Synthesis and evaluation of anti-tumor activities of N4 fatty acyl amino acid derivatives of 1-β-arabinofuranosylcytosine

Article abstract of DOI:10.1016/j.ejmech.2009.02.028

1-β-d-Arabinofuranosylcytosine (Ara-C, Cytarabine) is one of the drugs used for acute nonlymphocytic leukemia (ANLL). However, the bioavailability of Ara-C is relatively low due to its low lipophilicity. In order to improve the lipophilicity and bioavailability of Ara-C, a series of N⁴ derivatives of Ara-C, i.e., (fatty acid)-(amino acid)-Ara-C analogues, were prepared. The 15 derivatives synthesized were characterized by their melting points, optical rotations and partition coefficients. It was found that the Ara-C derivatives synthesized in this study were more lipophilic than Ara-C as determined by their partition coefficients. Their in vitro cytotoxicity and in vivo anti-tumor activity were determined and compared with that of Ara-C. It was found that the derivatives were more active than Ara-C in Hela cells, but not in HL-60 cells. The in vivo results showed that some of the derivatives were more effective than Ara-C in mice bearing S₁₈₀ tumor while others showed a decreased activity in comparison with Ara-C.

Full text of DOI:10.1016/j.ejmech.2009.02.028

European Journal of Medicinal Chemistry 44 (2009) 3596–3600
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of anti-tumor activities of N4 fatty acyl amino acid
derivatives of 1- -arabinofuranosylcytosine
b
,
b
Boyang Liu a, Chunying Cui a, Wei Duan a, Ming Zhao a, Shiqi Peng a, Lili Wang a
,
,
b
,
a,
**
Hu Liu a , Guohui Cui
*
a School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, China
b School of Pharmacy, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3V6
a r t i c l e i n f o
a b s t r a c t
Article history:
1-b-D-Arabinofuranosylcytosine (Ara-C, Cytarabine) is one of the drugs used for acute nonlymphocytic
Received 22 May 2008
Received in revised form
11 February 2009
Accepted 26 February 2009
Available online 6 March 2009
leukemia (ANLL). However, the bioavailability of Ara-C is relatively low due to its low lipophilicity. In
order to improve the lipophilicity and bioavailability of Ara-C, a series of N4 derivatives of Ara-C, i.e.,
(fatty acid)–(amino acid)–Ara-C analogues, were prepared. The 15 derivatives synthesized were char-
acterized by their melting points, optical rotations and partition coefficients. It was found that the Ara-C
derivatives synthesized in this study were more lipophilic than Ara-C as determined by their partition
coefficients. Their in vitro cytotoxicity and in vivo anti-tumor activity were determined and compared
with that of Ara-C. It was found that the derivatives were more active than Ara-C in Hela cells, but not in
HL-60 cells. The in vivo results showed that some of the derivatives were more effective than Ara-C in
mice bearing S180 tumor while others showed a decreased activity in comparison with Ara-C.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Ara-C
Ara-C derivatives
Anti-tumor
Hela cells
HL-60 cells
Leukemia
1. Introduction
effects including nausea, vomiting, inhibition of bone marrow and
loss of body weight [2,3]. The side effects following long term uses
1-
b
-
D
-Arabinofuranosylcytosine (Ara-C, Cytarabine), a pyrimi-
are of a special concern for pediatric patients [4]. Finally, drug
resistance is likely another reason for the limited success observed
with Ara-C clinically [5–7]. It has been reported that Ara-C is not
effective in solid tumors [8].
dine nucleotide analogue, is primarily used in the treatment of
various leukemias in conjunction with anthracycline antibiotics.
This cytidine-based antimetabolite undergoes initial phosphoryla-
tion by deoxycytidine kinase to monophosphate with subsequent
phosphorylations catalyzed by pyrimidine monophosphate and
diphosphate kinases. The active form, triphosphorylated Ara-C,
exhibits its anticancer activity via the inhibition of DNA polymerase
and/or DNA chain elongation [1]. It is one of the drugs used for
acute nonlymphocytic leukemia (ANLL). However, Ara-C is associ-
ated with relatively low lipophilicity and low bioavailability. It is
also known that Ara-C is quickly deactivated to uracil arabinoside
(Ara-U) by cytidine deaminase. Even its monophosphorylated form,
Ara-cytidine monophosphate, can be catabolized into uracil arabi-
notide by deoxycytidylate deaminase, resulting in the loss of
activity. In addition, Ara-C is known to be associated with side
The low lipophilicity of Ara-C is likely due to the presence of an
–NH2 and multiple –OH groups in its structure as shown in Fig. 1.
Therefore, there have been many reports where modifications at the
N4 and 30,50-OH positions [9–15] were made. Enocitabine and N4-
palmitoyl-Ara-C are two examples of derivatives with enhanced and
prolonged activities. Both of them are fatty acid derivatives of Ara-C
and are converted to Ara-C via metabolic biotransformation [16].
There have been many other reports on various N4 amide
derivatives of Ara-C. However, they seem to suffer from instability
problems. Previous reports by Aoshima et al. [17] and Bergman et
al. [18] on some of the N4 fatty acid derivatives of Ara-C suggested
that the length of fatty acids on the N4 position seemed to affect the
anti-tumor activity observed and found that Ara-C derivatives with
fatty acids of C18–C22 at the N4 position were most effective. As the
length of fatty acids further increased, their anti-tumor activity
decreased. They also found that the Ara-C derivatives showed
prolonged activities compared to Ara-C as the derivatives of Ara-C
gradually released Ara-C upon hydrolysis.
* Corresponding author. School of Pharmacy, Memorial University of
Newfoundland, St. John’s, NL, Canada A1B 3V6. Tel.: þ1 709 777 6382; fax: þ1 709
777 7044.
** Corresponding author. Tel.: þ86 10 83911538; fax: þ86 10 83911533.
E-mail addresses: hliu@mun.ca (H. Liu), cuiguohui@bjmu.edu.cn (G. Cui).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.028

B. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3596–3600
3597
(Tokyo, Japan). Microplate reader, BIO-RAD Model 680, was
purchased from Bio-Rad (Hercules, CA, USA). Solutions/suspensions
used for injection were autoclaved using a VARIOKLAVÒ steam
sterilizer manufactured by H þ P Labortechnik AG (Obers-
chleissheim, Germany). OLYMPUS IX71 inverted microscope was
purchased from Olympus Co. (Tokyo, Japan).
Cell culture media, RPMI 1640 and DMEM, were products of
GIBCO (Grand Island, NY, USA). Fetal bovine serum and trypsin were
purchased from HyClone (Logan, UT, USA). Penicillin, streptomycin
and MTT were products of Sigma, and DMSO was purchased from
ACROS ORGANICS (Geel, Belgium).
NH2
N
HO
N
OH
O
O
H
H
H
H
OH
Fig. 1. The structure of Ara-C.
Human leukemia cell line, HL-60, obtained from the School of
Pharmacy of Peking University Medical Center, was maintained in
RPMI 1640 containing 10% of fetal bovine serum, penicillin (100
units/mL) and streptomycin (100 units/mL). Hela cells were main-
tained in DMEM containing 10% of fetal bovine serum, penicillin
(100 units/mL) and streptomycin (100 units/mL).
Male Kunming mice purchased from the Animal Services,
Peking University Medical Center were supplied with food and
water ad libitum.
The purpose of this work was to introduce various fatty acyl
amino acids onto the N4 position (Fig. 2). Such a modification was
aimed to reduce the deamination associated with Ara-C in addition
to increased lipophilicity. Fatty acids of C10, C14 and C18 were used
and were connected to the N4 position of Ara-C via five different
amino acids, valine, methionine, tyrosine, arginine and glutamic
acid. The selected amino acids are associated with varied physico-
chemical properties. Valine and methionine are non-polar while
tyrosine is polar. Glutamic acid is acidic and arginine is alkaline. The
in vitro and in vivo anti-tumor activities of the derivatives synthe-
sized were evaluated and compared with that of Ara-C.
2.2. Chemistry
The Ara-C derivatives shown in Fig.
2 were synthesized
2. Experimental procedures
according to the scheme in Fig. 3. The respective fatty acids (1a–c)
with different carbon chain lengths (C-10, C-14 or C-18) were first
connected to the C-terminus protected amino acid chosen (Val,
Met, Arg, or Tyr) in the presence of DCC, HOBt and N-methyl-
morpholine (NMM) to yield the C-terminus protected fatty acyl
amino acid 1-methyl esters (3a–l). The de-protected fatty acyl
amino acids (4a–l) were then conjugated with Ara-C using EDC/
HOBt as the condensing agent in pyridine at 45 ꢀC to yield deriva-
tives, 6a–l, as shown in Fig. 3.
2.1. Materials
Ara-C (pharmaceutical grade) was obtained from Peking
University Pharmaceutical Ltd. (Beijing, China), while myristic acid
was obtained from Beijing Chaoyang Xudong Chemicals Inc. (Bei-
jing, China). Stearic acid and n-capric acid were purchased from
Tianjin Fuchen Chemicals Ltd. (Tianjin, China). All amino acids were
of L-configuration and were from Sichuan Gaosheng Inc. Ltd.
For glutamic acid containing derivatives, N-tert-butoxycarbonyl-
Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt)
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)$HCl
were from GL Biochem Ltd. (Shanghai, China). NaCl for injection
and CMC-Na were purchased from Shandong Qidu Pharmaceutical
Co. Ltd. (Zibo, China). All reagents were of chemical grade unless
otherwise specified.
Laborota 4000 rotary evaporator was from Heidolph Instru-
ments (Schwabach, Germany). Circulative multifunctional vacuum
pump (SHB IIIS) was a product of Zhengzhou Great Wall Industrial
Trade Ltd. (Zhenzhou, Henan, China). Hot plate with magnetic
stirrer, S21-2, was from Shanghai Si-le Instrument Ltd. (Shanghai,
China). Avance II 300 and 500 NMR spectrometers were purchased
from Bruker Biospin AG (Fa¨llanden, Switzerland). Quattro Micro
2000 mass spectrometer was from Waters (Milford, MA, USA).
Melting point apparatus, XT5, was purchased from Beijing Keyi
Electro-optical Instrument Ltd. (Beijing, China). Optical
rotation was determined using a polarimeter, P-1020, from Jasco
L-glutamic acid 5-benzyl ester (Boc-Glu-OBzl) was first conjugated
with N4-amine of Ara-C to yield 50. After selective de-protection of
N-Boc (500), the respective fatty acids (la–c) with different carbon
chain lengths (C-10, C-14, C-18) were connected to yield the
intermediates, 5m–o. The 5-benzyl protection was then removed to
obtain the glutamate derivatives (6m–o) as shown in Fig. 3.
2.3. Supplementary data [Details of synthetic experiments]
The data are available at the end of manuscript and are free of
charge via the Internet at http://www.sciencedirect.com.
2.4. Characterization of Ara-C derivatives synthesized
The structures of the derivatives synthesized, 6a–o, were
confirmed with mass-spectrometry (MS) and nuclear magnetic
resonance (NMR). Their melting points and optical rotations were
measured. The purity of the final compounds, 6a–o, were verified
by high pressure liquid chromatography (HPLC) using a C18 Sun-
FireÔ column (Waters). Analytical HPLC was performed on
a Waters 2695 system equipped with an UV detector set at 248 nm.
O
R
NH-AA
N
Compounds were dissolved in MeOH and injected through a 50 mL
loop. The eluent systems, A (H2O/CH3CN, 20:80) and B (pure CH3CN
100%), were used. HPLC retention times (tR) were obtained, at
a flow rate of 0.5 mL/min with 100% eluent A for the first 20 min
followed by gradient increase to 100% eluent B over the next
20 min. The partition coefficients of 6a–o were determined in
a water/1-octanol system by the shake-flask method. Briefly, about
1 mg of each of the compounds were dissolved in 5 mL of water-
saturated 1-octanol. An aliquot of this solution was diluted with
chloroform and the absorbance (A0) of the resultant solution at
N
OH
O
O
H
OH
H
H
H
OH
Fig. 2. The structures of N4 derivatives of Ara-C synthesized. R: –(CH2)8CH3;
–(CH2)12CH3; –(CH2)16CH3. AA: Val, Met, Arg, Tyr, Glu.

3598
B. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3596–3600
O
O
O
I
II
R-C AA-OCH3
3a-l
+
R C OH AA-OCH3
R-C-AA-OH
4a-l
1a-c
2a-d
O
R C
AA NH
N
Boc-Glu(OBzl)
N
NH
NH2
III
N
Boc-Glu(OBzl)-OH
OH
III
OH
OH
N
H
N
H
O
N
H
O
O
O
O
O
H
H
OH
H
H
OH
H
OH
H
H
H
H
OH
6a-l
5
OH
OH
5'
IV
O
O
R C
NH
N
Glu(OBzl)
N
R
AA NH
N
C
H-Glu(OBzl)
N
NH
O
C
V
OH
III
(1a-c)
R
OH
OH
OH
N
N
O
O
O
O
O
O
H
OH
H
OH
H
OH
H
H
H
H
H
H
H
H
OH
H
5m-o
OH
OH
5''
6m-o
Fig. 3. Synthesis of (fatty acid)–(amino acid)–Ara-C derivatives. I. DCC, HOBt and NMM; II. 2 N NaOH; III. EDC, HOBt and pyridine; IV. 4 N HCl-EtOAc; V. H2, Pd/C. R ¼ –(CH2)8CH3,
–(CH2)12CH3, –(CH2)16CH3 in 1a–c, 3a–l, 4a–l, 5m–o, 6a–l, 6m–o; AA ¼Val, Met, Tyr or Arg in 2a–d, 3a–l, 4a–l, 6a–l; AA ¼ Glu in 6m–o.
280 nm was determined using an UV-spectrophotometer. Five
milliliters of the above solution was mixed with 5 mL of 1-octanol-
saturated water in a 15 mL conical tube. Sample tubes were
vigorously shaken for 2 h at 25 ꢀC following which the mixture was
allowed to stand for 30 min. The 1-octanol layer was removed and
diluted in chloroform to determine the absorbance at 280 nm (Ax).
The partition coefficient was calculated as
incubation, 25 mL of 5 mg/mL of MTT were added to each well and
the plate was put back in the incubator for another 4 h at 37 ꢀC. The
plate was then subjected to centrifugation at 1000 r/min for 10 min
in the case of HL-60 cells, while no centrifugation was needed for
Hela cells. Supernatant was discarded, and 100 mL of DMSO were
added to each well and the plate was vortexed for 10 min to
dissolve the crystals formed. The OD values were obtained using
a microplate reader at 570 nm with a reference wavelength of
630 nm. The percentage of cell survival was calculated as OD value
of cells treated with test compound ꢁ OD value of culture medium/
(OD value of control cells ꢁ OD value of culture medium) ꢂ 100%
and IC50 was calculated as the concentration which resulted in 50%
of cell death.
nXAXVX
p ¼
nOAOVO ꢁ nXAXVX
where nx is the dilution factor of the 1-octanol layer prior to
measuring absorbance following partitioning; Vx is the aliquot
amount taken for the above dilution; and Ax is the absorbance of
the diluted 1-octanol layer following partitioning, while n0 is the
dilution factor of the 1-octanol sample prior to measuring absor-
bance before partitioning; A0 is the absorbance of the diluted
1-octanol sample before partitioning and V0 is the aliquot volume
of the 1-octanol sample used for dilution before partitioning.
2.6. In vivo anti-tumor activity
Kunming mice were used. Animal care was in accordance with
institutional guidelines. S180 tumor cells passaged in mice abdomen
were harvested on the eighth day and suspended at 2.0 ꢂ 107/mL.
The tumor cell preparation (0.2 mL) was injected to each mouse.
The mice inoculated with S180 tumor cells were randomly divided
into 17 different groups with 10 in each group.
Ara-C was dissolved in 0.5% CMC-Na and its derivatives
synthesized (6a–o) were suspended in 0.5% CMC-Na at 7.8 mmol/L.
Mice inoculated with S180 tumor cells were injected intraperito-
neally with 0.2 mL of the Ara-C solution or suspensions of its
derivatives daily for 7 days. At the end of 7 days, mice were sacri-
ficed and tumor was weighed. Tumor inhibition was calculated as:
(average tumor weight in the control group ꢁ average tumor
weight in drug treated group)/average tumor weight in the control
group ꢂ 100%.
2.5. Determination of in vitro cytotoxicity of Ara-C derivatives
The in vitro cytotoxicity of Ara-C derivatives synthesized was
evaluated in HL-60 and Hela cells, and was compared with that of
Ara-C.
HL-60 or Hela cells were suspended at 5 ꢂ104/mL and 100
mL of
the cell suspension were placed in each well of the 96-well culture
plate. Cells were incubated with various concentrations (0.01, 0.05,
0.10, 0.50 and 1.00
mg/mL for HL-60 cells, and 0.5, 1.0, 5.0, 10 and
50 g/mL for Hela cells) of test compounds in PBS containing 4%
m
DMSO and tests were in triplicates. Incubation was carried out at
37 ꢀC in an incubator supplied with 5% CO2 for 48 h. Following

B. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3596–3600
3599
Table 1
Table 3
Characterization of Ara-C and its derivatives.
The weight of tumor and percent tumor growth inhibition (%TGI) 7 days after
implantation of S180
.
Compound
Code
Melting
point (ꢀC)
Optical rotation
25, deg (c10,
Partition
coefficient (P)
log P
[
a
]
Compound
Code
Tumor weight (X ꢃ SD) g
TGI %
7.0 ꢃ 2.42
D
MeOH)
C10-Val-Ara-C
6a
6b
6c
6d
6e
6f
6g
6h
6i
6i
6k
6l
6m
6n
6o
1.181 ꢃ 0.308
1.163 ꢃ 0.656
0.523 ꢃ 0.095**
0.944 ꢃ 0.438
0.793 ꢃ 0.361*
0.438 ꢃ 0.102**
1.028 ꢃ 0.232
1.125 ꢃ 0.58
Ara-C
C10-Val-Ara-C
C14-Val-Ara-C
189–195
98–100
102–104
134–136
84–86
127
0.12
6.04
7.91
8.15
4.81
7.71
12.41
6.5
ꢁ0.94
0.78
0.9
0.91
0.68
0.88
1.09
0.81
0.9
0.97
0.76
0.45
0.61
ꢁ0.28
0.25
0.5
C14-Val-Ara-C
C18-Val-Ara-C
C10-Met-Ara-C
8.4 ꢃ 5.16
58.9 ꢃ 0.74
25.6 ꢃ 2.42
37.6 ꢃ 3.44
65.5 ꢃ 0.80
19.1 ꢃ 1.83
11.4 ꢃ 4.56
16.5 ꢃ 5.42
15.7 ꢃ 3.16
39.2 ꢃ 2.07
8.1 ꢃ 3.20
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
36.6
29.1
47.7
45.7
45.9
51.3
65.4
52.2
33.9
61.4
67.8
34.9
64.1
29.1
30.8
C18-Val-Ara-C
C14-Met-Ara-C
C10-Met-Ara-C
C14-Met-Ara-C
C18-Met-Ara-C
C10-Tyr-Ara-C
C14-Tyr-Ara-C
C18-Tyr-Ara-C
C10-Arg-Ara-C
C18-Met-Ara-C
C10-Tyr-Ara-C
C14-Tyr-Ara-C
C18-Tyr-Ara-C
C10-Arg-Ara-C
C14-Arg-Ara-C
C18-Arg-Ara-C
84–86
125–127
114–116
156–158
194–196
98–100
124–126
114–116
77–79
1.06 ꢃ 0.689
1.071 ꢃ 0.402
0.773 ꢃ 0.263*
1.168 ꢃ 0.406
1.072 ꢃ 0.451
0.958 ꢃ 0.226
1.106 ꢃ 0.439
0.891 ꢃ 0.272*
1.27 ꢃ 0.3
7.97
9.3
5.72
2.805
4.08
0.52
1.78
3.15
C14-Arg-Ara-C
C10-Glu-Ara-C
15.6 ꢃ 3.55
24.6 ꢃ 1.78
12.9 ꢃ 3.46
29.9 ꢃ 2.14
C18-Arg-Ara-C
C10-Glu-Ara-C
C14-Glu-Ara-C
C18-Glu-Ara-C
C14-Glu-Ara-C
C18-Glu-Ara-C
Ara-C in CMC-Na
CMC-Na
129–131
174–176
*Significant compared to control (P < 0.05); **Significant compared to Ara-C CMC-
Na (P < 0.05); n ¼ 10.
2.7. Statistics
3.2. In vitro cytotoxicity
Data are expressed as X ꢃ SD and statistical significance was
determined using unpaired two-sided Student’s t test.
The IC50 values of Ara-C and the derivatives synthesized in
HL-60 and Hela cells are shown in Table 2. It was found that Ara-C
was more effective in HL-60 than in Hela cell. The derivatives were
shown to have comparable IC50 values as that of Ara-C in HL-60
cells. It was also found that valine containing derivatives (6a, 6b, 6c)
had the lowest activity while derivatives containing methionine as
the linker (6d, 6e and 6f) showed the best results. The length of the
fatty acids seemed to play a role in the anti-tumor activity observed
as well and C10 fatty acid containing derivatives (6a, 6d, 6g, 6j and
6m) appeared to be more active. This is likely due to the more
balanced solubility of these derivatives and/or better affinity for
3. Results and discussion
3.1. Characterization of the Ara-C derivatives synthesized
Fifteen new derivatives of Ara-C (6a–o) were synthesized.
Analytical HPLC revealed that the purity of 6a–o was in the range of
95.56–99.15%. Details about the purity and retention time of 6a–o
are shown in the Supplementary data [Details of synthetic exper-
iments] section. Their melting points, optical rotation and partition
coefficients were determined, and the results are summarized in
Table 1.
membrane attributed to the medium chain fatty acid of C10
.
It was also found that the IC50 values of most of the derivatives
synthesized in Hela cells were higher than in HL-60 cells suggesting
that HL-60 cells are more sensitive than Hela cells. In this context,
previous studies have reported that the fatty acid derivatives of Ara-
C were found to be more active than Ara-C in L1210 cells [19,20]
while Bergman et al. [18] showed that the activity of fatty acid
derivatives of Ara-C increased as the length of fatty acids decreased.
As shown in Table 1, the log P of Ara-C was found to be ꢁ0.94
while the log P values of the derivatives were found to be higher
and were above 0 except for 6m. The lower lipophilicity associated
with 6m is probably due to the C10 fatty acid and the additional
hydroxyl group in glutamic acid in the molecule. As expected, as the
length of fatty acids increased, the lipophilicity of the resultant
derivatives increased. The increased lipophilicity associated with
the derivatives may lead to increased permeability across the cell
membranes and ultimately improved bioavailability.
3.3. In vivo anti-tumor activity
The in vivo anti-tumor results are shown in Table 3. Due to the
low water solubility of Ara-C derivatives, they were formulated into
their respective suspensions for injection. It was found that deriv-
atives 6c and 6f were more effective than Ara-C, and derivatives 6e,
6g and 6k showed similar activities as Ara-C. The rest of the
derivatives were less effective than Ara-C. The reduced anti-tumor
activity observed with most of the derivatives was likely due to
their poor water solubility.
Table 2
The IC50 values of Ara-C and its derivatives in HL-60 and Hela cells.
Compound
Code
IC50
(
m
mol/L)
HL-60
Hela
Ara-C
Ara-C
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
0.118 ꢃ 0.093
2.036 ꢃ 0.160
32.294 ꢃ 3.584
31.875 ꢃ 2.5
464.433 ꢃ 10.009
4.993 ꢃ 0.009
26.000 ꢃ 2.000
13.031 ꢃ 1.002
2.996 ꢃ 0.018
8.022 ꢃ 0.122
9.000 ꢃ 1.000
1.000 ꢃ 0.036
39.057 ꢃ 3.004
10.000 ꢃ 2.000
5.999 ꢃ 0.990
31.996 ꢃ 5.990
12.051 ꢃ 7.029
5.000 ꢃ 1.000
40.999 ꢃ 1.999
19.01 ꢃ 9.004
C10-Val-Ara-C
C14-Val-Ara-C
C18-Val-Ara-C
C10-Met-Ara-C
C14-Met-Ara-C
0.280 ꢃ 0.032
0.272 ꢃ 0.065
0.803 ꢃ 0.090
2.978 ꢃ 0.054
1.706 ꢃ 0.018
1.147 ꢃ 0.036
0.584 ꢃ 0.090
0.405 ꢃ 0.036
1.703 ꢃ 0.068
1.860 ꢃ 0.074
0.502 ꢃ 0.011
1.136 ꢃ 0.065
4. Conclusions
C18-Met-Ara-C
C10-Tyr-Ara-C
C14-Tyr-Ara-C
C18-Tyr-Ara-C
C10-Arg-Ara-C
C14-Arg-Ara-C
C18-Arg-Ara-C
C10-Glu-Ara-C
A
series of fatty acid–amino acid–Ara-C analogues were
synthesized. The fatty acids used were of C10, C14 and C18, and
amino acids include valine, methionine, tyrosine, glutamate and
arginine. The chemical structures of the derivatives and interme-
diates were confirmed using NMR and MS. The derivatives were
characterized by melting point, light rotation and partition coeffi-
cient. It was found that the derivatives synthesized were more
lipophilic than Ara-C with log P in the range of 0–1. Their
C14-Glu-Ara-C
C18-Glu-Ara-C

3600
B. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3596–3600
anti-tumor activity determined in HL-60 and Hela cells showed
that the derivatives were more active in Hela cells than Ara-C while
most of them demonstrated similar activities to Ara-C in HL-60
cells. Among the Ara-C derivatives synthesized, those containing
methionine demonstrated the best activity. In addition, the length
of fatty acids in the derivatives seemed to have an impact on the
activity observed and it was shown that the activity decreased as
the chain length of the fatty acid increased. However, their in vivo
anti-tumor activity determined in mice bearing S180 tumor was not
conclusive with some of the derivatives being more active while
others being less active than Ara-C.
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Appendix. Supplementary material
Supplementary data associated with this article can be found in
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