[GdPCP2A(H2O)2]-: A paramagnetic contrast agent designed for improved applications in magnetic resonance imaging

Article abstract of DOI:10.1021/jm000983a

A novel ligand based on a pyridine-containing macrocycle bearing two acetic and one methylenephosphonic arms (PCP2A) has been synthesized. An efficient synthesis of PCP2A is based on the macrocyclization reaction between 2,6-bis(chloromethyl)pyridine and

Full text of DOI:10.1021/jm000983a

J . Med. Chem. 2000, 43, 4017-4024
4017
[Gd P CP 2A(H2O)2]-: A P a r a m a gn etic Con tr a st Agen t Design ed for Im p r oved
Ap p lica tion s in Ma gn etic Reson a n ce Im a gin g
Silvio Aime,*,† Mauro Botta,‡ Luca Frullano,† Simonetta Geninatti Crich,† Giovanni Giovenzana,|
Roberto Pagliarin,§ Giovanni Palmisano, Federico Riccardi Sirtori,§ and Massimo Sisti
Dipartimento di Chimica I.F.M., Universita` di Torino, via P. Giuria 7, I-10125 Torino, Italy, Dipartimento di Scienze e
Tecnologie Avanzate, Universita` del Piemonte Orientale “Amedeo Avogadro”, Corso Borsalino 54, I-15100 Alessandria, Italy,
Dipartimento di Chimica Organica ed Industriale, Universita` di Milano, Viale Venezian 21, I-20133 Milano, Italy,
Dipartimento di SCAFF, Universita` del Piemonte Orientale, V.le Ferrucci 33, I-28100 Novara, Italy, and Dipartimento Scienze
CC.FF.MM, Universita` dell’Insubria, Via Lucini 3, I-22100 Como, Italy
Received May 10, 2000
A novel ligand based on a pyridine-containing macrocycle bearing two acetic and one
methylenephosphonic arms (PCP2A) has been synthesized. An efficient synthesis of PCP2A is
based on the macrocyclization reaction between 2,6-bis(chloromethyl)pyridine and a 1,4,7-
triazaheptane derivative bearing a methylenephosphonate group on N-4. The Gd(III) complex
of PCP2A displays characteristic properties which make it a very promising contrast agent for
improved applications in magnetic resonance imaging. In fact it shows (i) a very high stability
constant (log KGdPCP2A ) 23.4) which should guarantee against the in vivo release of toxic free
Gd(III) ions and free ligand molecules and (ii) a relaxivity that is about 2 times higher than
the values reported for contrast agents currently used in the clinical practice. Its high relaxivity
is the result of the presence of two water molecules in the inner coordination sphere and a
significant contribution from water molecule(s) hydrogen bonded to the phosphonate group.
Moreover, the inner sphere water molecules are involved in an exchange with the bulk water
which is relatively fast. This property is important for the attainment of an even higher
relaxivity once the molecular reorientation rate of the [GdPCP2A(H2O)2]- moiety is lengthened
by means of conjugation to a macromolecular substrate.
Sch em e 1
In tr od u ction
Recent years have seen an increased interest in the
study of Gd(III) complexes aimed at providing more
efficient contrast agents for magnetic resonance imaging
(MRI) applications.1-3 The basic requirements that a
Gd(III) complex should have in order to be considered
for these applications are high thermodynamic stability,
good water solubility, low osmolality, and a marked
ability to enhance the relaxation rate of solvent-water
protons.4,5 The latter property is usually evaluated “in
vitro” and expressed in terms of relaxivity (r1p), which
corresponds to the relaxation enhancement of water
protons promoted by the paramagnetic complex at a 1
mM concentration. Established theory provides a basis
for understanding the relationships between the struc-
tural and dynamic properties of a given Gd(III) complex
and its relaxivity. It has been shown that, at the
imaging fields, high relaxivities can be obtained in the
presence of long reorientational times (τr) of the complex
allied to long electronic relaxation times (τs) and to
exchange lifetimes of the coordinated water (τM) of the
order of a few tenths of nanoseconds.1,6 The molecular
motion of the complex can be slowed by the formation
of covalent or noncovalent conjugates between the Gd
chelate and a slowly moving substrate such as a
protein,7 micelle,8 polyamino acid,9 polysaccharide,10 or
dendrimer.11 Thus the first step usually deals with the
identification of a Gd(III) chelate containing an ap-
propriate synthon which will be successively conjugated
to a macromolecular substrate to fully exploit its
potential to relax water protons. It is then extremely
important to find Gd(III) complexes displaying optimal
values for hydration number q, τM, and τs because the
amplification effect associated with the formation of the
macromolecular adduct strictly depends on the relaxo-
metric properties of the free complex.12
* To whom correspondence should be addressed. Tel: (+39)-11-
6707520. Fax: (+39)-11-670-7855. E-mail: aime@ch.unito.it.
† Universita` di Torino.
Along this strategy, herein we report a novel Gd(III)
chelate based on a pyridine containing a macrocyclic
ligand with two acetic and one methylenephosphonic
arms (PCP2A ) pyridine containing a triaza macrocyclic
‡ Universita` del Piemonte Orientale “Amedeo Avogadro”.
§ Universita` di Milano.
| Universita` del Piemonte Orientale.
Universita` dell’Insubria.
10.1021/jm000983a CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/27/2000

4018 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Aime et al.
Sch em e 2
phosphonic diacetic acid, Scheme 1). The heptadenticity
of the ligand should allow the coordination of two water
molecules (q ) 2) in the inner sphere of the metal and
consequently a higher relaxivity with respect to the Gd
complexes with octadentate ligands (q ) 1). Moreover,
it has been previously shown that Gd complexes with
ligands bearing phosphonate groups such as GdDOTP
(H8DOTP ) 1,4,7,10-tetrakis(methylenephosphonic acid)-
1,4,7,10-tetraazacyclododecane) or GdPCTP-[12] (H3-
PCTP-[12] ) pyridine containing a triaza macrocyclic
triphosphonic acid) display an additional contribution
to the observed relaxivity arising from second-sphere
water molecules.13 This additional contribution is a
consequence of the formation of hydrogen bonds be-
tween solvent molecules and the oxygens of the meth-
ylenephosphonic group. Thus the relaxivity of GdPCP2A
should receive a significant contribution also from water
molecule(s) in the second coordination sphere. Another
interesting property of the Gd(III) complexes containing
heptadentate ligands should deal with a relatively fast
water-exchange rate, with respect to complexes with
octacoordinating ligands. In fact, both heptacoordinated
GdDO3A14 (H3DO3A ) 1,4,7,10-tetraazacyclododecane-
1,4,7-triacetic acid) and GdPCTA14 show τM values
which are significantly shorter than those of octacoor-
dinated GdDTPA and GdDOTA.15,16 In the presence of
short water-exchange lifetimes it should be possible to
attain high relaxivity values because the molecular
reorientational time (τr) of the complex will be drasti-
cally elongated upon the formation of a macromolecular
adduct. One may envisage a synthetic route to obtain
macromolecule targeting contrast agents through the
introduction of suitable substituents17-19 on either the
acetate or the phosphonic methylenic carbons once it
has been proven that the metal complex satisfies the
expectations at the basis of its design.
troduction of the methylenephosphonate moiety on the
previously described20 3,6,9,15-tetraazabicyclo[9.3.1]-
pentadeca-1(15),11,13-triene-3,9-diacetic acid and (ii) by
a macrocyclization reaction between 2,6-bis(chloro-
methyl)pyridine and a 1,4,7-triazaheptane derivative
bearing a methylenephosphonate group on N-4 (Scheme
1).
According to route i, the functionalized macrocycle 2
was reacted with phosphorous acid and paraformalde-
hyde in the presence of hydrochloric acid.21 Even chang-
ing the molar ratios of the reactants or varying the
volume of the reaction mixture, the acidity of the
medium, the rate of paraformaldehyde addition, or the
overall reaction time does not allow the recovery of the
PCP2A ligand in more than 5% yield, after a tedious
and time-consuming purification workup. Route ii
(Scheme 2) proved to work better, affording the ligand
PCP2A in 45% overall yield.
The use of N-sulfonylaziridines in the synthesis of
diethylenetriamine derivatives is well-documented in
the literature.22-24 Diethyl aminomethylphosphonate
was envisaged as a good candidate to induce the ring
opening of aziridines, leading to a 1,4,7-triazaheptane
derivative bearing a methylenephosphonate group on
N-4. Thus, N-tosylaziridine was reacted with diethyl
aminomethylphosphonate25 in refluxing toluene afford-
ing 1,7-ditosyl-4-diethoxyphosphorylmethyl-1,4,7-triaza-
heptane (3) in 80% yield after crystallization from
toluene.
The presence of two electron-withdrawing groups on
N-1 and N-7 facilitates the macrocyclization reaction
between the derivative 3 and the commercially available
bis-electrophilic reagent, 2,6-bis(chloromethyl)pyridine,
using the experimental conditions previously reported:
26
i.e. potassium carbonate as a base in anhydrous
acetonitrile in heterogeneous conditions. Under these
conditions, the macrocycle 4 was obtained in 75% yield
after TLC purification. Following the Richman and
Atkins protocol,27 compound 4 was obtained in less than
30% yield.
Syn th esis of P CP 2A
The synthesis of the ligand PCP2A has been pursued
through two alternative approaches: (i) by direct in-

[GdPCP2A(H2O)2]-
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4019
F igu r e 2. pH dependence of the 31P NMR resonance of the
ligand PCP2A recorded at 161.9 MHz and 25 °C.
10.9) likely as a consequence of hydrogen bond forma-
tion between -HN-6-+ and PO32- groups. Since meth-
ylenic resonances 4/8, 2/10 display an analogous, al-
though smaller, downfield shift in the same pH range,
it is reasonable to suppose that some positive charge is
located also on N-3 and N-9. In the pH range 5-8 there
is a downfield shift of resonances 4/8, 16/18 coupled to
an upfield shift of signal due to protons 17. This
behavior is consistent with the occurrence of two pro-
tonation steps characterized by similar pKa values (pK4
) 6.7; pK3 ) 6.4). The first step affords a bis-protonated
species on N-3 and N-9 following a shift of the former
proton from N-6 to N-3 or N-9. The second step involves
the protonation of one phosphonic oxygen. The overall
protonation scheme is confirmed by recording the pH
dependence of the 31P NMR resonance (Figure 2). At
the more basic pH, the protonation at N-6 causes a large
upfield shift of the 31P resonance whose position is (in
part) recovered at pH 4-5 following the deprotonation
of N-6 and the protonation of one of the oxygens of the
phosphonate group. At pH < 4 the simultaneous shift
of the 31P and the protons 17 resonances is a conse-
quence of the protonation of the second oxygen on the
phosphonic group (pK2 ) 2.42), whereas the last observ-
able protonation (pK1 ) 0.72) involves one acetic arm
as shown from the downfield shift of the protons 16/18.
Syn th esis a n d Rela xom etr ic Ch a r a cter iza tion of
th e Gd P CP 2A Com p lex. The Gd(III) complex was
synthesized by adding stoichiometric amounts of Gd-
(III) chloride to the aqueous solution of the PCP2A
ligand while maintaining the pH of the solution at 7.5
with 1 M NaOH. Formation of the complex has been
followed by measuring the solvent proton relaxation rate
(1/ T1). The presence of an excess of free Gd(III) ions,
which yields a noticeable increase of the observed
relaxation rate, can be easily removed by forming the
insoluble hydroxide at basic pH followed by centrifuga-
tion of the resulting suspension.
F igu r e 1. pH dependence of the 1H NMR resonance of the
ligand PCP2A recorded at 400 MHz and 25 °C.
Ch a r t 1
Removal of the tosyl groups and hydrolysis of the
diethoxyphosphoryl moiety were accomplished by heat-
ing 4 with concentrated sulfuric acid.28 The crude
compound 5‚nH2SO4 was alkylated by reaction with
chloroacetic acid in the presence of sodium hydroxide
at 100 °C and maintaining the pH of the solution at a
value of about 10. After cooling to room temperature,
the reaction mixture was treated with concentrated
hydrochloric acid (pH ) 0). The residue obtained after
evaporation of the solvent (and further purification)
corresponds to the pure ligand PCP2A as established
by NMR and MS determinations.
Resu lts a n d Discu ssion
Micr oscop ic P r oton a tion Sequ en ce of P CP 2A. It
is well-established that, with polyamino carboxylate
ligands, it is possible to assess the sequence of proto-
nation by NMR spectroscopy.29 This is due to the fact
that protonation of a basic site results in a deshielding
of the resonance of the adjacent nonlabile protons in the
1H NMR spectrum. The plot of 1H chemical shifts of the
PCP2A ligand, as a function of the pH (Figure 1), shows
that the first H+ addition mainly involves the nitrogen
opposite the pyridine (N-6, pK5 ) 12.8). In fact, the
resonance corresponding to protons 17 (Chart 1) dis-
plays the largest downfield shift at pH > 10.5. The
substitution of an acetic with a methylenephosphonic
arm causes an increase of the basicity of this nitrogen
(N-6) with respect to the PCTA-[12]26,30 ligand (pK1 )
The relaxivity (r1p) of GdPCP2A, measured at 20 MHz
and 298 K, is 8.3 mM-1 s-1. This value is significantly
higher than those reported for Gd complexes of similar
size and based on heptadentated ligands such as
GdDO3A and GdPCTA (r1p ) 6.0 and 6.9 mM-1 s-1
,
respectively).4 To characterize the different contribu-
tions to the observed relaxivity, the 1/ T1 NMRD profile
over an extended range of frequencies at 25 °C has been
measured (Figure 3). Fitting of the NMRD profile was
carried out by considering three contributions to the
observed relaxivity:31

4020 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Aime et al.
obs
1p
1st
1p
2nd
1p
os
1p
R
) R + R
+ R
(1)
1st
Where R represents the contribution from the water
1p
2nd
1p
molecule(s) directly coordinated to the metal ion, R
is the contribution from water molecules in the second
os
1p
coordination sphere, and R arises from the water
molecules that diffuse in the proximity of the paramag-
os
1p
netic complex. The outer sphere R term is rather
constant for different small-sized Gd complexes and at
20 MHz and 25 °C corresponds to ca. 2-2.5 mM-1 s-1 6
.
1st
1p
2nd
1p
As far as R and R
are concerned, both contribu-
tions can be evaluated on the basis of the Solomon-
Bloembergen-Morgan theory:
q
F igu r e 3. 1/T1 NMRD profiles of a 1 mM GdPCP2A solution
at pH 7 and 25 °C. The dashed curve through the data points
was calculated with the parameters reported in Table 1. The
lower solid line represents the outer sphere water contribu-
tions, whereas the upper dotted line corresponds to the sum
of second and outer sphere contributions.
R1p
)
(2)
55.5(T1M + τM)
1
k
r6
f(τc)
(3)
(4)
T1M
-1
-1
τc-1 ) τr-1 + τs + τM
Ta ble 1
2
∆
τv
τr
τM
∆Hm
∆Hv
complex
q
s-2/1019 (ps) (ps) (ns) (kJ mol-1
3.5a 22a 68a 60b 26b
)
(kJ mol-1
)
where q is the number of water molecules in the first
or second coordination layers of the Gd ion and T1M is
the longitudinal relaxation time of their protons which
is determined by the distance from the paramagnetic
center, r, and by the shorter of the three correlation
times, τr being the molecular reorientational time, τs the
electronic relaxation time, and τM the exchange lifetime
for the inner and second coordination layer waters,
respectively.
GdPC2AP
2
10b
a
Best-fitting parameters obtained from the analysis of the
NMRD profile by considering two inner sphere water molecules
(q) whose protons are at an average metal distance of 3 Å and one
second sphere water molecule whose protons are at a metal
b
distance of 3.9 Å. 17O NMR best-fitting parameters obtained
from the analysis of the temperature dependence of R2p for a 2.7
mM solution of GdPCP2A by using a Gd-17O scalar coupling
constant of -3.8 × 106 rad s-1 and a Gd-17O distance of 2.5 Å.
Table 1 shows the relaxometric parameters deter-
mined by fitting the NMRD profile to eqs 1-4 and to
Freed’s equation32 for the outer sphere term. On the
basis of the results obtained from the fitting procedure,
this Gd complex possesses two water molecules in the
inner coordination sphere whose protons are at a
distance of 3 Å from the Gd ion, similar to what was
previously found for the related compound with three
acetate arms (GdPCTA). The hindrance of only one
phosphonate group on the coordination cage does not
cause a reduction of the number of the metal-bound
water molecules as observed in the case of GdPCTP-
[12] which contains three phosphonate moieties. The
second sphere water molecules contribute ca. 10-15%
(at 20 MHz) to the overall relaxivity, but from the
available data we cannot determine their distance
without fixing their number. In Table 1 we report a
distance of 3.8 Å from the Gd(III) center obtained by
imposing the presence of only one water molecule bound
to the phosphonate group through a hydrogen bond. The
other relaxometric parameters determined for GdPCP2A
are very similar to those reported for related complexes
containing heptadentate ligands14 and q ) 2.
Figure 4 shows the r1p values measured at 20 MHz
and 25 °C as function of the pH of the solution. The
values of the observed relaxivities are constant in the
pH range 3.0-9.5. The increase in r1p observed at pH’s
lower than 3.0 caused by an increased hydration of the
resulting species may reflect effects associated with the
protonation of the phosphonate group. As previously
found14 for other Gd complexes with q > 1 (GdDO3A,
GdPCTA) at basic pH (>10), the relaxivity of the
GdPCP2A decreases by ca. 1.4 r1p units. This behavior
can be attributed to the formation of ternary complexes
with dissolved carbonate ions. This hypothesis has been
confirmed by measuring the pH dependence of the
relaxivity in a closed cell under N2 atmosphere and by
using CO2-free KOH (Figure 5). Under these conditions
r
1p remains rather constant up to pH values of 11.5. The
decrease of relaxivity observed at higher pH values is a
consequence of the partial displacement of the coordi-
nated water molecules by OH-.
In order to determine the exchange lifetime of the
coordinated water molecules, transverse 17O NMR
relaxation times of an aqueous solution of GdPCP2A as
a function of the temperature have been measured. In
fact, it is well-established that water 17O-R2p data fitted
to the values calculated on the basis of Swift-Connick
equations yield good estimates of this exchange lifetime
(τM).16 Figure 6 shows the 17O-R2p profile of a 2.7 mM
GdPCP2A solution recorded at 9.4 T and pH 7. It shows
a maximum of R2p at low temperature (ca. 280 K) typical
of a Gd complex with short τM values. At ambient
temperature the exchange lifetime obtained from the
analysis of the variable temperature profile (Table 1)
is ca. 60 ns, considering two water molecules in the
inner coordination sphere. It is interesting to note that
the substitution of an acetate arm with a phosphonate
one on the pyridine-containing macrocycle causes only
a slight effect on the exchange lifetime τM. In fact, the
GdPCTA-[12] complex shows a τM value of 70 ns.14 On
the other hand in the three-phosphonate derivative
(GdPCTP-[12]) only one, fast exchanging water molecule
has been found. Likely the short τM value (6 ns)14 found
in GdPCTP-[12] may be accounted for in terms of the

[GdPCP2A(H2O)2]-
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4021
F igu r e 4. pH dependence of the longitudinal water proton
relaxivity (r1p) for a 1 mM solution (20 MHz, 25 °C) of
GdPCP2A.
F igu r e 6. Temperature dependence of the transverse water
17O relaxation rate at 9.4 T and pH 7 for a 2.7 mM solution of
GdPCP2A. The solid curve through the data points was
calculated with the parameters reported in Table 1.
F igu r e 5. pH dependence of the longitudinal water proton
relaxivity (r1p) for a 1 mM solution of GdPCP2A with (b) or
without (O) carbonate.
F igu r e 7. Curves represent two NMRD profiles calculated
for immobilized GdDOTA (dotted line) and GdPC2AP systems.
τr has been set equal to 3 × 10-8 s. The other parameters used
for GdDOTA were taken from ref 4, whereas parameters for
GdPCP2A were taken from Table 1.
occurrence of an associative mechanism for the ex-
change of the coordinated water in this octacoordinated
complex. Thus, upon replacing all of the acetate arms
with the isosteric methylenephosphonic substituents,
there is a decrease in the hydration state of the
lanthanide ion analogous to what was observed on going
from the DOTA to DOTP complexes.
In summary, the presence of two water molecules in
the inner coordination sphere, which exchange faster
than in systems with q ) 1, coupled to a sufficiently
long electronic relaxation time make GdPCP2A a very
promising candidate for the attainment of very high
relaxivities upon conjugation to macromolecular sub-
strates.
The exchange lifetime τM of the GdPCP2A appears
short enough to avoid any quenching of the relaxivity
enhancement expected once this moiety would be en-
dowed with a long reorientation time (τr). To support
the latter statement, a NMRD profile has been calcu-
lated using the parameters reported in Table 1 but
whose τr value has been set equal to 3 × 10-8 s (the
reorientation time commonly assumed for human serum
albumin). As shown in Figure 7, the calculated profile
shows a relaxivity peak of ca. 120 mM-1 s-1 at the
imaging fields. For comparison, in the same figure, also
reported is an analogous NMRD profile calculated for
an analogously immobilized GdDOTA system which
shows a maximum relaxivity of 45 mM-1 s-1. Values of
such magnitude have already been experimentally
obtained in several systems based on macromolecular
conjugates of DOTA-like systems.17-19 The “quenching”
of the expected relaxation enhancement often shown
upon binding to a DOTA-like Gd complex (slowly moving
system) has been associated with the occurrence of a
long residence lifetime of the coordinated water (i.e. T1M
< τM in eq 2).
Deter m in a tion of th e Th er m od yn a m ic Sta bility
Con sta n t of th e Gd Com p lex. As anticipated in the
Introduction, one of the important requisites that a Gd
complex must have in order to be considered for in vivo
MRI applications is represented by a high stability
constant. In fact this is the requisite which is expected
to limit toxicity problems related to the release of free
Gd(III) ions and free ligand molecules as well. Deter-
mination of the GdPCP2A thermodynamic stability
constant has been carried out using an NMR method.33-35
This method is based on the measurement of the
relaxation rate (Ro1bs) of GdL aqueous solutions in the
presence of variable amounts of a ligand L′ which is able
to form a complex of similar stability but endowed with
a different relaxivity. The observed relaxation rates of
these solutions are then functions of the speciation of
Gd(III) complexes. The concentration of the various

4022 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Aime et al.
KL ) [KL′RL(CTL′r1pGdL × 103 - CTL′r1pGdL′
×
species present in these solutions depends on the
following equilibria:
103 + Ro1bs - r1pGdLCTM × 103 - 0.38)(CTMr1pML′
103 + Ro1bs - 0.38)]/[RL′(Ro1bs - r1pGdL × 103CTM
×
Gd + L a GdL
Gd + L′ a GdL′
-
0.38)(-CTLr1pGdL × 103 + CTLr1pML′ × 103 - r1pML′
×
103CTM + Ro1bs - 0.38)] (13)
GdL + H a GdHL
GdL′ + H a GdHL′
DTPA (log KGdDTPA ) 22.46, r1p ) 4.7 mmol-1 s-1at
25 °C and 20 MHz) was particularly suitable as a
competitive ligand for the determination of the GdPCP2A
stability constant. The solutions containing variable
GdPCP2A/DTPA ratios were allowed to equilibrate for
4-5 days at 50 °C before the relaxivity measurements
were carried out. Insertion of the observed Ro1bs data
into eq 13 yielded a log KGdPCP2A ) 23.4. The high
stability constant value found for this complex with
respect to other heptadentate ligands such as GdDO3A4
or GdPCTA26 (both showing a log KGdL ) 21) could be
ascribed to the presence of the methylenephosphonic
arm on the pyridine-containing tetraza macrocycle. This
observation is confirmed by an analogous difference
found between the stability constant of GdDOTP and
GdDOTA.
Another “in vitro” experiment which may anticipate
possible toxicological problems associated with the
release of Gd(III) ions is provided by the measurement
of the relaxivity of GdPCP2A in a 3 mM phosphate
buffer solution (pH ) 7; 25 °C). In fact, the decrease in
the relaxivity, observed when a Gd complex is dissolved
in a phosphate buffer with respect the pure water
solution, may be accounted for the precipitation of
GdPO4. The relaxivity for the GdPCP2A does not change
in the presence of phosphate ions, after 3 days; this
result anticipates its good stability in serum. On the
contrary, as shown by Rongved et al.,36 the relaxivity
of the less stable GdEDTA measured under the same
conditions is significantly lower with respect to that
observed in the absence of the competitive phosphate
ions.
Hn-1L + H a HnL
Hn-1L′ + H a HnL′
each equilibrium being characterized by the relative
constants KGdL, KGdHL, KGdL′, KGdHL′, KH L, KHL, KH
,
nL′
n
KHL′, etc. Working at pH ≈ 8 it has been possible to
neglect the formation of the species GdHL and GdHL′.
In fact, protonation of Gd complexes with heptadentate
or octadentate polyamino carboxylate ligands occurs
only at pH < 4. The pH dependence of the relaxivity of
the GdPCP2A complex gives support to this approxima-
tion. Figure 4 shows that the formation of the proto-
nated complex which is characterized by higher relax-
ivity occurs only at pH < 3.
On the basis of these assumptions, the system is
simply described by the following set of equations:
CTL ) [GdL] + [L]RL
(5)
(6)
CTL′ ) [GdL′]‚ + [L′]RL′
CTGd ) [GdL] + [GdL′]
(7)
(8)
R1obs ) r1pGdL[GdL] × 103 + R1w
Con clu sion s
[GdL]
[GdL′]
KGdL
)
; KGdL′
)
(9, 10)
GdPCP2A possesses a number of favorable properties
which make it a very promising complex in the develop-
ment of the field of contrast agents for MRI. First of
all, it displays one of the highest stability constants until
now reported for a Gd complex. This high stability is
strongly suggestive that no in vivo release of toxic free
Gd(III) ions and free ligand will occur. Next, its relax-
ivity is 2 times higher than those shown by the contrast
agents currently used in clinical practice (Figure 8). This
remarkable capability to relax water protons is the
result of the collective effects underlying the design of
GdPCP2A, namely (i) the presence of two water mol-
ecules in the inner coordination sphere; (ii) a nonneg-
ligible contribution from water molecule(s) in the second
coordination sphere thanks to the ability of the phos-
phonate group to form hydrogen bonds; and (iii) the
outer sphere contribution. Moreover GdPCP2A displays
a fast exchange of the coordinated water molecules, and
this property strongly suggests its use as a paramag-
netic synthon for conjugation to a macromolecular
substrate in order to obtain high relaxivities. There is
a clear need of systems endowed with powerful relaxo-
[Gd][L]
[Gd][L′]
where CTL, CTL′ and CTGd represent the total concentra-
tions of the ligand L, ligand L′, and metal ion, respec-
tively; RL and RL′ are given by:
RL ) 1 + KHL[H] + KHLKH L[H]2 +
2
KHLKH LKH L[H]3... (11)
2
3
RL′ ) 1 + KHL′[H] + KHL′KH L′[H]2 +
2
KHL′KH L′KH L′[H]3... (12)
2
3
In the calculation of RL and RL′ the PCP2A protonation
constants determined by NMR were used. r1pGdL and
r1pGdL′ are the millimolar relaxivities of the two Gd
complexes (mmol-1 s-1) and R1w is the diamagnetic
contribution to the observed relaxivity (0.38 s-1 at 20
MHz and 25 °C). By working out this set of equations
the final expression of the unknown stability constant
is:

[GdPCP2A(H2O)2]-
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4023
(AA’BB’ system, 4H), 6.03 (m, 2H, exchangeable with D2O),
3
3
4.16 (dq, 4H, J ) 7.3 Hz, J H-P ) 8.1 Hz), 2.93 (m, 4H), 2.81
(d, 2H, 2J H-P ) 9.9 Hz), 2.68 (m, 4H), 2.42 (s, 6H), 1.34 (t, 6H,
3J ) 7.3 Hz). 13C NMR (CDCl3): δ 142.9 (C), 137.0 (C), 129.5
2
(CH), 127.0 (CH), 62.3 (d, CH2, J C-P ) 6.9 Hz), 55.3 (d, CH2,
1
3J C-P ) 6.3 Hz), 49.6 (d, CH2, J C-P ) 165.1 Hz), 40.9 (CH2),
3
21.3 (CH3), 16.3 (d, CH3, J C-P ) 4.8 Hz). MS (EI): m/z 562
(M). Anal. (C23H36N3O7PS2) C: calcd, 49.18; found, 49.05. H:
calcd, 6.46; found, 6.56. N: calcd, 7.48; found, 7.52.
6-D ie t h o x y p h o s p h o r y lm e t h y l-3,9-d it o s y l-3,6,9,15-
tetr a a za bicyclo[9.3.1]p en ta d eca -1(15),11,13-tr ien e (4). To
a solution of 2,6-bis(chloromethyl)pyridine (5.14 g, 29.2 mmol),
in anhydrous acetonitrile (150 mL), compound 3 (14.89 g, 26.5
mmol) and potassium carbonate (14.00 g, 101.3 mmol) were
added and the mixture was refluxed under vigorous stirring
and nitrogen atmosphere. After 16 h (TLC, ethyl acetate as
eluant) the solvent was evaporated and the residue dissolved
in methylene dichloride (100 mL). The mixture was filtered
through Celite; the organic layer was washed with aqueous 2
N sodium hydroxide (40 mL), water (40 mL) and dried over
anhydrous sodium sulfate. After evaporation of the solvent,
the residue (17.6 g) was purified by flash silica gel chroma-
tography [ethyl acetate f ethyl acetate/methanol (9:1) as
eluant] to give pure compound 4 (14.52 g, 21.8 mmol, 75%) as
F igu r e 8. Comparison among the relaxivity (25 °C, 20 MHz)
shown by commercial Gd-based contrast agents (GdDTPA:
Magnevist, Shering; GdDOTA: Dotarem, Guerbet; GdDTPA-
BMA: Omniscan, Nycomed-Amersham; GdHP-DO3A: Pro-
Hance, Bracco) and GdPCP2A.
1
a white foam after high vacuum removal of the volatiles. H
NMR (CDCl3): δ 7.72 (AA’BB’ system and t, 5H, 3J ) 8.2 Hz),
7.33 (AA’BB’ system and d, 6H, 3J ) 8.2 Hz), 4.44 (s, 4H), 4.11
metric characteristics to be used as tracers to visualize
small tumor lesions37 and as receptors in neo-angiogen-
esis38 processes and, finally, to report successful trans-
fection in gene therapy.39
3
(dq, 4H, 3J ) 7.3 Hz, J H-P ) 8.0 Hz), 3.20 (m, 4H), 2.84 (d,
2
3
2H, J H-P ) 9.8 Hz), 2.54 (m, 4H), 2.45 (s, 6H), 1.33 (t, 6H, J
) 7.3 Hz). 13C NMR (CDCl3): δ 154.8 (C), 143.3 (C), 138.4 (C),
135.6 (CH), 129.9 (CH), 127.3 (CH), 123,7 (CH), 61.8 (d, CH2,
2J C-P ) 6.9 Hz), 54.3 (CH2), 52.8 (d, CH2, 3J C-P ) 5.3 Hz), 51.0
Exp er im en ta l Section
1
(d, CH2, J C-P ) 162.0 Hz), 44.5 (CH2), 21.3 (CH3), 16.3 (d,
1H and 13C NMR spectra were obtained on Brucker AC 200
(200 and 50.2 MHz, respectively) and J EOL EX-400 (400 and
100.4 MHz) spectrometers. 31P NMR spectra were run on
Varian XL200 (81.0 MHz) and J EOL EX-400 (161.9 MHz)
spectrometers. The NMR pH titration was made up in D2O
(99.8%), and the pD was adjusted with DCl or CO2-free NaOD
(Sigma). Mass spectra were recorded with a VG 7070 EQ
spectrometer (at 70 eV; m-nitrobenzyl alcohol as matrix in the
FAB+ ionization). Elemental analyses were performed with a
Perkin-Elmer 240 apparatus. Melting points were determined
with a Bu¨chi 520 apparatus and are uncorrected.
The longitudinal water proton relaxation rate was measured
on the Stelar Spinmaster spectrometer (Stelar, Mede (PV),
Italy) operating at 20 MHz, by means of the standard inver-
sion-recovery technique (16 experiments, 2 scans). A typical
90° pulse width was 3.5 µs and the reproducibility of the T1
data was (0.5%. The temperature was controlled with a Stelar
VTC-91 air-flow heater equipped with a copper-constantan
thermocouple (uncertainty of (0.1 °C). The NMRD profile was
measured on a continuum of magnetic field strength from
0.00024 to 0.24 T (corresponding to 0.01-10 MHz proton
Larmor frequency) on a Spinmaster FFC, fast field cycling
NMR relaxometer (Stelar, Mede (PV), Italy) installed at the
LIMA Laboratory (Bioindustry Park, Ivrea (TO), Italy). Vari-
able-temperature 17O NMR measurements were recorded on
a J EOL EX-400 (9.4 T) spectrometer, equipped with a 5-mm
probe, using D2O as external lock. Experimental settings
were: spectral width 10880 Hz, pulse width 7 µs, acquisition
time 10 ms, 256 scans, no sample spinning. Solutions contain-
ing 2.6% of 17O isotope (Yeda, Israel) were used. The observed
transverse relaxation rates (RO2obs) were calculated from the
signal width at half-height.
CH3, 3J C-P ) 5.4 Hz). MS (EI): m/z 665 (M). Anal. (C30H41N4O7-
PS2) C: calcd, 54.20; found, 54.15. H: calcd, 6.22; found, 6.30.
N: calcd, 8.43; found, 8.49.
3,9-Bis(car boxym eth yl)-6-dih ydr oxyph osph or ylm eth yl-
3,6,9,15-tetr aazabicyclo[9.3.1]pen tadeca-1(15),11,13-tr ien e‚
4HCl (P CP 2A). Compound 4 (2.98 g, 4.5 mmol) was dissolved
in concentrated sulfuric acid (15 mL) at 80 °C. The mixture
was heated to 200 °C in 9 min and left at this temperature for
1 more min. The resulting brown solution was cooled to 0 °C
and diethyl ether (60 mL) was added. The precipitate was
filtered and washed with diethyl ether (4 × 20 mL). After high
vacuum desiccation, compound 5‚nH2SO4 (1.45 g) was submit-
ted to the next step without further purification. 1H NMR
spectrum, registered in D2O, revealed the complete absence
of the tosyl group resonances. To a solution of compound 5‚
nH2SO4 (1.45 g) and chloroacetic acid (1.42 g, 15 mmol) in
water (25 mL) aqueous 15% sodium hydroxide (14 mL) and 2
drops of phenolphthalein were added. The reaction mixture
was heated at 100 °C for 24 h, maintaining a pH ) 10
(persistent pink color of phenolphthalein) with sodium hy-
droxide. The solution was then cooled to room temperature
and acidified with 4 N hydrochloric acid until pH ) 0. The
solvent was evaporated and the residue purified by column
chromatography on XAD 1600 [water f water/methanol (7:
3)]. PCP2A‚4HCl was obtained as a white solid after evapora-
tion of the solvent [mp ) 220 °C dec, 1.52 g, 2.7 mmol, 60%
1
3
overall yields from 4]. H NMR (D2O): δ 7.78 (t, 1H, J ) 8.0
3
Hz), 7.26 (d, 2H, J ) 8.0 Hz), 4.64 (s, 4H), 3.90 (s, 4H), 3.36
(m, 4H), 2.82 (m, 4H), 2.73 (m, 4H, 2J H-P ) 10.0 Hz). 13C NMR
(D2O): δ 172.9 (C), 153.1 (C), 144.3 (CH), 125.4 (CH), 61.4
1
(CH2), 59.1 (CH2), 55.7 (CH2), 54.5 (CH2), 52.5 (d, CH2, J C-P
) 147.6 Hz). 31P NMR (D2O): δ 22.6. MS (FAB+): m/z 439 (M
+ Na)+, 417 (M + H)+. Anal. (C16H29N4O7Cl4P) C: calcd, 34.18;
found, 34.31. H: calcd, 5.20; found, 5.11. N: calcd, 9.96; found,
9.89.
All chemical were of analytical grade and were purchased
from Sigma-Aldrich (St. Louis, MO).
4-Dieth oxyp h osp h or ylm eth yl-1,7-d itosyl-1,4,7-tr ia za -
h ep ta n e (3). A solution of diethyl aminomethylphosphonate
(6.80 g, 40.6 mmol) and N-tosylaziridine (20.32 g, 103.1 mmol)
in dry toluene (200 mL) was refluxed for 6 h while monitoring
by TLC on silica gel (ethyl acetate/methanol 9:1). The solvent
was evaporated under vacuum and the residue was crystallized
from toluene to afford pure 3 (mp 125 °C, 18.25 g, 32.5 mmol
80% yield). 1H NMR (CDCl3): δ 7.79 (AA’BB’ system, 4H), 7.31
Ack n ow led gm en t. Financial support from Bracco
S.p.A. (Milano), Italian CNR (P.F. Biotecnologie and
Programma Biotecnologie L. 95/95), and MURST (40%)
is gratefully acknowledged. The research has been
carried out under EU-COST-D18 Action.

4024 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Aime et al.
(22) Cernerud, M.; Skrinning, A.; Bergere, I.; Moberg, C. Bis-
(ethylsulfonamide)amines via nucleophilic ring-opening of chiral
aziridines. Application to Ti-mediated addition of diethylzinc to
benzaldehyde. Tetrahedron: Asymmetry 1997, 20, 3437-3441.
(23) Espana, E. G.; Micheloni, M.; Paoletti, P. Synthesis of the new
bis-azamacrocycle 1,2-bis(1,4,8,12-tetraazacyclotetradecanyl)-
ethane and of its dicopper(II)complex. Gazz. Chim. Ital. 1985,
115, 399-340.
(24) Hata, K.; Doh, M. K.; Kashiwabara, K.; Fujita, J . Preparation
and absorption and circular dichroism spectra of cobalt(III)
complexes with n,n,n’,n’-tetrakis(2-aminoethyl)-1,2-ethanedi-
amine, -1,3-propanediamine, -1,4-butanediamine, and -(R,R)-
and -(R,S)-pentanediamine. Bull. Chem. Soc. J pn. 1981, 54,
190-195. Gauss, W.; Moser, P.; Schwarzenbach, G.; N, N,N’,N’-
Tetrakis-(â-aminoa¨thyl)-a¨thylendiamin. Helv. Chim. Acta 1952,
35, 2359-2363.
(25) Ferrari, M.; J ommi, G.; Miglierini, G.; Pagliarin, R.; Sisti, M.
Asymmetric synthesis of diethyl R-amino-R-alkyl-phosphonates
by alkylation of the chiral Schiff base derived from (+)-ketopinic
acid and diethylaminomethyl phosphonate. Synth. Commun.
1992, 22, 107-123.
(26) Aime, S.; Botta, M.; Geninatti Crich, S.; Giovenzana, G. B.;
J ommi, G.; Pagliarin, R.; Sisti, M. Synthesis and NMR studies
of three pyridine-containing triaza macrocyclic triacetate ligands
and their complexes with lanthanide ions. Inorg. Chem. 1997,
36, 2992-3000.
(27) Richman, J . E.; Atkins, T. J . Nitrogen analogues of crown ethers.
J . Am. Chem. Soc. 1974, 96, 2268.
(28) Lazar, I. Rapid and high yield detosylation of linear and
macrocyclic p-toluenesulfonamides. Synth. Commun. 1995, 25,
3181-3185
(29) Sudmeier, J . L.; Reilley. Nuclear magnetic resonance studies of
protonation of some polyaminocarboxylate compounds containig
asymmetric carbon atoms. Anal. Chem. 1964, 36, 1707-1712.
(30) Geraldes, C. F. G. C.; Sherry, A. D.; Cacheris, W. P. Synthesis,
protonation sequence and NMR studies of polyazamacrocyclic
methylenephosphonates. Inorg. Chem. 1989, 28, 3336-3341.
(31) Banci, L.; Bertini, I.; Luchinat, C. Nuclear and electron relax-
ation; VCH: Weinheim, 1991.
(32) Freed, J . H. Dynamic effects of pair correlation functions on spin
relaxation by translational diffusion in liquids. II. Finite jumps
and independent T1 processes. J . Chem. Phys. 1978, 68, 4034-
4037.
(33) Cortes, S.; Brucher, E.; Geraldes, C. F. G. C.; Sherry, A. D.
Potentiometry and NMR Studies of 1,5,9,-triazacyclododecane-
N,N’,N’’’-triacetic acid and its metal ion complexes. Inorg. Chem.
1990, 29, 5-9.
(34) Brucher, E.; Cortes, S.; Chavez, F.; Sherry, A. D. Synthesis,
Equilibrium and kinetic properties of the Gadolinium(III) com-
plexes of three triazacyclodecanetriacetate ligands. Inorg. Chem.
1991, 30, 2092-2097.
(35) Sherry, A. D.; Ren, J .; Huskens, Brucher E.; Toth, E.; Geraldes,
C. F. C. G.; Castro, M. M. C. A.; Chacheris, W. P. Characteriza-
tion of lanthanide(III) DOTP complexes: Thermodynamics,
protonation and coordination to alkali metal ions. Inorg. Chem.
1996, 35, 4604-4612.
(36) Rongved, P.; Fritzell, T. H.; Strande, P.; Klaveness, J . Polysac-
charides as carriers for magnetic resonance imaging contrast
agents: synthesis and stability of a new amino acid linker
derivative. Carbohydr. Res. 1996, 287, 77-89.
(37) Wiener, E. C.; Konda, S.; Shadron, A.; Brechbiel, M.; Gansow,
O. Targeting Dendrimer-chelates to tumors and tumor cells
expressing the high affinity folate receptor. Invest. Radiol. 1997,
32, 748-754.
(38) Lemieux, G. A.; Yarema, K. J .; J acobs, C. L.; Bertozzi, C. R.
Exploiting differences in sialoside expression for selective target-
ing of MRI contrast agents. J . Am. Chem. Soc. 1999, 121, 4278-
4279.
(39) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats,
R.; J acobs, R. E.; Fraser, S. E.; Meade, T. J . In vivo visualisation
of gene expression using magnetic resonance imaging. Nat.
Biotechnol. 2000, 18, 321-325.
Refer en ces
(1) Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Lanthanide(III)
chelates for NMR applications. Chem. Soc. Rev. 1998, 27, 19-
29.
(2) Magnetic Resonance in Medicine; Rinck, P. A., Ed.; Blackwell
Scientific Publications: Oxford, 1993.
(3) Kumar, K.; Tweedle, M. F. Macrocyclic polyaminocarboxylate
complexes of lanthanides as magnetic resonance imaging con-
trast agents. Pure Appl. Chem. 1993, 65, 515-520.
(4) Caravan, P.; Ellison, J . J .; McMurry, T. J .; Lauffer, R. B.
Gadolinium(III) chelates as MRI contrast agents: Structure,
dynamic and applications. Chem. Rev. 1999, 99, 2293-2352.
(5) Peters, J . A.; Huskens, J .; Raber, D. J . Lanthanide induced shifts
and relaxation rate enhancements. Prog. NMR Spectrosc. 1996,
28, 283-350.
(6) Lauffer, R. B. Paramagnetic Metal Complexes as Water Proton
Relaxation Agents for NMR Imaging: Theory and Design. Chem.
Rev. 1987, 87, 901-927.
(7) Brasch, R. C. Rationale and applications for macromolecular Gd-
based contrast agents. Magn. Reson. Med. 1991, 22, 282-287.
(8) Aime, S.; Barbero, L.; Botta, M. Trends in NMR studies of
paramagnetic Gd(III) complexes as potential contrast agents in
MRI. Magn. Reson. Imaging 1991, 9, 843-847.
(9) Vexler, V. S.; Cle´ment, O.; Schmitt-Willich, H.; Brasch, R. C. J .
Effect of varying the molecular weight of the MR contrast agent
Gd-DTPA-polylysine on blood pharmacokinetics and enhance-
ment patterns. Magn. Reson. Imaging 1994, 4, 381-388.
(10) Rebizak, R.; Schaefer, M.; Dellacherie. Polymeric conjugates of
Gd3+-dietylenetriaminepentaacetic acid and dextran, Synthesis,
characterization and paramagnetic properties. Bioconjugate
Chem. 1997, 8, 605-610.
(11) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.;
Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C. Dendrimer-based
metal chelates: anew class of magnetic resonance imaging
contrast agents. Magn Reson. Med. 1994, 31, 1-8.
(12) Aime, S.; Botta, M.; Fasano, M.; Geninatti, Crich S.; Terreno,
E. 1H and 17O NMR relaxometric investigations of paramagnetic
contrast agents for MRI. Clues for higher relaxivities. Coord.
Chem. Rev. 1999, 185-186, 321-333.
(13) Aime, S.; Botta, M.; Geninatti, Crich S.; Giovenzana, G. B.;
Pagliarin, R.; Piccinini, M.; Sisti, M.; Terreno, E. Towards MRI
contrast agents of improved efficacy. NMR relaxometric inves-
tigations of the binding interactions to HSA of a novel hepa-
dentate macrocyclic triphosphonate Gd(III)-complex. J . Biol.
Inorg. Chem. 1997, 2, 470-479.
(14) Aime, S.; Botta, M.; Geninatti, Crich S.; Giovenzana, G. B.;
Pagliarin, R.; Sisti, M.; Terreno, E. NMR relaxometric studies
of Gd(III) complexes with heptadentate macrocyclic ligands.
Magn. Reson. Chem. 1998, 36, S200-S208.
(15) Mickei, K.; Helm, L.; Brucher, E.; Merbach, A. 17O NMR study
of water exchange on [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-
related to NMR imaging. Inorg. Chem. 1993, 32, 3844-3850.
(16) Powell, D. H.; Ni Dhubhghaill, O. M.; Pubanz, D.; Helm, L.;
Lebedev, Y. S.; Schlaepfer, W.; Merbach, A. E. Structural and
dynamic parameters obtained from 17O NMR, EPR, and NMRD
studies of monomeric and dimeric Gd3+ complexes of interest in
magnetic resonance imaging: an integrated and theoretically
self-consistent approach. J . Am. Chem. Soc. 1996, 118, 9333-
9346.
(17) Lauffer, R. B.; Parmelee, D. J .; Dunham, S. U.; Ouellet, H. S.;
Dolan, R. P.; Witte, S.; McMurry, T. J .; Walovitch, R. C. MS-
325: albumin-targeted contrast agent for MR angiography.
Radiology 1998, 207, 529-538.
(18) Toth, E.; Connac, F.; Helm, L.; Adzamli, K.; Merbach, A. E.
Direct assessment of water exchange on a Gd(III) chelate bound
to a protein. J . Biol. Inorg. Chem. 1998, 3, 606-613.
(19) Aime, S.; Botta, M.; Fasano, M.; Geninatti, Crich S.; Terreno,
E. Gd(III) complexes as contrast agents for magnetic resonance
imaging,: a proton relaxation enhancement study of the interac-
tion with human serum albumin. J . Biol. Inorg. Chem. 1996, 1,
312-319.
(20) Kim, W. D.; Hrncir, D. C.; Kiefer, G. E.; Sherry, A. D. Synthesis,
crystal structure and potentiometry of pyridine-containing tet-
raazamacrocyclic ligands with acetate pendant arms. Inorg.
Chem. 1995, 34, 2225-2232.
(21) Lazar, I.; Hrncir, D. C.; Kim, W. D.; Kiefer, G. E.; Sherry, A. D.
Optimized synthesis, structure, and solution dynamics of 1,4,7,-
10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephospho-
nic acid) (H8DOTP). Inorg. Chem. 1992, 31, 4422-4424.
J M000983A