Rigid Mn(II) chelate as efficient MRI contrast agent for vascular imaging

Article abstract of DOI:10.1039/c2dt31696j

The aza-semi-crown pentadentate ligand rigidified by pyridine and piperidine rings was designed and synthesized. It can react with Mn(II) in water to form complex with improved longitudinal relaxivity, leading to efficient signal intensity enhancement of vascular vessels under a clinical magnetic resonance imaging scanner.

Full text of DOI:10.1039/c2dt31696j

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Rigid Mn(II) chelate as efficient MRI contrast agent for vascular imaging†
Hongying Su,‡a Changqiang Wu,‡a Jiang Zhu,*b Tianxin Miao,a Dan Wang,a Chunchao Xia,c Xuna Zhao,d
Qiyong Gong,c Bin Songc and Hua Ai*a,c
Received 27th July 2012, Accepted 12th October 2012
DOI: 10.1039/c2dt31696j
residence life time), and the rotational correlation time of the
chelate (τR).12 Whereas optimal values of τR can be easily
achieved by designing slow-moving systems and increasing the
molecular weight of contrast agents.13–16 It has been recently
suggested that the rigidified backbone of a ligand with comp-
lementary cavity for metal ion is helpful for increasing the
complex stability, and favorable for improving longitudinal
relaxivity.17–20 Tóth et al. reported a series of 12/15-membered
pyridine based macrocyclic pentadentate ligands (12-PyDO1R,
15-PyN5, 15-PyN3O2) (Scheme 1A) which have one or two
inner-sphere water molecules in the manganese(II) complex.21–23
The presence of the pyridine rigidifies the macrocyclic cavity
and, therefore, increases the complex’s stability and relaxivity.
To ensure the safety and efficiency of manganese(II) chelates
as MRI CAs, highly coordinating ligand was used to maintain
the chelates with high thermodynamic stability and kinetic inert-
ness, with an inner-sphere of the complex having at least one
coordination site for water molecule. Normally, the coordination
number of manganese(II) is either 6 or 7.24 For these consider-
ations, the maximum number of donor atoms in an ideal ligand
to chelate manganese(II) as MRI CAs should not exceed 6. The
aza-crown ether is a useful structure for selective complexation
of metal ion in solution.25 Here, we have designed and syn-
thesized of an aza-semi-crown pentadentate ligand (DL-1,1′-
(pyridine-2,6-diylbis(methylene))bis(piperidine-2,1-diyl))diformic
acid, abbreviated as L), which contains pyridine and piperidine
rings to keep the ligand rigidified (Scheme 1B). The ligand will
The aza-semi-crown pentadentate ligand rigidified by pyri-
dine and piperidine rings was designed and synthesized. It
can react with Mn(II) in water to form complex with
improved longitudinal relaxivity, leading to efficient signal
intensity enhancement of vascular vessels under a clinical
magnetic resonance imaging scanner.
Magnetic resonance imaging (MRI) is one of the best noninva-
sive diagnostic modality today in medical imaging, but more
than 40% of all MRI examinations rely on contrast agent admin-
istration because of its low sensitivity.1 Complexes of paramag-
netic metals (Gd3+ or Mn2+) are widely used as longitudinal (T1)
contrast agents.1–4 Administration of Gd-based MRI contrast
agent can generate serious side effects such as nephrogenic sys-
temic fibrosis (NSF) in some patients,5 leading to numerous
interests involved in new MRI contrast agents (CAs) develop-
ment in recent years, such as iron oxide based imaging probes6,7
or manganese-enhanced magnetic resonance imaging
(MEMRI).5,8 Manganese(II) has five unpaired electrons, slow
electron relaxation, fast water exchange rate, and lower intrinsic
toxicity than Gd3+, which makes it an attractive alternative of
gadolinium(III) for enhanced MRI applications. Aime and Cara-
van’s work9,10 on Mn-EDTA (coordination number = 6, with
one water in the inner-sphere) has demonstrated that albumin-
bounding can improve the complex’s in vivo stability and
enhance its T1 relaxivity.
The efficiency of a T1 contrast agent is described by its proton
relaxivity (r1), originating from the inner- and outer-sphere
mechanisms.11 The following parameters usually have obvious
effects on r1: the number of coordinated water molecules
(hydration number, q), the exchange rate of water molecules
between inner- and outer-sphere (kex = 1/τm, in which τm is the
aNational Engineering Research Center for Biomaterials, Sichuan
University, Chengdu 610064, P. R. China. E-mail: huaai@scu.edu.cn;
Fax: +86-28-85413991; Tel: +86-28-85413991
bSichuan Key laboratory of Medical Imaging, North Sichuan Medical
College, Nanchong 637007, China. E-mail: zhujiang312@hotmail.com;
Tel: +86-817-2802315
cDepartment of Radiology, West China Hospital, Sichuan University,
Chengdu 610041, China
dPhilips Healthcare, Beijing 100020, China
†Electronic supplementary information (ESI) available: Experimental
details, spectral characterisation, and analytical data of reported com-
pounds. CCDC 876927. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2dt31696j
‡These authors contributed equally in this work.
Scheme 1 (A) 12/15-membered pyridine based macrocyclic penta-
dentate ligands; (B) illustration scheme of the ligand design and
complexation with Mn(II).
14480 | Dalton Trans., 2012, 41, 14480–14483
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Table 1 Stepwise protonationa constants of the ligand and stability
constantsb of its complex (I = 0.1 M KCl, 25 °C)
Stepwise protonation
constants
log KH1
log KH2
log KH3
log KH4
10.33
(0.55)
9.00
(0.55)
5.86
(0.58)
N.A.
Stability constants
log KML
log KMLH
log KMLH2
11.37
(0.18)
7.66
(0.15)
6.60
(0.15)
a Defined as KHi = [HiL]/[H+] × [Hi−1L] for i = 1–4. b Defined as KML
=
Fig. 1 ORTEP representations of the molecular structure of the
complex [Mn3L2(H2O)4Cl2]. All hydrogen atoms, noncoordinated
solvent molecules have been removed for clarity. All thermal ellipsoids
are drawn at 50% probability.
[ML]/[M] × [L]; KMLHi = [MLHi]/[ML] × [H+]i (charges were omitted
for simplicity).
act as a pentadentate chelator and allow coordination of at least
one water molecule in its manganese(II) complexes.
The synthesis and characterization details of the ligand and
the complex (1H-NMR, 13C-NMR, MS) can be found in the
ESI.† The single crystal of Mn-ligand complex suitable for
X-ray structure analysis was obtained by a slow vapor diffusion
of acetone into methanol solution (drops of H2O) of the Mn2+
complex (purified by CHCl3/MeOH extraction). In the structure
of the [Mn3L2(H2O)4Cl2]§ complex, it is interesting that two
common coordination states of Mn2+ were both found (coordi-
nation number 6 and 7). Two Mn2+ ions are heptadentated at the
center cavity of the pentadentate planar ligand in the same way,
with one water molecule and one chloride anion located on
apical positions of the ligand plane to complete the pentagonal-
bipyramidal coordination sphere (Fig. 1). This Mn2+ coordi-
nation state was also observed by Tóth’s group in studying of
their 15-membered pyridine based macrocyclic pentadentate
ligands (15-PyN5).21 The other Mn2+ is hexadentated with four
carboxyl oxygen atoms from two ligands and two water mole-
cules in the apical positions, forming a slightly distorted octa-
hedral coordination geometry. These two coordination states
were also confirmed by ESI Mass spectrum (Fig. S13†). The
extraction purified complex may contain excess amount of
MnCl2, which led to the Mn : L ratio as 3 : 2 in the crystal struc-
ture for close packing during crystal growth. The hexadentated
Mn(II) is not stable structure because the four carboxyl oxygen
atoms have no chelation effect. Further purification of the
complex through Sephadex LH-20 column can remove the
unstable Mn(II) species but led to unsuccessful crystal growth.
All these ligand and Mn–L complex in the following experi-
ments were purified through Sephadex LH-20 column before
use.
Fig. 2 Species distribution diagram of the Mn-Ligand system (I = 0.1
M KCl, 25 °C).
protonated piperidine nitrogen atoms. The protonation constant
related to pyridine nitrogen is too low to be detected in the
experimental conditions. Due to the lack of crystal-field stabiliz-
ation (d5 high-spin electron configuration of Mn(II)), lower posi-
tive charge and relatively bigger ionic radius, Mn(II) complexes
show lower thermodynamical stability comparing with their tran-
sition metal ion analogues (Irving–Williams rule26). The stability
constants of the Mn–L complex was determined to be log KML
=
11.37 (I = 0.1 M KCl, 25 °C). This is compatible with the Lukeš
and Toth’s work on 12/15-membered pyridine based macrocyclic
pentadentate ligands.21,22 The distribution diagram for Mn(II)-
ligand complex shows that the complete complex formation
above pH = 8.0 (Fig. 2).
To understand how the Mn–L complex performs as a contrast
agent for MRI, we studied its relaxivity under a 1.5 T clinical
MRI scanner. Manganese based MR contrast agents can shorten
the T1 (spin-lattice) relaxation time, and their net effectiveness is
expressed as T1 relaxivity (r1), representing the reciprocal of the
relaxation time per unit concentration of Mn, with unit of
Mn mM−1 s−1. Before relaxivity measurement, the complex was
purified by Sephadex LH-20 column. Fig. 3A displays the T1
relaxation rate over Mn concentration at the magnetic field of
1.5 T, 20 °C, and the Mn–L complex has a T1 relaxivity of
3.6 Mn mM−1 s−1 in water (pH = 7.3), higher than that of the
Mn-DPDP (Teslascan®, r1 = 2.1 mM−1 s−1, 20 °C, 1.5 T).27
The stepwise protonation constants of the ligand and stability
constant of its complex formed with Mn(II) were determined by
standard potentiometric titrations (Table 1). The first two proto-
nation constants belong to the addition of two protons to the two
piperidine amino groups (log KH1 = 10.33, log KH2 = 9.00). The
third protonation constant (log KH3 = 5.86) probably occurs on
one of the acetate groups. The fourth and fifth protonation
constant didn’t observed, maybe because the formation intra-
molecular hydrogen bonding between the last acetate group and
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contrast agent in SD rats. As shown in Fig. 4, signal intensity
enhancement in the T1-weighted images were observed at rat’s
chest and neck regions after intravenous injection of Mn–L
complex with a dosage of 0.5 mmol kg−1. MR signals of
vascular vessels including jugular vein, subclavian vein, and
aortic arch were significantly enhanced (Fig. 4B), which can be
maintained up to 20 min. In comparison, one can not differen-
tiate the vascular details in rat without the presence of Mn–L
complex (Fig. 4A). MRI scan of liver, spleen and kidney up to
24 hours showed dramatic decrease of image signal intensity at
these organ sites (Fig. S15†). Elemental analysis of tissue
revealed a lower Mn content expressed as percent injected dose
per gram of tissue comparing to Mn-DPDP complexes.29 Both
imaging and tissue Mn analysis studies indicate that Mn–L was
stable in vivo with low tissue uptake after 24 hours of injection.
In summary, we have designed and synthesized a rigid aza-
semi-crown ligand based on the coordination characters of
manganese(II). Two coordination states Mn2+ (heptadentated and
hexadentated) were found in crystal structure. T1 relaxivity
measurements indicate that the Mn2+ complex has a relatively
higher value of 3.6 Mn mM−1 s−1 (20 °C, 1.5 T). Efficient mag-
netic resonance signal intensity enhancement of vascular struc-
tures by the Mn–L complex in vivo has been demonstrated. The
complex may be considered as a potential alternative MRI con-
trast agent for vascular imaging with better biosafety.
Fig. 3 (A) T1 relaxation rate (1/T1, s−1) as a function of Mn concen-
tration (mM) at 20 °C, 1.5 T; (B) T1-weighted MRI images (1.5 T, spin-
echo sequence: TE = 5 ms, TR = 175 ms) of the above Mn–L complex
formulations.
This work was supported by the National Key Basic Research
Program of China (2013CB933903), New Century Excellent
Talents in University (NCET-06-0781), Doctoral Fund of Minis-
try of Education of China (20090181110068), Key Project of the
National Twelfth-Five Year Research Program of China
(2012BAI23B08), and National Natural Science Foundation of
China (20974065, 51173117 and 50830107). Dr Zhu thanks for
the Key Project of Chinese Ministry of Education (210188) and
the Program of Sichuan Key Laboratory of Medical Imaging
(KFJJ(10)-01) for the financial support.
Notes and references
§Crystal data for [Mn3L2(H2O)4Cl2] complex: C19H29ClMn1·5N3O6,
M = 513.31, a = 13.0690(4) Å, b = 13.0690(4) Å, c = 27.1747(9) Å,
α = 90.00°, β = 90.00°, γ = 120.00°, V = 4019.6(2) Å3, T = 145.0 K,
space group P3221, Refinement using the SHELXL-97 package on all
data converged at R1 = 0.0583, wR2 = 0.1454. Data were collected using
a Xcalibur Eos CCD diffractometer (Mo-Kα, λ = 0.794 Å). CCDC
876927.
Fig. 4 T1-weighted MR image (Philips Medical System, Intera 3 T, TR
= 5 ms, TE = 2 ms) of SD rat at pre-injection (A), (B) 2 min, (C) 5 min,
(D) 10 min, (E) 20 min and (F) 24 h after administration of Mn–L
complex (0.5 mmol kg−1). MR signal intensity in the jugular vein (1),
subclavian vein (2), and aortic arch (3) was enhanced significantly.
1 P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer, Chem. Rev.,
1999, 99, 2293–2352.
2 R. B. Lauffer, Chem. Rev., 1987, 87, 901–927.
3 P. Hermann, J. Kotek, V. Kubicek and I. Lukes, Dalton Trans., 2008,
3027–3047.
4 J. L. Major and T. J. Meade, Acc. Chem. Res., 2009, 42, 893–903.
5 D. Pan, S. D. Caruthers, A. Senpan, A. H. Schmieder, S. A. Wickline and
G. M. Lanza, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol., 2011,
3, 162–173.
6 J. W. M. Bulte and D. L. Kraitchman, NMR Biomed., 2004, 17, 484–499.
7 J. Xie, G. Liu, H. S. Eden, H. Ai and X. Chen, Acc. Chem. Res., 2011,
44, 883–892.
8 B. Drahoš, I. Lukeš and É. Tóth, Eur. J. Inorg. Chem., 2012, 1975–1986.
9 S. Aime, P. Anelli, M. Botta, M. Brocchetta, S. Canton, F. Fedeli,
E. Gianolio and E. Terreno, JBIC, J. Biol. Inorg. Chem., 2002, 7, 58–67.
10 J. S. Troughton, M. T. Greenfield, J. M. Greenwood, S. Dumas,
A. J. Wiethoff, J. Wang, M. Spiller, T. J. McMurry and P. Caravan, Inorg.
Chem., 2004, 43, 6313–6323.
The high T1 relaxivity of Mn–L complex is mainly associated
with the coordinated water molecule in complex’s inner-sphere,
leading to the exchange of water molecules with the surrounding
water. The rigidification of the chelate structure maybe favorable
for increasing the central metal’s electronic relaxation time and
shortening the residence life time of the inner-sphere water
molecules.28
Mn(II)-based contrast agents can enhance the image contrast
of tissue through substantial shortening T1 relaxation times,
resulting in hyperintense signals at locations where the probes
accumulate. The Mn–L complex has been evaluated as a T1 MRI
14482 | Dalton Trans., 2012, 41, 14480–14483
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11 É. Tóth, L. Helm and A. Merbach, Top. Curr. Chem., Springer, Berlin/
Heidelberg, 2002, 221, 61–101.
20 F. Dioury, C. Ferroud, A. Guy and M. Port, Tetrahedron, 2009, 65, 7573–
7579.
12 E. Toth, L. Helm, A. E. Merbach, J. A. McCleverty and T. J. Meyer,
Comprehensive Coordination Chemistry II, Pergamon, Oxford, 2003,
841–881.
13 J. M. Hooker, A. Datta, M. Botta, K. N. Raymond and M. B. Francis,
Nano Lett., 2007, 7, 2207–2210.
21 B. Drahoš, J. Kotek, P. Hermann, I. Lukeš and E. v. Tóth, Inorg. Chem.,
2010, 49, 3224–3238.
22 B. Drahos, M. Pniok, J. Havlickova, J. Kotek, I. Cisarova, P. Hermann,
I. Lukes and E. Toth, Dalton Trans., 2011, 40, 10131–10146.
23 B. Drahoš, J. Kotek, I. Císařová, P. Hermann, L. Helm, I. Lukeš and
É. Tóth, Inorg. Chem., 2011, 50, 12785–12801.
24 M.-N. Collomb and A. Deronzier, Encyclopedia of Inorganic Chemistry,
John Wiley & Sons, Ltd, 2006.
25 R. D. Hancock and A. E. Martell, Chem. Rev., 1989, 89, 1875–1914.
26 H. Irving and R. J. P. Williams, J. Chem. Soc. (Resumed), 1953, 3192–
3210.
27 E. Wiener, K. Woertler, G. Weirich, E. J. Rummeny and M. Settles,
Eur. J. Radiol., 2007, 63, 110–119.
14 Y. Song, E. K. Kohlmeir and T. J. Meade, J. Am. Chem. Soc., 2008, 130,
6662–6663.
15 J. J. Yang, J. Yang, L. Wei, O. Zurkiya, W. Yang, S. Li, J. Zou, Y. Zhou,
A. L. W. Maniccia, H. Mao, F. Zhao, R. Malchow, S. Zhao, J. Johnson,
X. Hu, E. Krogstad and Z.-R. Liu, J. Am. Chem. Soc., 2008, 130, 9260–9267.
16 R. C. Strauch, D. J. Mastarone, P. A. Sukerkar, Y. Song, J. J. Ipsaro and
T. J. Meade, J. Am. Chem. Soc., 2011, 133, 16346–16349.
17 R. S. Ranganathan, N. Raju, H. Fan, X. Zhang, M. F. Tweedle,
J. F. Desreux and V. Jacques, Inorg. Chem., 2002, 41, 6856–6866.
18 M. Port, I. Raynal, L. V. Elst, R. N. Muller, F. Dioury, C. Ferroud and
A. Guy, Contrast Media Mol. Imaging, 2006, 1, 121–127.
19 P. Caravan, Chem. Soc. Rev., 2006, 35, 512–523.
28 R. B. Shukla, K. Kumar, R. Weber, X. Zhang and M. Tweedle, Acta
Radiol. Suppl., 1997, 412, 121–123.
29 B. Gallez, C. Baudelet, J. Adline, M. Geurts and N. Delzenne, Chem.
Res. Toxicol., 1997, 10, 360–363.
This journal is © The Royal Society of Chemistry 2012
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