Gadolinium DO3A derivatives mimicking phospholipids; preparation and in vitro evaluation as pH responsive MRI contrast agents

Article abstract of DOI:10.1039/b100405k

A series of Gd-DO3A derivatives (8a-d) mimicking phospholipids have been prepared. Two of the complexes, Gd-HADB-DO3A (8a) and Gd-HADO-DO3A (8b), have been evaluated as pH responsive MRI contrast agents in vitro. The T₁-relaxivity (r₁

Full text of DOI:10.1039/b100405k

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Gadolinium DO3A derivatives mimicking phospholipids;
preparation and in vitro evaluation as pH responsive MRI contrast
agents
2
Ragnar Hovland,* Christian Gløgård, Arne J. Aasen and Jo Klaveness
Department of Medicinal Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1155,
Blindern, N-0317, Oslo, Norway
Received (in Cambridge, UK) 9th January 2001, Accepted 27th March 2001
First published as an Advance Article on the web 25th April 2001
A series of Gd-DO3A derivatives (8a–d) mimicking phospholipids have been prepared. Two of the complexes,
Gd-HADB-DO3A (8a) and Gd-HADO-DO3A (8b), have been evaluated as pH responsive MRI contrast agents
in vitro. The T1-relaxivity (r1) of Gd-HADO-DO3A (8b) increased 142% on changing the pH from 6 to 8. The pH
dependence is thought to arise from the formation of supramolecular structures caused by deprotonation of the
amphiphilic complex at alkaline pH.
mental data are inadequate. The procedure of Gever8 was, with
minor changes, used for the synthesis of these compounds
Introduction
Paramagnetic materials have been investigated as MRI contrast
agents (CAs) for more than two decades.1 These materials
enhance the contrast of the image indirectly by lowering the
magnetic relaxation time of the water protons in the surround-
ing tissues.2,3 Gd() is particularly suited for this purpose
because of its favourable magnetic properties (seven unpaired
electrons). The aqua ion itself is too toxic for human use. This
can be circumvented by chelation by a polydentate ligand. The
most frequently used Gd() based CAs are stable, hydrophilic
poly(aminocarboxylate) derived complexes with rapid extra-
cellular distribution and renal elimination. Depending on
the denticity one or more water molecules might be directly
coordinated to the paramagnetic centre.
Gd complexes with amphiphilic properties have previously
been synthesised and evaluated as blood-pool and liver imaging
agents. Long chain amides and esters of DTPA (diethylene-
triaminepentaacetic acid) are the most common.4 More recently
amphiphilic complexes based on the DO3A (1,4,7-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclododecane) structure have been
reported.5,6 These complexes are able to form supramolecular
systems such as micelles, mixed micelles and liposomes in
aqueous solutions, in the presence or absence of surfactants
and phospholipids. The formation of these systems increases the
efficacy (T1-relaxivity) of the contrast agent due to an increase
in the rotational correlation time (τR) of the Gd complex.
In the present work Gd–DO3A complexes mimicking the
phospholipid structure have been prepared in order to achieve
CAs able to form stable and rigid supramolecular systems.
Macrocyclic chelates were chosen for the studies because of
their favourable stability and hence reduced probability for
intracellular dechelation.7
(Scheme 1). Reaction between the dialkylamines 1a–d and
Scheme 1 Reagents and conditions: i, 1) epichlorohydrin, H2O, rt or ∆,
2) NaOH, H2O, rt.
epichlorohydrin, in the presence of small amounts of water,
followed by treatment with aqueous sodium hydroxide fur-
nished the oxiranes 2a–d in good yields. To obtain homo-
geneous reaction mixtures the temperature had to be raised in
correspondence with the melting points of the most lipophilic
dialkylamines.
The synthetic pathway to N-substituted DO3A derivatives
followed a method developed by Platzek et al.9 (Scheme 2). The
protected derivative 1,4,7,10-tetraazatricyclo[5.5.1.0]tridecane
(4) was obtained by reaction of 1,4,7,10-tetraazacyclododecane
(cyclen) (3) with dimethylformamide dimethyl acetal.10 The
protected cyclen 4 was reacted with the oxiranes 2a–d at 120 ЊC
in the absence of solvent. The formyl derivatives 5a–d were
isolated upon hydrolysis with aqueous methanol in 40–65%
yields after chromatography. The 13C NMR spectra showed a
higher number of signals than expected because of restricted
rotation around the amide bond.
Hydrolyses of the amides 5a–d were, except for the most
lipophilic compound 5d, performed with sodium hydroxide
in methanol–water. Poor solubility of the amide 5d resulted in
partial hydrolysis only. However, hydrolysis with hydrochloric
acid in methanol–water furnished the monosubstituted cyclen
6d in 71% yield.
Results and discussion
Synthesis
The oxiranes 2b–d have previously been reported in the liter-
ature.† However, especially for the oxiranes 2a–d the experi-
Carboxymethylation was achieved by reacting the mono-
substituted cyclen derivatives 6a–d with sodium chloroacetate
in water–ethanol at pH 10. The high lipophilicity of the DO3A
derivatives 7a–d made ion exchange purification difficult. How-
ever, pure materials were obtained by flash chromatography on
† A search for oxiranes 2a–d in Chemical Abstracts resulted in 21 refer-
ences distributed as follows: 2a: 17 references, 2b: 1 reference, 2c: 2
references and 2d: 1 reference. Of these 12 were patents with inadequate
experimental data, and the remaining 9 were papers in other languages
than English.
DOI: 10.1039/b100405k
J. Chem. Soc., Perkin Trans. 2, 2001, 929–933
This journal is © The Royal Society of Chemistry 2001
929

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Fig. 1 The pH dependence of the T1-relaxivity (r1) of Gd-HADB-
DO3A 8a (ᮀ) and Gd-HADO-DO3A 8b (᭿) (10 MHz, 25 ЊC).
purified by the addition of small amounts of anion and cation
exchange resin to an aqueous solution, followed by filtration.
The electrospray MS data confirmed the presence of the Gd
complexes.
In vitro relaxometry
The presence of a tertiary amino group in the side chains of the
complexes 8a–d led us to investigate the influence of pH on the
T1-relaxivities (r1) in aqueous solutions. Because of favourable
aqueous solubility Gd-HADB-DO3A (8a) and Gd-HADO-
DO3A (8b) were chosen for these studies. The complexes
were titrated in aqueous solution with HCl and NaOH. The
T1-relaxivities (r1) as a function of pH are shown in Fig. 1.
The T1-relaxivity (10 MHz, 25 ЊC) of Gd-HADB-DO3A (8a)
was found to be nearly constant at about 6.5 mMϪ1 sϪ1 in the
pH range 3–12, except for a small increase between pH 4.5
and pH 6. The T1-relaxivity at the maximum (pH 5) was
7.1 mMϪ1 sϪ1.
The T1-relaxivity (10 MHz, 25 ЊC) of Gd-HADO-DO3A (8b)
showed a marked pH dependence. At low pH (3–6) the relaxiv-
ity was about 7.9 mMϪ1 sϪ1. The slightly higher value compared
to Gd-HADB-DO3A might be attributed to an increase in the
rotational correlation time (τR) of this complex caused by a
higher molecular weight and/or possible formation of dimers or
small clusters.
At higher pH (8–10) the relaxivity was found to be about 19.1
mMϪ1 sϪ1, representing an increase of 142%. This large increase
is thought to arise from the formation of colloidal aggregates,
due to the higher lipophilicity of the deprotonated (neutral)
complex compared to the protonated (positively charged)
species. This will give rise to an increased τR of the complex and
thus a higher relaxivity.
Scheme 2 Reagents and conditions: i, dimethylformamide dimethyl
acetal, toluene, 120 ЊC; ii, 1) 2a–d, 120 ЊC, 2) MeOH, H2O, rt; iii, NaOH
or HCl, MeOH, H2O, ∆; iv, ClCH2COONa, EtOH, H2O, pH 10, 70 ЊC;
v, Gd2O3, H2O, 90 ЊC.
silica gel. The yields were between 40 and 45% for the
derivatives 7a–c. Due to the low yield of the derivative 7d
(16%), the carboxymethylation was performed with tert-butyl
bromoacetate followed by deprotection in neat TFA (Scheme 3).
At high pH (10–12) the relaxivity decreased. This is presum-
ably due to competition of hydroxide ions with water for the
last coordination site on the gadolinium.
The properties of Gd-HADO-DO3A make it a valuable
model for further development of pH responsive MRI contrast
agents. Such contrast agents will have a large potential because
of the reported reduced values of extracellular pH for tumours
compared to healthy tissue.11 Further experiments will be per-
formed to investigate the mechanisms of this pH dependence.
Scheme 3 Reagents and conditions: i, BrCH2COOC(CH3)3, Na2CO3,
THF, H2O, rt; ii, TFA, rt.
Complex patterns and overlapping peaks made the NMR
spectra difficult to interpret. Electrospray MS showed the
expected [M + H]+ ions.
The Gd complexes 8a–d were prepared by heating the corre-
sponding ligand with Gd2O3 in water to 90 ЊC. Purification of
all the complexes but the least lipophilic one, complex 8a, was
achieved by continuous extraction of alkaline (pH 10), aqueous
solutions with chloroform for 24 hours. Complex 8a was
Experimental
Synthesis
Reagents were obtained from Aldrich Chemical Co. Inc., USA
or Fluka Chemie AG, Switzerland and used as received.
1,4,7,10-Tetraazacyclododecane (cyclen) was purchased from
Macrocyclics Inc., USA. Dihexadecylamine (1d) was prepared
930
J. Chem. Soc., Perkin Trans. 2, 2001, 929–933

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by reacting hexadecylamine with palmitoyl chloride, followed
by reduction with lithium aluminium hydride.
and MeOH–water (3 : 1, 20 ml) was added. The mixture was
stirred at room temperature for 3 h and then the solvents were
evaporated in vacuo. The residue was purified by flash chroma-
tography (SiO2, EtOH–THF–NH3 (25%) 1 : 5 : 1) to yield the
products as oils.
NMR spectra were obtained on a Bruker Spectrospin Avance
DPX300 (Bruker GmbH, Germany). Electron impact (EI)
mass spectra were obtained using a Fisons VG ProSpec (70 eV
and 220 ЊC) (Micromass Ltd., England). Electrospray (ES)
FT-ICR-MS mass spectra were recorded on a Bruker BioApex
4.7 T (Bruker GmbH, Germany). Elemental analyses were
carried out by Ilse Beetz Microanalytisches Laboratorium,
Germany.
1-Formyl-7-[2-hydroxy-3-(N,N-dibutylamino)propyl]-1,4,7,
10-tetraazacyclododecane (5a). The reaction was performed
according to the general procedure with N,N-dibutyl(oxiranyl-
methyl)amine (3.06 g, 16.5 mmol). Yield: 3.52 g (61%). Mass
spectrum (EI): m/z (relative intensity) 385 (<1%) [M+, C20H43-
1
N5O2], 367 (8), 243 (100), 225 (47), 142 (52); H NMR (300
General procedure for the synthesis of oxiranes 2a–d. A mix-
ture of dialkylamine (100 mmol), epichlorohydrin (120 mmol)
and water (15 mmol) was stirred at 30–70 ЊC for 5–16 h. The
mixture was washed with aqueous potassium carbonate (20%,
20 ml). Sodium hydroxide (36%, 20 ml) was added to the oily
phase and the mixture was stirred at 30–70 ЊC overnight. The
phases were separated and the oil dissolved in diethyl ether
(100 ml), dried (MgSO4) and evaporated in vacuo to yield the
products as oils.
MHz, CDCl3): δ 0.81 (t, 7.2 Hz, 6H), 1.10–1.35 (m, 4H), 1.35–
1.50 (m, 4H), 2.10–2.40 (m, 9H), 2.45–2.70 (m, 10H), 3.00–3.70
(m, 9H), 8.05 (s, 1H); 13C NMR (75 MHz, CDCl3): δ 13.92,
20.41, 29.06, 43.89, 46.78, 46.89, 46.93, 47.41, 49.80, 50.52,
51.95, 54.03, 58.65, 59.54, 65.80, 164.58.
1-Formyl-7-[2-hydroxy-3-(N,N-dioctylamino)propyl]-1,4,7,
10-tetraazacyclododecane (5b). The reaction was performed
according to the general procedure with N,N-dioctyl(oxiranyl-
methyl)amine (3.27 g, 11.0 mmol). Yield: 3.26 g (65%). Mass
spectrum (EI): m/z (relative intensity) 497 (<1%) [M+,
C28H59N5O2], 479 (5), 284 (31), 280 (42), 254 (54), 243 (100); 1H
NMR (300 MHz, CDCl3): δ 0.81 (t, 6.7 Hz, 6H), 1.10–1.25
(s, 20H), 1.30–1.40 (m, 4H), 2.10–2.80 (m, 18H), 2.85–3.25
(m, 4H), 3.30–3.65 (m, 6H), 8.08 (s, 1H); 13C NMR (75 MHz,
CDCl3): δ 13.98, 22.53, 26.96, 27.36, 29.20, 29.46, 31.73, 43.96,
46.84, 47.01, 47.54, 49.85, 50.56, 52.08, 54.39, 58.73, 59.68,
65.83, 164.59.
N,N-Dibutyl(oxiranylmethyl)amine (2a). The reaction
between dibutylamine and epichlorohydrin was performed
according to the general procedure. Yield: 13.67 g (74%). Mass
spectrum (EI): m/z (relative intensity) 185 (7%) [M+,
1
C11H23NO], 142 (100), 100 (23); H NMR (300 MHz, CDCl3):
δ 0.84 (t, 7.2 Hz, 6H), 1.20–1.40 (m, 8H), 2.30–2.50 (m, 6H),
2.60–2.70 (m, 2H), 2.90–3.00 (m, 1H); 13C NMR (75 MHz,
CDCl3): δ 13.92, 20.50, 29.05, 45.17, 50.87, 54.22, 56.66.
N,N-Dioctyl(oxiranylmethyl)amine (2b). The reaction
between dioctylamine (12.07 g, 50 mmol) and epichlorohydrin
(5.55 g, 60 mmol) was performed according to the general pro-
cedure. Yield: 10.42 g (70%). Mass spectrum (EI): m/z (relative
intensity) 297 (1%) [M+, C19H39NO], 285 (88), 280 (100), 254
1-Formyl-7-[2-hydroxy-3-(N,N-didodecylamino)propyl]-1,4,7,
10-tetraazacyclododecane (5c). A solution of 1,4,7,10-tetra-
azacyclododecane (cyclen) (0.87 g, 5.1 mmol) and N,N-di-
methylformamide dimethyl acetal (0.69 g, 5.8 mmol) in toluene
(10 ml) was heated to 120 ЊC under argon. After 2 h the
toluene–MeOH azeotrope formed during the reaction was
distilled off. The remaining toluene was evaporated in vacuo at
70 ЊC. N,N-Didodecyl(oxiranylmethyl)amine (2.28 g, 5.6 mmol)
was added and the mixture was heated to 120 ЊC under argon.
After 16 h the reaction mixture was cooled to room temperature
and MeOH–water (3 : 1, 7 ml) was added. The mixture was
stirred at room temperature for 3 h and then the solvents were
evaporated in vacuo.
1
(40), 156 (34); H NMR (300 MHz, CDCl3): δ 0.84 (t, 6.7 Hz,
6H), 1.15–1.30 (m, 20H), 1.35–1.50 (m, 4H), 2.35–2.58 (m, 6H),
2.62–2.75 (m, 2H), 2.95–3.05 (m, 1H); 13C NMR (75 MHz,
CDCl3): δ 14.03, 22.61, 27.01, 27.48, 29.27, 29.52, 31.81, 45.32,
50.96, 54.67, 56.79.
N,N-Didodecyl(oxiranylmethyl)amine (2c). The reaction
between didodecylamine (2.48 g, 7.0 mmol) and epichloro-
hydrin (0.78 g, 8.4 mmol) was performed according to the
general procedure. Yield: 2.38 g (83%). Mass spectrum (EI):
m/z (relative intensity) 409 (3%) [M+, C27H55NO], 366 (10), 254
(100); 1H NMR (300 MHz, CDCl3): δ 0.84 (t, 6.7 Hz, 6H), 1.15–
1.30 (m, 36H), 1.35–1.50 (m, 4H), 2.30–2.55 (m, 6H), 2.60–2.80
(m, 2H), 2.95–3.05 (m, 1H); 13C NMR (75 MHz, CDCl3):
δ 14.10, 22.66, 26.91, 27.49, 29.34, 29.62, 31.89, 45.35, 50.95,
54.60, 56.75.
The residue was dissolved in water (50 ml) and extracted with
diethyl ether (3 × 50 ml). The combined organic extracts were
dried (MgSO4) and evaporated in vacuo. The residue was dis-
solved in n-hexane–ethyl acetate (3 : 2, 3 ml) and applied to a
short column of alumina (neutral). The column was washed
with n-hexane–ethyl acetate (3 : 2, 100 ml) and the product was
eluted with MeOH (70 ml). Yield: 1.26 g (41%) of a yellow oil.
Mass spectrum (EI): m/z (relative intensity) 609 (<1%) [M+,
N,N-Bis(hexadecyl)(oxiranylmethyl)amine (2d). The reac-
tion between dihexadecylamine (2.33 g, 5.0 mmol) and epichlo-
rohydrin (0.56 g, 6.0 mmol) was performed according to the
general procedure. The crude product was further purified by
applying it to a short column of silica gel. Elution with CHCl3–
MeOH–NH3 (25%) 90 : 10 : 1 yielded 2.18 g (84%). Mass spec-
trum (EI): m/z (relative intensity) 521 (3%) [M+, C35H71NO],
1
C36H75N5O2], 591 (10), 392 (25), 243 (77), 225 (100); H NMR
(300 MHz, CDCl3): δ 0.79 (t, 6.7 Hz, 6H), 1.10–1.20 (m, 36H),
1.25–1.35 (m, 4H), 2.15–2.55 (m, 8H), 2.56–2.80 (m, 7H), 2.85–
3.00 (m, 3H), 3.40–3.60 (m, 4H), 4.20–4.80 (m, 5H), 8.10 (s,
1H); 13C NMR (75 MHz, CDCl3): δ 13.94, 22.51, 26.80, 27.28,
29.18, 29.48, 29.50, 31.74, 43.93, 46.34, 46.64, 46.89, 47.94,
48.71, 50.93, 52.29, 54.15, 58.60, 60.65, 65.82, 164.52.
1
478 (12), 310 (100), 254 (88); H NMR (300 MHz, CDCl3):
δ 0.85 (t, 6.7 Hz, 6H), 1.10–1.30 (m, 52H), 1.35–1.50 (m, 4H),
2.30–2.55 (m, 6H), 2.60–2.75 (m, 2H), 2.95–3.05 (m, 1H); 13C
NMR (75 MHz, CDCl3): δ 14.08, 22.67, 26.98, 27.51, 29.35,
29.59, 29.65, 29.69, 31.91, 45.33, 50.93, 54.66, 56.79.
1-Formyl-7-[2-hydroxy-3-(N,N-dihexadecylamino)propyl]-
1,4,7,10-tetraazacyclododecane (5d). A solution of 1,4,7,10-
tetraazacyclododecane (cyclen) (627 mg, 3.6 mmol) and
N,N-dimethylformamide dimethyl acetal (499 mg, 4.2 mmol) in
toluene (10 ml) was heated to 120 ЊC under argon. After 2 h the
toluene–MeOH azeotrope formed during the reaction was
distilled off. The remaining toluene was evaporated in vacuo
at 70 ЊC. N,N-Dihexadecyl(oxiranylmethyl)amine (2.09 g, 4.0
mmol) was added and the mixture was heated to 120 ЊC under
argon. After 16 h the reaction mixture was cooled to room
temperature and MeOH–water (3 : 1, 8 ml) was added. The
mixture was stirred at room temperature for 3 h and then the
General procedure for the synthesis of formyl derivatives 5a
and 5b. A solution of 1,4,7,10-tetraazacyclododecane (cyclen)
(15.0 mmol) and N,N-dimethylformamide dimethyl acetal (17.3
mmol) in toluene (30 ml) was heated to 120 ЊC under argon.
After 2 h the toluene–MeOH azeotrope formed during the reac-
tion was distilled off. The remaining toluene was evaporated in
vacuo at 70 ЊC. N,N-Dialkyl(oxiranylmethyl)amine (16.5 mmol)
was added and the mixture was heated to 120 ЊC under argon.
After 16 h the reaction mixture was cooled to room temperature
J. Chem. Soc., Perkin Trans. 2, 2001, 929–933
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solvents were evaporated in vacuo. The residue was dissolved in
chloroform (75 ml) and washed with saturated brine (50 ml).
The aqueous phase was extracted with chloroform (50 ml) and
the combined organic extracts were dried (MgSO4) and evapor-
ated in vacuo. Flash chromatography (SiO2, EtOH–THF–NH3
(25%) 1 : 5 : 1) of the residue yielded an oil that was further
purified on a short column of alumina. Washing with n-hexane–
ethyl acetate 1 : 1 was followed by elution with EtOH–THF–
NH3 1 : 5 : 1 to yield the product as a yellow oil. Yield: 1.05 g
(40%). Mass spectrum (EI): m/z (relative intensity) 703 (<1%)
[M+ Ϫ 18], 477 (65), 379 (85), 254 (100), 225 (46); 1H NMR (300
MHz, CDCl3): δ 0.81 (t, 6.7 Hz, 6H), 1.10–1.20 (m, 52H), 1.25–
1.40 (m, 4H), 2.10–2.90 (m, 16H), 3.10–3.80 (m, 8H), 8.09
(s, 1H); 13C NMR (75 MHz, CDCl3): δ 13.98, 22.55, 27.00,
27.35, 29.24, 29.58, 30.19, 31.80, 43.97, 46.67, 46.95, 47.33,
47.45, 49.47, 50.80, 52.30, 54.32, 58.73, 60.11, 65.85, 164.46.
ated in vacuo. Yield: 300 mg (71%). Mass spectrum (EI): m/z
(relative intensity) 478 (6%), 464 (7), 268 (26), 254 (100); H
1
NMR (300 MHz, CDCl3): δ 0.84 (t, 6.7 Hz, 6H), 1.10–1.25 (m,
52H), 1.30–1.45 (m, 4H), 2.10–2.45 (m, 8H), 2.50–2.65 (m, 6H),
3.60–3.80 (m, 1H); 13C NMR (75 MHz, CDCl3): δ 14.05, 22.63,
27.00, 27.43, 29.31, 29.61, 29.65, 31.87, 45.16, 46.10, 46.91,
52.38, 54.17, 59.09, 59.34, 65.65.
General procedure for the synthesis of DO3A derivatives 7a–d.
Monosubstituted cyclen derivative (4.7 mmol) in EtOH (40 ml)
was added to a solution of sodium chloroacetate (23.5 mmol) in
water (40 ml). The pH of the solution was adjusted to 10 by the
addition of sodium hydroxide (1 M). The mixture was heated
to 70 ЊC with stirring for 48 h, maintaining a pH of 10. The
solvent was evaporated in vacuo and the residue was purified
by flash chromatography on silica gel to yield the products as
colourless solids.
General procedure for the synthesis of monosubstituted cyclen
derivatives 6a–c. Sodium hydroxide (61.0 mmol) was added to a
solution of the formyl derivative (6.1 mmol) in MeOH–water
(3 : 1, 15 ml) and the temperature was raised to 70 ЊC. After
16 h the solvents were evaporated in vacuo. The residue was
dissolved in water (10 ml) and extracted with dichloromethane
(50 ml + 2 × 20 ml). The combined organic extracts were
dried (MgSO4) and concentrated in vacuo to yield the products
as oils.
1-[2-Hydroxy-3-(N,N-dibutylamino)propyl]-1,4,7,10-tetra-
azacyclododecane (6a). The reaction was performed according
to the general procedure with 1-formyl-7-[2-hydroxy-3-(N,N-
dibutylamino)propyl]-1,4,7,10-tetraazacyclododecane. Yield:
1.96 g (90%). Mass spectrum (EI): m/z (relative intensity) 357
(<1%) [M+, C19H43N5O], 215 (53), 197 (100), 168 (47), 142 (82);
1H NMR (300 MHz, CDCl3): δ 0.80 (t, 7.2 Hz, 6H), 1.10–1.24
(m, 4H), 1.25–1.35 (m, 4H), 2.10–2.75 (m, 22H), 3.60–3.70 (m,
3H); 13C NMR (75 MHz, CDCl3): δ 13.92, 20.40, 29.09, 45.25,
46.06, 46.97, 52.30, 53.77, 58.96, 59.28, 65.48.
1-[2-Hydroxy-3-(N,N-dioctylamino)propyl]-1,4,7,10-tetra-
azacyclododecane (6b). The reaction was performed according
to the general procedure with 1-formyl-7-[2-hydroxy-3-(N,N-
dioctylamino)propyl]-1,4,7,10-tetraazacyclododecane (1.65 g,
3.1 mmol). Yield: 1.37 g (94%). Mass spectrum (EI): m/z
(relative intensity) 469 (1%) [M+, C27H59N5O], 284 (49), 280
(71), 254 (80), 215 (52), 197 (100); 1H NMR (300 MHz, CDCl3):
δ 0.80 (t, 6.6 Hz, 6H), 1.10–1.20 (m, 20H), 1.25–1.40 (m, 4H),
2.20–2.45 (m, 9H), 2.50–2.60 (m, 8H), 2.65–2.80 (m, 4H), 3.40–
3.70 (m, 5H); 13C NMR (75 MHz, CDCl3): δ 13.95, 22.51,
26.89, 27.32, 29.18, 29.45, 31.71, 44.98, 45.85, 46.73, 52.07,
54.09, 58.98, 59.22, 65.45.
1-[2-Hydroxy-3-(N,N-didodecylamino)propyl]-1,4,7,10-tetra-
azacyclododecane (6c). The reaction was performed according
to the general procedure with 1-formyl-7-[2-hydroxy-3-(N,N-
didodecylamino)propyl]-1,4,7,10-tetraazacyclododecane (1.15
g, 1.9 mmol). Yield: 0.85 g (77%). Mass spectrum (EI): m/z
(relative intensity) 581 (2%) [M+, C35H75N5O], 392 (41), 366
(57), 215 (63), 197 (100); 1H NMR (300 MHz, CDCl3): δ 0.80 (t,
6.7 Hz, 6H), 1.10–1.20 (m, 36H), 1.25–1.40 (m, 4H), 2.20–3.70
(m, 23H); 13C NMR (75 MHz, CDCl3): δ 13.99, 22.56, 26.99,
27.34, 29.23, 29.53, 29.55, 31.80, 45.34, 46.24, 47.09, 52.44,
54.11, 59.10, 59.33, 65.47.
1,4,7-Tris(carboxymethyl)-10-[2-hydroxy-3-(N,N-dibutyl-
amino)propyl]-1,4,7,10-tetraazacyclododecane (7a). The reac-
tion was performed according to the general procedure with
1-[2-hydroxy-3-(N,N-dibutylamino)propyl]-1,4,7,10-tetraaza-
cyclododecane. Mobile phase: CHCl3–MeOH–NH3 (25%)
9 : 4 : 1. Yield: 1.09 g (44%). Mass spectrum (ES): m/z 532
[[M + H]+, C25H50N5O7]; 1H NMR (300 MHz, CD3OD): δ 0.92
(t, 7.2 Hz, 6H), 1.20–1.35 (m, 4H), 1.36–1.50 (m, 4H), 2.00–2.20
(m, 5H), 2.21–2.35 (m, 4H), 2.36–2.60 (m, 6H), 2.65–2.90 (m,
5H), 2.91–3.05 (m, 2H), 3.10–3.30 (m, 3H), 3.40–3.60 (m, 2H),
3.61–3.70 (m, 1H), 4.20–4.30 (m, 1H); 13C NMR (75 MHz,
CD3OD): δ 14.46, 21.64, 30.05, 49.76, 51.91, 53.45, 54.30,
54.36, 55.42, 57.27, 60.39, 60.56, 60.65, 61.24, 65.87, 179.26,
179.43, 179.60.
1,4,7-Tris(carboxymethyl)-10-[2-hydroxy-3-(N,N-dioctyl-
amino)propyl]-1,4,7,10-tetraazacyclododecane (7b). The reac-
tion was performed according to the general procedure with
1-[2-hydroxy-3-(N,N-dioctylamino)propyl]-1,4,7,10-tetraaza-
cyclododecane (536 mg, 1.1 mmol). Mobile phase: CHCl3–
MeOH–NH3 (25%) 9 : 4 : 1. Yield: 300 mg (41%). Mass spec-
1
trum (ES): m/z 644 [[M + H]+, C33H66N5O7]; H NMR (300
MHz, CD3OD): δ 0.89 (t, 6.6 Hz, 6H), 1.20–1.35 (m, 20H),
1.40–1.55 (m, 4H), 2.00–2.20 (m, 4H), 2.21–2.35 (m, 3H), 2.40–
2.60 (m, 4H), 2.65–3.10 (m, 6H), 3.20–3.25 (m, 3H), 3.40–3.70
(m, 3H), 4.15–4.35 (m, 1H); 13C NMR (75 MHz, CD3OD):
δ 14.51, 23.73, 27.70, 28.55, 30.51, 30.67, 33.03, 51.89, 53.45,
54.34, 55.73, 57.20, 60.55, 61.27, 65.91, 179.36.
1,4,7-Tris(carboxymethyl)-10-[2-hydroxy-3-(N,N-didodecyl-
amino)propyl]-1,4,7,10-tetraazacyclododecane (7c). The reac-
tion was performed according to the general procedure with
1-[2-hydroxy-3-(N,N-didodecylamino)propyl]-1,4,7,10-tetra-
azacyclododecane (750 mg, 1.3 mmol). Mobile phase: MeOH.
Yield: 439 mg (45%). Mass spectrum (ES): m/z 756 [[M + H]+,
C41H82N5O7]; 1H NMR (300 MHz, CD3OD): δ 0.89 (t, 6.4 Hz,
6H), 1.20–1.35 (m, 38H), 1.40–1.70 (m, 4H), 2.00–2.40 (m, 8H),
2.45–3.00 (m, 16H), 3.10–3.60 (m, 4H); 13C NMR (75 MHz,
CD3OD): δ 14.56, 23.75, 25.67, 28.05, 30.50, 30.60, 30.69,
30.74, 30.78, 30.82, 33.09, 44.89, 51.54, 52.36, 53.46, 54.35,
54.71, 55.19, 59.70, 60.34, 60.57, 60.84, 65.40, 179.24, 179.53,
179.82.
1,4,7-Tris(carboxymethyl)-10-[2-hydroxy-3-(N,N-dihexa-
decylamino)propyl]-1,4,7,10-tetraazacyclododecane (7d). The
reaction was performed according to the general procedure
with 1-[2-hydroxy-3-(N,N-dihexadecylamino)propyl]-1,4,7,10-
tetraazacyclododecane (462 mg, 0.7 mmol). Mobile phase:
CHCl3–MeOH–NH3 (25%) 9 : 6 : 1. Yield 95 mg (16%). Mass
1-[2-Hydroxy-3-(N,N-dihexadecylamino)propyl]-1,4,7,10-
tetraazacyclododecane (6d). 1-Formyl-7-[2-hydroxy-3-(N,N-di-
hexadecylamino)propyl]-1,4,7,10-tetraazacyclododecane (440
mg, 0.6 mmol) was dissolved in MeOH (10 ml) and hydro-
chloric acid (37%, 1 ml) was added. The mixture was refluxed
for 16 h, cooled to room temperature and evaporated in vacuo.
Water (25 ml) was added to the residue and the pH was adjusted
to 12 by addition of sodium hydroxide (6 M). The aqueous
solution was extracted with dichloromethane (30 ml × 3), and
the combined organic extracts were dried (MgSO4) and evapor-
1
spectrum (ES): m/z 435 [[M + 2H]2+, C49H99N5O7]; H NMR
(300 MHz, CD3OD): δ 0.90 (t, 6.7 Hz, 6H), 1.20–1.40 (m, 52H),
1.60–1.80 (m, 4H), 2.00–2.40 (m, 5H), 2.50–2.90 (m, 7H), 3.00–
3.25 (m, 8H), 3.35–3.80 (m, 3H); 13C NMR (75 MHz, CD3OD):
δ 14.61, 23.79, 27.91, 30.42, 30.55, 30.76, 30.80, 30.85, 30.90,
33.13, 45.00–51.00 (small peaks), 51.19, 53.46, 54.15, 54.53,
55.78, 60.36, 60.74, 64.15, 178.84, 179.13.
932
J. Chem. Soc., Perkin Trans. 2, 2001, 929–933

View Article Online
1,4,7-Tris(tert-butoxycarbonylmethyl)-10-[2-hydroxy-3-(N,N-
dihexadecylamino)propyl]-1,4,7,10-tetraazacyclododecane (9).
tert-Butyl bromoacetate (328 mg, 1.7 mmol) was added to 1-[2-
hydroxy-3-(N,N-dihexadecylamino)propyl]-1,4,7,10-tetraaza-
cyclododecane (290 mg, 0.4 mmol) and sodium carbonate (178
mg, 1.7 mmol) in a mixture of THF and water (30 : 1, 7 ml).
The mixture was stirred overnight at room temperature and
filtered. The filtrate was evaporated in vacuo. The residue was
dissolved in dichloromethane (20 ml) and washed with aqueous
sodium carbonate (5%, 15 ml × 2). The organic phase was dried
(MgSO4) and evaporated in vacuo. The residue was submitted
to flash chromatography (SiO2, CH2Cl2–MeOH 9 : 1) to yield
130 mg (30%) of a yellow oil. Mass spectrum (ES): m/z 1036
Gadolinium
10-[2-hydroxy-3-(N,N-dihexadecylamino)-
propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate
(Gd-
HADHD-DO3A) (8d). The reaction was performed according
to the general procedure with 1,4,7-tris(carboxymethyl)-10-[2-
hydroxy-3-(N,N-dihexadecylamino)propyl]-1,4,7,10-tetraaza-
cyclododecane (92 mg, 0.11 mmol). Yield: 80 mg (71%). Mass
spectrum (ES): m/z 1024 [[M + H]+, C49H95N5O7Gd].
In vitro relaxometry
The relaxation measurements were performed at 10 MHz on
a Spinmaster FFC (Fast Field Cycling) NMR relaxometer
(Stelar s.r.l, Mede (PV), Italy). The T1 relaxation rates (R1) were
obtained by the inversion recovery method at 25 ЊC. The T1
relaxivity (r1) was obtained by the relationship given in eqn. (1),
1
[[M + H]+, C61H122N5O7]; H NMR (300 MHz, CDCl3): δ 0.78
(t, 6.7 Hz, 6H), 1.00–1.25 (m, 45H), 1.30–1.50 (m, 19H), 1.80–
2.40 (m, 9H), 2.60–3.25 (m, 11H), 3.40–4.20 (m, 4H); 13C NMR
(75 MHz, CDCl3): δ 13.95, 22.49, 27.72, 27.99, 29.17, 29.38,
29.47, 29.51, 31.72, 45.00–65.00 (small peaks), 81.49, 81.72,
84.33, 171.99.
(1)
where Ro1bs and Rm1 are the relaxation rates in sϪ1 of the sample
and the matrix respectively, and C is the Gd concentration in
mM.
The r1 values were plotted against pH to give the pH depend-
ent relaxivity relationship as shown in Fig. 1. Analyses were
performed in triplicate at each pH.
The measurements were performed on aqueous solutions
of Gd-HADB-DO3A (8a) and Gd-HADO-DO3A (8b) with
gadolinium concentrations of 1.49 mM and 1.52 mM, respec-
tively. The pH of the solutions was adjusted by addition of
minimal amounts of HCl or NaOH.
1,4,7-Tris(carboxymethyl)-10-[2-hydroxy-3-(N,N-dihexadecyl-
amino)propyl]-1,4,7,10-tetraazacyclododecane (7d). Trifluoro-
acetic acid (1 ml) was added to 1,4,7-tris(tert-butoxycarb-
onylmethyl)-10-[2-hydroxy-3-(N,N-dihexadecylamino)propyl]-
1,4,7,10-tetraazacyclododecane (110 mg, 0.1 mmol) under
argon. The solution was stirred at room temperature for 16 h
and then evaporated in vacuo. Water (10 ml) was added to the
residue and the resulting suspension was concentrated to dry-
ness in vacuo. This was repeated once and the residue was dried
under vacuum (50 ЊC). Yield 85 mg (98%).
Determination of gadolinium concentration
Gadolinium 10-[2-hydroxy-3-(N,N-dibutylamino)propyl]-1,4,
Nitric acid and water was added to the chelate solution to a
final concentration of 2.5 M HNO3. After incubation at ambi-
ent temperature for 24 h the T1 relaxivity was measured at 10
MHz (25 ЊC). The T1 relaxivity of a standard solution of 2.12
mM GdCl3 in 2.5 M HNO3 was measured at the same condi-
tions. The gadolinium concentration was then determined from
eqn. (1).
7,10-tetraazacyclododecane-1,4,7-triacetate
(Gd-HADB-
DO3A) (8a). A suspension of 1,4,7-tris(carboxymethyl)-10-[2-
hyd roxy-3-(N,N-dibutylamino)propyl]-1,4,7,10-tetraazacyclo-
dodecane (170 mg, 0.32 mmol) and gadolinium oxide (58 mg,
0.16 mmol) in water (10 ml) was heated to 90 ЊC for 7 h. Acti-
vated charcoal was added and the suspension filtered hot. Some
anion and cation exchange resin was added to the cooled solu-
tion and the pH adjusted to 7. The suspension was filtered
after 1 h and evaporated in vacuo to yield 140 mg (64%) of a
slightly yellow solid. Mass spectrum (ES): m/z 687 [[M + H]+,
C25H47N5O7Gd]; Anal. Calcd. (found) for C25H46N5O7Gdؒ
7H2O: C, 36.95% (36.94); H, 7.39 (7.06).
Acknowledgements
Financial support from the EU in the frame of the COST D8/
D18 action, and the contributions of J. Vedde, Department of
Chemistry and G. Stensrud, School of Pharmacy, University of
Oslo, are gratefully acknowledged. We also would like to thank
Professor Silvio Aime, University of Turin, for the use of the
NMR relaxometer located at the Bioindustry Park Canavese,
Italy.
General procedure for the synthesis of the Gd-complexes 8b–d.
A suspension of the DO3A derivative (0.23 mmol) and gado-
linium oxide (0.12 mmol) in water (10 ml) was heated to 90 ЊC
for 16 h. The mixture was cooled to room temperature, filtered
and the pH of the filtrate was adjusted to 10 by addition of 1 M
NaOH. The solution was continuously extracted with chloro-
form for 20 h. The organic phase was evaporated in vacuo to
yield the products as hygroscopic solids.
References
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Gadolinium 10-[2-hydroxy-3-(N,N-dioctylamino)propyl]-1,4,
7,10-tetraazacyclododecane-1,4,7-triacetate
(Gd-HADO-
DO3A) (8b). The reaction was performed according to the gen-
eral procedure with 1,4,7-tris(carboxymethyl)-10-[2-hydroxy-3-
(N,N-dioctylamino)propyl]-1,4,7,10-tetraazacyclododecane.
Yield: 120 mg (65%). Mass spectrum (ES): m/z 799 [[M + H]+,
C33H63N5O7Gd]; Anal. Calcd. (found) for C33H62N5O7Gdؒ
5.5H2O: C, 44.15% (44.11); H, 8.14 (7.52); N, 7.80 (8.00).
Gadolinium 10-[2-hydroxy-3-(N,N-didodecylamino)propyl]-
1,4,7,10-tetraazacyclododecane-1,4,7-triacetate
(Gd-HADD-
DO3A) (8c). The reaction was performed according to the gen-
eral procedure with 1,4,7-tris(carboxymethyl)-10-[2-hydroxy-3-
(N,N-didodecylamino)propyl]-1,4,7,10-tetraazacyclododecane
(197 mg, 0.26 mmol). Yield: 155 mg (65%). Mass spectrum
(ES): m/z 911 [[M + H]+, C41H79N5O7Gd]; Anal. Calcd. (found)
for C41H78N5O7Gdؒ3H2O: C, 51.01% (50.98); H, 8.78 (8.38); N,
7.26 (7.19).
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J. Chem. Soc., Perkin Trans. 2, 2001, 929–933
933