Binuclear gadolinium(III) complex based on DTPA and 1,3-bis(4-aminophenyl)adamantane as a high-relaxivity MRI contrast agent

Article abstract of DOI:10.1016/j.poly.2018.01.032

A novel dimeric gadolinium(III) complex, bis(4-Gd-DTPA-aminophenyl)adamantane (5), was successfully synthesized using bis(4-aminophenyl)adamantane to link two Gd-DTPA units. All the intermediates and bis(4-Gd-DTPA-aminophenyl)adamantane (5) have been characterized by ¹H NMR, ¹³C NMR, ES-API-MS, HR-ESI-MS, FT-IR and elemental analysis (EA). The longitudinal relaxivity of bis(4-Gd-DTPA-aminophenyl)adamantane is 8.43 mM^?1 Gd^?1 s^?1 at 0.43 T (32 °C, pH 7.0), and 4.89 mM^?1 Gd^?1 s^?1 at 3.0 T (32 °C, pH 7.4). The T₁-weighted imaging in vitro showed that bis(4-Gd-DTPA-acetylaminophenyl)adamantane can enhance the contrast of images distinctly. But most of all, the kinetic inertness of bis(4-Gd-DTPA-aminophenyl)adamantane (5) was comparable to that of [Gd(DTPA)(H₂O)]^2? (Magnetvist). Moreover, cytotoxicity tests indicated that the bis(4-Gd-DTPA-acetylaminophenyl)adamantane exhibited low toxicity to healthy liver cells (L-02 cells). These results demonstrate that the Gd(III) complex 5 may be a promising MRI contrast agent candidate.

Full text of DOI:10.1016/j.poly.2018.01.032

Accepted Manuscript
Binuclear gadolinium(III) complex based on DTPA and 1,3- bis(4-aminophen-
yl)adamantane as a high-relaxivity MRI contrast agent
Fuxian Wan, Lipeng Wang, Wenxin Xu, Changcheng Li, Ying Li, Chunbao
Zhang, Lin Jiang
PII:
S0277-5387(18)30062-7
https://doi.org/10.1016/j.poly.2018.01.032
POLY 13030
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
12 October 2017
29 January 2018
Please cite this article as: F. Wan, L. Wang, W. Xu, C. Li, Y. Li, C. Zhang, L. Jiang, Binuclear gadolinium(III)
complex based on DTPA and 1,3- bis(4-aminophenyl)adamantane as a high-relaxivity MRI contrast agent,
Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.01.032
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Binuclear gadolinium(III) complex based on DTPA and 1,3-
bis(4-aminophenyl)adamantane as a high-relaxivity MRI contrast agent
Fuxian Wana,*, Lipeng Wanga, Wenxin Xua,Changcheng Lia, Ying Lia, Chunbao
Zhangb, Lin Jianga,*
aCollege of Chemistry and material, Shan Dong Agricultural University, Taian 271018, PR China
bQuzhou Amk Biotech Co. Ltd., Quzhou 324000, PR China
ABSTRACT
A novel dimeric gadolinium(III) complex, bis(4-Gd-DTPA-aminophenyl)adamantane (5), was
successfully synthesized using bis(4-aminophenyl)adamantane to link two Gd-DTPA units. All the
1
intermediates and bis(4-Gd-DTPA-aminophenyl)adamantane (5) have been characterized by H
NMR,13C NMR, ES-API-MS, HR-ESI-MS, FT-IR and elemental analysis (EA). The longitudinal
relaxivity of bis(4-Gd-DTPA-aminophenyl)adamantane is 8.43 mM−1Gd-1s−1 at 0.43 T (32 °C, pH 7.0),
and 4.89 mM−1Gd-1s−1 at 3.0 T (32 °C, pH 7.4). The T1-weighted imaging in vitro showed that
bis(4-Gd-DTPA-acetylaminophenyl)adamantane can enhance the contrast of images distinctly.
But most of all, the kinetic inertness of bis(4-Gd-DTPA-aminophenyl)adamantane (5) was comparable
to that of [Gd(DTPA)(H2O)]2-(Magnetvist®). Moreover, cytotoxicity tests indicated that the
bis(4-Gd-DTPA-acetylaminophenyl)adamantane exhibited low toxicity to healthy liver cells (L-02 cells).
These results demonstrate that the Gd(III) complex 5 may be a promising MRI contrast agent candidate.
Keywords: adamantane, DTPA, rigid linker, MRI contrast agent, high relaxivity
* Corresponding author. Tel: +86 0538 8242251; Fax:+86 0538 8242251 E-mail address:
wfx@sdau.edu.cn (F.X. Wan) jiangl@sdau.edu.cn (L. Jiang)
1

1. Introduction
Nowadays, with medical imaging techniques, including ultrasonic diagnosis (US), computed
tomography (CT), single-photon emission computed tomography (SPECT), positron emission
tomography (PET) and magnetic resonance imaging (MRI), and various diseases can be diagnosed more
and more accurately [1-3]. Among these techniques, magnetic resonance imaging (MRI) shows
significant advantages, such as, non-invasiveness, high spatial resolution (<0.1 mm), 3D anatomical
images, temporal resolution, and deep tissue penetration [4,5]. Unfortunately, the contrast of MRI
images is often insufficient for diagnosing early or minimal lesions, thus preventing doctors from
making the accurate diagnosis decision and developing the right therapeutic schemes. With the help of
MRI contrast agents, the diagnostic accuracy can be improved distinctly in clinical practices [6-9], such
as [Gd(DTPA)(H2O)]2- (Magnetvist®, Schering AG, Germany), [Gd(DTPA-BMA)(H2O)] (Omniscan®,
Extracellular agent, Amersham-Nycomed, Norway) [Gd (BOPTA)(H2O)]-2 (Multihance®, Liver specific
agent, Bracco Imaging, Italy), and [Gd (HP-DO3A)(H2O)] (ProHance®, Extracellular agent, Bracco
Imaging, Italy) [10,11]. Annually, MRI contrast agents have been applied in more than 50% of MRI
diagnoses [12,13]. However, most commercial contrast agents possess several undesirable defects, such
as low longitudinal relaxivity, low tissue specificity, and rapid clearance [14-17]. These shortcomings
hindered the application of MRI in clinical practices and life science research. Therefore, many research
groups are committed to designing and preparing the better MRI contrast agents.
In a facile and wieldy adopted strategy to prepare new MRI contrast agent with significantly
enhanced contrast signals (high r1 value), the macromolecule framework was ornamented with small
molecular gadolinium complexes through non-covalent or covalent bond, such as Gd-DTPA,
Gd-DOTA, and Gd-DO3A [18,19]. Although macromolecular contrast agents possess high-performance
properties compared with traditional small molecular contrast agents, their applications in clinic is
limited by some drawbacks, such as toxicity, slow excretion rate, and immunogenicity [20-22].
Nephrogenic system fibrosis (NSF) as a rare systemic disease associated with exposure to gadolinium
based contrast agents (GBCAs) was first reported by T. Grobner in two case series in 2006 [23].
2

Subsequently, more and more cases were reported globally and the safety of GBCAs has been attracted
more attention [24]. Consequently, strict guidelines for use of GBCAs in MRI were enacted by Food
and Drug Administration(FDA) or European Medicines Agency(EMEA) [25]. In the process of the
development of new-generation of MRI CAs with mutifunction without impairing their high
thermodynamic and kinetic stability would be more attractive [26-28]. Thus, there is still an opportunity
for the development of small molecular imaging agents with high-performance properties. It is worth
noting that a number of multi-nuclear metal complexes with high longitudinal relaxivity have been
developed on the basis of increasing the rotational correlation times (τr) [29-36].
Adamantane and its derivatives have attracted more and more attention for their thermal stability,
good oxidation resistance, and highly lipophilic property. Particularly, in the field of medicine synthesis,
adamantane was often introduced into drug molecules (including magnetic resonance imaging contrast
reagents) because it can penetrate the lipid layers of membranes [37-40].
In our previous work, we reported several binuclear contrast agents with high relaxivity and
hepatic targeting compared to the Gd-DTPA [41-43]. In order to design and prepare more efficient
contrast agents, here we reported the synthesis and characterization of one novel chelate molecule and
its complex with gadolinium. The synthetic scheme of ligand 4 and complex 5 is shown in Scheme 1.
In this paper, two DTPA units were attached to adamantane scaffold by the amidation reaction of
bis(4-aminophenyl)adamantane (3) with diethylenetriaminepentaacetic dianhydride (DTPA), and then
corresponding Gd(III) complex was obtained. The longitudinal relaxivity (r1) , transmetalation kinetics
and T1-weighted imaging in vitro of bis(4-Gd-DTPA-acetylaminophenyl)adamantane (5) and Gd-DTPA
were evaluated and compared under the same conditions. Futhermore, the cytotoxicity in vitro of
bis(4-Gd-DTPA-acetylaminophenyl)adamantane (5) compared with Gd-DTPA was determined to assess
the toxicity of the agents.
2. Experimental
2.1. General information
Melting points were determined on a MP430 melting point apparatus (Jinan Hanon Instruments Co.,
3

13
Ltd., China) and are uncorrected.1H NMR and C NMR spectra were recorded on a Bruker AM-400
spectrometer using TMS as internal standard (1H NMR at 400 MHz, 13C NMR at 100 MHz). IR spectra
were recorded on a Bruker EQUINOX 55 (Bruker Optics Inc., Germany). Elemental analyses were
measured by an Elementary Vario EL III analyzer (Elementar, Germany). ES-API-Ms spectra were
obtained on a Angilent 6110 (Angilent Technologies, USA). HR-ESI-Ms spectra were obtained on a
Bruker micrOTOF ( Germany ).The longitudinal relaxation time (T1) was determined on a MicroMR
(Niumag Technology Co., Ltd., Suzhou, China) and Siemens Prisma 3.0 T MR scanner (Erlangen,
Germany).
1-adamantanol, diethylenetriaminepentaacetic dianhydride (DTPA), acetanilide were purchased
from Aladdin. All solvents and reagents were obtained from commercial sources and used without
further purification.
2.2. Synthesis
2.2.1. Synthesis of bis( 4-acetylaminophenyl)adamantane (2)
The synthesis of bis( 4-acetylaminophenyl)adamantane (2) was accroding to lierature method with
small changes [44]. 1-adamantanol (1) (3.75 g, 25 mmol) and acetanilide (7.8 g, 50 mmol) were
suspended in 38 mL of concentrated sulfuric acid (98%) under N2 atmosphere. After ultrasonic vibration
(100 W) for 4 h at room temperature, the mixture was added dropwise to the ice water (100 mL) with
vigorously stirring and the crude product precipitated out. The solid was obtained by vacuum filtration
and rinsed thoroughly with distilled water, then recrystallized from ethanol-water (1/3, v/v) to give
bis( 4-acetylaminophenyl)adamantane (2) as a white solid. Yield: 78%. mp 275–276.8 °C. FT-IR (KBr,
cm-1): 3297, 3256, 2908, 2849, 1666, 1601, 1535; 1H NMR (400 MHz, DMSO-d6, δ/ppm): 1.73 ( s, 2H ),
1.82-1.90 (m, 10H ) , 2.02 ( s, 6H ) , 2.23 ( s, 2H ), 7.31 ( d, J = 8.4 Hz, 4H) , 7.49 ( d, J = 8.4 Hz, 4H),
9.80( s, 2H ); 13C NMR ( 100 MHz, DMSO-d6, δ/ppm ): 24.38, 29.49, 35.75, 36.89, 42.22, 49.06,
119.32, 125.40, 137.38, 145.60, 168.50.
2.2.2. Synthesis of bis(4-aminophenyl)adamantane (3)[44]
To a solution of bis(4-acetylaminophenyl)adamantane (2) (4.03 g, 10 mmol) in EtOH (60 mL) was
4

added 10 mL aqueous solution of NaOH (12 M). The mixture was refluxed under N2 atmosphere for 12
h. After the solution was cooled down to room temperature, the mixture was added dropwise to the ice
water (150 mL) with vigorously stirring and the crude product precipitated out. The solid was obtained
by vacuum filtration and rinsed thoroughly with distilled water, then recrystallized from ethanol-water
(2/3, v/v) to give bis(4-aminophenyl)adamantane (3) as a white solid. Yield: 90%. mp 195-196.4 °C.
FT-IR (KBr, cm-1): 3429, 3346, 3020, 2909, 2847, 1605, 1514, 1433, 1271, 1178, 812, 650, 528; 1H
NMR (400 MHz, CDCl3, δ/ppm): 1.58( s, 2H ) , 1.85-1.98 ( m, 10H ) , 2.26 ( s, 2H ), 3.54( s, 4H ) , 6.66
13
(d, 4H, J = 8.4 Hz), 7.18( d, 4H, J = 8.4 Hz) (Fig. S1 in Supplementary Information); C NMR (100
MHz, CDCl3, δ/ppm): 141.37, 125.81, 125.72, 115.09, 49.40, 42.58, 36.53, 35.99, 29.70.
2.2.3. Synthesis of bis(4-DTPA-acetylaminophenyl)adamantane (4)
Diethylenetriaminepentaacetic dianhydride (1.78 g, 5 mmol) was dissolved in fresh distilled DMF
(50 mL) by heating at 60 °C, and then a catalytic amount of DMAP (4-dimethylaminopyridine) was
added. After the solution was cooled down to 20 °C, bis(4-aminophenyl)adamantane (3) (0.80 g, 2.5
mmol) was added into the solution to react for 24 h at 20 °C, and then, the solution was heated to 50 °C
for another 48 h. Subsequently, water (0.09 g, 5 mmol) was added to hydrolyze anhydride with
vigorous stirring for 2 h at 50 °C. After most of the solvent was removed under vacuum, crude product
was precipitated with anhydrous EtOH-Et2O (1/2, v/v). The crude product was recrystallized from
EtOH-H2O (1/3, v/v) to give bis(4-DTPA-acetylaminophenyl)adamantane (4) as a white solid. Yield:
73%. mp 185–187.5 °C. FT-IR (KBr, cm-1): 3424, 3024, 2908, 2851, 1631, 1454, 1385, 1144, 981, 952,
852, 692, 561; 1H NMR (400 MHz, DMSO-d6): 1.73 (t, 6H), 1.86 (t, 6H), 2.20 (d, J = 7.2 Hz, 2H), 2.89
(t, 3JH,H = 6.0 Hz, 8H) , 2.92 (t, 3JH,H =6.0 Hz, 8H), 3.03 (s, 8H), 3.43 (s, 8H), 3.56(s, 4H), 7.33-7.26 (m,
13
4H, Ar-H), 7.61-7.55 (m, 4H, Ar-H), 9.99 (s, 2H, -CONH-). C NMR (100 MHz, DMSO-d6): 173.38,
173.03, 169.53, 145.99, 136.72, 125.35, 119.78, 58.56, 55.53, 52.34, 50.92, 50.26, 48.99, 42.20, 36.91,
35.73, 29.47. Anal. Calcd for C50H68N8O18·2H2O: C, 54.34; H, 6.57; N, 10.14. Found: C, 54.42; H, 6.58;
N, 9.93. ES-API-MS (m/z):calcd for Calcd. for C50H68N8O18, 1069.5 ([M+H] +). Obsd. 1069.6 ([M+H]
+).
5

2.2.4. Synthesis of bis(4-Gd-DTPA-aminophenyl)adamantane (5)
Bis(4-DTPA-acetylaminophenyl)adamantane (4) (1.069 g, 1.0 mmol) was suspended in pure water
(10 mL), and then the aqueous solution of NaOH (1.0 M) was added dropwise until the suspended solids
exactly dissolves with stirring, a transparent solution was obtained. 5 mL aqueous solution of
GdCl3·6H2O (0.74 g, 2.0 mmol) was added dropwise to the solution, after which, the solution was
stirred at 80 °C for 24 h, the pH value was periodically maintained to 7.0 with NaOH (1.0 M). Until no
free gadolinium ions was detected by the xylenol orange test, and the white precipitate formed after
vacuum
evaporation,
washed
with
acetone,
and
dried
to
afford
bis(4-Gd-DTPA-aminophenyl)adamantane (5). Yield: 90%. mp > 250 °C. FT-IR (KBr, cm-1): 3422,
2910, 2852, 1610, 1403, 1125, 987, 952, 858, 541; Anal. calcd for C50H62Gd2N8ClNaO18.5: C, 41.59; H,
4.33; N, 7.76. found C, 41.54; H, 4.37; N, 7.74.
HR-ESI-MS (m/z):calcd for Calcd. for C50H62Gd2N8O18, 733.6066([M+K+Na+1.5H2O]2+),
1444.2176([M+K+1.5H2O]+). Obsd. 733.6173([M+K+Na+1.5H2O]2 +), 1444.2454([M+K+1.5H2O]+).
2.2.5. Relaxation Measurements
In this experiment, bis(4-Gd-DTPA-aminophenyl)adamantane (5) and Gd-DTPA were dissolved in
pure water to obtain different concentrations of the Gd(III) ion (0.2, 0.4, 0.8, 1.0, and 1.2 mM) and
arranged on the sample board according to the concentration of the Gd(III) ion, and T1 was measured
according to a standard inversion-recovery sequence at 32 °C and 18.169 MHz on a Meso PQOO1
(Niumag Technology Co., Ltd., China) or at 32 °C and 128 MHz on Siemens Prisma 3.0 T MR scanner
(Erlangen, Germany) with gradient strength up to 80 mT/m.
2.2.6. Transmetalation Kinetics
This test was performed according to the reference method [45]. The water proton paramagnetic
P
longitudinal relaxivity ( R1 ) of a buffered solution (phosphate buffer, pH 7.4) containing 2.5 mmol/L
gadolinium complex and 2.5 mmol/L ZnCl2 was measured during a period of 84 h. The measurements
were performed on a Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) with gradient strength up
to 80 mT/m.
6

2.2.7. In vitro cell cytotoxicity assay
The cytotoxicity of bis(4-Gd-DTPA-aminophenyl)adamantane (5) and Gd-DTPA toward healthy liver
cells
(L-02
cells)
was
evaluated
by
MTT
assays
(MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [46]. Healthy liver cells (L-02 cells)
were seeded in 96-well plates with a density of 4 × 104 cells/well, after incubated for 24 h at 37 °C in
5% CO2 atmosphere, and then various concentrations of the Gd (III) ion was added and co-incubated for
another 72 h under the same conditions. After washed with PBS three times, 0.5% MTT solution (5
mg/mL, 20 μL/well) was added to each well and incubated for another 4 h. The absorbance was
measured at 490 nm on a DG-5033A ELISA-Reader after adding 200 μL of DMSO. The L-02 cell
survival rate was calculated according to Formula (1).
Cell viability=(At-Abc)/(Anc-Abc)×100%,
(1)
Where At is the absorbance of the experimental group; Abc is the absorbance of the blank control group;
Anc is the absorbance of the negative control group. All the results are shown as the mean differences
and tested by a t-test to check for the significance. P<0.05 indicates the significant difference.
2.2.8. T1-weighted imaging in vitro
T1-weighted imaging in vitro of bis(4-Gd-DTPA-aminophenyl)adamantane (5) and Gd-DTPA was
obtained on a MR23-060H-I imaging & analyzing system (Niumag Technology Co., Ltd., China) at
32 °C. The MRI experimental parameters were set as: a 0.5T magnet, FOVRead=100mm,
FOVPhase=100mm, TR=200ms, TE=13.42ms, Slice Width=6.0mm, Slice Gap=2.0mm, NS=4, matrix
192 × 256.
3. Results and discussion
3.1. Synthesis of the Ligand and Complex
In order to synthesize a new MRI contrast agent with high longitudinal relaxivity, the ligand
bis(4-DTPA-acetylaminophenyl)adamantane (4), containing two DTPA units covalently appended onto
the bis(4-aminophenyl)adamantane framework through the carboxyl group of DTPA, was synthesized
via the amidation reaction of diethylenetraiaminepentaacetic dianhydride (DTPA) with
7

bis(4-aminophenyl)adamantane (3) in DMF catalyzed by 4-dimethylaminopyridine in a 2:1 molar ratio,
and then the bis(4-Gd-DTPA-aminophenyl)adamantane (5) complex was obtained easily. The two
Gd-DTPA units linked by bis(4-aminophenyl)adamantane is the highlight.
1
The structure of 4 was characterized by FT-IR, ES-API-MS, H NMR, 13C NMR (Fig. S2 in
Supplementary Information) and elemental analysis (EA). The peaks of NCH2COOH groups (DTPA),
aryl groups (benzene) and adamantane group of bis(4-acetylaminophenyl)adamantane structure were
1
obviously observed in H NMR spectra (Fig. S3 in Supplementary information), indicating that two
DTPA fragments were appended to bis(4-aminophenyl)adamantane (3) successfully. The MS spectra
(Fig. S4 in Supplementary information) further confirmed the given structure.
In the IR spectra of 4 (Fig. 1a), the broad and strong peaks around at 3424 cm-1 were assigned to the
N–H and O–H stretching vibration. The sharp peaks around at 1631 cm-1 were ascribed to the vibrations
of vs(-CONH-). In the IR spectra of bis(4-Gd-DTPA-acetylaminophenyl)adamantane (5) (Fig. 1b), the
broad bands around at 3422 cm-1 was ascribed to the vs(O-H) and vs(N-H) vibrations. The peaks at 1610
-
-
cm-1 and 1406 cm-1 were the result of the vas(-CO2 ) and vs(-CO2 ) vibrations, respectively. The
HR-ESI-MS spectra (Fig. S5 in Supplementary information) further confirmed the given structure.
3.2. Longitudinal Relaxivity
Longitudinal relaxation rate (r1) is a key factor for evaluating the performance of the synthesized
MRI contrast agent. Longitudinal relaxation rate (r1) can be calculated based on Formula 2 established
by Bloembergen and Solomon [47,48]:
r1[M] = 1/T1,obsd -1/T1,d
(2)
As shown in Fig. 2, the spin–lattice relaxivity value (r1) of bis(4-Gd-DTPA-acetylaminophenyl)
adamantane prepared in our present work was 8.43 mM−1s−1 and the r1 of [Gd(DTPA)(H2O)]2- was 3.86
mM−1s−1 at 18.168 Hz and 32°C. As shown in Fig. 3, the spin–lattice relaxivity value (r1) of
bis(4-Gd-DTPA-acetylaminophenyl) adamantane was 4.89 mM−1s−1 and the r1 of [Gd(DTPA)(H2O)]2-
was 3.60 mM−1s−1 at 128 Hz and 32°C. The longitudinal relaxivity is expressed per mol gadolinium of
8

CAs
under
different
conditions.
The
relaxation
rate
(r1)
of
bis(4-Gd-DTPA-acetylaminophenyl)adamantane was significantly higher than that of Gd-DTPA under
the same conditions. The difference may be interpreted as follows, the molecular weight of the dimer
was increased by rigid linker (adamantane and aryl group), thus appropriate rotational correlation time
(τr)(It is beneficial to obtain increased high field relaxivity). The results were consistent with previous
reports [30,35,36]. Furthermore, adamantane and its derivatives may be used as suitable moiety to
synthesize new paramagnetic MRI contrast agents.
3.3. Transmetalation Kinetics
p
The kinetic inertness for various Gd-Complexes can be evaluated directly by the evolution of R1
p
(t)/R1 (0) as a function of time ([Gd]0 and [ZnCl2 ]0 = 2.5 mM in PBS (pH 7.4) at 128 MHz (3 T) and
310 K). Among various endogenous ions, Zn2+ has been applied due to its higher concentration in
plasma than any other ions(Cu2+, Ca2+, K+) and high slectivity constant over gadolinium ions(Gd3+ to
form
GdPO4 precipitate).
As
shown
in
Fig.
4,
the
kinetic
stability
of
bis(4-Gd-DTPA-acetylaminophenyl)adamantane is comparable with Gd-DTPA, there were no
significant changes in relaxivity for as long as 48 hours. This indicated that
bis(4-Gd-DTPA-acetylaminophenyl)adamantane had a relative high kinetic stability toward
transmetallation by Zn2+.
3.4. In vitro MR imaging
Fig. 5 showed in vitro FLASH images obtained with bis(4-Gd-DTPA-acetylaminophenyl)
adamantane (5) (A) and Gd-DTPA (B) solutions in phantoms. T1-weighted phantom images of
bis(4-Gd-DTPA-acetylaminophenyl)adamantane (5) (A) exhibit a stronger positive contrast effect
(bright signal) than Gd-DTPA at a given Gd3+ concentration. The effect was significant when the
concentration was higher than 0.8 mM/L, indicating that bis(4-Gd-DTPA-aminophenyl)adamantane (5)
may be used as T1-MRI contrast agent with the superior performance.
3.5. In vitro cytotoxicity assay
The ability of Bis(4-Gd-DTPA-aminophenyl)adamantane (5) to induce cytotoxicity on the healthy
9

liver cells (L-02 cells) was evaluated with methylthiazole tetrazolium (MTT) assay. When the cells were
respectively incubated with bis(4-Gd-DTPA-acetylaminophenyl)adamantane (5) and Gd-DTPA at the
concentration of 400 µM/L in the growth medium, the viability of L-02 cells retained 87.50% and
85.17% compared to the negative control group (Fig. 6). When the concentration of Gd(III) ion was
below 400 µM, the survival rate of the L-02 cells was high; when the concentration of Gd(III) ion was
increased above 400 µM, the survival rate of the L-02 cells was significantly decreased.
Bis(4-Gd-DTPA-aminophenyl)adamantane possesses the obviously low cytotoxicity to L-02 cells and
may be used as a safe MRI contrast agent.
4. Conclusion
In the study, we have successfully synthesized
a
new gadolinium complex
(bis(4-Gd-DTPA-aminophenyl)adamantane) via a facile and efficient reaction. This Gd(III) complex
possesses high longitudinal relaxation rate (r1), high kinetic stability toward transmetallation by Zn2+,
low cytotoxicity, good water-solubility, easy to prepare, and the excellent T1-weighted imaging
performance in vitro. Based on the preliminary results, we will perform in vivo imaging and biological
assays with the new MRI contrast agent in the future.
Acknowledgements
This work was supported by the Natural Science Foundation of Shandong Province, China (No.
ZR2014BM030).
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12

Figure Captions:
Scheme 1. Synthesis of gadolinium complex 5
13

Fig. 1. FT-IR spectra of (a) 4 and (b) 5.
14

Fig. 2. 1/T1 vs the Gd3+ concentrations of complex 5 and Gd-DTPA in aqueous solution (pH 7.0)
at18.169 MHz (0.43 T) and 305 K.
Fig. 3. 1/T1 vs the Gd3+ concentrations of complex 5 and Gd-DTPA in PBS (pH 7.4)
at 128 MHz (3 T) and 305 K.
15

Fig. 4. Evolution of longitudinal relaxation rates R 1 p (t)/R 1 p(0) as a function of time for
various MRI CAs ([Gd]0 and [ZnCl2]0 = 2.5 mM in PBS (pH 7.4) at 128 MHz (3 T) and 310 K).
16

Fig. 5. T1-weighted phantom MR images for 5 (A) and Gd-DTPA (B) under a concentration of 0-1.2
mM at 0.5 T.
17

Fig. 6. Effect of Gd-DTPA (Magnevist) and 5 in the viability of L-02 cells (Human normal liver cells)
were assessed with the MTT assay after treating the cells with various concentrations of the complexes
for 144 h. Data represent mean values of two independent experiments.
18

A gadolinium(III) complex, bis(4-Gd-DTPA-aminophenyl)adamantane, the longitudinal
relaxivity of it is 8.43 mM−1Gd-1s−1, which is higher than that of [Gd(DTPA)(H2O)]2-
(3.86 mM−1s−1). The T1-weighted imaging showed that it can enhance the contrast of
images distinctly. Moreover, cytotoxicity tests indicated that the it exhibited less toxicity
to L-02 cells.
19