Preparation and in vitro evaluation of a novel amphiphilic GdPCTA-[12] derivative; a micellar MRI contrast agent.

Article abstract of DOI:10.1039/b211039c

A novel amphiphilic GdPCTA-[12] derivative has been prepared. The complex formed micelles in aqueous solution with a relatively low CMC, 0.15 mM (25 degrees C). The concentration dependent T1-relaxivity (r1) of the system has been described. The maximum T1-relaxivity, 29.2 s-1 mM-1 (20 MHz, 25 degrees C), was higher than for previously described micellar MRI contrast agents. This high T1-relaxivity is a consequence of the favourable water residence time (tau M) and the fact that the complex is heptadentate allowing two water molecules to coordinate to the gadolinium ion (q = 2).

Full text of DOI:10.1039/b211039c

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Preparation and in vitro evaluation of a novel amphiphilic
GdPCTA-[12] derivative; a micellar MRI contrast agent
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-0318 Oslo, Norway. E-mail: ragnar.hovland@farmasi.uio.no
Received 11th November 2002, Accepted 23rd December 2002
First published as an Advance Article on the web 29th January 2003
A novel amphiphilic GdPCTA-[12] derivative has been prepared. The complex formed micelles in aqueous solution
with a relatively low CMC, 0.15 mM (25 ЊC). The concentration dependent T1-relaxivity (r1) of the system has been
described. The maximum T1-relaxivity, 29.2 sϪ1 mMϪ1 (20 MHz, 25 ЊC), was higher than for previously described
micellar MRI contrast agents. This high T1-relaxivity is a consequence of the favourable water residence time (τM)
and the fact that the complex is heptadentate allowing two water molecules to coordinate to the gadolinium ion
(q = 2).
Introduction
Results and discussion
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 surrounding tissues.2,3 The most frequently used CAs are
stable gadolinium() (Gd) complexes with hydrophilic poly-
(aminocarboxylate) ligands resulting in rapid extracellular dis-
tribution and renal elimination. Gd() is preferred because of
its favourable magnetic properties (seven unpaired electrons).
Depending on the denticity of the ligand one or more water
molecules might be directly coordinated to the paramagnetic
centre.
Synthesis
The synthetic strategy was a slight modification of the pro-
cedure described by Aime et al.13 The trisubstituted pyridine 114
was selectively alkylated at the aromatic hydroxyl functionality
by dodecyl bromide in DMF in the presence of potassium
carbonate. The resulting dialcohol 2 was converted to the
dichloride 3 by treatment with thionyl chloride. The total yield
of this reaction sequence was 54% (Scheme 1).
Gd complexes with amphiphilic properties have pre-
viously been prepared and evaluated as blood-pool and liver
imaging agents. Long chain amides and esters of GdDTPA
(gadolinium diethylenetriamine pentaacetic acid) are the
most common.4 More recently amphiphilic gadolinium
complexes based on the DO3A (1,4,7-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododecane) ligand have been reported.5–7
In an aqueous environment these complexes are able to
form supramolecular systems such as micelles, or in the
presence of surfactants and phospholipids, mixed micelles
and liposomes. 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.
Scheme 1 Reagents and conditions: i, dodecyl bromide, K2CO3, DMF,
95 ЊC; ii, thionyl chloride, 40 ЊC.
The ring-forming reaction between the dichloride 3 and the
sulfonamide 415 was carried out under heterogeneous condi-
tions in acetonitrile in the presence of potassium carbonate.
The resulting macrocycle 5 was deprotected with thiophenol in
DMF to obtain the macrocycle 6. The total yield of these reac-
tions was 42% from the dichloride 3 (Scheme 2). Attempts to
However, the increase in T1-relaxivity of these octadentate
complexes is partly quenched by the long residence time (τM) of
the coordinated water molecule. These high τM values, 300 ns
(25 ЊC) for GdDTPA, are related to the dissociative exchange
mechanism of the water molecule from the coordination site to
the bulk.8 Being a heptadentate complex with two coordinated
water molecules (q = 2), GdPCTA-[12] (gadolinium 3,6,9,
15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tri-
acetate) is reported to have a considerably lower water
residence time, 70 ns (25 ЊC).9 The complex is also reported to
have an acceptable stability, in order not to release highly toxic
gadolinium ions in vivo.10
By introducing a lipophilic moiety onto the GdPCTA-[12]
structure, preferably on the pyridine unit, one would obtain
a Gd complex able to form micelles in aqueous solution.
This micellar MRI contrast agent would obtain a higher
T1-relaxivity above the CMC than the previously reported q = 1
complexes.5,11,12
Scheme 2 Reagents and conditions: i, 3, K2CO3, CH3CN, reflux;
ii, thiophenol, K2CO3, DMF, rt.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 4 4 – 6 4 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
644

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Table 1 The CMC value of the GdPCTA-[12] derivative 8 compared
to other relevant paramagnetic Gd complexes (25 ЊC)
prepare the macrocycle 6 by using the corresponding tosylated
intermediate was not successful. All methods employed for the
hydrolysis of the tosyl groups failed, because of incomplete
hydrolysis and/or cleavage of the ether bond.
Complex
CMC/mM
Carboxymethylation was achieved in low yield (21%) by
reacting macrocycle 6 with sodium chloroacetate in water–
ethanol at pH 10 (Scheme 3). Purification of the resulting
PCTA-[12] derivative 7 was accomplished by flash chromato-
graphy on silica gel, yielding the substance in a pure state. The
structure was confirmed by NMR (1H and 13C) and electrospray
MS.
8
0.15
2.0
0.10
4.45
0.06
GdHDDDO3A5
GdHHDDO3A5
GdDOTAC1212
GdDOTASAC1812
Fig. 1 The concentration dependence of the T1-relaxivity (r1) of the
GdPCTA-[12] derivative 8 (20 MHz, pH 7). ᭿ 25 ЊC, ᭝ 37 ЊC.
At 37 ЊC the minimum T1-relaxivity was 9.2 sϪ1 mMϪ1. The
maximum T1-relaxivity was 28.5 mMϪ1 Ϫ1. The shapes of the
s
two curves were very alike, with the data for 37 ЊC shifted
somewhat towards lower T1-relaxivity values, as expected from
the temperature dependence of τR.
Scheme 3 Reagents and conditions: i, ClCH2COONa, EtOH–H2O,
pH 10, 70 ЊC; ii, Gd2O3, H2O, 100 ЊC.
From the data in Fig. 1 it is also apparent that the CMC of
the substance is quite low. The exact values were determined by
the method developed for paramagnetic compounds by Gäelle
et al.12 At 25 ЊC the value was determined to 0.15 mM, and at
37 ЊC it was 0.16 mM. These values were considerably lower
than for earlier reported GdHPDO3A and GdDOTA com-
plexes with lipophilic side chains of about the same length
(C12).5,12 An explanation for this might be that the effective
chain length for the GdPCTA-[12] derivative 8 is larger because
of the hydrophobic character of the pyridine unit. In Table 1
the CMC values are compared to the values for other relevant
amphiphilic Gd complexes.
A Nuclear Magnetic Resonance Dispersion (NMRD) profile
of the GdPCTA-[12] derivative 8 (0.48 mM) is shown in Fig. 2.
The data were fitted, and the parameters obtained are presented
in Table 2. The value of τM was set to 100 ns, supposing that
the introduction of a lipophilic chain did not alter the water
The corresponding Gd complex 8 was prepared by heating
the ligand with Gd2O3 in water at 100 ЊC. Purification was
achieved by continuous extraction of an alkaline (pH 9)
aqueous solution with chloroform. The electrospray MS data
confirmed the presence of the Gd complex 8.
Relaxometric characterisation of the Gd complex
Because of the amphiphilic structure of the Gd complex it was
thought to form micelles in aqueous solution. The formation
of micelles would ideally result in a longer rotational corre-
lation time (τR) and thus higher T1-relaxivity. Earlier studies
on amphiphilic gadolinium 1,4,7,10-tetraazacyclododecane-
1,4,7, 10-tetraacetate (GdDOTA)- and gadolinium 10-(2-hydr-
oxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate
(GdHPDO3A) derivatives have shown an increase in the T1-
relaxivity explained by the formation of micelles.5,11,12 The
increase was, however, not large (r1 = 10–22 sϪ1 mMϪ1, 20 or
60 MHz, 25 ЊC).
A plot of the T1-relaxivity (20 MHz, pH 7) of the GdPCTA-
[12] derivative 8 as a function of the concentration at two
different temperatures (25 ЊC and 37 ЊC) is shown in Fig. 1.
At 25 ЊC the lowest T1-relaxivity, 11.9 sϪ1 mMϪ1, was reached
at concentrations below 0.05 mM. This value was somewhat
higher than reported for a monomeric GdPCTA-[12] derivative
(r1 = 8.3 sϪ1 mMϪ1) with about the same molecular weight.13 The
reason for this might have been that the complex was not
entirely in the monomeric form, even in diluted aqueous solu-
tions. Small clusters or dimers might have formed, resulting in a
slightly increased τR. Above this concentration the T1-relaxivity
increased until it reached a plateau of 29.2 sϪ1 mMϪ1 at about
1.50 mM. As expected, the favourable τM of the coordinated
water molecules in GdPCTA-[12] derivatives yielded a higher
T1-relaxivity than the corresponding GdDOTA and GdHP-
DO3A derivatives.
Fig. 2 NMRD profile of GdPCTA-[12] derivative 8 (0.48 mM, 25 ЊC).
The line represents the best fit.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 4 4 – 6 4 7
645

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Table 2 Relaxation parameters for the GdPCTA-[12] derivative 8
obtained by the fitting of the NMRD profile, compared to the values
for GdPCTA-[12]. The values in bold were fixed in the fitting procedure
(s, 2H), 4.69 (s, 2H), 7.17 (d, 8.5 Hz, 1H), 7.36 (d, 8.5 Hz, 1H).
13C NMR (75 MHz, CDCl3): δ 14.04, 22.62, 25.87, 28.91, 29.20,
29.27, 29.47, 29.50, 29.57, 29.58, 31.84, 42.08, 46.26, 68.66,
119.57, 124.09, 145.53, 147.28, 152.92.
Complex
q
∆2/sϪ2 × 1019
τv/ps
τR/ps
r/Å
12-Dodecyloxy-3,6,9-tris(2-nitrophenylsulfonyl)-3,6,9,15-tetra-
azabicyclo[9.3.1]pentadeca-1(15),11,13-triene (5)
8
2
2
2.3 0.1
2.8
43
28
2
473 18
70
3.10
3.10
GdPCTA-[12]
Potassium carbonate (3.32 g, 24 mmol) was added to a solution
of 3-dodecyloxy-2,6-bis(chloromethyl)pyridine (3) (2.16 g,
exchange rate to a great extent. This value could have been
determined by performing variable temperature O17 NMR
measurements. Unfortunately it was not possible to dissolve the
complex in the required concentration for this experiment.
The profile showed the expected T1-relaxivity peak around
20 MHz typical of complexes with an increased τR. The value
obtained for τR by fitting of the curve was 473 ps. This value is
nearly seven times the value for GdPCTA-[12], 70 ps.9
6
mmol) and 1,4,7-tris(2-nitrophenylsulfonyl)-1,4,7-triaza-
heptane (4) (3.95 g, 6 mmol) in acetonitrile (15 ml). The mixture
was refluxed for 5 h, cooled to room temperature and evapor-
ated in vacuo. The residue was partitioned between water
(100 ml) and dichloromethane (50 ml). The aqueous phase was
extracted with dichloromethane (3 × 50 ml). The combined
organic extracts were washed with water (100 ml), dried
(MgSO4) and evaporated in vacuo. The residue was submitted
to flash chromatography (SiO2, n-hexane–ethyl acetate 2 : 3) to
yield 2.89 g (51%) of a colourless solid. Mass spectrum (ES):
m/z 946 [[M ϩ H]ϩ, C41H52N7O13S3]. 1H NMR (300 MHz,
CDCl3): δ 0.84 (t, 6.6 Hz, 3H), 1.15–1.25 (m, 16H), 1.25–1.40
(m, 2H), 1.73 (quin., 6.9 Hz, 2H), 3.05–3.25 (m, 2H), 3.25–3.40
(m, 4H), 3.40–3.50 (m, 2H), 3.94 (t, 6.6 Hz, 2H), 4.50 (s, 2H),
4.68 (s, 2H), 7.21 (d, 8.5 Hz, 1H), 7.40 (d, 8.4 Hz, 1H), 7.50–
7.75 (m, 9H), 7.80–8.10 (m, 3H). 13C NMR (75 MHz, CDCl3):
δ 14.04, 22.60, 25.73, 28.72, 29.26, 29.44, 29.53, 29.56, 31.83,
45.61, 46.32, 49.61, 54.22, 68.79, 120.00, 124.18, 124.21, 124.29,
130.79, 131.01, 131.18, 131.80, 132.05, 132.12, 132.18, 133.68,
133.79, 133.89, 143.50 145.50, 147.96, 148.13, 153.73.
Experimental
Synthesis
Reagents were obtained from Aldrich Chemical Co. Inc., USA
or Fluka Chemie AG, Switzerland and used as received.
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)
FTICRMS 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).
12-Dodecyloxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),
11,13-triene (6)
3-Dodecyloxy-2,6-bis(hydroxymethyl)pyridine (2)
Thiophenol (1.13 g, 10.3 mmol) was added to a suspension of
12-dodecyloxy-3,6,9-tris(2-nitrophenylsulfonyl)-3,6,9,15-tetra-
azabicyclo[9.3.1]pentadeca-1(15),11,13-triene (5) (2.70 g,
2.9 mmol) and potassium carbonate (3.94 g, 28.5 mmol) in
DMF (30 ml). The mixture was stirred at room temperature
under argon. Hydrochloric acid (2 M, 100 ml) was added after
5 h, and the solution was extracted with dichloromethane
(3 × 50 ml). The pH of the aqueous phase was adjusted to 11 by
the addition of solid sodium hydroxide. The alkaline solution
was extracted with chloroform (5 × 60 ml). The combined
chloroform extracts were dried (MgSO4) and evaporated in
vacuo to yield 0.91 g (82%) of a yellow oil. Mass spectrum (EI):
m/z (relative intensity) 390 (15) [Mϩ, C23H42N4O], 334 (68), 322
(65), 149 (60), 122 (63), 43 (100). 1H NMR (300 MHz, CDCl3):
δ 0.78 (t, 6.5 Hz, 3H), 1.10–1.20 (m, 16H), 1.20–1.40 (m, 2H),
1.70 (quin., 6.8 Hz, 2H), 2.45–2.60 (m, 4H), 2.80–3.00 (m, 4H),
3.83 (t, 6.5 Hz, 2H), 3.89 (s, 2H), 3.94 (s, 2H), 4.60–5.20 (m,
3H), 6.88 (d, 8.3 Hz, 1H), 6.97 (d, 8.3 Hz, 1H). 13C NMR (75
MHz, CDCl3): δ 13.92, 22.47, 25.85, 28.89, 29.13, 29.35, 29.39,
29.42, 29.44, 31.69, 47.07, 47.37, 47.46, 47.87, 48.25, 52.16,
68.16, 118.17, 119.70, 148.78, 149.51, 151.06.
To a suspension of 3-hydroxy-2,6-bis(hydroxymethyl)pyridine
(1) hydrochloride (4.79 g, 25 mmol) and potassium carbonate
in ethanol (100 ml) were added a few drops of water. The
mixture was stirred for 3 h at room temperature and filtered.
The filtrate was evaporated in vacuo and dodecyl bromide
(6.23 g, 25 mmol), potassium carbonate (6.90 g, 50 mmol) and
DMF (100 ml) were added to the residue. The mixture was
stirred at 95 ЊC under an argon atmosphere for 16 h, cooled to
room temperature and filtered. Water (50 ml) was added to
the filtrate and the solution was extracted with diethyl ether
(3 × 80 ml). The combined organic extracts were dried (MgSO4)
and evaporated in vacuo. The residue was recrystallised from
cyclohexane to yield 4.84 g (60%) of a colourless solid. Mass
spectrum (EI): m/z (relative intensity) 323 (100) [Mϩ, C19H33-
1
NO3], 292 (38), 155 (89). H NMR (300 MHz, CDCl3): δ 0.84
(t, 6.7 Hz, 3H), 1.20–1.25 (m, 16H), 1.27–1.45 (m, 2H), 1.74
(quin., 6.5 Hz, 2H), 3.93 (t, 6.5 Hz, 2H), 4.06 (s, 2H), 4.64 (s,
2H), 4.70 (s, 2H), 7.07 (d, 8.4 Hz, 1H), 7.15 (d, 8.4 Hz, 1H). 13
C
NMR (75 MHz, CDCl3): δ 14.01, 22.58, 25.90, 28.93, 29.24,
29.46, 29.49, 29.53, 29.56, 31.82, 59.92, 64.36, 68.32, 118.31,
119.90, 147.65, 149.37, 150.92.
3-Dodecyloxy-2,6-bis(chloromethyl)pyridine (3)
12-Dodecyloxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),
11,13-triene-3,6,9-triacetic acid (7)
Thionyl chloride (10 ml) was added dropwise to 3-dodecyloxy-
2,6-bis(hydroxymethyl)pyridine (2) (3.88 g, 12 mmol) at 0 ЊC.
After complete addition the temperature was raised to 40 ЊC.
After 4 h the mixture was cooled to room temperature and
evaporated in vacuo. Water (20 ml) was added to the residue and
the solution was neutralised by addition of aqueous sodium
carbonate (10%). This solution was extracted with dichloro-
methane (3 × 40 ml) and the combined organic extracts were
dried (MgSO4) and evaporated in vacuo. Yield: 3.89 g (90%) of
a slightly yellow solid. Mass spectrum (EI): m/z (relative inten-
12-Dodecyloxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),
11,13-triene (6) (0.85 g, 2.2 mmol) in ethanol (20 ml) was added
to a solution of sodium chloroacetate (1.52 g, 13.1 mmol) in
water (20 ml). The pH was adjusted to 10 by the addition of
sodium hydroxide (1 M). The solution was heated at 70 ЊC for
48 h, maintaining a pH of 10. The solution was evaporated in
vacuo and the residue was submitted to flash chromatography
(SiO2, CHCl3–CH3OH–NH3 (25%) 9 : 6 : 1) yielding 265 mg
(21%) of a slightly yellow solid. Mass spectrum (ES): m/z 565
[[M ϩ H]ϩ, C29H49N4O7]. 1H NMR (300 MHz, CD3OD): δ 0.89
(t, 6.2 Hz, 3H), 1.20–1.40 (m, 16H), 1.45–1.60 (m, 2H), 1.75–
1.90 (m, 2H), 1.90–2.20 (m, 2H), 2.21–2.50 (m, 3H), 2.51–2.65
sity) 359 (89) [Mϩ, C19H31Cl2NO], 191 (100). H NMR (300
MHz, CDCl3): δ 0.85 (t, 6.7 Hz, 3H), 1.20–1.35 (m, 16H), 1.40–
1.50 (m, 2H), 1.81 (quin., 6.5 Hz, 2H), 4.00 (t, 6.4 Hz, 2H), 4.61
1
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 4 4 – 6 4 7
646

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(m, 2H), 2.66–2.80 (m, 1H), 2.90–3.05 (m, 1H), 3.10–3.27 (m,
2H), 3.35–3.50 (m, 3H), 3.55–3.70 (m, 1H), 3.90–4.20 (m, 5H),
7.23 (d, 8.3 Hz, 1H), 7.45 (d, 8.4 Hz, 1H). 13C NMR (75 MHz,
CD3OD): δ 14.53, 23.73, 27.19, 30.13, 30.48, 30.73, 30.76,
30.79, 33.06, 55.03, 55.58, 56.40, 57.04, 59.39, 61.14, 62.11,
62.74, 63.32, 64.73, 69.94, 121.89, 122.86, 149.40, 150.05,
153.10, 179.91.
The Gd concentrations were determined by mineralisation
with hydrochloric acid (37%), followed by T1-measurements.
The T1-relaxivity of Gd() under these conditions is 13.5 sϪ1
mMϪ1
.
The NMRD profile was recorded at 25 ЊC by a Spinmaster
FFC, fast field cycling relaxometer (Stelar, Mede (PV), Italy)
installed at the LIMA laboratory (Bioindustry Park, Ivrea
(TO), Italy). The relaxation parameters obtained from the
experimental NMRD data were calculated using a programme
written by E. Terreno (University of Turin, Italy) and the
computer software Origin (MicrocalTM Origin, Northampton,
MA).
Gadolinium(III) 12-dodecyloxy-3,6,9,15-tetraazabicyclo[9.3.1]-
pentadeca-1(15),11,13-triene-3,6,9-triacetate (8)
Gadolinium()oxide (36 mg, 0.1 mmol) was added to a solu-
tion of 12-dodecyloxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-
1(15),11,13-triene-3,6,9-triacetic acid (7) (113 mg, 0.2 mmol) in
water (10 ml), and the pH was adjusted to 4 by addition of
acetic acid. The suspension was heated to 100 ЊC for 24 h. The
resulting clear solution was cooled to room temperature and the
pH adjusted to about 9 by addition of aqueous sodium hydrox-
ide (1 M). The solution was then continuously extracted with
chloroform for 16 h. The chloroform extract was dried (MgSO4)
and evaporated in vacuo to yield 100 mg (69%) of a colourless
solid. Mass spectrum (ES): m/z 780 [M ϩ CH3COOH ϩ H]ϩ,
720 [[M ϩ H]ϩ, C29H46N4O7Gd]. Anal. Calcd. (found) for
C29H45N4O7Gdؒ9H2O: C, 39.50 (39.18); H, 7.15 (6.88); N, 6.35
(6.10)%.
Acknowledgements
Financial support from the EU in the frame of the COST D8/
D18 action and from Amersham Health A/S (Oslo, Norway) is
gratefully acknowledged. The authors thank Mr J. Vedde,
Department of Chemistry, University of Oslo (MS) and
Professor S. Aime and Dr E. Gianolio, University of Turin
(NMRD) for their contributions.
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(2)
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.
A stock solution of 1.50 mM was prepared by dissolving
the complex in water and adjusting the pH to 7.0. The other
solutions were prepared by diluting the stock solution. In
addition a solution of 3.25 mM was prepared.
The r1 values were plotted against the concentration as
shown in Fig. 1. Analyses were performed in triplicate at each
concentration.
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 4 4 – 6 4 7
647