Selective bone targeting 5-fluorouracil prodrugs: Synthesis and preliminary biological evaluation

Article abstract of DOI:10.1016/j.bmc.2011.05.004

Bone tumor is a notoriously difficult disease to manage, requiring frequent and heavy doses of systemically administered chemotherapy. Targeting anticancer drug to the bone after systemic administration may provide both greater efficacy of treatment and l

Full text of DOI:10.1016/j.bmc.2011.05.004

Bioorganic & Medicinal Chemistry 19 (2011) 3750–3756
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective bone targeting 5-fluorouracil prodrugs: Synthesis and preliminary
biological evaluation
Liang Ouyang a,b, , Dongsheng He b, , Jing Zhang c, Gu He a,b, Bo Jiang b, Qiang Wang a,b, Zhengjun Chen a,b
,
⇑
,
Junzhu Pan b, Yanhua Li b, Li Guo b,
⇑
a State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China
b Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University,
Chengdu 610041, PR China
c Feed Quality Inspection Center of Agriculture Ministry, Chengdu 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bone tumor is a notoriously difficult disease to manage, requiring frequent and heavy doses of systemi-
cally administered chemotherapy. Targeting anticancer drug to the bone after systemic administration
may provide both greater efficacy of treatment and less frequent administration. In this paper, a series
of bone targeting Asp oligopeptides 5-fluorouracil conjugates have been synthesized in a convergent
approach and well characterized by NMR and MS techniques. Their hydroxyapatite (HAP) affinity, drug
release and cytotoxicity characteristics were evaluated in in vitro conditions. All the prodrugs were water
soluble and exhibited high affinity to HAP .The efficient release of the active drug moiety occurring by the
cleavage of different linkage in physiological conditions significantly reduced the number of viable
human cancer cells. From in vivo distribution, we get these compounds with high bone-selectivity and
long halflife. These results provided an effective entry to the development of new bone targeting chemo-
therapeutic drugs.
Received 26 March 2011
Revised 2 May 2011
Accepted 2 May 2011
Available online 8 May 2011
Keywords:
Bone targeting
5-Fluorouracil
Asp oligopeptides
Drug release
Prodrugs
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
structures of bone targeting oligopeptides were shown in Fig. 1)
In addition, unlike the P–C–P bond of bisphosphonates, the oligo-
Bone tumors are classified as ‘primary tumors’ which originate
in the bone, including osteoma, osteoid osteoma, osteochondroma,
osteoblastoma, enchondroma, giant cell tumor of bone, etc. and
‘secondary tumors’ which originate elsewhere. Nowadays, bone tu-
mors especially bone metastases become one of the most common
cancers in the world.1,2 However, it is generally difficult to treat
through drug chemotherapy because of the special component of
bone tissue.3 Bones which are mostly composed of the inorganic
compound hydroxyapatite (HAP), lack circulating systems and
have a very low blood flow. Current methodology using selective
bone targeting drug delivery through binding HAP is able to specif-
ically activate prodrugs at precise bone locations and looks very
promising.4–6 Therefore, some scientists developed a series of drug
targeting systems using bisphosphonates and other bone targeting
moieties and achieved some results.7–9 Recently, linear acidic
oligopeptides which posses Asp repetitive sequences have been
identified as high-affinity binding site for HAP.10 (The chemical
peptides are biologically labile and enzymatically degraded. The
advantageous property can make drug release more efficient and
no unexpected long-term effects.11 Bone targeting prodrugs based
on Asp oligopeptides have been studied in the treatment of bone
diseases by Miyamoto and co-workers,12,13 Wang et al.14,15 and
our group16,17 such as osteoporosis and osteomyelitis. As parts of
our continuing efforts, in order to construct more effective bone
targeting prodrugs based on Asp oligopeptides and find out a po-
tential bone site-specific chemotherapeutic drug treatment, we
chose a well-known broad spectrum anti-cancer drug 5-fluoroura-
cil (5-FU) as a model drug and hope we can prepare a new kind of
selective bone targeting 5-fluorouracil prodrugs bearing Asp oligo-
peptides and find out its use in bone tumor treatment. Like other
bone-targeting drug delivery systems, this prodrug strategy is hop-
ing to reduce the chemotherapeutic drugs administration dose and
reduce the side effect in normal tissues.
A classic way of prodrug design is that the active molecules
should be linked by labile and hydrolyzable bonds.18 The active
drugs were easily cleavable: the removal of the prodrug periphery
groups, liberation of the terminal and subsequent unzipping of the
scaffold lead to release the parent drugs. To create a 5-FU-aspatic
oligopeptides conjugate, it was of primary importance to deter-
mine the manner in which 5-FU attached to the exposed amino
⇑
Corresponding authors. Tel.: +86 028 85503817; fax: +86 028 85164060 (L.O.);
tel.: +86 028 85503777; fax: +86 028 85502629 (L.G.).
E-mail addresses: ouyangliang@scu.edu.cn (L. Ouyang), rosaguoli2000@yahoo.
com.cn (L. Guo).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.004

L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
3751
O
O
O
O
O
O
O
O
O
O
O
NH
O
O
NH
O
O
O
O
O
NH
O
O
O
O
H2N
H2N
NH
O
NH
O
O
O
NH
O
NH
O
O
1b
1a
O
O
O
O
O
O
O
O
O
NH
O
O
O
NH
O
O
O
O
H2N
NH
O
NH
O
NH
O
1c
Figure 1. Structures of the fully protected Asp4–6 (1a–c).
of Asp. In the most case of 5-FU prodrugs designing, attaching
directly to the N-1 of 5-FU by the use of a carbonate or carbamate
linker was chosed.19,20 However, some recent studies have shown
that the N–C bond of N-1 alkyl-substituted 5-FU derivates was
too stable to release and such prodrugs exhibited low therapeutic
effect when tested against human tumor cell lines.21,22 To solve
this problem, experts proposed the concept of adding a labile long-
er carbon chain or more cleavable chemical bond as a self-immola-
was outlined in Scheme 2. 5-Fluorouracil reacted with formalde-
hyde to give N, N-1, 3-dimethylol-5-fluorouracil, the N-3 substi-
tuted 5-FU derivatives were relatively unstable and C–N bond
was easily cleavable, and then condensed with benzyl succinate
in the presence of DCC (dicyclohexylcarbodiimide) and DMAP
(dimethylamino-pyridine) to obtain 5-fluorouracil derivatives
compound 5 with a labile ester bond linkage. After purified by sil-
ica gel chromatography column and removed N-3 substituted co-
products, we use the method of Pd/C catalyzed hydrogenation to
obtain compound 7. Then these derivatives condensed with the
protected aspartic acid oligopeptide 1a–c under the condition of
IBCF and NMM, and finally the target compounds 6a–c were
obtained after removal of the protecting groups of the peptide.
The high degree of symmetry in these molecules enabled facile
confirmation of both structure and purity by NMR techniques. For
example, in the 1H NMR spectrum of compound 4a–c, the 5-FU lin-
ker protons observed the resonance signals at 7.69 (d) and 4.12 (s)
were clearly distinguishable from the resonances arising from the
Asp oligopeptides at 2.72 (m) and 4.90 (m) ppm. In the 1H NMR
spectrum of compound 6a–c, succinate protons were observed
the resonance signals at 2.56 (br s). Integration of the respective
areas of all the protons confirmed the complete coupling and the
purity of the target compounds. In the 13C NMR spectrum of all tar-
get molecules, 5-FU signals at near 124.5, 139.0, 145.7, 147.5 ppm.
Furthermore, the structures of these compounds were further
verified by electrospray ionization–mass spectrometry (ESI-MS).
All the spectra displayed a very prominent peak corresponding to
the compounds complexed with protons or sodium cation. More-
over, elemental analysis was also in good agreement with those
of the signed structures.
tive moiety which was further attached through
a stable
carbamate linkage to the amine group.23 Based on the above view-
points, we introduced a longer carbon chain to the N-1 of 5-FU: a
succinate chain which was used as a simple self-immolative linker
was connected between the drug and oligopeptides. We could
compare the different characteristics of drug release and cytotoxic-
ity of prodrugs with the two different linkers.
2. Results and discussion
2.1. Synthesis and characteristic of the prodrugs
The fully protected bone-targeting peptides 1a–c were synthe-
sized by a conventional liquid-phase peptide synthetic method
from Boc–Asp–OBzl utilizing IBCF (isobutyl chloroformate) and
NMM (N-methyl morpholine) (mixed anhydrides method) by a
series of segment condensation which was described in our previ-
ous work.16,17
The synthetic procedure of the N-1 acetic acid modified 5-
fluorouracil prodrugs was outlined in Scheme 1. First, the bromine
acetic acid was conjugated to 5-FU through a simple substitution
reaction to obtain the compound 2. The protected 5-FU-oligopep-
tides conjugates 3a–c was obtained by divergent synthesis of
1a–c with 2 in the presence of IBCF and NMM in anhydrous tetra-
hydrofuran. The activation of the focal point was done by the
removal of benzyl group by catalytic hydrogenolysis giving the tar-
get compound 4a–c.
2.2. Biological evaluation
To demonstrate in vivo activity, the 5-FU prodrugs need not
only to bind to bone but also to release the active 5-fluorouracil.24
These two requirements were evaluated in vitro to predict the
therapeutic potential of these compounds. To study the binding
of these conjugates to bone, an in vitro HAP binding assay was
The synthetic procedure of the N-1 succinate acid modified
5-fluorouracil prodrugs involved several iterative protection–
deprotection steps of orthogonally protected building blocks and

3752
L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
O
O
N
F
O
O
O
HN
F
3a n=3
3b n=4
3c n=5
F
HN
BrCH2COOH
KOH
1a-c,
HN
IBCF,NMM
O
O
OBzl
OBzl
O
N
H
O
N
O
N
H
NH
OBzl
n
OH
O
O
5-FU
3
2
O
F
O
HN
Pd/C, H2
4a n=3
4b n=4
4c n=5
O
OH
OH
O
N
H
N
NH
OH
n
O
O
O
4
Scheme 1. The synthesis of compound 4a–c.
O
O
O
N
F
F
HOH2C
N
F
HN
HN
PhCH2OOCCH2CH2COOH
HCHO
O
N
O
O
N
H
5-FU
O
DCC,DMAP
O
CH2OH
O
5
O
N
O
O
F
O
HN
F
HN
Pd/C, H2
1a-c
, IBCF,NMM
O
OBzl
OBzl
O
O
H
N
8a n=3
8b n=4
8c n=5
O
N
O
NH n
OBzl O
O
OH
O
O
O
8
7
O
O
Pd/C, H2
F
O
O
HN
O
O
OH
OH
O
N
O
6a n=3
6b n=4
6c n=5
H
N
NH n
OH
O
O
6
Scheme 2. The synthesis of compound 6a–c.
Table 1
set up using in vitro HAP binding methods described by Wang
et al.25 The conjugates were dissolved in water with various precise
concentrations and the adsorption amounts were determined by a
UV spectrophotometer at 254 nm to obtain the A–C linear regres-
sion equation. Tetracycline was taken as a positive control in this
experiment. The bound percentage was calculated and the result
was presented in Table 1. From The data presented, all conjugates
of 5-FU exhibited HAP binding capability and the binding could
take effect rapid within 0.5 h. Non-modified 5-FU hardly showed
any binding demonstrating that oligopeptides played an important
role in the HAP binding process. The prodrugs with Asp6 (4c and
6c) were found to be more effective in binding HAP comparing
the well-known bone-targeting substance: tetracycline. The bind-
ing trend was Asp6 >> Asp4 > or = Asp5, the preferential binding
to bone of 4c and 6c exhibit the oligopeptides with six amino acid
residues were more effective to be a bone-targeting moiety. The
structures with multi-carboxy groups provide ionic interaction be-
tween negative charges and calcium ions in the bone mineral com-
ponent. Therefore, we chose 4c and 6c in further biological
evaluation.
Relative binding of analogs 4a–c, 6a–c to hydroxyapatite compared to 5-fluorouracil
and tetracycline binding
Compound
Binding indexa (%)
0.5 h
Over 24 h
5-FU
Tetracycline
0
0
24.1 2.6
14.3 2.4
11.6 0.7
31.4 2.6
16.6 3.2
13.4 0.9
37.8 1.7
56.2 1.8
40.2 3.2
19.8 1.7
62.7 4.3
42.5 1.5
25.5 2.6
69.7 4.3
4a
4b
4c
6a
6b
6c
a
The data represent the mean SD (n = 3).
Another important factor to be considered for the conjugate is
the drug releasing profile.26 Since the conjugate contains different
covalent bond between the drug and the bone-targeting peptides,
it should be enzymatically and/or hydrolytically degradable to

L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
3753
release the drug molecules to bone tissue and the different in vitro
release capabilities of the two types of 5-FU prodrugs can be com-
pared. The precipitate (4c and 6c) was incubated in PBS pH 7.4 or
50% (v/v in PBS) human plasma (with a small amount of DMSO as
hydrotropy agent) at 37 °C and the hydrolytic release of 5-FU in the
solution was monitored by RP-HPLC.
The results from these in vitro assays (Fig. 2) suggest several
trends. First, two different types of prodrugs were able to release
the parent drugs in vitro. Specifically, the drug released rapidly
from the compound 4c in 50% human plasma, reaching 47.4% after
60 h and almost 60% within five days, whereas the drug release at
PBS was much slower, implying 3.2% and 5% in the same period,
respectively. Second, in 50% human plasma, the drug released from
the compound 6c reaching 60.3% after 60 h and almost 80% within
five days. The release property of compound 6c was much better
than compound 4c.
The trends observed with in vitro drug release data would sug-
gest that the two linkages were labile under blood circulation con-
ditions and relatively stable in PBS (pH 7.4) to allow the transport
of the prodrug to bone issues and effectively release the free active
drug from the carrier. Compound 6c with succinate ester had a
more rapid activation pathway, possibly because it had a longer
and more labile carbon chain or the eliminate process was acceler-
ating by a self-immolative disassembly pathway or the ester bond
was instable than the amide bond.27,28 Anyway, 5-FU prodrugs
with succinate linkage were better substrates of enzymes in hu-
man plasma. The results shown in this study provide a proof of
concept that succinate ester linkages modified bone-targeting 5-
FU prodrugs had more effective release profiles and this concept
could guide us to further prodrugs design.
a
70
60
50
40
30
20
10
0
PBS 7.4
50% plasma
0
24
48
72
96
120
Time (h)
80
70
60
50
40
30
20
10
0
b
PBS 7.4
50% plasma
0
24
48
72
Time (h)
96
120
Figure 2. Release of 5-FU from compound 4c (Fig. 2a) and compound 6c (Fig. 2b) in
physiological conditions. The percentage of drug release was deduced from the
difference between the initial amounts of 4c or 6c and that of regenerated drug 5-
FU. Error bars represented the mean and standard deviation of three independent
experiments.
Finally, the in vitro cytotoxicities of the compounds 4c and 6c in
the 0% and 50% human plasma lipid were estimated using two 5-
FU-sensitive cancer cell lines: human epithelial carcinoma cell line
(HeLa) and human osteosarcoma cell line (MG63). In order to prove
120
a
120
5-FU
5-FU
110
4c
6c
110
4c+plasma
6c+plasma
100
90
80
70
60
50
40
30
20
100
90
80
70
60
50
40
30
20
0
8
16
24
32
40
48
56
64
0
8
16
24
32
40
48
56
64
Concentration (uM)
Concentration (uM)
b
120
110
100
90
5-FU
4c
120
110
100
90
5-FU
6c
6c+plasma
4c+plasma
80
80
70
70
60
60
50
50
40
40
30
30
0
8
16
24
32
40
48
56
64
0
8
16
24
32
40
48
56
64
Concentraion (uM)
Concentration (uM)
Figure 3. Cytotoxicities of the prodrugs, with and without human plasma, as functions of the human epithelial carcinoma cell line (HeLa) (a), and the human osteosarcoma
cell line (MG63) (b). Error bars represented the mean and standard deviation of three independent experiments.

3754
L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
Table 2
Pharmacokinetic parameters of 5-FU-oligopeptides conjugate 4c, 6c and 5-FU in mice
Compound
AUC (mgÃh/L)
T1/2 (h)
V1/F (L/kg)
Cmax (mg/ml)
MRT
4c
6c
5-FU
76.55 8.61
90.08 9.64
31.17 4.79
4.36 0.62
6.75 1.22
0.64 0.13
24.12 2.41
32.18 1.45
64.74 2.14
42.45 5.41
51.08 4.78
78.54 8.11
7.41 1.06
8.25 1.26
1.61 0.31
AUC: area under (the concentration time) curve; T1/2: biological halflife; V1/F: apparent volume of distribution; Cmax: maximum concentration; MRT: mean retention time.
Data are represented as mean SD.
5-FU
5
10
9
8
7
6
5
4
3
2
1
0
b
5-FU
4c
a
4c
6c
4
3
2
1
0
6c
N.D
blood
kidney
liver
femur
blood
kidney
liver
femur
Figure 4. The biodistribution of the 5-FU oligopeptides conjugates with different linkers 4c, 6c and naked 5-FU. The biodistribution was analyzed at (a) 1 h and (b) 12 h after
oral administration. Error bars represented the mean and standard deviation of three independent experiments. N.D. cannot be detected.
that the two conjugates are active through the cleavage by
enzymes in human plasma, we performed a standard MTT cell-
growth inhibition assay: cells were challenged for three days with
free 5-FU, conjugate 4c, conjugate 6c, or the control conjugate over
a range of concentrations. Cell viability was measured using color-
imetric assay based on the MTT. The data were presented in Figure
3. Proliferation of HeLa cells were inhibited by free 5-FU, conjugate
4c and conjugate 6c (at 5-FU-equivalent concentrations) with
concentration of 5-FU in bone delivered by Asp oligopeptides is
much higher than that by uncoupled control during 24 h. The
results clearly show that, in contrast to nontargeted (no oligopep-
tides) controls, oligopeptides containing conjugates have a tendency
of targeting and accumulation to the bone. From the in vivo data, the
two linker’s biodistribution is no significant difference, but com-
pared to naked 5-FU, this bone-targeting prodrug strategy can re-
duce drug administration dose, reach a higher bone tissue drug
accumulation and reduce side effects in other organs by chemother-
apy drugs.
IC50s of 4
MG63 cells were inhibited with the same trend: 8
and >64 M, respectively. 5-FU prodrugs 4c and 6c exhibited sig-
l
M, >64
lM and >64
lM, respectively. Proliferation of
l
M, >64
lM
l
3. Conclusion
nificantly reduced toxicity than free 5-FU in the absence of plasma.
There was no noticeable change in the cytotoxicity of the conjugate
4c and 6c in the 0% plasma lipid throughout all concentration
range. However, the cytotoxicities of the conjugates in 50% plasma
lipid were significantly enhanced as the concentration of 5-FU
increased. This result exhibited these bone targeting prodrugs
themselves did not have cytotoxicity, but could be activated by
enzymes in human plasma, the amide or esters were able to appre-
ciably release their anticancer moieties and the process at least
required the participation of enzymes.
An in vivo pharmacokinetic and biodistribution assay was
taken to further exhibit the targeting ability of the prodrugs.
The pharmacokinetic parameters for conjugates 4c and 6c were
summarized in Table 2. The AUC and T1/2 value of conjugate 4c
in blood were 76.55 mgÃh/L and 4.36 h, respectively, values much
larger than those of 5-FU (31.17 mgÃh/L and 0.64 h, resp.). The
longer circulation time of conjugate 4c in the blood stream may
be attributable to its larger molecular size, resulting in a lower
glomerular filtration rate in the kidney. From this point of view,
5-FU-oligopeptides conjugates, with a longer retention time and
a higher therapeutic window, was better than 5-FU for bone
targeting chemotherapy.
Site-specific bone drug delivery via a prodrug approach has
generated considerable interest for enhancing the potency and
diminishing the side effects of a drug. Effective release of chemo-
therapeutic drugs from a prodrug system is important if a high
concentration of active drug is needed at the bone tissue. In
conclusion, a number of methods were presented to tether a
bone-targeting Asp oligopeptides group to the different carboxylic
acid functionality of 5-fluorouracil to construct prodrugs as novel
potential bone-targeting therapeutics. The resulting 5-FU oligo-
peptide conjugates were evaluated for the affinity to bone and
the ability to release parent drug once bound to bone. In vitro
investigations highlighted the strong affinity of Asp oligopeptide
derivatives for bone, as opposed to the negligible bone affinities
of the parent drugs. The bounded compound 4c and 6c could
slowly release parent drug in physiological condition and signifi-
cantly reduce the number of tumor cells. The in vivo biodistribu-
tion and pharmacokinetic data obtained support the effective
targeting of 5-FU oligopeptides conjugates to bone with long half-
life and the clearance rate of the conjugates from the organism
possibly was dependent on molecular size and the different link-
ers. The preliminary results seem to be very promising and we
are continuing to develop more suitable animal models for
in vivo comprehensive evaluation of osteosarcoma treatment
and possible harmful adverse effect.
Figure 4 shows the accumulation of the conjugates in the skele-
ton (represented by femur). All conjugates reached the skeleton
within 1 h after administration. After 12 h, the amount of
deposited conjugates began to decrease. It is obvious that the

L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
3755
ESI-MS (m/z): calcd for 763.55, obsd 763.41 ([M]+). Anal. Calcd
for C26H30FN7O19: C, 40.90; H, 3.96; N, 12.84. Found: C, 41.09; H,
3.88; N, 12.67.
4. Experimental
4.1. Chemistry
4.1.2.3. Compound 4c.
0.5 mmol).1H NMR (400 MHz, D2O): 2.64–2.96 (br s, 12H, Asp–
bCH2), 4.15 (s, 2H, N–CH2), 4.43–4.96 (m, 6H, Asp– CH), 7.77 (d,
White wax (0.26 g, 56%) from 2 (0.1 g,
General: All reactions requiring anhydrous conditions were per-
formed under an Ar or N2 atmosphere. Chemicals and solvents
were either A.R. grade or purified by standard techniques. Thin
layer chromatography (TLC): silica gel plates GF254; compounds
were visualized by irradiation with UV light and/or by treatment
with a solution of phosphomolybdic acid (20% wt in ethanol) fol-
lowed by heating. Column chromatography was performed by
using silica gel with eluent given in parentheses. 1H NMR and 13C
NMR analysis was performed using CDCl3 or D2O as a solvent at
room temperature. The chemical shifts are expressed in relative
to TMS (= 0 ppm) and the coupling constants J in Hz. Hydroxyapa-
tite (HAP) were purchased from Shanghai Institute of Biochemistry
a
1H, J = 5.2 Hz, 5-FU–CH). 13C NMR (100 MHz, D2O): 35.1, 35.4,
35.9, 36.7, 37.1, 37.3, 51.3, 51.7 52.4, 53.0, 53.5, 54.2, 55.4 125.5,
140.0, 149.7, 153.5, 169.1, 172.3, 172.5, 172.7, 173.1, 173.4. ESI-
MS (m/z): calcd for 878.64, obsd 903.49 ([M+Na]+). Anal. Calcd
for C30H35FN8O22: C, 41.01; H, 4.02; N, 12.75. Found: C, 41.39; H,
3.78; N, 12.28.
4.1.3. General procedure for the synthesis of compound 5
To the solution of 37% formaldehyde/water (5 ml), 5-fluoroura-
cil (0.52 g, 4 mmol) was added. The mixture was stirred at 60 °C to
make the solid part dissolved completely, after 50 min, a colorless
transparent thick oil 1,3-dimethylol-5-fluorouracil was obtained.
Then anhydrous acetonitrile (12 ml), benzyl succinate (1.16 g,
5.6 mmol), DCC(1.15 g, 5.6 mmol) and DMAP(0.032 g, 0.26 mmol)
was added under ice-bath, after stirred at 0 °C for 1 h, the mixture
was then stirred at room temperature overnight. The dicyclohexyl-
urea (DCU) was removed by filtration, then evaporated under re-
duced pressure. After concentration, the residue was taken up in
50 ml ethyl acetate and washed with 1 M HCl, 1 M NaHCO3, and
brine. The organic layer was dried and evaporated to give the crude
product. The crude product was purified by silica gel column chro-
matography using petroleum ether–acetone as an eluent to yield
compound 5 1.00 g as a white solid-like substance. The total yield
after two steps was about 71%. 1H NMR (400 MHz, CDCl3): 2.70 (s,
4H, SA-CH2 Â 2), 5.14 (s, 2H, N–CH2), 7.30–7.50 (m, 5H, ph-H), 7.54
(d, 1H, J = 5.2 Hz, 5-FU-CH). ESI-MS (m/z): calcd for 350.30, obsd
373.34 ([M+Na]+).
with surface area 9.12 m2/g and average particle size 15
lm.
4.1.1. General procedure for the synthesis of compound 2
KOH (2.56 g, 45 mmol) and 5-fluorouracil (1.34 g, 10 mmol) dis-
solved in a clean flask, then 5 ml aqueous solution of bromoacetic
acid (1.7 g, 18 mmol) was added under the temperature of 40 °C
while stirred smoothly. The reaction mixture was stirred at 60 °C
for 5 h and then cooled by ice-bath, adjusted to pH 5.5 with hydro-
chloric acid, after filtration through a membrane, the crude product
was recrystallized by water, and compound 2 was obtained as
white solid 1.53 g. Yield 85%, mp 255–257 °C. 1H NMR (400 MHz,
D2O): 4.41 (s, 2H, N–CH2), 7.54 (d, 1H, J = 5.2 Hz, 5-FU-H), ESI MS
(m/z): calcd for 188.11. obsd 189.01 ([M+H]+)
4.1.2. General procedure for the synthesis of compound 4
Compound 2 (0.1 g, 0.5 mmol) dissolved in anhydrous THF was
cooled by ice-salt bath, then NMM (0.05 ml, 0.5 mmol) and IBCF
(0.08 ml, 0.5 mmol) was added, after stirred for 30 min then, the
protected oligopeptides NH2–Asp(4–6) 1 (1a, 0.46 g, 0.5 mmol; 1b,
0.57 g, 0.5 mmol; 1c, 0.67 g, 0.5 mmol) in THF was added. The mix-
ture was stirred for 3 h. After evaporated under reduced pressure,
the residue was taken up in ethyl acetate and washed with 1 M
HCl, 1 M NaHCO3, and brine each for twice. The organic layer
was dried and evaporated to give the crude product. The crude
product was purified by silica gel column chromatography using
DCM–methanol as an eluent to yield a ceraceous solid (Compound
3).
A mixture of the obtained compound 3 and 10% Pd/C (10 mg) in
CH3OH (10 ml) was stirred at room temperature under a H2 atmo-
sphere. After 24 h, the mixture was passed through a membrane
filter to remove the catalyst and then evaporated under reduced
pressure to give the target molecules compound 4. The total yield
after two steps was about 56–68%.
4.1.4. General procedure for the synthesis of compound 7
The obtained compound 5 (0.6 g, 1.71 mmol) was then dis-
solved in methanol (15 ml), 10%Pd/C (60 mg) was added. The mix-
ture was stirred at room temperature under a H2 atmosphere
overnight. The Pd/C was removed by filtration, and then the mix-
ture was evaporated under reduced pressure to give a colorless
wax. The crude product was purified by silica gel column chroma-
tography using petroleum ether–acetone as an eluent to yield a
white ceraceous solid compound 7 (0.39 g). Yield 91%. 1H NMR
(400 MHz, CDCl3): 2.64 (s, 4H, SA-CH2 Â 2), 5.31 (s, 2H, N–CH2),
7.62 (d, 1H, J = 5.2 Hz, 5-FU–CH). ESI-MS (m/z): calcd for 260.18,
obsd 283.20 ([M+Na]+).
4.1.5. General procedure for the synthesis of compound 6
Same procedure as described above for preparation of the com-
pound 4. The total yield after two steps was about 47–61%.
4.1.2.1. Compound 4a.
White wax (0.23 g, 68%) from 2 (0.1 g,
0.5 mmol).1H NMR (400 MHz, D2O): 2.72–2.92 (br s, 8H, Asp–
4.1.5.1. Compound 6a.
White wax (0.15 g, 56%) from com-
bCH2), 4.04 (s, 2H, N–CH2), 4.56–4.90 (m, 4H, Asp–aCH), 7.69 (d,
pound 7 (0.1 g, 0.4 mmol).1H NMR (400 MHz, D2O): 2.59 (br s,
4H, SA-CH2 Â 2), 2.76–3.04 (br s, 8H, Asp–bCH2), 4.66–4.97 (m,
1H, J = 5.2 Hz, 5-FU–CH). 13C NMR (100 MHz, D2O): 36.2, 36.6,
37.5, 51.0, 52.4, 53.0, 53.5, 54.2, 124.5, 139.0, 145.7, 147.5, 169.1,
172.3, 172.5, 172.7, 173.1, 173.4. ESI-MS (m/z): calcd for 648.46,
obsd 671.65 ([M+Na]+). Anal. Calcd for C22H25FN6O16: C, 40.75; H,
3.89; N, 12.96. Found: C, 40.11; H, 4.02; N, 12.17.
4H, Asp–aCH), 5.50 (s, 2H, N–CH2), 7.85 (d, 1H, J = 5.2 Hz, 5-FU–
CH). 13C NMR (100 MHz, D2O): 27.1, 29.8, 35.2, 36.5, 52.0, 52.4,
53.5, 54.2, 76.4, 126.5, 139.4, 149.7, 154.5, 169.1, 171.3, 171.4,
172.3, 173.1, 174.4. ESI-MS (m/z): calcd for 720.53, obsd 719.38
([MÀH]À). Anal. Calcd for C25H29FN6O18: C, 41.67; H, 4.06; N,
11.66. Found: C, 41.11; H, 4.48; N, 11.07.
4.1.2.2. Compound 4b.
White wax (0.25 g, 62%) from 2 (0.1 g,
0.5 mmol). 1H NMR (400 MHz, D2O): 2.69–2.90 (br s, 10H, Asp–
bCH2), 4.12 (s, 2H, N–CH2), 4.44–4.91 (m, 5H, Asp–aCH), 7.71 (d,
4.1.5.2. Compound 6b.
White wax (0.15 g, 47%) from
1H, J = 5.2 Hz, 5-FU–CH). 13C NMR (100 MHz, D2O): 35.2, 35.7,
36..3, 37.4, 52.4, 52.3, 52.9, 53.4, 54.5, 55.1, 126.1.6, 139.9, 147.7,
149.5, 169.1, 171.2, 172.0, 172.5, 172.9, 173.3, 173.9, 174.2.
compound 7 (0.1 g, 0.4 mmol).1H NMR (400 MHz, D2O): 2.47 (br
s, 4H, SA–CH2 Â 2), 2.74–3.06 (br s, 10H, Asp–bCH2), 4.76–5.01
(m, 5H, Asp–aCH), 5.48 (s, 2H, N–CH2), 7.76 (d, 1H, J = 5.2 Hz,

3756
L. Ouyang et al. / Bioorg. Med. Chem. 19 (2011) 3750–3756
5-FU–CH). 13C NMR (100 MHz, D2O): 28.1, 29.3, 34.7, 35.1, 35.7,
36.6, 53.3, 53.4, 53.5, 54.7, 77.1, 127.4, 140.2, 150.1, 157.1, 169.9,
170.1, 170.2, 170.3, 171.1, 171.4, 172.1, 173.1, 173.4. ESI-MS (m/
z): calcd for 835.61, obsd 834.42 ([MÀH]À). Anal. Calcd for
4.2.5. Biodistribution in mice tissue in vivo
According to the requirements of the National Act on the usage
of experimental animals (PR China), the Sichuan University Animal
Ethical Experimentation Committee, approved all procedures of
our in vivo studies. At the indicated time, the animals were sacri-
ficed and blood samples were collected from the ocular artery di-
rectly after removing eyeball. Then the animals were dissected
and each tested organ was removed, including kidney, liver, and fe-
mur. Organs were rinsed with cold normal saline, blotted dry with
a paper towel, extracted with methanol, diluted, centrifuged, and
dispensed in plastic sample vials. Both the samples were centri-
C
29H34FN7O21: C, 41.68; H, 4.10; N, 11.73. Found: C, 41.92; H,
3.99; N, 11.41.
4.1.5.3. Compound 6c.
White wax (0.22 g, 61%) from com-
pound 7 (0.1 g, 0.4 mmol).1H NMR (400 MHz, D2O): 2.56 (br s,
4H, SA–CH2 Â 2), 2.72–3.01 (br s, 12H, Asp–bCH2), 4.64–4.99 (m,
6H, Asp–aCH), 5.74 (s, 2H, N–CH2), 7.86 (d, 1H, J = 5.2 Hz, 5-FU–
CH). 13C NMR (100 MHz, D2O): 29.0, 31.3, 35.1, 35.2, 35.3, 35.5,
36.1, 53.0, 53.4, 53.5, 53.6, 53.7, 54.1, 78.2, 128.5, 140.4, 150.7,
158.5, 169.9, 170.0, 170.5, 170.6, 171.3, 171.5, 172.8, 173.7,
174.5, 175.4. ESI-MS (m/z): calcd for 950.70, obsd 951.43
([M+H]+). Anal. Calcd for C33H39FN8O24: C, 41.69; H, 4.13; N,
11.79. Found: C, 40.98; H, 4.25; N, 11.28.
fuged at 3500 rpm for 15 min. After that, 20 lL of the supernatants
were removed and the concentration of 5-FU was analyzed using
the HPLC conditions mentioned above.
Acknowledgments
Financial support from National Natural Science Foundation of
China (No. 20472055), Youth Foundation of Sichuan University
(No. 2010SCU11067) and The Open Drug Research Fund of the
State Key Laboratory of Biotherapy is gratefully acknowledged.
4.2. Biological evaluation
4.2.1. Hydroxyapatite (HAP) binding study
The conjugates were dissolved in water with various precise
concentrations and the adsorption amounts were determined by
a UV spectrophotometer at 254 nm to obtain the A–C linear regres-
sion equation. In tubes 25 mg/ml HAP was added to 5 ml solutions
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followed by supersonic shake for 5 min, then placed in a water
bath at 37 °C for 1 h and over 24 h. After the prescribed time, tubes
were centrifuged for 1 min at 5000 rpm. The adsorption amounts
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were determined by UV at 254 nm and the
the special equation. The bound percentage was calculated by
(C0 À C)/C0Ã100%.
DC was obtained by
D
4.2.2. HPLC analysis
The HPLC system consisted of an SPD-10A variable UV–vis det-
etor and a set of Model LC-10AT liquid chromatograph includin a
manometric module as well as a dynamic mixer from Agilent
1100 HPLC system. The mobile phase consists of pure water which
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lm) was eluted
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with the mobile phase at flow rate of 1.0 mL/min. The eluate was-
monitored bymeasurin the absorption at 256 nm with a sensitivity
of AUFS 0.01 at 25 °C. The retention time (RT) of 5-FU is 7.265 min,
and the retention time of 4c and 6c was 5.465 min and 5.898 min.
4.2.3. Drug release study
The conjugates(compound 4c and 6c) were incubated in PBS pH
7.4 or 50% (v/v in PBS) human plasma (with a small amount of
DMSO as hydrotropy agent) at 37 °C and the hydrolytic release of
5-FU in the solution was monitored by RP-HPLC. The concentration
of 5-FU was analyzed using the HPLC conditions mentioned above.
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4.2.4. In vitro cytotoxicity assay
The cytotoxic effects of 5-FU, compound 4c, and compound 6c
in the absence or presence of human plasma was determined using
the standard MTT assay. MG-63 or HeLa cells were harvested from
culture flasks, resuspended in cell culture medium, and plated at a
density of 5Ã103cells/well in 200
Cells were challenged with prodrug 4c or prodrug 6c (1–64
in the presence or absence of human plasma enzyme and incu-
bated for 72 h (5% CO2). Activation solution (20 L) was added to
l
L onto 96-well culture plate.
l
M)
l
MTT reagent. The reaction solutions were added to each well.
The plate was incubated for 2 h, shaken gently to evenly distribute
the dye in the wells. Absorbance was measured at a wavelength of
490 nm.
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