Bone targeting prodrugs based on peptide dendrimers, synthesis and hydroxyapatite binding in vitro

Article abstract of DOI:10.2174/157017809788489981

Novel bone targeting naproxen prodrugs with poly(aspartic acid) moieties and with two and three poly(aspartic acid) sequences peptide dendrimers were synthesized using a conventional method. The modified naproxen conjugates were incubated with hydroxyapatite in PBS at physiological conditions over 16h. The study revealed the hydroxyapatite binding properties of poly(aspartic acid) and it was found that the peptide dendrimer prodrugs exhibited a faster initial binding and a greater total binding. The obtained binding data in vitro indicated that the peptide dendrimers with poly(aspartic acid) sequences were useful for the development of new bone targeting molecules for drug delivery to bone.

Full text of DOI:10.2174/157017809788489981

272
Letters in Organic Chemistry, 2009, 6, 272-277
Bone Targeting Prodrugs Based on Peptide Dendrimers, Synthesis and
Hydroxyapatite Binding In Vitro
Liang Ouyanga, Wencai Huangb, Gu Hea and Li Guo*,a
aKey Laboratory of Drug Targeting of Education Ministry West China School of Pharmacy, Sichuan University,
Chengdu 610041, P.R. China
bSchool of Chemical Engineering, Sichuan University, Chengdu, 610064, P.R. China
Received July 08, 2008: Revised March 04, 2009: Accepted April 09, 2009
Abstract: Novel bone targeting naproxen prodrugs with poly(aspartic acid) moieties and with two and three poly(aspartic
acid) sequences peptide dendrimers were synthesized using a conventional method. The modified naproxen conjugates
were incubated with hydroxyapatite in PBS at physiological conditions over 16h. The study revealed the hydroxyapatite
binding properties of poly(aspartic acid) and it was found that the peptide dendrimer prodrugs exhibited a faster initial
binding and a greater total binding. The obtained binding data in vitro indicated that the peptide dendrimers with
poly(aspartic acid) sequences were useful for the development of new bone targeting molecules for drug delivery to bone.
Keywords: Bone-targeting, poly(aspartic acid), peptide dendrimers, hydroxyapatite, in vitro.
INTRODUCTION
hoped that they could show selective targeting to HAP and
we could investigate the different HAP binding rates of the
different length of the oligopeptides’ chain (Asp4, Asp5,
Asp6). The structures of conjugates were shown in Fig. (1).
Nowadays, musculoskeletal diseases such as ostearthritis
and osteoporosis are recognized as major public health prob-
lems in the world. There is a great interest in the develop-
ment of drug delivery systems that could allow an efficient
transport of drugs to the bone. Bone differs from the rest of
the body by the presence of hydroxyapatite(HAP). We have
known that tetracyclines and bisphosphonates exhibit a
strong affinity to HAP and can be useful as targeting moie-
ties to bone [1,2].
Furthermore, on the basis of peptide dendrimers: peptide
dendrimers are synthetic, highly branched, spherical, mono-
disperse macromolecules. They have a protein-like globular
structure, good biocompatibility and biodegradability which
make them macromolecular vehicles for drug delivery pur-
poses and other biomedical application [9]. A dendritic
linker approach will increase the molar ratio of Aspn, which
may increase the bone affinity and more negative charges in
dendrimer periphery increase the binding capacity to the
HAP crystal surface [10,11]. The increased molar ratio can
be controlled by varying the size or generation of the den-
dritic molecule, which will allow investigations into relation-
ship between the molar ratio of targeting moieties and bone
targeting abilities of conjugates.
In recent years, acidic oligopeptides such as poly-aspartic
acid (Aspn) have been reported as novel bone targeting drug
carriers [3,4]. Unlike the P-C-P bond of bisphosphonates, the
Aspn are biologically labile and enzymatically degraded. The
advantageous property can make more efficient drug release
and there is no unexpected long-term effects of the bone tar-
geting moiety [5]. Therefore, the Aspn seems very attractive
as an alternative bone targeting carrier to bisphosphonates.
Previous work has been conducted that estradiol modified
with Asp6 exhibited high accumulation in bone in vivo [6],
while polymers PEG and HPMA conjugates to Asp8 showed
greater than 80% binding to HAP in vitro [7]. However,
there is few literature for the relationship between the num-
bers of the negatively charged amino acid residues in Aspn
and their respectively HAP binding rates.
In this manuscript, we reported the design and synthesis
of the first generation dendritic compounds with two or three
Asp(4-6) fragments. We employed the polyamide and poly-
ether-amide dendrimers as scaffolds which were successfully
used in the RGD and CCK peptide dendrimers in our previ-
ous work [12]. These dendrimers have an flexible scaffold,
biocompatibility and low cytotoxicity and these characters
were helpful in the drug delivery. The structure of these pep-
tide dendrimers were shown in Fig. (1). We aimed to in-
crease the bone-targeting potency of the peptide dendrimers
with a dendritic approach. The HAP binding assay was es-
tablished in vitro condition and the affinity of the targeting
conjugates for HAP was reported here.
Anti-inflammatory drugs (NSAIDs) are clinically used in
ostearthritis. However, they have been reported to cause a
number of side effects, in particular gastrointestial toxicity
[8]. In this present study, to improve the therapeatic index of
NSAIDs, we selected Asp(4-6) as bone targeting moieties and
developed
a
type of conjugates using
a
NSAID
naproxen(NAP) as prodrugs through an amide linkage. We
EXPERIMENTAL SECTION
General Remarks
*Address correspondence to this author at the Key Labratory of Drug Taget-
ing of Education Ministry, West China School of Pharmacy, Chengdu,
610041, P.R. China; Tel: +86(028)85503777;
All starting materials, unless otherwise specified, were
used as received. The compounds 13,14 were synthesized
E-mail: rosaguoli2000@yahoo.com.cn
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

Synthesis of Bone Targeting Prodrugs
Letters in Organic Chemistry, 2009, Vol. 6, No. 4
273
O
O
O
O
O
O
O
OH
NH
O
O
OH
NH
O
O
O
OH
NH
O
O
OH
OH
H
N
H
N
NH
OH
NH
OH
OH
OH
NH
OH
NH
OH
O
O
O
O
O
O
O
O
O
Ib
Ia
O
O
O
O
OH
NH
O
O
OH
NH
O
O
OH
OH
H
N
NH
OH
NH
OH
NH
OH
O
O
O
O
O
COOH
O
Ic
OH
n
COOH
HN
O
O
OH
NH
COOH
O
O
O
O
n
OH
H
N
N
O
N
H
O
O
O
O
n
O
O
O
O
HN
O
HN
OH
n
OH
n
COOH
COOH
n=4,5,6
IIIa n=4
IIIb n=5
IIIc n=6
IIa n=4
IIb n=5
IIc n=6
Fig. (1). The structures of the bone targeting prodrugs.
according to the literature method, respectively [13]. TLC
was performed on glass-backed silica plates.1H NMR analy-
sis was performed by the INOVA VARIAN 400 MHz spec-
trometer using CDCl3 or D2O as a solvent at room tempera-
ture. Mass spectra were recored on a Agilent 1946B ESI-MS
instrument. The synthetic procedure was outline in Scheme 1
and Scheme 2.
6c: ESI-MS(m/z): calcd. for 1339.5 ([M+H]+), obsd.
1339.7
Synthetic Procedure for Compound 8
A mixture of ethyl acrylate (10g,100mmol), benzylamine
(4g, 37mmol) in CH3OH (10ml) was stirred in room tem-
perature for 48 hours. The solvent was evaporated and the
residue was diluted with ethyl acetate (50ml). After washed
with saturated NaCl(15mlꢀ3), the organic layer was dried
over anhydrous Na2SO4 and evaporated to yield a light-
yellow oil (8) 9.6g, yield 84%.
Synthetic Procedure for Asp(4-6) Peptide 6a-c
The bone-targeting peptides were synthesized by a
conventional liquid phase peptides synthetic method from
Boc-Asp-Obzl utilizing IBCF(isobutyl chloroformate) and
NMM(N-methyl morpholine)(mixed anhydrides method) by
a series of segment condensation. The -COOH part dissolved
in anhydrous THF was cooled at -15˚C, then added 1eq.
NMM and IBCF, after stirred for 5 min, the -NH2 part in
THF was added to the reaction mixture. After the coupling
reaction was completed at room temperature. The mixture
was purified by recrystallisation or silica column chromatog-
raphy using CHCl2 and CH3OH as an eluent, then depro-
tected in trifluoroacetic acid to give the peptides, total yield:
6a, five steps reactions 47%; 6b, seven steps reactions, 34%;
6c, nine steps reactions 23%.
Synthetic Procedure for Compound 9
Compound 8 (5g,16mmol) was dissolved in anhydrous
CH3OH (20ml), ammonium formate(5g,79mmol) and 10%
Pd/C(450mg) was added.The mixture was refluxed for 2h.
Then it was allowed to attain room temperature and filter.
Following removal of the solvent, a colorless oil(9) was ob-
tained 3.0g, yield 85%.
Synthetic Procedure for Compound 10
A stirred solution of naproxen (2.3g, 10mmol) in THF
(10ml) was added DCC (2.06,10mmol) and HoSu
(1.15g,10mmol) at 0˚C, after stirred for 3h, 9(2.17g,
10mmol) was added at room temperature for 24h.The dicy-
Data: 6a: ESI-MS(m/z): calcd. for 929.4 ([M+H]+), obsd.
929.3
6b: ESI-MS(m/z): calcd. for 1133.4 ([M+H]+), obsd.
1134.2

274 Letters in Organic Chemistry, 2009, Vol. 6, No. 4
Ouyang et al.
BzlO
H2N
O
O
BzlO
HN
O
O
OBzl
OH
a,b
1
+
OBzl
H2N
O
O
O
O
O
O
O
O
O
4
BzlO
NH2
Boc
COOBzl
BzlO
N
H
O
O
OBzl
NH
O
O
OBzl
OBzl
a,b
a,b
+
OBzl
n
2
2
NH
NH
OBzl
O
OH
H2N
HO
O
repeat
steps
O
OBzl
O
BzlO
BzlO
NH2
OH
HN
OBzl
O
3
a
+
(n=5-6)
6b-c
O
6a
O
Boc
O
BzlO
N
H
Boc
5
N
H
2
Scheme 1. Synthetic procedure of the Asp(4-6) peptides.
Reagents and Conditions: a) IBCF, NMM, THF; b)TFA, CH2Cl2.
OH
g
Ia-c
O
O
NAP
O
O
O
OEt
OEt
OEt
OEt
b
a
c
N
N
HN
CO2Et
OEt
O
OEt
O
O
O
O
8
9
10
7
d
O
OH
g
N
IIa-c
OH
O
O
O
O
11
O
OEt
OEt
CN
O
O
O
O
OH
O
H
N
e
c
f
O
O
OEt
H2N
H2N
H2N
O
CN
CN
OH
OH
O
OEt
O
O
O
O
15
OEt
OEt
14
13
O
12
d
O
O
OH
O
O
H
g
N
IIIa-c
O
OH
O
O
O
OH
16
O
Scheme 2. Synthetic procedure of the Nap-Peptide dendrimers
Reagents and Conditions: a) BnNH2, CH3OH, r.t; b) ammonium formate, CH3OH, reflux; c) Naproxen, DCC, HoSu, THF, r.t; d) 2N
NaOH/CH3OH, r.t; e) acrylonitrile, 40%KOH, 0˚C; f) H2SO4, C2H5OH, reflux; g) i, 6a-c, DCC, HoBt, THF, r.t; ii, 10% Pd/ C, CH3OH, r.t.

Synthesis of Bone Targeting Prodrugs
Letters in Organic Chemistry, 2009, Vol. 6, No. 4
275
clohexylurea (DCU) was removed by filtration. The filtrate
was purified by silica colummn chromatography using EA
and PE as an eluent to afford a colorless oil (10), 2.8g, yield
66%.
Data: 1HNMR (400MHz, CDCl3): ꢀ=1.28 (t, 6H, J=6.4Hz
OCH2CH3ꢁ2), 1.52(d, 3H, J=7.2Hz, NAPCH3), 2.38 (t, 4H,
J=6, CH2COꢁ2), 3.52(t, 4H, J=6Hz, N-CH2ꢁ2), 3.89(brs,
4H, NAPOCH3+CH), 4.17 (m, 4H, OCH2CH3ꢁ2), 7.10-7.7
(m, 6H, NAPph-H).
ESI-MS(m/z): calcd. for 691.6 ([M+H]+), obsd. 691.9
Anal. calcd. for C30H34N4O15: C, 52.17, H, 4.96, N, 8.11.
found: C, 52.35, H, 4.89, N, 8.02;
Ib: 1HNMR(400MHz, D2O): ꢀ=1.52(d, 3H, J=7.2Hz
NAPCH3), 2.69-2.92(brs, 10H, Aspꢂ-CH2), 3.91(brs, 4H,
NAPCH3O+CH), 4.44-4.91(m, 5H, Aspꢃ-CH), 7.21-
7.72(m, 6H, NAPph-H). ESI-MS(m/z): calcd. for 805.7
([M]+), obsd. 805.1
Ic: 1HNMR(400MHz, D2O): ꢀ=1.52(d, 3H, J=7.2Hz,
NAPCH3), 2.64-2.92(brs, 12H, Aspꢂ-CH2), 3.91(brs, 4H,
NAPCH3O+CH), 4.34-4.91(m, 6H, Aspꢃ-CH), 7.21-
7.72(m, 6H, NAPph-H). ESI-MS(m/z): calcd. for 919.8 ([M-
H]-), obsd. 919.5
Synthetic Procedure for Compound 11
Compound 10 (1.0g, 2mmol) was dissolved in
CH3OH(10ml), aqueous NaOH(2mol/L) was added. The
mixture was stirred for 5h, then acidified by HCl(2mol/L) to
pH<3. The solvent was evaporated and the residue was di-
luted with ethyl acetate. (15mlꢁ3). The organic layer was
dried over anhydrous Na2SO4 and evaporated to yield a
white powder(11), 0.77g, yield: 90%.
Data: 1HNMR (400MHz, CDCl3): ꢀ=1.53(d, 3H,
J=7.2Hz, NAPCH3), 2.41(t, 4H, J=6Hz CH2COꢁ2), 3.47 (t,
4H, J=6Hz N-CH2ꢁ2), 3.89(brs, 4H, NAPCH3O+CH), 7.10-
7.7(m, 6H NAPph-H).
Synthetic Procedure for IIa-c
Compound 11 (0.075g, 0.2mmol) dissolved in anhydrous
THF. Activated Asp(4-6) peptide 6a-c (6a, 0.46g, 0.5mmol;
6b, 0.56g, 0.5mmol; 6c; 0.67g, 0.5mmol) and HOBt (0.068g,
0.5mmol) were added to the reaction mixture. DCC (0.103g,
0.5mmol) dissolved in THF was slowly dropped into the
solution. After the coupling reaction was completed at room
temperature, the solution was filtered twice to remove the
DCU. The filtrate was purified by silica column chromatog-
raphy using CHCl2 and CH3OH as an eluent, then depro-
tected the -Obzl with Pd catalyst by hydrogen in CH3OH at
room temperature to afford IIa-c. (IIa, 0.17g, yield 66%;
IIb, 0.18g, yield 59%;IIc, 0.20g, yield 58%) as white pow-
ders.
Data: IIa: 1HNMR (400MHz, D2O): ꢀ=1.48(d, 3H,
J=7.2Hz, NAPCH3), 2.60(brs, 4H, CH2COꢁ2), 2.72-
2.99(brs, 16H, Aspꢂ-CH2), 3.57(brs, 4H, N-CH2ꢁ2),
3.98(brs, 4H NAPCH3O+ CH), 4.52-4.92(m, 8H, Aspꢃ-
CH), 7.24-7.80(m, 6H, NAPph-H). ESI-MS(m/z): calcd. for
1294.1([M]+), 1317.1([M+Na]+), 648.0([M+H]2+), obsd.
1294.8, 1316.6, 648.1 Anal. calcd. for C52H63N9O30: C,
48.26, H, 4.91, N, 9.74 found: C, 48.08, H, 5.01, N, 9.82;
ESI-MS(m/z): calcd. for 374.4 ([M+H]+), obsd. 374.1
Synthetic Procedure for Compound 15
Same procedure as describe above for preparation of the
compound 10. A colorless oil, yield: 71%.
1
Data: HNMR(400MHz, CDCl3): ꢀ=1.31(t, 9H, J=6.4,
OCH2CH3ꢁ3), 1.52(d, 3H, J=7.2Hz, NAPCH3), 2.38(brs,
6H, CH2COꢁ3), 3.52-3.7(m, 6H, OCH2ꢁ3), 3.91(brs, 4H,
NAPCH3O+CH), 4.14(m, 6H, OCH2CH3ꢁ3), 7.10-7.7(m,
6H NAPph-H)
Synthetic Procedure for Compound 16
Same procedure as describe above for preparation of the
compound 11. A colorless oil. yield 95%.
1
1
IIb: HNMR(400MHz, D2O): ꢀ=1.47(d, 3H, J=7.2Hz,
Data: HNMR(400MHz, CDCl3): 1.52(d, 3H, J=7.2Hz,
NAPCH3), 2.58(brs, 4H, CH2COꢁ2), 2.71-3.04(brs, 20H,
Aspꢂ-CH2), 3.62(brs, 4H, N-CH2ꢁ2), 3.97 (brs, 3H,
NAPCH3O+CH), 4.52-4.92 (m, 10H, Aspꢃ-CH), 7.25-7.86
(m, 6H, NAPph-H). ESI-MS(m/z): calcd. for 1525.3
([M+H]+), obsd.1525.5;
NAPCH3), 2.41 (brs, 6H, CH2COꢁ3), 3.42-3.7(m, 6H,
OCH2ꢁ3), 3.91(brs, 4H, NAPCH3O+CH), 7.10-7.7(m, 6H,
NAPph-H).
ESI-MS(m/z): calcd. for 550.6 ([M+H]+), obsd. 550.4
IIc: 1HNMR(400MHz, D2O): ꢀ=1.48(d, 3H, J=7.2Hz
NAPCH3), 2.60(brs, 4H, CH2COꢁ2), 2.74-2.92(brs, 24H,
Aspꢂ-CH2), 3.62(brs, 4H, N-CH2ꢁ2), 3.92(brs, 4H,
NAPCH3O+CH), 4.50-4.94(m, 12H, Aspꢃ-CH), 7.27-
7.88(m, 6H, NAPph-H). ESI-MS(m/z): calcd. for 1778.3
([M+Na]+), obsd. 1776.9.
Synthetic Procedure for Ia-c
The Asp(4-6) peptides 6a-c(6a, 0.9g, 1.0 mmol; 6b, 1.1g,
1.0 mmol; 6c, 1.3g, 1.0mmol) dissolved in anhydrous
THF(10ml). Naproxen (0.23g, 1.0mmol) and HOBt (0.15g,
1.1mmol) were added to the reaction mixture. DCC (0.23g,
1.1mmol) dissolved in THF was slowly dropped into the
solution. After the coupling reaction was completed at room
temperature, the solution was filtered twice to remove the
DCU. The filtrate was purified by silica column chromatog-
raphy using CHCl2 and CH3OH as an eluent, then depro-
tected the -Obzl with Pd catalyst by hydrogen in CH3OH at
room temperature to afford Ia-c (Ia, 0.44g, yield 63%; Ib,
0.45g, yield 56%;Ic, 0.53g, yield 58%) as white powders.
Synthetic Procedure for IIIa-c
Compound 16 (0.055g, 0.1mmol)as a core part and DCC
(0.08g, 0.4 mmol), HOBt (0.054g, 0.4mmol) were dissolved
in anhydrous THF, then the mixture was stirred for 1 h. Ac-
tivated Asp(4-6) peptide 6a-c (6a, 0.37g, 0.4mmol; 6b, 0.45g,
0.4mmol; 6c; 0.54g, 0.4mmol) was added to the solution
under nitrogen atmosphere. After the reaction was com-
pleted, DCU was filtered twice and the filtrate was purified
with silica column chromatography with CHCl2 and MeOH
as an eluent, then deprotected the –Obzl with Pd catalyst by
hydrogen in CH3OH at room temperature to afford IIIa-c
Data: Ia: 1HNMR (400MHz, D2O): ꢀ=1.52(d, 3H,
J=7.2Hz, NAPCH3), 2.72-2.92 (brs, 8H, Aspꢂ-CH2),
3.91(brs, 4H, NAPCH3O+CH), 4.56-4.92(m, 4H, Aspꢃ-
CH), 7.21-7.72 (m, 6H, NAPph-H).

276 Letters in Organic Chemistry, 2009, Vol. 6, No. 4
Ouyang et al.
(IIIa, 0.10g, yield 49%; IIIb, 0.10g, yield 43%; IIc, 0.11g,
yield 42%) as white powders.
Data: IIIa: 1HNMR (400MHz, D2O): ꢀ=1.55(d, 3H,
J=6.0Hz NAPCH3), 2.44(brs, 6H, CH2COꢁ3), 2.77-2.94(br,
24H, Aspꢂ-CH2), 3.57-3.8 (brs, 12H, OCH2ꢁ3+OCH2ꢁ3),
3.98(brs, 4H, NAPCH3O+CH), 4.60-4.96(m, 12H, Aspꢃ-
CH), 7.24-7.88 (m, 6H, NAPph-H).
ESI-MS(m/z): calcd. for 1928.5 ([M-H]-) 964.3([M-H]2-),
obsd. 1928.3, 963.9 Anal. calcd. for C75H95N13O47: C, 46.66,
H, 4.96, N, 9.43. found: C, 46.49, H, 4.81, N, 9.61;
1
IIIb: HNMR (400MHz, D2O): ꢀ=1.50 (d, 3H, J=6.0Hz
NAPCH3), 2.42(brs, 6H, CH2COꢁ3), 2.77-2.94(br, 30H,
Aspꢂ-CH2ꢁ5ꢁ3), 3.54-3.82 (brs, 12H, OCH2ꢁ3+OCH2ꢁ3),
3.98(brs, 4H, NAPCH3O+CH), 4.55-4.96(m, 15H, Aspꢃ-
CH), 7.24-7.88(m, 6H, NAPph-H).
Fig. (3). Percentage of conjugates bound to HAP at 16h.Error bars
represent the mean and standard deviation of three independent
experiment.
ESI-MS(m/z): calcd. for 2276.9 ([M+H]+), obsd. 2276.6;
1
IIIc: HNMR (400MHz, D2O): ꢀ=1.56(d, 3H, J=6.0Hz
NAPCH3), 2.45(brs, 6H, CH2COꢁ3), 2.70-2.99(brs, 36H,
Aspꢂ-CH2), 3.69 (brs, 12H, OCH2ꢁ3+OCH2ꢁ3), 3.99(brs,
4H, NAPCH3O+CH), 4.55-4.96(m, 18H, Aspꢃ-CH), 7.26-
7.89(m, 6H, NAPph-H).
ESI-MS(m/z): calcd. for 2620.7 ([M+H]+), obsd. 2620.7
In Vitro HAP Binding Assay
Conjugates were dissolved in PBS(pH7.4, 5ml, concen-
tration 100 μg/ml) and incubated with HAP(10mg) in a
shaker bath at 37ꢀ. Each formulation was cultured for 0.5h
and 16h. After the prescribed time, conjugate solutions were
removed from the water bath and centrifuged for 2 min at
2500rpm. Standard conjugate solutions were also prepared
from 16μg/ml to 400μg/ml to generate a concentration cali-
bration curve. Concentrations of non-bound conjugates were
determined by UV spectrophotometry absorbance at 273nm.
A control was tested in Ca2+ containing solution to corrobo-
rate the HAP binding further. The result was shown in Figs.
(2 and 3).
Fig. (4). Change of percent conjugates Ia, IIa, IIIa bound to HAP.
RESULTS AND DISCUSSION
We designed and synthesized Naproxen-Peptide-
Monomer and Naproxen-Peptide-Dendrimer as bone target-
ing prodrugs by a conventional coupling strategy. All modi-
fied naproxen conjugates exhibited some binding to HAP in
vitro and the naproxen-peptide-dendrimers showed a more
greater binding rate(up to 90%)than the monomers after 16h.
Unmodified naproxen showed no greater than 2% mean
binding.
In the synthesis of the naproxen peptide dendrimers, us-
ing IBCF/NMM was more difficult to achieve than previous
amidation reactions. To overcome the problem of steric hin-
drance, several other coupling reagents(DCC, CDI, EDCI,
etc.) were used and we found DCC/Hobt method afforded
the product in a relatively high yield.
Fig. (2). Percentage of conjugates bound to HAP at 0.5h.Error bars
represent the mean and standard deviation of three independent
experiment.
The high degree of symmetry in these dendrimers en-
abled facile confirmation of both structure and purity by
1
After that assay, we chose three conjugates (the mono-
mer, dimer and trimer) which displayed a good HAP affinity
and determined 0.5, 1, 2, 4, 8, 16 h’ HAP binding with the
same method respectively. The result was shown in Fig. (4).
NMR techniques. For example in the HNMR spectrum of
dendrimer III, the core protons observed the resonance sig-
nals at 1.44 (s), 2.45 (brs), and 3.69 (brs) ppm were clearly
distinguishable from the resonances arising from the periph-

Synthesis of Bone Targeting Prodrugs
Letters in Organic Chemistry, 2009, Vol. 6, No. 4
277
ery (Asp oligopeptides) at 2.71 (m) and 4.84 (m) ppm. Inte-
gration of the respective areas of the core protons and ꢀ-
methine protons of Asp oligopeptides confirmed the com-
plete coupling of the central cores and peptides 6. Further-
more, the structures of these dendrimers were further verified
by ESI MS and GPC. The GPC results were outlined in
Table 1. All of the spectra displayed a very prominent peak
corresponding to the dendrimers complexed with protons or
sodium cation. Moreover, elemental analysis was also in
good agreement with those of the signed structures.
readily synthesized and characterized. Both prodrugs bound
to HAP, a model for bone. The peptide dendrimers with
higher numbers of total poly(aspartic acid) groups attached
per naproxen than naproxen-peptide monomer exhibited
their intended effect, faster initial binding and greater total
binding rates. Although the water solubility of naproxen was
poor, these prodrugs showed a good solubility in water.
Therefore, based on the in vitro data presented here, we sug-
gested that the Asp peptide-dendrimer had potential for drug
delivery to bone. We are currently exploring higher genera-
tions of dendritic molecules bearing more Aspn sequences
and developing a suitable animal model for in vivo evalua-
tion.
Table 1. Molecular Weights of Synthesized Dendrimers
Measured by GPC
Compound
RetTimea
Mwa
Mw/Mna
m/zb
ACKNOWLEDGEMENT
Ia
17.045
16.490
16.238
1233
2728
3318
1.05
1.38
1.18
691
1294
1928
This work was supported by the National Natural Science
Foundation of China. (Project 20472055 supported by
NSFC).
IIa
IIIa
aMeasured by GPC (polystyrene standards) with THF as an eluent.
bMeasured by ESI mass spectroscopy.
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Fig. (4) described that the dimer and trimer exhibited
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CONCLUSIONS
In summary, the naproxen-peptide monomer and the
naproxen-peptide dendrimer as bone targeting prodrugs were