Synthesis and evaluation of fatty acyl ester derivatives of cytarabine as anti-leukemia agents

Article abstract of DOI:10.1016/j.ejmech.2010.07.024

Cytarabine is a chemotherapeutic agent predominately used for the treatment of acute myeloid leukemia and lymphoblastic leukemia. Cytarabine is a polar nucleoside, has a short plasma half-life, and its use is associated with severe side effects. Fatty acyl derivatives of cytarabine were synthesized with the expectation to improve cellular uptake and generate derivatives with a longer duration of action. Multi-step protection and deprotection reactions of hydroxyl and amino groups and conjugation with a fatty acid (i.e., myristic acid and 12-thioethyldodecanoic acid) afforded 5′-O-substituted, 2′-O-substituted, and 2′,5′-disubstituted fatty acyl derivatives of cytarabine. 2′,5′-Dimyristoyl derivative of cytarabine was found to inhibit the growth of CCRF-CEM cells by approximately 76% at concentration of 1 μM after 96 h incubation.

Full text of DOI:10.1016/j.ejmech.2010.07.024

European Journal of Medicinal Chemistry 45 (2010) 4601e4608
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of fatty acyl ester derivatives of cytarabine as
anti-leukemia agents
*
Bhupender S. Chhikara, Deendayal Mandal, Keykavous Parang
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytarabine is a chemotherapeutic agent predominately used for the treatment of acute myeloid leukemia
and lymphoblastic leukemia. Cytarabine is a polar nucleoside, has a short plasma half-life, and its use is
associated with severe side effects. Fatty acyl derivatives of cytarabine were synthesized with the
expectation to improve cellular uptake and generate derivatives with a longer duration of action. Multi-
step protection and deprotection reactions of hydroxyl and amino groups and conjugation with a fatty
acid (i.e., myristic acid and 12-thioethyldodecanoic acid) afforded 50-O-substituted, 20-O-substituted, and
20,50-disubstituted fatty acyl derivatives of cytarabine. 20,50-Dimyristoyl derivative of cytarabine was
Received 12 May 2010
Received in revised form
12 July 2010
Accepted 13 July 2010
Available online 21 July 2010
Keywords:
Cytarabine
Leukemia
Fatty acids
Cellular uptake
Prodrug
found to inhibit the growth of CCRF-CEM cells by approximately 76% at concentration of 1
incubation.
mM after 96 h
Ó 2010 Elsevier Masson SAS. All rights reserved.
Nucleoside
1. Introduction
acidefatty acidecytarabine [8], cytarabine phosphate derivatives
[9], and 50-O-unsaturated fatty acid derivatives [10].
Cytarabine (cytosine arabinoside, 1-
b
-
D
-arabinofuranosyl-
ValCytarabine [7,11] and CP-4055 as amino acid and fatty acid
prodrugs of cytarabine, respectively, have been evaluated for the
treatment of leukemia and solid tumors. CP-4055, an elaidic acid
derivative of cytarabine (ara-C-50-elaidic acid ester), enhanced the
cellular uptake of ara-C in tumor cells possibly via passive diffusion
through the cellular membrane [12]. Repeated treatment with
CP-4055 in human leukemia and in solid tumor models in vivo
enhanced the antitumor effect [13]. CP-4055 [14] is currently
undergoing phase 2 studies in patients with solid tumors under the
brand name ElacystÔ. Among other lipophilic derivatives, a simple
stearyl phosphate diester prodrug, cytarabine ocfosfate, was
approved in 1992 for leukemia treatment [15]. Phosphoramidate
cytosine, ara-C), a pyrimidine nucleoside analog, is predominantly
used against acute myelogenous leukemia and non-Hodgkin’s
lymphoma [1,2]. It is also used in combination with other anti-
cancer drugs for the treatment of leukemia and solid tumors [3,4].
Cytarabine is a polar nucleoside and has a short plasma half-life.
The low bioavailability of cytarabine is created by its low perme-
ability across membrane and rapid conversion into inactive 1-b-D-
arabinofuranosyluracil. Thus, continuous intravenous infusion of
higher doses is required to maintain constant plasma level of the
drug in 8e24 h. The higher doses of cytarabine lead to toxicity to
normal organs and side effects [5].
Recently there has been emphasis on the development of
cytarabine derivatives to obtain compounds with a higher thera-
peutic index for the treatment of leukemia and lymphoma. Among
the explored alternatives, the prodrug strategy by introducing
modifications on the parent drug to enhance plasma half-life or
delivery to cancer cells is a subject of major interest. These design
approaches included designing amino acidecytarabine [6,7], amino
derivative of cytarabine metabolite 2-
has also been evaluated [16].
b-D-arabinouridine (AraU)
The biological activities of synthesized conjugates depended
upon the rate of intracellular release of cytarabine. Once inside the
cells, cytarabineis convertedintocytarabinetriphosphate derivative
to show its cytotoxic effect. Cytarabine triphosphate is incorporated
into the growing DNA chain by DNA polymerases and leads to the
termination of chain elongation process of DNA synthesis [17]. The
phosphorylation process occurs on the 50-hydroxyl group of the
arabinose sugar. Prodrug strategies used either the substitution at
amine group of cytosine base or the protected 50-monophosphate
* Corresponding author. Tel.: þ1 401 874 4471; fax: þ1 401 874 5787.
E-mail address: kparang@uri.edu (K. Parang).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.024

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derivatives have resulted in compromised activities against various
cancers and generally leading to reduced anticancer activity [15].
Alternatively, cytarabine encapsulation in delivery systems [18]
like nanoparticles [19], chitosan [20], hydrogels [21], niosome
vesicles of tween 20 [22,23], and liposomes [24] has been explored.
Liposomal cytarabine was approved by the FDA for the treatment of
lymphomatous meningitis [25]. Lipophilic liposomal encapsulated
formulation of cytarabine passes across the blood brain barrier,
enhances delivery of the drug, and thus therapeutic effect. Our
earlier efforts of designing lipophilic antifungal and antiviral agents
indicated that designed lipophilic prodrugs, by conjugating of
parent analogs with fatty acid derivatives, led to enhanced bio-
logical activities of the respective parent drugs [26e30]. Herein, we
report the synthesis of lipophilic fatty acyl derivatives of cytarabine
with the expectation to improve the cellular uptake. Furthermore,
intracellular hydrolysis of the conjugate may result in the sustained
release of cytarabine and increasing the duration of biological
activity. Three classes of 50-O-substituted, 20-O-substituted, and
20,50-disubstituted fatty acyl derivatives of cytarabine were
synthesized. The anti-leukemia activity of compounds were eval-
uated and compared in CCRF-CEM cells.
20-hydroxyl group prevents the proximity of bulky HBTU-activated
fatty acid. On the other hand, smaller size acyl chloride was able to
form a conjugate ester with 20 hydroxyl group to yield 11. Dimyr-
istoyl substituted cytarabine 13 was obtained from 11 by depro-
tection of N4-DMTr and 30-O-TBDMS groups with acetic acid/TFA
and TBAF, respectively (Scheme 2).
2.1.3. Synthesis of 20-O-fatty acyl derivatives of cytarabine
Similar to the 20,50-disubstitution, the fatty acyl chloride was
chosen to achieve the esterification at 20-hydroxyl group. Reaction
of 3 with the fatty acyl chloride in the presence of DMAP afforded
14 and 15. Excess of DMAP base was maintained during the
coupling (esterification) reaction with 3 to avoid cleavage of DMTr
because of the formation of HCl during the reaction. The DMTr
group was removed by using acetic acid and 1e2% trifluoroacetic
acid. While removing the DMTr group from product 14, the
50-TBDMS group was also cleaved as indicated by the observed MS
peak at 568 [M þ 1]þ to generate 16. Similar results were observed
during deprotection of 15 [32]. Compounds 16 and 17 were treated
with TBAF in THF to afford 20-O-(fatty acyl) ester derivatives of
cytarabine (18 and 19) (Scheme 3). Myristoyl chloride is commer-
cially available. 12-Thioethyldodecanoic acid was synthesized from
12-bromododecanoic acid and thioethanol as described previously
[33]. 12-Thioethyldodecanoic acid was treated with oxalyl chloride
under nitrogen atmosphere to afford crude 12-thioethyldodecanoyl
chloride in situ after removal of excess oxalyl chloride and was
immediately used in the esterification reaction with 3.
2. Results and discussion
2.1. Chemistry
Three classes of 50-O-substituted, 20-O-substituted, and 20,50-
disubstituted fatty acyl derivatives of cytarabine were synthesized
from cytarabine (1) using multi-step protection and deprotection
reactions. Two fatty acids, myristic acid and 12-thio-
ethyldodecanoic acid, were used for the conjugation with
cytarabine.
2.1.4. Purification and characterization
The final products were purified on a reverse phase high
performance liquid chromatography (HPLC) using a C18 column.
The chemical structures of the synthesized fatty acyl derivatives of
cytarabine and intermediate products were confirmed with mass-
spectrometry (MS) and/or nuclear magnetic resonance (NMR). The
purity of the final products (9, 10, 13, 18, 19) was in the range of
95.0e99.1% as determined by the analytical HPLC using water/
acetonitrile as eluents with a flow rate of 1 mL/min.
2.1.1. Synthesis of 50-O-fatty acyl derivatives of cytarabine
Scheme 1 depicts the synthesis of 50-O-fatty acyl ester deriva-
tives of cytarabine. Subsequent protection of 50- and 30-hydroxyl
groups with tert-butyldimethylsilyl chloride (TBDMS-Cl, 2.5 equiv)
in the presence of imidazole as a base afforded 50,30-TBDMS-pro-
tected cytarabine (2) in a controlled reaction. The 20-hydroxyl
group of cytarabine was unaffected, but using a larger amount of
TBDMS-Cl led to protection of all three hydroxyl groups. 4-Amino
group of 2 was protected with the 4,40-dimethoxytrityl (DMTr)
protecting group in the presence of DMTr-Cl and pyridine to afford
3. Selective deprotection of 50-O-TBDMS group in 3 in the presence
of 0.1 Methanolic potassium hydroxide solution [31] generated 4.
The potassium salt was neutralized with dilute acetic acid. The
neutralization was carried out carefully in a controlled fashion and
addition of excess acetic acid was avoided, thus highly acid labile
DMTr group remained intact under these conditions. Conjugation
reaction of 4 with the fatty acid (i.e., myristic acid and 12-thio-
ethyldodecanoic acid) in the presence of 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hyd-
roxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIPEA)
afforded protected fatty acyl derivatives of cytarabine, 5 and 6.
Deprotection of 4-amino and 30-hydroxyl groups in the presence of
acetic acid and tetrabutylammonium fluoride (TBAF), respectively,
generated 50-O-(fatty acyl) ester derivatives of cytarabine 9 and 10.
2.2. Biological activity
The effect of the compounds (1
leukemia cells was evaluated in CCRF-CEM cell line after 24 h and
96 h (Fig. 1). Cytarabine (1 M) and DMSO were used as the positive
mM) on the cell proliferation of
m
and negative controls, respectively [34,35].
The cytotoxic mechanism of cytarabine involves phosphory-
lation of 50-hydroxyl into triphosphate, which on incorporation in
nucleotide chain synthesis results into termination of chain
elongation and thus inhibition of ribonucleotide synthesis and
ultimately cell division inhibition [36]. The effectiveness of the
cytarabine and cytarabine derivatives depends on the concen-
tration of drug delivered intracellularly and availability of 50-
hydroxyl group. 50-Monosubstituted fatty acyl derivatives of
cytarabine (9 and 10) were not able to inhibit significantly the
cell proliferation even after 96 h. The cell proliferation inhibitory
activity at 24 h and after 96 h was not significantly different for
both derivatives, suggesting that the compounds were probably
stable intracellularly and did not release cytarabine effectively.
Because of high lipophilicity, these compounds were expected to
have enhanced cellular uptake. Thus, the lack of inhibitory
activity is postulated to be because of limited intracellular
hydrolysis of 50-ester.
2.1.2. Synthesis of 20,50-dimyristoyl derivative of cytarabine
The synthesis of 20,50-dimyristoyl derivative of cytarabine was
initiated with the reaction of 4 with an excess amount of myristoyl
chloride in the presence of 4-dimethylaminopyridine (DMAP) to
generate 11. When the coupling reaction of 4 with myristic acid was
carried out using HBTU and HOBt, only the 50-monosubstituted
analog was obtained possibly because the sterical hindrance at
On the other hand, 20,50-dimyristoyl derivative of cytarabine (13)
and 20-fatty acyl derivatives of cytarabine (18 and 19) were able to
inhibit the growth of cancer cells by approximately 36e76% at
a concentration of 1 m
M after 96 h incubation. The 20-fatty acyl

B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
4603
Scheme 1. Synthesis of 50-O-fatty acyl derivatives of cytarabine.
derivatives (18 and 19) showed better inhibitory activity when
compared to 50-fatty acyl derivatives (9 and 10) probably because of
readily available 50-hydroxyl for phosphorylation and eventual
cytotoxic activity.
20,50-Disubstituted derivative 13 showed comparable activity to
that of parent drug 1 and physical mixtures after 96 h and signifi-
cantly higher activity when compared with less lipophilic mono-
substituted analogs, 9,10,18, and 19. The inhibitory activity for 13 at
Scheme 2. Synthesis of 20,50-dimyristoyl derivative of cytarabine.

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B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
Scheme 3. Synthesis of 20-O-fatty acyl derivatives of cytarabine.
a
120
100
80
60
40
20
0
DMSO
1
1 + Myr. 1 + 12-
Acid Thio.
Acid
9
10
13
18
19
120
100
80
60
40
20
0
b
DMSO
1
1 + Myr. 1 + 12-
Acid Thio. Acid
9
10
13
18
19
Fig. 1. Inhibition of CCRF-CEM cell proliferation by the compounds (1
mM) and cytarabine (1 mM) after (a) 24 h and (b) 96 h. The results are shown as the percentage of the control
that has no compound (set at 100%). All the experiments were performed in triplicate.

B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
4605
24 h and 96 h suggests that the intracellular hydrolysis of the
prodrug to cytarabine leads to improved antiproliferative activity
after longer incubation period. Enhanced activities of 13, 18, and 19
after 96 h compared to 24 h indicate that the conjugates release
cytarabine slowly and may behave as a prodrug for sustained
delivery of the parent nucleoside. Although the anticancer activities
of these compounds were only comparable to that of cytarabine
after 96 h, their sustained release effect may be beneficial similar to
liposomal cytarabine. Furthermore, these prodrugs are expected to
have higher cellular uptake when compared to cytarabine, because
of the presence of lipophilic fatty acyl chains.
(0e10%) as the eluents to afford product 2 (178 mg, 92%). 1H NMR
(CD3OD, 400 MHz,
d
ppm): 7.71 (d, J ¼ 10 Hz, 1H, H-6), 6.20 (d,
J ¼ 5 Hz, 1H, H-10), 5.86 (d, J ¼ 10 Hz, 1H, H-5), 4.31 (dd, J ¼ 5 Hz,
J ¼ 5 Hz,1H, H-20), 4.09 (t, J ¼ 5 Hz,1H, H-30), 3.93e3.97 (m,1H, H-40),
3.85e3.92 (m, 2H, H-50), 0.96 (s, 9H, SiC(CH3)3), 0.83 (s, 9H, SiC
(CH3)3), 0.15 (s, 3H, SiCH3), 0.13 (s, 3H, SiCH3), 0.11 (s, 3H, SiCH3),
ꢁ0.05 (s, 3H, SiCH3); 13C NMR (CD3OD, 100 MHz,
d ppm): 161.93 (C-
4), 149.38 (C-2), 147.18 (C-6), 93.84 (C-5), 88.17 (C-10), 86.71 (C-40),
77.42 (C-30), 77.06 (C-20), 63.01 (C-50), 26.65, 26.38 (2 ꢀ SiC(CH3)3),
19.16, 18.97 (2 ꢀ SiC(CH3)3), ꢁ4.07, ꢁ4.41, ꢁ5.08, ꢁ5.13 (2 ꢀ Si
(CH3)2); HR-MS (ESI-TOF) (m/z): calcd for C21H41N3O5Si2: 471.2585;
found, 472.2620 [M þ 1]þ, 943.5228 [2M þ 1]þ.
3. Conclusions
4.2.2. 1-[30,50-Di(O-tert-butyldimethylsilyl)-N-(4,40-
Three classes of fatty acyl derivatives of cytarabine were
synthesized as fatty acyl prodrugs and evaluated for the inhibition
of CCRF-CEM cell proliferation. The data indicate that designing
molecules with comparable anti-leukemia activity to cytarabine
with sustained release effect is possible by structure modification.
A number of compounds may have potential application for slow
delivery of cytarabine. Further optimization is required to generate
lead compounds with improved cell proliferation inhibition versus
cytarabine.
dimethoxytrityl)-b-D-arabinofuranosyl]cytosine (3)
Compound 2 (170 mg, 0.36 mmol) was dissolved in dry pyridine
(5 mL). To the solution was added 4,40-dimethoxytrityl chloride
(DMTr-Cl, 166 mg, 0.49 mmol) dissolved in dry pyridine (10 mL)
dropwise at 0 ꢂC. The mixture was stirred for 4 h at 0 ꢂC and then for
an additional 14 h at room temperature. The solution was then
neutralized with aqueous sodium bicarbonate (5.0%) and extracted
with dichloromethane (3 ꢀ 50 mL). The organic layer was dried
with anhydrous sodium sulfate, and the solvent was removed
under reduced pressure. The crude compound was purified by
column chromatography over silica gel using hexane/dichloro-
methane containing 1.0% triethylamine as the eluents to yield 3
4. Experimental protocols
4.1. Materials and methods
(240 mg, 86%). 1H NMR (CDCl3, 500 MHz,
d
ppm): 7.82 (d, J ¼ 7.7 Hz,
1H, H-6), 7.40e7.53 (m, 5H, DMTr-Ar-H), 7.32 (br s, 2H, DMTr-Ar0-
C2-H), 7.31 (br s, 2H, DMTr-Ar0-C6-H), 7.01 (br s, 2H, DMTr Ar’-C5-
H), 7.00 (s, 2H, DMTr Ar’-C3-H), 6.28 (d, J ¼ 4.7 Hz,1H, H-10), 5.21 (d,
J ¼ 7.7 Hz, 1H, H-5), 4.46e4.53 (m, 1H, H-20), 4.27e4.32 (m, 1H, H-
30), 4.04e4.08 (m, 2H, H-50), 3.90 (s, 6H, 2 ꢀ OCH3), 3.86e3.92 (m,
1H, H-40), 1.07 (s, 9H, SiC(CH3)3), 0.97 (s, 9H, SiC(CH3)3), 0.31 (s, 3H,
Cytarabine, HBTU, HOBt, anhydrous dichloromethane, anhy-
drous pyridine, N,N-dimethylformamide (DMF) and other chem-
icals and reagents were purchased from SigmaeAldrich Chemical
Co. (Milwaukee, WI). 12-Thioethyldodecanoic acid was synthesized
from 12-bromododecanoic acid and thioethanol as described
previously [33]. HBTU and HOBt in N,N-dimethylformamide (DMF)
were used as coupling and activating reagents, respectively. The
chemical structures of final products were characterized by nuclear
magnetic resonance spectra (1H NMR, 13C NMR) determined on
a Bruker NMR spectrometer (400 MHz) or a Varian NMR spec-
trometer (500 MHz). 13C NMR spectra are fully decoupled. Chemical
shifts were reported in parts per million (ppm) using deuterated
solvent peak as the standard. The chemical structures of final
products were confirmed by a high-resolution Biosystems QStar
Elite time-of-flight electrospray mass spectrometer. Details of
procedures and spectroscopic data of the respective compounds are
presented below. Final compounds were purified on a Phenomenex
SiCH3), 0.27 (s, 3H, SiCH3), 0.20 (s, 3H, SiCH3), 0.17 (s, 3H, SiCH3); 13
NMR (CDCl3, 125 MHz, ppm): 165.63 (C-4), 158.80 (DMTr Ar-C4-
C
d
O), 156.22 (C-2), 144.73 (DMTr-C1-Ar), 142.04 (C-6), 136.44 (DMTr-
C1-Ar0-OCH3), 130.24, 130.13, 128.75, 128.41, 127.90, 127.52, 113.68
(DMTr-Ar carbons), 94.24 (C-5), 87.29 (C-10), 84.64 (C-40), 78.33 (C-
30), 75.86 (C-20), 70.43 (DMTr C-Ar3), 62.07 (C-50), 55.38 (DMTr Ar-
O-CH3), 26.03, 25.92 (TBDMS 2 ꢀ SiC(CH3)3), 18.37, 18.14 (TBDMS
2 ꢀ SiC(CH3)3), ꢁ4.16, ꢁ4.92, ꢁ5.47, ꢁ5.48 (TBDMS 4 ꢀ SiCH3); HR-
MS (ESI-TOF) (m/z): calcd for C42H59N3O7Si2: 773.3892; found,
774.0265 [M þ 1]þ.
4.2.3. 1-[30-O-(tert-Butyldimethylsilyl)-N-(4,40-dimethoxytrityl)-
-arabinofuranosyl]cytosine (4)
Compound 3 (200 mg, 0.26 mmol) was treated with 0.1 M
b-
Prodigy 10
m
m ODS reversed-phase column (2.1 cm ꢀ 25 cm) with
D
a Hitachi HPLC system using a gradient system of acetonitrile or
methanol and water (CH3CN/CH3OH/H2O, 0ꢁ100%, pH 7.0, 60 min).
The purity of final products (>99%) was confirmed by analytical
HPLC. The analytical HPLC was performed on the Hitachi analytical
solution of potassium hydroxide in ethanol (20 mL). The mixture
was stirred for 1 h at room temperature. After completion of
reaction, the solution was neutralized with 0.01% acetic acid in
ethanol to pH 7 at 0 ꢂC. The solvent was evaporated under
reduced pressure and the crude sticky product was purified on
column chromatography over silica gel using dichloromethane/
methanol containing 1.0% triethylamine as the eluents to yield 4
HPLC system using a C18 Shimadzu Premier 3
mm column
(150 cm ꢀ 4.6 mm) using two different isocratic systems, and a flow
rate of 1 mL/min with a UV detection at 265 nm.
4.2. Chemistry
(152 mg, 89%). 1H NMR (CD3OD, 400 MHz,
d ppm): 7.58 (d,
J ¼ 8.0 Hz, 1H, H-6), 7.25e7.40 (m, 5H, DMTr-Ar-H), 7.17 (br s, 4H,
DMTr-Ar0-C2-H, DMTr-Ar0-C6-H), 6.88 (br s, 4H, DMTr Ar’-C3-H,
DMTr Ar’-C5-H), 6.05e6.20 (m, 1H, H-10), 5.22 (d, J ¼ 8.0 Hz, 1H,
H-5), 4.08e4.13 (m, 2H, H-20, H-30), 3.81e3.83 (m, 1H, H-40), 3.79
(s, 6H, 2 ꢀ OCH3), 3.69e3.75 (m, 2H, H-50), 0.93 (s, 9H, SiC
4.2.1. 1-[30,50-Di(O-tert-butyldimethylsilyl)-
b-D-arabinofuranosyl]
cytosine (2)
tert-Butyldimethylsilyl chloride (TBDMS-Cl) (247 mg,1.64 mmol)
and imidazole (112 mg, 1.64 mmol) were added to a solution of
cytarabine (100 mg, 0.41 mmol) in dry DMF (20.0 mL). The reaction
mixture was stirred at room temperature for 14 h. After the
completion of the reaction, the solvent was removed under reduced
pressure, and the crude compound was purified by column
chromatography over silica gel using dichloromethane/methanol
(CH3)3), 0.13 (s, 6H, Si(CH3)2); 13C NMR (CD3OD, 100 MHz,
d,
ppm): 165.9 (C-4), 158.96, 158.68 (DMTr Ar-C4-O), 156.21 (C-2),
144.57 (DMTr-C1-Ar), 142.92 (C-6), 136.16, 136.07 (DMTr-C1-Ar-
OCH3), 130.13, 129.88, 129.79, 128.41, 127.93, 127.11, 113.19,
112.61, 112.37 (DMTr-Ar carbons), 94.42 (C-5), 87.23 (C-10), 86.21

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B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
(C-40), 78.12 (C-30), 75.41 (C-20), 70.13 (DMTr C-Ar3), 61.10 (C-50),
54.38 (DMTr Ar-O-CH3), 54.31 (DMTr Ar0-O-CH3), 24.96 (TBDMS
SiC(CH3)3), 17.14 (TBDMS SiC(CH3)3), ꢁ5.92, ꢁ6.18 (TBDMS
2 ꢀ SiCH3); HR-MS (ESI-TOF) (m/z): calcd for C36H45N3O7Si:
659.3027; found, 660.0500 [M þ 1]þ, 697.9557 [M þ K]þ,
1319.0863 [2M þ 1]þ.
removal of dichloromethane, the crude product was directly carried
forward to the next deprotection reaction. HR-MS (ESI-TOF) (m/z):
calcd for C50H71N3O8SSi: 901.4731; found, 902.1630 [M þ 1]þ.
4.2.8. 1-[30-O-(tert-Butyldimethylsilyl)-50-O-(12-
thioethyldodecanoyl)-b-D-arabinofuranosyl]cytosine (8)
Synthesis of 8 followed the same procedure as that of 7 as
described above. HR-MS (ESI-TOF) (m/z): calcd for C29H53N3O6SSi:
599.3424; found, 600.0838 [M þ 1]þ.
4.2.4. 1-[30-O-(tert-Butyldimethylsilyl)-50-O-tetradecanoyl-N-(4,40-
dimethoxytrityl)-b-D-arabinofuranosyl]cytosine (5)
Compound 4 (100 mg, 0.15 mmol) was dissolved in dry DMF
(20 mL). Myristic acid (34.8 mg, 0.15 mmol), HBTU (115 mg,
0.30 mmol), and HOBt (40.9 mg, 0.30 mmol) were added to the
reaction mixture. DIPEA (100 mg, 0.77 mmol) was added to the
reaction mixture at room temperature. The mixture was stirred for
10 h under nitrogen atmosphere. After completion of the reaction,
the solvent was removed under vacuum. Water (50 mL) was added
to the mixture and the crude product was extracted with
dichloromethane (3 ꢀ 40 mL). After removal of dichloromethane,
the crude product was directly carried forward to the next depro-
tection steps. HR-MS (ESI-TOF) (m/z): calcd for C50H71N3O8Si:
869.5010; found, 870.2031 [M þ 1]þ.
4.2.9. 1-[50-O-(12-Thioethyldodecanoyl)-
b-D-arabinofuranosyl]
cytosine (10)
A similar procedure to that of 9 was used for the synthesis and
purification of 10 using 8 as the starting material instead of 7 as
described above. 1H NMR (CD3OD, 500 MHz,
d, ppm): 7.57 (d,
J ¼ 7.5 Hz,1H, H-6), 5.97 (d, J ¼ 4.2 Hz,1H, H-10), 5.70 (d, J ¼ 7.5 Hz,1H,
H-5), 3.92e3.96 (m, 1H, H-20), 3.81e3.86 (m, 1H, H-30), 3.69e3.75 (m
1H, H-40), 3.53e3.58 (m, 2H, H-50), 2.35e2.40 (m, 4H, CH2SCH2), 2.11
(t, J ¼ 7.4 Hz, 2H, CH2CO), 1.40e1.48 (m, 4H, SCH2CH2, CH2CH2CO),
1.15e1.30 (br m,14H, methylene protons),1.09 (t, J ¼ 7.4 Hz, 3H, CH3);
13CNMR(CD3OD,125MHz,
d, ppm):173.50(COO),166.21(C-4),155.91
(C-2),143.37 (C-6), 93.94 (C-5), 86.27 (C-10), 84.91 (C-40), 76.31 (C-20),
74.99 (C-30), 64.17 (C-50), 31.03 (CH2COO), 29.18, 29.13, 28.98, 28.85,
28.80, 28.44, 28.31, 25.36, 24.85 (methylene eCH2e carbons), 15.06
(CH3); HR-MS (ESI-TOF) (m/z): calcd for C23H39N3O6S: 485.2560;
found, 485.4508 [M]þ.
4.2.5. 1-[30-O-(tert-Butyldimethylsilyl)-50-O-tetradecanoyl-
b-D-
arabinofuranosyl]cytosine (7)
Compound 5 was treated with acetic acid (30 mL) and was
stirred for 3 h under nitrogen atmosphere. After the completion of
the reaction, acetic acid was removed under vacuum and concen-
trated crude product was directly carried forward to the next
reaction. HR-MS (ESI-TOF) (m/z): calcd for C29H53N3O6Si: 567.3704;
found, 568.0838 [M þ 1]þ.
4.2.10. 1-[30-O-(tert-Butyldimethylsilyl)-20,50-di(O-tetradecanoyl)-
N-(4,40-dimethoxytrityl)-
b-D-arabinofuranosyl]cytosine (11)
Compound 4 (65 mg, 0.1 mmol) and 4-dimethylaminopyridine
(DMAP, 50 mg, 0.41 mmol) were dissolved in dry dichloromethane
(5 mL) under nitrogen atmosphere. Myristoyl chloride (60 mg,
0.24 mmol) diluted with dry DCM (5 mL) was added dropwise to
the reaction mixture at room temperature. The reaction mixture
was stirred for 16 h under nitrogen atmosphere. After completion
of the reaction, additional DCM (30 mL) was added to the reaction
mixture, which was washed with 0.1% HCl (20 mL ꢀ 2) and then
with water (20 mL ꢀ 1). The organic layer was dried over Na2SO4
and evaporated under reduced pressure. After removal of
dichloromethane, the crude product was directly carried forward to
the next deprotection reaction. HR-MS (ESI-TOF) (m/z): calcd for
C64H97N3O9Si: 1079.6994; found, 1080.7453 [M þ 1]þ.
4.2.6. 1-[50-O-Tetradecanoyl-
b-D-arabinofuranosyl]cytosine (9)
Compound 7 was treated with tetrabutylammonium fluoride
(TBAF,1 M) solution in tetrahydrofuran (20 mL) and stirred for 6 h at
room temperature under nitrogen atmosphere. After completion of
the reaction, the solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography over
silica gel using hexane/dichloromethane and then dichloro-
methane/methanol (0e10%) as the eluents to afford product 9
(21 mg, 27% yield from 4). The product was further purified on
a reverse phase HPLC using C18 column and gradient methanol/
water as mobile phase as described above. 1H NMR (CD3OD,
500 MHz,
d
, ppm): 7.84 (d, J ¼ 7.5 Hz,1H, H-6), 6.18 (d, J ¼ 3.9 Hz,1H,
H-10), 5.84 (d, J ¼ 7.5 Hz,1H, H-5), 4.18 (dd, J ¼ 3.9 Hz, J ¼2.4 Hz,1H, H-
20), 4.08 (t, J ¼ 2.8 Hz, 1H, H-30), 3.94e3.98 (m, 1H, H-40), 3.76e3.86
(m, 2H, H-50), 2.28 (t, J ¼ 7.5 Hz, 2H, CH2CO), 1.56e1.67 (m, 2H,
CH2CH2CO), 1.28 (br m, 20H, methylene protons), 0.89 (t, J ¼ 6.9 Hz,
4.2.11. 1-[30-O-(tert-Butyldimethylsilyl)-20,50-di(O-tetradecanoyl)-
b-D
-arabinofuranosyl]cytosine (12)
Compound 11 was treated with acetic acid (30 mL) and trifluoro-
acetic acid (1 mL). The reaction mixture was stirred for 4 h under
nitrogen atmosphere. After the completion of the reaction, acetic
acid/TFA was removed under vacuum and concentrated product was
directly carried to next reaction. HR-MS (ESI-TOF) (m/z): calcd for
C43H79N3O7Si: 777.5687; found, 778.6055 [M þ 1]þ.
3H, CH3); 13C NMR (CD3OD, 100 MHz,
d, ppm): 173.81 (COO), 166.22
(C-4), 156.76 (C-2), 143.12 (C-6), 93.21 (C-5), 87.36 (C-10), 82.83 (C-
40), 77.15 (C-20), 74.91 (C-30), 64.30 (C-50), 34.01 (CH2COO), 31.65,
29.36, 29.33, 29.27, 29.17, 29.05, 29.97, 29.89, 28.71, 24.87, 22.31
(methylene eCH2e carbons), 13.00 (CH3); HR-MS (ESI-TOF) (m/z):
calcd for C23H39N3O6: 453.2839; found, 454.3192 [M þ 1]þ.
4.2.12. 1-[20,50-di(O-Tetradecanoyl)-
b-D-arabinofuranosyl]cytosine
(13)
4.2.7. 1-[30-O-(tert-Butyldimethylsilyl)-50-O-(12-
thioethyldodecanoyl)-N-(4,40-dimethoxytrityl)-
b-D-
arabinofuranosyl]cytosine (6)
Compound 12 was treated with TBAF (1 M solution inTHF, 20 mL).
The mixture was stirred for 12 h at room temperature under nitrogen
atmosphere. After completion of the reaction, the solvent was evap-
orated under reduced pressure. The liquid crude product compound
was purified by column chromatography over silica gel using hexane/
dichloromethane and then dichloromethane/methanol (0e10%) as
the eluents to afford product 13 (18 mg, 27.6% yield from 4). The
product was further purified on a reverse phase HPLC using C18
column and gradient water/acetonitrile as mobile phase as described
Compound 4 (100 mg, 0.15 mmol), 12-thioethyldodecanoic acid
(39.0 mg, 0.15 mmol), HBTU (115 mg, 0.30 mmol), HOBt (40.9 mg,
0.30 mmol) were dissolved in dry DMF (20 mL). DIPEA (100 mg,
0.77 mmol) was added to the reaction mixture at room tempera-
ture. The mixture was stirred for 12 h under nitrogen atmosphere.
After the completion of reaction, the solvent was removed under
vacuum. Water (50 mL) was added to the mixture. The crude
product was extracted with dichloromethane (3 ꢀ 40 mL). After
above.1H NMR (DMSO, 500 MHz,
d
, ppm): 7.69 (d, J ¼ 7.5 Hz,1H, H-6),
6.09e6.12 (br s, 1H, H-10), 5.81 (d, J ¼ 7.5 Hz, 1H, H-5), 4.10e4.23 (m,

B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
4607
1H, H-20), 3.97e4.05 (m,1H, H-30), 3.96e3.97 (m,1H, H-40), 3.66e3.70
(m, 2H, H-50), 2.22e2.28 (m, 4H, 2 ꢀ CH2CO), 1.50e1.57 (m, 4H,
2 ꢀ CH2CH2CO), 1.10e1.35 (br m, 40H, methylene protons), 0.92 (dt,
chloride was removed under vacuum. Dry DCM (20 mL) was added
and evaporated under vacuum again to remove traces of oxalyl
chloride. 12-Thioethyldodecanoyl chloride obtained was diluted
with dry DCM (10 mL) and was added dropwise at room temper-
ature to the mixture of compound 3 (120 mg, 0.16 mmol) and DMAP
(50 mg, 0.41 mmol) dissolved in dry DCM. The reaction mixture was
stirred for 7 h at room temperature. After the completion of the
reaction, the crude product was washed with 0.1% HCl (2 ꢀ 20 mL)
and then with water (2 ꢀ 20 mL). The organic layer was dried over
sodium sulpfate and evaporated under reduced pressure. The crude
product 15 thus was carried to the next deprotection reaction
without further purification. HR-MS (ESI-TOF) (m/z): calcd for
C56H85N3O8SSi: 1015.5596; found, 1016.6177 [M þ 1]þ.
J ¼ 6.7 Hz, 6H, 2 ꢀ CH3); 13C NMR (CDCl3, 100 MHz,
d, ppm): 173.86
(COO), 166.23 (C-4), 156.76 (C-2), 143.13 (C-6), 93.21 (C-5), 87.35
(C-10), 82.83 (C-40), 77.15 (C-20), 74.93 (C-30), 62.09 (C-50), 31.65
(CH2COO), 29.19, 29.33, 29.27, 29.17, 29.05, 29.97, 29.89, 28.83, 24.81,
22.45 (methylene eCH2e carbons),14.33 (CH3); HR-MS (ESI-TOF) (m/
z): calcd for C37H65N3O7: 663.4823; found, 664.5155 [M þ 1]þ.
4.2.13. 1-[30,50-di-(O-tert-Butyldimethylsilyl)-20-O-myristoyl-N-
(4,40-dimethoxytrityl)-
b-D-arabinofuranosyl]cytosine (14)
Compound 3 (120 mg, 0.16 mmol) and DMAP (45 mg, 0.37 mmol)
were dissolved in dry dichloromethane (15 mL) under nitrogen
atmosphere. Myristoyl Chloride (60 mg, 0.24 mmol) diluted with dry
DCM (5 mL) was added dropwise to the reaction mixture at room
temperature. The mixture was stirred for 18 h under nitrogen
atmosphere. After the completion of the reaction, additional DCM
(30 mL) was added to the reaction mixture, which was washed with
0.1% HCl (20 mL ꢀ 2) to remove DMAP and then with water
(20 mL ꢀ 1). The organic layer was dried over Na2SO4 and evapo-
rated under reduced pressure. After removal of dichloromethane,
product was directly carried forward to the next deprotection reac-
tion. HR-MS (ESI-TOF) (m/z): calcd for C56H85N3O8Si2: 983.5875;
found, 984.6040 [M þ 1]þ.
4.2.17. 1-[30-O-(tert-Butyldimethylsilyl)-20-O-(12-
thioethyldodecanoyl)-b-D-arabinofuranosyl]cytosine (17)
Crude compound 17 was synthesized by a similar procedure
described above for that of 16. HR-MS (ESI-TOF) (m/z): calcd for
C29H53N3O6SSi: 599.3424; found, 600.3065 [M þ 1]þ.
4.2.18. 1-[20-O-(12-Thioethyldodecanoyl)-
b-D-arabinofuranosyl]
cytosine (19)
Compound 19 wassynthesizedand purified bya similarprocedure
described above for that of 18. 1H NMR (CD3OD, 500 MHz,
d, ppm):
7.82 (d, J ¼ 7.6 Hz, 1H, H-6), 6.16 (d, J ¼ 4.2 Hz, 1H, H-10), 5.93 (d,
J ¼ 7.6 Hz,1H, H-5), 4.22e4.27 (m,1H, H-20), 4.05e4.10 (m, J ¼ 3.6 Hz,
1H, H-30), 3.92e3.96 (m,1H, H-40), 3.75e3.87(m, 2H, H-50), 2.47e2.56
(m, 4H, CH2SCH2), 2.28 (t, J ¼ 7.4 Hz, 2H, CH2CO),1.50e1.63 (m, 2H, m,
4H, SCH2CH2, CH2CH2CO), 1.36e1.41 (m, 2H, CH2CH2CH2CO),
1.25e1.35 (br m,12H, methylene protons),1.23 (t, J ¼ 7.4 Hz, 3H, CH3);
4.2.14. 1-[30-O-(tert-Butyldimethylsilyl)-20-O-tetradecanoyl-
b-D-
arabinofuranosyl]cytosine (16)
DMTr group was removed from compound 14 by adding acetic
acid (20 mL) and stirring at room temperature for 3 h. Evaporation
of acetic acid under reduced pressure gave crude product 16, which
was directly carried further to the next reaction without further
purification. HR-MS (ESI-TOF) (m/z): calcd for C29H53N3O6Si:
567.3704; found, 568.3944 [M þ 1]þ.
13CNMR(CD3OD,125MHz,
d, ppm):173.81(COO),166.21(C-4),156.77
(C-2),143.14 (C-6), 93.90 (C-5), 86.66 (C-10), 84.44 (C-40), 76.31 (C-20),
75.31 (C-30), 61.10 (C-50), 30.94 (CH2COO), 29.11, 29.03, 29.00, 28.84,
28.73, 28.70, 28.31, 25.24, 24.75 (methylene eCH2e carbons), 13.86
(CH3); HR-MS (ESI-TOF) (m/z): calcd for C23H39N3O6S: 485.2560;
found, 486.2244 [M þ 1]þ.
4.2.15. 1-[20-O-Tetradecanoyl-
b-D-arabinofuranosyl]cytosine (18)
Crude compound 16 was treated with TBAF (1 M solution in THF,
20 mL). The reaction mixture was stirred for 8 h at room temper-
ature under nitrogen atmosphere. After completion of the reaction,
THF was removed under reduced pressure and the liquid crude
product was purified by column chromatography over silica gel
using hexane/dichloromethane and then dichloromethane/meth-
anol (0e20%) as eluents to afford white solid product 18. The
product was further purified on reverse phase HPLC using C18
column and gradient water/methanol as mobile phase as described
above. Overall yield obtained from 3 (22 mg, 31.4%). 1H NMR
4.3. Cell culture
Human leukemia cell line CCRF-CEM was obtained from
American Type Culture Collection (ATCC no. CCL-119). Cells were
grown on 75 cm2 cell culture flasks with RPMI-16 medium, sup-
plemented with 10% Fetal bovine serum (FBS), and 1% pen-
icillinestreptomycin solution (10,000 units of penicillin and 10 mg
of streptomycin in 0.9% NaCl) in a humidified atmosphere of 5% CO2,
95% air at 37 ꢂC.
(CD3OD, 500 MHz,
d
, ppm): 7.68 (d, J ¼ 10 Hz, 1H, H-6), 6.16 (d,
J ¼ 5 Hz, 1H, H-10), 5.88 (d, J ¼ 10 Hz, 1H, H-5), 4.22e4.25 (m, 1H,
H-20), 4.11e4.17 (m, 1H, H-30), 4.01e4.07 (m, 1H, H-40), 3.95e4.00
(m, 1H, H-500), 3.75e3.80 (m, 1H, H-50), 2.21 (t, J ¼ 7.5 Hz, 2H,
CH2CO), 1.50e1.62 (m, 2H, CH2CH2CO), 1.24 (br m, 20H, methylene
4.4. Cell proliferation assay
Cell proliferation assay of cytarabine derivatives synthesized
was evaluated in CCRF-CEM cells, and was compared with that of
cytarabine. Cell proliferation assay was carried out using CellTiter
96 aqueous one solution cell proliferation assay kit (Promega,
USA). Briefly, CCRF-CEM cells were suspended at 5 ꢀ 105/mL and
protons), 0.85 (t, J ¼ 6 Hz, 3H, CH3); 13C NMR (CD3OD, 125 MHz,
d,
ppm): 173.81 (COO), 166.22 (C-4), 156.76 (C-2), 143.12 (C-6), 93.21
(C-5), 87.36 (C-10), 82.83 (C-40), 77.15 (C-20), 74.91 (C-30), 64.30 (C-
50), 34.01 (CH2COO), 31.65, 29.36, 29.33, 29.27, 29.17, 29.05, 29.97,
29.89, 28.71, 24.87, 22.31 (methylene eCH2e carbons), 13.00 (CH3);
HR-MS (ESI-TOF) (m/z): calcd for C23H39N3O6: 453.2839; found,
454.2857 [M þ 1]þ.
100 mL of the cell suspension were placed in each well of the 96
well culture plate. Cells were incubated with cytarabine and fatty
acyl derivatives (1 mM) in 4% DMSO and tests were in triplicates.
Incubation was carried out at 37 ꢂC in an incubator supplied with
4.2.16. 1-[30,50-Di(O-tert-butyldimethylsilyl)-20-O-(12-
5% CO2 for 24e96 h. At the end of the sample exposure period
(24e96 h), 20 mL CellTiter 96 aqueous solution was added. The
thioethyldodecanoyl)-N-(4,40-dimethoxytrityl)-
b-
D-
arabinofuranosyl]cytosine (15)
plate was returned to the incubator for 1 h in a humidified
atmosphere at 37 ꢂC. The absorbance of the formazan product was
measured at 490 nm using microplate reader. The percentage of
cell survival was calculated as OD value of cells treated with test
12-Thioethyldodecanoic acid (70 mg, 0.24 mmol) was added to
oxalyl chloride (20 mL, 210.4 mmol). The reaction mixture was
stirred for 2 h at room temperature and then at 60 ꢂC for 4 h. Oxalyl

4608
B.S. Chhikara et al. / European Journal of Medicinal Chemistry 45 (2010) 4601e4608
[15] M.H. Gouy, L.P. Jordheim, I. Lefebvre, E. Cros, C. Dumontet, S. Peyrottes,
C. Perigaud, Bioorg. Med. Chem. 17 (2009) 6340e6347.
[16] Y. Mehellou, R. Valente, H. Mottram, E. Walsby, K.I. Millls, J. Balzarini,
C. McGuigan, Bioorg. Med. Chem. 18 (2010) 2439e2446.
compound ꢁ OD value of culture medium/(OD value of control
cells ꢁ OD value of culture medium) ꢀ 100%.
[17] W. Plunkett, J.O. Liliemark, E. Estey, M.J. Keating, Semin. Oncol. 14 (1987)
159e166.
Acknowledgement
[18] S.K. Dubey, A. Pandey, R. Mishra, N. Kapoor, A. Tiwari, K. Misra, Indian J.
Biotech. 6 (2007) 159e174.
We acknowledge the financial support from the American
[19] K. Rukmani, M. Sivakumar, S.S. Kumar, J. Biomed. Nanotech. 3 (2007) 90e96.
[20] M.D. Blanco, C. Gomez, R. Olmo, E. Muniz, J.M. Teijon, Int. J. Pharm. 202 (2000)
29e39.
[21] R. Mulik, V. Kulkarni, R.S.R. Murthy, Drug Dev. Ind. Pharm. 35 (2009) 49e56.
[22] G.H. Sagar, M.A. Arunagirinathan, J.R. Bellare, Indian J. Exp. Biol. 45 (2007)
133e159.
[23] K. Ruckmani, S.K. Ghosal, S.T.P. Pharma Sci. 11 (2001) 301e303.
[24] M. Benesch, C. Urban, Expert Opin. Pharmacother. 9 (2008) 301e309.
[25] M. Kripp, R.-D. Hofheinz, Int. J. Nanomedicine 3 (2008) 397e401.
[26] N.H. Nam, S. Sardari, M. Selecky, K. Parang, Bioorg. Med. Chem. 12 (2004)
6255e6269.
[27] K. Parang, E.E. Knaus, L.I. Wiebe, Antivir. Chem. Chemother. 9 (1998) 311e323.
[28] K. Parang, L.I. Wiebe, E.E. Knaus, J. Pharm. Pharmacol. 50 (1998) 989e996.
[29] K. Parang, L.I. Wiebe, E.E. Knaus, J.S. Huang, D.L. Tyrrell, J. Pharm. Pharm. Sci. 1
(1998) 107e113.
[30] K. Parang, L.I. Wiebe, E.E. Knaus, Curr. Med. Chem. 7 (2000) 995e1039.
[31] L.L.H. Fallois, J.-L. Décout, M. Fontecave, Tetrahedron Lett. 36 (1995)
9479e9480.
[32] X.-F. Zhu, H.J. Williams, A.I. Scott, Synth. Commun. 33 (2003) 2011e2016.
[33] N.S. Kishore, T. Lu, L.J. Knoll, A. Katoh, D.A. Rudnick, P.P. Mehta, B. Devadas,
M. Huhn, J.L. Atwood, S.P. Adams, G.W. Gokel, J.I. Gordon, J. Biol. Chem. 266
(1991) 8835e8855.
Cancer Society Grant # RSG-07-290-01-CDD.
References
[1] E. Jabbour, J.E. Cortes, F.J. Giles, S. O’Brien, H.M. Kantarjian, Cancer 109 (2007)
2171e2181.
[2] M. Shah, B. Agarwal, Indian J. Pediat. 75 (2008) 831e837.
[3] J. Pinilla-Ibarz, C. Bello, Curr. Oncol. Rep. 10 (2008) 365e371.
[4] I. Matsumura, J. Clin. Hemat. 50 (2009) 1389e1400.
[5] R.L. Capizzi, J.C. White, B.L. Powell, F. Perrino, Semin. Hematol. 28 (1991) 54e69.
[6] M.-J. Jin, J.H. Hong, H.-K. Han, J. Kor. Pharm. Sci. 38 (2008) 255e259.
[7] Y. Sun, J. Sun, L. Shi, Y. Jing, S. Yin, Y. Chen, G. Li, Y. Xu, Z. He, Mol. Pharmacol.
6 (2009) 315e325.
[8] B. Liu, C. Cui, W. Duan, M. Zhao, S. Peng, L. Wang, H. Liu, G. Cui, Eur. J. Med.
Chem. 44 (2009) 3596e3600.
[9] L.W. Peterson, C.E. McKenna, Expert Opin. Drug Deliv. 6 (2009) 405e420.
[10] A.M. Bergman, C.M. Kuiper, D.A. Voorn, E.M. Comijn, F. Myhren, M.L. Sandvold,
H.R. Hendriks, G.J. Peters, Biochem. Pharmacol. 67 (2004) 503e511.
[11] F. Li, J. Maag, H. Alfredson, J. Pharm. Sci. 97 (2008) 1109e1134.
[12] K. Breistøl, J. Balzarini, M.L. Sandvold, F. Myhren, M. Martinsen, E. De Clercq,
O. Fodstad, Cancer Res. 59 (1999) 2944e2949.
[13] A.M. Bergman, C.M. Kuiper, F. Myhren, D.A. Voorn, E.M. Comijn, F. Myhren,
M.L. Sandvold, H.R. Hendriks, G.J. Peters, Nucleosides Nucleotides Nucleic
Acids 23 (2004) 1523e1526.
[14] S. Dueland, S. Aamdal, M.J. Lind, H. Thomas, M. Liland Sandvold, J.-M. Gaullier,
W. Rasch, Acta Oncol. 48 (2009) 137e145.
[34] P.B. Koneru, E.J. Lien, V.I. Avramis, Pharm. Res. 10 (1993) 515e520.
[35] D.Y. Bouffard, J. Jolivet, L. Leblond, B. Hamelin, F. Ouellet, S. Barbeau,
A. Richard, H. Gourdeau, Cancer Chemother. Pharmacol. 52 (2003) 497e506.
[36] L.B. Crisp, S.M. Smith, M.A.G. Mathers, G.A.R. Young, S.D. Lyons,
R.I. Christopherson, J. Biochem. Cell Biol. 28 (1996) 1061e1069.