3.88 (m, 2ꢇ, ꢇ-5’, ꢇ-5”), 3.74 (m, 2ꢇ, 2ꢇ-3 Gly),
2.23 (m, 2ꢇ, ꢃꢇ3(CH2)11CH2CO), 2.21 (m, 4ꢇ,
ꢃꢇ3(CH2)11CH2CO), 1.46 (m, 4ꢇ, 2 ꢃꢇ3(CH2)10CH2CH2CO),
1.28–1.15 (m, 40ꢇ, 2 ꢃꢇ3(CH2)10C2H4CO), 0.85 (t, 6ꢇ, 2
ꢃꢁ3(CH2)12CO). 13C NMR (DMSO-d6): ꢄ 172.53 (CH2OC(O)),
172.27 (CHOC(O)), 156.78, 153.28, 150.16, 139.91, 117.31
(Ade), 94.95 (d, JC,F 193.0 Hz, C(2’)), 81.89 (C(4’)), 81.29 (d,
JC,F 16.0 Hz, C(1’)), 73.03 (d, JC,F 25.0 Hz, C(3’)), 70.41 (C-2
Gly), 63.59 (d, JC,P 2.5 Hz, C(5’)), 62.64 (C-3 Gly), 62.29 (C-1
Gly), 33.56, 33.38 (CH2CO), 31.31 (CH3CH2CH2), 29.05, 28.93,
28.73, 28.43, 28.42 (CH3(CH2)2(CH2)8(CH2)2CO), 24.44, 24.40
(CH2CH2CO), 22.10 (CH3CH2), 13.93 (CH3). 31P NMR (DMSO-
d6): ꢄ -0.62. Anal. calcd for C41H69ClFN5NaO10P‡ꢇ2ꢁ, (918.444),
%: C 53.62, H 7.79, N 7.63; found, %: C 53.46, H 7.99, N 7.07.
ESI-MS: [MH]+: 900.
1H NMR (CDCl3): ꢄ 8.14 (d, J 3.0 Hz, ꢇ-8), 8.13 (d, J 3.0
Hz, ꢇ-8), 6.52 (dd, J1’,2’ 2.5 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.51 (dd,
J1’,2’ 2.5 Hz, J1’,F 22.5 Hz, ꢇ-1’), 6.44 (br s, NH2), 5.12 (m, J2’,F
51.0 Hz, ꢇ-2’), 4.82-4.77 (m, ꢇ-3’, SiOH), 4.45-4.19 (m,
CH2CH2CN, ꢇ-1 Gly, ꢇ-2 Gly, ꢇ-3 Gly, ꢇ-4’, H-5’, H-5”), 2.77
(m, ꢃꢇ2ꢃꢁ2ꢃN), 2.33 (m, ꢃꢇ3(CH2)11CH2CO), 1.60 (m,
ꢃꢇ3(CH2)10CH2CH2CO), 1.26 (m, ꢃꢇ3(CH2)10C2H4CO), 1.09-
0.95 (m, i-Pr), 0.88 (t, ꢃꢁ3(CH2)12CO). 13C NMR (CDCl3): ꢄ
173.23 (CH2OC(O)), 156.20, 154.36, 150.56, 140.46 (Ade, C(6),
C(2), C(4), C(8)), 117.87, 116.09 (C(5), ꢃꢇ2ꢃꢇ2ꢂN), 94.41 (d,
JC,F 192.5 Hz, C(2’)), 94.36 (d, JC,F 193.0 Hz, C(2’)), 84.51 (m,
C(4’)), 83.80 (d, JC,F 15.5 Hz, C(1’)), 75.15, 74.93 (C(3’), C-2
Gly), 66.80 (m, C(5’)), 62.41 (m, C-1 Gly, C-3 Gly), 33.95,
33.93 (CH2CO), 31.89, 29.62, 29.44, 29.32, 29.25, 29.10, 24.75,
22.65 (CH3(CH2)11CH2CO), 19.55 (d, J 7.0 Hz, ꢃꢇ2ꢂꢇ2ꢃN),
17.27, 17.21, 17.09, 14.07, 13.39, 12.97, 12.78 (i-Pr,
ꢂꢇ3(CH2)12CO). 31P NMR (CDCl3): ꢄ -0.40 (m). ESI-MS:
[MH]+: 1192.
4.2.9. {2-Chloro-9-[2-deoxy-2-fluoro-3-O-(1-hydroxy-1,1,3,3-
tetraisopropyldisiloxane-3-yl)-ꢀ-D-arabinofuranosyl]-
4.2.12. [2-Chloro-9-(2-deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-
adenine]-5’-(1,2-di-ꢀ-palmitoyl-sn-glycer-3-yl)phosphate,

sodium salt, 15
956.
1H NMR (DMSO-d6): ꢄ 8.22 (s, 1ꢇ, ꢇ-8), 7.84 (br s, 2ꢇ,
NH2), 6.34 (br s, 1ꢇ, 3’-ꢁꢇ), 6.30 (dd, 1ꢇ, J1’,2’ 4.5 Hz, J1’,F 13.5
Hz, ꢇ-1’), 5.21 (dt, 1ꢇ, J2’,F 53.0 Hz, ꢇ-2’), 5.04 (m, 1ꢇ, ꢇ-2
Gly), 4.44 (m, 1ꢇ, J3’,F 18.5 Hz, ꢇ-3’), 4.28 (dd, 1ꢇ, J1,2 3.0 Hz,
J1,1 12.0 Hz, ꢇ-1 Gly), 4.06 (dd, 1ꢇ, J1,2 7.0 Hz, J1,1 12.0 Hz, ꢇ-1
Gly), 3.93 (m, 1ꢇ, ꢇ-4’), 3.88 (m, 2ꢇ, ꢇ-5’, ꢇ-5”), 3.74 (m, 2ꢇ,
2ꢇ-3 Gly), 2.23 (m, 2ꢇ, ꢃꢇ3(CH2)13CH2CO), 2.20 (m, 2ꢇ,
ꢃꢇ3(CH2)13CH2CO), 1.47 (m, 4ꢇ, 2ꢃꢇ3(CH2)12CH2CH2CO),
1.28–1.15 (m, 48ꢇ, 2ꢃꢇ3(CH2)12C2H4CO), 0.85 (t, 6ꢇ,
2ꢃꢁ3(CH2)14CO). 13C NMR (DMSO-d6): ꢄ 172.37 (CH2OC(O)),
172.11 (CHOC(O)), 156.69, 153.20, 150.10, 139.75, 117.26
(Ade), 94.85 (d, JC,F 193.0 Hz, C(2’)), 81.87 (d, J 4.0 Hz, C(4’)),
81.20 (d, JC,F 17.0 Hz, C(1’)), 73.14 (d, JC,F 22.0 Hz, C(3’)), 70.42
(d, J 8.0 Hz, C-2 Gly), 63.42 (br s, C(5’)), 62.50 (d, J 5.0 Hz, C-3
Gly), 62.27 (C-1 Gly), 33.51, 33.33 (CH2CO), 31.19
Acknowledgments
The authors are grateful for the financial support from the
National Academy of Sciences of Belarus within the research
project number 4.19 of ChemPharmSynthesis Program.
The authors are greatly indebted to Dr. G.G. Sivets for
providing
2-chloro-9-(2-deoxy-2-fluoro-ꢀ-D-
arabinofuranosyl)adenine (clofarabine).
References
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Galmarini, C.M.; Mackey, J.R.; Dumontet, C. Lancet Oncol.,
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2
3
Jones, R. J.; Bischofberger, N. Antiviral Res, 1995, 27, 1.
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(CH3CH2CH2),
28.94,
28.81,
28.60,
28.33
(CH3(CH2)2(CH2)10(CH2)2CO), 24.35, 24.30 (CH2CH2CO), 21.98
(CH3CH2), 13.80 (CH3). 31P NMR (DMSO-d6): ꢄ -0,62. Anal.
calcd for C45H77ꢃlFN5NaO10P‡ꢇ2ꢁ, (974.551), %: C 55.46, H
8.17, N 7.19; found, %: C 55.56, H 8.19, N 7.01. ESI-MS:
[MH]+: 956.
4
5
6
7
8
9
MacCoss, M.; Ryu, E.K.; Matsushita, T. Biochem. Biophys.
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Ryu, E.K.; Ross, R.J.; Matsushita, T.; MacCoss, M.; Hong,
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Maccoss, M.; Edwards, J.J.; Seed, T.M.; Spragg, S.P.
Biochim. Biophys. Acta, 1982, 719, 544.
4.2.13. [2-Chloro-9-(2-deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-
adenine]-5’-(1,3-di-ꢀ-myristoylglycer-2-yl)phosphate, sodium
salt, 16
Rahman, Y.E.; Patel, K.R.; Cerny, E.A.; Maccoss, M. Eur. J.
Cancer Clin. Oncol., 1984, 20, 1105.
Hong, C.I.; Kirisits, A.J.; Buchheit, D.J.; Nechaev, A.; West,
C.R. Canc. Drug Deliv., 1986, 3, 101.
1H NMR (DMSO-d6): ꢄ 8.22 (s, 1ꢇ, ꢇ-8), 7.88 (br s, 2ꢇ,
NH2), 6.32 (dd, 1ꢇ, J1’,2’ 5.0 Hz, J1’,F 14.0 Hz, ꢇ-1’), 5.23 (dt,
1ꢇ, J2’,F 53.0 Hz, ꢇ-2’), 4.42 (m, 1ꢇ, J3’,F 17.0 Hz, ꢇ-3’), 4.30
(m, 1H, ꢇ-2 Gly), 4.08 (m, 4ꢇ, 2ꢇ-1 Gly, 2H-3 Gly), 3.97 (m,
1ꢇ, ꢇ-4’), 3.91 (m, 2ꢇ, ꢇ-5’, ꢇ-5”), 2.23 (m, 4ꢇ,
2ꢃꢇ3(CH2)11CH2CO), 1.44 (m, 4ꢇ, 2ꢃꢇ3(CH2)10CH2CH2CO),
1.22–1.18 (m, 40ꢇ, 2ꢃꢇ3(CH2)10C2H4CO), 0.84 (t, 6ꢇ,
2ꢃꢁ3(CH2)12CO). 13C NMR (DMSO-d6): ꢄ 172.37 (CH2OC(O)),
156.65, 153.15, 150.02, 139.72, 117.19 (Ade), 95.30 (d, JC,F
192.0 Hz, C(2’)), 81.83 (br s, C(4’)), 81.28 (d, JC,F 17.0 Hz,
C(1’)), 72.69 (d, JC,F 23.0 Hz, C(3’)), 69.41 (d, J 6.0 Hz, C-2
Gly), 63.62 (C(5’)), 62.97, 62.91 (C-1 Gly, C-3 Gly), 33.21
(CH2CO), 31.17 (CH3CH2CH2), 28.23, 28.03, 28.77, 28.69, 28.32
(CH3(CH2)2(CH2)8(CH2)2CO), 24.24 (CH2CH2CO), 21.97
(CH3CH2), 13.79 (CH3). 31P NMR (DMSO-d6): ꢄ -0,62. Anal.
calcd for C41H69ClFN5NaO10P‡ꢇ2ꢁ, (918.444), %: C 53.62, H
7.79, N 7.63; found, %: C 53.56, H 8.03, N 7.67. ESI-MS:
[MH]+: 900.
Hong, C.I.; West, C.R.; Bernacki, R.J.; Tebbi, C.K.; Berdel,
W.E. Lipids, 1991, 26, 1437.
10 Schwendener, R.A.; Supersaxo, A.; Rubas, W.; Weder, H.C.;
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20 Heckl-Östreicher, G.; Müller, C.; Lutz, C.; Kulke, M.;
Obermeier, J.; Minchinton, A.; Wehr, R.; Anderl, J. In:
Proceedings of the 100th Annual Meeting of AACR 2009, Apr
18-22; Denver, CO. Philadelphia (PA): AACR; 2009.
Abstract Nr 4523.
4.2.14. [2-Chloro-9-(2-deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-
adenine]-5’-(1,3-di-ꢀ-palmitoylglycer-2-yl)phosphate, sodium
salt, 17
1H NMR (DMSO-d6): ꢄ 8.21 (s, 1ꢇ, ꢇ-8), 7.89 (br s, 2ꢇ,
NH2), 6.31 (dd, 1ꢇ, J1’,2’ 5.0 Hz, J1’,F 14.0 Hz, ꢇ-1’), 5.21 (dt, 1ꢇ,
J2’,F 53.0 Hz, ꢇ-2’), 4.44 (m, 1ꢇ, J3’,F 19.0 Hz, ꢇ-3’), 4.32 (m,
1H, ꢇ-2 Gly), 4.08 (m, 4ꢇ, 2ꢇ-1 Gly, 2H-3 Gly), 3.95 (m, 1ꢇ,
ꢇ-4’), 3.93 (m, 2ꢇ, ꢇ-5’, ꢇ-5”), 2.24 (m, 4ꢇ,
2ꢃꢇ3(CH2)13CH2CO), 1.46 (m, 4ꢇ, 2ꢃꢇ3(CH2)12CH2CH2CO),
1.27–1.19 (m, 48ꢇ, 2ꢃꢇ3(CH2)12C2H4CO), 0.85 (t, 6ꢇ,
2ꢃꢁ3(CH2)14CO). 13C NMR (DMSO-d6): ꢄ 172.49 (CH2OC(O)),
156.77, 153.28, 150.15, 139.85, 117.31 (Ade), 94.89 (d, JC,F
192.0 Hz, C(2’)), 81.92 (br s, C(4’)), 81.34 (d, JC,F 17.0 Hz,
C(1’)), 73.11 (d, JC,F 23.0 Hz, C(3’)), 69.53 (d, J 6.0 Hz, C-2
Gly), 63.74 (C(5’)), 62.99, 62.93 (C-1 Gly, C-3 Gly), 33.33
(CH2CO), 31.30 (CH3CH2CH2), 29.01, 29.04, 28.89, 28.71, 28.44
(CH3(CH2)2(CH2)10(CH2)2CO), 24.36 (CH2CH2CO), 22.09
(CH3CH2), 13.91 (CH3). 31P NMR (DMSO-d6): ꢄ -0,62. Anal.
calcd for C45H77ꢃlFN5NaO10P‡ꢇ2ꢁ, (974.551), %: C 55.46, H
8.17, N 7.19; found, %: C 55.34, H 8.29, N 7.35 ESI-MS: [MH]+:
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2000, 41, 9541.
24 Sivets, G.G.; Boghok, T.B.; Kalinichenko, E.N. In: IRT 2010
- XIX International Roundtable on Nucleosides, Nucleotides
and Nucleic Acids: Abstracts, Aug. 29 - 3 Sept. 3, 2010,
Lyon, France, 92.

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