Lanthanide(III) Complexes of Novel Mixed Carboxylic-Phosphorus Acid Derivatives of Diethylenetriamine: A Step towards More Efficient MRI Contrast Agents

FULL PAPER

Lanthanide(iii) Complexes of Novel Mixed Carboxylic-Phosphorus Acid

Derivatives of Diethylenetriamine: A Step towards More Efficient

MRI Contrast Agents

Jan Kotek,^{[a, b]}Petra Lebdusœkovµ,^{[a, b]}Petr Hermann,^[b]Luce Vander Elst,^[c]Robert

N. Muller,^[c]Carlos F. G. C. Geraldes,^[d]Thomas Maschmeyer,^[a]Ivan Lukesœ,*^[b]and

Joop A. Peters*^[a]

Abstract: Three novel phosphorus-con-

taining analogues of H₅DTPA (DTPA

plexes studied have one coordinated

water molecule. The residence times

(t²_M⁹⁸) of the coordinated water mole-

cules in the gadolinium(iii) complexes

of H₆L¹and H₅L²are 88 and 92 ns, re-

spectively, which are close to the opti-

mum. This is particularly important

upon covalent and noncovalent attach-

ment of these Gd³⁺chelates to poly-

mers. The relaxivity of the complexes

studied is further enhanced by the

presence of at least two water mole-

cules in the second coordination sphere

of the Gd³⁺ion, which are probably

bound to the phosphonate/phosphinate

moiety by hydrogen bonds. The com-

plex [Gd(L³)(H₂O)]^2ꢀshows strong

binding ability to HSA, and the adduct

has a relaxivity comparable to MS-325

(40 s^ꢀ1mm^ꢀ1at 40 MHz, 378C) even

though it has a less favourable t_Mvalue

(685 ns). Transmetallation experiments

with Zn²⁺indicate that the complexes

have a kinetic stability that is compara-

ble to–or better than–those of

=

diethylenetriaminepentaacetate)

were synthesised (H₆L¹, H₅L², H₅L³).

These compounds have -CH₂-

a

P(O)(OH)-R function (R = OH, Ph,

CH₂NBn₂) attached to the central ni-

trogen atom of the diethylenetriamine

backbone. An NMR study reveals that

these ligands bind to lanthanide(iii)

ions in an octadentate fashion through

the three nitrogen atoms,

a

PꢀO

oxygen atom and four carboxylate

oxygen atoms. The complexed ligand

occurs in several enantiomeric forms

due to the chirality of the central nitro-

gen atom and the phosphorus atom

upon coordination. All lanthanide com-

Keywords: chelates

¥

imaging

¥ NMR

agents

¥

lanthanides

spectroscopy

complexes

plexes

¥

phosphinate

[Gd(dtpa)(H₂O)]^2ꢀ

bma)(H₂O)].

and

[Gd(dtpa-

¥

phosphonate com-

Introduction

Metal chelates of the polyaminocarboxylates DTPA^5ꢀ

œ

[a] Dr. J. A. Peters, J. Kotek, P. Lebduskovµ, Prof. Dr. T. Maschmeyer

Laboratory for Applied Organic Chemistry and Catalysis

Delft University of Technology

(DTPA^5ꢀ= diethylenetriamine-N,N,N’,N’’,N’’-pentaacetate),

DOTA^4ꢀ(DOTA^4ꢀ

=

1,4,7,10-tetraazacyclododecane-

Julianalaan 136, 2628BL Delft (The Netherlands)

Fax : (+31)152-784-289

E-mail: J.A.Peters@tnw.tudelft.nl

1,4,7,10-tetraacetate) and derivatives thereof have found

widespread use in medical diagnosis (e.g. Magnetic Reso-

nance Imaging, MRI; Positron Emission Tomography, PET;

or Single-Photon Emission Computed Tomography, SPECT)

œ

[b] Prof. Dr. I. Lukes, J. Kotek, P. Lebduskovµ, Dr. P. Hermann

Department of Inorganic Chemistry, Charles University

Hlavova 2030, 12840 Prague (Czech Republic)

E-mail: lukes@natur.cuni.cz

3]

and in radiotherapy.^[1These complexes have high thermo-

dynamic and kinetic stabilities, essential features for in vivo

applications, since the metal aqua ions as well as their li-

gands are toxic, whereas the complexes are not. The applica-

bility of radioactive complexes also requires that complexa-

tion should be rapid enough to allow radiolabelling by a

simple procedure just prior to the diagnostic procedure or

the treatment. Complexes of DTPA^5ꢀmeet this require-

ment, while the formation of complexes of DOTA^4ꢀis usu-

ally very slow.

[c] Prof. Dr L. Vander Elst, Prof. Dr. R. N. Muller

NMR Laboratory, Department of Organic Chemistry

University of Mons Hainaut, 7000 Mons (Belgium)

[d] Prof. Dr. C. F. G. C. Geraldes

Departamento de BioquÌmica

Faculdade de CiÜncias e Tecnologica, e Centro de NeurociÜncias

Universidade de Coimbra, 3049 Coimbra (Portugal)

Supporting information for this article is available on the WWW

under http://www.chemeurj.org/ or from the author.

5899

Chem. Eur. J. 2003, 9, 5899 5915

DOI: 10.1002/chem.200305155

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER

MRI contrast agents are mostly Gd³⁺complexes, as this

paramagnetic ion has a relatively long electronic relaxation

time, which leads to high nuclear relaxation efficiency. This

is usually expressed as the relaxivity, r₁, which is the en-

hancement of the water proton relaxation rate in s^ꢀ1mm^ꢀ1.

Other important parameters governing the relaxivity are the

rotational correlation time (t_R), the number of Gd³⁺-bound

water molecules (q), their residence time (t_M) and the elec-

tron spin relaxation times (T_ie, i = 1,2). Theory predicts op-

timal efficiency for high-molecular-weight gadolinium(iii)

chelates if the residence time, t_M, is in the range of 20 50

ns.^[4]All current commercial Gd³⁺-based contrast agents

have low molecular weights and are hydrophilic. Conse-

quently, these compounds are distributed rather unselective-

ly over the extracellular fluids. More efficient contrast

agents are being developed that may be directed to targets

of interest, thereby achieving higher local concentrations at

lower dosages.^[5]These agents usually are conjugates of one

or more Gd³⁺chelates and a targeting vector. The criterion

regarding the water exchange rate is particularly critical to

achieve optimal efficiency for this new class of compounds.

The current commercial Gd³⁺chelates show water exchange

rates that are an order of magnitude lower than the optimal

value.^[1,2,4,6]Recently, it was shown that phosphorus-contain-

ing analogues of the commercially used [Gd(dota)(H₂O)]^ꢀ

complex have faster water exchange than the parent system.

Scheme 1. Molecular structures of a) the ligands discussed and of b)

H₅DTPA, c) atom-labelling scheme for NMR assignment.

followed by deprotection of the tosyl groups and alkylation

with ethyl bromoacetate to give a H₅DTPA analogue with a

N-benzyl-protected central amino group of the skeleton (5)

were not successful due to the formation of a very stable

lactam (6) after debenzylation (Scheme 2). This lactam was

found to be extremely stable towards hydrolysis; it could be

hydrolysed only under very harsh conditions (i.e., 20%

NaOH, 908C, overnight), affording the tetraacetic derivative

(7). Therefore, it was decided first to attach the phosphorus-

containing moiety to the diethylenetriamine backbone. This

was achieved by a Mannich-type reaction between N,N’’-

bis(phthaloyl)diethylenetriamine (8), paraformaldehyde and

the appropriate phosphorus derivative, followed by depro-

tection of phthaloyl moieties with hydrazine. Then, alkyla-

tion of intermediate (10) with ethyl bromoacetate and hy-

drolysis of the ester groups afforded the desired compounds

H₆L¹, H₅L²and H₅L³in overall isolated yields of 50 80%

(Scheme 3).

[7]

Similar results were observed on pyridine-containing mac-

rocyclic ligands with phosphonic acid pendant arms.^[8,9]

Moreover, these compounds show rapid complex formation,

which makes them suitable for radiodiagnostic and radio-

therapeutic applications. The phosphorus-containing arm

can be functionalised (e.g. with an ester moiety or some

alkyl or aryl group) to afford bifunctional ligands that can

be easily linked to a biologically active compound that de-

termines the biodistribution of the final complex. The inter-

action of a paramagnetic Gd³⁺complex with a macromole-

cule results in an increase in relaxivity due to the elongation

of t_R.

Determination of the ligand-protonation constants by using

¹H and ³¹P NMR chemical-shift titrations: Since the thermo-

dynamic stability of the Ln³⁺complexes of aminocarboxy-

We have extended these studies to phosphorus-containing

analogues of open-chain DTPA^5ꢀcomplexes. In this paper,

we describe the synthesis and physicochemical characterisa-

tion of lanthanide complexes of three novel DTPA^5ꢀderiva-

tives with a phosphorus acid pendant arm on the central ni-

trogen atom of the diethylenetriamine backbone, H₆L¹, H₅L²

and H₅L³(see Scheme 1). Ligand H₆L¹is the parent struc-

ture, while H₅L²is a ligand that, after appropriate substitu-

tion of the phenyl group, can be linked covalently to a poly-

mer. Ligand H₅L³has a dibenzylamino moiety attached to

the phosphorus function, a structural motif that has some

similarity with that occurring in MS-325.^[10]The Gd³⁺com-

plex of the latter ligand is known to be a very efficient

blood-pool contrast agent due to its ability to bind noncova-

lently to human serum albumin (HSA).

lates is related to the summed protonation constants of the

13]

free ligand,^[11

insight into the structural effects on these

constants is desirable. Therefore, the protonation constants

of all the ligands were determined by using the pH depend-

1

ence of H and ³¹P NMR chemical shifts. The chemical shift

curves (see Figure 1) display sharp changes at several ranges

of pH values; they may be ascribed to the shift dependence

on the changes of the protonation state of the ligand con-

cerned.

Since the protonation equilibria are fast on the NMR

timescale, the chemical shift of each signal can be given as a

weighted average of the shifts of the various protonated spe-

cies (see [Eq. (1)]).^[14]

X

d_obs

¼

X_iÁ d_i

ð1Þ

Here d_obsis the observed chemical shift of a given signal,

X_iis the molar fraction of species i and d_iis its chemical

Results and Discussion

1

shift. The observed H and ³¹P chemical shifts were fitted si-

Synthesis of the ligands: Attempts to build up the ligands

from benzylamine (2) by treatment with tosylaziridine (1),

multaneously according to Equation (1) by using the dissoci-

ation constants (pK_ai) and the values of d_ias adjustable pa-

5900

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Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

Scheme 3. Synthesis of H₆L¹(R = OH), H₅L²(R = Ph) and H₅L³(R =

CH₂N(CH₂Ph)₂); reagents: i) HP(O)(R)(OEt), CH₂O; ii) N₂H₄; iii)

BrCH₂COOEt; iv) HCl or NaOH.

Scheme 2. Unsuccessful approach to ligand synthesis–formation of

lactam (6), reagents: i) HBr; ii) BrCH₂COOEt, NaOH; iii) H₂, Pd/C; iv)

NaOH.

procedure of Sudmeier and Reilley.^[14]This assumes that the

chemical shifts of methylene protons in aminocarboxylates

can be estimated by considering the effects of protonation

of various basic sites to be additive and characteristic for

the position of the given methylene group with respect to

the protonation site, as expressed in Equation (2).

rameters. The fits of experimental data points are shown in

Figure 1, and the resulting pK_aand d_ivalues are compiled in

Table 1 and the Supporting Information (Tables S1 S3), re-

spectively. For comparison, pK_avalues for H₅DTPA report-

ed in the literature^[15]are included in Table 1.

X

Ddⁿ_i

¼

C_Nf_N

þ

C_N₀f_N

þ

C_Of_O

þ

C_Pf_P

ð2Þ

The ¹H NMR chemical shifts, Ddⁿ_i, calculated for each

proton H_iat the various degrees of protonation of the li-

gands (L¹)^6ꢀ, (L²)^5ꢀand (L³)^5ꢀ(see Tables S1 S3) were then

used to evaluate the protonation fractions f_i(i = 1 5) at the

nitrogen and oxygen basic sites of the ligands (Scheme 1c)

for their successive protonated forms, with the empirical

The protonation shifts of the methylene groups of the di-

ethylenetriamine backbone reflect the protonation fractions

of each of the terminal N(1) (f₁) and the central N(2) (f₂) ni-

trogen atoms of the ligands through the shielding constants

C_Nand C_N’for the protonation of those amino groups, when

they are at an a or b position,

respectively, relative to those

Table 1. Dissociation constants (pK_a) of the ligands studied (0.1m, 258C) and comparison with those of

methylene groups (values of C_N

0.75 ppm and C_N’0.35

H₅DTPA.

=

pK_a1

pK_a2

pK_a3

pK_a4

pK_a5

ꢀpK_a

ppm have been listed).^[14,15]The

protonation fractions of the

oxygen atoms at the terminal

carboxylates O(3) (f₃) and at

the central phosphonate group

H₆L¹

10.747(5)

9.60(5)

10.1(1)

10.2

7.88(2)

9.10(4)

9.4(2)

8.6

6.92(7)

2.63(19)

7.13(2)

4.2

2.7(2)

2.15(12)

1.32(5)

2.9

2.17(6)

0.72(14)

30.4

24.2

H₅L²

H₅L³

28.0 (20.8)^[a]

27.7

H₅DTPA^[15]

1.8

[a] Values in parenthesis exclude the pK_avalue associated with protonation of side-chain nitrogen atom.

5901

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œ

I. Lukes, J. A. Peters et al.

FULL PAPER

Figure 1. ¹H and ³¹P NMR chemical-shift titration curves of 0.1m solutions of H₆L¹(a,b), H₅L²(c,d) and H₅L³(e,f) in H₂O/D₂O (9:1 v/v) at 258C and 7T.

~

&

^

*

^

Vertical lines mark the dissociation constants (for values, see Table 1). For labelling of hydrogen atoms see Scheme 1c: a ( ), b ( ), c ( ), d ( ), e ( ), f

~

).

(

O(4) (f₄) were also calculated by using shielding constants

in aminomethylcarboxylates.^[13]However, aminomethylphos-

phinates are less basic than the corresponding aminomethyl-

carboxylates; this explains why the central nitrogen atom

(f₂) of the phenylphosphinic derivative (L²)^5ꢀis only poorly

protonated in the monoprotonated species (HL²)^4ꢀ. In all

cases, the protons of the (H₂L)^xꢀspecies are located mainly

on the outer nitrogen atoms (f₁), this can be rationalised by

the electrostatic repulsion between the two incoming protons.

For n>2, the protonation also involves the basic atoms

located at the pendant arms of the ligands. For the ligand

(L¹)^6ꢀ, the f_ivalues show that the third protonation step

mainly occurs at the phosphonate moiety; this is in agree-

ment with its value of pK_a3(6.92) being close to the pK_a

values commonly observed for phosphonates.^[18]The next

C_O= 0.20 ppm for a-carboxylate protonation^[15]and C_P

0.20 ppm for a-phosphonate/phosphinate protonation.^[16,17]

=

The results are given in Table 2 together with data for

DTPA^5ꢀreported previously.^[15]It can be seen that the first

two protons bind exclusively to the backbone nitrogens in

the three cases. The first protonation of the phosphonate

ligand (HL¹)^5ꢀtakes place mainly on the central nitrogen

atom of the backbone (f₂), whereas in HDTPA^4ꢀand

(HL³)^4ꢀ, the preference for the central nitrogen is somewhat

less, so that the central nitrogen atom is protonated to about

the same extent as the sum of the two terminal ones. This is

in agreement with the basicity of the nitrogen atom in ami-

nomethylphosphonates, which is generally higher than that

5902

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Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

Table 2. Percent protonation fractions of the different basic sites of the

ligands (L¹)^6ꢀ, (L²)^5ꢀ, (L³)^5ꢀand DTPA^5ꢀin the protonated forms H_nL^xꢀ

at increasing values of n (for DTPA^5ꢀ, f₃and f₄correspond, respectively,

to the terminal and central carboxylates. The errors in f_ivalues are

Æ10%).

complexes of the first two ligands will be comparable with

those of the corresponding H₅DTPA complexes, whereas the

protonation of the side-chain nitrogen of the third ligand is

expected to lead to a somewhat reduced stability of the cor-

responding complexes.

H_nL^xꢀ

f₁

f₂

f₃

f₄

f₅

ꢀ

(H_nL¹)^(nꢀ6)

n=1

The hydration numbers of the lanthanide complexes of

13

7

4

0

H₆L¹, H₅L²and H₅L³as determined from the lanthanide-in-

duced water ¹⁷O NMR shifts: The ¹⁷O NMR chemical shifts

for water oxygen in 0.2m solutions of complexes of all li-

gands with 14 different Ln³⁺ions were measured at 40.7

MHz, 708C and pH 5 6. Under these conditions, the ex-

change between the Ln³⁺-bound and bulk-water protons

was rapid on the NMR timescale. Therefore, the observed

chemical shifts with respect to free water (d_obs, see Table S4

n=2

n=3

n=4

n=5

92

100

16

170

20

76

0

83

20

31

0

100

ꢀ

(H_nL²)^(nꢀ5)

n=1

46

100

8

0

8

0

23

38

0

n=2

n=3

n=4

48

(H_nL³)^(nꢀ5)

n=1

in the Supporting Information) are related to those of Ln³⁺

-

23

94

98

54

12

4

0

100

bound water (d_M) by Equation (3), in which q is the number

of water molecules in the first coordination sphere of Ln³⁺

and 1_wis the Ln³⁺/water molar ratio in the sample. The

bound shifts comprise diamagnetic (d_d), contact (d_c) and

pseudocontact contributions (d_p) [see Eq. (4)].^[20]

6

n=2

n=3

n=4

100

18

16

^ꢀ[15]

(H_nDTPA)^(nꢀ5)

n=1

26

8716

80

41

64

0

7

n=2

n=3

5

0

d_obs¼ q Á 1_wÁ d_M

ð3Þ

d_M¼ d_dþ d_cþ d_p

ð4Þ

proton is mainly equally distributed over the four carboxy-

late oxygens, while the fifth shows a preference for the cen-

tral nitrogen. In the cases of the phosphinate derivative li-

gands, (L²)^5ꢀand (L³)^5ꢀ, the central phosphinate oxygen is

never protonated (f₄= 0) under the conditions applied (2<

pH<13), but the value of pK_a3of the second ligand (7.13)

corresponds to protonation of the dibenzylamino moiety, as

shown by the value f₅= 100% (Table 2) and the shifts of

the protons e and f next to the N(5) atom of its side chain

(Figure 1f). Thus, for the (L²)^5ꢀligand, the third proton is

about equally distributed over the four carboxylate oxygens,

while the fourth binds preferentially to the central nitrogen.

However, in the case of (L³)^5ꢀ, the fourth proton is almost

equally distributed over the four carboxylate oxygens and

the central nitrogen atom.

This protonation scheme is qualitatively confirmed by the

³¹P NMR titration curves (Figure 1a,c,e). Protonation of

N(2) dramatically affects the charge density of the phospho-

rus atom of the phosphonate group, and to a lesser extent

that of the phosphinate group. This can be ascribed to for-

mation of a strong intramolecular hydrogen bond between

N(2)H⁺and the phosphonate O^ꢀ, forming stable five-mem-

bered rings and resulting in a shift of the ³¹P resonance to

low frequency.^[17,19]This can be seen in Figure 1, where low-

frequency ³¹P shifts are observed when N(2) and/or N(5) are

protonated. Deprotonation of N(2) or protonation of the

phosphonate/phosphinate group leads to high-frequency ³¹P

shifts, as both processes lead to the disappearance of the in-

ternal NH⁺¥¥¥O^ꢀbonds.^[17]

The contact contribution is the result of a through-bond

transmission of unpaired-electron-spin density from the cen-

tral ion to the ligand nucleus, and the pseudocontact contri-

bution results from a dipolar interaction between the mag-

netic moment of the central ion and the nucleus in question.

Both paramagnetic contributions, d_cand d_p, can be ex-

pressed as the product of lanthanide-dependent but ligand-

independent constants (hS_zi and C_D, respectively) and terms

characteristic for the nucleus under study (F and G, respec-

tively) as given in Equation (5).

D ¼ d_obs=1_w¼ qðhS_zi Á F þ C_DÁ G þ d_dÞ

ð5Þ

Values for hS_zi and C_Dare tabulated in the literature.^[21

For isostructural complexes, the ligand-dependent parame-

ters F and G for the water ¹⁷O nucleus are the same for all

paramagnetic Ln³⁺ions, and thus Equation (5) can be line-

arised in two different ways [Eqs. (6) and (7), in which D’ =

Dꢀq¥d_d].^[20]In other words, if the observed data afford

linear plots according to Equations (6) and (7), it may be

concluded that the complexes concerned are isostructural.

25]

Dꢀq Á d_d

D0

hS_zi

ð6Þ

ð7Þ

¼

q Á F þ q Á G

C_D

Dꢀq Á d_d

hS_zi

D0

C_D

¼ q Á F þ

q Á G

hS_zi

The sum of the pK_avalues of the atoms of the ligand that

are involved in binding to the Ln³⁺ion of H₆L¹, H₅L²and

H₅L³(for the last one, thus excluding the pK_avalue associat-

ed with the protonation of the side-chain nitrogen atom,

Table 1) is a good indication of the thermodynamic stability

of the lanthanide complexes. Their values indicate that the

It has previously been shown that the value of parameter

F is in the narrow range of 70Æ11 for one coordinated

oxygen-donor atom, independent of the nature of that atom

and of the other ligands present in the Ln³⁺complex.^[20]

Thus, the slopes of the plots of the experimental data ac-

cording to Equation (6) are proportional to the number of

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œ

I. Lukes, J. A. Peters et al.

FULL PAPER

water molecules coordinated in the inner sphere of the Ln³⁺

ion.

ꢀ3600 ppm; this reflects the substitution of 7coordinated

water molecules by the organic ligand, H₅L². Between pH

2.5 and 9.5, D is invariant, and no precipitation of hydrox-

ides is observed; once again, this demonstrates the high sta-

bility of the complex formed.

The observed chemical shifts for the diamagnetic La³⁺

and Lu³⁺complexes of the ligands under study were taken

as q¥d_d. Plots of the values of D’ for the Ln³⁺complexes of

the ligands studied according to Equation (6) were perfectly

linear (see Figure 2a,c,e). The slopes of the lines and thus

the values of q¥F are ꢀ59, ꢀ65 and ꢀ70 for complexes of

H₆L¹, H₅L²and H₅L³, respectively. The values of q¥F are

close to that observed for H₅DTPA (ꢀ53),^[26]in agreement

with the presence of one water molecule in the inner coordi-

nation sphere of the Ln³⁺ion for these complexes. This

structural feature is the most common motif in the chemis-

try of Ln³⁺complexes of H₅DTPA and related ligands.^[1]

The plots according to Equation (7) (see Figure 3b,d,f)

show a break between lighter (Ln = Ce!Eu) and heavier

(Ln = Tb!Yb) Ln³⁺ions, whereas the plots according to

Equation (6) are linear. Such a break, observed exclusively

in the former plots, can be ascribed to some small gradual

changes of complex geometry^[27]and/or to a change of crys-

tal-field parameters along the lanthanide series.^[28]

Structure of the lanthanide complexes of H₆L¹and H₅L²in

solution as determined from ¹³C and ³¹P relaxation enhance-

ments: The coordination number of Ln³⁺ions in complexes

of polyaminocarboxylates is, in general, nine. The ¹⁷O NMR

measurements described above show that one water mole-

cule is bound in the first coordination sphere of the Ln³⁺

ion in the complexes of H₆L¹, H₅L²and H₅L³. Therefore, it

is most likely that these ligands are bound in an octadentate

fashion, with binding occurring through the three nitrogen

atoms of the backbone, four carboxylate oxygen atoms and

one phosphonate/phosphinate oxygen atom. This is a bind-

ing mode similar to that of H₅DTPA itself. The NMR spec-

tra of the Ln³⁺complexes all displayed multiple resonances

for the various types of nuclei. For example, the ¹³C NMR

spectrum of the diamagnetic complexes [Y(L¹)(H₂O)]^3ꢀand

[Y(L²)(H₂O)]^2ꢀat 258C showed two resonances in the car-

boxylate region (intensities 1:1) and some broad signals for

the aliphatic ¹³C nuclei. This indicates that several isomers

of these complexes exist in solution and are in exchange

with each other.

The pH dependence of the ¹⁷O NMR shifts was studied in

some detail for the Dy³⁺complex of H₅L². A plot of the D

values for this system as a function of the pH (see Support-

ing Information Figure S1) shows that, at pH close to 0, the

value of D (ꢀ21000 ppm) is almost the same as that ob-

served for the free Dy³⁺aqua ion (ꢀ21685 ppm), which has

eight water molecules coordinated to the Dy³⁺ion. Upon

increase of the pH to 2.5, the value of D changes to about

To support the coordination mode proposed, we evaluated

the Nd³⁺ꢀC and Nd³⁺ꢀP distances from the ¹³C and ³¹P par-

amagnetic lanthanide-induced longitudinal-relaxation-rate

enhancements. The Nd³⁺ion

was selected for this purpose,

since it has the longest elec-

tron-spin-relaxation

times

among the light Ln³⁺ions (Ln

= Ce!Eu). The measurements

were performed at 808C, at

which temperature the spectra

displayed relatively sharp lines

(Dn_1/2<10 Hz). In order to cor-

rect for diamagnetic contribu-

tions, the relaxation rates of the

corresponding La³⁺complexes

were subtracted from the meas-

ured values for the Nd³⁺com-

plexes. Under the conditions

employed, the ¹³C NMR spectra

of the Nd³⁺complexes of H₆L¹

and H₅L²showed two carboxyl

resonances, two resonances of

carboxymethyl methylenes, two

signals of the diethylenetria-

mine backbone and one dou-

blet for the central methylene

carbon, whereas the ³¹P NMR

spectra showed a single reso-

nance. Since the outer-sphere

contribution to longitudinal re-

laxation rates (1/T_1,OS) becomes

significant only for remote

Figure 2. Linearisation of lanthanide-induced ¹⁷O NMR shifts observed in D₂O solutions of the Ln³⁺com-

plexes of H₆L¹(a,b), H₅L²(c,d) and H₅L³(e,f) according to Equations (6) and (7).

5904

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

nuclei, this was neglected. From the electron-spin relaxation

for Nd³⁺(T_1eꢁ10^ꢀ13s),^[29]it can be estimated that the con-

tact contribution to the paramagnetic relaxation is negligi-

ble. Therefore, two contributions are of importance: the di-

polar relaxation and the Curie relaxation. These are repre-

sented by a combination of a simplified Solomon Bloember-

gen equation with one for Curie relaxation, giving Equation

(8):^[20]

Interconversion between isomers of the Ln³⁺complexes of

the ligands under study: Upon binding of the ligands H₆L¹,

H₅L²and H₅L³to a Ln³⁺ion in an octadentate fashion

through the diethylenetriamine nitrogen atoms, a phospho-

nate/phosphinate oxygen atom and four carboxylate oxygen

atoms, the central nitrogen atom and the phosphorus atom

become chiral. An inspection of crystal structures of Ln³⁺

complexes of DTPA derivatives has shown that the two eth-

ylene moieties can adopt either a ll or a dd conforma-

tion.^[32,33]Therefore, this is most likely to also be the case

for the presently studied ligands. Then, four enantiomers

(two diastereomeric pairs) are possible: llR, llS, ddR and

ddS, where R and S denote the chirality of the phosphorus

atom. In a static situation, all ¹³C nuclei in an isomer are

chemically different. Therefore, for example four carboxy-

late resonances should be expected for each diastereomeric

pair, leading to an expected total number of eight carboxy-

late resonances.

ꢀ

ꢁ

ꢃ

ꢂ

¼

²Á m²Á g²_IÁ b²Á T_1e

1

T₁

4

3

m₀

4p

ð8Þ

ꢁ

ꢂ

ꢄ

ꢅ

6

5

m₀²g²_IÁ H²₀Á m⁴Á b⁴

1

r⁶

þ

Á t_R

2

4p

ð3k_BTÞ

Here m₀/4p is the permeability of a vacuum, m is the effec-

tive magnetic moment of Nd³⁺, g_Iis the gyromagnetic ratio

of the nucleus under study (¹³C and ³¹P), b is the Bohr mag-

[29]

neton, T_1eis electronic spin relaxation time for Nd³⁺

,

H₀

The variable-temperature behaviour of the ¹³C NMR

spectrum of the diamagnetic [Y(L²)(H₂O)]^2ꢀcomplex was

studied in some detail. At 0.58C, four carboxylate resonan-

ces of about equal intensity were observed. Upon increasing

the temperature, these resonances broadened and coalesced

to two resonances at about 98C, and sharpened again upon

further temperature increase. Similar behaviour was ob-

served for the other ¹³C resonances; this indicates that a rac-

emisation process becomes rapid on the NMR timescale.

Racemisation of the central nitrogen atom can be achieved

by a wagging motion of the diethylenetriamine moiety,

which interconverts its ll and dd conformations. The phos-

phorus atom can racemise by decoordination of the phosphi-

nate moiety followed by ™inversion∫ of the phosphorus

atom, that is, rotation around the CH₂ꢀP bond and recoordi-

nation. Apparently, one of these two racemisation processes

is already rapid on the NMR timescale at 0.58C, whereas

the other becomes fast above 98C. From the coalescence

temperature of the carboxylate resonances (98C), the free

enthalpy of activation of the exchange process concerned

(DG₂₈₂) can be estimated to be 56Æ3 kJmol^ꢀ1. The value is

in the range of DG values generally found for the racemisa-

is the strength of the magnetic field, k_Bis the Boltzmann

constant, T is the temperature, t_Ris the rotational correla-

tion time for the complex species and r is the distance of

Nd³⁺to the nuclei in the complex. This equation can be

used to calculate the Nd³⁺ꢀC and Nd³⁺ꢀP distances r. For

these calculations, values of t_Rat 808C of the corresponding

Gd³⁺complexes (23.1 ps for [Nd(L¹)(H₂O)]^3ꢀand 33.2 ps

for [Nd(L²)(H₂O)]^2ꢀ) were employed. These values were

evaluated from variable-concentration ²H NMR data per-

formed on the deuterated La³⁺complexes by using the acti-

vation energy for t_Ras obtained from the fitting of the ¹⁷O

and nuclear magnetic relaxation dispersion (NMRD) data

(see below).Table 3 lists the experimental values of longitu-

dinal relaxation rates observed for Nd³⁺and La³⁺com-

plexes, together with the Nd³⁺ꢀC and Nd³⁺ꢀP distances cal-

culated from them by using Equation (8). For comparison,

results obtained for complexes of H₅DTPA and its bis(ami-

de)derivatives reported previously^[26,30,31]have been included

in the Table. The similarity of these values confirms the pro-

posed octadentate binding mode (similar to structures of

well-known complexes of H₅DTPA) of the new ligands in

their lanthanide complexes.

Table 3. Observed longitudinal relaxation rates in La³⁺and Nd³⁺complexes of ligands H₆L¹and H₅L²and calculated nonbonding distances r_(Nd-P)and

r_(Nd-C)

.

longitudinal relaxation rates 1/T₁[s^ꢀ1

]

distances from Nd³⁺[ä]

H₅DTPA^[a]

atom

LaꢀH₆L¹

NdꢀH₆L¹

LaꢀH₅L²

NdꢀH₅L²

H₆L¹

H₅L²

3.52

H₃DTPA-bis(amides)^[b]

P

CO

N-CH₂-CO

N-CH₂-P

0.356

0.17^[c]

2.56^[c]

3.00^[e]

3.11

10.06

0.291

0.10^[c]

2.62^[c]

1.72

9.73

3.49

6.02 6.25^[c]

7.04 7.46^[c]

5.39^[e]

6.29 8.93^[c]

7.52 7.69^[c]

8.71

3.22 3.24^[c]

3.33 3.38^[c]

3.76^[e]

3.03 3.22^[c]

3.33 3.35^[c]

3.15

3.15 3.20

3.14 3.30

3.20 3.59

3.14 3.30^[f]

3.04 3.48^[g]

3.04 3.48

[d]

3.17^[f]

3.48^[g]

3.21

CH₂-N-CH₂-P

CH₂-N-CH₂-CO

P-C(arom)

C(arom-o)

C(arom-m)

C(arom-p)

6.99

3.10

7.52

3.47

3.41

3.30

2.82

7.09

3.11

8.40

0.11

0.72

0.70

1.28

1.96

1.29

0.89

1.41

3.94

4.79

5.75

6.13

[a] Taken from ref. [26]. [b] Taken from ref. [31]. [c] Two different signals of equal intensity are found in ¹³C NMR spectra. [d] Not determined. [e] The

signal has low intensity and overlaps others; this makes it unsuitable for the determination of relaxation rates. [f] Value corresponding to the signal of an

acetate pendant moiety bound to the central nitrogen atom. [g] Value corresponding to the signal of a backbone carbon atom bound to the central nitro-

gen atom.

5905

Chem. Eur. J. 2003, 9, 5899 5915

www.chemeurj.org

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

œ

I. Lukes, J. A. Peters et al.

FULL PAPER

tion of the central nitrogen atom in Ln³⁺complexes of

H₅DTPA and its derivatives;^[32,33]this suggests that the ex-

change process observed here can be assigned to such a rac-

emisation. The racemisation at the phosphorus atom has a

considerably lower barrier, as at 08C the exchange between

the corresponding two enantiomeric forms is already rapid

on the NMR timescale.

These results were confirmed by a variable-temperature

1

and variable-pH study of the H and ³¹P NMR spectra of a

0.1m aqueous solution of the diamagnetic [La(L³)(H₂O)]^2ꢀ

1

complex. The ³¹P and H NMR signals of the complex were

quite broad at 258C, but considerably sharpened at 608C.

The proton resonances were assigned with the aid of a

COSY spectrum at 608C, pH 6.4. A value of pK_a= 6.58(4)

was obtained from fitting the pH dependence of the ³¹P and

H_eand H_fresonances of the [La(L³)(H₂O)]^2ꢀcomplex, with

the protonation shift values indicating that the process

occurs at the side-chain N(5) atom. At 608C, two resonances

of about equal intensity were observed for each of the back-

bone (H_c,c’, H_d,d’) and acetate (H_a,a’, giving two AB patterns

Figure 3. Rotational correlation times at 298 K (t²_R⁹⁸) obtained from solu-

tions of [La([D₈]L¹)(H₂O)]^3ꢀ(&), [La([D₈]L²)(H₂O)]^2ꢀ

(

and

^

)

[La([D₈]L³)(H₂O)]^2ꢀ

(

~

) in H₂O at different concentrations.

Evaluation of the parameters governing the relaxivity from

1

a variable-temperature ¹⁷O NMR and H NMRD study on

the Gd³⁺complexes: From a comparison of the observed

longitudinal (T₁) and transversal (T₂) relaxation times and

the frequencies (w) of the ¹⁷O NMR signal of water in the

presence of Gd³⁺complexes and the same parameters of

the signal of pure water, the corresponding reduced parame-

ters T_1r, T_2rand Dw_rwere calculated by using Equations (10)

and (11):

2

with J_HHvalues of 16.6 Hz) protons of the complex, while

only one sharp resonance was observed for each of the side-

2

chain protons (singlet H_f, doublets H_eand H_bwith J_PH

values of 9.8 and 8.0 Hz, respectively). This again indicates

that at 608C the racemisation processes for the central nitro-

gen and the phosphorus atom are rapid on the NMR time-

scale.

1=T_ir¼ 1=P_mð1=T_iꢀ1=T_iwÞ; i ¼ 1; 2

Dw_r¼ 1=P_m:ðwꢀw_wÞ

ð10Þ

ð11Þ

2

Evaluation of rotational correlation times by H NMR: The

Here, the index ™w∫ denotes the variable corresponding

to pure water and P_mis the molar fraction of coordinated

water. The calculated reduced variables are plotted in

Figure 4a f and listed in the Supporting Information

(Tables S5 S7).

rotational correlation time, t_R, is one of the parameters gov-

erning the relaxivity of a Gd³⁺complex. Usually, a relatively

large discrepancy exists between the t²_R⁹⁸values evaluated

1

from the H and ¹⁷O NMR data. Therefore, we decided to

determine the rotational correlation times independently

using the deuterium longitudinal relaxation rates of the deu-

terated ligands [D₈]H₆L¹, [D₈]H₅L²and [D₈]H₅L³in their dia-

magnetic La³⁺complexes.^[34]In such a diamagnetic system,

the deuterium relaxation depends only on quadrupolar in-

teractions and is given by Equation (9):

The magnetic-field dependence of the proton longitudinal

1

relaxation was recorded as H NMRD profiles at 5, 25 and

378C. The relaxation rates are, as usual, expressed in terms

of relaxivity (r₁) in s^ꢀ1mm^ꢀ1(see Figure 4g i).

1

The ¹⁷O NMR and H NMRD data obtained were fitted

with the sets of equations usually used to predict variable-

temperature ¹⁷O NMR data, with the Solomon Bloember-

gen Morgan equations (which describe the field dependency

of the inner-sphere relaxivity, r₁) and with the Freed equa-

tion for the outer-sphere contribution of the relaxivity.^[35]

The set of equations used is given in the Supporting Infor-

mation.

ꢁ

ꢂ

2

1

T₁

3

8

e²qQ

ꢁh

ð9Þ

R₁

¼

t_R

The quadrupolar coupling constant (e²qQ/ꢁh) has a value

of 170î2p kHz for an sp³-hybridised Cꢀ H bond. It has

2

1

been demonstrated that t_Rvalues obtained in this way agree

¹⁷O NMR and H NMRD data are influenced by a large

well with those obtained from H NMRD measurements.^[34]

number of parameters, many of which are common for these

measurements. The fittings of these data were performed si-

multaneously; this has the advantage of putting constraints

on these common parameters. Further constraints were ach-

ieved by fixing some of the parameters. It was assumed that

the number of inner-sphere water molecules, q, is 1, the

value that was obtained from the analyses of the Ln³⁺-in-

duced ¹⁷O NMR shifts of bound water in the complexes

under study (see above). The distance between Gd³⁺and

the oxygen atom of the coordinated water molecule, which

is usually not dependent on the nature of the coordinated

ligand,^[36]was fixed at 2.5 ä. The distance between Gd³⁺

1

2

The 1/T₁values and, therefore, also the t_Rvalues for H in

samples of the La³⁺complexes of the deuterated ligands

were found to be dependent on the concentration of the

complex for concentrations varying between 4 and 200 mm

(Figure 3). Extrapolation of the curves in Figure 3 to the

concentration used in the NMRD measurements (1 mm, see

below) gave estimated values of 86, 110 and 121 ps for t_R²⁹⁸

of the Gd³⁺complexes of [D₈]H₆L¹, [D₈]H₅L²and [D₈]H₅L³,

respectively. The trend of these t²_R⁹⁸values agrees with the

expected increase of the rotational correlation time upon in-

crease of the molecular volume.

5906

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

Figure 4. Simultaneously fitted data from ¹⁷O NMR and ¹H NMRD measurements of [Gd(L¹)(H₂O)]^3ꢀ(a,d,g), [Gd(L²)(H₂O)]^2ꢀ(b,e,h) and

[Gd(L³)(H₂O)]^2ꢀ(c,f,i). First row (a,b,c): the temperature dependence of logarithms of the reduced relaxation rates (upper line ( ) corresponds to T_2r,

~

&

^

lower line ( ) corresponds to T_1r). Second row (d,e,f): the temperature dependence of the reduced frequency ( ). Third row (g,h,i): proton-relaxivity de-

~

&

^

pendence on the magnetic field at 378C ( ), 258C ( ) and 58C ( ).

and a water proton was fixed at 3.1 ä, whereas the distance

of closest approach of a water molecule to Gd³⁺, a^H, was

fixed at 3.5 ä. The value of E_V, the activation energy of the

correlation time t_v, was fixed at 1 kJmol^ꢀ1. Attempts to

unfix this parameter led to negative values of the activation

energy. The hyperfine coupling constants A/hꢁ were fixed at

the values calculated from the F values obtained from the

¹⁷O NMR studies described above and by using Equation

(12), in which b is the Bohr magneton, k is the Boltzmann

constant and g_Iis the ¹⁷O magnetogyric ratio. Furthermore,

the quadrupolar coupling constant of the bound water,

c(1+h²)^1/2, was taken equal to that determined recently for

the complex [Gd(dota)(H₂O)]^ꢀof 5.2 MHz.^[37]

urements (1 mm) were carried out (see above).^[34]Further-

1

more, the ¹⁷O and H relaxation rates are modulated by ro-

tation of the GdꢀO and the GdꢀH vectors, respectively. It

may be expected that these rotations have different correla-

tion times.^[37]Therefore, two rotational correlation times

were taken into consideration, t^H_Rand t_R^O. The parameter t^H_R

2

was fixed at the values obtained from the H NMR meas-

urements (see above).

A comparison of the values of the fitted parameters of

the complexes under study with those of [Gd(dtpa)(H₂O)]^2ꢀ

(see Table 4) reveals significant differences in the parame-

ters related to the electronic relaxation (the square of the

zero-field-splitting tensor, D², and the corresponding correla-

tion time, t_vand, particularly, in the diffusion coefficient,

D_GdH, (see Table 4), which is unexpectedly low. D_GdHde-

pends on the self-diffusion coefficients of the Gd³⁺complex

concerned, D_complex, and that of water, D_water[Eq. (13)].

b

A

ð12Þ

F ¼

10⁶

3kTg_Iꢁh

Fitting of the data with a single rotational correlation

time resulted in bad fits, whereas separate fitting of the ¹⁷O

and NMRD data resulted in good fits, but with different t_R

values. This may be ascribed to the large difference in con-

centration at which the ¹⁷O (200 mm) and the NMRD meas-

D_GdH¼ D_complexþ D_water

ð13Þ

Since D_waterat 298 K is 2.23î10^ꢀ9m²s^ꢀ1,^[38]it should be ex-

298

GdH

pected that D

is larger than this value. The relatively low

5907

Chem. Eur. J. 2003, 9, 5899 5915

www.chemeurj.org

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

œ

I. Lukes, J. A. Peters et al.

FULL PAPER

1

Table 4. Parameters for Gd³⁺complexes as obtained from the simultaneous fitting of ¹⁷O NMR and H NMRD data by using a model including second-

sphere water molecules and a model without second-sphere water molecules (see text), compared with literature values for [Gd(dtpa)(H₂O)]^2ꢀcomplex.

Parameter

[Gd(L¹)(H₂O)]^3ꢀ

[Gd(L²)(H₂O)]^2ꢀ

[Gd(L³)(H₂O)]^2ꢀ

[Gd(dtpa)(H₂O)]^{2ꢀ [a]}

no 2nd sphere

2nd sphere

no 2nd sphere

2nd sphere

no 2nd sphere

2nd sphere

t²_M⁹⁸[ns]

62Æ20

38Æ10

86.4^[b]

88Æ26

41Æ8

74Æ25

36Æ9

109.5^[b]

15Æ3

2.5Æ0.4

34Æ3

1^[b]

92Æ29

37Æ8

543Æ120

29Æ10

121.0^[b]

20Æ3

685Æ297303

37Æ9

DH^#[kJmol^ꢀ1

]

51.6

58

17.3

t²_R⁹⁸[ps]

86.4^[b]

109.5^[b]

19Æ3

121.0^[b]

23Æ3

H

E_R[kJmol^ꢀ1

t²_R⁹⁸_O/t²⁹⁸

]

14Æ2

21Æ3

2.3Æ0.4

30Æ3

2.7Æ0.4

22Æ3

2.7Æ0.4

26Æ3

3.5Æ0.4

31Æ3

3.7Æ0.4

25Æ3

H

R

t²_V⁹⁸[ps]

25

1.6

0.46

1.2

E_V[kJmol^ꢀ1

1^[b]

D²[10²⁰s^ꢀ2

]

0.29Æ0.03

0.45Æ0.09

0.22Æ0.02

0.32Æ0.06

0.26Æ0.03

0.36Æ0.02

dg_L²[10^ꢀ2

]

5Æ2

6Æ2

8Æ2

8Æ3

12Æ2

11Æ2

Ah^ꢀ1[10⁶rads^ꢀ1

]

ꢀ3.28^[b]

154.3Æ0.722.7

28.7Æ3

2.50^[b]

ꢀ3.28^[b]

ꢀ4.2

ꢀ3.61^[b]

22.75^[c]

ꢀ3.69^[b]

12.4Æ0.6

25Æ3

ꢀ3.69^[b]

22.75^[c]

ꢀ3.8

298

GdH

[c]

D

[10^ꢀ10m²s^ꢀ1

]

13.2Æ0.5

39.6Æ3

2.50^[b]

5.2^[b]

20

E_DGdH[kJ mol^ꢀ1

r_GdO[ä]

]

19.4

2.20

14

2.50^[b]

5.2^[b]

2.50^[b]

5.2^[b]

2.50^[b]

5.2^[b]

c(1+h²/3)^1/2[MHz]

5.2^[b]

q_2s

0

2.2Æ0.4

0

2.0Æ0.3

0

1.9Æ0.3

r_GdH2s[ä]

3.5^[b]

298

M2s

t

[ps]

35Æ8

36Æ11

50Æ9

48Æ12

60Æ10

35Æ10

DH_2s[kJ mol^ꢀ1

]

ln(1/T_2e) exp^[d]

23.65

22.53

23.4723.25

22.24

ln(1/T_2e) calcd^[e]

22.35

[a] Taken from ref. [36]. [b] Parameters were fixed during the fitting. [c] Calculated with Equation (14), [d] Determined from EPR line widths; [e] Calcu-

lated by using fitted parameters.

298

GdH

values obtained for D

indicate that the outer-sphere con-

ed for [Gd(dtpa)(H₂O)]^2ꢀ.^[36]The residence times of these

298

tribution to the total relaxivity is overestimated in the calcu-

lations. Most likely, this is due to an unaccounted contribu-

tion of water molecules in the second coordination sphere

of Gd³⁺, which may be bound to the ligand through hydro-

gen bonds to, for example, the negatively charged phosphi-

nate/phosphonate group.^[39]To account for such a contribu-

tion of second-sphere water molecules, we included a series

of equations that are similar to those for the inner-sphere

contribution (see Supporting Information). It should be

noted, however, that it is very difficult to evaluate the

second-sphere parameters because strong correlations exist

among some of them. Moreover, the second-sphere water

protons probably do not occupy a unique location but may

exchange among various sites. We fixed the distance be-

tween the Gd³⁺ion and the protons of the second-sphere

water molecules, r^H_2s, at 3.5 ä in the fitting procedure. Fur-

thermore, the values of D^T_waterat various temperatures were

calculated with the semiempirical relationship proposed by

Hindman [Eq. (14)].^[38]The size of the Gd³⁺complexes of

H₆L¹, H₅L²and H₅L³is much larger than that of water and,

consequently, the self-diffusion constants of these complexes

will be much smaller than that of water. In the present fit-

tings D_complexwas, therefore, neglected.

second-sphere water molecules (t

= 35 60 ps) are of the

M2s

41]

same magnitude as those obtained for other systems.^[39

Although the accuracy of the second-sphere parameters ob-

tained may be low due to the many assumptions made, it is

clear that at least two second-sphere water molecules, with a

residence time that is sufficiently long to be detected, have

to be included in the model to adequately explain the

NMRD profile. Aime et al.^[42]have reported that two

second-sphere water molecules are present in [Gd(pcp2a)-

(H₂O)₂]^ꢀ, a complex of a pyridine containing macrocycle

bearing one methylenephosphonic and two acetate arms.

Apparently, phosphonate/phosphinate groups are capable of

forming hydrogen bonds to two water molecules. Previously,

it has been shown that second-sphere water molecules con-

tribute to the relaxivity of several other phosphonate-bear-

ing ligands including [Gd(dotp)]^5ꢀ(H₈DOTP = 1,4,7,10-tet-

raazacyclododecane-1,4,7,10-tetrakis(methylphosphonic

acid)).^[42]

While the results of simultaneous fits of the present data

gave a t²_R⁸⁹_O/t²⁸⁹ratio of 2.7 3.0, the concentration depend-

H

R

2

ence of t_Ras determined from H NMR (see above) gave

an estimate of this ratio of about 1.4 for equal concentra-

tions. The latter value (1.4) is in agreement with that report-

ed by Dunand et al. for the [Gd(dota)(H₂O)]^ꢀcomplex.^[37]

The differences between t²_R⁸⁹and t_R²⁸⁹may be ascribed to

ð5:06258Â10³=TÞ

ꢀln D^T_water¼ ln ½3:11815 Â 10^ꢀ4

e

H

O

differences in the rotation rates of the Gd³⁺ꢀH and

ð14Þ

3

þ 1:54792 Â 10²e^{ð1:62931Â10 =TÞ}

Gd³⁺ꢀO vectors.

The residence time of the Gd³⁺-bound water molecule,

t_M, is an important parameter with regard to the efficiency

of an MRI contrast agent. The theoretical curve of the re-

laxivity as a function of t_Mhas a sharp maximum between

20 and 50 ns.^[1,2,4]The values of t_M²⁹⁸for the [Gd(L¹)(H₂O)]^3ꢀ

and [Gd(L²)(H₂O)]^2ꢀsystems (88 and 92 ns, respectively)

A good fit was obtained by assuming about two second-

sphere water molecules (q_2sꢁ2). The resulting optimised pa-

rameters are included in Table 4, and the results are also

shown as curves in Figure 4g i. Now, the optimised parame-

ters obtained all compare well with those previously report-

5908

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

are close to the optimum value. These t_Mvalues are lower

than those of the current commercial contrast agents. For

example, [Gd(dtpa)(H₂O)]^2ꢀand [Gd(dota)(H₂O)]^ꢀhave

t²_M⁹⁸values of 303 and 243 ns, respectively.^[36]Consequently, it

may be expected that very efficient MRI contrast agents can

be obtained from [Gd(L²)(H₂O)]^2ꢀby increasing its t_Rvalue

through covalent or noncovalent binding to macromolecules.

Unfortunately, the t²_M⁹⁸value of the [Gd(L³)(H₂O)]^2ꢀcom-

plex is considerably higher (685 ns) and, thus, it may be ex-

pected that for this complex t_Mis limiting the relaxivity

upon binding to a high-molecular-weight compound.

Evaluation of the electron-spin relaxation times T_2eof the

Gd³⁺complexes from EPR measurements: The X-band

(0.34 T) EPR spectra of the Gd³⁺complexes in aqueous sol-

ution at 298 K give approximately Lorentzian lines of g~2.0.

The transverse electronic relaxation rates (1/T_2e) were calcu-

lated from the experimental peak-to-peak line widths, DH_pp,

by using Equation (15), in which the symbols have their

usual meaning.^[43]

pﬃﬃﬃﬃﬃ

ð15Þ

1=T_2e¼ ðg_Lm_Bp 3hÞDH_pp

It has been shown that the water exchange in Gd³⁺-polya-

minocarboxylates with one Gd³⁺-bound water generally

takes place through a dissociative mechanism.^[35]Then,

steric strain at the water site may increase the energy of the

initial state and, therefore, decrease the activation energy.

The decrease in t²_M⁹⁸upon replacement of the central ꢀ

The experimental values of DH_ppobtained at 298 K were

([Gd(L¹)(H₂O)]^3ꢀ),

0.122Æ0.05 mT

0.103Æ0.05 mT

([Gd(L²)(H₂O)]^2ꢀ) and 0.182Æ0.04 mT ([Gd(L³)(H₂O)]^2ꢀ).

The corresponding values of ln(1/T_2e)_expare compared in

Table 4 with the ln(1/T_2e)_calcdvalues calculated by using

Equation (S9) (see Supporting Information), from the

values of the parameters D²and t_V²⁹⁸obtained from the si-

2ꢀ

CH₂COO^ꢀmoiety in [Gd(dtpa)(H₂O)]^2ꢀby a ꢀCH₂PO₃to

1

multaneous fitting of the ¹⁷O NMR and H NMRD data. Al-

form [Gd(L¹)(H₂O)]^3ꢀmay be rationalised by an increase in

steric strain around the Gd³⁺-bound water molecule due to

though the relative experimental and calculated 1/T_2evalues

follow very similar trends in the three Gd³⁺complexes, the

experimental values are systematically larger by a factor of

about three. This discrepancy has been noted before^[36,44,45]

and corrected by introducing both static and dynamic zero-

2ꢀ

the relatively large size of the ꢀPO₃function compared

with the ꢀCOO^ꢀfunction. An inspection of molecular

models shows that the phenyl group in [Gd(L²)(H₂O)]^2ꢀis

in the proximity of the Gd³⁺-bound water. Most likely,

[Gd(L³)(H₂O)]^2ꢀhas a preferred conformation with the

phenyl groups at large distances from the water site. The ni-

trogen atom of the dibenzylamino moiety is protonated

field-splitting effects in the electronic relaxation mechanisms

[46]

of Gd³⁺

.

(pK_a

=

6.58 for the corresponding La³⁺complex, see

Interaction of [Gd(L³)(H₂O)]^2ꢀwith human serum albumin:

To study the interaction between [Gd(L³)(H₂O)]^2ꢀand

HSA, a solution of the complex was added stepwise to a

4% solution of HSA in water. A nonlinear increase of the

water-proton paramagnetic longitudinal relaxation rate was

observed (see Figure 6) when plotted as a function of the

concentration of the Gd³⁺complex. The paramagnetic re-

above) and is in close proximity of the Gd³⁺-bound water

molecule. Possibly, the positive charge and the hydrogen

bonding between these functions may slow down the water

exchange rate and thus explain the rather long t²_M⁹⁸for this

complex.

The temperature dependence of the NMRD profiles usu-

ally gives a good indication of the parameter limiting the

proton relaxivity. If the relaxivity at high field (>10 MHz)

increases with increasing temperature, it is limited by slow

water exchange, whereas in the opposite case fast rotation is

the limiting factor. From the temperature dependence of re-

laxivity at constant magnetic field (20 MHz, see Figure 5) it

can be concluded that the total relaxivity decreases with in-

laxation rate of

a

solution containing 0.81 mm of

[Gd(L³)(H₂O)]^2ꢀin 4% HSA is 3.9 times higher than that of

0.81 mm of [Gd(L³)(H₂O)]^2ꢀin pure water; this indicates a

strong interaction between the complex studied and HSA.

This interaction is characterised by a stability constant (K_AS

)

of an adduct of HSA with the Gd³⁺complex [Eq.

creasing temperature, mainly because of a decrease of t_R²⁹⁸

.

Figure 6. Proton longitudinal paramagnetic relaxation rates in solutions

containing 4% HSA and increasing amounts of [Gd(L³)(H₂O)]^2ꢀ

(

^

)

Figure 5. Relaxivity at 20 MHz as

a

(

function of temperature for

measured at 20 MHz and 310 K. The full line corresponds to the fitting

of the data, and the dashed line represents R^p₁in an aqueous solution in

the absence of albumin.

2

2ꢀ

3

2ꢀ

[Gd(L¹)(H₂O)]^3ꢀ

), [Gd(L )(H₂O)]

) and [Gd(L )(H₂O)] ( ). Ex-

~

&

^

(

perimental data were not fitted–the lines are only guides for the eye.

5909

Chem. Eur. J. 2003, 9, 5899 5915

www.chemeurj.org

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

œ

I. Lukes, J. A. Peters et al.

FULL PAPER

(16)].^[47]The proton relaxivity data obtained in HSA, R^p₁^obs

,

were fitted to Equation (17)]; here p⁰is the protein concen-

tration, s⁰is the concentration of the paramagnetic complex,

n is the number of independent interaction sites and r^c₁and

r^f₁are the relaxivities of the [Gd(L³)(H₂O)]^2ꢀcomplex when

noncovalently bound to HSA and free, respectively. In this

fitting procedure, the association constant, K_AS, and r^c₁were

used as adjustable parameters.

½ðGdLÞ_nꢀHSA

n GdL þ HSA Ð ðGdLÞ_nꢀHSA K_AS

¼

n

½GdL ½HSA

ð16Þ

ð17Þ

ꢀ

ꢃ

1

2

R^p₁^obs¼ 1000 Â ðr₁^fÁ s⁰Þ þ ðr^c₁ꢀr₁^fÞ ðn Á p⁰Þ þ s⁰

ꢄꢅ

qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

ꢀ1

2

þ K

ꢀ

ððn Á p⁰Þ þ s⁰þ K Þ ꢀ4 N Á s⁰Á p⁰

AS

A good fit between experimental and calculated values

was obtained by using a model for one binding site (n = 1)

with an association constant K_AS= 4500Æ175m^ꢀ1. The re-

laxivity of noncovalently bound complex (r^c₁) was calculated

to be 43Æ0.4 s^ꢀ1mm^ꢀ1, while the value of the relaxivity of

free complex (r^f₁) is 5.9Æ0.3 s^ꢀ1mm^ꢀ1(see above). Thus, in a

solution containing 0.81 mm of [Gd(L³)(H₂O)]^2ꢀand 4%

(0.6 mm) HSA, 48.4% of Gd³⁺complex interacts with the

protein.

Figure 7. a) Proton-relaxation rate (R^p₁) of [Gd(L³)(H₂O)]^2ꢀ(0.81 mm)

Longitudinal relaxation rates of the solution containing

4% HSA and [Gd(L³)(H₂O)]^2ꢀ(0.81 mm) were measured at

310 K over the range of magnetic fields 4î10^ꢀ47.05 T. The

corresponding NMRD profile (Figure 7a) shows the expect-

ed hump, characteristic for interactions with macromole-

cules, appearing in the high-frequency part (ꢁ20 MHz) of

the recorded profile; this represents the combined contribu-

tions of the bound and free Gd³⁺complex in the solution.

*

dissolved in 4% HSA ( ). The dotted line corresponds to the proton-re-

laxation rate of [Gd(L³)(H₂O)]^2ꢀin pure water at the same concentra-

tion. b) Calculated theoretical ¹H NMRD profile of the complex

[Gd(L³)(H₂O)]^2ꢀfully bound to human serum albumin at 310 K ( ), ¹H

&

NMRD profile of the complex simulated with the parameters obtained

from simultaneous fit and optimal t_R= 14 ns.

1

The theoretical H NMRD profile of the [Gd(L³)(H₂O)]^2ꢀ-

HSA adduct was then calculated from the known NMRD

profile of free [Gd(L³)(H₂O)]^2ꢀand the concentrations of

free and bound complex obtained from the estimated stabili-

ty constant of the adduct (see above) (Figure 7b). The relax-

ivities in the frequency region of importance for MRI (20

100 MHz) are similar to those of MS-325.^[48]The NMRD

curve of the [Gd(L³)(H₂O)]^2ꢀHSA adduct could only be

fitted with a model that assumed ten water molecules in the

second sphere of the Gd³⁺ion (see Figure 7b). A lower

number of second-sphere water molecules always resulted in

calculated relaxivities too low for the low-field part (<10

MHz) of the NMRD curve. The results of the fitting possi-

bly reflect the presence of mobile HSA protons that are di-

polarly relaxed by the proximity of the Gd³⁺ion.^[6]Alterna-

tively, reduced mobility of solvent molecules in the second

coordination sphere of Gd³⁺upon noncovalent binding of

[Gd(L³)(H₂O)]^2ꢀto HSA can explain this result.

taining complexes studied, we performed some transmetalla-

tion studies with Zn²⁺according to a previously described

protocol.^[49]

Samples containing the Gd³⁺complexes of H₆L¹, H₅L²,

H₅L³and a phosphate buffer containing ZnCl₂were moni-

tored by measuring the ¹H relaxivity at 20 MHz. Upon

transmetallation with Zn²⁺, the free Gd³⁺formed immedi-

ately precipitated as the phosphate salt and, therefore, did

not contribute to the total relaxivity any more. The resulting

decrease in the proton-relaxation rate observed is a good es-

timate of the extent of transmetallation and, therefore, also

for the kinetic stability of the Gd³⁺complex. The results of

the transmetallation experiments are displayed in Figure 8,

while Table 5 shows the percentage of Gd³⁺complexes left

in the solution after 3 d. Thus, the kinetic stability de-

creases in the following order: [Gd(dtpa)(H₂O)]^2ꢀ

@

[Gd(L¹)(H₂O)]^3ꢀꢂ[Gd(L³)(H₂O)]^2ꢀꢂ[Gd(dtpa-

Transmetallation: An important parameter determining the

toxicity of Gd³⁺-based contrast agents is the kinetic stability

of the complexes. Transmetallation by endogenous metal

ions may afford free Gd³⁺ion, which is highly toxic. To get

an impression of the kinetic stability of the phosphorus-con-

bma)(H₂O)]>[Gd(L²)(H₂O)]^2ꢀ. Therefore, all complexes

studied are less stable towards Zn²⁺transmetallation than

[Gd(dtpa)(H₂O)]^2ꢀ,

but

[Gd(L¹)(H₂O)]^3ꢀ

and

[Gd(L³)(H₂O)]^2ꢀare slightly more kinetically stable than

[Gd(dtpa-bma)(H₂O)].

5910

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

actions was obtained from Acros. Bis(phthaloyl)diethylenetriamine (8),^[50]

ditosylethanolamine,^[51]N-tosylaziridine (1),^[52]and ethyl phenylphosphi-

nate^[53]were prepared by published methods. Paraformaldehyde was ob-

tained by filtration of aged aqueous formaldehyde solutions and was

dried in a desiccator over concentrated sulfuric acid.

¹H (300 MHz), ²H (46.1 MHz), ¹³C (75.5 MHz), ¹⁷O (40.7MHz) and ³¹P

NMR spectra (121.5 MHz) were recorded on a Varian INOVA-300 spec-

trometer with 5 mm sample tubes. Unless stated otherwise, NMR experi-

ments were performed at 258C. Chemical shifts are reported as d values

and are given in ppm. For measurements in D₂O, tert-butyl alcohol was

used as an internal standard with the methyl signal calibrated at 1.2 ppm

(¹H) or 31.2 ppm (¹³C). Deuterium oxide (100%) was used as an external

chemical-shift reference for ¹⁷O resonances. The ³¹P chemical shifts were

measured with respect to 1% H₃PO₄in D₂O as an external standard

(substitution method). The pHs of the samples were measured at ambient

temperature by using a Corning 125 pH meter with a calibrated micro-

combination probe from Aldrich. The pH values of the solutions were

adjusted by using dilute solutions of NaOH and HCl. The variable-tem-

perature ¹⁷O measurements were performed at a magnetic field of 7.05 T

on a Varian INOVA-300 spectrometer equipped with a 5 mm probe.

Thin-layer chromatography was performed on silica-coated aluminium

sheets (Silufol, Kavalier, Czech Republic).

Figure 8. Evolution of the relative water proton paramagnetic longitudi-

nal relaxation rate R₁^p(t)/R₁^p(0) vs. time for [Gd(dtpa)(H₂O)]^2ꢀ

(

^

),

3

2ꢀ

[Gd(L¹)(H₂O)]^3ꢀ(&), [Gd(L²)(H₂O)]^2ꢀ( ) and [Gd(L )(H₂O)]

( ). The

~

&

lines are only guides for the eye. The solution initially contained Gd³⁺

complex (2.5 mm), ZnCl₂(2.5 mm) and phosphate (H₂PO₄^ꢀ, HPO₄

PO₄^3ꢀ, 67 mm).

,

2ꢀ

Table 5. Percentage of remaining Gd³⁺complexes after 3 d of transme-

Mass spectra were obtained with a VG AUTOSPEC 6F mass spectrome-

ter (VG Analytical, Manchester, UK) or on a Bruker ESQUIRE3000

with ion-trap detection in positive or negative modes.

tallation with Zn²⁺

.

p

Complexes

R_{1 (t=3d)}/R₁

[%]

(t=0)

Preparation of N’-benzyldiethylenetriamine (4): N-tosylaziridine (1)

(32.0 g, 162 mmol) was dissolved in ethanol (150 mL). Benzylamine (2)

(8.00 g, 75 mmol) was added, and the solution was heated under reflux

for 3 days. Then, the mixture was cooled and concentrated aqueous am-

monia (5 mL) was added to quench the excess of tosylaziridine. The mix-

ture obtained was heated under reflux for 15 min. Solvents were re-

moved, and the residual brown oil was dissolved in a mixture of concen-

trated HBr/AcOH (300 mL, 1:1, v/v). The solution was heated under

reflux for 24 h. After cooling, the reaction mixture was evaporated to

dryness leaving a brown oil, which solidified upon cooling. This solid was

dissolved in NaOH (100 mL, 15%), and the solution obtained was ex-

tracted with chloroform (7î50 mL). The organic phases were combined

and evaporated to dryness to leave the mixture of monotosylated inter-

mediate and the required product as a yellow oil. These compounds were

separated by chromatography on silica by using gradient elution with in-

creasing concentration of concentrated ammonia in ethanol from a ratio

of 1:25 (mixture A) to 1:5 (mixture B), detection by ninhydrine (purple

spots). Note: extension of the reaction period to 36 48 h reduces the

yield of the final product due to decomposition.

[Gd(L¹)(H₂O)]^3ꢀ

[Gd(L²)(H₂O)]^2ꢀ

[Gd(L³)(H₂O)]^2ꢀ

[Gd(dtpa)(H₂O)]^2ꢀ

13

1.9

11

49

9

[Gd(dtpa-bma)(H₂O)]

Conclusion

A useful synthetic approach for a new class of H₅DTPA-

based ligands in which the central pendant arm has a ꢀ

P(OH)(O)R moiety is reported. Their Ln³⁺complexes show

structural features analogous to the H₅DTPA complexes, in-

cluding the presence of one water molecule coordinated in

the first coordination sphere of the metal ion. The phospho-

nate (H₆L¹) and the phenylphosphinate derivatives (H₅L²)

have t²_M⁹⁸values that are close to optimal (88 and 92 ns, re-

spectively). Therefore, these complexes are very suitable for

attachment to polymers, which should result in compounds

with very high relaxivity. The relaxivity will be less limited

by water exchange than in most conjugates of chelates of

the H₅DTPA or H₄DOTA type. Furthermore, an additional

increase in the relaxivity is obtained by virtue of the pres-

ence of two water molecules in the second coordination

sphere and which are probably bound to the phosphonate/

phosphinate moiety through hydrogen bonds. The

[Gd(L³)(H₂O)]^2ꢀcomplex has a less favourable water-ex-

change rate (t²_M⁹⁸= 685 ns), but it has a high affinity to

HSA and a relaxivity that is comparable with that of the

well-known blood pool contrast agent MS-325.

Yield: 8.25 g (57%), R_f(mixt. A) = 0.1, R_f(mixt. B) = 0.3; ¹H NMR

(CDCl₃): d = 2.05 (br, 4H; NH₂), 2.52, 2.75 (2m, 4H; NCH₂CH₂N), 3.58

(s, 2H; NCH₂Ph), 7.31 (m, 5H; Ph); ¹³C NMR (CDCl₃): d = 34.58 (2C),

57.05 (2C, NCH₂CH₂N), 59.14 (1C, PhCH₂N), 127.02 (1C), 128.30 (2C),

128.86 (2C) and 139.49 (1C, all Ph); ESI-MS: positive m/z: 194.0

[M+H]⁺.

N-Tosyl-N’-benzyldiethylenetriamine: Yield: 4.55 g (18%); R_f(mixt. A)

= 0.8, R_f(mixt. B) = 0.9; ¹H NMR (CDCl₃): d = 2.40 (s, 3H; CH₃),

2.49 (m, 2H), 2.58 (m, 2H), 2.76 (m, 2H) and 2.95 (m, 2H; all

NCH₂CH₂N), 3.05 (br, 1H; NH), 3.52 (s, 2H; NCH₂Ph), 7.20 (m, 2H;

Ts), 7.27 (m, 5H; Ph), 7.73 (m, 2H; Ts); ¹³C NMR (CDCl₃): d = 22.12

(1C, CH₃), 39.73, 42.11, 53.14, 55.99 (4î1C, NCH₂CH₂N), 59.83 (1C,

NCH₂Ph), 127.67 (2C), 127.82 (1C), 129.01 (2C), 129.45 (2C), 130.20

(2C), 137.82 (1C), 139.51 (1C), 143.58 (1C); ESI-MS: positive m/z: 348.3

[M+H]⁺.

Preparation of N’-benzyldiethylenetriamine-N,N,N’’,N’’-tetraacetic acid

(5): N’-benzyldiethylenetriamine (4) (1.00 g, 5.2 mmol) was dissolved in

water (5 mL) and then 1 mL of a solution of NaOH (2.5 g, 62.5 mmol,

dissolved in 10 mL of water) was added. The mixture was heated to

908C. Ethyl bromoacetate (4.34 g, 26 mmol) and the remaining NaOH

solution were added in 6 portions during 1.5 h. The solution was then

heated under reflux for 24 h to hydrolyse the ester groups. After cooling,

the mixture was poured onto a column containing a strong cation ex-

change resin (150 mL, Dowex50) in the H⁺form. The column was

washed with water and the product was eluted with diluted aqueous am-

monia (1:3). The eluate was evaporated to dryness to leave the crude

Experimental Section

Materials and methods: Commercially available benzylamine (2), diethy-

lenetriamine, phthalanhydride, ethyl chloroformate, ethyl bromoacetate,

diethylphosphite and phenylphosphinic acid had synthetic purity and

were used as received. The 10% Pd/C catalyst for the hydrogenation re-

5911

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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

œ

I. Lukes, J. A. Peters et al.

FULL PAPER

product as a yellow oil. This product was purified by chromatography on

a strong anion exchanger (Dowex1) in the acetate form. After washing

with 10% AcOH, the product was eluted with 5% HCl. The product was

precipitated as a nonstoichiometric hydrochloride (2.5 3 equiv HCl per

tions. The fractions containing the product were evaporated to dryness

leaving a glassy solid, which was dissolved in small amount of water and

crystallised by standing for several days. The white solid obtained was fil-

tered, washed with acetone and air-dried. Yield: 10.15 g (76%); m.p.

133 1358C (dec.); ¹H NMR (D₂O, pD 0.7): d = 3.01 (2H; NCH₂P), 3.22

(4H; NCH₂CH₂N) 3.39 (4H; NCH₂CH₂N), 3.96 (8H; NCH₂CO); ¹³C

NMR (D₂O, pD 0.7): d = 49.64 (d, ¹J_CP=147Hz, 1C, N CH₂P), 50.89

(2C, PCH₂NCH₂), 51.86 (2C, PCH₂NCH₂CH₂N), 55.30 (4C, NCH₂CO),

169.56 (4C, CO); ³¹P NMR (D₂O, pD 0.7): d = 15.75; elemental analysis

calcd (%) for H₆L¹¥HCl¥H₂O (C₁₃H₂₇ClN₃O₁₂P, M = 483.79): C 32.27, H

5.63, Cl 7.33, N 8.69; found: C 31.90, H 5.49, Cl 7.33, N 8.45; ESI-MS:

positive m/z: 430.3 [M+H]⁺; negative m/z: 428.3 [MꢀH]^ꢀ.

Preparation of diethylenetriamine-N’-methylene(phenyl)phosphinic-

N,N,N’’,N’’-tetraacetic acid (H₅L²): Freshly prepared ethyl phenylphos-

phinate^[53](13.80 g, 81.1 mmol, 2.9 equiv) was transferred into a 250 mL

flask and dissolved in a mixture of toluene (150 mL) and dry ethanol (30

mL). Bis(phthaloyl)diethylenetriamine (8) (10.00 g, 27.5 mmol) was

added, and then the mixture was heated under reflux with a Dean Stark

trap. During the next 6 h, paraformaldehyde (2.48 g, 82.5 mmol, 3 equiv)

was added in portions. The solvent in the trap was removed, and then the

mixture was heated for another 14 h at 1008C. The mixture was cooled

and filtered. Ethyl hydroxymethyl(phenyl)phosphinate formed as a by-

product (d = 40.1 ppm in ³¹P NMR spectra) and some starting ethyl phe-

nylphosphinate were extracted with water (10î50 mL), the organic layer

was dried over Na₂SO₄and then evaporated to dryness leaving a yellow

oil. ³¹P NMR spectra indicated that the product (9) was contaminated

with a small amount of starting ethyl phenylphosphinate. ¹³C NMR

(CDCl₃): d = 16.32 (2C, CH₃), 35.30 (2C, NCH₂CH₂NPht), 52.89 (d,

³J_CP=5.4 Hz, 2C, NCH₂CH₂NPht), 52.96 (d, ¹J_CP=112 Hz, 1C, NCH₂P),

60.65 (d, ²J_CP=6.6 Hz, 2C, POCH₂), 123.01, 132.04 and 133.75 (all Pht),

128.39 (d, ³J_CP=12.1 Hz, 2C, Ph), 130.32 (d, ¹J_CP=119 Hz, 1C, Ph),

131.76 (d, ²J_CP=9.4 Hz, 2C, Ph), 132.30 (1C, Ph), 167.97 (4C, CO); ³¹P

NMR (CDCl₃): d = 40.18.

1

ligand molecule) upon trituration in acetone. Yield: 2.10 g (~75%); H

NMR (D₂O, pD 2.5): d = 2.95 and 3.17(br, 8H; NC H₂CH₂N), 3.57(s,

8H; NCH₂CO), 3.62 (s, 2H; NCH₂Ph), 7.36 (m, 5H; Ph); ¹³C NMR

(D₂O, pD 2.5): d = 48.60 (2C, NCH₂CH₂N), 53.70 (2C, NCH₂CH₂N),

58.26 (4C, NCH₂CO), 59.30 (1C, NCH₂Ph), 129.54 (1C, Ph), 130.42 (2C,

Ph), 131.07(2C, Ph), 138.50 (1C, Ph), 171.53 (4C, CO).

Preparation of the lactam of diethylenetriamine-N,N,N’’,N’’-tetraacetic

acid (6): N-benzylated tetraacetate (5) (1.00 g of the hydrochloride) was

dissolved in water (10 mL). Acetic acid (5 mL) was added, followed by

10% Pd/C catalyst (0.10 g). The mixture was stirred at room temperature

under a hydrogen atmosphere for 48 h. Then the catalyst was removed

by filtration, and all solvents were evaporated in vacuo. The product was

purified by chromatography on Dowex 1 in the acetate form (75 mL).

Some unidentified impurities were eluted with acetic acid (1 5% in

water) and, subsequently, the required product was eluted with 10%

AcOH. Evaporation of solvents afforded the product as yellowish oil

(0.56 g, 95%). ¹H NMR (D₂O, pD 3.5):

d = 2.5 2.7(br, 8H;

NCH₂CH₂N), 3.03 (2H), 3.13 (s, 2H; CH₂CO), 3.18 (s, 4H; CH₂CO); ¹³C

NMR (D₂O, pD 3.5): d = 44.40, 44.90, 50.41, 54.22 (4î1C, NCH₂CH₂N),

56.19 (1C, NCH₂CO), 56.36 (2C, NCH₂CO), 57.33 (1C, NCH₂CO),

166.56 (1C, CON), 168.63 (1C) and 169.51 (2C, COO).

Preparation of diethylenetriamine-N’-methylenephosphonic-N,N,N’’,N’’-

tetraacetic acid (H₆L¹): Bis(phthaloyl)diethylenetriamine (8) (10.00 g,

27.5 mmol) and diethylphosphite (11.4 g, 82.5 mmol) were dissolved in a

mixture of toluene (100 mL) and dry ethanol (30 mL), then the mixture

was heated under reflux with a Dean Stark trap. Over a period of 6 h,

paraformaldehyde (2.48 g, 82.5 mmol) was added in portions. The reac-

tion mixture was heated under reflux for another 12 h. After cooling, the

mixture was filtered and the solvent was evaporated under vacuum.

¹³C NMR of intermediate (9) (CDCl₃): d = 16.08 (2C, CH₃), 35.00 (2C,

NCH₂CH₂NPht), 48.24 (d, ¹J_CP=150 Hz, 1C, NCH₂P), 52.47(2C,

NCH₂CH₂NPht), 61.45 (2C, POCH₂), 122.66, 131.79 and 133.61 (all Pht),

167.64 (4C, CO). Signals due to the excess of diethylphosphite overlap-

ped the ethoxy resonances of the product in spectra of the crude mixture.

Furthermore, an additional signal of diethyl hydroxymethylphosphonate

(doublet of NCH₂P centred at 57.5 ppm) was observed.

The material obtained was dissolved in dry ethanol (80 mL). Hydrazine

hydrate (3.44 g, 68 mmol) was added, and the mixture was heated under

reflux for 10 h. Precipitation of phthalhydrazide began after about 10 min

of reflux. The mixture was cooled, and the phthalhydrazide formed was

filtered off. The ethanol was evaporated, and water was removed by co-

distillation with dry ethanol. The residue (intermediate (10)) was dis-

solved in DMF (80 mL). Then, BrCH₂CO₂Et (36.7g, 220 mmol, 8 equiv)

and K₂CO₃(30.4 g, 220 mmol) were added. The mixture was stirred at

room temperature for 24 h. After filtering off the solids, the reaction mix-

ture was diluted with a saturated aqueous solution of NaHCO₃(50 mL),

and the product was extracted into toluene (150 mL). The organic layer

was washed with a saturated solution of NaHCO₃(2î150 mL) and with

brine (1î100 mL). The toluene phase was dried (Na₂SO₄), filtered and

evaporated to dryness. NMR data of intermediate (11): ¹H NMR

(CDCl₃): d = 1.26 (m, 15H; CH₃), 2.73 (m, 8H; backbone CH₂), 3.10 (d,

²J_CP=10 Hz, 2H; NCH₂P), 3.48 (s, 8H; CH₂CO₂), 4.15 (m, 10H; OCH₂),

7.50, 7.84 (m, 5H; Ph); ³¹P NMR (CDCl₃): d = 40.48, and small impuri-

ties at ~20, 22, 35 and 40 ppm (<10%).

The material obtained was dissolved in dry ethanol (60 mL). Hydrazine

hydrate (3.44 g, 68 mmol) was added, and the mixture was heated under

reflux for 6 h. Precipitation of phthalhydrazide began after ~10 min of

reflux. The mixture was cooled, and phthalhydrazide was filtered off. The

ethanol was evaporated, and water was removed by co-distillation with

dry ethanol.

The intermediate obtained (10) was dissolved in DMF (70 mL), and then

BrCH₂CO₂Et (36.7g, 220 mmol, 8 equiv) and K ₂CO₃(30.4 g, 220 mmol)

were added. The mixture was stirred at room temperature for 18 h. The

solids were filtered off, after which the reaction mixture was diluted with

a saturated aqueous solution of NaHCO₃(70 mL). Subsequently, the

product was extracted into toluene (200 mL). The organic layer was

washed with a saturated solution of NaHCO₃(70 mL) and of brine (70

mL). The toluene phase was dried over Na₂SO₄, filtered and then evapo-

The crude intermediate (11) obtained above was dissolved in diluted HCl

(250 mL, 1:1) and the mixture was heated under reflux for 18 h. During

this time some precipitate formed. After cooling, the suspension was fil-

tered, and nonpolar impurities were extracted with CHCl₃(3î50 mL).

Then, the aqueous phase was evaporated to dryness. The residue was dis-

solved in water and poured onto a cation-exchange column (Dowex 50,

170 mL, H⁺form). The nonbasic impurities were removed by elution

with water. A yellow fraction containing the desired product was ob-

tained by elution with a diluted ammonia (1:3) solution. This fraction

was evaporated to dryness, redissolved in water and chromatographed on

an anion exchanger (Dowex 1, 250 mL, OH^ꢀform). After washing with

water, some yellow-coloured impurities were removed with 10% acetic

acid. The product was collected by elution with HCl (1:3). The solvents

were evaporated off, and the product was dissolved in concentrated HCl

(30 mL). Treatment with acetone (800 mL) gave a light yellow oil, which

solidified upon standing. The white precipitate was filtered off, washed

with acetone and dried at 808C. Yield: 8.63 g (54%); m.p. 132 1348C

(dec.); ¹H NMR (D₂O, pD 0.5): d = 3.07and 3.12 (10H; unresolved mul-

tiplets, NCH₂CH₂N and NCH₂P), 4.02 (s, 8H; NCH₂CO), 7.58, 7.63 and

7.80 (m, 5H; Ph); ¹³C NMR (D₂O, pD 0.5): d = 47.19 (d, ³J_CP=6.0 Hz,

1

rated to dryness. NMR of intermediate (11): H NMR (CDCl₃): d = 1.16

(m, 18H; CH₃), 2.80 (m, 8H; backbone CH₂), 3.04 (d, ²J_CP=10 Hz, 2H;

NCH₂P), 3.51 (s, 8H; CH₂CO₂), 4.15 (m, 12H; OCH₂); ³¹P NMR

(CDCl₃): d = 26.00. ESI-MS: positive m/z: 598.4 [M+H]⁺.

The crude ethyl ester (11) obtained above was dissolved in diluted HCl

(250 mL, 1:1), and the mixture was heated under reflux for 18 h. During

this time, some precipitate (probably remains of phthalic acid) was

formed. The mixture was filtered and then evaporated to dryness.

The residue was dissolved in water and poured onto a column of cation

exchange resin (Dowex 50, 170 mL, H⁺form). Nonbasic compounds

were removed by elution with water. The crude product was eluted using

a diluted ammonia (1:3) solution. The yellow fraction was evaporated to

dryness, dissolved in water and then purified by chromatography on an

anion exchange column (Dowex 1, 250 mL, acetate form). After being

washed with water, some yellow impurities were removed by elution with

10% acetic acid. The product was collected in diluted HCl (3%) frac-

5912

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2003, 9, 5899 5915

Lanthanide Complexes of Diethylenetriamine

5899 5915

2C, PCH₂NCH₂), 49.83 (2C, PCH₂NCH₂CH₂N), 52.10 (d, ¹J_CP=111 Hz,

1C, NCH₂P), 56.69 (4C, NCH₂CO), 125.89 (d, ³J_CP=12.1 Hz, 2C, Ph),

128.57(d, ²J_CP=9.1 Hz, 2C, Ph), 128.71 (1C, Ph), 133.03 (d, ¹J_CP=122

Hz, 1C, Ph), 167.97 (4C, CO); ³¹P NMR (D₂O, pD 0.5): d = 31.63; ESI-

MS: positive m/z: 490.3 [M+H]⁺, negative m/z: 488.3 [MꢀH]^ꢀ; elemen-

(CDCl₃): d = 1.08 (br, 3H; CH₃), 1.75 (br, 4H), 2.45 (br, 8H), 2.50 (br,

8H), 2.68 (br, 8H), 3.60 (s, 4H; CH₂Ph), 4.00 (br, 2H; OCH₂), 7.10 (br,

10H; Ph); ¹³C NMR (CDCl₃): d = 16.98 (1C, CH₃), 39.82 (2C, CH₂Ph),

50.59 (d, ¹J_CP=113 Hz, 1C, CH₂P), 51.99 (d, ¹J_CP=116 Hz, 1C, CH₂P),

59.17and 59.91 (2î2C, CH₂CH₂), 60.90 (d, ²J_CP=7Hz, 1C, O CH₂),

127.45 (2C, Ph), 128.49 (4C, Ph), 129.35 (4C, Ph), 138.59 (2C, Ph); ³¹P

NMR (CDCl₃): d = 49.69.

tal analysis calcd (%) for H₅L²¥2HCl¥H₂O (C₁₉H₃₂Cl₂N₃O₁₁P,

M =

580.35; based on ¹H NMR, the product is slightly contaminated by ace-

tone) C 39.32, H 5.56, Cl 12.22, N 7.24; found: C 39.82, H 5.43, Cl 11.50,

N 7.17.

Compound (10) (5.90 g, 14.1 mmol) was stirred with BrCH₂COOEt (11.8

g, 70 mmol) and K₂CO₃(9.7g, 70 mmol) in DMF (50 mL) at room tem-

perature for 24 h. The reaction mixture was then extracted with toluene

(100 mL), and the organic phase was extracted aqueous NaHCO₃(3î

100 mL). After evaporation, a solution of NaOH (10 g) in water (50 mL)

was added. Then, EtOH (ꢁ50 mL) was added to obtain a homogeneous

solution. This solution was stirred at room temperature for two days. The

³¹P NMR spectrum showed that the reaction was complete (one major

peak at 35.9 ppm). The reaction mixture was evaporated until dryness.

The residue was dissolved in a minimum amount of water and then puri-

fied on a column of a strong cationic exchanger (Dowex 50, 150 mL,

After evaporation of the mother liquor, a second crop of the product

(1.02 g) was isolated in the same way.

Preparation of (N,N-dibenzylamino)methylphosphinic acid: Dibenzyla-

mine (5.00 g, 25.3 mmol) was dissolved in ethanol (50 mL, 96%). Two

equivalents of paraformaldehyde (1.52 g, 50.7mmol) were added, and the

mixture was heated to 608C. Hypophosphorus acid (10.0 g of 50% aq.

solution, 76.0 mmol) was added, and the reaction mixture was stirred at

608C for 24 h. Then, chromatography on a strong cation exchanger in the

H⁺form (Dowex 50, 200 mL, elution with water/ethanol 1:1 v/v and di-

luted ammonia), followed by chromatography on a strong anion exchang-

er (Dowex 1, 250 mL, acetate form, elution with water and 10% acetic

acid), afforded the product as slightly yellow oil, which crystallised upon

standing at room temperature. Yield: 6.30 g (85%); m.p. 66 688C; ¹H

+

NH₄form), with water (ꢁ1000 mL) as the eluent. After elution, a ³¹P

NMR spectrum of the eluate was taken and showed the presence of

H₅L³. The fractions concerned were evaporated to obtain a yellow oil,

which was crystallised at room temperature from an ethanol solution

containing a small amount of acetone. The crystallisation started immedi-

ately. The product was filtered, washed with small amounts of ethanol

and acetone, and dried at 808C. Yield: 4.1 g (47%); m.p. 139 142 8C

NMR (CDCl₃): d

=

2.91 (d, ²J_PH=7.2 Hz, 2H; CH₂P), 4.27(s, 4H;

CH₂Ph), 7.31 (d, ¹J_PH=534 Hz, 1H; Ph), 7.36 (m, 10H; Ph), 7.51 (m,

10H; Ph); ¹³C NMR (CDCl₃): d = 50.60 (d, ¹J_PC=84 Hz, CH₂P), 58.36

3

(d, J_PC=4.6 Hz, NCH₂Ph), 129.04, 129.56, 129.73 and 131.37 (all Ph); ³¹P

(dec.); ¹H NMR (D₂O, pD 8.02): d = 2.66 (dd, ²J_HP=9.6, 8.0 Hz, 4H;

NMR (CDCl₃): d = 7.97 (dm, ¹J_PH=537Hz); ESI-MS: negative m/z:

274.1 [MꢀH]^ꢀ; elemental analysis calcd (%) for monohydrate

(C₁₅H₂₀NO₃P, M = 293.30) C 61.43, H 6.87, N 4.78; found: C 61.24, H

6.69, N 4.96.

3

NCH₂PCH₂N), 2.85 (t, ³J_HH=6.8 Hz, 4H; NCH₂CH₂N), 3.07(t, J_HH

=

6.8 Hz, 4H; NCH₂CH₂N), 3.58 (s, 8H; NCH₂COOH), 3.72 (s, 4H;

NCH₂Ph), 7.33 (m, 10H; Ph), 7.40 (m, 10H; Ph); ¹³C NMR (D₂O, pD

8.02): d = 51.50 (d, ³J_CP=5 Hz, 2C, NCH₂CH₂NCH₂CH₂N), 53.06 (d,

¹J_CP=29.8 Hz, 1C, PCH₂NCH₂CH₂N), 53.58 (d, ¹J_CP=75.5 Hz, 1C,

PCH₂NBn), 58.89 (2C, NCH₂CH₂NCH₂COOH), 60.63 (d, ³J_CP=6.8 Hz,

2C, NCH₂Ph), 71.34 (4C, NCH₂COOH), 129.18 (Ph), 130.18 (Ph), 131.65

Preparation of ethyl (N,N-dibenzylamino)methylphosphinate: (N,N-di-

benzylamino)methylphosphinic acid (3.03 g, 11.0 mmol) was suspended in

chloroform (50 mL), and ethyl chloroformate (1.31 g, 12.1 mmol) was

added. After 15 min, pyridine (0.96 g, 12.1 mmol) was added drop-wise

(CO₂was evolved immediately, the mixture remained heterogeneous).

After 24 h at room temperature, a sample for ³¹P NMR was filtered off.

It showed only 65% conversion of acid to ester. Therefore, new portions

of chloroformate (1 g) and pyridine (1.5 g) were added. After another 24

h, only one signal belonging to the desired ethyl ester was observed in

the ³¹P NMR spectrum (~35 ppm). The reaction mixture was washed

with water (3î30 mL), dried (Na₂SO₄) and evaporated to dryness. The

residue, a yellowish oil, was redissolved in toluene and evaporated in

order to remove any excess of chloroformate. Yield: 3.30 g (99%); ¹H

NMR (CDCl₃): d = 1.34 (t, 3H; CH₃), 2.99 (m, 2H; NCH₂P), 3.78 (4H;

AB-system, CH₂Ph), 4.07(m, OC H₂), 6.97(m, P- H, ¹J_PH=540 Hz, 1H),

7.35 (m, 10H; Ph); ¹³C NMR (CDCl₃): d = 16.45 (d, ³J_CP=6.0 Hz, 1C,

CH₃), 51.42 (d, ¹J_CP=114 Hz, 1C, NCH₂P), 60.01 (d, ³J_CP=7.7 Hz, 2C,

CH₂Ph), 62.45 (d, ³J_CP=7.7 Hz, 1C, OCH₂), 127.58 (2C), 128.58 (4C),

129.20 (4C) and 138.37(2C; all Ph); ³¹P NMR (CDCl₃): d = 36.85 (dm,

¹J_PH=547Hz).

(Ph), 139.73 (Ph), 174.14 (4C, CO); ³¹P NMR (D₂O, pD 8.02): d

=

34.54; elemental analysis calcd (%) for H₅L³¥2NH₃¥0.5H₂O

(C₂₈H₄₆N₆O_10.5P, M = 565.68) C 50.52, H 6.97, N 12.62; found: C 50.38,

H 6.69, N 12.68; ESI-MS: positive m/z: 623.4 [M+H]⁺; negative m/z:

621.7[ MꢀH]^ꢀ.

pH Dependence of ¹H and ³¹P NMR spectra: For these experiments,

0.1m solutions of ligands H₆L¹and H₅L²in H₂O/D₂O (9:1) and H₅L³and

[La(L³)(H₂O)]^2ꢀin D₂O were prepared. The pH was adjusted by stepwise

addition of a solution of NaOH or HCl (both prepared in H₂O/D₂O (9:1)

for H₆L¹and H₅L²and in pure D₂O for H₅L³and [La(L³)(H₂O)]^2ꢀ). The

pH values reported for H₅L³were corrected for the deuterium effect by

using the relationship pD=pH + 0.4.^[54]A drop of tBuOH (d

= 1.2

ppm) was added to the samples as internal reference for ¹H. The assign-

ment of the backbone hydrogen atoms was done by using selective ¹H-

decoupled ¹³C NMR spectroscopy for the free ligands and COSY spectra

for [La(L³)(H₂O)]^2ꢀ. The calculations were performed by using the com-

puter programs Micromath Scientist, version 2.0 (Salt Lake City, UT,

USA) or OPIUM.^[55]Both of them resulted in the same values of pK_a

(within standard errors).

Preparation of diethylenetriamine-N’-methylene(dibenzylaminomethyl)-

phosphinic-N,N,N’’,N’’-tetraacetic acid (H₅L³): The freshly prepared ethyl

(N,N-dibenzylamino)methylphosphinate (3.30 g, 10.9 mmol, 2 equiv) was

dissolved with bis(phthaloyl)diethylenetriamine (8) (2.00 g, 5.5 mmol) in

a mixture of toluene (50 mL) and ethanol (30 mL). The mixture was

heated under reflux with a Dean Stark trap. Over a period of 6 h, para-

formaldehyde (0.50 g, 16.7mmol, 3 equiv) was added in portions. The re-

action mixture was refluxed overnight. An undecoupled ³¹P NMR spec-

trum showed some unreacted PꢀH precursor (doublet at ~33 ppm) to-

gether with some phosphonic acid (~15 ppm) and two signals at ~48

ppm (probably the desired product (9) and hydroxymethyl(N,N-dibenzyl-

amino)methylphosphinate). More paraformaldehyde was added, and the

mixture was heated under reflux for another 12 h. During that time all of

the PꢀH precursor disappeared. The reaction mixture was evaporated to

dryness and re-dissolved in dry ethanol (40 mL). Hydrazine hydrate

(0.83 g, 16.5 mmol, 3 equiv) was added, and the mixture was heated

under reflux overnight. Precipitation of phthalhydrazide began after 10

min of reflux. The mixture was cooled, phthalhydrazide was filtered off,

and the solvent was evaporated, yielding a yellowish oil. The product

(10) was separated by chromatography on silica by using an aqueous am-

monia/ethanol gradient (1:20 to 1:5). Yield 1.45 g (63%); ¹H NMR

Analysis of induced shifts in ¹⁷O NMR spectra of lanthanide(iii) com-

plexes: Samples with a complex concentration of about 0.2m were pre-

pared. The solid lanthanide(iii) chlorides and solid ligands (~10%

excess) were dissolved in a weighed amount of D₂O in a small vial con-

taining a stirring bar and a microscopic grain of methyl red indicator.

The vials were capped with a septum, and then a solution of 10 15%

sodium hydroxide in D₂O was added dropwise from a syringe with stir-

ring till a colour change of the indicator (pH~4 6). Then the vial was

weighed again. From the increase of the weight, the molar ratio of ex-

changeable oxygen atoms per Ln³⁺ion was calculated.

Relaxation enhancements in ¹³C and ³¹P NMR spectra of lanthanide(iii)

complexes: For these experiments, the samples prepared for ¹⁷O NMR

experiments described above were used. The longitudinal relaxation

rates were measured at 808C by using the inversion-recovery method.^[56]

Variable temperature ¹⁷O NMR study of Gd³⁺complexes: Solutions of

Gd³⁺complexes of H₆L¹, H₅L²and H₅L³with a concentration of about

0.15m were prepared by dissolution of exactly weighed solid GdCl₃¥6H₂O

and the solid ligands (H₆L¹and H₅L²as the hydrochloride salts, ~10%

5913

Chem. Eur. J. 2003, 9, 5899 5915

www.chemeurj.org

¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

œ

I. Lukes, J. A. Peters et al.

FULL PAPER

1

excess) in a weighed amount of deionised water. Calculated amounts of

sodium hydroxide (9 and 8 equivalents for H₆L¹and H₅L²or H₅L³, re-

spectively, as 10% solution) were added dropwise with stirring. After the

mixture had stood for 30 min at room temperature, the pH was checked.

All samples gave a negative Xylenol orange test for the presence of free

Gd³⁺. All NMR spectra were conducted without a frequency lock. To

correct the ¹⁷O NMR shift for the contribution of the bulk magnetic sus-

ceptibility (BMS), the difference between chemical shifts of proton sig-

nals of acetone (or tert-butanol) in the paramagnetic sample and in pure

water was used.^[57]Longitudinal (1/T₁) and transversal (1/T₂) relaxation

rates were obtained by the inversion-recovery method^[56]and the Carr -

Purcell Meiboom Gil pulse sequence,^[58]respectively. Experimental data

were fitted with a computer program written by Dr. …. TÛth and Dr. L.

Helm (EPFL Lausanne, Switzerland) using the Micromath Scientist pro-

gram, version 2.0 (Salt Lake City, UT, USA).

The transmetallation was followed by the H longitudinal-relaxation rates

of the water at Bruker Minispec 20 MHz (Bruker, Karlsruhe, Germany)

at 378C.

Acknowledgement

This work was performed within the framework of the EU COST Action

D18: Lanthanide chemistry for diagnosis and therapy. Thanks are due to

the EU for financial support through a Marie Curie training site host fel-

lowship (QLK5-CT-2000-60062) and to Kristina Djanashvili for some

NMR measurements. The work was supported by the Grant Agency of

the Czech Republic (203/02/0493 and 203/03/0168). J.K. thanks the

Charles University Grant Agency (250/2003/B-Ch).

Concentration dependence of ²H NMR longitudinal-relaxation time:

Deuterium-containing ligands were prepared by H D exchange from

H₆L¹, H₅L²and H₅L³in D₂O/K₂CO₃(pD 10.5) by heating mixtures at

958C for 5 d, analogously to the method reported in the literature.^[31]The

ligand and LaCl₃¥7H₂O (equivalent amounts) were dissolved in deuteri-

um-depleted water (1 mL). Then the pH was adjusted to a value close to

neutral by addition of small portions of solid LiOH¥H₂O. The transversal-

relaxation rates were measured by using the inversion-recovery pulse se-

quence.

EPR Measurements: EPR spectra of the Ln^IIIcomplexes in aqueous sol-

ution were recorded on a Bruker ESP300E spectrometer operating at

9.43 GHz (0.34 T, X-band). 5 mm aqueous solutions of the complexes

were measured at 298 K in a quartz flat cell. Typical parameters used

were: sweep width 40 mT, microwave power 20 mW, modulation ampli-

tude 0.32 mT and time constant 0.02 s. The frequency was calibrated with

diphenylpicrylhydrazyl (dpph) and the magnetic field with Mn²⁺in MgO.

[1] The Chemistry of Contrast Agents in Medical Magnetic Resonance

Imaging (Eds.: A. E. Merbach, …. TÛth), Wiley, Chichester, 2001.

[2] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev.

1999, 99, 2293.

[3] S. Liu, D. S. Edwards, Bioconjugate Chem. 2001, 12, 7 .

[4] R. B. Lauffer, Chem. Rev. 1987, 87, 901.

[5] V. Jacques, J. F. Desreux, Top. Curr. Chem. 2002, 221, 123.

[6] S. Aime, M. Botta, M. Fasano, E. Terreno, Chem. Soc. Rev. 1998, 27,

19.

œ

¬

[7] I. Lukes, P. CÌgler, J. Kotek, J. Rudovsky, P. Hermann, J. Rohovec, P.

œ

VojtÌsek, J. Inorg. Biochem. 2001, 86, 68.

[8] S. Aime, M. Botta, S. G. Crich, G. Giovenzana, R. Pagliarin, M.

Sisti, E. Terreno, Magn. Reson. Chem. 1998, 36, S200.

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4017.

[10] D. J. Parmelee, R. C. Walovitch, H. S. Ouellet, R. B. Lauffer, Invest.

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[11] G. R. Choppin, P. A. Bertand, Y. Hasegawa, E. N. Rizkalla, Inorg.

Chem. 1982, 21, 3722.

Measurements of NMRD profiles: The samples were prepared by mixing

the ligand under study with a slight excess of solid GdCl₃¥6H₂O, followed

by dissolution in water. The pH values of the solutions were adjusted to

about 7with a NaOH solution. The solutions were then stirred overnight

in the presence of Chelex 20 to remove the remaining free Gd³⁺. The

solids were removed in a centrifuge, and the remaining solutions were

freeze-dried. The solid complexes were dissolved in an appropriate

amount of water. The absence of free Gd³⁺was checked with an Arsena-

zo III indicator. The concentrations of Gd³⁺in the samples were deter-

mined from proton-relaxivity measurements at 20 MHz and 378C after

complete hydrolysis. The purity of complex solutions was checked by

[12] G. R. Choppin, J. Less-Common Met. 1985, 112, 193.

œ

[13] I. Lukes, J. Kotek, P. VojtÌsek, P. Hermann, Coord. Chem. Rev. 2001,

216 217, 287, and references therein.

[14] J.L. Sudmeier, C. N. Reilley, Anal. Chem. 1964, 9, 1698.

[15] C. F. G. C. Geraldes, A. M. Urbano, M. C. Alpoim, A. D. Sherry, K.-

T. Kuan, R. Rajagopalan, F. Maton, R. N. Muller, Magn. Reson.

Imaging 1995, 13, 401.

ESI-MS; [Gd(L¹)]^3ꢀ

:

m/z: 651 [M+3Na]⁺, 67 3 M[+4Na]⁺, 695

[M+5Na]⁺, 730 [M+5Na+K]⁺; [Gd(L²)]^2ꢀ: m/z: 711 [M+3Na]⁺, 7 69

[M+2Na+K]⁺; [Gd(L³)]^2ꢀ: m/z: 822 [M+2Na]⁺; 844 [M+3Na]⁺.

The complexes [Gd(L¹)(H₂O)]^3ꢀ, [Gd(L²)(H₂O)]^2ꢀand [Gd(L³)(H₂O)]^2ꢀ

were studied by ¹H longitudinal-relaxation-time measurements. NMRD

profiles were measured at 5, 25 and 378C at magnetic field strengths be-

tween 4.7î10^ꢀ4and 0.35 T (Stelar SpinMaster FFC-2000). Measurements

at 0.47, 1.42 and 7.05 T were performed on Bruker Minispec 20 and 60

MHz and on a Bruker AMX-300 spectrometer (Bruker, Karlsruhe, Ger-

many), and were included in the profiles. The experimental data were

fitted simultaneously with ¹⁷O NMR data, ¹H NMRD data and data ob-

tained from the temperature dependence of relaxivity at constant mag-

netic field (20 MHz), by using a least-squares fitting procedure with the

Micromath Scientist program version 2.0 (Salt Lake City, UT, USA).

Interaction of [Gd(L³)(H₂O)]^2ꢀwith HSA: For human serum albumin in-

teraction studies, a stock solution of [Gd(L³)(H₂O)]^2ꢀin water was pre-

pared as described in the preceding section. The dependence of the

proton-relaxation rate on the concentration of the Gd³⁺complex at con-

stant HSA concentration (4%) was measured by using a Minispec PC-20

at a constant magnetic field of 0.47T and a constant temperature of 310

K. The concentrations of [Gd(L³)(H₂O)]^2ꢀvaried from 0.038 to 0.81 mm.

Longitudinal-relaxation rates of the solution containing 4% HSA and

0.81 mm complex were measured at 310 K over the range of magnetic

fields 4î10^ꢀ47.05 T.

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Received: May 19, 2003 [F5155]

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Chem. Eur. J. 2003, 9, 5899 5915

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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Article Doi

Lanthanide(III) Complexes of Novel Mixed Carboxylic-Phosphorus Acid Derivatives of Diethylenetriamine: A Step towards More Efficient MRI Contrast Agents

DOI: 10.1002/chem.200305155

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