a bis(phosphonate)mono-amide analogueof dota

A bis(phosphonate) mono-amideanalogue of DOTA: a potential agent

for bone-targeting

Charles University, Prague Delft University of Technology

Bis(phosphonates)

PO3H2

PO3H2

R1

R2

OPO3H2

PO3H2

Pyrophosphate

Geminal bis(phosphonate)

Commercial name R1 R2

Aledronate

Clodronate

Etidronate

Ibadronate

Pamidronate

Risedronate

Tiludronate H

Cl Cl

OH

OH

OH

OH

OH

(CH3)3 NH2

CH3

(CH3)2 N(CH2)4

CH3

CH3

(CH3)2 NH2

CH2

N

S Cl


Ligand BPAMD

Bis(phosphonic acid) group – bone targeting group

DOTA monoamide – chelating group for lanthanide(III) ions

N

N

N

NHOOC

HOOC

PO3H2

PO3H2HN

O

COOH


Synthesis

HPO(OEt)2PO3Et2

PO3Et2Bn2NHC(OEt)3 HNBn2 ++

PO3Et2

PO3Et2H2N

N

N

N

Nt-BuOOC

t-BuOOC

PO3Et2

PO3Et2HN

O

COOt-Bu

PO3Et2

PO3Et2HN

O

Cl

N

N

N

NHOOC

HOOC

PO3H2

PO3H2HN

O

COOH

ClCH2COCl

t-Bu3DO3A


Complexation of Ln(III) ions

The signals of excess of the free ligand are marked with asterisks

3 step process:

• pH 2–4: only phosphonates are coordinated

• pH 7–9: “out of cage” complex (A)

• After heating: DOTA-like complex (B)


Ln-BPAMD complexes

The signals of excess of the free ligand are marked with asterisks

• All pendants coordinated

• One molecule of water in the inner coordination sphere

• Bis(phosphonic acid) group is not coordinated

• Phosphorus atoms are nonequivalent


Relaxometry of Gb-BPAMD

a Powell, D. H.; Dhubhghaill, O. M. N.; Pubanz, D.; Helm, L.; Lebedev, Y. S.; Schlaepfer, W.; Merbach, A. E. J. Am. Chem. Soc. 1996, 118, 9333–9346b Aime, S.; Botta, M.; Garino, E.; Geninatti Crich, S.; Giovenzana, G.; Pagliarin, R.; Palmisano, G.; Sisti, M. Chem. Eur. J. 2000, 6, 2609–2617

• Slow water exchange

• High value of relaxivity

• Expected second hydration sphere


LigandΔ2

[s–2/1019]τv[ps]

τr[ps]

τM[μs]

r1 (20 MHz)[s–1 mM–1]

BPAMD 3.7 ± 0.2 17 ± 1 88 ± 3 1.18 ± 0.6 5.3

DOTAa 1.6 11 77 0.244 4.8

DOTA-bis(methylphosphonic) monoamideb 1.8 21 97 1.6 6.2

Relaxometry of Gb-BPAMD

pH dependence of relaxivity

• Two steps in relaxivity: pH = 2–3 and 6–8

protonation of bis(phosphonic) acid group

• Increase in basic region:

prototropic exchange

5

5.5

6

6.5

7

7.5

2 4 6 8 10 12pH

r 1 [s

-1m

M-1

]


8

10

12

14

16

18

20

0 1 2 3 4 5 6 7ratio Ca/Gd

17O

1/T

1ir1

05 [s-1

]

• 1:1 complex formation – logβ = 2.2

• 2 times shorter 17O T1ir increase of rotation correlation time

• Formation of 2+2 complexes – 8 member ring typical for phosphonate complexes

P

O

OR

O

P

O

O R

O

M

M

Interaction of Gd-BPAMD with Ca(II) ions


0

4

8

12

16

0.01 0.1 1 10 100Larmor frequency[MHz]

r 1[s

-1m

M-1

]

without Ca(II)with Ca(II)

Interaction of Gd-BPAMD with Ca(II) ions

12

13

14

15

2.7 2.9 3.1 3.3 3.51000K/T

17O

ln(1

/T2i

r)

9

10

11

12

13

2.7 2.9 3.1 3.3 3.51000K/T

17O

ln(1

/T1i

r)

• Increase of relaxivity upon interaction with Ca(II) ions

• Unusually flat 17O temperature relaxation profiles

• Different complexing mode at higher temperature


Sorption of Tb-BPAMD complexon the hydroxyapatite (HA)

0

10

20

30

40

50

0 0.5 1 1.5

c [mmol dm-3]

X [m

mol

g-1

]

K = X/(c.(Xm – X))

0

10

20

30

40

0 0.5 1 1.5c [mmol dm-3]

c/X

[g d

m-3

]

c/X = 1/KXm + c/Xm

c – equilibrium concentration; X – specific adsorbed amount; Xm – maximum adsorption capacity; K – affinity constant

• Fast and fully reversible sorption

• Maximum adsorption capacity Xm = 4.0×10-5 mol g-1 (specific surface of HA 53 m2g-1)indicates monomolecular layer formation

• Affinity constant K = 2.1×105 dm3mol-1


Sorption of Tb-BPAMD complexon the hydroxyapatite (HA)

0

20

40

60

80

100

0.0 1.0 2.0 3.0 4.0 5.0

overall concentration of BPAMD [mmol dm-3]

activ

ity in

solu

tion

[%]

• Very strong interaction of Tb-BPAMD complex with HA surface

• Identical sorption abilities of free ligand BPAMD and its Tb(III) complex

• α-amido-bis(phosphonic acid) group – responsible for exceptional properties

bis(phosphonate)affinity constant

K ×103 [mol–1dm–3]HEDPa,b 3.3

MDPa,b 0.7

Tb-BPAMD 210a HEDP - 1-hydroxy-ethane-1,1-bis(phosphonic acid)

MDP – methanebis(phosphonic acid)b Claessens, R. A. M. J.; Kolar, I. Z. Langmuir 2000, 16, 1360–1367


Relaxometry of the hydroxyapatite slurry

0

2

4

6

8

buffer Gd-BPAMD Gd-DTPA

1 H R

1 [s-1

]

SolutionSlurry

Solution

Solution

SlurrySuspension

Slurry

• Gd-BPAMD complex is fully adsorbed in the slurry

• Value of the relaxivity of adsorbed Gd-BPAMD comparable with that obtained for the slurry containing Gd-DTPA complex

• Same concentration of Gd-DTPA in the solution and in the slurry different relaxivities due to physicochemical conditions


Relaxometry of the hydroxyapatite slurry

0

102030

40

5060

70

80

0.01 0.1 1 10 100

Proton Larmor frequency [MHz]

R1 [

s-1]

(Gd-BPAMD)-HA

0

102030

40

5060

70

80

0.01 0.1 1 10 100


R1 [

s-1]

(Gd-BPAMD)-HA

the diamagnetic contribution

0

102030

40

5060

70

80

0.01 0.1 1 10 100


R1 [

s-1]

(Gd-BPAMD)-HA

the paramagnetic contribution

the diamagnetic contribution

0

102030

40

5060

70

80

0.01 0.1 1 10 100


R1 [

s-1]

the paramagnetic contribution

Gd-BPAMD in ageous solution

• Superposition of a paramagnetic contribution from the complex and a diamagnetic contribution coming from HA

• Paramagnetic contribution shows a maximum at 10 – 20 MHz typical for Gd(III)complexes with slow rotation of the molecule

• 3-5 times higher milimolar relaxivity than the Gd-BPAMD complex in the solutionas result of the hindered rotation


Conclusion

• new bis(phosphonate) containing DOTA monoamide

• three step lanthanide complexation

• DOTA-like Ln(III) complexes

• interaction with Ca(II) ions

• fast and strong adsorption of Ln(III)complexes on hydroxyapatite

• increase of rotation corelation time and relaxivity upon binding on hydroxyapatite


Financial support• Marie Curie training site host fellowship (QLK5-CT-2000-60062) • Grant Agency of the Czech Republic (No. 203/03/0168) • COST D18 Action • EU Network of Excellence (NoE) “European Molecular Imaging Laboratory” (EMIL).

PragueIvan Lukeš

Petr Hermann

Jakub Rudovský

Jan Kotek

DelftJoop A. Peters

Hubert Th. Wolterbeek

Zvonimir I. Kolar

Kristina Djanashili

MonsRobert N. Muller

Luce Vander Elst

Sophie Laurent

Acknowledgement


a bis(phosphonate)mono-amide analogueof dota

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