Novel liver-specific cholic acid-cytarabine conjugates with potent antitumor activities: Synthesis and biological characterization

Article abstract of DOI:10.1038/aps.2011.7

Aim: Cytarabine is an efficient anticancer agent for acute myelogenous leukemia, but with short plasma half-life and rapid deamination to its inactive metabolite. The aim of this study was to design and synthesize novel cholic acid-cytarabine conjugates to improve its pharmacokinetic parameters. Methods: The in vitro stability of novel cholic acid-cytarabine conjugates was investigated in simulated gastric and intestinal fluid, mouse blood and liver homogenate using HPLC. The portacaval samples of the conjugates were examined in male Sprague-Dawley rats using LC/MS, and in vivo distribution was examined in male Kunming mice using LC/MS. Antitumor activities were tested in HL60 cells using MTT assay. Results: Cholic acid-cytarabine compounds with four different linkers were designed and synthesized. All the four cholic acid-cytarabine conjugates could release cytarabine when incubated with the simulated gastric and intestinal fluid, mouse blood and liver homogenate. The conjugates 6, 12, and 16 were present in the portacaval samples, whereas the conjugate 7 was not detected. The conjugates 6 and 16 showed high specificity in targeting the liver (liver target index 34.9 and 16.3, respectively) and good absorption in vivo, as compared with cytarabine. In cytarabine-sensitive HL60 cells, the conjugates 6, 12, and 16 retained potent antitumor activities. Conclusion: Three novel cholic acid-cytarabine conjugates with good liver-targeting properties and absorption were obtained. Further optimization of the conjugates is needed in the future.

Full text of DOI:10.1038/aps.2011.7

Acta Pharmacologica Sinica (2011) 32: 664–672
© 2011 CPS and SIMM All rights reserved 1671-4083/11 $32.00
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npg
Original Article
Novel liver-specific cholic acid-cytarabine conjugates
with potent antitumor activities: Synthesis and
biological characterization
*
Dan-qi CHEN, Xin WANG, Lin CHEN, Jin-xue HE, Ze-hong MIAO, Jing-kang SHEN
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
Aim: Cytarabine is an efficient anticancer agent for acute myelogenous leukemia, but with short plasma half-life and rapid deamination
to its inactive metabolite. The aim of this study was to design and synthesize novel cholic acid-cytarabine conjugates to improve its
pharmacokinetic parameters.
Methods: The in vitro stability of novel cholic acid-cytarabine conjugates was investigated in simulated gastric and intestinal fluid,
mouse blood and liver homogenate using HPLC. The portacaval samples of the conjugates were examined in male Sprague-Dawley rats
using LC/MS, and in vivo distribution was examined in male Kunming mice using LC/MS. Antitumor activities were tested in HL60 cells
using MTT assay.
Results: Cholic acid-cytarabine compounds with four different linkers were designed and synthesized. All the four cholic acid-cytarabine
conjugates could release cytarabine when incubated with the simulated gastric and intestinal fluid, mouse blood and liver homoge-
nate. The conjugates 6, 12, and 16 were present in the portacaval samples, whereas the conjugate 7 was not detected. The conju-
gates 6 and 16 showed high specificity in targeting the liver (liver target index 34.9 and 16.3, respectively) and good absorption in vivo,
as compared with cytarabine. In cytarabine-sensitive HL60 cells, the conjugates 6, 12, and 16 retained potent antitumor activities.
Conclusion: Three novel cholic acid-cytarabine conjugates with good liver-targeting properties and absorption were obtained. Further
optimization of the conjugates is needed in the future.
Keywords: cytarabine; cholic acid; liver; antitumor drug
Acta Pharmacologica Sinica (2011) 32: 664–672; doi: 10.1038/aps.2011.7; published online 25 Apr 2011
activity[6]. For example, ancitabine (cyclocytidine), enocitabine
Introduction
1-β-D-Arabinofuranosylcytosine (cytarabine, Ara-C) is an and fostabine have been used clinically in Japan (Figure 1)[7–9]
.
efficient anticancer agent for acute myelogenous leukemia[1, 2]
.
The conjugation of cytarabine through the formation of a
However, its usefulness is limited by its short plasma half-life bond with its amino group at the N4 position may protect the
and rapid deamination to the biologically inactive metabolite, amino group from deamination and change the pharmacoki-
1-β-D-arabinofuranosyluracil (Ara-U) by cytidine deaminase[3]. netic parameters of the cytarabine because new molecules are
Cytidine deaminase is a ubiquitous enzyme found in high often more lipophilic. Behenoyl-Ara-C (Enocitabine, 4), one
concentrations in the intestine, liver, spleen, and kidneys[4]. of the N4-acyl derivatives of saturated fatty acids, has been
When administered by iv, systemic elimination is biphasic found to be superior to its parent compound, cytarabine,
with an initial plasma half-life (t1/2α) of 7–20 min and a termi- likely because of the resistance to cytidine deaminase and the
nal half-life (t1/2β) of 2–3 h[5]. Cytarabine derivatives and con- liberation of Ara-C over a period of time[8]. N4-peptidyl-Ara-
jugates have been designed with the intention of increasing C, like L-valyl-Ara-C, was reported to increase cellular uptake
its stability and lipophilicity as well as avoiding rate-limiting of cytarabine through the involvement of various transport-
cytarabine phosphorylation while maintaining its biological ers[10]. Relatively high activity of cytarabine conjugates was
achieved through the coupling of amino groups of cytarabine
with carboxyl groups of polysaccharide molecules (eg, poly-
galacturonic acid (PGA) and carboxymethylated yeast beta-
D-glucan (CMG)); the in vitro antileukemic activity of these
*
To whom correspondence should be addressed.
E-mail jkshen@mail.shcnc.ac.cn
Received 2010-11-02 Accepted 2011-01-18

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conjugates was comparable or higher than the activity of non- of cytarabine because the activation of cytarabine to triphos-
conjugated/free cytarabine combined with a polysaccharide[11] phate occurs at this position; thus, if the 5’-hydroxyl group
(Figure 2).
is blocked by conjugation, it would prevent its phosphoryla-
The 5’-hydroxyl group is essential for the biological activity tion until free cytarabine is liberated, which would result in
changed pharmacokinetic parameters. The attachment of a
lipidic structure to cytarabine is usually achieved through
the remains of phosphates as in Ara-CDP (arabinosylcytosine
diphosphate), which is a normal cytarabine metabolite preced-
ing activation to its active metabolite Ara-CTP. Steroid-cytar-
abine conjugates are small molecular conjugates that deserved
attention. For example, conjugating cytarabine with predniso-
lone or prednisone by forming a phosphodiester bond at the 5’
position has produced increased activities in L1210 leukemia
models[12]
.
Individual bile acids are endogenous markers of liver trans-
port and synthesis function. The enterohepatic circulation of
bile acid makes it an attractive tool in designing pharmacolog-
ical hybrid molecules and prodrugs with the view of improv-
ing intestinal absorption, increasing the metabolic stability
of pharmaceuticals, specifically targeting drugs to organs
involved in enterohepatic circulation, and sustaining therapeu-
tically reasonable systemic concentrations of active agents[13–19]
.
In light of these characteristics of bile acids, cholic acid was
chosen to modify cytarabine to improve its in vivo function
and to achieve improved liver-targeting, which would make it
possible to use cytarabine for treating liver cancer.
Herein, the synthesis of several cholic acid-cytarabine con-
jugates is described with different linkers. In vitro stability,
portacaval samples and in vivo distribution were tested to
investigate the metabolic process. Furthermore, the antitumor
activities of the conjugates were also tested.
Figure 1. Cytarabine and its derivatives.
Materials and methods
Chemistry
Conjugate 6 consisted of cholic acid linked to cytarabine with
a direct amide bond from cholic acid to cytarabine via ethyl
chloroformate[20]. Cholic acid was linked to cytarabine with a
direct ester bond in conjugate 7 and was synthesized via gen-
eral conditions using dicyclohexylcarbodiimide and 4-dimeth-
ylaminopyridine (Scheme 1).
Conjugates 12 and 16 were designed to have a β-Alanine as
the linker between cholic acid and cytarabine. The N-terminal
of β-Alanine was attached to cholic acid with an amide bond,
whereas the carboxyl group was linked to cytarabine via an
ester bond or an amide bond to form 12 and 16, respectively.
As shown in Scheme 2, commercially available β-Alanine was
directly converted to an ester by thionyl chloride and EtOH.
The resulting ester was coupled to cholic acid through isobu-
tyl chloroformate to yield 10. After the hydrolysis of 10 by
NaOH, compound 12 was then synthesized in similar man-
ner as 7 using the corresponding acid, 11. Cytarabine was
first coupled to a Boc-protected β-Alanine to obtain 14. After
deprotection by HCl in EtOH, the resulting compound, 15,
was reacted with cholic acid to produce 16.
The formation of the amide and ester bonds was deter-
1
Figure 2. Conjugates of cytarabine with other moleculars.
mined by H-NMR. The chemical shift, δ, of hydrogen on
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Scheme 1. Reagents and conditions:
(a) ethyl chloroformate, triethylamine,
N,N-dimethylformamide, -15 ºC to
RT; (b) dicyclohexylcarbodiimide, 4-
dimethylaminopyridine, N,N-dimethyl-
formamide.
Scheme 2. Reagents and
conditions: (a) thionyl chlo-
ride, EtOH; (b) 5, isobutyl chlo-
roformate, triethylamine, N,N-
dimethylformamide, -15 ºC to
RT; (c) 10% NaOH, EtOH; (d)
1, dicyclohexylcarbodiimide,
4-dimethylaminopyridine, N,N-
dimethylformamide; (e) ethyl
chloroformate, triethylamine,
N,N-dimethylformamide,
-15 ºC to RT; (f) HCl in EtOH;
(g) 5, ethyl chloroformate,
triethylamine, N,N-dimethylfor-
mamide, -15 ºC to RT.
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the 5-, 6-, and 5’-carbon of cytarabine was 6.0, 7.78, and 3.85,
Cytarabine, 6, 7, 12, and 16 were weighed (2 mg each), dis-
respectively. When N4 was coupled with the carboxyl group solved in 50 μL DMSO and added to an 800 μL matrix solution
o
of cholic acid (compounds 6 and 7), δ of 5- and 6-hydrogen described above. The mixtures were incubated at 37 C, and
moved to 7.4 and 8.2, respectively; when the 5’-OH was cou- the samples were obtained at different time points (gastric and
pled with the carboxyl group of cholic acid, δ of 5’-H shifted to intestinal fluid: 0, 2, 5, and 24 h; blood and liver homogenate:
4.2 (Figure 3). Similar changes were observed in compounds 0, 1, 3, 5, and 24 h). All target compounds were analyzed
12 and 14.
using an HPLC system (HP1100) with UV-detection at 214 nm.
The separation was performed using a ZORBAX SB-C18 (5 µm,
4.6 mm×150 mm) with a 5 mmol/L ammonium acetate solu-
tion/methanol gradient at a flow rate of 1.0 mL/min (column
temperature was set to 35 oC).
Portacaval sampling
It has been reported that cytarabine deaminates rapidly in the
liver[4]. To determine the forms of the conjugates after intes-
tinal absorption and before reaching liver, portacaval blood
samples were investigated by LC/MS after oral administra-
tion.
Four male Sprague-Dawley rats, weighing 200 to 230 g,
were orally administered conjugates 6, 7, 12, and 16 separately
(each compound was equivalent to 50 mg/kg cytarabine).
The compounds administered were prepared by dissolving
the compounds in propanediol and diluting them with the
same volume of 0.5% carboxyl methyl cellulose (CMC) to 35
mmol/L. The rats were anaesthetized by barbital and the
abdominal cavity was opened. The portavacal vein was intu-
bated and blood samples were collected at 0.25, 0.5, and 1 h.
All of the samples were examined by LC-MS/MS. The analy-
sis was performed using a LCQ Deca XP ion trap mass spec-
trometer (ThermoFinnigan, San Jose, CA, USA), an Atlantis
analytical HILIC Silica column (2.1 mm×100 mm, 5 µm) from
Waters (made in Ireland); a mixture of methanol, 5 mmol/L
ammonium acetate solution, water and acetonitrile contain-
ing 0.1% acetic acid was used as the mobile phase [Mobile
phases A and B consisted of methanol and 5 mmol/L ammo-
mium acetate; Mobile phases C and D consisted of water and
acetonitrile containing 0.1% acetic acid, respectively. Gradient
chromatographic separation using mobile phases A, B, C, and
D was as follows: 0 min (2% A, 5% B, and 93% D), 2.1 min (2%
A, 5% B, and 93% D) , 2.5 min (2% A, 5% B, 60% D, and 33%
C), 3.0 min (2% A, 5% B, 60% D, and 33%C), 3.5 min (2% A, 5%
B, and 93% D) and finished at 6.5 min at a constant flow rate
of 0.3 mL/min]. The MS/MS reactions selected to monitor
Figure 3. 1H-NMR spectrum of cytarabine, compound 6 and 7.
In vitro stability
Taking into account the possibility of the oral administration cytarabine and Acyclovir (internal standard) were the transi-
of the conjugates, the stability of all the newly synthesized tions from m/z 244 → m/z 112 and m/z 226 → m/z 152 with col-
compounds was tested in simulated gastric and intestinal lision energies of 32% and 30% of the instrumental maximum
fluids, mouse blood and liver homogenate. After incubation for MS2 experiments, respectively.
with the four matrixes, samples at different time points were
subjected to HPLC to determine the extent of metabolism.
In vivo distribution
Simulated gastric and intestinal fluids were prepared Male Kunming mice, weighing 18 to 20 g, were administered
according to the Chinese Pharmacopoeia. Mouse blood was cytarabine (50 mg/kg), compound 6 or 16 orally, which was
collected, and heparin was added; the solution was then incu- equivalent to 50 mg/kg cytarabine. Blood and liver samples
o
bated at 37 C. Mouse liver homogenate was prepared using were collected at different time points. All of the samples
a buffer containing 50 mmol/L HEPES (pH 7.4), 150 mmol/L were examined by LC/MS.
NaCl, 10% glycerol, 1% Tween 20, 1.5 mmol/L MgCl2, 10
The liver target index was calculated by the following
formula:
[21]
mmol/L Na3PO4, 100 mmol/L NaF, and 2 mmol/L Na3VO4
.
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AUC0–t in liver
AUC0–t in plasma
compounds for 72 h. At the end of the treatment, 20 μL of
MTT (5 mg/mL) was added to each well and the plates were
incubated at 37 ºC for 4 h. Then “triplex solution” (10% SDS-
5% isobutanol-HCl 12 mmol/L) was added, and the plates
were incubated at 37 ºC for 12–20 h. The OD value was read
on a plate reader at 570 nm. The inhibition rate was calcu-
lated by the following equation: inhibition rate of proliferation
(%)=(ODcontrol–ODexperiment)/ODcontrol×100%.
Liver target index (LTI)=
Normal, non-fasted male Kuming mice were administered
ig with a dose of 50 mg/kg of cytarabine, 130 mg/kg of 6 and
145 mg/kg of 16, respectively. At specific times after admin-
istration, mice were anesthetized, and blood was drawn. The
liver was removed, rapidly frozen on crushed ice and then
stored at -20 °C. The livers were slit, added to methanol with
THU (final concentration of 0.4 mmol/L) and homogenized.
The centrifugation was carried out at 3500 revolutions per
minute and 4 °C for 5 min. A 100 μL aliquot of supernatant
was added to 25 μL of the internal standard (Acyclovir) and
100 μL of acetonitrile and mixed by vortexing for 1 min. After
centrifugation, the supernatant was transferred to a glass tube
and evaporated until dry using an evaporator at 40 °C under
a stream of nitrogen. The dried extract was then reconstituted
in 100 μL of the mobile phase and a 20 μL aliquot was injected
into the LC-MS/MS system for quantitative analysis.
Blood was added to THU (final concentration of 0.1
mmol/L) and chilled on crushed ice. The plasma was then
separated by centrifugation (11 000 revolutions per minute,
4 °C), frozen and stored at -20 °C. The mouse plasma samples
were prepared using the protein precipitation technique. A
100 μL aliquot of an acetonitrile solution containing 25 μL of
internal standard (2 μg/mL, Acyclovir) was added to 25 μL of
plasma, and mixed by vortexing for 1 min. After centrifuga-
tion, the supernatant was transferred to a glass tube and evap-
orated at 40 °C under a stream of nitrogen. The dried extract
was then reconstituted in 100 μL of mobile phase, and a 20
μL aliquot was injected into the proposed LC-MS/MS system
for quantitative analysis. The analysis was performed using
a triple quadrupole mass spectrometer, TSQ Quantum Ultra
(ThermoFinnigan, San Jose, CA, USA), equipped with an ESI
source. Cytarabine and internal standard (IS) were analyzed
by a Waters HILIC column (150 mm×2.1 mm id, 5 μm) and
protected by a SecurityGuard C18 guard column (4 mm×3.0
mm id). A mixture of acetonitrile-10 mmol/L ammonium
acetate-formic acid (90:10:0.1, v/v/v) was used as the mobile
phase at a flow rate of 0.70 mL/min. The retention time of
cytarabine was approximately 6.6 min. Compounds 6, 16, and
IS were analyzed by a Venusil ASB-C18 column (150 mm×4.6
mm id, 5 μm) and protected by a SecurityGuard C18 guard col-
umn (4 mm×3.0 mm id). A mixture of acetonitrile-10 mmol/L
ammonium acetate-formic acid (70:30:0.1, v/v/v) was used as
the mobile phase at a flow-rate of 0.60 mL/min. The retention
time of 6 and 16 was approximately 4 min. The quantification
was performed using the selective reaction monitoring (SRM)
mode to study parent → product ion (m/z) transitions for
cytarabine (244 → 112 and 212), 6 (634 → 112, 337, and 502), 16
(705 → 426 and 573) and IS (227 → 163), respectively.
Results and discussion
The in vitro stability test showed that all of the cholic acid-
cytarabine conjugates might release cytarabine when incu-
bated with the four matrixes (Table 1). According to the
results, gradual degradations of 6 and 16, which contain
amide bonds, were found in acid gastric fluid (t1/2<8 h);
released cytarabine was detected in the sample of conjugate
12 in mouse blood and liver homogenate (t1/2>8 h). Conju-
gate 7, which had cytarabine directly linking to cholic acid via
an ester bond, was completely converted into cytarabine in
mouse blood within one hour.
The conjugated forms of 6, 12, and 16 were observed in
portacaval samples, among which 16 had the most obvious
MS characteristic peak. A small amount of cytarabine was
detected at one hour only after the administration of conju-
gate 12. Neither the conjugate nor released cytarabine was
observed when conjugate 7 was administered. The results
showed that either conjugate 7 was not absorbed by the intes-
tine or was metabolized to an unknown form (Figure 4).
Table 2 showed in vivo pharmacokinetics results of cytara-
bine, 6, and 16. The concentration of the compounds in the
plasma, after the administration of cytarabine, reached their
peaks at 0.5 h, and only trace amounts were left in the blood
after 4 h. The Cmax was 1.14 μg/mL. The Cmax, Tmax, and Auc0–t
of the livers were all lower than those of the plasma; addi-
tionally, the elimination rate in liver was found to be much
faster (Figure 5A). The released cytarabine of conjugate 6 was
observed in the plasma samples but not in the liver. The Cmax
of the conjugate in the plasma and liver were 0.091 and 3.25
μg/mL respectively, and 0.188 μg/mL for released cytarabine
in plasma (Figure 5B). Conjugate 16 also released cytarabine
in vivo. The Cmax of the conjugate in plasma and liver were
0.826 and 14.868 μg/mL, whereas released cytarabine lev-
els in plasma and liver were 0.097 and 0.455 μg/mL (Figure
5C), respectively. Although 12 was present in the portacaval
samples, only a small amount of the conjugate form could be
detected in the liver, and it was not found in plasma. Released
cytarabine was not detected in the liver or plasma. Thus, data
on conjugate 12 were not provided. According to the calcula-
tion of AUC0–t, the liver targeting index was 0.17 for cytara-
bine, 34.9 for 6, and 16.3 for 16.
Both conjugates 6 and 16 showed good liver targeting com-
pared to cytarabine. The LTI of 6 reached 34.9, which was
200 times more than cytarabine. The LTI of 16 also reached
16.3, and the Cmax was 14.868 μg/mL, which demonstrated
good absorption. The stability of the amide bond prevented
the conjugates from degradation through oral absorption,
Antitumor activity
Compounds were tested for their cytotoxic effects against a
leukemia HL60 cell line by methyl-thiazol-tetrozolium (MTT)
assays[22]. Cells were treated with different concentrations of
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Table 1. Results of the in vitro stability test.
Conditions
Tested
Samples
Simulated gastric fluid
2 h 5 h 24 h 0 h 2 h 5 h
100 100 100 100 100 100 100 100
Simulated intestinal fluid
24 h 0 h
100 100
100 100
Mouse blood
3 h 5 h
100
Mouse liver homogenate
0 h
1 h
24 h 0 h 1 h
3 h
5 h
100
24 h
Ara-C
100
100
100 100
100
66.89
6
7
6
Ara-C
100 84.51 50.71 34.30 100 100 100 100
15.49 49.29 65.70
100
0
100
0
100
0
100 95.15 91.74 91.40 87.88
4.85 8.26 8.60 12.12
0
0
0
0
0
0
0
0
7
100 100 100
78.14 100 100 100 100
21.86
100
0
0
100
0
100
NT
NT
NT
NT
100 100
100
0
100
0
100
0
Ara-C
0
0
0
0
0
0
0
0
0
12 12
Ara-C
100 100 100
90.66 100 100 100 80.40 100 81.66 62.84 55.07 16.87 100 82.51 NT
9.34 19.60 18.34 37.16 44.93 83.13 17.49 NT
78.83 72.25
21.17 27.75
0
0
0
0
0
0
0
0
16 16
Ara-C
100 78.48 56.12 21.34 100 100 100 100
21.52 43.87 78.66
100 100
100
0
100
0
100
0
100 NT
100
0
100
0
83.29
16.71
0
0
0
0
0
0
0
0
NT
NT: not tested.
*
Table 2. Pharmacokinetic parameters of cytarabine, 6 and 16 after oral administration to Kunming mice.
tmax
h
Cmax
μg/mL
AUC0–t
μg·h/mL
AUC0–∞
μg·h/mL
MRT
h
t1/2
h
Compound
Ingredients
Matrix
LTI
Cytarabine
Cytarabine
Plasma
Liver
0.50
0.25
1.14
0.361
1.94
0.339
1.95
0.562
1.22
1.59
0.55
1.02
0.17
6
6
Plasma
Liver
Plasma
1.0
1.0
0.50
–
0.091
3.25
0.188
–
0.177
6.17
0.371
–
0.188
6.22
0.532
–
1.67
1.50
3.00
–
0.86
0.79
1.97
–
34.9
–
Cytarabine
**
Liver
16
16
Plasma
Liver
Plasma
Liver
0.083
0.083
2.0
0.826
14.868
0.097
0.455
0.472
7.716
0.387
0.485
0.491
7.962
0.443
0.542
2.79
1.37
3.27
1.61
3.47
1.73
2.35
1.45
16.3
1.25
Cytarabine
0.5
*
: All the pharmacokinetic parameters were calculated using DAS 2.0.
**
: Not detected.
whereas in vivo, the bond could be metabolized, which made it
an appropriate link. The Cmax of 16 was approximately 4 times
larger than conjugate 6, which showed that using β-Alanine as
the linker might increase cytarabine’s absorption.
The antitumor activities of conjugates 6, 16, and 12 were
tested on HL60 cells (Table 3). All three compounds showed
potent antitumor activities on cytarabine-sensitive HL60 cells,
which indicated that conjugating cytarabine to cholic acid
allowed its antitumor activities to be retained.
In conclusion, a new series of cholic acid-cytarabine con-
jugates with antitumor activities have been described. Some
of them exhibited excellent targeting of the liver and good
absorption compared to cytarabine alone. Further studies will
Figure 4. All the samples were detected by LC/MS. The ordinate represen-
ted the peak-area ratio of the conjugates to the internal standard (Acyclo-
vir).
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Figure 5. A) Plasma and liver concentrations following oral administration of Ara-C (50 mg/kg). B) Plasma and liver concentrations following oral
administration of 6 (130 mg/kg). C) Plasma and liver concentrations following oral administration of 16 (145 mg/kg). Mean±SD. n=3.
o
Table 3. Antitumor activities on HL60 cells after 72 h. Mean±SD. n=3.
(DMF, 20 mL). The mixture was cooled to -15 C, and ethyl
chloroformate (0.31 mL, 3 mmol) was then added. After being
Compounds
IC50 (μmol/L)
stirred for 20 min, cytarabine (0.69 mg, 3 mmol) in 15 mL DMF
was added the mixture, dropwise. The reaction mixture was
stirred at -15 oC for 1 h and at rt overnight. The mixture was
filtered, and the filtrate was concentrated. The residue was
subjected to silica gel chromatography (dichloromethane/
methanol, 10:1 to 4:1) to yield 6 as a white solid (1.34 g, 75%).
mp 190–192 oC.
Cytarabine
0.19±0.13
2.09±1.23
0.99±0.65
2.96±2.03
6
12
16
IR (KBr cm-1): 3396, 2933, 2868, 1711, 1649, 1566, 1493, 1389,
1315, 1117, 1076, 1047, 980, 951, 804, 609, 559.
continue by developing more conjugates with different link-
ers. Biological evaluations will be conducted on conjugates 6
and 16 in the near future.
1H-NMR (CD3OD, 300 MHz): δ 8.22 (d, 1H, J=7.6 Hz), 7.4 (d,
1H, J=7.1 Hz), 6.2 (d, 1H, J=3.8 Hz), 4.25 (m, 1H), 4.08 (t, 1H),
4.01 (m, 1H), 3.94 (s, 1H), 3.8 (m, 3H), 2.6–1.05 (m, 25H), 1.02
(d, 3H, J=6.2 Hz), 0.9 (s, 3H), 0.7 (s, 3H).
Experimental
13C NMR (100 MHz, d6-DMSO) δ 174.5, 162.3, 154.7, 146.7,
94.5, 87.1, 85.3, 76.2, 74.7, 71.1, 70.6, 66.4, 61.1, 46.3, 45.8, 41.6,
41.5, 35.4, 35.3, 34.9, 34.5, 33.7, 31.0, 30.4, 28.6, 27.4, 26.3, 22.9,
22.7, 17.1, 12.4.
The reagents were purchased from Alfa-Aesar (Karlsruhe,
Germany), Acros (Geel, Belgium) and Shanghai Chemical
Reagent Company (Shanghai, China) and were used without
further purification. Analytical thin-layer chromatography
was performed on HSGF 254 (150–200 μm thickness; Yantai
Huiyou Company, Yantai, Shandong, China). The IR spectra
were recorded on a Nicolet Magna 750 FTIR spectrometer, and
the 1H NMR (300 MHz or 400 MHz) spectra were recorded on
MS (ESI): m/z 634 (M+H)+; HRMS calcd for C33H51N3O9Na:
656.3523 (M+Na)+; Found: 656.3536.
(4R)-((2R,3S,4S)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydro-
a Varian Mercury-300 or a 400 High Performance Digital FT- xytetrahydrofuran-2-yl)methyl-4-((3R,5S,7R,9S,10S,12S,13R,
NMR using tetramethylsilane as an internal standard. The 13
14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-
NMR (100 MHz) spectra were determined using a Varian Mer- cyclopenta[a]phenanthren-17-yl)pentanoate (7)
C
cury-400 High Performance Digital FT-NMR. HPLC analysis
was performed using a Gilson HPLC system with UV detec-
tion at 214 and 254 nm. UV scanning was performed on an
Agilent HP1100. LC-MS spectra were obtained using a LCQ
Deca XP ion trap mass spectrometer (ThermoFinnigan, San
Jose, CA, USA). Accurate mass measurements were carried
out on a Q-TOF ultima Globe mass spectrometer (Micromass,
Manchester, UK).
Cytarabine (1.46 g, 6 mmol) was dissolved in dry DMF (30
mL) and heated to 60 C. Cholic acid (3.67 g, 9 mmol), dicy-
o
clohexylcarbodiimide (DCC, 1.79 g, 9 mmol) and 4-dimeth-
ylaminopyridine (DMAP, 109 mg, 0.9 mmol) were added.
The mixture was cooled to rt and stirred for 2 d. Then, a
second addition of cholic acid (3.67 g, 9 mmol), DCC (1.79 mg,
9 mmol) and DMAP (109 mg, 0.9 mmol) was made. The mix-
ture was stirred continuously for 4 d. The reaction mixture
was filtered, and the filtrate was concentrated. The residue
was subjected to silica gel chromatography (dichloromethane/
methanol, 15:1) to yield 7 as a white solid (310 mg, 10%).
mp 204–205 oC.
(4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((3R,5S,7R,12S,
13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-17-yl)pentanamide (6)
Cholic acid (1.16 g, 3 mmol) and triethylamine (TEA, 0.39
mL, 3 mmol) were dissolved in dry N,N-dimethylformamide
IR (KBr cm-1): 3423, 2929, 2868, 1730, 1649, 1491, 1377, 1290,
1076, 1045, 795, 735, 609.
1H-NMR (CD3OD, 300 MHz): δ 7.76 (d, 1H, J=7.8 Hz), 6.22
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Chen DQ et al
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671
(d, 1H, J=3.9 Hz), 5.86 (d, 1H, J=7.5 Hz), 4.57 (m, 1H), 4.20 (m, 13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
2H), 4.07 (m, 1H), 4.01 (m, 1H), 3.94 (brs, 1H), 3.78 (d, 1H, J=2.8 1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propanoate
Hz), 3.37 (m, 1H), 2.50–2.15 (m, 4H), 2.05–1.10 (m, 20H), 1.0 (d, (12)
3H, J=6.5 Hz), 0.9 (s, 3H), 0.65 (s, 3H).
Cytarabine (324 mg, 1.33 mmol) was dissolved in dry DMF
o
13C NMR (100 MHz, CD3OD) δ 176.2, 168.0, 158.6, 145.3, 95.3, (12 mL) and heated to 60 C. Compound 11 (0.96 g, 2 mmol),
89.2, 84.8, 78.9, 76.8, 74.5, 73.3, 69.5, 65.3, 48.6, 48.0, 43.7, 43.5, DCC (453 mg, 2.2 mmol) and DMAP (24.4 mg, 0.2 mmol) were
41.4, 41.0, 37.0, 36.4, 32.8, 32.6, 31.7, 30.1, 29.2, 28.4, 24.7, 23.7, then added. The mixture was cooled to rt and stirred for
18.0, 13.4.
2 d. Another addition of 11 (0.96 g, 2 mmol), DCC (453 mg,
MS (ESI): m/z 634 (M+H)+; HRMS calcd for C33H51N3O9Na: 2.2 mmol) and DMAP (24.4 mg, 0.2 mmol) was made. The
656.3523 (M+Na)+; Found: 656.3531.
mixture continued to stir for 2 d and was then filtered. The
filtration was concentrated, and the residue was subjected to
Ethyl 3-((4R)-4-((3R,5S,7R,9S,10S,12S,13R,14S,17R)-3,7,12-tri- silica gel chromatography (dichloromethane/methanol, 15:1)
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan- to yield 12 as a white solid 200 mg (15%).
thren-17-yl)pentanamido)propanoate (10)
Thionyl chloride (20 mL) was added dropwise to anhydrous
ethanol (80 mL), and the temperature did not exceed 25 C. (d, 1H, J=3.5 Hz), 5.87 (d, 1H, J=7.6 Hz), 4.51 (m, 1H), 4.27 (m,
β-Alanine (9 g, 0.1 mol) was then added. After stirring at 50 oC 1H), 4.18 (m, 1H), 4.08 (m, 1H), 4.02 (m, 1H), 3.93 (brs, 1H),
for 3 h, β-Alanine dissolved. The mixture was stirred at rt 3.78 (m, 1H), 3.44 (t, 2H, J=6.9 Hz), 2.58 (t, 2H, J=6.3 Hz), 2.4–
overnight. The solvent was evaporated and ether was added 1.1 (m, 25H), 0.99 (d, 3H, J=6.4 Hz), 0.90 (s, 3H), 0.69 (s, 3H).
mp 174–176 oC.
1H-NMR (CD3OD, 300 MHz): δ 7.72 (d, 1H, J=7.4 Hz), 6.20
o
to the residue. A white solid was formed, filtered and then
13C NMR (100 MHz, CD3OD) δ 177.5, 173.6, 168.0, 158.6,
dried (Na2SO4) to yield 16 g 9 (100%). Cholic acid (5.57 g, 13.6 145.0, 95.3, 89.2, 84.6, 79.0, 76.8, 74.5, 73.3, 69.5, 65.6, 55.3, 48.4,
mmol) and triethylamine (TEA, 1.92 mL, 15 mmol) were dis- 47.9, 43.6, 43.4, 41.5, 40.9, 37.3, 36.9, 36.8, 36.4, 36.3, 35.4, 34.5,
solved in dry DMF (45 mL). The mixture was cooled to -15 oC, 33.7, 31.6, 30.0, 29.2, 28.3, 24.7, 23.7, 18.2, 13.5.
and isobutyl chloroformate (1.78 mL, 17 mmol) was added.
MS (ESI): m/z 705 (M+H)+; HRMS calcd for C36H56N4O10Na:
After stirring for 5 min, 9 (2.3 g, 20 mmol) and TEA (3.84 mL, 727.3894 (M+Na)+; Found: 727.3913.
30 mmol) were combined with 10 mL DMF, which was added
o
dropwise. The reaction mixture was stirred at -15 C for 1 h tert-butyl 3-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetra-
and at rt overnight. The mixture was filtered, and the filtra- hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylamino)-3-oxopro-
tion was concentrated. The residue was dissolved in ethyl pylcarbamate (14)
acetate and washed with water 4 times (water/ethyl acetate, Boc-β-Ala-OH (945 mg, 5 mmol) and triethylamine (0.6 mL, 5
1:2). The organic layer was combined and washed with 0.5 mmol) were dissolved in dry DMF (30 mL). The mixture was
o
mol/L NaOH, 0.5 mol/L HCl and brine; it was then dried and cooled to -15 C, and ethyl chloroformate (0.48 mL, 5 mmol)
concentrated to yield 10 as a white solid 5.67 g (87%).
was added. After stirring for 30 min, cytarabine (1.22 g, 5
1H-NMR (CD3OD, 300 MHz): δ 4.12 (m, 2H), 3.94 (brs, 1H), mmol) in 10 mL DMF was added dropwise to the mixture.
3.78 (brs, 1H), 3.4 (m, 3H), 2.5 (t, 2H, J=6.6 Hz), 2.4–1.2 (m, The reaction mixture was stirred at -15 oC for 30 min and then
24H), 1.24 (t, 3H, J=7.2 Hz), 1.0 (d, 3H, J=6.4 Hz), 0.9 (s, 3H), at rt overnight. The reaction mixture was filtered, and the
0.7 (s, 3H).
filtration was concentrated and subjected to silica gel chroma-
tography (dichloromethane/methanol, 20:1) to yield 14 as a
white solid (1.7 g, 82%).
MS (ESI): m/z 1015 (2M+H)+.
3-((4R)-4-((3R,5S,7R,9S,10S,12S,13R,14S,17R)-3,7,12-tri-
1H-NMR (CD3OD, 300 MHz): δ 8.24 (d, 1H, J=7.5 Hz), 7.42
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan- (d, 1H, J=7.5 Hz), 6.19 (d, 1H, J=3.9 Hz), 4.25 (m, 1H), 4.08 (t,
thren-17-yl)pentanamido)propanoic acid (11)
1H, J=2.4 Hz), 4.01 (m, 1H), 3.81 (d, 2H, J=4.8 Hz), 3.36 (t, 2H,
Compound 10 (3.9 g, 7.7 mmol) was dissolved in ethanol (30 J=6.6 Hz), 2.62 (t, 2H, J=6.3 Hz), 1.41 (s, 9H).
mL) and added to a solution of 10% NaOH (30 mL). The reac-
tion mixture was stirred at rt overnight and then poured
MS (ESI): m/z 415 (M+H)+.
into ice water and acidified with 1 mol/L HCl, which was (4R)-N-(3-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetra-
extracted with ethyl acetate 3 times. The organic layer was hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylamino)-3-oxopro-
combined and washed with brine, dried (Na2SO4) and concen- pyl)-4-((3R,5S,7R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-
trated to yield 11 as a white solid 3.2 g (87%).
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
1H-NMR (CD3OD, 300 MHz): δ 3.95 (brs, 1H), 3.8 (brs, 1H), yl)pentanamide (16)
3.4 (t, 3H, J=7 Hz), 2.5 (t, 2H, J=6.8 Hz), 2.4–1.05 (m, 24H), 1.0 Compound 14 (700 mg, 1.7 mmol) was dissolved in saturated
(d, 3H, J=6.1 Hz), 0.9 (s, 3H), 0.7 (s, 3H).
HCl in ethanol (5 mL) and stirred at rt overnight. The
solvent was evaporated and methanol (10 mL) was added to
the residue then stirred for 1 h. The solid was filtered and
MS (ESI): m/z 959 (2M+H)+.
((2R,3S,4S)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxy- washed with methanol. The white solid was dried, and the
tetrahydrofuran-2-yl)methyl- 3-((4R)-4-((3R,5S,7R,9S,10S,12S, crude product 15 (400 mg) was obtained, which was used in
Acta Pharmacologica Sinica

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Chen DQ et al
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5
6
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the next step without purification.
Cholic acid (408 mg, 1 mmol) and triethylamine (0.12 mL, 1
mmol) were dissolved in dry DMF (6 mL). The mixture was
o
cooled to -15 C, and ethyl chloroformate (0.1 mL, 1 mmol)
was added. After stirring for 20 min, compound 15 (351 mg,
1.1 mmol) was added to the mixture by a dropwise addition
in 5 mL DMF. The reaction mixture was stirred at -15 C for
30 min and at rt overnight. The reaction mixture was filtered
and concentrated. Subsequently, the residue was subjected to
silica gel chromatography (dichloromethane/methanol, 20:1 to
5:1) to yield 16 as a white solid (300 mg, total yield: 82%).
mp 178–180 oC.
7
o
8
9
1H-NMR (CD3OD, 300 MHz): δ 8.24 (d, 1H, J=7.8 Hz), 7.46
(d, 1H, J=7.7 Hz), 6.19 (d, 1H, J=4.1 Hz), 4.28 (m, 1H), 4.09 (m,
1H), 4.01 (m, 1H), 3.89 (s, 1H), 3.8 (m, 3H), 3.47 (t, 2H, J=6.4
Hz), 2.66 (t, 2H, J=6.2 Hz), 2.4-1.01 (m, 25H), 0.98 (d, 3H, J=6.3
Hz), 0.9 (s, 1H), 0.65 (s, 1H).
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macol 2006; 58: 927–32.
11 Kery V, Novotny L, Tihlarik K, Haplová J, Kacuráková M, Sandula J, et
al. Preparation, properties and antileukemic activity of arabinosylcyto-
sine polysaccharide conjugates. Int J Biochem 1990; 22: 1203–7.
12 Hong CI, Nechaev A, West CR. Nucleoside conjugates as potential
antitumor agents. 2. Synthesis and biological activity of 1-(beta-D-ar-
abinofuranosyl)cytosine conjugates of prednisolone and prednisone.
J Med Chem 1979; 22: 1428–32.
13 Sievanen E. Exploitation of bile acid transport systems in prodrug de-
sign. Molecules 2007; 12: 1859–89.
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15 Tolle-Sander S, Lentz KA, Maeda DY, Coop A, Polli JE. Increased acy-
clovir oral bioavailability via a bile acid conjugate. Mol Pharm 2004; 1:
40–8.
13C NMR (100 MHz, d6-DMSO) δ 172.9, 172.2, 162.1, 154.6,
146.7, 94.5, 87.1, 85.8, 76.2, 74.6, 71.1, 70.5, 66.4, 61.1, 46.2, 45.8,
41.6, 41.4, 36.5, 35.4, 35.2, 34.9, 34.5, 32.5, 31.8, 30.8, 30.4, 28.5,
27.4, 26.3, 22.9,22.7, 17.2, 12.4.
MS (ESI): m/z 705 (M+H)+; HRMS calcd for C36H56N4O10Na:
727.3894 (M+Na)+; Found: 727.3898.
Acknowledgements
This work was financially supported by National Science &
Technology Major Project “Key New Drug Creation and Man-
ufacturing Program” of China (No 2009ZX09301-001).
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17 Gabano E, Ravera M, Osella D. The drug targeting and delivery ap-
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18 Jurcek O, Wimmer Z, Svobodova H, Bennettova B, Kolehmainen E,
Drasar P. Preparation and preliminary biological screening of cholic
acid-juvenoid conjugates. Steroids 2009; 74: 779–85.
Author contribution
Jing-kang SHEN and Dan-qi CHEN designed research; Dan-
CHEN, Xin WANG, Lin CHEN, Jin-xue HE and Ze-hong
MIAO performed research; Dan-qi CHEN and Xin WANG
contributed new analytical tools and reagents; Dan-qi CHEN
and Xin WANG analyzed data; and Dan-qi CHEN wrote the
paper.
19 Rohacova J, Marin ML, Martinez-Romero A, Diaz L, O’Connor JE,
Gomez-Lechon MJ, et al. Fluorescent benzofurazan-cholic acid conju-
gates for in vitro assessment of bile acid uptake and its modulation by
drugs. ChemMedChem 2009; 4: 466–72.
20 Hu P, Hou S, Jin H, Zhang J, Wang L. Synthesis and stability of bile
acid conjugate lamivudine as a liver target prodrug. Central South
Pharmacy 2005; 3: 13–5.
21 Ju J, Gitcho MA, Casmaer CA, Patil PB, Han D, Spencer SA, et al. Po-
tentiation of insulin-stimulated glucose transport by the AMP-activated
protein kinase. Am J Physiol Cell Physiol 2007; 292: 564–72.
22 Qing C, Miao ZH, Tong LJ, Zhang JS, Ding J. Actinomycin D inhibiting
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Acta Pharmacologica Sinica