Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent

Article abstract of DOI:10.1002/chem.200800402

The design of effective pH responsive MRI contrast agents is a key goal in the development of new diagnostic methods for conditions such as kidney disease and cancer. A key factor determining the effectiveness of an agent is the difference between the rel

Full text of DOI:10.1002/chem.200800402

DOI: 10.1002/chem.200800402
Synthesis and Relaxometric Studies of a Dendrimer-Based pH-Responsive
MRI Contrast Agent
M. Meser Ali,[a] Mark Woods,[a, b] Peter Caravan,[c] Ana C. L. Opina,[a] Marga Spiller,[d]
James C. Fettinger,[e] and A. Dean Sherry*[a, f]
Abstract: The design of effective pH
responsive MRI contrast agents is a
key goal in the development of new di-
agnostic methods for conditions such
as kidney disease and cancer. A key
factor determining the effectiveness of
an agent is the difference between the
relaxivity of the “on” state compared
to that of the “off” state. In this paper,
we demonstrate that it is possible to
improve the pH-responsive action of a
low molecular weight agent by conju-
gating it to a macromolecular con-
struct. The synthesis of a bifunctional
pH responsive agent is reported. As
part of that synthetic pathway we ex-
amine the Ing–Manske reaction, identi-
fying an undesirable by-product and es-
tablishing effective conditions for pro-
moting a clean and effective reaction.
Reaction of the bifunctional pH re-
sponsive agent with a G5-PAMAM
dendrimer yielded a product with an
average of 96 chelates per dendrimer.
The relaxivity of the dendrimer conju-
gate rises from 10.8 mmꢀ1 sꢀ1 (pH 9) to
24.0 mmꢀ1 sꢀ1 (pH 6) per Gd3+ ion. This
more than doubles the relaxivity pH
response, Dr1, of our agent from just
51% for the original low molecular
weight chelate to 122% for the dendri-
mer.
Keywords: dendrimers
agents lanthanide complexes
MRI contrast agents
·
imaging
·
·
Introduction
Diseases, such as kidney disorders and cancer, often result
in a significant reduction in the extracelluar pH of these tis-
sues.[1–3] Thus, imaging tissue pH could be quite useful in
clinical diagnose of these and other conditions. Current
methods for assessing tissue pH involve relatively invasive
procedures and typically can assess pH only from a limited
number of locations.[2–4] Less invasive techniques such as
31P NMR NMR spectroscopy[4] can provide a direct measure
of pH but the concentrations of the endogenous phosphorus
metabolites that respond to tissue pH are relatively low so
pH measurements can only be taken from relatively large
volumes of tissue. pH sensitive fluorescence dyes are quite
sensitive but applications of optical techniques in human
imaging is limited to tissues near the surface of skin where
sufficient light penetration can be achieved.[4] A pH respon-
sive T1-shortening contrast agent could offer a minimally in-
vasive and highly practical approach to mapping of tissue
pH so it is hardly surprising that a large number of pH sen-
sitive MRI contrast agents have been reported.[5–13]
[a] Dr. M. M. Ali, Dr. M. Woods, A. C. L. Opina, Prof. A. D. Sherry
Department of Chemistry, University of Texas at Dallas
P.O. Box 830660, Richardson, Texas 75083 (USA)
E-mail: dean.sherry@utsouthwestern.edu
sherry@utdallas.edu
[b] Dr. M. Woods
Macrocyclics, 2110 Research Row, Suite 425
Dallas, Texas 75235 (USA)
[c] Prof. P. Caravan
A.A. Martinos Center for Biomedical Imaging
Department of Radiology
Massachusetts General Hospital and Harvard Medical School
Charlestown, MA 02129 (USA)
[d] M. Spiller
Department of Radiology, New York Medical College
Valhalla, New York 10595 (USA)
[e] Dr. J. C. Fettinger
Department of Chemistry, University of California at Davis
One Shields Avenue, Davis, CA 95616-5298 (USA)
[f] Prof. A. D. Sherry
Advanced Imaging Research Center
University of Texas Southwestern Medical Center
5323 Harry Hines Boulevard, Dallas, Texas 75390 (USA)
Fax : (+1)214-645-2744
Typically, Gd3+-based complexes used to relax bulk water
have relatively low T1 relaxivities. Relaxivity is defined as
the increase in water proton relaxation rate per unit concen-
tration of contrast agent and is a measure of the effective-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800402.
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FULL PAPER
ness of a contrast agent. At typical magnetic fields used for
clinical imaging, the relaxivity of a Gd3+ complex is primari-
ly determined by three factors: q, the number of water mol-
ecules in the inner coordination sphere of Gd3+; tM, the resi-
dence lifetime of these water molecule on Gd3+; and tR, the
rotational correlation time, or how fast the complex tumbles
in solution.[14–18] Examples of contrast agents that respond to
pH through changes in each of these parameters have been
reported,[5] but the only agent actually used to image tissue
pH to date is Gd1 (see below).[12,13] Although Gd1 exhibits a
groups. The increase in relaxivity of Gd1 on passing from
pH 9 to 6 was found to be the result of a combination of
these two effects.[12]
Despite the demonstrated utility of Gd1, the maximum in-
crease in relaxivity with changing pH, Dr1, is relatively
modest, rising from 3.5 mmꢀ1 sꢀ1 at pH 9.5 to 5.3 mmꢀ1 sꢀ1 at
pH 6.3; a Dr1 of just 51%.[12] The relaxivity arising from an
exchanging inner-sphere water molecule, r1i.s., is given by
Equation (1), where tM is the lifetime of the inner-sphere
bound water molecule and T1M is the T1 of the inner-sphere
water protons. The relaxivity arising from water molecules
in the second hydration sphere follows a similar relationship.
From Equation (1) it can be seen that relaxivity will be
higher if T1M is shorter, but the rapid rotational dynamics of
Gd1 cause T1M to be long thereby limiting relaxivity.
ꢀ
ꢁ
1
q
i:s:
r1
¼
ð1Þ
55:6 T1M þ tM
Slowing molecular rotation is most easily achieved by conju-
gating the contrast agent to a macromolecule, such as a den-
drimer. However, improving relaxivity alone will not im-
prove the function of Gd1 as a pH responsive contrast
agent. To do that it is necessary to accentuate the difference
between the high and low relaxivity regimes. We hypothe-
sized that T1M could be reduced by conjugating Gd1 to a
dendrimer which would render the system more sensitive to
changes in tM. At higher pH values, the relaxivity would be
expect to be limited by slow exchange whereas at lower pH
values, catalysis of inner-sphere proton exchange by the
phosphonate groups should lift the limiting effect of ex-
change upon relaxivity (T1M > tM). Overall, coupling Gd1
to the dendrimer should improve both the absolute relaxivi-
ty, r1, and the change in relaxivity, Dr1, on going from low to
high pH. We therefore set out to conjugate Gd1 to a larger
macromolecule, in this case an ethylenediamine core G5-
PAMAM dendrimer, in order to reduce the rate of rotation
and improve the pH responsive characteristics of the com-
plex.
relatively small a change in relaxivity over a physiologically
relevant pH range, it has been used successfully in vivo to
generate pH maps of kidneys and tumors in small ani-
mals.[19–21] The mechanism by which Gd1 operates as a pH
responsive agent is unique among responsive Gd3+ agents
and stems from the presence of the phosphonate groups of
the pendant arms.[12] The sole inner-sphere water molecule
of Gd1 is in slow exchange with the bulk solvent which, or-
dinarily, would limit relaxivity. However, the phosphonate
groups of Gd1 are able to catalyse the exchange of the pro-
tons of this single Gd3+-bound water molecule with bulk sol-
vent protons. The effectiveness of the phosphonates at cata-
lysing proton exchange is dependent upon their protonation
state so as the four phosphonates become successively pro-
tonated at the pH falls below ꢁ8.5, the rate of proton ex-
change increases and the paramagnetic relaxation effects of
Gd3+ are transferred to the bulk solvent protons. In addition
the phosphonates can organise a number of other water
molecules into a second hydration sphere through hydro-
gen-bonding interactions.[22,23] The extent, and proton resi-
dence lifetime, of this second hydration sphere is also likely
to fluctuate with the protonation state of the phosphonate
Results and Discussion
Synthesis: As the phosphonate groups of the pendant arms
were found to be responsible for the pH responsive nature
of Gd1,[12] it was important to maintain this structural fea-
ture when the complex was modified to facilitate attachment
to the dendrimer. Accordingly, the complex was modified by
incorporating a functionalized benzyl group onto the macro-
cyclic backbone of the complex, leaving the four phospho-
nate groups intact. The functionalized complex Gd2 was
prepared following the same synthetic procedure used for
the preparation of Gd1[12] simply substituting (S)-2-(p-nitro-
benzyl) cyclen for cyclen (Scheme 1).
Compound 3 was prepared by the Michaelis–Arbuzov re-
action of triethylphosphite with bromomethyl phthalimide.
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A. D. Sherry et al.
Scheme 2.
effective the reaction.[27] Thus, if the product amine is signifi-
cantly more basic than hydrazine, it is likely to compete
with hydrazine in attacking the phthalimide 3 to yield signif-
icant quantities of compound 5.
The identity of compound 5 was established by NMR
spectroscopy and mass spectrometry and, although attempts
to remove compound 5 from the crude reaction mixture by
crystallisation were unsuccessful, compound 5 readily crys-
tallises once purified. X-ray quality crystals could be grown
at room temperature from a solution of 5 in dichlorome-
thane by addition of diethyl ether and hexanes. This allowed
the structure of compound 5 to be confirmed by X-ray crys-
tallography (Figure 1). The production of compound 5
Scheme 1. Synthesis of a functionalised pH responsive contrast agent. i)
(OEt)3/D; ii) H2NNH2/EtOH; iii) BrCH2COBr/K2CO3/CH2Cl2; iv) (S)-2-
(p-nitrobenzyl) cyclen/K2CO3/MeCN/608C; v) 30% HBr in AcOH.
P
ACHTREUNG
The key intermediate 4 was then obtained by removal of the
phthalimide protecting group by an Ing–Manske[24] reaction
with 1.2 equivalents of hydrazine in ethanol. Normally this
type of deprotection reaction proceeds in good yields;[24]
however, in our case the yields were moderate at best.[12]
Furthermore, the presence of significant quantities of a reac-
tion by-product meant that column chromatography was
necessary to purify amine 4, an oil at room temperature.
Amines can often be purified by an acid/base extraction but
4 shows good water solubility, even at high pH, and this pre-
cluded purification by extraction. Low yielding reactions
that require complicated purification procedures are unsuit-
able for the scale-up necessary for the production of large
quantities of an MRI contrast agent. We therefore under-
took an investigation into the reasons for the low reaction
yield of this step.
1H NMR analysis of the crude reaction mixture indicated
that the primary contaminant of the product 4 was a single
reaction by-product. This compound was isolated by column
chromatography and characterized. Rather surprisingly, this
by-product was identified as compound 5, the product of a
reaction between the starting material 3 and the intended
reaction product 4 (Scheme 2). A significant effort has been
applied to understanding the mechanism and intermediates
of the Ing–Manske reaction,[25–28] however, this particular re-
action pathway is rarely included in these discussions.[29] The
quantities of 5 produced suggest that this side reaction may,
on occasion, be more important than generally described.
The mechanism of the Ing–Manske reaction is quite com-
plex[26–29] but it is initiated by nucleophilic attack of hydra-
zine at one of the carbonyls of the phthalimide. The effec-
tiveness of an amine at removing phthalimide protecting
groups has been shown to relate to the protonation constant
of the attacking amine; the more basic the amine, the more
Figure 1. ORTEP rendering of the crystal structure of 5 showing 50%
ellipsoids. Hydrogen atoms have been omitted for clarity.
during the deprotection of the phthalimide 3 is doubly detri-
mental to the yield of 4 since two molecules of 4 are taken
up into one molecule of 5; this is in addition to the need for
chromatography. Thus, eliminating compound 5 from the re-
action would both improve yield and simplify purification
since the other reaction by-product, the insoluble phthalhy-
drazide, can be removed by filtration. A number of im-
provements to the Ing–Manske reaction have been suggest-
ed, most notably raising the reaction pH through addition of
sodium hydroxide.[26,27] However, concerns over the lability
of the phosphonate diester moiety towards base-catalysed
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hydrolysis precluded this as a solution to this problem. We
investigated a number of the other Ing–Manske reaction
conditions to assess whether the reaction conditions could
be improved.
1
Compounds 4 and 5 are easily distinguished by H NMR
spectroscopy; a shift difference of almost 1 ppm is observed
for the methylene resonance a to the phosphorous. This res-
onance appears as a doublet at 2.9 ppm (coupled to phos-
phorous) in 4 and as a doublet of doublets at 3.8 ppm (cou-
pled to phosphorous and the amide proton) in 5. A series of
experiments were performed in which various reaction con-
ditions were altered and the ratio of the two reaction prod-
1
ucts in the crude reaction mixture determined by H NMR
spectroscopy. Neither increasing the reaction temperature
from room temperature to reflux nor increasing the reaction
time from 6 h to 5 d was found to have a significant effect
upon the distribution of reaction products. However, when
the amount of hydrazine used in the reaction was increased
the amount of 4 obtained from the reaction was greatly in-
creased at the expense of 5. Increasing the amount of hydra-
zine from one to four equivalents greatly reduced the
amount of 5 produced in the reaction. At six equivalents of
hydrazine the yield of 5 was <5%, and ten equivalents of
hydrazine eliminated all traces of 5 from the reaction
1H NMR spectrum. This observation does not indicate that
5 is not produced during the reaction. Indeed, when an puri-
fied sample of 5 was treated with ten equivalents of hydra-
zine it was found to undergo a quantitative conversion to 4,
Scheme 3. Synthesis of the dendrimer-based pH responsive contrast agent
Gd11. i) H2/Pd on C/H2O; ii) SCCl2/CHCl3/H2O pH 2; iii) G-5 PAMAM
dendrimer/H2O pH 8; iv) 30% HBr in AcOH.
1
as determined by H NMR (Scheme 2). Thus, 5 may still be
and repulsion between conjugated and incoming phospho-
nates. The ethylenediamine core G5-PAMAM dendrimer se-
lected as the basis of our macromolecular construct has 128
primary amine groups on its surface. The dendrimer was re-
acted with 256 equivalents of 9 for 24 h at 408C followed by
a further 256 equivalents for 48 h. The reaction pH was
maintained at 9 throughout by addition of a 1m solution of
NaOH. The reaction was analysed by HPLC using a Phe-
nomenex BIOSEP SEC S-3000 size exclusion column (5–
700 kD, PBS buffer, pH 7.4). The chelate–dendrimer conju-
gate was purified by repeated diafiltration using a Centricon
C-30 membrane with a 30 kD cut-off (Millipore) until no
low molecular weight materials could be detected by HPLC.
HPLC analysis of the resulting dendrimer indicated that an
average hydrodynamic volume equating to a molecular
weight of about 140 kD was achieved. This corresponds to
an average of 75% coverage or 96 ligands per dendrimer. A
ligand/dendrimer coverage ratio of 97:1 was confirmed by
elemental analysis of the carbon and sulfur content of the
produced in the reaction but, importantly, it is reactive
under these conditions and does not persist as a reaction
product.
Changing the conditions of the Ing–Manske reaction by
increasing to ten the equivalents of hydrazine allowed pu-
rification to be simplified from column chromatography to
filtration to remove the phthalhydrazide. It also improved
the reaction yield to 97% and rendered this process suitable
for scale-up. Condensation of the amine 4 with bromoacetyl
bromide afforded the bromoacetamide 6 which was used to
alkylate (S)-2-(p-nitrobenzyl) cyclen in acetonitrile with
K2CO3 as base (Scheme 1). Subsequent deprotection of the
phosphonate esters of 7 with HBr in AcOH afforded the
ligand 2 a functionalized analogue of 1 that preserved the
integrity of the four phosphonate groups.
It was later found that conjugation of the bifunctional
ligand to the PAMAM dendrimer was more efficient if the
protected ester ligand 7 was used instead of the free acid.
Reduction of the nitro group with hydrogen and palladium
catalyst afforded the corresponding amine 8 in 72% yield
(Scheme 3). The amine was then converted to the isothio-
cyanate 9 by reaction with thiophosgene in a biphasic reac-
tion at pH 2. The isothiocyanate group is ideal for conjuga-
tion with primary amines, such as those that decorate the
surface of the PAMAM dendrimer, under mild conditions.
The phosphonate ester was preferred for this conjugation re-
action in order to minimize non-reactive salt formation be-
tween the amines of the dendrimer and phosphonic acids
1
conjugate. Similar loading values were obtained by H NMR
analysis of the aromatic and alkyl protons, however, the re-
producibility of loading values determined by this method
was poorer than SEC and combustion analysis. The conjuga-
tion reaction was performed in H2O, DMSO and mixtures
of the two, the extent of ligand/dendrimer ratio was found
to be unaffected by the choice of solvent. The phosphonate
esters of the conjugate 10 were finally removed under iden-
tical conditions to those used in the preparation of the
ligand 2, HBr and AcOH, to afford the conjugate 11.
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A. D. Sherry et al.
Formation of the gadolinium complexes of 2 and 11 also
requires special attention. With most DOTA–tetraamide li-
gands, this step is relatively simple; however, we have re-
cently reported that the phosphonate groups of 1 can inter-
fere with these complexation reactions[12] so care was taken
to perform the complexation reactions of both 2 and 11 at
pH 9 in order to ensure that the gadolinium ion was bound
by the macrocyclic coordination cage. In the case of ligand 2
equimolar amounts of ligand and gadolinium chloride hexa-
hydrate were reacted together at pH 9 and 608C in aqueous
solution. No further purification was undertaken. However,
1.2 equivalents of gadolinium chloride were used in the re-
action with 11 to ensure complete reaction of the ligands.
After 48 h at 408C and pH 9 in aqueous solution the excess
gadolinium was removed by addition of EDTA followed by
dialysis in water (12 kD molecular weight cut-off, Sigma Al-
drich). The conjugate Gd11 was then further purified by dia-
filtration with Centricon C-10 (10 kD cut-off) in water at
pH 7.4. Although addition of the Gd3+ ion into each ligand
of the conjugate would lead to a substantial increase in mo-
lecular weight, this increase would be expect to have little
or no effect upon the hydrodynamic volume of the conju-
gate. Thus, size exclusion HPLC was used to verify that the
apparent molecular weight remained near 140 kD.
(Figure 2); here the advantage of conjugating the low mo-
lecular weight chelate to a dendrimer is immediately appar-
ent. The relaxivity of Gd11 changes over the same physio-
logically relevant pH range as that of Gd1 and Gd2 but is
much higher on a per Gd3+ basis, rising from 10.8 mmꢀ1 sꢀ1
at pH 9.5 to 24.0 mmꢀ1 sꢀ1 at pH 6. (On a per molecule basis
relaxivity rises from 1037 mmꢀ1 sꢀ1 to 2304 mmꢀ1 sꢀ1.) This
equates to a relaxivity pH response, Dr1, of 122% on passing
from pH 9.5 to 6.0, more than doubling the Dr1 of Gd1 and
Gd2, Dr1 =51 and 59%, respectively, over the same pH
range. Although the pH profiles of Gd1 and Gd2 exhibit a
drop in relaxivity on passing below pH 6 the profile of Gd11
cannot be measured below this pH since the dendrimer con-
jugate precipitates from solution immediately below pH 5.9.
This is presumably the result of the high molecular weight
conjugate suddenly reaching its isoelectric point.
The difference in relaxivity between the “on” (pH 6) and
“off” (pH 9.6) states of the dendrimer-based pH responsive
agent was improved by more than a factor of 2 by slowing
molecular rotation. This should render the dendrimer-conju-
gate, Gd11, a more effective pH responsive agent for imag-
ing tissue pH. In order to assess the origins of this improve-
ment in pH responsive behaviour, nuclear magnetic reso-
nance dispersion (NMRD) profiles of Gd11 were recorded
at high and low pH values (Figure 3). At both pH 6.5 and at
pH 9.3, the relaxivity increased at lower temperatures indi-
Relaxometric studies: The relaxivity pH profile of Gd2 was
recorded and compared with that of Gd1 to ensure that the
introduction of the benzylic function on the macrocyclic ring
did not negatively impact the pH responsive properties of
the complex. The relaxivity pH profiles of Gd1 and Gd2
(Figure 2) are comparable, rising and falling to approximate-
ly the same relaxivity at approximately the same pH. The
only slight difference between the two profiles is that the re-
laxivity of Gd2 is slightly higher than that of Gd1 between
pH 4 and 6. This may be a reflection of slight changes in the
protonation constants of the phosphonates, but nonetheless
indicates that introduction of the nitrobenzyl substituent
does not negatively impact the behaviour of Gd2. Like Gd1
the relaxivity of Gd2 changes over a physiologically relevant
pH range. The relaxivity pH profile of Gd11 is also shown
[12]
^
)
*
*
Figure 2. Relaxivity pH profiles of Gd1 (
Gd2 ( ) and Gd11 ( ) re-
Figure 3. NMRD profiles of Gd11 recorded at pH 6.5 (top) and pH 9.3
corded at 20 MHz and 298 K. Relaxivity is expressed per Gd3+ ion.
(bottom); : 5, : 15, : 25, : 358C.
^
^ *
*
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Dendrimer-Based MRI Contrast Agents
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cating that the observed relaxivities are not limited by slow
water proton exchange between complex and bulk water.
Comparing the high field (1–100 MHz) regions of NMRD
profiles recorded at the same temperature we can see that
at higher pH (9.3) the curve is flatter and has a lower mag-
nitude than at lower pH (6.5). This indicates that at pH 9.3
the effective correlation time, tC, which is responsible for
modulating relaxation, is shorter than it is at pH 6.5.
Owing to the large number of ionizable phosphonate and
dendrimer amine groups on the dendrimer, multiple proton-
ated species must exist at each pH value. This complicates
any attempt to “fit” these NMRD profiles in a quantitative,
meaningful way. Nonetheless, it is useful examine the pa-
rameters that influence relaxivity in a qualitative sense in
order to probe which factors are responsible for the ob-
served behaviour. One may assume that each of the 96 Gd3+
chelates on the dendrimer surface has one water molecule
in its inner coordination sphere. In addition, the phospho-
nate groups of each pendant arm may form hydrogen-bond-
ing interactions with nearby water molecules forming a
second hydration sphere. The extent of this second hydra-
tion sphere is likely to vary as the phosphonates are proton-
ated or deprotonated with changing pH. Over a certain pH
range it has also been shown that these phosphonates can
catalyse exchange of protons from the coordinated water
molecule to the bulk solvent.[12] Finally there is an outer-
sphere contribution to relaxivity resulting from the diffusion
water molecules of the bulk solvent close by the slowly tum-
bling Gd3+ chelates. This outer-sphere effect depends pri-
marily on the rate of diffusion of water and is insensitive to
changes in pH.
Figure 4. Effect of proton residence lifetime (tM) on the relaxivity of
Gd3+-G5 PAMAM dendrimer conjugates in the fast (top) and slow
(bottom) exchange regimes. The simulated profiles are plotted at same
scale using parameters: tg =4.5 ns, tl =0.15 ns, S2 =0.5, tv =20 ps, D=2”
109 s.
Conjugating Gd3+ complexes to dendrimers is a common
strategy for slowing the rotational dynamics of paramagnetic
complexes.[30–37] In order to quantitatively describe the rota-
tional dynamics of these dendrimer systems, the Lipari–
Szabo approach is typically used.[38–43] This model employs
two correlation times; a long correlation time that defines
the global motion of the entire dendrimer conjugate, tg, and
a second shorter correlation time, tl, that reflects the local
motion of the metal complex about its point of attachment
to the dendrimer. This fast local motion is superimposed
upon the slower global motion of the dendrimer. For sys-
tems conjugated to G5-PAMAM dendrimers the global cor-
relation time, tg, has been reported on the order of 4–
5 ns.[42,44] The correlation time describing the local motion,
tl, of the complex range from 0.07–0.76 ns.[41–44] The relative
weighting of these two correlation times is given by an
order parameter, S2, that can range from 0 (where local
motion is dominant) to 1 (where isotropic, global motion is
dominant). For dendrimers modified with Gd3+ chelates S2
was found to range from 0.28–0.5.
1st or 2nd hydration sphere) and the bulk solvent is ex-
tremely fast (short tM) the high field NMRD profile is flat
and relaxivity low (Figure 4a). This is because these protons
have a low probability of being relaxed before they ex-
change back into the bulk solvent. At the other extreme,
very slow exchange that is, very long tM, the profile is also
flat and relaxivity low (Figure 4b) because these protons,
once relaxed by Gd3+, remain on the complex preventing
others from being relaxed. So in both cases relaxation is not
effectively transferred to the bulk solvent and the T1 of the
bulk remains long. Between these two extremes, where 1 ns
< tM < 1000 ns, the profile is characterized by higher relax-
ivities and a “hump” between 10 and 60 MHz (Figure 4).
The magnitude of the relaxivity in the profiles will depend
on these exchange kinetics, but also on the number of ex-
changeable protons and their distance from Gd3+. However,
these latter two parameters will not affect the shape of the
NMRD profile.
We assumed that similar rotational dynamics applied to
Gd11 and these parameters to simulate the high field region
(1–100 MHz) of an NMRD profile (Figure 4). Electron-spin
relaxation parameters were fixed at values similar to those
used elsewhere.[40–44] When the rate of proton exchange be-
tween water molecules associated with the complex (either
In order to mimic the flat, field independent behaviour
observed for Gd11 at pH 9.3, the proton residence lifetime,
tM, must be either very short (<1 ns) or very long (>1 ms).
The rate of water proton exchange is temperature depen-
dent; as the temperature is lowered tM becomes longer. This
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A. D. Sherry et al.
increase in tM applies to both water molecules in the inner
and second hydration spheres; however, it has an opposing
effect on the relaxivity of each. The water protons of the
inner-sphere water molecule of Gd1 were found to be in
slow exchange with the bulk,[12] and so these protons con-
tribute poorly to the overall relaxivity. In contrast, water
protons of a second hydration sphere are known to undergo
very rapid exchange that also limits their contribution to re-
laxivity.[22] Whereas making tM of the inner sphere longer
(lower temperature) will not result in an increase in relaxivi-
ty, making tM of the second sphere longer could bring these
protons into a range where they are able to contribute more
substantially to relaxivity (Figure 4a). Inspecting the NMRD
profile of Gd11 recorded at pH 9.3 and 358C, the high field
region is flat suggesting that exchange of inner-sphere water
protons is too slow and exchange of second sphere water
protons too fast for a relaxivity enhancement “hump” to be
observed at high field. As the temperature is taken down to
58C the profile begins to take on the appearance of a small
“hump” at high field. Clearly exchange from the inner-
sphere water protons will continue to be too slow to provide
a contribution to relaxivity at lower temperatures. The small
increase in relaxivity must therefore be the result of slowing
the exchange rate of second-sphere water protons into a
range that allows some contribution to relaxivity. This
second-sphere contribution to relaxivity is not observed for
either Gd1 or Gd2 because their rotational dynamics are
too rapid. Given the small size of the high field relaxivity
hump, exchange of second-sphere water molecules is likely
to be in the range of tM =0.5–5 ns.
Over a certain pH range, the phosphonate groups of the
pendant arms of the low molecular weight complexes Gd1
and Gd2, catalyse exchange of inner-sphere water protons
with the bulk solvent.[12] The fast rotational dynamics (tg
ꢁ0.1 ns) of these low molecular weight chelates limits their
relaxivity but conjugation to the dendrimer lifts this restric-
tion in Gd11 and so the observed relaxivity is higher by a
factor of almost 5. At pH 6.5 and 358C the NMRD profile
of Gd11 already has a slight high field “hump” which be-
comes more pronounced as the temperature is lowered.
From studies on Gd1, a contribution to this high field relax-
ivity from the inner-sphere proton exchange is expected.
However, a contribution from protons in the second hydra-
tion sphere is also apparent. This is most clearly seen when
the temperature is lowered. The tM of inner-sphere water
protons of Gd1 was found to be on the order of microsec-
onds[12] and so as the temperature is lowered the inner-
sphere relaxivity should decrease as exchange becomes in-
creasingly limited (cf. Figure 4b). The observed relaxivity in-
creases with decreasing temperature indicating that a sub-
stantial second-sphere component must be present (Fig-
ure 4a). The fact that the increase in high field relaxivity
with decreasing temperature is larger at pH 6.5 than it is at
pH 9.3 suggests that either the second hydration sphere is
larger or it is more ordered, leading to longer tM values, at
pH 6.3 than at 9.3. Gd11 would seem to be a rare example
of a system in which relaxivity is limited both by prototropic
exchange in the 2nd-sphere that is too fast and by water ex-
change in the inner-sphere that is too slow.
It is worth noting that a third factor may also play a role
in improving the relaxivity pH response of Gd11. Gd3+ com-
plexes that exhibit no pH response have been found to
behave as pH responsive agents once conjugated to
PAMAM dendrimers.[44] The origin of this phenomenon is
thought to be changes in the internal motion of the dendri-
mer itself as the pH changes. Protonation of amines within
the body of the dendrimer is believed to make the dendri-
mer more rigid making tR longer at lower pH. Thus the re-
laxivity of agents conjugated to these dendrimers has been
found to increase as the pH drops. It is likely that, in addi-
tion to the interplay of inner-sphere and second-sphere
water proton exchange rates, a third contribution to the pH
responsive behaviour of Gd11 arises from changes in the ri-
gidity of the dendrimer with changes in pH.
Conclusions
It is possible to take advantage of the interplay between mo-
lecular reorientation and water proton exchange kinetics to
enhance the response behaviour of complexes that exhibit
changes in relaxivity arising from changes in water or
proton exchange kinetics. Our initial hypothesis was that
coupling the pH-responsive agent Gd1 to a dendrimer
would increase both the overall relaxivity, r1, the responsive-
ness of relaxivity to pH, Dr1. This was achieved by slowing
rotation via conjugation to a dendrimer, increasing Dr1 by
more than a factor of 2. This enhancement should enable
Gd11 to serve as an effective pH responsive MRI contrast
agent. Furthermore, this dendrimer system has provided fur-
ther insights into the mechanism by which this class if pH
responsive agent, Gd1, Gd2 and Gd11, operates. The pH re-
sponse is the result of a complex interplay between the rate
of proton exchange between the bulk solvent and water
molecules in the inner and second hydration spheres. The
overall relaxivity is ultimately limited by the slow exchange
kinetics of protons in the inner hydration sphere and rapid
exchange kinetics of protons in the second hydration sphere.
From an imaging point of view the substantial improve-
ments in both r1 and Dr1 afforded by Gd11 should allow im-
proved determination of in vivo pH by MRI. However, it is
worth noting that improving Dr1 through increased molecu-
lar weight may also negatively impact the effectiveness of
such agents. Large molecules, such as dendrimers, remain in
vasculature longer than discrete agents, such as Gd1, which
are better able to diffuse into all extracellular space. Fur-
thermore, large molecules, such as Gd11, tend to clear more
slowly from the body as a result of increased liver uptake.
This extends the retention time of Gd3+ in the body. Further
studies into the in vivo behaviour of dendrimer-based MRI
contrast media will be required to establish if this approach,
which is successful for increasing both r1 and Dr1, will yield
agents that can actually be applied in vivo.
7256
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Chem. Eur. J. 2008, 14, 7250 – 7258

Dendrimer-Based MRI Contrast Agents
FULL PAPER
carbon (0.12 g) was added. The reaction mixture was shaken on a Parr
hydrogenation apparatus for 12 h under H2 (25 psi). The catalyst was re-
moved by filtration and the solvents removed by lyophilization to afford
the title compound as a colourless solid (0.35 g, 72%). ESI+: m/z (%):
1106 (65) [M+H]+, 1128 (100) [M+Na]+.
Experimental Section
General remarks: All reagents and solvents were purchased from com-
mercial sources and used as received unless otherwise stated. The ethyl-
ene core G5-PAMAM dendrimer from Dendritech was purchased
through Sigma-Aldrich as a 5% solution in methanol. Prior to use the
solvents were removed under vacuum and the dendrimer redissolved in
the reaction solvent. 1H NMR spectra were recorded on a JOEL Eclipse
270 spectrometer operating at 270.17 MHz, a Varian Mercury 300 spec-
trometer operating at 299.95 MHz and a Bruker Avance III spectrometer
operating at 400.13 MHz. 13C NMR spectra were obtained using a Bruker
Avance III spectrometer operating at 100.61 MHz. Longitudinal relaxa-
tion times were measured using the inversion recovery method on a
(S)-2-(p-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetamidomethylene-(diethyl)phosphonate (9): Amine
8
(0.50 g,
0.45 mmol) was dissolved in water (5 mL) and the pH of the resulting so-
lution adjusted to 2 by addition of a dilute HCl solution. Chloroform
(15 mL) was added to the reaction which was then stirred vigorously at
room temperature. Thiophosgene (0.052 mg, 0.45 mmol) was added to
the reaction which was then stoppered and stirred vigorously for 18 h at
room temperature. The reaction mixture was then transferred to a sepa-
ratory funnel and the chloroform layer was allowed to run off. The aque-
ous layer was then washed with chloroform (2”20 mL). The aqueous
layer was then collected and the solvents removed under reduced pres-
sure to afford the title compound as a colourless solid (0.48 g, 93%).
ESI+: m/z (%): 1148 (100) [M+H]+, 1170 (78) [M+Na]+.
ˇ
MRS-6 NMR analyzer from the Institut “Jozef Stefan”, Ljubljana, Slov-
enjia operating at 20 MHz. The pH of samples for relaxivity measure-
ments was adjusted by addition of either lithium hydroxide monohydrate
or p-toluenesulfonic acid monohydrate in order to avoid dilution. Melting
points were determined on a Fisher-Johns melting point apparatus and
are uncorrected. NMRD profiles between 0.01 and 50 MHz were record-
ed using the field cycling relaxometer at New York Medical Collge, Val-
hala NY.
(S)-2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacet-
amidomethylene phosphonic acid (2): Octaethyl ester
7 (0.75 g,
0.66 mmol) was dissolved in a 30% solution of HBr in acetic acid
(8 mL). The resulting solution was stirred at room temperature for 18 h.
The solvents were removed under reduced pressure and the residue
taken up in EtOH (20 mL), the solvents were again removed under re-
duced pressure. The solid residue was then taken up into MeOH (10 mL)
and the title compound precipitated by dropwise addition of Et2O. The
title compound was isolated by filtration, dissolved in water and the sol-
vents removed by lypholization to afford a tan solid (0.49 g, 81.4%).
Synthesis: The synthesis of diethyl phthalimidomethylphosphonate (3)
and diethyl bromoacetamidomethylphosphonate (6) have been reported
previously.[12]
Diethyl aminomethylphosphonate (4): Hydrazine (81.6 mL, 168.2 mmol)
was added to a solution of the phthalimide 3 (50.0 g, 1.68 mol) in dry eth-
anol (500 mL). The reaction was stirred at room temperature for 18 h.
The solvents were removed under reduced pressure and the residue
placed under high vacuum to remove as much excess hydrazine as possi-
ble. To the residue was added diethyl ether (750 mL) which was then fil-
tered to remove the precipitate. The precipitate was washed with diethyl
ether (2000 mL). The solvents were removed from the filtrate under re-
duced pressure to afford the title compound as a colourless oil (41.9 g,
97%). Characterisation data is identical to that reported previously.[12]
ESI+: m/z (%): 511 (100) [H4L+5Na]2+
.
G5-PAMAM dendrimer–ligand phosphonate diethyl ester conjugate
(10): G5-PAMAM dendrimer (40 mg, 1.4 mmol) was dissolved in DMSO/
water 1:1 (4 mL). Isothiocyanate 9 (0.41 g, 0.36 mmol) was added and the
pH of the resulting solution raised to 9 by addition of 1m NaOH solution.
The reaction was then stirred at 408C for 24 h with the pH maintained at
9 by addition of NaOH before a solution of 9 (0.41 g, 0.36 mmol) in
DMSO (1 mL) was added. The reaction was stirred for a further 48 h at
pH 9. The reaction mixture was then transferred to a Centircon C-30 dia-
filtration cell with a 30 kD molecular weight cut-off. Diafiltration was re-
peated until SEC-HPLC revealed that no further low molecular weight
material was present, the solvents were then removed by lyophylisation
to afford a colourless solid (0.11 g). Anal found C 28.63, S 1.34%, or
56.975 carbon atoms per sulfur atom. The ligand formula C44H81N9O16P4S
equates to 1 ligand per 12.975 carbon atoms of the dendrimer. The den-
drimer formula C1262H2528N506O252 affords a ratio 1262/12.975 or 97.26 li-
gands per dendrimer.
N’,N’’-Bis-(diethyl)methylphosphonate phthalamide (5): The title com-
pound was isolated as a by-product of the reaction used to synthesise
4.[12] X-ray quality crystals were grown by dissolving 5 (0.5 g) in a mini-
mum of dichloromethane in a 20 mL scintillation vial. Diethyl ether
(8 mL) was added and the components mixed thoroughly. Hexanes
(9 mL) were then added slowly such that a layer of hexanes lay on top of
the solution of 5. The vial was sealed and the layers were allowed to dif-
fuse together at room temperature, affording high quality crystals of 5.
M.p. 119–1208C; 1H NMR (400 MHz, CDCl3): d=1.21 (t, 12H, 3J-
(H,H)=7 Hz, CH3), 3.73 (dd, 4H, 2J
N
G
3
CH2P), 3.98 (q, 4H, J
OCH2), 7.36 (dd, 2H, 3J
3J(H,H)=6 Hz, 4J(H,H)=3 Hz, Ar), 7.70 ppm (dd, 2H, 3J
NH); 13C NMR (100 MHz, CDCl3): d=16.3 (3J
(C,P)=6 Hz, CH3), 35.2
(1J(C,P)=156 Hz, CH2P), 62.4 (2J
(C,P)=6 Hz, OCH2), 128.4 (3-Ar),
130.1 (2-Ar), 134.5 (1-Ar), 168.7 (3J
(C,P)=5 Hz, C=O); IR (KBr disc):
G
G5-PAMAM dendrimer–ligand phosphonic acid conjugate (11): Conju-
gate 10 (0.11 g) was dissolved in a 30% solution of HBr/glacial acetic
acid (3 mL) under an argon atmosphere. The reaction was stirred at
room temperature for 18 h. The solvents were then removed under re-
duced pressure and residue taken up into methanol (10 mL) which was
then also removed under reduced pressure. Dissolution and evaporation
of methanol was performed a further three times to removes as much
excess acetic acid as possible. The residue was then washed with diethyl
and vacuum dried to afford the title compound as a colourless solid
(0.09 g).
A
ACHTREUNG
E
A
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
nmax = 3251 (NH), 3063, 2984, 2931, 2910, 2236 (PO), 1661 (CO) 1595,
1539, 1479, 1444, 1369, 1319, 1217 (PO), 1163, 1098, 1023 (PO), 973, 786,
730 cmꢀ1; ESMS-: m/z (%): 463 (100) [MꢀH+]ꢀ; elemental analysis calcd
(%) for C18H30N2O8P2: C 46.6, H 6.5, N 6.0; found: C 46.1, H 6.4, N 5.9.
SEC-HPLC analysis: Size-exclusion HPLC analysis of 10 and Gd11 were
performed on a phenomenex BIOSEP SEC S-3000 size exclusion column
(5–700 kD) eluting with PBS buffer at pH 7.4. The column was standar-
dised using commercially available protein (globular) molecular weight
standards: cytochrome C (12.4 kD), carbonic anhydrase (29 kD), bovine
serum albumin (66 kD), g-globulins (160 kD), apoferritin (480 kD), dex-
tran blue (2000 kD).
(S)-2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacet-
amidomethylene-(diethyl)phosphonate (7): (S)-2-(p-Nitrobenzyl) cyclen
(0.48 g, 1.56 mmol) was dissolved in acetonitrile (20 mL). Potassium car-
bonate (1.45 g, 11.0 mmol) and bromoacetamide 6 (2 g, 6.94 mmol) were
added and the reaction mixture stirred for 72 h at 608C. The reaction
mixture was filtered and the solvents removed under reduced pressure.
The residue was purified by column chromatography over silica gel elut-
ing with 10% methanol in dichloromethane to afford the title compound
as pale yellow solid (0.53 g, 30%). ESI+: m/z (%): 1136 (35) [M+H]+,
1158 (100) [M+Na]+, 1174 (60) [M+K]+.
Crystal structure determination of 5
Data collection: A colourless irregular block with approximate orthogo-
nal dimensions 0.46”0.41”0.25 mm was placed and optically centered on
the Bruker SMART1000 CCD system (Bruker, SMART, Version 5.054
(2004) and SAINT, Version 7.23 A, Bruker AXS Inc.) at ꢀ1838C. The in-
itial unit cell was indexed using a least-squares analysis of a random set
(S)-2-(p-Aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacet-
amidomethylene-(diethyl)phosphonate (8): Nitro compound 7 (0.50 g,
0.44 mmol) was dissolved in water (15 mL) and 10% palladium on
Chem. Eur. J. 2008, 14, 7250 – 7258
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7257

A. D. Sherry et al.
of reflections collected from three series of 0.38 wide w scans, 10 seconds
per frame, and 25 frames per series that were well distributed in recipro-
cal space. Five w-scan data frame series were collected (MoKa) with 0.38
wide scans, 20 seconds per frame and 606 frames collected per series at
varying f angles (f=0, 72, 144, 216, 2888). The crystal to detector dis-
[10] M. Woods, G. E. Kiefer, S. Bott, A. Castillo-Muzquiz, C. Eshelbren-
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679.
tance was 4.123 cm, thus providing a complete sphere of data to 2qmax
60.488.
=
Structural determination and refinement: A total of 88185 reflections
were collected and corrected for Lorentz and polarization effects and ab-
sorption using Blessingꢁs method[45] as incorporated into the program
SADABS (Sheldrick, G.M., SADABS, Version 2.10 (2003), “Siemens
Area Detector Absorption Correction” Universität Gçttingen, Gçttin-
gen, Germany) with 13581 unique. The SHELXTL (G. M. Sheldrick,
SHELXTL, Version 6.1, 2002, Bruker AXS Inc.) program package was
implemented to determine the probable space group and set up the ini-
tial files. System symmetry, systematic absences, and intensity statistics in-
dicated the non-centrosymmetric orthorhombic space group Pna21 (no.
33). The structure was determined by direct methods with the successful
location of a majority of the molecule within the asymmetric unit using
the program XS (G. M. Sheldrick, SHELXS97 and SHELXL97, 1997),
which was also used to refine the structure. The data collected were
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merged based upon identical indices yielding 53260 data [R(int)=0.0267]
G
that were truncated to 2qmax =60.008 resulting in 51060 data that were
further merged during least-squares refinement to 13322 unique data [R-
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A
required to locate the remaining non-hydrogen atoms and optimize the
various disorders present for the two molecules in the asymmetric unit.
All full occupancy non-hydrogen atoms were refined anisotropically. Hy-
drogen atoms were idealized throughout the final refinement stages. The
final structure was refined to convergence with R(F)=6.55%, wR
12.39%, GOF=1.118 for all 13322 unique reflections [R(F)=5.72%,
wR
(F2)=12.02% for those 11744 data with Fo > 4s(Fo)]. The final dif-
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correct and complete. An empirical correction for extinction was also at-
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indicating that racemic twinning is present.
CCDC 680275 (5) contains the supplementary crystallographic data for
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Received: March 5, 2008
Published online: July 7, 2008
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Chem. Eur. J. 2008, 14, 7250 – 7258