Manganese Complex of Ethylenediaminetetraacetic Acid (EDTA)-Benzothiazole Aniline (BTA) Conjugate as a Potential Liver-Targeting MRI Contrast Agent

Article abstract of DOI:10.1021/acs.jmedchem.6b01799

A novel manganese(II) complex based on an ethylenediaminetetraacetic acid (EDTA) coordination cage bearing a benzothiazole aniline (BTA) moiety (Mn-EDTA-BTA) was designed and synthesized for use as a liver-specific MRI contrast agent with high chelation stability. In addition to forming a hydrophilic, stable complex with Mn²⁺, this new Mn chelate was rapidly taken up by liver hepatocytes and excreted by the kidneys and biliary system. The kinetic inertness and R₁ relaxivity of the complex were much higher than those of mangafodipir trisodium (MnDPDP), a clinically approved liver-specific MRI contrast agent. The diagnostic utility of this new Mn complex in MRI was demonstrated by high-sensitivity tumor detection in an animal model of liver cancer.

Full text of DOI:10.1021/acs.jmedchem.6b01799

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Article
Manganese Complex of Ethylenediaminetetra acetic
acid (EDTA)-Benzothiazole Aniline (BTA) Conjugate
as a Potential Liver-Targeting MRI Contrast Agent
Md. Kamrul Islam, Soyeon Kim, Hee-Kyung Kim, Subin Park, Gang Ho Lee, Hyo
Jeung Kang, Jae Chang Jung, Joon-Suk Park, Tae-Jeong Kim, and Yongmin Chang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01799 • Publication Date (Web): 16 Mar 2017
Downloaded from http://pubs.acs.org on March 17, 2017
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Manganese Complex of Ethylenediaminetetra
acetic acid (EDTA)ꢀBenzothiazole Aniline (BTA)
Conjugate as a Potential LiverꢀTargeting MRI
Contrast Agent
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Md. Kamrul Islam,1,‡ Soyeon Kim,1, ‡ Hee-Kyung Kim,2 Subin Park,3 Gang-Ho Lee,4 Hyo
Jeung Kang,5 Jae-Chang Jung,3 Joon-Suk Park,8 Tae-Jeong Kim,6,* and Yongmin Chang,1,2,7,*
1Department of Medical & Biological Engineering, 2Department of Molecular Medicine &
BK21 Plus KNU Biomedical Convergence Program, 3Department of Biology, 4Department of
Chemistry, 5Department of Pharmacy, 6Institute of Biomedical Engineering Research,
7Department of Radiology, Kyungpook National University, 80 Daehakro, Bukgu, Daegu
702ꢀ701, Korea, 8Laboratory Animal Center, DaeguꢀGyeongbuk Medical Innovation
Foundation 41061, Chumbokꢀro 80, Dongꢀgu, Daegu, Korea
KEYWORDS: Manganese, Contrast agent, MRI, Liver, Chelate
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ABSTRACT: A novel manganese (II) complex based on an ethylenediaminetetraacetic acid
(EDTA) coordination cage bearing a benzothiazole aniline (BTA) moiety (MnꢀEDTAꢀBTA)
was designed and synthesized for use as a liverꢀspecific MRI contrast agent with high
chelation stability. In addition to forming a hydrophilic, stable complex with Mn2+, this new
Mn chelate was rapidly taken up by liver hepatocytes and excreted by the kidneys and biliary
system. The kinetic inertness and R1 relaxivity of the complex were much higher than those
of mangafodipir trisodium (MnDPDP), a clinically approved liverꢀspecific MRI contrast
agent. The diagnostic utility of this new Mn complex in MRI was demonstrated by highꢀ
sensitivity tumor detection in an animal model of liver cancer.
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INTRODUCTION
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Manganese (Mn) carries five unpaired electrons and is one of the earliest paramagnetic metal
ions reported to effectively enhance positive contrast in magnetic resonance imaging
(MRI).1,2 As a clinical MRI contrast agent (CA), gadolinium (Gd) is mostly used. However,
Gdꢀcomplex is linked with nephrogenic systemic fibrosis (NSF).3,4 NSF is a rare, idiopathic
systemic fibrosing disorder and can be critical to patients with acute or chronic kidney
disease (CKD) with severely impaired renal function.5 Furthermore, it has been recently
reported that intravenously administered Gd accumulates in the brains of patients with normal
renal function.6 With increasing safety concerns over potential toxicities associated with Gd
retention in the human body, alternative approaches based on nonꢀlanthanide metals,
particularly Mn, are receiving more attention for use in MRI.7,8
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Mn plays a critical role in cell function.9 It is a natural cellular constituent and is
required for mitochondrial function. Hepatocytes are mitochondriaꢀrich cells; thus, Mn is an
excellent CA for MRI of the liver.10 Although small amounts are essential in humans,
overexposure to free Mn ions may result in neurotoxicity.11 It has been known for many years
that chronic exposure to Mn in certain occupational settings leads to a neurological syndrome
known as manganism, which is similar to Parkinson’s disease.12 The neurological symptoms
of manganism correlate with accumulation of Mn in the basal ganglia, which can often be
seen as hyperꢀintensity on a T1ꢀweighted MRI owing to contrast enhancement from the Mn
accumulation.13 Furthermore, although no association has been found between Mn and NSF
so far, potential harmful effects of exposure to free Mn ions at a high concentration remain a
concern with regard to its use as a CA. Indeed, MnDPDP (mangofodipir trisodium), which
was approved by the FDA for use as a T1 MR CA for liver imaging, releases free Mn in
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plasma after intravenous (IV) injection.14 When an MRI dose (i.e. 5–10 mmol/kg) is injected
into a human patient, only about 20% remains bound to the chelator. Release of paramagnetic
Mn2+ occurs through doseꢀdependent transmetalation, in which Mn2+ is displaced by
endogenous Zn2+.15 Zn2+ has roughly 1000 times higher affinity than Mn2+ does for DPDP.
Free Mn2+ ions dissociated from MnDPDP are in fact responsible for the T1 contrast
enhancement of MnDPDP.16 Although free Mn2+ ions are known to accumulate primarily in
the liver, pancreas, and heart, one report showed Mn2+ accumulation in patients’ brains,
suggesting possible neurotoxicity.17
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Several efforts have been made to design stable Mn(II) complexes for MRI. Recently,
Caravan et al. synthesized an Mn(II) complex of [Mn(PyC3A)(H2O)]− as an alternative CA to
Gd. This complex is one of the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40)
and has a possible application as a molecular agent for vascular thrombi.18 More recently,
Lattuada et al. synthesized a new Mn(II)ꢀEDTAꢀdeoxycholic acid conjugate as a potential
MRI blood pool agent.19 However, design of stable liverꢀspecific Mn(II) complexes remains a
challenge. In the current study, we report the design and synthesis of a new liverꢀspecific,
highly stable Mn chelate for liver MRI. Specifically, we designed a novel Mn(II) complex
based on an EDTA coordination cage bearing a benzothiazole aniline (BTA) moiety with
high chelation stability for use as a liverꢀspecific MRI CA. BTA derivatives have high
lipophilicity and sufficient proteinꢀbinding affinity.20 We therefore hypothesized that BTA
will contribute to the increased lipophilicity of MnꢀEDTAꢀBTA, which will enhance its
ability to target liver cells. In case of liverꢀspecific Gd agents such as gadolinium
dimeglumine ethoxybenzyl (GdꢀEOBꢀDTPA), lipophilicity is known to enhance liver
uptake.21
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In addition to forming a hydrophilic, stable complex with Mn2+, this new hepatobiliary
Mn chelate is rapidly taken up by liver hepatocytes. We believe this complex is the first
example of a small Mnꢀchelateꢀbased liver agent with high in vivo stability. This new family
of CAs is highly suitable for liver imaging applications such as liver cancer imaging.
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RESULTS AND DISCUSSION
Synthesis. The methods for synthesis of the chelate conjugate and its Mn(II) complex
(abbreviated as L and MnꢀEDTAꢀBTA, respectively) are depicted in Scheme 1. Synthesis of
the ligand, L started with the commercially available D,Lꢀ2,3ꢀdiaminopropionic acid
monohydrobromide. Compound 1 was synthesized following a published method,22 and was
conjugated to BTA in the presence of triphenyl phosphite to form a white solid 2. tertꢀbutyl
was cleaved using a mixture of trifluoroacetic acid (TFA) and dichloromethane, to yield a
paleꢀyellow product 3 after precipitation. Alkylation with tertꢀbutyl bromoacetate under
conditions reported previously23 resulted in a high yield of the protected EDTAꢀBTA
compound 4 after column chromatography. After deprotection with hydrochloric acid, the
ligand L was purified from the reaction mixture by precipitation at pH 2. The Mn complex
MnꢀEDTAꢀBTA was prepared by reaction of L with a stoichiometric equivalent of
MnCl2.4H2O at pH 6 followed by removal of inorganic impurities by reverseꢀphase
chromatography. The pure chelate was isolated as a sodium salt in moderate yield by
lyophilization. The formation of the ligand and its Mnꢀcomplex was confirmed by
1
microanalysis and spectroscopic techniques including H NMR, HRꢀFABꢀMS and elemental
analysis (Supporting Information).
Kinetic inertness. Zn is the second most abundant trace metal in the human body and can
thus displace more Mn2+ ions than other endogenous ions such as Cu2+ and Ca2+. The rate of
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transmetalation of an Mn ligand chelate is inversely correlated with the stability of the
complex. The relative stability of CAs can therefore be measured by determining the kinetics
of their transmetalation with Zn2+. Transmetalation rates are often represented by plotting the
evolution of the normalized longitudinal (R1) or transverse (R2) relaxivity as a function of
time.24,25 In the current study, the kinetic inertness of MnꢀEDTAꢀBTA is represented by a
change in transverse relaxivity (ꢁR2(t) = R2(t) – R2(0)) as a function of time. Here, R2(t) at
any time t is a good estimator of the extent of transmetalation of Mn by Zn. MnDPDP, Mnꢀ
EDTA, and GdꢀDTPA were also examined for comparison (Figure 1). We prepared the
complex under two conditions following a published method with slight modifications.18
Transmetalation was evaluated by measuring ꢁR2(t) of each MnꢀEDTAꢀBTA complex
incubated with Zn2+ (10 or 25 equivalents) in pH 6.0 MES buffer. The results were
consistently similar in all cases, and a representative set is presented here (10 mM Zn2+). The
same method was used to evaluate transmetalation of MnDPDP, MnꢀEDTA, and GdꢀDTPA.
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The ꢁ
MnꢀEDTAꢀBTA rapidly increased at first, but subsequently slowed to reach saturation at a
much lower ꢁ 2(t) value compared to that of MnDPDP. Therefore, compared to MnDPDP,
R2(t) of MnDPDP rapidly increased and reached saturation. In contrast, the ꢁR2(t) of
R
MnꢀEDTAꢀBTA is significantly more inert to Mn2+ transmetalation. Figure 1 also shows that
MnꢀEDTAꢀBTA is kinetically more stable than MnꢀEDTA is, suggesting the possible role of
the BTA moiety in stabilizing Mn chelation. Furthermore, the pattern of ꢁR2(t) for Mnꢀ
EDTAꢀBTA is almost identical to that for GdꢀDTPA, the widely used clinical MRI CA,
indicating that MnꢀEDTAꢀBTA is as stable as GdꢀDTPA is.
Relaxivity and lipophilicity. The relaxivity values of MnꢀEDTAꢀBTA are summarized in
Table 1 along with those of MnDPDP, MnꢀEDTA, and GdꢀDTPA for comparison. Of the 3
complexes, MnꢀEDTAꢀBTA had the highest relaxivity (Table 1). Although Mn2+ (S=5/2) has
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fewer unpaired electrons than Gd3+ (S=7/2) does, the lipophilic moiety BTA has a slower
tumbling rate and might contribute to high relaxivity relative to that reported for GdꢀDTPA or
MnꢀEDTA. In addition, the shorter distance between Mn2+ and water may contribute to the
increased relaxivity of MnꢀEDTAꢀBTA.21 We also measured relaxivity in an aqueous
solution of human serum albumin (HSA). In this solution as well, the relaxivity values of
MnꢀEDTAꢀBTA were higher than those of GdꢀDTPA, and indicated an interaction between
HSA and MnꢀEDTAꢀBTA. The binding constant (Ka), reflecting the interaction of Mnꢀ
EDTAꢀBTA with HSA was determined following a previously published method.26 The Ka of
MnꢀEDTAꢀBTA binding to HSA (95 Mꢀ1) was higher than that of GdꢀDOTA binding to HSA
(21 Mꢀ1).27 Figure 2 shows the result of fitting data obtained by measuring the proton
longitudinal relaxation rate as a function of the concentration of MnꢀEDTAꢀBTA, at a fixed
concentration of HSA. To estimate lipophilicity, we determined the octanolꢀwater
partitioning coefficient (logP). The logP value for MnꢀEDTAꢀBTA (logP = ꢀ1.84) was higher
than those of MnDPDP (logP = ꢀ3.07), MnꢀEDTA (logP = ꢀ2.72), and GdꢀDTPA (logP = ꢀ
3.16), demonstrating the higher lipophilicity of MnꢀEDTAꢀBTA (Table 1).
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In vitro cytotoxicity. Tests of MnꢀEDTAꢀBTA and GdꢀDTPA cytotoxicity were
performed on the human prostate cancer cell line DU 145, and the mouse liver cell line
NCTC 1469. Figure 3 shows that viability of DU 145 cells was above 88%, and that of
NCTC 1469 cells was above 86% when incubated with various concentrations of MnꢀEDTAꢀ
BTA up to 50 ꢂM. As shown in Figure 3, MnꢀEDTAꢀBTA showed lesser cytotoxicity than
GdꢀDTPA did at all concentrations. These observations indicate that MnꢀEDTAꢀBTA has
negligible cytotoxicity in the concentration range needed to sufficiently enhance signal
intensity in MRI.
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In vivo MRI and in vivo biodistribution. In vivo MRI using MnꢀEDTAꢀBTA was
performed by obtaining T1ꢀweighted MR images of 6ꢀweekꢀold male mice of the Institute for
Cancer Research strain (ICR mice) after a bolus injection of MnꢀEDTAꢀBTA through the tail
vein (Figure 4). The most characteristic MR feature of MnꢀEDTAꢀBTA is that it shows
strong contrast enhancement in the liver and kidney after injection, and subsequently in the
gallbladder and intestine as well. In vivo MRI therefore indicates that MnꢀEDTAꢀBTA is
eliminated via hepatobiliary and renal pathways. In the hepatobiliary pathway, MnꢀEDTAꢀ
BTA is eliminated via the bile duct following hepatobiliary uptake. Interestingly, this dual
elimination property of MnꢀEDTAꢀBTA is similar to that of the clinically approved Gdꢀbased
liverꢀspecific agent GdꢀDTPAꢀEOB.21 In vivo biodistribution of MnꢀEDTAꢀBTA was
quantitatively measured with an inductivelyꢀcoupled plasma (ICP) spectrophotometer (Figure
5). The data show the highest Mn(II) accumulation in the liver and intestine, indicating Mnꢀ
EDTAꢀBTA excretion via the hepatobiliary pathway. In addition to the liver, the kidney
shows relatively high Mn(II) accumulation, suggesting glomerular excretion via the renal
pathway. Together with the MRI data, the in vivo biodistribution data strongly suggest that
MnꢀEDTAꢀBTA is eliminated via dual pathways (renal and hepatobiliary).
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Hepatocyte targeting. Figure 6 shows stronger signal enhancement in the nucleus rather
than at the plasma membrane or in the cytosol of NCTC 1469 cells (a normal mouse
hepatocyte cell line), demonstrating the subcellular preferential targeting of MnꢀEDTAꢀBTA.
These experiments provide direct evidence that the liver MR signal enhancement is a result of
hepatocyte targeting by MnꢀEDTAꢀBTA. However, a further study is warranted to
investigate the detailed mechanism of hepatocyte targeting of MnꢀEDTAꢀBTA, although its
lipophilicity may be partly responsible for this. In the case of GdꢀDTPAꢀEOB, the active
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transport system on the hepatocyte membrane plays an important role. Specifically, Gdꢀ
DTPAꢀEOB enters hepatocytes through two different organic anion transport systems.21 In
addition, in future studies, it will be important to identify more efficient alternatives to BTA,
for hepatocyte targeting.
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Tumor imaging: Distinction between normal liver and tumor tissue. Because Mnꢀ
EDTAꢀBTA behaves as a liverꢀspecific agent, we performed an in vivo test to determine
whether it can distinguish between normal and tumor liver tissue. Figure 7a and 7b show T2ꢀ
weighted and T1ꢀweighted MR images from a HepG2 xenograft mouse model before and
after injection of MnꢀEDTAꢀBTA or MnDPDP. T2ꢀweighted images without CA injection
clearly showed the tumor location and size. In the case of MnꢀEDTAꢀBTA, greater signal
enhancement is seen in normal liver tissue than in tumor tissue. However, in the case of
MnDPDP, positive signal enhancement is seen in both normal liver and tumor tissue,
suggesting that normal hepatocytes and tumor cells efficiently take up MnDPDP. MnꢀEDTAꢀ
BTA thus showed a higher difference in contrastꢀtoꢀnoise ratio (∆CNR) between tumor and
normal liver tissue (Figure 7c) than MnDPDP did, suggesting a significant improvement in
tumor detection and characterization. From the kinetic stability data (Figure 1), one possible
mechanism for a significant improvement in contrast between tumor and normal liver tissue
is that MnꢀEDTAꢀBTA remains as a stable complex and thus is taken up only by normal liver
cells. However, MnDPDP is dissociated into free Mn2+ ions and DPDP, and free Mn2+ ions
enter all cells with equal efficiency, enhancing both tumor and normal liver tissue equally.
In summary, MnꢀEDTAꢀBTA shows good potential as a diagnostic agent for liver cancer.
It should be mentioned that although the injection dose of MnDPDP used in the current study
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was lower than that of MnꢀEDTAꢀBTA, based on the suggested clinical dose of MnDPDP,28
the main reason for the low ∆CNR of MnDPDP between tumor and normal liver tissue was
not the low dose but the uptake of MnDPDP both in tumor and normal liver tissue.
Furthermore, the injection dose of MnDPDP could not be increased because of possible
toxicity in vivo, especially due to neurotoxicity of Mn2+ released from DPDP.
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CONCLUSIONS
In the current study, we synthesized and characterized an MnꢀEDTA complex
incorporating a BTA functionality, and investigated its possible diagnostic utility in liver
MRI. The kinetic inertness of this complex was much higher than those of MnDPDP and Mnꢀ
EDTA, and was comparable to that of stable GdꢀDTPA. Furthermore, its R1 relaxivity (3.47
mM−1 s−1) was higher than those of MnDPDP (R1 = 2.8 mM−1 s−1) and GdꢀDTPA (R1 = 3.3
mM−1 s−1). In vivo biodistribution and in vivo MRI patterns effectively demonstrated that Mnꢀ
EDTAꢀBTA is a liverꢀspecific MRI CA that utilizes both renal and hepatobiliary elimination
pathways. This elimination pattern is similar to that of Gdꢀbased hepatobiliary agents such as
GdꢀDTPAꢀEOB and GdꢀBOTPA. Finally, in an animal model of liver cancer, MnꢀEDTAꢀ
BTA significantly improved tumor detection and characterization compared to that observed
with MnDPDP, suggesting that MnꢀEDTAꢀBTA may be a good diagnostic MRI agent for
liver cancer.
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EXPERIMENTAL SECTION
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General remarks. D,Lꢀ2,3ꢀdiaminopropionic acid monohydrobromide, diꢀtertꢀbutyl dicarbonate,
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triphenyl phosphite, N,Nꢀdiisopropylethylamine, and ethylenediaminetetraacetic acid manganese
disodium salt hydrate (MnꢀEDTA) were purchased from Tokyo Chemical Industry (Tokyo, Japan).
tertꢀButylꢀbromoacetate was obtained from AlfaꢀAesar (Ward Hill, Massachusetts, USA). Sodium
bicarbonate (NaHCO3) was purchased from Daejung Chem. (Korea). Potassium iodide (KI) and
sodium sulfate (Na2SO4) anhydrous were purchased from Duksan Scientific Corp. (Korea). All other
commercial reagents were purchased from SigmaꢀAldrich (St. Louis, MO, USA) and used without
further purification unless otherwise stated. Solvents were purified and dried using standard
procedures. Deionized water was used for all experiments. 1H NMR spectra was recorded on a Bruker
Advance 500 MHz spectrometer at the Center for Instrumental Analysis, Kyungpook National
University (KNU). Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as
the internal standard. Coupling constants are in Hz. Elemental analyses (EA) were performed by the
Center for Instrumental Analysis, KNU. Highꢀresolution fast atom bombardment mass spectra (HRꢀ
FABꢀMS) were obtained using a JMSꢀ700 model (Jeol, Japan) mass spectrophotometer at the Korea
Basic Science Institute (KBSI). An HPLC (highꢀpressure liquid chromatography, LCꢀForte/R, YMC,
Japan) system equipped with a Luna C18 column (250 x 21.2 mm, Phenomenex Inc. USA) was used
for the purification and purity tests at room temperature. The methods used in the purity test were as
follows: (method A) eluent A: 0.1% TFA in water, B: 0.1% TFA in ACN; gradient: 5% B to 97% B in
20 min; flow rate 12 mL/min, (method B) eluent A: 10 mM ammonium acetate in water, B: 10 mM
ammonium acetate in ACN ; gradient: 5% B to 40% B in 3 min, 40% B to 80% B in 25 min, 80% B
to 100% B in 3 min; flow rate 12 mL/min. The purity of all products was determined using elemental
analysis, or a reverseꢀphase HPLC with UVꢀvis detection at 320 nm.
Synthesis and Characterization.
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2,3-Bis-tert-butoxycarbonylamino-propionic acid (1). The title compound was prepared
according to the literature method with little modification.22 Yield: 7.8 g (95%). 1H NMR (CDCl3): δ
= 1.45 (s, 18H, CH3), 3.55 (m, 2H, CH2), 4.12 (s, 1H, CH), 4.27–5.17 (m, 1H, NH), 5.85 (s, 1H, OH),
2.05 (s, 1H, NH). Anal. Calcd for C13H24O6N2·2EtOAc: C, 51.71; H, 8.04; N, 8.10. Found: C, 51.87;
H, 8.15; N, 7.75.
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[2-(4-Benzothiazol-2-ylphenylcarbamoyl)-2-tert-butoxycarbonylamino-ethyl]-carbamic acid
tert-butyl ester (2). 2,3ꢀBisꢀtertꢀbutoxycarbonylaminoꢀpropionic acid (7.00 g, 23.01 mmol) was
dissolved in pyridine (40 ml) and benzothiazole aniline (BTA) (5.20 g, 23.01 mmol) in pyridine (20
ml) was added slowly. Resulting mixture stirred for 30 min and triphenyl phosphite (7.13 ml, 23.01
mmol) was added drop wise. The solution was stirred for 3 h at 80 °C and then overnight at RT. Solid
obtained was filtered and washed with D.I water and acetone. Crude product was recrystallized in
1
absolute acetonitrile. The desired product was obtained as white powder. Yield : 9.6 g (81.4%). H
NMR (CDCl3): δ = 9.28 (s, 1H, NH) 8.06−8.01 (m, 3H, BTA), 7.89−7.85 (d, 1H, BTA), 7.68−7.64 (d,
2H, BTA), 7.49−7.44 (t, 1H, BTA), 7.38−7.33 (t, 1H, BTA), 5.97ꢀ5.93 (s, 2H, NH), 4.02−3.98 (d, 1H,
CH), 3.57ꢀ3.41 (m, 2H, CH2), 1.50ꢀ1.40 (d, 18H,CH3). Anal. Calcd for C26H32N4O5S: C, 60.92; H,
6.29; N, 10.93; S, 6.26. Found: C, 60.55; H, 6.36; N, 10.60; S, 5.98. HRꢀFABꢀMS (m/z) for
C26H33N4O5S): calcd, 513.2172 [M+H]+; found, 513.2170 [M+H]+.
2,3-Diamoniumtrifluoroacetate-N-(4-benzothiazol-2-yl-phenyl)-propionamide (3). TFA (10
ml) was added drop wise into solution of 2 (1.5 g, 2.92 mmol) in CH2Cl2 at 0 °C. Resulting yellow
solution was stirred until starting was consumed. Solvent was removed and Et2O was added to obtain
precipitate. The solid was filtered and washed three times with Et2O then dried to give the title
compound as a pale yellow solid in quantitative yield. Yield: 0.82 g (90%), 1H NMR (MeOHꢀd4): δ =
8.75 (s, 1H, NH), 8.01−7.99 (d, 2H, BTA), 7.9−7.89 (d, 2H, BTA), 7.82−7.78 (d, 2H, BTA), 7.45 (t,
1H, BTA), 7.35 (t, 1H, BTA), 4.43 (t, 1H, CH), 3.62−3.55 (dd, 1H, CH2), 3.51ꢀ3.44 (dd, 2H, CH2),
1.94 (s, 2H, NH). Anal. Calcd for C16H16N4OSꢃ3CF3COOH: C, 40.37; H, 2.93; N, 8.56; S, 4.90.
Found: C, 40.74; H, 2.68; N, 8.86; S, 5.26. HRꢀFABꢀMS (m/z) for C16H17N4OS: calcd, 313.1123
[M+H]+; found, 313.1121 [M+H]+.
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{[1-(4-Benzothiazol-2-yl-phenylcarbamoyl)-2-(bis-tert-butoxycarbonylmethyl-amino)-ethyl]-
tert-butoxycarbonylmethyl-amino}-acetic acid tert-butyl ester (4). Compound 3 (2 g, 3.70 mmol),
N, Nꢀdiisopropylethylamine (7.66 mL, 59.2 mmol) and KI (1.84 g, 11.1 mmol) were dissolved in
DMF (20 mL) and the solution warmed to 45 °C. tertꢀButylbromoacetate (5.77 ml, 29.6 mmol) was
added drop wise into this solution over 30 min. Resulting solution was stirred for 4 h at 110 °C and
cooled to RT. The solvent was removed and the residue was partitioned between 10% NaHCO3 and
ethyl acetate. The aqueous layer was extracted three more times with EtOAc. Combined organic layer
was then washed with brine and dried over Na2SO4. Evaporation gave a dark brown oil that was
purified by column chromatography (silica, hexanes/EtOAc, 9:1). The desired product 4 was obtained
as a light brown oily solid. Yield: 3.37 g (82%). 1H NMR (CDCl3): δ = 10.85 (s, 1H, NH), 8.08−8.01
(m, 3H, BTA), 7.91−7.82 (dd, 3H, BTA), 7.47 (t, 1H, BTA), 7.36 (t, 1H, BTA), 3.75 (t, 1H, CH),
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t
3.62−3.40 (m, 8H, CH2ꢀtBu), 3.04ꢀ2.96 (dd, 2H, CH2), 1.46 (s, 36H, Bu). Anal. Calcd for
C40H56N4O9S·3H2O: C, 58.37; H, 7.59; N, 6.81; S, 3.90; Found: C, 58.61; H, 7.31; N, 6.46; S, 3.58.
HRꢀFABꢀMS (m/z) for C40H57N4O9S: calcd, 769.3846 [M+H]+; found, 769.3842 [M+H]+.
[{1-(4-Benzothiazol-2-yl-phenylcarbamoyl)-2-(bis-carboxymethyl-amino)-ethyl]-
carboxymethyl-amino}-acetic acid] (L). Compound 4 (1.5 g, 1.95 mmol) was dissolved in ACN (30
mL) and conc. HCl (15 mL) was added at 0 °C. Resulting solution was stirred for 20 h. An additional
portion of HCl (5 mL) was added and the solution was stirred for one more hour. The volume was
reduced by approximately 75% by rotary evaporation. The residue was diluted with water (30 mL)
and the pH was adjusted to 2 by the addition of sodium hydroxide. The white precipitate appears
which was collected by filtration and washed with D.I Water (pH 2.0), ethyl acetate (30 mL), and
ether (30 mL). The desired product, L was obtained as a white powder. The purity also confirmed
using HPLC (method A) (Figure S10). Yield: 0.89 g (83%). 1H NMR (CDCl3): δ = 8.06−8.01 (m, 2H,
BTA), 7.99−7.96 (d, 2H, BTA), 7.85−7.83 (d, 2H, BTA), 7.52ꢀ7.49 (t, 1H, BTA), 7.42−7.39 (t, 1H,
BTA), 5.50ꢀ5.48 (s, 2H, OH), 4.02−3.98 (d, 1H, CH), 3.96−3.76 (m, 8H, CH2), 3.57ꢀ3.41 (m, 2H,
CH2), Anal. Calcd for C24H24N4O9S·H2O: C, 51.24; H, 4.66; N, 9.96; S, 5.70. Found: C, 51.02; H,
4.65; N, 9.64; S, 5.54. HRꢀFABꢀMS (m/z) for C24H25N4O9S: calcd, 545.1342 [M+H]+; found,
545.1340 [M+H]+.
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[Mn(EDTA-BTA)(H2O)]2-. Compound L (1 g, 1.8 mmol) was dissolved in MeOH (30 mL) and the
pH of the solution adjusted to pH 6 with NaOH (1.0 M), then the mixture changed to suspension
including the offꢀwhite precipitation. To this mixture, MnCl2ꢃ4H2O (0.36 g, 1.8 mmol) in MeOH (3
mL) was slowly added and stirred at RT for overnight, during this time the mixture was reꢀadjusted to
pH 6. The precipitate was filtered, washed with cold methanol and dried under vacuum. Which was
dissolved in a minimum amount of water and more purified by flash column chromatography (C18,
95:5 to 85:15, water:MeOH) to yield a white solid. Further purification carried out using preparative
HPLC (method B) and also purity was confirmed (Figure S11). Yield: 0.35g (32%). HRꢀFABꢀMS
(m/z) for C24H20MnN4O9S: calcd, 595.0331 [M]; Found: 595.0327 [M].
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Transmetalation kinetics. This experiment was performed following published literature with a
slight modification.18 About 20 ꢂL of a 50 mM MES (2ꢀ(Nꢀmorpholino)ethanesulfonic acid)ꢀbuffered
solution (pH 6.0) of ZnCl2 was added to 1 mL of a buffered solution of 1 mM metal complex. The
mixture was shaken briefly, and immediately used for measuring solvent T2 as a function of time.
Control studies were also conducted with MnDPDP (Teslascan), GdꢀDTPA (Magnevist), and Mnꢀ
EDTA for comparison. The measurements were performed on a 3 Tesla (T) whole body system
(Discovery MR750w 3.0T, GE healthcare) at room temperature. The graph was plotted using the
equation ꢁR2(t) = R2(t) – R2(0), as function of time.
Relaxivity. Using an inversion recovery method, T1 measurements were made with a variable
inversion time (TI) at 1.5T (64MHz, GE Healthcare Milwaukee, WI, USA). Magnetic resonance (MR)
images were acquired at 35 different TI values over the frequency range 50 to 1750 msec. T1
relaxation times were achieved from the nonꢀlinear least square fit of the signal intensity measured at
each TI value. In place of T2 measurements, the CPMG (CarrꢀPurcellꢀMeiboonꢀGill) pulse sequence
ware adapted for multiple spinꢀecho measurements. Thirtyꢀfour images were achieved with 34
different echo time (TE) values ranging from 10 to 1900 msec. T2 relaxation times were attained from
the nonꢀlinear least squares fit of the mean pixel values of multiple spinꢀecho measurements at each
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echo time. Relaxivities (R1 and R2) were then measured as an inverse of relaxation time per mM. The
determined relaxation times (T1 and T2) and relaxivities (R1 and R2) were finally imageꢀprocessed to
provide the relaxation time map and relaxivity map, respectively.
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Octanol-water partition coefficients. This experiment was performed following published
literature.29 Manganese(II) complex (1 mg) was dissolved in 2 mL of a 1:1 mixture of water and 1ꢀ
octanol. The solution was shaken for 30 s, then the vial containing the mixture was placed on a rotator
for gentle mixing to equilibrate for 48 h. The sample was then allowed to settle at room temperature
for 24 h. Mn(II) concentrations of each layer were determined by ICPꢀMS (Inductively Coupled
Plasma Mass Spectrometry). Partition coefficients were calculated from the equation log P = log
(Co/Cw), where log P is the logarithm of the partition coefficient, Co is the concentration of Mn in the
1ꢀoctanol layer, and Cw is the concentration of Mn in the water layer.
Determination of binding constants. The binding constants of several CAs with HSA were
measured following published literature.26,27 The nonꢀlinear increase of the proton paramagnetic
relaxation rates measured at 64 MHz in solutions containing 0.67 mM HSA, were fitted using
equation 2, where Ka is the binding constant of the interaction with HSA, p0 is the concentration of the
HSA, s0 is the paramagnetic complex concentration, N is the number of independent interaction sites
c
f
(N was set to 1), and r1 and r1 are the relaxivities of the complex HSAꢀcontrast agent and of the free
contrast agent, respectively.
ꢃꢄꢅ
1
ꢕ
ꢓꢁ
ꢉ
ꢁ
ꢀꢁꢂ = 1000 × ꢆꢇꢈꢉ × ꢊꢋꢌ + (ꢈꢁ − ꢈ ) ꢎ ꢏ × ꢐ + ꢊ + ꢑꢒ
ꢇ ꢏ × ꢐ + ꢊꢋ + ꢑꢒ ꢌ − 4 × ꢏ × ꢊꢋ × ꢐꢋꢖꢗ
ꢔ
( )
−
ꢍ
ꢋ
ꢋ
ꢓꢁ
ꢋ
(
)
ꢁ
2
(1)
Cell culture. The culture medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM,
Gibco Invitrogen, Carlsbad, CA) or Roswell Park Memorial Institute (RPMI, Gibco Invitrogen,
Carlsbad, CA), 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) penicillinꢀstreptomycin. Cells were
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plated at a density of 2 × 105 cells/35 mm dish, incubated overnight for stabilization and then treated
with Mn for 24 h in serumꢀdepleted media.
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Liver tumor model. An orthotopic xenograft mouse liver tumor model was approved by the
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Institutional Animal Care and Use of Committee of DaeguꢀGyeongbuk Medical Innovation
Foundation (DGMIF). Fiveꢀweekꢀold male nude mice (BALB/c nu/nu) were purchased from Orient
Bio (Seongnam, Korea) and housed in a specific pathogenꢀfree facility at the Laboratory Animal
Center of DGMIF before use. The mice were inoculated with HepG2ꢀluc2 cells (1 × 106 cells in 50 ꢂl
HBSS) in the subcapsular parenchyma of the left liver lobe. The HepG2ꢀluc2 cell line was purchased
from Perkin Elmer Inc. Four weeks after inoculation, mice were imaged by the IVIS system to check
tumor induction (Figure S11 in Supporting Information).
Bioluminescence imaging. Bioluminescence images were acquired using the IVIS Lumina
system (Perkin Elmer). Mice were intraperitoneally administered firefly DꢀLuciferin potassium salt
(Perkin Elmer) at a dose of 150 mg/kg body weight in Dulbecco’s phosphateꢀbuffered saline. During
image acquisition, anesthesia was maintained with 2% isoflurane. Analysis was performed with
Living Image® software by measuring the photon flux (measured in photons / [sec • cm2 • steradian])
using a region of interest manually drawn over the body of the mouse. Signals were measured for
approximately 1 h.
Cell viability assay (in vitro growth inhibition). Human prostate cancer cell line DU 145,
and mouse liver cell line NCTC 1469 were plated at a density of 1 × 104 in 96ꢀwell plates. The
DMEM or RPMI growth medium was removed and the cells were incubated with GdꢀDTPA or Mnꢀ
EDTAꢀBTA in DMEM or RPMI serumꢀdepleted medium for 24 h. Cell viability was assessed using
the CCKꢀ8 kit (Dojindo, Sunnyvale, CA) according to the manufacturer’s protocol. In brief, 10 ꢂL of
CCKꢀ8 solution was added to each well and the samples were incubated for 4 h before the absorbance
was measured at 450 nm.
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Cell fractions for MRI. NCTC 1469 cells were plated at a density of 2 × 105 in 35 mm dishes.
The DMEM growth medium was removed, and the cells were incubated with MnꢀEDTAꢀBTA (100
ꢂM) in DMEM serumꢀdepleted media for 12 h. The cells were washed with phosphate buffered saline
(PBS) and harvested. For cell fractionation, cancer cells were lysed with three cycles of freezing and
thawing in PBS, and lysates centrifuged at 1000 rpm for 5 min at 4 °C. The supernatant was the
cytosolic fraction, and the first pellet was reꢀsuspended in radioimmunoprecipitation assay (RIPA)
buffer for 1 h at 4 °C and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant corresponded
to RIPA bufferꢀsoluble membrane fraction and the final pellet contained nuclei and cell organelles.
Collected cells were subject to MRI using a 1.5T MRI scanner.
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In vitro MRI. In vitro MR images were obtained with a 1.5 T (T) MR unit (GE Healthcare,
Milwaukee, WI, USA) equipped with a homemade small animal RF coil. The imaging parameters for
SE (Spin Echo) were as follows: repetition time (TR) = 500 ms; echo time (TE) = 13.6 ms; 10 mm
field of view (FOV); 192 × 128 matrix size; 1.0 mm slice thickness; number of acquisition (NEX) =
15.
In vivo MRI. All animal experiments were approved by and performed in accordance with the
rules of, Kyungpook National University animal care committee. Sixꢀweekꢀold male ICR mice
weighing 25ꢀ30 g were used for the MRI study. The mice were anesthetized with 1.5% isoflurane in
oxygen. Measurements were made before and after tail vein injection of paramagnetic complexes.
After each measurement, the mice were revived from anesthesia and placed in cages with free access
to food and water. During these measurements, the animals were maintained at room temperature.
Whole body MR images were obtained with a 1.5 T MR unit (GE healthcare) equipped with a
homemade small animal RF coil. The coil was of receiver type bird cage with an inner diameter of 50
mm. The imaging parameters for spin echo (SE) T1ꢀweighted images were as follows: repetition time
(TR) = 300 ms; echo time (TE) = 12 ms; 11 mm field of view (FOV); 192 x 128 matrix size; 1.2 mm
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slice thickness; number of acquisition (NEX) = 8. The imaging parameters for fast spin echo (FSE)
T2ꢀweighted images were as follows: TR = 2000 ms; TE = 40 ms; 11 mm FOV; 192 x 128 matrix size;
1.2 mm slice thickness; NEX = 8.
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Image analysis. Anatomical positions with enhanced contrast were identified with respect to
heart, liver, gallbladder, kidney, and bladder on postꢀcontrast MR images. For quantitative
measurement, signal intensities in specific regions of interest (ROI) were measured using the image
processing program Image J (National Institutes of Health, USA). The CNR (contrastꢀtoꢀnoise ratio)
was calculated using equation 1, where SNR is the signal to noise ratio.
CNR = SNRpost – SNRpre
(2)
Biodistribution. MnꢀEDTAꢀBTA was administered intravenously as a bolus (0.05 mmol/kg) via
tail veins of four normal male mice (ICR mice; 25ꢀ30 g) for each time point. The mice were
anesthetized and killed by exsanguination from the vena cava at each time point (after 30 min, 1, 6, 12
and 24 h injection time). The Mn concentration was measured in various tissues (brain, heart, liver,
gall bladder, spleen, intestine, bladder, kidney, blood, and lung) by digesting the tissues with HNO3
(70%) and H2O2 (30%) at 180 ℃ for 3 h, and measuring the concentration in the clear diluted solution
with an inductively coupled plasma spectrophotometer (ICP Spectrophotometer, Optima 7300DV,
Perkin Elmer, USA). The detection limit of this method is 0.01 ppm.30
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ASSOCIATED CONTENT
Supporting information
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The Supporting Information is available free of charge on the ACS Publications website at
DOI:
1H NMR and HRꢀFABꢀMS data of synthesized complexes, HPLC spectra of L and Mnꢀ
EDTAꢀBTA and bioimage of liver tumor model (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*For Y.C.: Phone, (+)82ꢀ53ꢀ420ꢀ5471; Eꢀmail, ychang@knu.ac.kr
*For T.J.K.: Phone, (+)82ꢀ53ꢀ950ꢀ4127; Eꢀmail, tjkim@knu.ac.kr
Notes
‡These authors contributed equally to the work.
*Authors to whom correspondence should be addressed.
Author Contributions
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The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. ‡These authors contributed equally to the
work
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ACKNOWLEDGMENTS
This work was supported by the Basic Research Laboratory (BRL) Program
(2013R1A4A1069507) through the National Research Foundation funded by the Ministry of
Science, ICT and Future Planning.
ABBREVIATIONS
EDTA, ethylenediaminetetraacetic acid; BTA, benzothiazole aniline; MRI, magnetic
resonance imaging; MnDPDP, mangafodipir trisodium; NSF, nephrogenic systemic fibrosis;
CA, contrast agent; GdꢀDTPA, gadopentetic acid; HSA, human serum albumin; ICR,
Institute of Cancer Research; CNR, contrast to noise ratio; GdꢀEOBꢀDTPA, Gadoxetic acid;
GdꢀBOPTA, Gadobenic acid; RT, room temperature; HPLC, high pressure liquid
chromatography;
MES,
(2ꢀ(Nꢀmorpholino)ethane
sulfonic
acid);
MnꢀEDTA,
ethylenediaminetetraacetic acid manganese disodium salt hydrate; TI, inversion time; CPMG,
CarrꢀPurcellꢀMeiboonꢀGill pulse sequence; TE, echo time; TR, repetition time; SE, spin echo;
FSE, fast spin echo; FOV, field of view; NEX, number of acquisition; ROI, regions of
interest; DMEM, Dulbecco’s Modified Eagle’s Medium; RPMI, Roswell Park Memorial
Institute; FBS, fetal bovine serum; HSA, human serum albumin; PBS, phosphate buffered
saline; HBSS, Hank’s Balanced Salt Solution; IVIS, in vivo imaging instruments; RIPA,
radioimmunoprecipitation assay; CCKꢀ8, cell counting kitꢀ8.
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biphenylꢀ2,2‘ꢀbisamides as strong bloodꢀpool magnetic resonance imaging contrast agents. J.
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Table 1. Relaxivity and octanolꢀwater partition coefficients data of MnꢀEDTAꢀBTA, Mnꢀ
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EDTA, MnDPDP and GdꢀDTPA in water (64 MHz, 297 K).
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r
1 (mM-1sec-1)
r
2 (mM-1sec-1)
logPoct/wat
water
3.5±0.1
1.9±0.1
2.8
HSAc
water
4.9±0.1
3.7±0.1
3.7
HSAc
MnꢀEDTAꢀBTA
MnꢀEDTA
15.1±1.9
34.5±3.9
ꢀ1.84
ꢀ2.72
ꢀ3.07
ꢀ3.16
ꢀ
ꢀ
ꢀ
ꢀ
MnDPDPa
GdꢀDTPAb
3.3
4.3
3.9
4.4
aData obtained from ref 7, bData obtained from ref 31. C[HSA] = 0.67mM in water.
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Figure 1. Transmetalation of 1 mM MnDPDP (△), MnꢀEDTA (▽), GdꢀDTPA (□) and Mnꢀ
EDTAꢀBTA (●) by 10 mM Zn2+ plotted by ꢁR2 as a function of time at 3T and 293 K. (ꢁR2(t)
= R2(t) – R2(0)).
Figure 2. Proton longitudinal paramagnetic relaxation rates of MnꢀEDTAꢀBTA as a function
of [Mn] in aqueous solution of HSA (0.67 mM) at 64 MHz and 293 K, fitted using eq. 1.
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Figure 3. Relative proliferation (%) of (a) mouse liver cells (NCTC 1469) and (b) the human
prostate cancer cells (DU 145) after treatment with various concentrations of MnꢀEDTAꢀ
BTA (black bars) or GdꢀDTPA (gray bars).
Figure 4. In vivo T1ꢀweighted spin echo (SE) MR images of normal ICR mice obtained after
tail vein injection of MnꢀEDTAꢀBTA (0.05 mmol/kg).
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Figure 5. Biodistribution of MnꢀEDTAꢀBTA (0.05 mmol Mn/kg body weight) in normal
ICR mice represented by Mn percentage in each tissue. Groups of mice (n = 4) were
sacrificed at 30 min, 1 h, 6 h, 12 h, and 24 h.
Figure 6. T1ꢀweighted MR images of NCTCꢀ1469 cell fractions incubated with MnꢀEDTAꢀ
BTA (100ꢂM) for 12h.
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