Highly-iodinated fullerene as a contrast agent for X-ray imaging

Article abstract of DOI:10.1016/S0968-0896(02)00252-3

The first fullerene-based X-ray contrast agent (CA) has been designed, synthesized, and characterized. The new CA is an externally functionalized derivative of C₆₀ that is conceptually based on contemporary X-ray CA, all of which use iodine as the X-ray attenuating vehicle and are based on the 2,4,6-triiodinated-benzene-ring substructure. Using a modified Bingel-type reaction, a single addend containing 6 iodine atoms and 8 protected hydroxyl groups was appended to C₆₀ followed by the addition of 4 more addends each containing 4 protected hydroxyl groups. Final deprotection afforded the highly water-soluble (>460 mg/mL), non-ionic, highly-iodinated (24% I) fullerene for application as an X-ray contrast agent.

Full text of DOI:10.1016/S0968-0896(02)00252-3

Bioorganic & Medicinal Chemistry 10 (2002) 3545–3554
Highly-Iodinated Fullerene as a Contrast Agent For
X-ray Imaging
Tim Wharton and Lon J. Wilson*
Department of Chemistry and the Center for Nanoscale Science and Technology, MS-60 Rice University,
Houston, TX 77251-1892, USA
Received 25 March 2002; accepted 24 May 2002
Abstract—The first fullerene-based X-ray contrast agent (CA) has been designed, synthesized, and characterized. The new CA is an
externally functionalized derivative of C60 that is conceptually based on contemporary X-ray CA, all of which use iodine as the
X-ray attenuating vehicle and are based on the 2,4,6-triiodinated-benzene-ring substructure. Using a modified Bingel-type reaction,
a single addend containing 6 iodine atoms and 8 protected hydroxyl groups was appended to C60 followed by the addition of 4 more
addends each containing 4 protected hydroxyl groups. Final deprotection afforded the highly water-soluble (>460 mg/mL), non-
ionic, highly-iodinated (24% I) fullerene for application as an X-ray contrast agent.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Since the fortuitous discovery of X-rays by Wilhelm C.
Roentgen in 1895, X-ray radiography has evolved into
the foundation of contemporary medical imaging.
Although the past two decades have experienced an
explosive growth in the ultrasound and magnetic reso-
nance imaging (MRI) modalities (due largely to the
advent of the microchip), fully 75–80% of all imaging
procedures still entail the use of X-rays.1
Figure 1. The structure of commercially-available Iohexol1 based on
the tri-iodinated benzene ring with typical 1,3-diol water-solubilizing
substituents.
imaging of the blood vessels and/or the urinary system.
Typically, an aqueous CA commercial formulation is
injected rapidly via catheter directly into the blood
stream followed by immediate imaging. Contrast is
enhanced by the increased X-ray attenuation caused by
the multiple heavy iodine atoms. Other less common
applications include imaging of the spinal fluid, the
nasal sinuses, and the salivary gland ducts.8
Contemporary X-ray radiography is intimately depen-
dent upon the use of X-ray contrast agents (also called
X-ray contrast media, radiopaque agents, and roent-
genographic agents). With the exception of BaSO4 slur-
ries for gastrointestinal imaging, all commonly
employed X-ray contrast agents (CA) are based on the
2,4,6-triiodinated-5-aminoisophthalic acid substructure.1ꢀ4
The structure of a typical, commercially-available X-ray
CA, Iohexol,5 is shown in Figure 1. Such iodinated CA
are currently used in approximately 20 million proce-
dures in the US annually.6,7
Upon injection, the agents diffuse throughout the
bloodstream and are rapidly excreted (typically, t1/2=
1.5–2 h)9 primarily through the kidneys by glomerular
filtration which allows imaging of the urinary system.
Iodinated, water-soluble X-ray CA like Iohexol (Fig. 1)
are technically known as uroangiographic agents since
they are most commonly used (and best suited) for
Today’s X-ray CA have evolved to the point where it is
unlikely that a simple modification of the R groups in
Figure 1 will lead to any significant improvement in
tolerability or performance. Accordingly, much of the
current research in X-ray CA is focused on novel sys-
tems such as tungsten clusters,10,11 metal chelates,12,13
*Corresponding author. 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)00252-3

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T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
hydrolytically-stable malonodiamide functionality. Pre-
liminary model-compound studies28 established the via-
bility of appending the malonodiamide group to C60.
The preparation of the highly-iodinated addend 7 is shown
in Scheme 1. Starting from 5-aminoisophthalic acid, three
iodine atoms were introduced with KICl2 in water29 fol-
lowed by treatment with SOCl2 to give diacid chloride 2.
Diacid chloride 2 was then directly condensed with
malonyl dichloride to give the tetraacid chloride 3 in
83% yield.30 Condensation with malonyl dichloride is
favored over polymerization due to the steric congestion
caused by the large flanking iodine atoms.29
A suitable method for the O,O0-protection of serinol
was developed based on the reported stability of both
cis- and trans-5-amino-2-phenyl-1,3-dioxane.31 Serinol
was first N-protected with benzylchloroformate and
then O,O0-protected by condensation with benzaldehyde
(cat. H2SO4) to give an equilibrium mixture of cis:trans
substituted 1,3-dioxane in a ratio of 8:2.
Figure 2. The C60-based X-ray contrast agent.
iodinated dicarbon carborane cages,14,15 and fullerenes
(this work).
The use of C60-derived materials for biological appli-
cations is currently an active area of research and sev-
eral reviews have been written on the subject.16ꢀ18 The
idea of using the easily derivatized, non-toxic scaffold-
ing19,20 of C60 for the development of a new X-ray con-
trast agent has not previously been explored. However,
preliminary results of this work have been commu-
nicated21,22 and a patent application has been filed.23
The cis isomer selectivity can be rationalized by the
axial preference for the N-benzyl carbamate group due
to the presence of intramolecular N–HÁÁÁÁO hydrogen
bonding.32 In addition, the nucleophilicity of the cis
isomer of 5-amino-2-phenyl-1,3-dioxane has been
shown to be approximately twice that of the trans iso-
mer.33 Therefore, the cis isomer was chosen as the more
desirable protected-serinol isomer.
The target compound 12 is shown in Figure 2. Like
other contemporary CA agents, 12 uses iodine as the
active X-ray attenuating vehicle. However, 12 also con-
tains the unique and synthetically versatile molecular
scaffolding of C60.
Removal of the benzoxycarbonyl from the nitrogen of 5
under catalytic hydrogenation conditions (300 psi H2,
Pd/C) proceeded smoothly to give pure 6. Condensation
of 6 with tetraacid chloride 3 in DMA at room tem-
perature proceeded in excellent yield (95%) to give the
malonodiamide 7.
The spheroidal, truncated-icosahedral structure of C60
confers a globular shape to it’s highly-functionalized
derivatives. The advantage of a more globular shape
(rather than the disk-like shape of a contemporary CA)
is reduced viscosity in clinical formulations, which
allows a more rapid intravenous injection of the agent.3
A C60-derived agent could also be expected to benefit
from an enhanced masking effect in which one side of
the tri-iodinated phenyl ring may be blocked by the
fullerene core from having hydrophobic interactions
with blood plasma proteins. This would lead to
decreased protein binding and increased in vivo toler-
ability (this is known as the ‘hydrophilic sphere con-
cept’).24 Additionally, the possibility of having more
than 3 iodine atoms per molecule would allow a solu-
tion of lower molarity to be used in CA injections.
The characterization of addend 7 was complicated by
the presence of up to 12 hindered rotations at room
temperature caused by the sterically congested environ-
ment of the hexasubstituted benzene rings.34 The large
number of atropisomers caused the room temperature
1H NMR to be broadened and complex. However,
1
interpretable 500 MHz H and 13C NMR spectra were
obtained at 70 ꢁC.
To prepare the C60 monoadduct of the malonodiamide 7,
the method of Hirsch for the in situgeneration (with CBr 4
and DBU) of the active brominated intermediate was
used.35 The reaction, as shown in Scheme 2, was found to
proceed nicely at room temperature in toluene/pyridine, in
which both 7 and C60 were suitably soluble, to give the
monoadduct 8.21 The relatively good yield of 77% was
somewhat surprising, considering the steric bulk of addend
7, but indicates that the central methylene (methine after in
situbromination) in 7 is available for reaction with C60 in
its more favored room temperature conformer(s).
Results and Discussion
Of the efficient methods available to functionalize C60,25
one of the best approaches is nucleophilic cyclopropa-
nation or the Bingel reaction.26 However, the typical
malonodiester fullerene adducts are easily hydrolyzed
to give the diacid product,27 an undesirable attribute
for materials requiring high in vivo stability. Accord-
ingly, the C60-based CA design incorporates the more
The FTIR spectrum of C60-monoadduct 8 was nearly
superimposable on the IR spectrum of addend 7, bu t
additionally had the characteristic C60-monoadduct

T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
3547
Scheme 1. Synthesis of the hexa-iodinated, O,O0-protected malondiamide addend 7. Reagents and conditions: (i) KICl2, H2O, 55 ꢁC, 24 h; (ii)
SOCl2, reflux 6 h; (iii) CH2(COCl)2, THF, reflux 6 h; (iv) benzyl chloroformate, NEt3, EtOH, 2.25 h; (v) PhCHO, cat. H2SO4, toluene, reflux 6 h; (vi)
300 psi H2, Pd/C, EtOH, 50 min; (vii) DMA, NEt3, 24 h.
Scheme 2. Synthesis of monoadduct 8. Reagents and conditions: (i) CBr4, DBU, 25:14 toluene:pyridine.
band at 526 cmꢀ1 36,37
.
A molecular ion peak at 2547.5
low and the signals too broadened to interpret, indicat-
ing that some of the rotations of the addend 7 are even
more hindered upon addition to C60. Higher tempera-
ture experiments were prohibited due to the thermal
decomposition of the benzylidene acetal groups.
was observed in the MALDI-TOF MS ([M]ꢀ calcd for
C119H50N6O14I6, 2547.8), with the base peak at 2453.5
corresponding to the [MꢀI+S]ꢀ ion.38 Additionally, a
single band was observed by thin-layer chromatography.
The NMR characterization of 8, as with compound 7,
was hampered by the presence of multiple atropisomers.
In the case of 8, however, even after 17 h of data collec-
tion at 70 ꢁC, the S/N ratio of the 13C NMR spectrum
(500 MHz, pyridine-d5, 0.05 M, 6128 scans) was still too
A qualitative in vitro determination of the degree of
X-ray attenuation produced by hexa-iodinated
showed, as expected based purely on the iodine content
(30%), significantly enhanced X-ray absorption relative
to the blank.21
8

3548
T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
When C60 was treated with an excess of addend 7 (ꢂ3-
fold relative to C60), only the monoadduct formed in
good yield as determined by TLC and MALDI-TOF
MS. Although some bisadduct product was observed by
MS, no higher adducts were detected, and the mono-
adduct was the major product. Apparently, the bulki-
ness of the addend does not allow the formation of
adducts higher than the bis-, whose formation is also
greatly inhibited.
This result is not surprising considering the highly
amphiphilic nature of the material. Therefore, given the
very high water solubility requirements for X-ray CA,
and the lack of formation of the highly functionalized
adducts (Tris-, tetrakis-, etc.), attention turned to water-
solubilization of 8 by additional functionalization. This
was easily accomplished using the smaller mal-
onodiamide group BrCH(COSer)2 (10, Ser=2-amino-
1,3-propanediol) which was previously shown to be
extremely effective at water-solubilizing native C60
(>240 mg C60 mLꢀ1).22 The synthesis of 10 is shown in
Scheme 3.
Additionally, the deprotection of monoadduct 8 resul-
ted in a material that is only sparingly soluble in water.
Scheme 3. Synthesis of BrCH(COSer)2, 10, (Ser=2-amino-1,3-propanediol). Reagents and conditions: (i) 225 ꢁC, 45 min; (ii) Ac2O, pyridine, 18 h;
(iii) Br2, EtOAc, NEt3.
Scheme 4. Assembly and deprotection of X-ray CA 12. Reagents and conditions: (i) CHCl3, DBU, 40 h; (ii) BF3Et2O, CHCl3; (iii) Na2CO3, MeOH, H2O.

T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
3549
To accomplish water solubilization, 8 was treated with
an 8-fold excess of 10 to give 11 as shown in Scheme 4.
(All attempts to control the regiochemistry of the addi-
tions with the template dimethyl anthracene39 were
unsuccessful.) The orange-red product mixture, after
workup, consisted of predominantly the pentaadduct
(addition of four water-solubilizing groups, 10, to
monoadduct 8), with small amounts (<10%) of the
tetraadduct present, as determined by MALDI-TOF
MS.
steps involving any C60 material, which in general pro-
vides overall improved yields.
Animal studies to determine the efficacy of 12 as an
X-ray CA are currenty being performed.
Experimental
All compounds used were reagent grade or better, sol-
vents were used as received unless otherwise specified.
For anaerobic reactions, solvents were degassed by
bubbling dry N2 or Ar. The following reagents were
used as received: C60 (99.5+%, MER Corp.), 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (Aldrich), diethyl malonate
(Aldrich), Br2 (Aldrich), 2-amino-1,3-propanediol
(Aldrich), Pd/C (Pd 10%, Aldrich), iodine mono-
chloride (Aldrich), 5-amino-isophthalic acid (Aldrich),
malonyl dichloride (Aldrich), benzyl chloroformate
(Aldrich), benzaldehyde (EM Science), CBr4 (Aldrich),
BF.3Et2O (Aldrich), The following reagents were pur-
ified as described: NEt3 (Acros) was refluxed and dis-
tilled from CaH2, SOCl2 (Acros) was distilled from
linseed oil (Aldrich, 20% v/v) and used immediately,
acetic anhydride (Fisher) was distilled.
A molecular ion peak for the deprotonated sodium
adduct of 12 was observed at m/e 3211.1 (calcd 3211.1).
The lack of peaks corresponding to residual protected
species indicated complete alcohol deprotection. The
clear presence of two higher mass peaks at
[M+Na+16]+ and [M+Na+32]+ indicates the addi-
tional presence of oxygenated species (epoxides) which
are commonly observed in C60-derived materials.40
Although very small oxygenated species peaks were
observed in the mass spectra of the precursor com-
pounds 8 and 11, the much greater intensity of the peaks
in the mass spectrum of 12 indicate that epoxide for-
mation most likely occurs predominantly during the
deprotection step (despite anaerobic conditions). Alter-
natively, the oxygen may have been introduced during
the mass spectrum sample preparation. Additionally,
fragment peaks corresponding to the loss of up to all 6
iodine atoms are clearly seen with the loss of one iodine
atom [MꢀI+Na]+ being the base peak.
The following solvents were purified as described: tolu-
ene (Fisher) was refluxed and distilled from Na (with
benzophenone indicator), hexanes (Fisher) were dried
by contact with CaCl2 and either distilled or micro-
filtered, CS2 (Fisher) was stirred with CaCl2 4 h then dis-
tilled from P2O5, CHCl3 (Fisher) was refluxed and
distilled from CaCl2, pyridine (Fisher) was refluxed and
distilled from Na or KOH, EtOAc (Fisher) was distilled
from MgSO4, THF (Fisher) was distilled from a K/Na
alloy (made by heating 1:1 Na and K metal until molten),
EtOH (Pharmco) was refluxed and distilled from CaSO4,
and methanol (Fisher) was distilled. Silica gel (Aldrich),
grade 62, 62–200 mesh, 150 A was used for column
chromatography after activation at 200 ꢁC for at least 24 h.
TLC analyses were carried out on Whatman 250 mm layer
silica gel with fluorescent indicator on polyester backing
(PE SIL G/UV). Cation exchange resin (Bio-Rad) AG
50W-X2 (H+ form) was used for the removal of cations
from the fullerene materials. The resin was prepared and/
or converted to H+ form by rinsing with at least 5 bed
volumes of each: deionized (DI) H2O, 1 M HCl, DI H2O.
The UV–vis spectra of 12 (0.62 M) has no maxima as
1
expected for a complex mixture of isomers. In the H
NMR of 12, complete deprotection is apparent and only
the methine and methylenes of the serinol residues are
observed. The elemental analysis is consistent with the
structure of 12 and the characteristic amide carbonyl
(1652 cmꢀ1) and hydroxy (3382 cmꢀ1) stretches were
observed in the FT-IR spectrum.
Several water-soluble, amphiphilic C60 derivatives have
been shown to form aggragates in aqueous solution.41,42
However, dynamic light scattering experiments with 12
indicated that the compound does not form aggragates
in the detection range of 3–3000 nm in aqueous solution
(pH ꢃ7) at concentrations of ꢄ2 mM. This result is
indicative of the hydrophobic shielding expected by
multiple strongly hydrophilic addends, and at con-
centrations of around 2 mM, 12 exists as a monomer in
solution.
NMR spectra were obtained on a Bruker Avance 250,
400, or 500 MHz NMR (for VT experiments) spectro-
meter. FT-IR spectra were collected on a Perkin Elmer
Paragon 1000 PC spectrometer as KBr pellets.
Additionally, the n-octanol/water partition coefficent
(KOW) was determined, and within detection limits,
KOW=0 for 14. This result compares well with the very
small KOW values (<10ꢀ3) of currently-used X-ray
contrast agents which are also very hydrophilic.
Mass spectra were collected on either a Finnigan Mat
95 mass spectrometer or a Bruker Biflex III MALDI-
TOF mass spectrometer. For the MALDI spectra,
unless otherwise specified, an elemental sulfur matrix
deposited from a slurry in CS2 was used. Where resolu-
tion permitted, the isotopic distribution of the mass
spectral peaks was compared to theory.
Conclusion
The first fullerene-based X-ray contrast agent has been
designed, synthesized, and characterized. The synthetic
pathway was designed to minimize the number of synthetic
HPLC analyses were performed on Hitachi l-6200A
Intelligent Pump HPLC system with a Hitachi Model

3550
T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
l-3000 UV–vis photodiode array detector. Elemental
analyses were carried out commercially by Galbraith
Laboratories (Knoxville, Tennessee). Melting points
were determined on a Mel-Temp apparatus and are
uncorrected.
(1C, C–NH2), 170.1 (2C, COOH); FT-IR (KBr) n
(cmꢀ1) 3629, 3442, 3378, 3305 (N–H), 2990, 2480
(COO–H), 1719 (C¼O); MALDI-TOF MS (MeOH, +
ion) calcd for C8H4O4NI3 (M+) 558.7, found 558.6.
For anhydrous reactions, all glassware was either flame-
dried or dried at 200 ꢁC for 24 h.
5-Amino-2,4,6-triiodo-1,3-benzenedicarbonyl dichloride
(2). In 400 mL of freshly purified SOCl2, 52 g 1 (93
mmol) was refluxed for 6 h. The SOCl2 was then
removed under reduced pressure. The product was dis-
persed in 400 mL EtOAc by sonication (1 min). The
EtOAc was removed under reduced pressure to give a
dark yellow solid. The crude material was again dis-
persed in 425 mL EtOAc (the material dissolved fully
upon washing) and washed with 40 mL portions of a
saturated solution of 250 g NaCl/100 g NaHCO3 in 1.25
L H2O until the aqueous phase remained basic (5Â),
followed by satd NaCl. The clear dark yellow-orange
organic phase was flash chromatographed on toluene
slurry packed SiO2 (10 cm heightÂ7.9 cm ID) with tol-
uene and rotovapped to give 49.1 g (83 mmol) of a pure
beige solid, yield 89.3%. TLC Rf 0.62 in 1:3 C6:EtOAc;
HPLC (anal. C-8) detection at 285 nm, retention time
7.80 min in 8:2 AcCN:H2O, flowrate 0.25 mL/min; 13C
NMR (400 MHz, acetone-d6, solvent ref) d 64.8 (1C, C-
I) 77.0 (2C, C-I), 150.3 (1C, C–NH2), 150.8 (2C, C–
COCl), 170.0 (2C, COCl); FT-IR (KBr) n (cmꢀ1) 3469,
3370 (N–H), 1769 (C¼O); MALDI-TOF MS (acetone,
ꢀion) calcd for C8H2O2NI3Cl2 [(M–H)ꢀ] 593.7, found
593.3.
For the dynamic light scattering experiment, a Coulter
N4 Plus dynamic light scattering particle sizer was used.
Data were taken at 90ꢁ.
The n-octanol/water partition coefficient (KOW) of 12
was determined at 25 ꢁC by the method of Leo.43
Accordingly, a solution (ꢅ1 mM) of 12 in water (pre-
saturated with n-octanol) was vigorously stirred for 5
min with an equal volume of n-octanol, then cen-
trifuged. The absorbance in the UV–vis spectra of the
n-octanol ‘extracted’ solutions was compared to the
spectra of the starting solutions.
The qualitative determination of X-ray attenuation of
compound 8 was carried out as follows: In 1 mL syr-
inges, a 0.197 M (150 mg I/mL) solution of 8 in 5:1
DMF:toluene, 4 standard aqueous solutions of Omni-
paque1 at concentrations of 300, 225, 150, and 75 mg
I/mL (diluted with saline), and 2 samples containing the
saline (0.9% NaCl) and the DMF:toluene backgrounds
were exposed to diagnostic wavelength (0.087–8.5 nm)
X-rays in a Faxitron 43855A imager. The X-ray image
was captured on standard Kodak MIN-R 2000 film.
N,N0-Bis(2,4,6-triiodo-3,5-benzenedichlorocarbonyl)-mal-
onamide (3). In 200 mL anhydrous THF, 25.7 g 2 (43
mmol) was dissolved (500 mL rbf) and brought to reflux
under argon. To the solution, 2.0 mL (21 mmol) mal-
onyl dichloride was added dropwise over 5 min and the
orange solution refluxed for 6 h (under Ar sparge).
Anhydrous hexanes were added until slight precipita-
tion was noted (ꢅ10 mL). Stirring was stopped and the
heat removed. Crystals formed when the flask was
allowed to cool overnight. It was then placed on an ice
bath for 3 h and filtered to give 19.65 g (16 mmol) of a
fluffy, white, microcrystalline solid. The mother liquor
was stirred and precipitated with hexanes followed by
filtration to give crude product. The crude tan solid was
dissolved in minimal boiling THF and hexanes added
until near precipitation. Upon cooling to room tem-
perature, followed by an ice bath (2 h), a second crop of
1.91 g (1.5 mmol) pure material was obtained. An
additional 2.53 g of crude material was obtained upon
full precipitation and filtration of the mother liquor.
Pure yield 83%. TLC Rf 0.66 in 2:1 EtOAc:C6s; HPLC
(anal. SiO2) in 4:1 PhCH3:THF, detection at 300 nm,
retention time 5.92 min, flowrate 1.0 mL/min; 1H NMR
(400 MHz, acetone-d6, TMS ref) d 3.85 (s, 2H, CH2),
10.0 (bs, 2H, NH); 13C NMR (400 MHz, acetone-d6,
solvent ref) d 43.0 (1C, CH2), 83.1 (2C, C-I), 98.3 (4C,
C-I), 146.2 (2C, C–NH2), 151.8 (4C, C–COCl), 166.1
5-Amino-2,4,6-triiodo-1,3-benzenedicarboxylic acid (1).
The preparation of 1 was modified from a literature
procedure.29 Thus, 98.11 g of ICl (600 mmol) was added
to a solution of 72.11 g KCl (970 mmol) in 300 mL H2O
and stirred at room temperature for 5 min. The mixture
was filtered to remove insolubles, and the orange solu-
tion of KICl2 was added via addition funnel over 1 h to
a stirring slurry of 33.22 g of 5-aminoisophthalic acid
(180 mmol) in 1100 mL DI H2O at 55 ꢁC. After com-
plete addition, the tan slurry was stirred at 55 ꢁC for 24
h. The slurry was cooled on an ice bath for 3 h and fil-
tered to give a light tan solid. The crude product was
rinsed on a fritted glass filter 2Â with 40 mL H2O, 1Â20
mL H2O, 2Â20 mL 0.5 N NaHSO3, 2Â10 mL H2O,
dissolved in minimal 0.66 M KOH to give a clear-brown
solution, neutralized with concd HCl, and treated
repeatedly with 0.5 g activated charcoal at 40–50 ꢁC for
10 min with stirring and filtered until nearly colorless
(6–8Â). The product was then precipitated with concd
HCl with stirring until no further precipitation was
observed, allowed to stand for several hours, then fil-
tered and dried at 100ꢁC under vacuum to give 73.68 g
(130 mmol) of analytically pure off-white tan solid, 73%
yield. TLC Rf 0.60 in 2:1 MeOH:EtOAc; HPLC (anal.
C-8) detection at 255 nm, retention time 2.79 min in
phosphate buffer (pH 7): MeOH (7:3), flowrate 0.3 mL/
min; 13C NMR (400 MHz, DMSO-d6, solvent ref) d 70.6
(1C, C-I) , 77.9 (2C, C-I), 147.7 (2C, C–COOH), 148.9
(2C, CONH), 169.9 (4C, COCl); FT-IR (KBr) n (cmꢀ1
3190 (N–H), 2960 (C–H), 1760 (C¼O, acid chloride),
)

T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
3551
1650 (C¼O, amide); MALDI-TOF MS (acetone, ꢀion)
calcd for C19H4O6N2I6Cl4 [(M–H)ꢀ] 1256.3, found
1256.7.
anhydrous EtOH in a 50 mL Erlenmeyer flask. 0.14 g of
10% Pd/C was added. The slurry was stirred at room
temperature under 300 psi H2 (in a Parr hydrogenator)
for 50 min. The catalyst was removed by syringe
microfiltration, followed by careful solvent removal
under reduced pressure (bp 22: 50 ꢁC, 0.65 Torr44). The
colorless residue was dissolved in minimal anhydrous
Et2O (ꢅ5–10 mL) and microfiltered to remove inso-
lubles. The Et2O was removed under reduced pressure
to yield 0.72 g of pure 6 as a colorless oil (89%) which
was used immediately. 1H NMR (400 MHz, CDCl3,
TMS ref) d 1.98 (bs, 2H, NH2), 2.77 (m, 1H, CHNH2),
4.03–4.16 (m, 4H, CH2), 5.55 (s, 1H, ArCH), 7.30–7.40
(m, 3H, ArH), 7.48–7.51 (m, 2H, ArH); 13C NMR
(400 MHz, CDCl3, solvent ref) d 45.96 (1C, CHNH2),
73.49 (2C, CH2), 101.97 (1C, acetal CH), 126.06 (2C,
ArC), 128.44 (2C, ArC), 129.14 (1C, ArC), 138.36 (1C,
ArC).
(2-Hydroxy-1-hydroxymethyl-ethyl)-carbamic acid benzyl
ester (4). In 60 mL dry EtOH, 11.03 g of serinol (120
mmol) and 18.5 mL NEt3 were dissolved in a 150 mL
rbf and placed on an ice bath. Benzyl chloroformate
(19.2 mL) was added via an addition funnel over 15 min
without allowing the temperature to rise above 35 ꢁC.
After complete addition, stirring was continued for 2 h
at room temperature with the formation of a colorless
ppt (NEt3HCl). The mixture was filtered and con-
centrated to 50 mL. Allowed to stand at room tem-
perature overnight, the product crystallized to give
26.72 g. A second crop of 2.5 g was obtained by seeding
the mother liquor and cooling at ꢀ10 ꢁC 18 h. Pure
product was obtained by recrystallization from H2O in
two crops to give colorless crystals, 20.64 g, 76% yield.
Mp 104–105 ꢁC; TLC Rf 0.58 in 10:1 EtOAc:MeOH; 1H
NMR (400 MHz, acetone-d6, TMS ref) d 2.91 (s, 2H,
OH), 3.66 (m, 4H, CH2), 3.86 (p, 1H, CH), 5.06 (s, 2H,
CH2), 6.03 (bs, 2H, NH), 7.28–7.40 (m, 5H, ArH); 13C
NMR (400 MHz, acetone-d6, solvent ref) d 55.70 (1C,
CH), 62.39 (2C, CH2), 66.60 (1C, CH2), 128.66, 128.71,
129.26, 138.45 (ArC), 157.21 (1C, C¼O); FT-IR (KBr) n
(cmꢀ1) 3320 (O–H), 1695 (C¼O); EI-MS calcd for
C11H15N1O4 (M+) 225.1, found 225.1.
N,N0-Bis[2,4,6-triiodo-N,N0-bis-[cis-(2-phenyl-[1,3]dioxan-
5-yl)]-3,5-benzene-dicarbamoyl]-malonamide (7). In a
typical preparation, 0.94 g 3 (0.75 mmol) was dissolved
in 40 mL anhydrous DMA. A solution of 0.72 g of 6
(4.0 mmol, 1.33 molar excess) and 560 mL anhydrous
NEt3 (4.0 mmol) in 10 mL anhydrous DMA was added
via a cannulating needle. A white ppt slowly formed as
the colorless solution was stirred for 20 h under Ar at
room temperature. After filtration, the DMA was
removed under reduced pressure to give a colorless
solid. The material was flash chromatographed on a
slurry packed column (silica, THF) with 10:3 THF:pyr-
idine, the solvent removed under reduced pressure and
flash chromatogpraphed a second time on a slurry
packed column (silica, THF) with pure THF. Solvent
removal under reduced pressure gave 1.30 g (0.71 mmol)
of an analytically pure colorless to very pale yellow
cis-(2-Phenyl-[1,3]dioxan-5-yl)-carbamic acid benzyl ester
(5). To a refluxing solution of 17.13 g 4 (76 mmol) in a
500 mL flask in 290 mL toluene was added 7.34 mL
benzaldehyde (72 mmol). 15 mL of concd H2SO4 was
suspended in 3 mL toluene by sonication and added to
the flask. After 1.5 h at reflux, 25 mL of the reaction
sovent was distilled off to azeotropically remove water.
A second aliquot of 15 mL concd H2SO4 suspended in 3
mL toluene was added and reflux was continued for 3 h
and an additional 45 mL distilled off. The heat was
removed and crystals allowed to form by cooling the
flask to room temperature overnight under in Ar
atmoshpere. The colorless crystals were filtered to give
11.5 g of 80–90% starting material 19 and 10–20%
trans-(2-phenyl-[1,3]dioxan-5-yl)-carbamic acid benzyl
1
solid, yield 94.7%. TLC Rf 0.50 in 1:1 PhCH3:THF; H
NMR (500 MHz, DMSO-d6, 70 ꢁC, solvent ref) d 3.57
(s, 2H, –COCH2CO–), 3.99 (distorted d, J=6.5 Hz, 4H,
–NHCH(CH2–)2), 4.22 (distorted q, J=8.4 Hz, 16H,
–CH(CH2O–)2), 5.63 (s, 4H, acetal CH), 7.35 (m, 12H,
ArH), 7.50 (m, 8H, PhH), 7.98 (bs, 1H, NH), 8.7–9.3
(bm, 3H, NH), 10.08 (s, 2H, NH); 13C NMR (500 MHz,
DMSO-d6, 70 ꢁC, solvent ref) d 41.95 (1C, –COCH2CO–
), 43.70 (4C, –NHCH(CH2–)2, 68.45 (8C, –CH(CH2O–
)2), 90.21, 98.10 (ArC), 100.40 (4C, acetal CH), 126.14,
127.40, 128.23, 138.27, 142.07, 149.22 (ArC), 164.55,
168.90 (C¼O); FT-IR (KBr) n (cmꢀ1) 1654 (s), 1103 (s);
UV lmax 285; MALDI-TOF MS calcd for
C59H52N6O14I6 [M]ꢀ 1829.8, found 1828.7 [M–H]ꢀ;
anal. (C59H52N6O14I6) calcd: I, 41.60; C, 38.71; H, 2.86;
N, 4.59, found: I, 41.86; C, 39.17; H, 3.34; N, 4.41.
1
ester as determined by H NMR. The filtrate was trea-
ted with 0.5 g MgSO4, filtered, and the solvent removed
under reduced pressure to give a clear oil which was
recrystallized from 9:1 EtOH (200 proof): H2O in two
crops to give 6.13 g of pure 20 as colorless crystals. Mp
67.0–69.0 ꢁC; TLC Rf 0.39 in 7:3 C6s:EtOAc; mp 67–
69 ꢁC; H NMR (400 MHz, CDCl3, TMS ref) d 3.76 (d,
1
J=8.8 Hz, 1H, CH), 4.14 (m, 4H, CH2), 5.18 (s, 2H,
CH2), 5.55 (s, 1H, CH), 5.95 (bd, 1H, NH), 7.36–7.45
(m, 8H, ArH), 7.54 (m, 2H, ArH); 13C NMR (400 MHz,
CDCl3, solvent ref) d 46.04 (1C, NCH), 67.31 (1C,
CH2), 71.14 (2C, CH2), 102.11 (1C, acetal CH), 126.38,
128.62, 128.76, 129.02, 129.56, 136.87, 138.31 (12C,
ArC), 156.40 (1C, C¼O); FT-IR (KBr) n (cmꢀ1) 3337
(N–H), 1685 (C¼O), 1524; EI-MS calcd for
C18H19N1O4 (M–H)+ 312.1, found 312.
10,10-Bis[N-[2,4,6-triiodo-N,N0-bis-[cis-(2-phenyl-[1,3]dioxan
-5-yl)]-3,5-benzene-dicarbamoyl]carbamoyl]-1,2-methano
[60]fullerene (8). All of the solvents in the preparation
and purification of 8 were degassed. In a 2000 mL rbf,
800 mg of C60 (1.1 mmol) was added to 500 mL PhCH3
and 200 mL pyridine and stirred until dissolved (>2 h).
2.0 g of 7 (1.1 mmol) in 80 mL pyridine was then added.
0.36 g of CBr4 (1.1 mmol) was added followed by 245
mL DBU (1.6 mmol). A darkening of the color of the
solution was noticeable almost immediately. Under N2
cis-5-Amino-2-phenyl-1,3-dioxane (6). In a typical pre-
paration, 1.4 g of 5 (4.5 mmol) was dissolved in 50 mL

3552
T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
sparge, the reaction mixture was stirred 14 h at room
temperature. The solvent was removed and 150 mL
CHCl3 and 10 mL MeOH added to the dark solid. After
stirring and 20 s sonication, the mixture was filtered and
the solution flash chromatographed on SiO2 (slurry
packed with CHCl3, 10Â7.7 cm ID) with 30:1
CHCl3:MeOH (degassed) and the solvent removed
under reduced pressure. The dark product was redis-
solved in ꢅ100 mL CHCl3 and rechromatographed on
SiO2 (slurry packed with CHCl3, 10Â7.7 cm ID) first
with CHCl3 to remove C60 and then with 30:1
CHCl3:MeOH. After solvent removal, 2.17 g of dark
brown solid obtained (0.85 mmol), 77% yield (based on
C60). TLC Rf 0.75 in 1:1 THF:toluene; 1H NMR
(400 MHz, CDCl3, solvent ref) d 4.0–4.5 (bm, 20H,
–NHCH(CH2O–)2, –NHCH(CH2O–)2), 5.6 (bs, 4H,
PhCH(O–)2), 7.2–7.6 (bm, 20H, ArH), 8.89 (NH);
MALDI-TOF MS (CHCl3, ꢀion) calcd for
C119H50N6O14I6 [M]ꢀ 2547.8, found 2547.5; FT-IR
(KBr) n (cmꢀ1) 1654 (s), 1103 (s), 526 (m); UV lmax
(nm) 273, 328; anal. (C119H50N6O14I6) calcd: C, 56.07;
H, 1.98; N, 3.30; O, 8.79; I, 29.87; found: C, 54.76; H,
2.60; N, 3.05; O, 8.34; I, 31.26 (O by subtraction).
added with large amounts of salt precipitation and a
return to a colorless mixture. After filtration, analysis
1
by H NMR indicated ꢅ77% mono-bromination with
no dibromo compound formed. The residue was redis-
solved in 150 mL EtOAc with warming and, after cool-
ing to room temperature, 282 mL Br2 added (5.5 mmol,
1.35 molar excess relative to unbrominated material)
with stirring. After 15 min, 765 mL NEt3 (5.5 mmol) was
added. The slurry was filtered and rotavapped as before.
1H NMR analysis of the residue indicated ꢅ90%
monobromination and ꢅ10% dibromination. Recrys-
tallization from 25:16 EtOAc:hexanes in two crops gave
6.75 g of pure 10 (14 mmol), 79% yield. Mp 111.0–
112.5 ꢁC; TLC Rf 0.10 in 1:1 EtOAc:hexanes; H NMR
1
(400 MHz, CDCl3, TMS ref) d 2.10 (s, 12H, CH3), 4.15–
4.26 (m, 8H, CH2), 4.42–4.43 (m, 2H, CH), 4.71 (s, 1H,
CH), 7.36 (br d, J=5.25 Hz, 2H, NH); 13C NMR
(400 MHz, CDCl3, solvent ref) d 20.90 (4C, CH3), 43.02
(1C, CH2), 48.46 (2C, CH), 62.39, 62.43 (4C, CH2, sig-
nal split), 165.66 (2C, NC¼O), 170.91, 170.96 (4C,
OC¼O, signal split); FT-IR (KBr) n (cmꢀ1) 3247 (m, N–
H), 1741 (s, ester C¼O), 1665 (s, amide C¼O), 1225 (s,
asym. –OCOCH3); HPLC (anal. C-8 column) in 7:3
AcCN:H2O, retention time 3.30 min, detection at 250
nm; UV (7:3H3CCN:H2O) lmax 225 nm; EI-MS calcd
for C17H25N2O10Br (M+) 496.1, found 496.1 (M+),
423.0 (–CH2OCOCH3), 357.1 (base peak –Br,
–OCOCH3).
N,N0-Bis[2-(acetyloxy)-1-[(acetyloxy)methyl]ethyl]-malo-
namide (9). In a loosely capped 16.5Â3.7 mm ID Pyrex
tube, 10.59 g of serinol (2-amino-1,3-propanediol, 120
mmol) and 7.94 mL diethyl malonate (52 mmol) were
combined and heated with vigorous stirring at 200–
225 ꢁC for 45 min. After this time, the loose cap was
removed the EtOH was allowed to boil off. The heat
was removed and, upon cooling, the solid, colorless
residue was treated with 40 mL each of freshly distilled
Ac2O and pyridine and stirred 18 h at room tempera-
ture. Methanol (20 mL) was added carefully with stir-
ring and cooling at 0 ꢁC. The sovents were removed and
the residue disssolved in 75 mL of EtOAc and extracted
with dilute CuSO4 (5Â10 mL) followed by H2O, and
saturated NaCl. The organic phase was dried by contact
with MgSO4 and filtered. Recrystallization from EtOA-
c:hexanes (ꢅ2:1) in two crops gave 11.57 g (28 mmol) of
pure 9 as a colorless crystalline solid, yield 53%. Mp
91.5–92.5 ꢁC; TLC Rf 0.58 in 1:1 EtOAc:C6s; mp 91.5–
92.5 ꢁC; HPLC (anal. C-8) in 7:3 AcCN:H2O, detection
at 240 nm, retention time 3.06 min, flowrate 0.30 mL/
Compound 11. All of the solvents in the preparation and
purification of 11 were degassed. In a typical prepara-
tion, 1.28 g 8 (0.50 mmol) was dissolved in 250 mL
CHCl3 (degassed). To the solution, 1.61 g of 10 (3.2
mmol) was added. DBU (616 mL, 4.1 mmol) in 50 mL
CHCl3 was added dropwise via addition funnel over 1 h.
The brown solution was stirred under N2 for 40 h at
room temperature. After removal of the solvent under
reduced pressure, the reddish-brown residue was dis-
solved in 50 mL acetone and then 25 mL toluene was
added followed by chromatography on SiO2 (slurry
packed with toluene, 10Â7.7 cm ID) first with 1:5 acet-
one:toluene causing a small amount of colored material
to elute which was discarded. The eluent was then
changed to 1:1 toluene:acetone and 1200 mL of a brown
solution was collected. The solvent was removed under
reduced pressure to give 1.4 g of brownish solid. 66%
1
min; H NMR (400 MHz, CDCl3, TMS ref) d 2.08 (s,
1
12H, CH3), 3.21 (s, 2H, CH2), 4.18 (m, 8H, CH2), 4.43
(m, 2H, CH), 7.50 (d, J=8.36, 2H, NH); 13C NMR
(400 MHz, CDCl3, solvent ref) d 20.70 (4C, CH3), 42.49
(1C, CH2), 47.42 (2C, CH), 62.47 (4C, CH2), 167.27
yield (calculated for pentaadduct). H NMR (400 MHz,
CDCl3, solvent ref) d 1.9–2.2 (multiple bs, CH3), 4.1–4.5
(bm, serinol CH and CH2), 5.6 (bs, 4H, PhCH(O–)2),
7.1 (bd, NH), 7.3–7.5 (bm, ArH), 7.6 (bd, NH); FT-IR
(KBr) n (cmꢀ1) 1740 (ester C¼O), 1675 (amide C¼O),
1234 (asym –OCOCH3); MALDI-TOF MS avg calcd
for pentaadduct C187H146N14O54I6 4214.7, found
4214.4, avg calcd for tetraadduct C170H122N12O44I6
3798.3, found 3798.5; anal calcd for pentaadduct
C187H146N14O54I6: C, 53.3; H, 3.49; N, 4.65; O, 20.5; I,
18.1, found: C, 49.6; H, 4.45; N, 5.40; I, 18.6; O, 22.0 (O
by difference).
(2C, C¼O), 170.68 (4C, C¼O); FT-IR (KBr) n (cmꢀ1
)
3254 (N–H), 3085, 1740 (C¼O, ester), 1646 (C¼O,
amide), 1241 (asym. –OCOCH3); EI-MS calcd for
C17H26N2O10 (M+) 418.2, found 418.1.
2-Bromo-N,N0-bis[2-(acetyloxy)-1-[(acetyloxy)methyl]ethyl]
-malonamide (10). In a typical bromination, 7.19 g of 9
(17 mmol) was dissolved in 140 mL EtOAc. With stir-
ring at room temperature, four 299 mL aliquots of Br2
(23 mmol, 1.35 molar excess) were added in 4 min
intervals with the orange color disappearing each time.
After complete addition, some yellow color remained
and 3.22 mL of distilled NEt3 (23 mmol) was quickly
Compound 12. All of the solvents in the preparation and
purification of 12 were degassed. In a typical prepara-
tion, 421.7 mg of 11 was dissolved in 50 mL CHCl3 in a
100 mL rbf. The dark reddish-brown solution was

T. Wharton, L. J. Wilson / Bioorg. Med. Chem. 10 (2002) 3545–3554
3553
cooled for 5 min on an acetone/dry ice bath but not
allowed to freeze. 210 mL BF.3ET2O was added with
stirring under N2. After 15 min, the flask was allowed to
room to room temperature over 1 h during which some
orange ppt formed. With stirring, an additional 110 mL
of BF.3Et2O was then added. Increased precipitation
occurred and stirring was continued at room tempera-
ture for 2.5 h. The product was collected by centrifuge
and washed with 3 aliquots of fresh CHCl3 (10 mL) by
repeated sonication and centrifugation to give the acetal
6. Editorial staff Diagnostic Imaging Industry Report: Update
1996; Miller Freeman: San Francisco, 1996.
7. POV Reports The Diagnostic Imaging Marketplace in the
US; POV Inc: Cedar Grove, NJ, 1998.
8. Scally, P. Medical Imaging; Oxford University: Oxford, 1999.
9. Krause, W.; Schuhmann-Gampieri, G. In Contrast Media
in Practice; Dawson, P., Clauss, W., Eds.; Springer-Verlag:
Berlin, 1993; Chapter 1.6, pp 24–34.
10. Yu, S.-B.; Droege, M.; Segal, B.; Kim, S.-H.; Sanderson,
T.; Watson, A. D. Inorg. Chem. 2000, 39, 1325.
11. Yu, S.-B.; Droege, M.; Downey, S.; Segal, B.; Newcomb,
W.; Sanderson, T.; Crofts, S.; Suravajjala, S.; Bacon, E.; Ear-
ley, W.; Delecki, D.; Watson, A. D. Inorg. Chem. 2001, 40,
1576.
12. Quinn, A. D.; O’Hare, N. J.; Wallis, F. J.; Wilson, G. F.
J. Comput. Assist. Tomogr. 1994, 18, 634.
13. Schmitz, S. A.; Wagner, S.; Schumann-Giampieri, G.
Radiology 1997, 202, 407.
14. Srivastava, R. R.; Hamlin, D. K.; Wilbur, D. S. J. Org.
Chem. 1996, 61, 9041.
15. Meyer, D.; Spielvogel, B., World Patent WO9308122.
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17. Jensen, A. W.; Wilson, S. R.; Schuster, D. I. Bioorg. Med.
Chem. 1996, 4, 767.
18. Da Ros, T.; Prato, M. Chem. Commun. 1999, 663.
19. Schuster, D. I.; Wilson, S. R.; Schinazi, R. F. Bioorg.
Med. Chem. Lett. 1996, 1253.
20. Moussa, F.; Pressac, M.; Hadchouel, M.; Arbeille, B.;
Chretien, P.; Trivin, F.; Ceolin, R.; Szwarc, H. Proc. Electro-
chem. Soc. Recent Advances in the Chemistry and Physics of
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21. Wharton, T.; Wilson, L. J. Tetrahedon Lett. 2002, 43, 561.
22. Wharton, T.; Kini, V. U.; Mortis, R. S.; Wilson, L. J.
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1
deprotected intermediate (as verified by H NMR). The
brown solid was then washed with 5 aliquots of ice cold
H2O (3 mL) until neutral (5Â). The brown solid was
then dissolved in 12 mL of 4:1 MeOH:H2O. A solution
of 200 mg Na2CO3 in 3 mL H2O was then added. After
1 h of stirring at room temperature under N2, the dark
reddish-brown solution was diluted up to 75 mL with 50
mL H2O and 10 mL MeOH and stirred with ꢅ1 g (dry
weight) cation exchange resin (H+ form) over 1 h. The
solution was filtered and the cation exchange resin
reconverted to H+ form with 1 M HCl. The solution
was again treated with the same resin (to minimize pro-
duct adsorption, which caused loss of material) for 1 h.
After this time, the complete removal of Na+ was ver-
ified by flame test on a Pt wire. The solution was filtered
and the solvent was removed to give 106 mg of depro-
tected material 12. Yield for two steps 33%. 400 MHz
1H NMR (D2O, solvent ref) d 3.5–4.2 (bm, CH, CH2);
FT-IR (KBr) n (cmꢀ1) 3382 (O–H), 1652 (amide C¼O);
MALDI-TOF MS (2,5-dihydroxy benzoic acid matrix
from 1:1 H3CCN:H2O) avg. calcd for pentaadduct
C127H98N14O38I6 [M–H+Na]+ 3211.1, found 3211.1,
base peak [M–I+Na]+ 3085.1; anal. calcd for pentaad-
duct C127H98N14O38I6: C, 47.8; H, 3.10; N, 6.15; I, 23.9;
O*, 19.1, found: C, 43.8; H, 3.20; N, 5.80; I, 27.6; O,
19.6 (O by difference).
24. Meyer, D. H. In Textbook of Contrast Media; Dawson, P.,
Cosgrove, D. O., Grainger, R. G., Eds.; Isis Medical Media:
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27. Lamparth, I.; Hirsch, A. J. Chem. Soc., Chem. Commun.
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Acknowledgements
The authors wish to thank Dr. Larry Alemany at Rice
University for his help with the collection of the NMR
data and Dr. Ken Wright and Irene A. Szwarc at the M.
D. Anderson Cancer Center, Houston, Texas, for their
assistance with the in vitro X-ray experiments. This
research was supported by The Robert A. Welch Foun-
dation (C-0627) and C-Sixty, Inc., Toronto, Canada.
TW also thanks The Hasselman Foundation for a
graduate student fellowship.
28. Wharton, T.; Alford, J. M.; Husebo, L. O.; Wilson, L. J.
Proc. Electrochem. Soc. 2000, 9, 258.
29. Pillai, K. M. R.; Diamantidis, G.; Duncan, L.; Ranga-
nathan, R. S. J. Org. Chem. 1994, 59, 1344.
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