Novel dendritic naproxen prodrugs with poly(aspartic Acid) Oligopeptide: Synthesis and hydroxyapatite binding in vitro

Article abstract of DOI:10.1080/00397911.2011.584259

Novel bone-targeting prodrugs containing dendritic naproxen and poly(aspartic acid) oligopeptide were synthesized in a convergent approach and were characterized by NMR, mass spectral, and elemental analysis techniques. The modified naproxen prodrugs showed a high affinity to hydroxyapatite in vitro and provided an effective entry for the synthesis of a dendritic naproxen-poly(aspartic acid) oligopeptide conjugates used for bone targeting.

Full text of DOI:10.1080/00397911.2011.584259

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Novel Dendritic Naproxen Prodrugs
with Poly(aspartic Acid) Oligopeptide:
Synthesis and Hydroxyapatite Binding in
Vitro
Junzhu Pan a , Lifang Ma b , Bo Li a , Yanhua Li a & Li Guo a
a Key Laboratory of Drug Targeting and Drug Delivery System of
Education Ministry, Department of Medicinal Chemistry , West China
School of Pharmacy, Sichuan University , Chengdu , China
b School of Chemical Engineering, Sichuan University , Chengdu ,
China
Accepted author version posted online: 11 Nov 2011.Published
online: 02 Aug 2012.
To cite this article: Junzhu Pan , Lifang Ma , Bo Li , Yanhua Li & Li Guo (2012) Novel Dendritic
Naproxen Prodrugs with Poly(aspartic Acid) Oligopeptide: Synthesis and Hydroxyapatite Binding in
Vitro, Synthetic Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 42:23, 3441-3454, DOI: 10.1080/00397911.2011.584259
To link to this article: http://dx.doi.org/10.1080/00397911.2011.584259
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Synthetic Communications1, 42: 3441–3454, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.584259
NOVEL DENDRITIC NAPROXEN PRODRUGS WITH
POLY(ASPARTIC ACID) OLIGOPEPTIDE: SYNTHESIS
AND HYDROXYAPATITE BINDING IN VITRO
Junzhu Pan,1 Lifang Ma,2 Bo Li,1 Yanhua Li,1 and Li Guo1
1Key Laboratory of Drug Targeting and Drug Delivery System of Education
Ministry, Department of Medicinal Chemistry, West China School of
Pharmacy, Sichuan University, Chengdu, China
2School of Chemical Engineering, Sichuan University, Chengdu, China
GRAPHICAL ABSTRACT
Abstract Novel bone-targeting prodrugs containing dendritic naproxen and poly(aspartic acid)
oligopeptide were synthesized in a convergent approach and were characterized by NMR, mass
spectral, and elemental analysis techniques. The modified naproxen prodrugs showed a high
affinity to hydroxyapatite in vitro and provided an effective entry for the synthesis of a dendritic
naproxen–poly(aspartic acid) oligopeptide conjugates used for bone targeting.
Keywords Bone-targeting; convergent synthesis; dendrimer; drug delivery; poly(aspartic
acid) oligopeptide
INTRODUCTION
Musculoskeletal diseases such as osteoarthritis and osteoporosis seriously
threaten human health, and they are recognized as major public health problems
worldwide. Bone is composed of unique materials such as the inorganic compound
hydroxyapatite (HAp) and bone matrix proteins, which are quite different from
the components of other tissues. Thus, the properties of bone (hardness, poor
permeability, and special biochemical process) limit drug efficacy in bone. Increasing
Received December 1, 2010.
Address correspondence to Li Guo, 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, China. E-mail: rosaguoli2000@yahoo.com.cn
3441

3442
J. PAN ET AL.
the dosage can make the drug concentration available at the bone for treatment but
increases systemic toxic possibility. For instance, anti-inflammatory drugs (NSAIDs,
e.g., naproxen, ibuprofen) are clinically used in osteoarthritis. However, they have
been reported to cause a number of side effects at high dosage, in particular gastro-
intestinal toxicity. Hence, study of drug delivery systems that could allow efficient
transport of drugs to the bone will be one of the hotspots in drug-targeting research.
Dendrimers are new artificial macromolecules that have highly branched struc-
ture and globular shape. To date, dendrimers have been widely applied in medical and
biomedical fields, such as drug delivery,[1] gene delivery,[2] and gene transfection.[3]
Dendrimers conjugated with multiple copies of drugs have displayed remarkably
increased drug action and specificity relative to the monomer by virtue of cooperativ-
ity effects. Furthermore, the specific properties of dendrimers give them superior
application in drug delivery, for example, well-defined and large globular structure,
mono- or low polydispersity, and multivalency.[4] On the basis of reported works,
dendrimers provide a novel approach for both multidrug-carrying and drug-targeting
purposes.[5] The biodendrimers reported by Grinstaff’s group are a special type of
dendrimer family[6–8] and elements of the biodendrimers include the following: (i):
The building blocks are compounds known to be biocompatible or degradable, or
natural metabolites in vivo; and (ii): these building blocks are assembled by biodegrad-
able chemical bonds (commonly ester linkage and amide linkage).[6] Besides common
advantages of dendrimer, the prominent superiority of the biodendrimer is that the
macromolecules will eventually degrade under physiological conditions through
hydrolytic scission to building blocks that will be metabolized or excreted on the time
scale appropriate for a beneficial medical outcome. Consequently, biodendrimers are
used as absorbable temporary scaffolds for tissue engineering[6,7] and drug delivery.[8]
Poly(aspartic acid) oligopeptides (Aspn) are novel bone-targeting moieties. Asp
pentapeptid (Asp5) and Asp hexapeptide (Asp6) are oligopeptides with five to six
consecutive negatively charged amino acid residues that readily adsorb certain
calcium-containing surfaces and exhibit a remarkable affinity to bonehydroxyapatite.
(The carboxyl group–protected Asp5-6 1a and b are shown in Fig. 1).[9] Unlike the tra-
ditional bone-targeting moieties (tetracycline, bisphosphonate, and their derivates),
the oligopeptides are biologically labile and enzymatically degradable.[10] Even more,
the oligopeptides can alter the pharmacokinetic and biological properties, including
the blood clearance, distribution, and biological activity.[11] Therefore, poly(aspartic
Figure 1. Structure of the fully protected Asp5-6 1a and b.

BONE-TARGETING NAPROXEN DENDRIMER
3443
acid) oligopeptides seem very attractive as alternative bone targeting carriers. Previous
work has been conducted on small-molecule drugs (e.g., estradiol[12] or quinolones[13]
)
modified with Asp6, which exhibited high accumulation in bone in vivo. However,
there are few reports on poly(aspartic acid) oligopeptides used as bone-targeting
moiety for drug delivery based on dendrimer (a macromolecule). Also, how the
number of the negatively charged amino acid residues in Aspn and the generation of
dendrimers influence the conjugates’ HAp binding rates have never been elaborated.
In this manuscript, we designed and synthesized of two series of novel bone-
targeting prodrugs. These prodrugs were composed of two fragments: the first- or
second- generation biodendrimer with naproxen surface and the poly(aspartic acid)
oligopeptides (Asp5-6) as bone-targeting moiety. A hydroxyapatite (HAp) binding
assay was established under in vitro conditions, and the affinity of the bone-targeting
conjugates for HAp is reported here. Preliminary study of drug release, biodistribu-
tion, and pharmacokinetic analysis of these conjugates for delivering naproxen into
the bone in vivo is now under way.
RESULTS AND DISCUSSION
The Bone-Targeting Prodrug Design
Based on the concept of biodendrimer, glycerol and succinic acid are included in
natural metabolites in vivo of human, and diethanolamine is a known biocompatible
compound which is widely used in pharmaceutical.[14] Therefore, these compounds
are suitable building blocks for biodendrimer. Furthermore, ester bond linkage to
NSAIDs does not weak its anti-inflammatory activity. On the contrary, compared
with the free drug, the active of NSAIDs would be decreased with amide conjugates.[15]
Following the principle of building biodendrimer and the previous research on
NSAIDs’ prodrug, we designed two monomers [4-(1,2-dihydroxyethoxy)-4-oxobuta-
noic acid) (DHEOBA) and (4-(N,N-bis(2-hydroxyethyl)amino)-4-oxobutanoic acid)
(bis-HEAOBA)] for building dendrimers (Fig. 2). These two monomers were com-
posed of two hydroxyl (for loading drugs and generating the dendrimer) and a car-
boxyl (for generating the dendrimer and connecting Aspn). By convergent synthesis,
Figure 2. Structure of the dendrimers’ monomers.

3444
J. PAN ET AL.
Scheme 1. Preparation of the core 4 and 5; Conditions: (a) Benzyl alcohol, DMAP, THF, 50 ꢀC. (b) (i)
2,2-Dimethyl-1,3-dioxolane-4-methanol, EDCI=DMAP, CH2Cl2, rt; (ii) HOAc=H2O, 50 ꢀC. (c) Diethano-
lamine, NMM, IBCF, acetone, ꢁ15 ꢀC to rt.
two series of drug-tagged biodendrimers, dendritic (DHEOBA) dendrimer (Scheme 2)
and dendritic (bis-HEAOBA) dendrimer (Scheme 3) with naproxen periphery, were
prepared. Then, the dendrimer fragment was functionalized with poly(aspartic acid) oli-
gopeptide by amide bond. Overall, the oligopeptide–dendrimer conjugates are biologi-
cally labile and enzymatically degraded, and there are no unexpected long-term effects.
Synthesis
Two series of novel bone-targeting prodrugs containing dendritic naproxen
and poly(aspartic acid) oligopeptide were synthesized. The synthetic route is outlined
in Schemes 2 and 3.
Scheme 2. Preparation of the Aspn-[Gm]-dendri(DHEOBA)-Napx; Conditions: (a) Naproxen, EDCI=
DMAP, CH2Cl2, rt. (b) (i) H2, Pd-C (10%, wt%), MeOH, rt; (ii) 4, EDCI=DMAP, CH2Cl2, rt. (c) (i)
H2, Pd-C (10%, wt%), MeOH, rt; (ii) 1a=1b, EDCI, HoBt, CH2Cl2, rt; (iii) H2, Pd-C (10%, wt%), MeOH,
rt. (d) (i) H2, Pd-C (10%, wt%), MeOH, rt; (ii) 1a=1b, HBTU, DIPEA, CH2Cl2, rt; (iii) H2, Pd-C (10%,
wt%), MeOH, rt.

BONE-TARGETING NAPROXEN DENDRIMER
3445
Scheme 3. Preparation of the Aspn-[Gm]-dendri(bis-HEAOBA)-Napx; Conditions: (a) Naproxen, EDCI=
DMAP, CH2Cl2, rt. (b) (i) H2, Pd-C (10%, wt%), MeOH, rt; (ii) 5, EDCI=DMAP, CH2Cl2, rt. (c) (i) H2,
Pd-C (10%, wt%), MeOH, rt; (ii) 1a=1b, EDCI, HoBt, CH2Cl2, rt; (iii) H2, Pd-C (10%, wt%), MeOH, rt.
(d) (i) H2, Pd-C (10%, wt%), MeOH, rt; (ii) 1a=1b, HBTU, DIPEA, CH2Cl2, rt; (iii) H2, Pd-C (10%, wt%),
MeOH, rt.
Two types of dendritic naproxen were prepared through a convergent synthesis.
First, the bifunctional core 4=5 was coupled with naproxen in the presence of EDCI ꢂ
HCl and DMAP to afford [G1]-dendri (DHEOBA)-Nap2 6 and [G1]-dendri(bis-
HEAOBA)-Nap2 10, respectively. Formation of the second-generation dendritic
naproxen was achieved in a two-step sequence involving Pd=C-mediated removal
of the benzyl groups from [G1] dendrimer 6=10, and subsequent generation with core
(4=5)=EDCI=DMAP to afford [G2]-dendri(DHEOBA)-Nap4 7 and [G2]-dendri(bis-
HEAOBA)-Nap4 11 respectively.
To functionalize dendritic naproxen with poly(aspartic acid) oligopeptides, the
benzyl group of the dendritic naproxen 6=7=10/11 was removed via hydrogenolysis,
and the resultant carboxylic acid was treated with peptide 1 in the presence of a coup-
ling reagent. We attempted to use several coupling reagents, such as isobutyl
chloroformate=N-methylmorpholine (mixed anhydride method), N,N0-dicyclohexyl-
carbodiimide (or 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride,
N,N0-methanetetraylbis(1-methylethylamine))=dimethylamino pyridine (or N-hydro-
xysuccinimide, 1-hydroxy benzotriazole). To load poly(aspartic acid) oligopeptides
on the first-generation dendrtic naproxen, using IBCD=NMM and DCC (EDCI)=
HOBt could give the product in acceptable yields, but EDCI=HOBt provided the best
yield. For the similar reaction between the oligopeptides and the second-generation
dendrtic naproxen, steric hindrance had become the major problem with the

3446
J. PAN ET AL.
increased generation of dendrimer. Using EDCI=HOBt did not give the product in
acceptable yield, and other coupling reagents mentioned previously gave similarly
poor results. Only the solid-phase peptide coupling reagent o-benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium hexafluorophosphate=N,N-diisopropylethylamine
afforded the products in good yield. Finally, the conjugates could show affinity to
HAp only after the benzyl group of the poly(aspartic acid) oligopeptides was
removed.[16] The deprotection was accomplished by hydrogenolysis over 10% pal-
ladium on charcoal, and it required only filtration and evaporation to provide isolated
products.
The high degree of symmetry in these dendrimers and conjugates enabled facile
confirmation of both structure and purity by NMR techniques. For example, in the
1H NMR spectrum of dendrimers 6, 7, 10, and 11, the core protons at the resonance
signals at 5.10 (s) ppm were clearly distinguishable from the resonances arising from
the periphery (naproxen) at 1.36–1.42 (m) and 3.78–3.83 (m) ppm. Integration of the
respective areas of the core protons (ph-CH2) and naproxen protons (–CHCH3) con-
1
firmed the complete coupling. In the H NMR spectrum of oligopeptide–dendrimer
conjugates 8, 9, 12, and 13, the dendritic naproxen protons at the resonance signals
at 1.36–1.42 (m) ppm were clearly distinguishable from the poly(aspartic acid) oligo-
peptide at 4.49–4.57(m) ppm. Integration of the respective areas of the Asp-a -CH
protons and –CHCH3 protons from dendritic naproxen confirmed the jointing of
the oligopeptides and dendritic naproxen. Furthermore, the structures of these
dendrimers and conjugates were further verified by electrospray ionization (ESI) mass
spectrometry (MS) or turnover frequency (TOF) MS. Moreover, elemental analyses
were also in good agreement with those of the target structures.
HAp Binding Assay in Vitro
In Figures 3 and 4, two general characteristics of these compounds were
revealed. First, all of the compounds exhibited some HAp affinity, whereas the parent
drug naproxen was at best negligibly bound. The binding rates of these compounds
approximate, or evidently exceed, 50%, and moreover, 13a and 13b show dramatic
binding rates of more than 85%. The other distinguishing feature is that the binding
rates culminate rapidly. To compare certain compounds’ HAp binding rates of 1 h
and 24 h, they do not mark any divergence in the statistics (t > 0.05). This comparison
indicated that the compounds loaded with poly(aspartic acid) oligopeptide can bind to
HAp and approach their binding plateaus in a short time (1 h), which is in agreement
with our group’s previous work.[17]
To draw a comparison with the same generation and peptide between
Aspn-[Gm]-dendri(DHEOBA)-Napx and Aspn-[Gm]-dendri(bis-HEAOBA)-Napx,
Aspn-[Gm]-dendri(bis-HEAOBA)-Napx performs with better HAp affinity in general.
Nonetheless, the difference of their binding rates is less than 10% (except for 12a ver-
sus 8a, which is more than 20%). For the compounds with the same dendritic frame-
work, the second generation of the compounds provided better binding rates than the
first generation. All of the [G2] compounds exhibited notable affinity to HAp (>80%),
whenas the [G1] compounds’ binding rates are 40% to 70%. The precise mechanism of
how the compounds with greater generation of dendrimer display better affinity (the
dendrimer itself has no affinity) is not yet understood. However, it could inspire us to

BONE-TARGETING NAPROXEN DENDRIMER
3447
Figure 3. Percentage of conjugates Aspn-[Gm]-dendri(DHEOBA)-Napx bound to HAp. Error bars
represent the mean and standard deviation of three independent expriments.
Figure 4. Percentage of conjugates Aspn-[Gm]-dendri(bis-HEAOBA)-Napx bound to HAp. Error bars rep-
resent the mean and standard deviation of three independent expriments.
combine the poly(aspartic acid) oligopeptides with greater generation dendrimer.
Besides, comparison of compounds with different peptides, and conjugates contain-
ing Asp5 or Asp6 shows no differences in HAp affinity.
EXPERIMENTAL
All reactions requiring anhydrous conditions were performed under an Ar or N2
atmosphere. Chemicals and solvents were either analytical-reagent grade or purified
by standard techniques. Thin-layer chromatography (TLC) used silica-gel plates
GF254; compounds were visualized by irradiation with ultraviolet light and=or by
treatment with a solution of phosphomolybdic acid (20% wt in ethanol) followed
by heating. Column chromatography was performed by using silica gel with eluent

3448
J. PAN ET AL.
given in parentheses. 1H NMR analysis was performed by the Inova Varian 400-MHz
spectrometer using CDCl3 or dimethylsulfoxide (d6-DMSO) as a solvent at room tem-
perature. The chemical shifts are expressed in relative to tetramethylsilene (TMS)
(¼0 ppm) and the coupling constants J are in hertz (Hz). MS spectra were
performed on Agilent 1964B spectrometer. MALDI-TOF MS were recorded with
Bruker Biflex III (matrix was sinapic acid SA). Ultraviolet spectroscopy were per-
formed on Perkin-Elmer Lambda 35 instrument. Melting points (ꢀC) were determined
with X-4 digital melting-point apparatus and are uncorrected. Elemental analysis was
performed by Atlantic Microlab, Atlanta, GA, USA. Hydroxyapatite (HAp) was
purchased from Shanghai Institute of Biochemistry with surface area 9.12 m2=g and
average particle size 15 mm.
Synthesis of Peptide 1a and b
Peptides 1a, and 1b (Fig. 1), were synthesized by literature methods.[17]
Synthesis of Succinic Acid Monobenzyl Ester 3
Succinic acid monobenzyl ester 3 was prepared by the literature methods.[18]
General Procedure for the Synthesis of the Core 4
Succinic acid monobenzyl ester 3 (10 mmol, 2.0 g) was dissolved in CH2Cl2
(30 ml), and 2,2-dimethyl-1,3-dioxolane-4-methanol (11 mmol), EDCI ꢂ HCl
(12 mmol), and DMAP (1 mmol) were added to the solution. The reaction mixture
was stirred at room temperature for 24 h. The solution was then concentrated under
vacuum, and the residue was dissolved in HOAc–H2O (1:4 v=v, 30 ml). The reaction
mixture was stirred at 50 ꢀC for 5 h. Saturated aqueous K2CO3 was added to quench
the reaction and make the pH 8. The aqueous phase was extracted with CHCl3
(3 ꢃ 15 mL). The combined organic portions were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified on a silica-gel
chromatography column (CH2Cl2=MeOH) to obtain 4 (1.6 g) as white waxy solid.
1
Yield: 72%. H NMR (CDCl3) d: 2.31 (brs, 2H, glycerol-OH ꢃ 2), 2.66–2.74 (m,
4H, CH2CH2), 3.56–3.69 (m, 2H, CH2OH), 3.88–3.93 (m, 1H, CHOH), 4.15–4.23
(m, 2H, COOCH2), 5.14 (s, 2H, ph-CH2), 7.26–7.40 (m, 5H, ph-H).
General Procedure for the Synthesis of the Core 5
Succinic acid monobenzyl ester 3 (10 mmol, 2.0 g) dissolved in anhydrous
acetone (25 ml) was cooled at ꢁ15 ꢀC, and then NMM (10 mmol) and IBCF
(10 mmol) were added. After stirring for 10 min, diethanolamine (15 mmol, 1.6 g) dis-
solved in anhydrous acetone (10 ml) was added dropwise to the reaction. The reac-
tion mixture was vigorously stirred at room temperature for 2 h. The solution was
then concentrated under vacuum, and the residue was taken up in ethyl acetate
(30 ml) and washed with 1 M HCl (10 ml), 1 M NaHCO3 (10 ml), and brine
(10 ml). The organic layer was dried over anhydrous Na2SO4. After concentration,
the residue was purified by silica-gel chromatography column (CH2Cl2=MeOH) to

BONE-TARGETING NAPROXEN DENDRIMER
3449
1
obtain 5 (2.1 g) as a colorless oil. Yield: 70%. H NMR (CDCl3) d: 2.77 (brs, 4H,
succinic acid-CH2CH2), 3.55–3.59 (m, 4H, NCH2 ꢃ 2), 3.81–3.86 (m, 4H,
CH2OH ꢃ 2), 5.16 (s, 2H, ph-CH2), 7.37–7.40 (m, 5H ph-H).
General Procedure for the Synthesis of [G1]-Dendri(DHEOBA)-Nap2 6
Core 4 (1.41 g, 5 mmol) was dissolved in CH2Cl2 (30 ml), and EDCI ꢂ HCl
(15 mmol), DMAP (1 mmol), and naproxen (2.2 g, 10.5 mmol) were added to the
solution. The reaction mixture was stirred under N2 at room temperature for 24 h.
The solution was then concentrated under vacuum, and the residue was taken up
in ethyl acetate (30 ml) and washed with 1 M HCl (10 ml), 1 M NaHCO3 (10 ml),
and brine (10 ml). The organic layer was dried over anhydrous Na2SO4. After con-
centration, the residue was purified by silica-gel chromatography column (petroleum
ether=ethyl acetate) to obtain 6 (2.65 g) as a white solid. Yield: 75%, mp. 52–56 ꢀC.
1H NMR (CDCl3) d: 1.40–1.56 (m, 6H, CHCH3 ꢃ 2), 2.22–2.57 (m, 4H, CH2CH2),
3.88–3.90 (m, 8H, OCH3 ꢃ 2 þ CHCH3 ꢃ 2), 3.96–4.24 (m, 4H, glycerol-CH2 ꢃ 2),
5.08–5.13 (m, 2H, ph-CH2), 5.17–5.24 (m, 1H, glycerol-CH), 7.06–7.26 (m, 5H,
ph-H), 7.32–7.38 (m, 6H, Nap-phH), 7.59–7.71 (m, 6H, Nap-phH). ESI MS (m=z):
calcd. for C42H42O10: 706.28; obsd. 729.26 ([M þ Na]þ).
General Procedure for the Synthesis of [G2]-Dendri(DHEOBA)-Nap4 7
The mixture of 6 (1.77 g, 2.5 mmol) and 10% Pd=C (100 mg) in methanol–CH2Cl2
(5:1 v=v 30 ml) was stirred at room temperature under a hydrogen atmosphere. After
24 h, the mixture was passed through a membrane filter to remove the catalyst and then
evaporated under reduced pressure to afford a white foam. Without further purifi-
cation, the obtained white foam was dissolved in CH2Cl2 (30 ml), and EDCI ꢂ HCl
(5 mmol), DMAP (1 mmol), and core 4 (1.2 mmol) were added into the solution.
The reaction mixture was stirred under N2 at room temperature for 24 h. The solution
was then concentrated under vacuum, and the residue was taken up in ethyl acetate
(30 ml) and washed with 1 M HCl (10 ml), 1 M NaHCO3 (10 ml), and brine (10 ml).
The organic layer was dried over anhydrous Na2SO4. After concentration, the residue
was purified by silica-gel chromatography column (petroleum ether=ethyl acetate) to
1
obtain 7 (0.76 g) as a colorless oil. Yield: 40%. H NMR (CDCl3) d: 1.36–1.55 (m,
12H, CHCH3 ꢃ 4), 2.15–2.52 (m, 12H, CH2CH2 ꢃ 3), 3.89 (m, 16H, OCH3 ꢃ
4 þ CHCH3 ꢃ 4), 4.09–4.34 (m, 12H, glycerol-CH2 ꢃ 6), 5.11 (s, 2H, ph-CH2),
5.17–5.24 (m, 3H, glycerol-CHꢃ3), 7.07–7.24 (m, 11H, ph-HþNap-phH), 7.32–7.36
(m, 6H, Nap-phH), 7.44–7.49 (m, 2H, Nap-phH), 7.58–7.70 (m, 10H, Nap-phH).
ESI MS (m=z): calcd. for C84H86O24: 1478.55; obsd.1500.44 ([M þ Na-H]þ).
General Procedure for the Synthesis of Aspn-[G1]-Dendri(DHEOBA)-
Nap2 8a and 8b
The mixture of 6 (0.25 mmol) and 10% Pd=C (100 mg) in methanol–CH2Cl2
(5:1 v=v 30 ml) was stirred at room temperature under hydrogen atmosphere. After
24 h, the mixture was passed through a membrane filter to remove the catalyst, and
then evaporated under reduced pressure to afford a white foam. Without further

3450
J. PAN ET AL.
purification, the obtained white foam was dissolved in CH2Cl2 (30 ml), and
EDCI ꢂ HCl (0.25 mmol), HOBt (0.25 mmol), and 1a and 1b separately (0.25 mmol)
were added to the solution. The reaction mixture was stirred under N2 at room tem-
perature for 24h. The solution was then concentrated under vacuum, and the residue
was taken up in ethyl acetate (30 ml) and washed with 1 M HCl (10 ml), 1 M NaHCO3
(10 ml), and brine (10 ml). The organic layer was dried over anhydrous Na2SO4. After
concentration, the residue was purified by silica-gel chromatography column (pet-
roleum ether=acetone) to obtain a white waxy solid. Next, 10% Pd=C (100mg) was
added to a solution of the obtained white waxy solid in methanol–ethyl acetate
(3:1 v=v 30 ml). The reaction mixture was stirred under hydrogen atmosphere for
24 h, filtered through a membrane filter, and concentrated under reduced pressure to
afford 8a and b.
Compound 8a. White foam (110 mg, yield: 37%) from 6 (0.25 mmol).
1H NMR (d6-DMSO) d: 1.24–1.44 (m, 6H, CHCH3 ꢃ 2), 2.44–2.55 (m, 9H,
CH2CH2 þ Asp-b-CH2=2 ꢃ 5), 2.61–2.72 (m, 5H, Asp-b-CH2=2 ꢃ 5), 3.57–3.67 (m,
2H, CHCH3 ꢃ 2), 3.85 (s, 6H, OCH3 ꢃ 2), 3.92–4.32 (m, 4H, glycerol-CH2 ꢃ 2),
4.47–4.61 (m, 5H, Asp-a -CH ꢃ 5), 5.13–5.21 (m, 1H, glycerol-CH), 7.13–7.56 (m,
6H, Nap-phH), 7.65–8.08 (m, 6H, Nap-phH), 12.54 (brs, 6H, Asp-COOH ꢃ 6). ESI
MS (m=z): calcd. for C55H61N5O25: 1191.37; obsd.1215.00 ([M þ Na]þ). Anal. calcd.
for C, 55.41; H, 5.16; N, 5.87; O, 33.55. Found: C, 55.22; H, 5.21; N, 5.77; O, 33.67.
Compound 8b. White foam (111 mg, yield: 34%) from 6 (0.25 mmol). 1H NMR
(d6-DMSO) d: 1.25–1.44 (m, 6H, CHCH3 ꢃ 2), 2.44–2.53 (m, 10H, CH2CH2 þ Asp-b-
CH2=2 ꢃ 6), 2.62–2.72 (m, 6H, Asp-b-CH2=2 ꢃ 6), 3.62–3.67 (m, 2H, CHCH3 ꢃ 2),
3.85 (s, 6H, OCH3 ꢃ 2), 3.92–4.36 (m, 4H, glycerol-CH2 ꢃ 2), 4.49–4.54 (m, 6H,
Asp-a-CH ꢃ 6), 5.12–5.20 (m, 1H, glycerol-CH), 7.12–7.55 (m, 6H, Nap-phH),
7.65–8.04 (m, 6H, Nap-phH), 12.47 (brs, 7H, Asp-COOH ꢃ 7). ESI MS (m=z): calcd.
for C59H66N6O28: 1306.39; obsd.1329.03 ([M þ Na]þ). Anal. calcd. for C, 54.21; H,
5.09; N, 6.43; O, 34.27. Found: C, 54.46; H, 5.15; N, 6.50; O, 34.13.
General Procedure for the Synthesis of Aspn-[G2]-Dendri(DHEOBA)-
Nap4 9a and 9b
The same procedure as described for preparation of 8a and b was used.
1
Compound 9a. White foam (78 mg, yield: 40%) from 7 (0.1 mmol). HNMR
(d6-DMSO) d: 1.24–1.43 (m, 12H, CHCH3 ꢃ 4), 2.48–2.55 (m, 17H, CH2CH2 ꢃ
3 þ Asp-b-CH2=2 ꢃ 5), 2.68–2.76 (m, 5H, Asp-b-CH2=2 ꢃ 5), 3.84 (s, 12H, OCH3 ꢃ
4), 4.10–4.21 (m, 12H, glycerol-CH2 ꢃ 6), 4.47–4.55 (m, 5H, Asp-a-CHꢃ5),
5.12–5.19 (m, 3H, glycerol-CHꢃ3), 7.12–7.34 (m, 11H, Nap-phH), 7.64–7.77 (m,
9H, Nap-phH), 7.99–8.25 (m, 4H, Nap-phH), 12.46 (brs, 6H, Asp-COOH ꢃ 6).
ESI MS (m=z): calcd. for C97H105N5O39: 1963.64; obsd. 1963.67 ([M]ꢁ). Anal. calcd.
for C, 59.29; H, 5.39; N, 3.56; O, 31.76. Found: C, 59.05; H, 5.34; N, 3.62; O, 31.60.
Compound 9b. White foam (73 mg, yield: 35%) from 7 (0.1 mmol). 1H NMR
(d6-DMSO) d: 1.23–1.43 (m, 12H, CHCH3 ꢃ 4), 2.40–2.55 (m, 18H, CH2CH2 ꢃ 3 þ
Asp-b-CH2=2 ꢃ 6), 2.68–2.76 (m, 6H, Asp-b-CH2=2 ꢃ 6), 3.56–3.67 (m, 4H,
CHCH3 ꢃ 4), 3.84 (s, 12H, OCH3 ꢃ 4), 4.10–4.30 (m, 12H, glycerol-CH2 ꢃ 6),

BONE-TARGETING NAPROXEN DENDRIMER
3451
4.47–4.55 (m, 6H, Asp-a-CH ꢃ 6), 5.15 (m, 3H, glycerol-CH ꢃ 3), 7.12–7.45 (m, 11H,
Nap-phH), 7.66–7.79 (m, 9H, Nap-phH), 8.01–8.24 (m, 4H, Nap-phH), 12.46 (brs,
7H, Asp-COOH ꢃ 7). ESI MS (m=z): calcd. for C101H110N6O42: 2078.67; obsd.
2079.19 ([M]þ). Anal. calcd. for C, 58.32; H, 5.33; N, 4.04; O, 32.31. Found: C,
58.47; H, 5.39; N, 4.10; O, 32.47.
General Procedure for the Synthesis of [G1]-Dendri(bis-HEAOBA)-
Nap2 10
The same procedure as described for preparation of 6 was used. White solid
(2.38 g, yield: 66%) from core 5 (1.48 g, 5 mmol), mp ¼ 107–110 ꢀC. 1H NMR (CDCl3)
d: 1.50–1.53 (m, 6H, CHCH3 ꢃ 2), 2.22–2.36 (m, 4H, succinct acid-CH2CH2),
2.93–3.39 (m, 4H, NCH2 ꢃ 2), 3.84–3.89 (m, 8H, OCH3 ꢃ 2 þ CHCH3 ꢃ 2), 3.98–
4.14 (m, 4H, NCH2CH2 ꢃ 2), 5.08 (s, 2H, ph-CH2), 7.06–7.13 (m, 5H, ph-H),
7.29–7.37 (m, 6H, Nap-phH), 7.58–7.66 (m, 6H, Nap-phH). ESI MS (m=z): calcd.
for C43H45NO9: 719.31; obsd. 742.52 ([M þ Na]þ).
General Procedure for the Synthesis of [G2]-Dendri(bis-HEAOBA)-
Nap4 11
The same procedure as described for preparation of 7 was used. Colorless oil
1
(760 mg, yield: 41%) from 10 (2.5 mmol). H NMR (CDCl3) d: 1.50–1.52 (m, 12H,
CHCH3 ꢃ 4), 2.21–2.68 (m, 12H, succinct acid–CH2CH2 ꢃ 3), 2.92–3.73 (m, 12H,
NCH2 ꢃ 6), 3.82–3.89 (m, 16H, OCH3 ꢃ 4 þ CHCH3 ꢃ 4), 3.99–4.17 (m, 12H,
NCH2CH2 ꢃ 6), 5.10 (s, 2H, ph-CH2), 7.06–7.66 (m, 7H, ph-H þ Nap-phH), 7.26–
7.32 (m, 10H, Nap-phH), 7.59–7.66 (m, 12H, Nap-phH). ESI MS (m=z): calcd. for
C87H95N3O21: 1517.65; obsd. 1539.42 ([M þ Na-H]þ).
General Procedure for the Synthesis of Aspn-[G1]-Dendri
(bis-HEAOBA)-Nap2 12a and 12b
The same procedure as described for preparation of 8a and b was used.
Compound 12a. White foam (132 mg, yield: 44%) from 10 (0.25 mmol).
1H NMR (d6-DMSO) d: 1.42–1.44 (m, 6H, CHCH3 ꢃ 2), 2.47–2.56 (m, 9H, succinic
acid CH2 ꢃ 2 þ Asp-b-CH2=2 ꢃ 5), 2.69–2.75 (m, 5H, Asp-b-CH2=2 ꢃ 5), 3.25–3.27
(m, 4H, NCH2 ꢃ 2), 3.81–3.88 (m, 8H, OCH3 ꢃ 2 þ CHCH3 ꢃ 2), 3.94–4.07 (m, 4H,
NCH2CH2 ꢃ 2), 4.49–4.56 (m, 5H, Asp-a-CHꢃ5), 7.13–7.34 (m, 6H, Nap-phH),
7.67–7.77 (m, 6H, Nap-phH), 12.51 (brs, 6H, Asp-COOHꢃ6). ESI MS (m=z): calcd.
for C56H64N6O24: 1204.40; obsd. 1204.21 ([M]þ). Anal. calcd. for C, 55.81; H, 5.35; N,
6.97; O, 31.86. Found: C, 55.73; H, 5.31; N, 6.90; O, 31.99.
Compound 12b. White foam (135 mg, yield: 41%) from 10 (0.25 mmol).
1H NMR (d6-DMSO) d: 1.42–1.44 (m, 6H, CHCH3 ꢃ 2), 2.47–2.57 (m, 10H, succinic
acid-CH2 ꢃ 2 þ Asp-b-CH2=2 ꢃ 6), 2.69–2.75 (m, 6H, Asp-b-CH2=2 ꢃ 6), 3.25–3.27
(m, 4H, NCH2 ꢃ 2), 3.85–3.90 (m, 8H, OCH3 ꢃ 2 þ CHCH3 ꢃ 2), 3.94–4.03(m, 4H,
NCH2CH2 ꢃ 2), 4.50–4.51 (m, 6H, Asp-a-CHꢃ6), 7.13–7.40 (m, 6H, Nap-phH),
7.67–7.77 (m, 6H, Nap-phH), 12.68 (brs, 7H, Asp-COOHꢃ7). ESI MS (m=z): calcd.

3452
J. PAN ET AL.
for C60H69N7O27: 1319.42; obsd. 1318.49 ([M-H]ꢁ). Anal. calcd. for C, 54.58; H, 5.27;
N, 7.43; O, 32.72. Found: C, 54.71; H, 5.33; N, 7.37; O, 32.65.
General Procedure for the Synthesis of Aspn-[G2]-Dendri(bis-
HEAOBA)–Nap4 13a and 13b
The same procedure as described for preparation of 8a and b was used.
Compound 13a. White foam (76 mg, yield: 38%) from 11 (0.1 mmol).
1H NMR (d6-DMSO) d: 1.40–1.42 (m, 12H, CHCH3 ꢃ 4), 2.26–2.54 (m, 17H,
succinic acid-CH2 ꢃ 6 þ Asp-b-CH2=2 ꢃ 5), 2.68–2.76 (m, 5H, Asp-b-CH2=2 ꢃ 5),
3.17–3.34 (m, 12H, NCH2 ꢃ 6), 3.78–3.84 (m, 16H, OCH3 ꢃ 4 þ CHCH3 ꢃ 4),
3.88–4.11(m, 12H, NCH2CH2 ꢃ 6), 4.50–4.57 (m, 5H, Asp-a-CHꢃ5), 7.11–7.33 (m,
12H, Nap-phH), 7.64–7.77 (m, 12H, Nap-phH), 12.41 (brs, 6H, Asp-COOHꢃ6).
MALDI-TOF MS (m=z) calcd. for C100H114N8O36: 2002.73; obsd. 2025.62
([M þ Na]þ). Anal. calcd. for C, 59.93; H, 5.73; N, 5.59; O, 28.74. Found: C, 59.79;
H, 5.68; N, 5.62; O, 28.85.
Compound 13b. White foam (66 mg, yield: 31%) from 11 (0.1 mmol).
1H NMR (d6-DMSO) d: 1.36–1.42 (m, 12H, CHCH3 ꢃ 4), 2.26–2.58 (m, 18H, suc-
cinic acid-CH2 ꢃ 6 þ Asp-b-CH2=2 ꢃ 6), 2.68–2.72 (m, 6H, Asp-b-CH2=2 ꢃ 6),
3.17–3.56 (m, 12H, NCH2 ꢃ 6), 3.78–3.83 (m, 16H, OCH3 ꢃ 4 þ CHCH3 ꢃ 4),
3.86–4.11(m, 12H, NCH2CH2 ꢃ 6), 4.49–4.57 (m, 6H, Asp-a-CHꢃ6), 7.11–7.33 (m,
12H, Nap-phH), 7.64–7.77 (m, 12H, Nap-phH), 12.39 (brs, 7H, Asp-COOHꢃ7).
MALDI-TOF MS (m=z) calcd. for C104H119N9O39: 2117.76; obsd. 2140.31
([M þ Na]þ). Anal. calcd. for C, 58.95; H, 5.66; N, 5.95; O, 29.45. Found: C, 58.77;
H, 5.60; N, 5.99; O, 29.33.
In Vitro HAp Binding Assay
Conjugates (8a and b, 9a and b, 12a and b, 13a and b) were dissolved in anhy-
drous MeOH (10 ml, concentration 200 mg=ml) and incubated with HAp (20 mg) in a
shaker bath at 37 ꢀC. Each formulation was cultured for 1 h and 24 h. After the pre-
scribed time, conjugate solutions were removed from the water bath and centrifuged
for 2 min at 3000 rpm. Standard conjugate solutions were also prepared from
25 mg=ml to 400 mg=ml to generate a concentration calibration curve. Concentrations
of conjugates were determined by ultraviolet spectrophotometry absorbance at
273 nm. The results are shown in Figs. 3 and 4.
CONCLUSIONS
In conclusion, we have prepared a series of the first- and second-generation
monodisperse naproxen dendrimer–peptide conjugates as novel potential bone-
1
targeting dendritic drug delivery systems. The H NMR, MS, and elemental analysis
results supported the formation of the targeting peptide–dendrimer conjugates. All
of the conjugates exhibited their intended effects, with faster initial binding and greater
total binding rates. We are continuing to explore higher generations of dendritic mole-
cules bearing naproxen and develop a suitable animal model for in vivo evaluation.

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3453
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