Synthesis and evaluation of potential CT (computer tomography) contrast agents for bone structure and microdamage analysis

Article abstract of DOI:10.1039/b606976b

The design and synthesis of several novel X-ray contrast agents 1-3, developed for targeting bone structures, and in particularly microcracks in bones, using CT (Computer Tomography) detection is described. These contrast agents are based on the use of the well known triiodobenzene platform, which was conjugated into one or more phenyliminodiacetate moieties, which can be used to 'lock' onto bone matrices. Compounds 1-3 were all tested for their ability to visualise cracks in bone structures (bovine bones) using μ-CT imaging. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b606976b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and evaluation of potential CT (computer tomography) contrast
agents for bone structure and microdamage analysis†
Raman Parkesh,a,b Wolfgang Gowin,c T. Clive Leeb and Thorfinnur Gunnlaugsson*a
Received 16th May 2006, Accepted 7th July 2006
First published as an Advance Article on the web 15th August 2006
DOI: 10.1039/b606976b
The design and synthesis of several novel X-ray contrast agents 1–3, developed for targeting bone
structures, and in particularly microcracks in bones, using CT (Computer Tomography) detection is
described. These contrast agents are based on the use of the well known triiodobenzene platform, which
was conjugated into one or more phenyliminodiacetate moieties, which can be used to ‘lock’ onto bone
matrices. Compounds 1–3 were all tested for their ability to visualise cracks in bone structures (bovine
bones) using l-CT imaging.
who have developed water-soluble, biocompatible X-ray contrast
Introduction
agents for use in the vascular, gastrointestinal and nervous systems.
In medical diagnosis, contrast can be used to distinguish an
However, to the best of our knowledge, no X-ray targeting contrast
organ or part of the body, via opacification or visualisation,
thus providing important structural information. Despite the
agents have been made specifically to characterize bone structures
and bone damage, although recently such MRI contrast agents
have been developed.11
fact that Magnetic Resonance Imaging (MRI) has revolutionized
medical imaging in recent times, X-ray based imaging is still one
of the most employed medical diagnoses, with over 80% of all
diagnostic imaging today being based on such radiation.1 Contrast
agents are essential parts of radiology and are routinely used
in modern medicine.2 As in the case of MRI,3 contrast agents
designed to target particular organs, tissues, etc., can play a
major role in X-ray based imaging.4 Currently, two types of X-
ray contrast agents are generally employed in medicine. These are
based upon the use of barium sulfate suspensions (predominantly
used for gastrointestinal tract imaging) and iodinated organic
compounds.1,5,6 At present, most available organic X-ray contrast
agents are based on the use of single triiodobenzene derivatives
or where two such aromatic rings have been connected to each
other by a covalent spacer.6,7 The introduction of an N-acetoxy or
N-methylcarbamyl functionality into the triiodobenzene structure
has been shown to greatly improve both the water solubility and
biological safety of such molecules. Such modifications also allow
for the introduction of other functionalities for achieving more
targeted diagnostics, for instace, of particular organs, tissues,
etc. Examples of such modifications have been carried out by
Ranganathan and co-workers,8 Bo¨hle et al.9 and Rongved et al.10
Bone is continuously renewing and repairing itself from the
ravages of time and injury. As bone is under constant stress due
to the repetitive loading during normal day-to-day activity, this
results in the generation of microcracks or microdamage. The
problem with the imaging of bones is their inherent contrast due
to their crystalline hydroxyapaptite and organic matrix.12 Thus it
is difficult to distinguish the contrast agent from the surrounding
bone matrix. One way of approaching this problem is to increase
the degree of opacity at the damaged site.13 We are interested in
the targeting of biologically important molecules using various
spectroscopic methods14 and we have recently achieved such bone
targeting by using fluorescent molecules that have binding sites
(chelators) for ions such as Ca(II) or Mg(II).15 However, to date, the
analysis of such bone structures has not been achieved using either
iodine contrast agents or dual based iodine-fluorescent contrast
agents. Consequently, we set out to develop such contrast agents
(Fig. 1). Our objective was to synthesize organic-iodine based
conjugates that could ‘lock’ onto bone cracks or microdamaged
sites. To achieve this, such a structure would need to possess the
specific ability to penetrate the bone matrix and incorporate into
the free lattice, where the weak ionic interactions with the lattice
can provide additional modes of binding. Hence, such contrast
agents would need to have a receptor moiety conjugated into the
iodine moiety with the aim of maximising the interactions with
ions such as Ca(II) that are exposed on the surfaces on the damaged
sites. Such structural designs could thus provide a non-invasive
method of detecting and quantifying microdamage in patients.
Herein, we describe the synthesis of three such first-generation
ion-receptor based contrast agents 1–3 which are based on the use
of one or two triiodobenzene moieties within a single structure
and the analysis of these to detect microcracks in bovine bones
using micro computer tomography (lCT) imaging.16
aSchool of Chemistry, Centre for Synthesis and Chemical Biology (CSCB),
Trinity College Dublin, Dublin 2, Ireland. E-mail: gunnlaut@tcd.ie; Fax: 00
353 1 671 2826; Tel: 00 353 1 608 3459
bDepartment of Anatomy, Royal College of Surgeons in Ireland, St. Stephen’s
Green, Dublin 2, Ireland
cCenter of Muscle and Bone Research, Department of Radiology and Nuclear
Medicine, Charite´-University Medicine Berlin, Campus Benjamin Franklin,
Berlin, Germany
† Electronic supplementary information (ESI) available: 1H NMR spectra
of compounds 1–3. See DOI: 10.1039/b606976b
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receptors and iodinated phenyl rings. In molecules 1 and 4, a single
phenyliminodiacetate unit was attached to the iodinated aromatic
ring, via an amide spacer, while 2 has two such ion chelator units
attached meta to each other. The dimer 3 contains, however, six
iodine atoms and four chelator units in meta configuration, and
as such would be expected to have the highest binding affinity
as well as giving rise to the most efficient imaging. Moreover, as
their sodium salts, these contrast agents would be expected to be
highly water-soluble.1 These compounds also have incorporated
solubilising groups in the form of the amides that will render them
safer and less viscous.7b
Scheme 1 Synthesis of 1 from 5 and 6.
As discussed above, all of the four compounds are based on
a triiodo moiety connected to the phenyliminodiacetate (made
as the corresponding diethyl ester). Consequently, all of these
were synthesized by making the phenyliminodiacetate moiety and
connecting that into the relative carboxylic acid derivative of the
triiodo compounds. As a starting point for this investigation,
we synthesized 1 first as shown in Scheme 1. This involved the
coupling of 2,3,5-triiodobenzoyl chloride 5, which was synthesized
from the commercially available carboxylic acid using thionyl
chloride in 85% yield after recrystallization from a chloroform–
hexane (1 : 1, v/v) mixture. The phenyliminodiacetate ethyl ester
6 was formed in four steps from aniline, involving the dialkylation
using ethyl bromoacetate in the presence of K2HPO4 and in
refluxing CH3CN which gave the desired product in 90% yield.
This was followed by nitration using 70% nitric acid (15 min)
followed by recrystallization of the crude product from 2-propanol
which gave 8 (not shown) in 76% yield. Hydrogenation on 8 using
10% Pd/C proceeded smoothly to give 6 in almost quantitative
yields. Condensation of 5 with 6 was achieved in THF using Et3N
and resulted in the formation of the desired diester 7, in 80%
yield after purification by silica flash column chromatography
(using 2 : 5 ethyl acetate–hexane as eluant). The alkaline hydrolysis
(NaOH) of 7 to give 1 was achieved in MeOH–H2O (10 : 1). The
purification of 1 involved the evaporation of the solvent mixture
to dryness, and the resulting residue was dissolved in a minimal
amount of distilled water, followed by dilution with methanol. The
resulting mixture was then heated at reflux in the presence activated
charcoal, followed by cooling to room temperature. After standing
at room temperature for a further two days, it was filtered and
evaporated to dryness, diluted with a minimal amount of water
and added dropwise to a stirred solution of ethanol to precipitate
1. This purification method removed all of the coloured impurities
and resulted in the isolation of 1 in 92% yield (see 1H NMR of 1
in ESI†).
Fig. 1 Compounds 1–4.
Results and discussion
Design and synthesis of 1–4
Compounds 1–4 shown in Fig. 1, are all based on the use of
phenyliminodiacetate as an ion chelator linked via an amide
linkage to a triiodo benzene skeleton.17 We choose the phenylimin-
odiacetate moiety as a potential ion-receptor or chelator for Ca(II)
and or Mg(II). Here the aim was that this simple receptor would
lock or bind to these ions within the cracks of the bone structure,
which would then allow the use of CT for the selective imaging
of the ion-bound contrast agents.‡ As the objective of the current
study was the ‘proof of principle’ of selective CT imaging of such
bones, we chose to use bovine bones for our imaging studies (see
later). This would then allow us to wash off any excess contrast
agents (non-bound) prior to the CT imaging, allowing us to image
only those agents that had bound to exposed damaged bone such
as in scratches or microcracks.
Several types of contrast agent were developed for this study,
simply by varying the combination/numbers of both the ion-
‡ For the future development of this technique, it is important to account
for the fact that the concentrations of these ions within the bones
would be significantly greater than found in both extra-and intra-cellular
environments. Consequently, at the same time as the receptors should be
Ca(II) selective, they would also need to have a low affinity for these ions to
prevent any interference from background signals arising from the binding
of these agents to free Ca(II) in blood or in cells. Because of this we chose
to use the simple phenyliminodiaetate moiety as a receptor as it would
hopefully fulfil these criteria.
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Scheme 2 Synthesis of 2 from 6 and 10.
The contrast agent 2 was based on similar design strategy as
developed for 1 and is outlined in Scheme 2. We predicted that by
using two phenyliminodiacetate moieties in a meta configuration
to each other, this could possibly result in an increased ionic
interaction and stronger binding at the unsaturated lattice sites
in damaged bone. Furthermore, the primary aromatic amine
in 2 was introduced as an extra functionality with the aim of
attaching a fluorescent tag such as fluorescein at this position. This
would result in the formation of a dual-functionalised molecule
consisting of an iodinated contrast agent and a histological stain.
The synthesis of 2 was achieved by firstly converting the
commercially available 5-aminoisophthalic acid into the desired
three iodine derivative 9 using a modified synthetic method
developed by Larsen et al. using KICl2–H2O, generated in situ
by combining potassium chloride (KCl) and iodine monochloride
(ICl).18 The purification of 9 was achieved by firstly converting
the dicarboxylic acid to its potassium salt using 1 M KOH. The
resulting solution was then decolourised with activated charcoal
by heating at 70–80 ◦C for 30 min, followed by filtration. The
solution was then acidified using conc. HCl to give 9 in 75% yield.
The conversion of 9 to the corresponding bis-acid chloride 10 was
accomplished in 85% yield using freshly distilled thionyl chloride.19
The condensation of 10 and 6 in DMA, using Et3N, resulted in
the formation of 11 in 80%, after recrystallization from ethanol.
Alkaline hydrolysis of 11 resulted in an almost quantitative yield
of 2, which was purified in a similar manner as described for 1 (see
ESI for 1H NMR of 2†).
The synthesis of the hexa-iodo derivative 3 is illustrated in
Scheme 3. The synthesis involved the use of the bis-acid chloride 10
synthesized above, which was first reacted with malonyl dichloride
using an established literature method. This involved the refluxing
of the two reagents in anhydrous THF, giving the desired product
12 in 75% yield.19 The steric crowding of the bulky iodine atoms
hindered the polymerisation of 10 and 12 was therefore obtained as
the major single product. The condensation of 12 with 5 equiv. of
6 in DMA at 30 ◦C for 24 h in the presence of Et3N resulted in the
formation of the octa-ester 13 in 80% yield after recrystallization
from ethanol. The subsequent basic (NaOH) hydrolysis of 13 in
MeOH–H2O afforded the target molecule 3 which was purified in
a similar manner to that described for 1 above in 92% yield (see
ESI for 1H NMR of 3†).
Scheme 3 Synthesis of compound 3.
Hexa-substituted benzene compounds such as those synthesized
above have been studied extensively for the phenomenon of con-
formational equilibrium analysis. Bradmante and Vittadini have
carried out such comprehensive NMR studies on the steric effects
of highly crowded molecules based on the 1,3,5-triiodobenzene
platform, which were found to give rise to atropisomerism.20 As
in their cases, all of the positions in 1–3 are substituted and hence
would thus also be expected to be sterically congested and lead to
atropisomerism due to the lack of rotation around the Ar–CO, the
Aryl–N and the CO–N bond of the isophthalimide moieties, and
the N–CO bonds of the amide moieties. However, in 1–3 the 13C
NMR and 1H NMR exhibited simple splitting patterns indicating
that all the carbonyl functionalities were equivalent. Moreover,
the aromatic and methylene protons of the phenyliminodiacetate
chelators were found to be equivalent. The corresponding esters
also exhibited similar spectral patterns, indicating that all these
molecules existed as single isomers. The probable explanation for
this could be the effect of stabilisation of one isomer induced by
extended conjugation. Similar observations were made in natural
products with extended conjugation such as biphyscion.21
The final compound in this study, 4 (see Fig. 1), was designed to
represent a simple amide analogue of 1, based on the substitution
pattern employed in 2 and 3. The attempted synthesis is shown in
Scheme 4, which involved the iodination of 2,5-diaminobenzoic
acid using KICl2–H2O followed by purification as described above
for 9 to give 14 in 65% yield.22 The acetylation of the 14 was
achieved in 90% yield to give 15 using acetic anhydride in the
presence of a small amount of conc. H2SO4.23 The chlorination
of 15 to give the acid chloride 16 was achieved in a yield of 71%
by refluxing 15 in freshly distilled SOCl2 for 3 h. The final step
involved the formation of 17 from the condensation of 16 with
6. As before, this condensation was first attempted in DMA and
Et3N, but the reaction was unsuccessful. Alternate reactions such
as the use of HMPT and Et3N; DMF and Et3N, and THF and
Et3N were attempted but on all occasions the desired ester 17 was
not formed. Because of this we did not achieve the synthesis of 4
as planned.
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sagittal and transparent three-dimensional images clearly show
that these contrast agents were able to absorb the X-rays more
effectively than the bone, thus providing better contrast from the
bone matrix. This is not unexpected as both 2 and 3 have a higher
iodine content and as such should give rise to stronger imaging.
After performing qualitative studies with these contrast agents
in powdered form, the next stage was to label bone scratches
with 10−3 M aqueous solutions of the contrast agents 1–3, using
bovine tibiae, and image the resulting bone specimens using l-
CT. The preliminary l-CT studies described below were performed
in a similar manner as before. Even though the bone specimen
labelled with compound 1 indicated that the scratch was visible,
the background contrast for both the scratch depth and the
surrounding bone matrix almost overlapped with that observed
form 1 (Fig. 3). Consequently, it was difficult to differentiate
between bone and contrast agent. Similar effects were observed
for 2 and 3. The most likely reason for this may be from the
beam hardening effect, which masks the imaging from the agents.§
Nevertheless, these results clearly indicate that our agents can bind
to bone cracks (microcracks) and as such the results obtained from
1–3 are important for the future development of bone-targeting
CT imaging agents. We are currently working towards achieving
that objective.
Scheme 4 Attempted synthesis of 17 from 6 and 16.
Evaluation of contrast agents 1–3 in solid (powder) form and in
solution using l-CT
Upon computer tomography imaging using l-CT, compounds 1–
3 synthesized above were all able to provide good contrast from
the surrounding bone matrix as powdered substances.24 A two-
dimensional sagittal reconstruction was performed in order to
evaluate the contrast ability of the agents, in comparison with the
surrounding bone matrix (see Experimental for further details).
The first of those compounds to be tested was contrast agent 1,
which was applied to the right-hand side of the bone specimen
as shown in the red box in the sagittal reconstruction in Fig. 2A.
For comparison the left-hand side of the bone, within the blue
box has no contrast agent applied. Both the bone and 1 gave an
image that is predominantly white. However, areas where 1 has
been applied appear to be more bright due to its higher X-ray
absorption power, and hence it is distinct from the bone surface.
A three-dimensional transparent section was also reconstructed
to further illustrate the contrast ability of 1 (Fig. 2B). Here, it
is clearly possible to distinguish the internal architecture of the
bone in the transparent reconstructed section, as well as Haversian
and Volkmann’s canals and the resorption cavities due to the
contrast provided by 1. In a similar way, compounds 2 and 3 were
tested under identical conditions. Similar results where obtained to
those seen in Fig. 2. However, the reconstructed two-dimensional
Fig. 3 Three-dimensional axial reconstruction of the bone immersed in
an aqueous solution of 1 (1 mM). A scratch can be seen on the left-hand
side (yellow arrow). The top of the axial plane shows a vessel canal (red
arrow) within the bone tissue.12a
Conclusion
We have demonstrated the synthesis of several new bone-targeting
contrast agents for CT imaging based on the use of the triiodoben-
zene framework, and we have carried out a preliminary evaluation
of these to quantify microdamage in bone. All three contrast agents
exhibited good water solubility and stability. Preliminary imaging
studies of these contrast agents on bovine cortical bone specimens,
using cone-beam l-CT, demonstrated that agents in powder form
are able to provide good contrast from the surrounding bone
§ Such beam hardening arises from X-ray photons striking the bone surface
at different energy so that the photons of lower energy are absorbed with a
higher probability than those of a higher energy. As a result, the attenuation
of the X-ray is not homogenous and is dependent upon the energy of the
radiation, the type of object used in the imaging and the direction of
projection.
Fig. 2 Computer tomography images of 1: (A) two-dimensional and (B)
three-dimensional sagittal sections of a cortical bone specimen labelled
with contrast agent 1. The white powder on the bone edges is marked by a
black arrow. Bars are in lm.
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matrix. Solution phase studies of these contrast agents was,
however, not as successful, possibly due to a beam hardening effect.
We are currently working towards synthesising related structures
of 1–3 with the aim of achieving such imaging in solution. We are
currently modifying the structure of 2 with the aim of achieving
dual CT and fluorescence analysis of bone structures.
After 15 min, the reaction mixture was poured over ice, filtered
and the resulting solid recrystallized from ethanol. This gave 8 as
yellow brown needles in 76% yield. Mp = 160–162 ◦C MS (ES+)
m/z = 310 (M+). Anal. calc. for C14H18N2O6: C, 54.19; H, 5.85;
N, 9.03. Found: C, 54.12; H, 5.85; N, 8.98%. 1H NMR (400 MHz,
CDCl3): d 1.30 (t, 6H, J = 7.0 Hz, NHCH2CO2CH2CH3),
4.21 (s, 4H, NHCH2CO2CH2CH3), 4.27 (q, 4H, J = 7.0 Hz,
NHCH2CO2CH2CH3), 6.6 (d, 2H, J = 9.5 Hz, Ar–H), 8.13 (d, 2H,
J = 9.5 Hz, Ar–H). 13C NMR (100 MHz, CDCl3): d 13.74, 53.00,
61.29, 110.82, 125.55, 138.52, 152.23, 168.86. IR (mmax, KBr, cm−1):
3474, 3121, 3098, 2976, 2908, 2696, 2614, 2426, 2231, 1918, 1893,
1751, 1591, 1516, 1420, 1272, 1117, 1026, 961, 918, 871, 828, 757,
736, 696, 632, 586, 535, 559.72.
Experimental
General
Reagents (obtained from Aldrich) and solvents were purified using
standard techniques. Solvents were dried over the appropriate
drying agent before use using standard procedures. Melting points
were determined using a GallenKamp melting point apparatus.
Infrared spectra were recorded on a Mattson Genesis II FTIR
spectrophotometer equipped with a Gateway 2000 4DX2-66
workstation. Oils were analysed using NaCl plates; solid samples
were dispersed in KBr and recorded as clear pressed discs.
1H NMR spectra were recorded at 400 MHz using a Bruker
Spectrospin DPX-400 instrument. 13C NMR were recorded at
100 MHz using a Bruker Spectrospin DPX-400 instrument.
[(4-Aminophenyl)ethoxycarbonylmethylamino]acetic acid ethyl
ester (6). A solution of 8 (1.0 g, 3.22 mmol) in methanol (50 mL)
was hydrogenated at 1 atm for 15 min using Pd–C (10% w/w,
0.07 g). After completion the catalyst was filtered off and the
solvent removed under reduced pressure to yield 9 (0.85 g, 95%)
as a brown liquid. MS (ES+) m/z = 281 (MH)+. Anal. calc. for
C14H20N2O4: C, 59.99; H, 7.19; N, 9.99. Found: C, 59.25; H, 7.03;
N, 9.84%. 1H NMR (400 MHz, CDCl3): d 1.28 (t, 6H, J = 6.8 Hz,
NCH2CO2CH2CH3), 4.09 (s, 4H, NCH2CO2CH2CH3), 4.18–4.23
(q, 4H, J = 6.8 Hz, NCH2CO2CH2CH3), 6.57 (d, 2H, J = 8.9 Hz,
Ar–H), 6.66 (d, 2H, J = 8.9 Hz, Ar–H). 13C NMR (100 MHz,
CDCl3): d 170.88, 140.94, 138.02, 116.26, 114.54, 60.49, 53.68,
13.79. IR (mmax, NaCl, cm−1): 3330, 2981, 2930, 2355, 1733, 1616,
1519, 1448, 1412, 1347, 1255, 1188, 1097, 1025, 974, 918, 817,
729, 521.
Materials and methods for CT imaging
The soft tissue was removed from all bones and they were stored
at −20 ◦C until required. Longitudinal sections of cortical bones
with dimensions of 2 × 2 mm from the mid-diaphysis were
cut into 1 cm beams using a band saw and polished using
emery paper (grade 400). All machining was carried out in wet
conditions and the bones were not allowed to dry at any time.
After machining the specimens were stored at −20 ◦C prior to
CT imaging. The sample was mounted on the examination table
and rotated 180◦ and a total of 360 projections were recorded.
At a 5 s exposure time for each projection the entire scanning
lasted 1 h. The data stored in the computer were analysed and
reconstructed using the Amira software. The reconstruction lasted
approximately 1.5 h. By manipulating 3D data-sets, it was possible
to observe the canals, osteons and resorption cavities in the
bones. The smallest volume element (voxel) achieved was 4 lm
and the images were reconstructed using a Shep-Logan filtered
modified Feldkamp algorithm. The X-ray absorption value for
each and every point in space was reconstructed. The different
absorption values (CT number or Hounsfield value) resulted
in a two- or three-dimensional image of the scanned object.
Reconstruction of images along sagittal (a two-dimensional plane
going from the left to the right through the specimen), coronal
(a two-dimensional plane going from the front to the back of
the specimen) and transaxial planes (a two-dimensional plane
going from the top to the bottom of the specimen) were made
possible using Amira visualisation software. The two- and three-
dimensional representation of the image is given in terms of pixel
and voxel respectively.
Ethoxycarbonylmethyl - [4 - (2,3,5 - triiodobenzoylamino)phenyl]-
amino}acetic acid ethyl ester (7). 2,3,5-Triodobenzoyl chloride,
5 (0.5 g, 0.96 mmol), was dissolved in anhydrous THF (20 mL).
To this solution was added 6 (0.27 g, 0.96 mmol) in 10 mL of
THF and freshly distilled Et3N (0.15 g, 0.20 mL, 1.2 mmol). The
resulting solution was kept at reflux for 24 h, after which the THF
was removed under reduced pressure and the resulting residue
extracted several times with chloroform. The combined organic
layers were washed with HCl (1 M), NaHCO3 (1 M) and brine,
dried over MgSO4 and the solvent removed under reduced pressure
to give a white solid which was further purified using flash-column
chromatography (chloroform). ◦This gave 7 as a white solid in 90%
yield (0.67 g). Mp = 174–176 C. MS (ES+) m/z = 762 (MH)+.
Anal. calc. for C21H21I3N2O5: C, 33.10; H, 2.78; N, 3.68. Found:
1
C, 33.40; H, 2.81; N, 3.51%. H NMR (400 MHz, DMSO-d6): d
1.20 (t, 6H, J = 7.0 Hz, NHCH2CO2CH2CH3), 4.12 (q, 4H, J =
7.0 Hz, NHCH2CO2CH2CH3), 4.18 (s, 4H, NHCH2CO2CH2CH3)
7.44 (d, 2H, J = 9.0 Hz, Ar–H), 7.69 (d, 2H, J = 9.0 Hz, Ar–H),
8.31 (d, 1H, J = 2.0 Hz, Ar–H), 10.19 (s, 1H, CONH). 13C NMR
(100 MHz, DMSO-d6): d 170.52, 165.73, 147.51, 146.06, 144.55,
134.50, 129.19, 121.02, 111.96, 113.30, 107.94, 95.51, 60.39, 52.81,
14.14. IR (mmax, KBr, cm−1): 3261, 2977, 1638, 1595, 1520, 1410,
1324, 1261, 1186, 1024, 974, 910, 866, 816, 765, 716, 567, 513.
Ethoxycarbonylmethyl - [4 - (2,3,5 - triiodobenzoylamino)phenyl]-
Synthesis
amino}acetic acid sodium salt (1). Compound
7 (1.5 g,
[Ethoxycarbonylmethyl-(4-nitrophenyl)amino]acetic acid ethyl
ester (8). [Ethoxycarbonylmethyl-(phenylamino)]acetic acid
ethyl ester (6.00 g, 22.72 mmol) was taken into 50 ml of acetic
acid and stirred at 0 ◦C. To this, 3 mL of HNO3 was added.
1.97 mmol), was dissolved in methanol (20 mL). To this was added
NaOH (2 mL, 3 M) and the resulting solution refluxed for 2 h.
Addition of ethanol resulted in the precipitation of a white solid,
which was collected by filtration and dried under vacuum to afford
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1 (1.4 g, 95%) as a yellow solid. MS (ES+) m/z = 767 (M + H2O).
Anal. calc. for C17H11I3N2Na2O5·H2O: C, 26.59; H, 1.71; N, 3.65.
Found: C, 26.67; H, 1.52; N, 3.48%. 1H NMR (400 MHz, CDCl3):
d 3.82 (s, 4H, NCH2CO2Na), 6.46 (d, 2H, J = 8.9 Hz, Ar–H),
7.2 (d, 2H, J = 8.9 Hz, Ar–H), 7.68 (s, 1H, Ar–H), 8.32 (s, 1H,
CONH). 13C NMR (100 MHz, D2O): d 178.89, 168.66, 147.35,
146.65, 144.43, 134.40, 124.36, 123.56, 118.86, 112.24, 105.92,
94.00, 55.22. IR (mmax, KBr, cm−1): 3587, 3379, 3099, 1690, 1520,
1477, 1418, 1319, 1269, 1247, 1171, 1125, 1098, 975, 894, 865, 819,
729, 703, 612, 525.
concentrated and ethanol added. The resulting precipitate was
collected by filtration and dried under vacuum to afford 2 as a
white solid (2.27 g) in 93% yield. MS (ES+) m/z = 1106 (M +
Na)+. Anal. calc. for C28H20I3N5Na4O10·6H2O: C, 28.81; H, 2.76;
N, 6.00. Found: C, 28.11; H, 2.22; N, 5.6%. 1H NMR (400 MHz,
D2O): d 3.83 (s, 8H, NCH2CO2Na), 6.5 (d, 2H, J = 9.0 Hz,
Ar–H), 7.3 (d, 2H, J = 9.0 Hz, Ar–H). 13C NMR (100 MHz,
D2O): d 178.95, 170.83, 170.77, 147.96, 147.25, 147.21, 146.87,
123.72, 123.55, 111.17, 79.53, 72.56, 55.18. IR (mmax, KBr, cm−1):
3548, 3279, 3199, 1677, 1620, 1577, 1518, 1319, 1209, 1107, 1001,
975, 864, 805, 729, 713, 610, 565.
5-Amino-2,4,6-triiodoisophthalic acid (9). ICl (9.8 g,
100 mmol) was added to a solution of KCl (7.2 g 9.70 mmol)
in H2O (100 mL) and stirred at room temperature for 5 min.
The mixture was filtered to remove any insoluble materials. The
resulting orange solution (of KICl2) was added dropwise over 1 h
to a solution of 5-aminoisophthalic acid (3.32 g. 1.80 mmol) in
H2O (110 mL) at 55◦ C. After complete addition the mixture was
stirred at 60 ◦C for a further 24 h and finally at 90 ◦C for 1 h. On
cooling to room temperature, the resulting solid was dissolved
in 20 ml of 1 M KOH and treated four times with activated
charcoal at 40–50 ◦C for 30 min. The resulting product◦was then
precipitated using conc. HCl, filtered and dried at 100 C under
vacuum. This gave 9 as a yellow solid in 75% yield (3.68 g). Mp =
N,N-Bis(2,4,6-triiodo-3,5-benzenedichlorocarbonyl)malonamide
tetraphenyliminodiethyl diester (13). Compound 12 (1.09 g,
0.86 mmol) was dissolved under argon in anhydrous
dimethylacetamide (20 mL). To this solution 6 (1.052 g,
3.75 mmol) was added in 15 mL of dimethylacetamide, via a
syringe, and freshly distilled Et3N (0.38 g, 0.51 mL, 3.75 mmol).
The resulting solution was stirred at room temperature for 24 h,
after which the solvent was removed by vacuum distillation. The
resulting residue was extracted three times with chloroform, and
the combined organic layer was washed with HCl (1 M), NaHCO3
(1 M) and brine. The organic layer was dried over MgSO4 and
the solvent removed under reduced pressure to give a white solid
which was further purified by recrystallization from ethanol. This
gave 13 as a white solid (1.19 g) in 80% yield. MS (ES+) m/z
1117 (M+/2). Anal. calc. for C75H80I6N10O22: C, 40.31; H, 3.61; N,
◦
260 C (decomp.). MS (ES+), m/z = 559 (M + H+). Anal. calc.
for C8H4I3NO4·H2O: C, 16.66; H, 1.05; N, 2.43. Found: C, 16.86;
H, 0.78; N, 2.16%. 13C NMR (400 MHz, DMSO-d6): d 170.11,
148.91, 147.78, 77.93, 70.62. IR (mmax, KBr, cm−1): 3629, 3442,
3378, 3305, 2894, 2600, 1785, 1600, 1367, 1290, 1009, 987, 867,
765.
1
6.27. Found: C, 40.78; H, 3.98; N, 6.41%. H NMR (400 MHz,
DMSO-d6): d 1.20 (t, 24H, J = 7.0 Hz, NHCH2–CO2CH2CH3),
4.12 (q, 8H, J = 7.0 Hz, NHCH2CO2CH2CH3), 4.19 (s, 8H,
NHCH2CO2CH2CH3), 6.55 (d, 4H, J = 8.5 Hz, Ar–H), 7.43–7.46
(m, 4H, Ar–H), 10.24 (brs, 2H, –CONH). 13C NMR (100 MHz,
DMSO-d6): d 170.55, 167.08, 150.06, 144.54, 142.75, 129.28,
129.24, 121.03, 121.06, 112.01, 111.98, 91.04, 60.40, 52.85, 14.15.
IR (mmax, KBr, cm−1): 3243, 2982, 1735, 1650, 1530, 1186, 1024,
970, 815.
4-Amino-N,N-bis(2,4,6-triiodo) bis-phenyliminoacetic acid di-
ethyl ester (11). 5-Amino-2,4,6-triiodoisophthaloyl dichloride
10 (1.5 g, 2.5 mmol) was dissolved with stirring in anhydrous
dimethylacetamide (35 mL) under argon. To this, 6 (1.43 g,
5.11 mmol) was added in 15 ml of dimethylacetamide, via a
syringe, and freshly distilled Et3N (0.51 g, 0.68 mL, 2.4 mmol).
The resulting solution was stirred at room temperature for 24 h,
after which the solvent was removed by vacuum distillation and
the resulting residue extracted three times with chloroform. The
combined organic layers were washed with HCl (1 M), NaHCO3
(1 M) and brine, dried over MgSO4 and finally the solvent was
removed under reduced pressure to give a white solid which was
further purified using flash-column chromatography (chloroform)
and finally recrystallized from ethanol to give 11 as a white solid
(2.3 g) in 85% yield. Mp = 187–189 ◦C. MS (ES+) m/z = 1083 (M
+ H)+. Anal. calc. for C36H40I3N5O10·2C2H5OH: C, 40.87; H, 4.46;
N, 5.96. Found: C, 40.83; H, 4.13; N, 6.46%. 1H NMR (400 MHz,
DMSO-d6): d 1.20 (t, 24H, J = 6.5 Hz), 4.14 (q, 16H, J = 6.5
Hz), 4.20 (s, 4H, 16H) 6.56 (d, 8H, J = 7.5 Hz), 7.69 (m, 8H),
10.27–10.43 (m, 6H, –NH). 13C NMR (100 MHz, DMSO-d6):
d 170.53, 165.73, 147.51, 146.06, 144.55, 14.14, 134.50, 129.19,
N,N-Bis(2,4,6-triiodo-3,5-benzenedichlorocarbonyl)malonamide
tetraphenyliminoacetic acid sodium salt (3). Compound 13
(2.7 g, 1.21 mmol) was dissolved in methanol (20 mL), and to this
solution was added NaOH (3 mL, 6 M). The resulting solution
was refluxed for 3 h, after which the solution was concentrated
and ethanol added. The resulting precipitate was collected by
filtration and dried under vacuum, which gave 3 as an off-white
◦
solid (2.43 g) in 92% yield. Mp = 400 C (decomp.). Anal. calc.
for C59H40I6N10Na8O22·8H2O: C, 30.41; H, 2.42; N, 6.01. Found:
1
C, 27.55; H, 2.05; N, 5.28. H NMR (400 MHz, D2O): d 3.93 (s,
16H, NHCH2CO2Na), 3.82 (s, 2H, CH2), 6.49 (d, 8H, J = 8.5 Hz,
Ar–H). 13C NMR (100 MHz, D2O): d 178.95, 170.38, 170.29,
147.96, 146.90, 123.81, 123.63, 111.22, 95.31, 56.96, 55.20, 53.94,
16.36. IR (mmax, KBr, cm−1): 3239, 1671, 1518, 1404, 1308, 1183,
976, 909, 820, 610.
121.02, 113.30, 111.96, 107.94, 95.51, 60.39, 52.81, 14.14. IR (mmax
,
2,4,6-Triiodo-3,5-diacetamidobenzoylchloride
(15). Com-
KBr, cm−1): 3233, 2962, 1745, 1640, 1590, 1066, 1024, 976, 815,
730, 645.
pound 14 (6 g, 9.78 mmol) was added to freshly distilled SOCl2
(60 ml) and the mixture was refluxed for 3 h, after which the
SOCl2 was removed. The resulting residue was washed three times
with hexane, which gave 15 as a yellow solid, which was then
filtered to give the desired product in 71% yield (4.48 g). MS (ES+)
m/z = 654 (MNa)+. Anal. calc. for C11H8ClI3N2O2: C, 20.89;
4-Amino-N,N-bis(2,4,6-triiodo) bis-phenyliminoacetic acid
sodium salt (2). Compound 11 was dissolved in methanol
(20 mL). To this solution was added NaOH (2 ml, 6 M) and the
resulting solution refluxed for 3 h, after which the solution was
3616 | Org. Biomol. Chem., 2006, 4, 3611–3617
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1
H, 1.28; N, 4.43. Found: C, 20.69; H, 0.87; N, 3.80%. H NMR
11 V. Kubicek, J. Rudovsky, J. Kotek, P. Hermann, L. V. Elst, R. N. Muller,
Z. I. Kolar, H. Th. Wolterbeek, J. A. Peters and I. Lukes, J. Am. Chem.
Soc., 2005, 127, 16477 and refs. cited therein.
12 (a) T. C. Lee, A. Staines and D. Taylor, J. Anat., 2002, 201, 437;
(b) T. C. Lee, F. J. O’Brien and D. Taylor, Int. J. Fatigue, 2000, 22,
847.
13 F. J. O’Brien, D. Taylor and T. C. Lee, J. Biomech., 2003, 36, 973.
14 T. Gunnlaugsson and J. P. Leonard, Chem. Commun., 2005, 3114;
J. P. Leonard and T. Gunnlaugsson, J. Fluoresc., 2005, 15, 595;
T. Gunnlaugsson and J. P. Leonard, Dalton Trans., 2005, 3204; T.
Gunnlaugsson, P. E. Kruger, P. Jensen, J. Tierney, H. D. P. Ali and
G. M. Hussey, J. Org. Chem., 2005, 70, 10875; T. Gunnlaugsson, A. P.
Davis, G. M. Hussey, J. Tierney and M. Glynn, Org. Biomol. Chem.,
2004, 2, 1856; T. Gunnlaugsson, P. E. Kruger, T. C. Lee, R. Parkesh,
F. M. Pfeffer and G. M. Hussey, Tetrahedron Lett., 2003, 35, 6575; T.
Gunnlaugsson, T. C. Lee and R. Parkesh, Org. Biomol. Chem., 2003, 1,
3265.
15 T. C. Lee, S. Mohsin, D. Taylor, R. Parkesh, T. Gunnlaugsson, F. J.
O’Brien, M. Giehl and W. Gowin, J. Anat., 2003, 203, 161; T. C. Lee,
E. R. Myers and W. C. Hayes, J. Anat., 1998, 193, 179.
16 Some of the l-CT imaging work from this research programme
has recently been communicated, see: R. Parkesh, T. C. Lee, T.
Gunnlaugsson and W. Gowin, J. Biomech., 2006, 39, 1552.
17 This receptor has also been used for targeting Cd(II), see: T. Gunnlaugs-
son, T. C. Lee and R. Parkesh, Tetrahedron, 2004, 49, 11239; T.
Gunnlaugsson, T. C. Lee and R. Parkesh, Org. Lett., 2003, 5,
4065.
(400 MHz, DMSO-d6) d 2.04 (s, 6H, NHCOCH3), 10.09 (s, 1H,
–NHCOCH3), 10.16 (s, 1H, NHCOCH3). 13C NMR (100 MHz,
CDCl3): d 167.92, 167.70, 149.03, 145.02, 111.02, 95.13, 22.98,
22.9. IR (mmax, KBr, cm−1): 3242, 3007, 2058, 1889, 1675, 1498,
1345, 1299, 1007, 850, 782, 650.
Acknowledgements
We thank TCD, RCSI and HRB for financial support.
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