Synthesis and in vitro characterization of a tissue-selective fullerene: vectoring C(60)(OH)(16)AMBP to mineralized bone.

Article abstract of DOI:10.1016/S0968-0896(02)00049-4

A tissue-vectored bisphosphonate fullerene, C(60)(OH)(16)AMBP [4,4-bisphosphono-2-(polyhydroxyl-1,2-dihydro-1,2-methanofullerene[60]-61-carboxamido)butyric acid], designed to target bone tissue has been synthesized and evaluated in vitro. An amide bisphosphonate addend, in conjunction with multiple hydroxyl groups, confers a strong affinity for the calcium phosphate mineral hydroxyapatite of bone. Constant composition crystal growth studies indicate that C(60)(OH)(16)AMBP reduces hydroxyapatite mineralization by 50% at a concentration of 1 microM, following a non-Langmuirian mechanism. Parallel studies with C(60)(OH)(30) also indicate an affinity for hydroxyapatite, but at a reduced level (28% crystal growth rate reduction at 1 microM) compared with C(60)(OH)(16)AMBP. This study is the first to demonstrate that a fullerene-based material can be successfully targeted to a selected tissue as a step toward the development of such materials for medical purposes, in general.

Full text of DOI:10.1016/S0968-0896(02)00049-4

Bioorganic & Medicinal Chemistry 10 (2002) 1991–1997
Synthesis and In Vitro Characterization of a
Tissue-Selective Fullerene: Vectoring C60(OH)16AMBP
to Mineralized Bone
Kelly A. Gonzalez,a Lon J. Wilson,a,* Wenju Wub and George H. Nancollasb
aDepartment of Chemistry and the Center for Nanoscale Science and Technology and the Laboratory for
Biochemical and Genetic Engineering, MS-60, Rice University, PO Box 1892, Houston, TX 77251-1892, USA
bDepartment of Chemistry, State University of New York at Buffalo, Buffalo, NY 14214-3001, USA
Received 18 October 2001; accepted 10 December 2001
Abstract—A tissue-vectored bisphosphonate fullerene, C60(OH)16AMBP [4,4-bisphosphono-2-(polyhydroxyl-1,2-dihydro-
1,2-methanofullerene[60]-61-carboxamido)butyric acid], designed to target bone tissue has been synthesized and evaluated in vitro.
An amide bisphosphonate addend, in conjunction with multiple hydroxyl groups, confers a strong affinity for the calcium phos-
phate mineral hydroxyapatite of bone. Constant composition crystal growth studies indicate that C60(OH)16AMBP reduces
hydroxyapatite mineralization by 50% at a concentration of 1 mM, following a non-Langmuirian mechanism. Parallel studies with
C60(OH)30 also indicate an affinity for hydroxyapatite, but at a reduced level (28% crystal growth rate reduction at 1 mM) compared
with C60(OH)16AMBP. This study is the first to demonstrate that a fullerene-based material can be successfully targeted to a selected tissue
as a step toward the development of such materials for medical purposes, in general. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
HAP bind to the surface of the crystals at kinks and
dislocations, blocking crystallization. Using carefully
designed experiments, the extent of crystal growth inhi-
bition by a bone-vectored compound can then be used to
estimate the compound’s affinity for bone tissue in vivo.13
Fullerene- and metallofullerene-based materials offer
promise as new diagnostic and therapeutic drugs.1 This
promise is based on the facts that such materials are
proving to be relatively non-toxic,2ꢀ5 non-metaboliz-
able,6 and capable of in vivo delivery of radiolabeled
and non-labeled metal ions commonly used in diag-
nostic and therapeutic medicine. To fulfill their promise,
however, such materials must also be capable of being
selectively delivered to specific tissue upon demand.
Crystal growth inhibition technology is especially
important because bone-vectored compounds typically
target areas of bone undergoing formation and resorp-
tion processes.14 The vectored compounds are attracted
to the active growth sites of HAP, and thus bind in
greatest concentration to the metabolically-active por-
tions of bone. Where bone tissue is diseased, a high rate
of bone metabolism exists, and this activity attracts sui-
tably-derivatized compounds to the diseased site. If, for
instance, a radionuclide-containing fullerene deriva-
tive15 could be successfully vectored to these diseased
sites in bone, an opportunity exists for tissue-specific
radiation therapy. High tissue specificity reduces the
dose level required by the subject, and thereby reduces
treatment costs and potential harm to non-diseased tis-
sues. As yet, no tissue-vectored fullerene derivatives
have been reported. This study is the first to specifically
target a fullerene-based material to a particular tissue
Bone tissue is an especially appealing target for vectored
pharmaceuticals because its primary inorganic compo-
nent, hydroxyapatite (HAP), offers a multitude of
binding sites for structurally suitable compounds.
Compounds with functional groups such as hydroxyls
and carboxylic and phosphonic acids are capable of
forming ionic and hydrogen bonds to the mineral
portion of bone.7ꢀ9 The interactions between a bone-
vectored compound and the mineralized tissue may be
modeled in vitro using HAP crystal growth inhibition
studies,10ꢀ12 whereby compounds with high affinity for
type
with
a
bisphosphonate-C60
derivative,
C60(OH)16AMBP (Fig. 1), being targeted to mineralized
bone in vitro.
*Corresponding author. Tel.: +1-713-348-3268; fax: +1-713-348-
5155; e-mail: durango@rice.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00049-4

1992
K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
Table 1. The rate of HAP crystallization decreases with increasing
concentration of water-soluble fullerene derivatives
C60(OH)30
(10ꢀ6 mol Lꢀ1
Rate
(10ꢀ8 mol minꢀ1 mꢀ2
C
60(OH16AMBP)
(10ꢀ6 mol Lꢀ1
Rate
(10ꢀ8 mol minꢀ1 mꢀ2
)
)
)
)
0
9.09
8.31
7.8
6.55
5.94
5.7
4.65
4.07
3.97
3.85
2.64
2.49
0
9.09
7.67
6.51
4.55
3.77
2.83
2.46
1.92
1.57
1.23
0.41
0.81
1.22
1.63
2.03
2.44
2.85
3.25
3.66
4.07
4.47
0.39
0.77
1.16
1.54
1.93
2.32
2.7
Figure 1. C60(OH)16AMBP, the bone-vectored fullerene derivative.
3.09
3.47
Results and Discussion
Constant composition growth experiments were first
performed in pure supersaturated solutions of HAP and
then in the presence of C60(OH)16AMBP and
C60(OH)30. The results are presented in Table 1.
Figures 2 and 3 show typical plots of titrant volume
required to maintain the supersaturation as a function
of time at different fullerene concentrations. For com-
parison, a curve of titrant consumption for the growth
of HAP crystals in pure supersaturated solutions is
included (open symbols). All the rate curves show a
rapid titrant addition immediately following the intro-
duction of seed crystals. This frequently observed
phenomenon, usually attributed to conditioning of the
surface of the seed crystals in the supersaturated solu-
tion, may reflect ion exchange involving solution and
surface cations and protons, or the removal of active
growth sites on the seed crystals due to the rapid crys-
tallization of high energy sites.16,17 Significant reduc-
tions of the initial surges observed in the presence of the
fullerenes suggest their adsorption at the growth sites on
the HAP seed crystals, thus their competition with other
initial surface processes. It can be seen in Figure 2 that
linearity of the rate plots of HAP crystal growth
(reflected by the constant slopes of volume versus time
curves) was usually achieved 10–20 min after seed
introduction to the supersaturated solution in experi-
ments performed in the absence and presence of full-
erenes. It should be noted that at high concentrations of
inhibitors, HAP crystals appear to grow only during the
initial reaction stage.
Figure 2. Titrant volume added as a function of time during constant
composition HAP crystal growth inhibition experiments with
C60(OH)30
.
It is quite well established that strong inhibitors of crystal
growth, such as the phosphonates, act by blocking,
through adsorption, active growth sites at the crystal
surfaces.16,18 Commonly, inhibition kinetics data are
interpreted in terms of a simple Langmuir adsorption
model. Assuming that the adsorbed fullerene molecules
occupy a fraction, y, of the active growth sites, thereby
preventing them from participating in the growth, the
growth rate R can be written in terms of the uninhibited
rate R0 (eq 2)
Figure 3. Titrant volume added as a function of time during constant
composition HAP crystal growth inhibition experiments with
C60(OH)16AMBP.
C60(OH)16AMBP are plotted in Figure 4 as a function
of additive concentration.
R0=R ¼ 1 þ KLC
ð2Þ
Comparison of the two curves shows that both com-
in which KL is the adsorption affinity constant with
units of liters per mole. Crystal growth rates R mea-
sured in the presence of C60(OH)30 and
pounds
C60(OH)16AMBP being the more effective inhibitor. At
inhibit
HAP
crystal
growth,
with
a concentration of 3.1ꢁ10ꢀ5 M, C60(OH)30 reduces the

K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
1993
Table 2. Data from HAP crystal growth inhibition studies with
C60(OH)30, C60(OH)16AMBP, and CH3C(OH)[P(O)(OH)2]2
Compound
Concentration
(mol/L)
Percent
reduction in
Affinity
constant
growth rate (L molꢀ1
)
C60(OH)30
1.0ꢁ10ꢀ6
1.ꢁ10ꢀ6
28
50
87
69a
4.14ꢁ105
C60(OH)16AMBP
C60(OH)16AMBP
CH3C(OH)[P(O)(OH)2]2
—
—
3.5ꢁ10ꢀ6
1.0ꢁ10ꢀ6
13.3ꢁ105a
aRef 29.
Figure 4. Rate of HAP crystal growth in the presence of C60(OH)30
(&) and C60(OH)16AMBP (*).
Under such conditions, an equilibrium distribution may
be impossible, thereby preventing the molecules from
effectively inhibiting mineralization. In experiments
with higher inhibitor concentrations, molecular aggre-
gation can develop more easily, thus making the
decrease in inhibitory activity more apparent. Such
aggregation may result from enhanced intermolecular
hydrogen bonding interactions among the hydroxyl and
carboxylic and phosphonic acid groups of the fullerene
material.
Table 2 compares the percent reductions in growth rate of
the two fullerenol derivatives to that for 1-hydroxyethyl-
idene-1,1-diphosphonic acid, CH3C(OH)[P(O)(OH)2]2,
a commercially-available bone-vectored compound used
in the treatment of osteoporosis.20 At 1.0ꢁ10ꢀ6
M
Figure 5. Plot of R0/R versus concentration for C60(OH)30 (&) and
C60(OH)16AMBP (*).
inhibitor concentration, neither C60(OH)30 nor
C60(OH)16AMBP is as effective as CH3C(OH)-
[P(O)(OH)2]2 at inhibiting HAP crystal growth.
C60(OH)30 also has a lower affinity constant, suggesting
that the compound binds to the surface less strongly
than CH3C(OH)[P(O)(OH)2]2. The lower binding con-
stant for C60(OH)30 is not surprising given that the full-
erenol does not contain a bisphosphonic acid moiety.
The hydroxyl functionalities present in C60(OH)30 are
capable of hydrogen bonding to the surface, but they do
not provide the stronger ionic interactions that are pre-
sent with bisphosphonic acid groups.
HAP crystal growth by 58%, while C60(OH)16AMBP
reduces the rate by 87%. The increase in inhibition is
linear with concentration for C60(OH)30, but not for the
bisphosphonate derivative, C60(OH)16AMBP.
Following eq 2, plots of R0/R as a function of con-
centration for each compound are shown below in
Figure 5.
The linear relation shown for C60(OH)30 indicates that
the compound inhibits HAP crystal growth by the
mechanism described by the Langmuir formalism.
From the slope, the affinity constant is 4.14 105 L molꢀ1
(R2=0.97). In fact, this affinity of fullerenol materials
for HAP has also been observed previously in an in vivo
Comparison of C60(OH)16AMBP to CH3C(OH)-
[P(O)(OH)2]2 is somewhat problematic because the latter
molecule inhibits HAP crystal growth by a mechanism
that appears strictly Langmuirian, with the affinity con-
stant being independent of concentration. As shown in
Figure 4, such independence is not the case for
C60(OH)16AMBP.
mouse
model
study
using
radiolabeled
166Ho@C60(OH)x.15
The plot of R0/R versus concentration for
C60(OH)16AMBP lacks linearity, which suggests that
the mechanism of inhibition is different from that
described by the Langmuir model. The model relies on
the assumptions that the inhibitor reversibly binds to
the mineral surface at active growth sites and that the
surface-bound molecules do not interact with one
another.16,19 For C60(OH)16AMBP, these assumptions
may be invalid. The curvature observed in Figure 4 for
C60(OH)16AMBP at higher concentrations may be
caused by lateral interference among the surface-bound
molecules. Once adsorbed to a surface, molecules are
capable of lateral movement and surface aggregation.
Despite uncertainty in the inhibition mechanism, the
results demonstrate that both C60(OH)30 and
C60(OH)16AMBP inhibit HAP crystal growth from
supersaturated calcium phosphate solutions and that
both compounds have relatively strong affinities for
HAP. The greater crystal growth inhibition by
C60(OH)16AMBP stresses the importance of incorpor-
ating bisphosphonate moieties into bone-vectored full-
erene derivatives. Further explanation for the
diminished rate inhibition observed at higher
C60(OH)16AMBP concentrations will require studies of
other fullerene compounds, especially those having multi-
directional surface-binding functionalities such as

1994
K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
C60C6(COOH)1221,22 and others. As the first investigation,
however, the present study demonstrates the potential
usefulness of fullerene-based materials in tissue-targeting
technologies and lays the ground-work for in vivo
studies using radiolabeled bisphosphonate materials as
potential bone therapeutic agents.
Solutions were prepared using triply distilled carbon
dioxide-free water and filtered before use through
washed 0.22 mm filters (Millipore, Bedford, MA, USA).
Calcium concentrations were determined either com-
plexometrically by EDTA titration with Eriochrome
Black-T as indicator, or by atomic absorption (Perkin-
Elmer, model 3100, Norwich, CT, USA). Carbon dioxide-
free potassium hydroxide solutions were prepared from
washed reagent grade pellets in a nitrogen atmosphere.
Experimental
Materials
Crystal growth experiments were made in magnetically-
stirred water-jacketed Pyrex vessels at 37.0ꢃ0.05 ꢂC
with ionic strength, I=0.15 mol Lꢀ1, adjusted by the
addition of sodium chloride. Supersaturated solutions
were prepared by introducing calcium chloride solution,
followed by potassium dihydrogen phosphate solution.
The pH was adjusted to the required value by the slow
addition of potassium hydroxide solutions. During the
reactions, carbon dioxide was excluded by bubbling
with presaturated nitrogen gas. After equilibration, 0.5
mL of the HAP slurry was introduced to initiate the
reaction. Since the nucleation and growth of crystals
consume solution lattice ions, the lowering of pH was
used to trigger the addition of two titrant solutions that
served to maintain constant the pH, the concentrations
of calcium and phosphate and the ionic strength of the
solution. A glass electrode (Orion, model 9101), stan-
dardized using two NBS buffer solutions at pH 7.386
and 4.028 at 37 ꢂC, was used to control titrant addition
through a potentiostat. The total calcium concentration
in all experiments was 6.00ꢁ10ꢀ4 mol Lꢀ1 with a cal-
cium/phosphate molar ratio of 1.67, so as to achieve a
supersaturation with respect to HAP of s=5.55 (as
defined in eq 1), as computed from mass balance, pro-
ton dissociation, electroneutrality, and equilibrium
expressions involving calcium and phosphate species.
Reagent grade solvents and electrolytes (Fisher) were
used without purification unless stated otherwise.
Anhydrous solvents were obtained by distillation from
appropriate drying agents under inert atmosphere. Pet-
roleum ether and bromobenzene were each pre-dried
with NaSO4 and then refluxed over and distilled from
sodium. Benzene, toluene, and ethyl ether were each
pre-dried with CaCl2 and then refluxed over and dis-
tilled from sodium in the presence of sodium benzo-
phenone ketyl. Chloroform, stabilized with 0.75%
ethanol, was used as received. Triethylamine was dis-
tilled from KOH under inert atmosphere.
Silica gel (Aldrich, grade 62, 60–200 mesh, 150 A) was
activated at 130 ꢂC for a minimum of 12 h before use.
Sephadex G-25 (Aldrich, 20–80 m) was equilibrated in
DI H2Ofor 24 h at room temperature prior to use.
Physical and spectroscopic methods
Unless otherwise noted, residual solvent signals were
used for spectral reference in the 13C and 1H NMR
spectra (DMSO-d6, 2.50 and 39.1 ppm; CDCl3, 7.26 and
77.0 ppm; D2O, 4.70 ppm). Phosphoric acid (85%, 0
ppm) was used as an external reference for 31P NMR
spectra. Signals that were shifted upfield from H3PO4
were assigned positive values; signals downfield from
H3PO4 were assigned negative values. For each set of
phosphorus NMR data, the upfield or downfield shift is
stated for clarity.
Â
Ã1=v
ꢀ ¼ S ꢀ 1 ¼ IP=KSO ꢀ1
ð1Þ
In eq 1, v is the number of ions per formula unit of
precipitating phase and IP and KSO are the ionic and
solubility products, respectively, of HAP. The addition
of the fullerene derivatives at micromolar levels to the
reaction solutions did not affect the established super-
saturation.
Mass spectra were measured on a Finnigan MAT 95
GC-MS analyzer using electron ionization (EI, 70 eV)
or atmospheric pressure chemical ionization (APCI).
High-resolution APCI peak matching spectra were col-
lected using Gramocidin S as the peak reference at
1141.71376 amu in 50/50 CHCl3/MeOH.
During the reactions, samples were withdrawn periodi-
cally, filtered (0.22 mm Millipore filter) and analyzed for
calcium by atomic absorption and for phosphate spec-
trophotometrically (Varian, Cary 210) as the phospho-
vanadomolybdate complex in order to verify the
constancy of the solution composition. Solid phases
were examined by X-ray powder diffraction, XRD
(Siemens Nicolet/Nic spectrometer, CuK radiation with
Ni filter=1.540; 2 from 3 to 45ꢂ), by scanning electron
microscopy (SEM at 20 kV; JEOL JSM-5300, Noran
Instrumental Inc. Middleton, WI, USA) and by diffuse
reflectance infrared Fourier transform spectroscopy
(FTIR, Perkin-Elmer 1760X FT-IR spectrometer).
Plots of mineralization were calculated from plots of
titrant volumes as a function of time as described
previously.23
Elemental analyses were obtained commercially from
Galbraith Laboratories, Inc., Knoxville, TN, USA.
Materials and Methods for HAP crystal growth
inhibition studies
Hydroxyapatite seed crystals were prepared from calcium
nitrate and potassium dihydrogen phosphate, as detailed
elsewhere.23 The specific surface area, 34.9 m2 gꢀ1, was
determined by BET nitrogen adsorption using a 30/70 N2/
He mixture (Monosorb, Quantachrome Corp). HAP
crystals in the form of a suspension in water (41.8 g Lꢀ1
were used for the crystal growth experiments.
)

K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
1995
In order to investigate the uptake of fullerene deriva-
tives by HAP surfaces, an equilibrium adsorption
experiment was performed in which 0.0209 g of HAP in
its saturated solution was equilibrated with various
concentrations of these additives. A UV–visible spec-
trophotometer (Perkin-Elmer model 3100) was used for
the analysis of fullerene derivative concentrations. Since
the z-potential of HAP surfaces was markedly influ-
enced by the presence of the additives, this parameter
was used as an indication of the extent of adsorption.
The z-potential of HAP surfaces for the same suspen-
sions in the presence of these additives was measured
using a Malvern Zetasizer IIc (Malvern, UK).
IR (neat): 2984 (C–H), 2935 (C–H), 2910 (C–H), 1636
(C¼C), 1251, (P¼O), 1024 (C–O), 974 (P–C–P bend),
803 cmꢀ1 (P–O).
Benzylidene glycine ethyl ester. In a typical synthesis,26
the hydrochloride salt of glycine ethyl ester (15.0 g,
107.5 mmol) was dissolved in 125 mL CH2Cl2. Treat-
ment with freshly distilled NEt3 (21.8 g, 30 mL, 215.8
mmol) resulted in the formation of a small amount of
white precipitate. Benzaldehyde (7.60 g, 7.28 mL, 71.6
mmol) was then added to the reaction at room tem-
perature followed by 6 g MgSO4 to remove the water
by-product. After 10 h stirring, the solution was filtered
and reduced under vacuum to give a yellow oil. This
compound was dissolved in 100 mL Et2O, washed with
satd aq NaCl (6ꢁ50 mL) and dried (MgSO4). Solvent
removal under vacuum at 25 ꢂC gave a light yellow oil
that was used without further purification. The com-
pound was stored at 10 ꢂC. Yield: 13.42 g, 98%. 1H
NMR (250 MHz, CDCl3, TMS ref) d 8.30 (s, 1H), 7.78
(m, 2H), 7.42 (m, 3H), 4.40 (s, 2H), 4.24 (q, 2H, J=7.1
Hz), 1.31 ppm (t, 3H, J=7.1 Hz); 13C NMR (250 MHz,
CDCl3) d 169.99, 165.27, 135.45, 131.07, 128.46, 128.35,
61.94, 60.94, 14.07. IR (neat): 3063 (Ar C–H), 3029 (Ar
C–H), 2983 (C–H), 2938 (C–H), 2975 (C–H), 2903 (C–H),
2854 (C–H), 1735 (C¼O), 1646 (C¼N), 1200 (C–O),
1027 (C–O), 759 (Ar–H out-of-plane), 694 cmꢀ1 (Ar–H
out-of-plane).
Syntheses
1,2-Dihydro-1,2-methanofullerene[60]-61-carboxylic acid,
C60-CHCOOH. 1,2-Dihydro-1,2-methanofullerene[60]-
61-carboxylic acid was synthesized according to the
procedure by Isaacs and Diederich.24
Tetraethyl ethenylidenebisphosphonate. In
a typical
synthesis,25 paraformaldehyde (2.60 g, 86.7 mmol) and
diethylamine (1.79 mL, 1.27 g, 17.4 mmol) were com-
bined in MeOH (25 mL) with gentle warming to aid
dissolution. The mixture was then cooled to room tem-
perature and 5.0 g (17.3 mmol) tetraethyl methylenedi-
phosphonate was added with stirring. The reaction was
refluxed for 24 h under atmospheric conditions and then
diluted with 25 mL MeOH and concentrated under
vacuum at 35 C. Toluene (50 mL) was added to the flask
and the contents were again concentrated under vacuum
at 35 C. This step was repeated a second time to ensure
that all of the MeOH had been removed. The inter-
mediate (tetraethyl 2-methoxyethylenebisphosphonate)
was then placed under vacuum at room temperature for
Ethyl N - benzylidene - 2 - amino - 4,4 - bis(diethoxyphos-
phoryl)butyrate. In a typical synthesis,27 benzylidene
glycine ethyl ester (1.91 g, 10.0 mmol) was added to a
solution of NaOEt (1 mmol) in 20 mL of fresh absolute
EtOH at ꢀ8 ꢂC (ice-salt bath). Tetraethyl ethenylidene-
bisphosphonate (3.00 g, 10.0 mmol) was added drop-
wise over 3 min with vigorous stirring. The reaction was
stirred for 30 min at 25 ꢂC and then neutralized with
satd aq NH4Cl (ca. 3 mL). Removal of the EtOH (ca.
25 ꢂC) at reduced pressure left a paste-like residue that
was extracted with CHCl3 (3ꢁ20 mL). The organic
fraction was dried over MgSO4, filtered and evaporated
at reduced pressure to give a light-yellow oil that was
stored in the freezer. Yield: 4.72 g, 96%. 1H NMR
(250 MHz, CDCl3, TMS ref) d 8.38 (s, 1H, N=CHPh),
7.76 (m, 2H), 7.44 (m, 3H), 4.46 (dd, 1H, J=4.9, 8.8
Hz), 4.16 (m, 10H), 2.7–2.3 (m, 3H), 1.32 (m, 15H); 13C
NMR (250 MHz, CDCl3) d 171.08, 165.47, 135.54,
131.18, 128.49, 128.44, 69.81 (dd, J=4.4, 10.5 Hz), 62.5
(m), 61.11, 32.73 (t, J=133.1 Hz), 28.83, 16.30 (d,
3JC-P=24.3 Hz), 14.05; 31P NMR (250 MHz, CDCl3):
23.66, 23.93 ppm, downfield from H3PO4. IR (neat):
2984 (C–H), 2933 (C–H), 2907 (C–H), 2872 (C–H),
1737 (ester C¼O), 1641 (CH=N), 1280 (shoulder;
P¼O), 1253 (P¼O), 1027 (C–O), 970 (P–C–P), 754 cmꢀ1
(P–O).
1
3 h. H NMR (250 MHz, CDCl3) d 4.15 (m, 8H), 3.84
(td, 2H, JP-H=16.3 Hz, JH-H=5.4 Hz), 3.33 (s, 3H), 2.65
(tt, 1H, JP-H=23.8 Hz, JH-H=5.4 Hz), 1.30 (t, 12H,
J=7.0 Hz).
The reaction flask was attached to a septum-capped
soxlet extractor containing 4 A molecular sieves and the
entire system was flushed with Ar for 15 min. Approxi-
mately 125 mL anhydrous toluene was then added
through the condenser. p-Toluenesulfonic acid mono-
hydrate (0.20 g, 1.1 mmol) was added under Ar sparge
and the flask was wrapped with aluminum foil. The
reaction was refluxed under inert atmosphere for 14 h.
After solvent removal, the remaining light yellow oil
was redissolved in CHCl3, washed with three 50 mL
portions of DI H2O, and dried over MgSO4. The CHCl3
was then removed, leaving a yellow oil that distilled
over at 139 C/1 torr as a colorless liquid. An analytically
pure sample was prepared by column chromatography
on SiO2 with 50:50 hexanes/acetone eluent. Yield: 4.0 g,
Ethyl 2-amino-4,4-bis(diethoxyphosphoryl)butyrate. In a
typical synthesis,27 ethyl N-benzylidene-2-amino-
4,4-bis(diethoxyphosphoryl)butyrate (3.00 g, 6.1 mmol)
was dissolved in 10 mL DI H2Oat room temperature.
p-Toluenesulfonic acid monohydrate (1.74 g, 9.2 mmol)
was then added and the mixture was stirred for 30 min
at room temperature. After extraction with Et2O(4 ꢁ15
1
77%. H NMR (250 MHz, CDCl3) d 6.96 (distorted dd,
JP-H=37.6 Hz, JP-H=34.1 Hz), 4.14 (m, 8H), 1.33 (t,
JH-H=7.1 Hz, 12H); 13C NMR (250 MHz, CDCl3, TMS
ref) d 149.28, 131.80 (t, JP-C=166.5 Hz), 62.60 (t, JP-
C=2.8 Hz), 16.20 (t, JP-C=3.3 Hz); 31P NMR
(250 MHz, CDCl3) 13.73 ppm downfield from H3PO4.

1996
K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
mL) to remove benzaldehyde, the aqueous solution was
rendered alkaline by addition of satd aq NaHCO3 (ca.
pH 8.1) and then extracted with CHCl3 (4ꢁ10 mL). The
organic layer was dried over MgSO4, filtered and eva-
porated under reduced pressure to give a light yellow oil
that was used without further purification. The com-
pound was stored in the freezer and checked by NMR
to ascertain purity before each use. Yield: 2.3 g, 94%.
1H NMR (200 MHz, CDCl3, TMS ref) d 4.2 (m, 10H),
3.77 (dd, J=4.4,0 10.6 Hz, 1H), 3.14–2.84 (dddd,
4,4 - Bisphosphono - 2 - (1,2 - dihydro - 1,2 - methanofuller-
ene[60]-61-carbox-amido)butyric acid, C60AMBP. C60-
AMBP-protected (0.160 g, 0.137 mmol) was dissolved in
28
125 mL anhydrous toluene in a glove box. I-Si(CH3)3
(0.196 mL, 0.275 g, 1.37 mmol) was added drop-wise at
room temperature over 30 s. After 72 h stirring at 50 ꢂC,
the reaction was filtered to remove a dark insoluble
product. The filtrate was then removed from the glove
box and quenched with 1 mL MeOH. An insoluble
brown precipitate formed immediately. It was collected in
a centrifuge tube, washed consecutively with CHCl3 and
Et2O, and then dried under vacuum at 65 ꢂC for 8 h. This
compound was insoluble in all common solvents. Yield:
127 mg, 90%. IR (KBr): 3600–2500 (br, P–OH), 2923 (C–
H), 1719 (acid C¼O), 1650 (amide I band), 1540 (amide II
band), 1241–1024 (envelope P¼O , C–O , O H bend), 928
(P–C–P), 526 cmꢀ1 (fullerene resonance).
3
2
3JHH=3.3 Hz, J HH=8.9 Hz, JHP=23.2 Hz, and
2J0HP=24.7 Hz, 1H, PCHP), 2.5–2.2 (m, 1H), 2.1–1.8
(m, 1H), 1.3 (m, 15H); 13C NMR (200 MHz, CDCl3) d
175.50, 62.5 (m), 61.02, 52.77 (dd, J=10.3, 3.1 Hz),
32.77 (t, J=133.9 Hz), 30.49, 16.39 (d, J=6.1 Hz),
14.22; 31P NMR (250 MHz, CDCl3,): 24.43, 24.16 ppm,
downfield from H3PO4. IR (neat): 3476 (br, N–H), 2984
(C–H), 2934 (C–H), 2910 (C–H), 1733 (ester C¼O),
1646 (NH2 deformation), 1277 (shoulder, P¼O), 1245
(P¼O), 1020 (C–O), 972 (P–C–P), 838 (P–O), 799 cmꢀ1
(P–O).
The intermediate tetra-silyl ester, produced by the reac-
tion of ITMS with the four phosphonate ester groups
1
1
was characterized by H NMR. H NMR (200 MHz,
CD2Cl2) d NH not observed, 5.26 (s, 1H), 5.04 (m, 1H),
4.37 (q, 2H), 2.75–2.15 (br m, 3H), 1.35 (t, 3H), 0.52 and
0.43 (br s, 36H). CH3CH2I is also present in the sample
as a by-product of the deprotection reaction.
Ethyl 4,4-bis(diethoxyphosphoryl)-2-(1,2-dihydro-1,2-me-
thanofullerene[60]-61-carboxamido)butyrate, C60AMBP-
protected. C60-CHCOOH (0.100 g, 0.128 mmol) and
1-hydroxybenzotriazole, BtOH, (0.035 g, 0.256 mmol)
were combined in 30 mL PhBr. Approximately 0.104 g
(0.256 mmol) ethyl 2-amino-4,4-bis(diethoxypho-
sphoryl)butyrate, AMBP, was added via pipette. Imme-
4,4-Bisphosphono-2-(polyhydroxyl-1,2-dihydro-1,2-metha-
nofullerene[60]-61-carboxamido)butyric acid, C60(OH)16-
AMBP. A less vigorous hydroxylation procedure was
used for C60(OH)16AMBP than for C60(below) to
ensure survival of the bisphosphonate substituent.
C60AMBP (0.050 g, 0.05 mmol) was dissolved in 0.5 mL
40% tetra n-butylammonium hydroxide and then dilu-
ted to 10 mL with 1 M KOH. The mixture was stirred
for 24 h at room temperature and then chromato-
graphed on Sephadex G-25 (fine) size exclusion gel (2.5
inchesꢁ5 inches). The product eluted as a well-defined
brown-orange band followed later by a volume of col-
orless basic eluent. Between aliquots, the column was
rinsed thoroughly with DI H2Ountil the pH of the elu-
ent was no longer basic. After three passes, the pH of
the collected sample fractions was ca. 6, suggesting that
most of the base had been removed. The collected frac-
tions were reduced to 10 mL under vacuum with very
gentle heating (T<30 ꢂC) and then dried under slow air
flow overnight to remove the remaining solvent. The
resulting flaky, black solid was collected and dried at
100 ꢂC/1 torr over P2O5 for 12 h. 37 mg
C60(OH)16AMBP isolated. 1H NMR (250 MHz, D2O) d
4.15, 3.95, 3.56, 2.25, 1.9–0.9 (all br signals); 31P NMR
(250 MHz, D2O): 12.4 ppm upfield from H3PO4 (br
weak singlet). IR (KBr): 3358 (v br, O-H), 2922 (ali-
phatic C–H), 1717 (shoulder, carboxy C¼O), 1595 (v br,
amide C¼O), 1387 (v br, O-H bend), 1239 (P¼O), 1072
(s, C–O), 1045 (s, C–O). UV–vis (H2O): No maxima
were observed; the absorption curve decreases gradually
toward the visible region. To describe the absorption
strength, measurements were taken at 300, 400, and 500
nm. The molar extinction coefficients at these wave-
lengths are 22.9ꢁ106, 7.57ꢁ106 and 2.08ꢁ106 cm2/mol,
respectively. MALDI-MS: no peaks observed other
than 720 (C60). Anal. Calcd for C66H27NO25P2
(C60(OH)16AMBP) C, 61.18; H, 2.10; N, 1.09; P, 4.78;
diately, 0.0530
g (0.256 mmol) 1,3-dicyclohexyl-
carbodiimide (DCC) was added to the reaction flask.
Stirring for 5 days at room temperature under inert
atmosphere yielded a dark cranberry colored solution.
The product was purified by column chromatography
on a 10ꢁ1 inch SiO2 column using toluene to elute PhBr
and a 1% MeOH/CHCl3 solution to elute the cranberry
colored product. Two precipitations from CHCl3 with
Et2Ogave a brown solid that was collected in a cen-
trifuge tube and dried overnight under vacuum at room
1
temperature. Yield: 0.103 g, 69%. H NMR (200 MHz,
CDCl3) d 8.93 (br d, 1H, J=7.8), 4.85 (m, 1H), 4.79
(s, 1H), 4.27 (m, 10H), 2.71–2.46 (m, 3H), 1.43 (m,
15H); 13C NMR (200 MHz, CDCl3) d 170.74, 165.37, 34
out of 60 fullerene resonances observed ꢀ148.53,
148.36, 146.48, 146.17, 145.59, 145.50, 145.08, 144.89,
144.80, 144.61, 144.43, 144.31, 144.24, 144.02, 143.76,
143.70, 143.49, 143.44, 143.10, 142.86, 142.77, 142.72,
142.55, 142.24, 141.98, 141.91, 141.85, 140.87, 140.58,
140.20, 140.10, 136.20, 136.10, 71.56 (2ꢁ; sp3 C60
bridgehead carbon atoms), 63.33 (m), 61.93, 53.13,
41.11, 33.34 (t, J=132.1 Hz), 26.72 (br s), 16.48 (br d),
14.23; 31P NMR (400 MHz, CDCl3, H3PO4): 24.51 (d,
J=5.2 Hz), 24.48 ppm downfield from H3PO4 (d,
J=5.2 Hz). IR (KBr): 3244 (N-H), 2977 (C–H), 2900 (C–
H), 1734 (carboxy ester C¼O), 1684 (amide I band), 1540
(amide II band), 1249 (P¼O), 1023 (C–O), 972 (P–C–P),
798 (P–O) 528 cmꢀ1 (fullerene stretch). UV–vis (CHCl3,
l nm, (e M-1 cmꢀ1)): 326 (3.4ꢁ104), 402 (4.1ꢁ103), 416
(3.4ꢁ103), 427 (3.7ꢁ103), 479 (1.7ꢁ103), 690 (2.1ꢁ102).
High res. APCI-MS (50:50 CHCl3/MeOH): 1164.153200
(M++1). Anal. Calcd for C76H31NO9P2: C, 78.42; H,
2.68; N, 1.20; O, 12.37; P, 5.32. Found: C, 77.42; H, 3.19;
N, 1.19; (O, 12.89); P, 5.31.

K. A. Gonzalez et al. / Bioorg. Med. Chem. 10 (2002) 1991–1997
1997
O, 30.87. Found: C, 60.38; H, 2.77; N, 1.27; P, 4.86; (O,
30.72 by difference). Analysis for potassium showed
only a trace amount (<0.37%).
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alyst. Decoloration of the toluene layer occurred within
min accompanied by formation of a black semi-solid at
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perature for 10–12 h to allow further reaction and to
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¨
amount of insoluble material. The filtrate was con-
centrated under reduced pressure with gentle warming
until precipitate started to form. MeOH was then added
to complete precipitation. A mixture of brown and
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indicated presence of base (if the pH >6 when saturated
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decreases gradually toward the visible region. To
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C60H30O30 2H2O , [C (OH)30 2H2O] C, 56.88; H, 2.71; O,
60
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The Robert A. Welch Foundation (Grant C-0627) is
gratefully acknowledged for support of this work.
K.A.G. also thanks the National Institutes of Health
for support through a biotechnology training fellowship
and Dr. F.F. (Russ) Knapp Jr. for helpful discussions.