Article

pubs.acs.org/jmc

Manganese Complex of Ethylenediaminetetraacetic 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*,†,‡,∇
†
Department of Medical and Biological Engineering, ‡Department of Molecular Medicine and BK21 Plus KNU Biomedical
Convergence Program, §Department of Biology, ∥Department of Chemistry, ⊥Department of Pharmacy, #Institute of Biomedical
Engineering Research, and ∇Department of Radiology, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 702-701, Korea
○
Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Chumbok-ro 80, Dong-gu, Daegu 41061, Korea
*
S Supporting Information

ABSTRACT: A novel manganese(II) complex based on an ethylene-

diaminetetraacetic acid (EDTA) coordination cage bearing a benzothia-
zole 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.

■ INTRODUCTION
Manganese (Mn) carries five unpaired electrons and is one of
weighted MRI owing to contrast enhancement from the Mn
accumulation.13 Furthermore, although no association has been
the earliest paramagnetic metal ions reported to effectively found between Mn and NSF so far, potential harmful effects of
enhance positive contrast in magnetic resonance imaging exposure to free Mn ions at a high concentration remain a
(MRI).1,2 As a clinical MRI contrast agent (CA), gadolinium concern with regard to its use as a CA. Indeed, MnDPDP
(Gd) is mostly used. However, Gd complex is linked with (mangofodipir trisodium), which was approved by the FDA for
nephrogenic systemic fibrosis (NSF).3,4 NSF is a rare, use as a T1 MR CA for liver imaging, releases free Mn in plasma
idiopathic systemic fibrosing disorder and can be critical to after intravenous (iv) injection.14 When an MRI dose (i.e., 5−
patients with acute or chronic kidney disease (CKD) with 10 mmol/kg) is injected into a human patient, only about 20%
severely impaired renal function.5 Furthermore, it has been remains bound to the chelator. Release of paramagnetic Mn2+
recently reported that intravenously administered Gd accumu- occurs through dose-dependent transmetalation, in which Mn2+
lates in the brains of patients with normal renal function.6 With is displaced by endogenous Zn2+.15 Zn2+ has roughly 1000
increasing safety concerns over potential toxicities associated times higher affinity than Mn2+ does for DPDP. Free Mn2+ ions
with Gd retention in the human body, alternative approaches dissociated from MnDPDP are in fact responsible for the T1
based on non-lanthanide metals, particularly Mn, are receiving contrast enhancement of MnDPDP.16 Although free Mn2+ ions
more attention for use in MRI.7,8 are known to accumulate primarily in the liver, pancreas, and
Mn plays a critical role in cell function.9 It is a natural cellular heart, one report showed Mn2+ accumulation in patients’ brains,
constituent and is required for mitochondrial function. suggesting possible neurotoxicity.17
Hepatocytes are mitochondria-rich cells; thus, Mn is an Several efforts have been made to design stable Mn(II)
excellent CA for MRI of the liver.10 Although small amounts complexes for MRI. Recently, Caravan et al. synthesized an
are essential in humans, overexposure to free Mn ions may Mn(II) complex of [Mn(PyC3A)(H2O)]− as an alternative CA
result in neurotoxicity.11 It has been known for many years that to Gd. This complex is one of the most stable Mn(II)
chronic exposure to Mn in certain occupational settings leads to complexes at pH 7.4 (log KML = 11.40) and has a possible
a neurological syndrome known as manganism, which is similar application as a molecular agent for vascular thrombi.18 More
to Parkinson’s disease.12 The neurological symptoms of
manganism correlate with accumulation of Mn in the basal Received: January 11, 2017
ganglia, which can often be seen as hyperintensity on a T1- Published: March 16, 2017

© 2017 American Chemical Society 2993 DOI: 10.1021/acs.jmedchem.6b01799

J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

Scheme 1. Synthesis of Mn-EDTA-BTA

recently, Lattuada et al. synthesized a new Mn(II)-EDTA- including 1H NMR, HR-FAB-MS, and elemental analysis
deoxycholic acid conjugate as a potential MRI blood pool (Supporting Information).
agent.19 However, design of stable liver-specific Mn(II) Kinetic Inertness. Zn is the second most abundant trace
complexes remains a challenge. In the current study, we report metal in the human body and can thus displace more Mn2+ ions
the design and synthesis of a new liver-specific, highly stable than other endogenous ions such as Cu2+ and Ca2+. The rate of
Mn chelate for liver MRI. Specifically, we designed a novel transmetalation of an Mn ligand chelate is inversely correlated
Mn(II) complex based on an EDTA coordination cage bearing with the stability of the complex. The relative stability of CAs
a benzothiazole aniline (BTA) moiety with high chelation can therefore be measured by determining the kinetics of their
stability for use as a liver-specific MRI CA. BTA derivatives transmetalation with Zn2+. Transmetalation rates are often
have high lipophilicity and sufficient protein-binding affinity.20 represented by plotting the evolution of the normalized
We therefore hypothesized that BTA will contribute to the longitudinal (R1) or transverse (R2) relaxivity as a function of
increased lipophilicity of Mn-EDTA-BTA, which will enhance time.24,25 In the current study, the kinetic inertness of Mn-
its ability to target liver cells. In the case of liver-specific Gd EDTA-BTA is represented by a change in transverse relaxivity
agents such as gadolinium dimeglumine ethoxybenzyl (Gd- (ΔR2(t) = R2(t) − R2(0)) as a function of time. Here, R2(t) at
EOB-DTPA), lipophilicity is known to enhance liver uptake.21 any time t is a good estimator of the extent of transmetalation
In addition to forming a hydrophilic, stable complex with of Mn by Zn. MnDPDP, Mn-EDTA, and Gd-DTPA were also
Mn2+, this new hepatobiliary Mn chelate is rapidly taken up by examined for comparison (Figure 1). We prepared the complex
liver hepatocytes. We believe this complex is the first example under two conditions following a published method with slight
of a small Mn-chelate-based liver agent with high in vivo modifications.18 Transmetalation was evaluated by measuring
stability. This new family of CAs is highly suitable for liver ΔR2(t) of each Mn-EDTA-BTA complex incubated with Zn2+
imaging applications such as liver cancer imaging. (10 or 25 equiv) in pH 6.0 MES buffer. The results were

■ RESULTS AND DISCUSSION

Synthesis. The methods for synthesis of the chelate
consistently similar in all cases, and a representative set is
presented here (10 mM Zn2+). The same method was used to

conjugate and its Mn(II) complex (abbreviated as L and Mn-

EDTA-BTA, respectively) are depicted in Scheme 1. Synthesis
of the ligand L started with the commercially available D,L-2,3-
diaminopropionic acid monohydrobromide. Compound 1 was
synthesized following a published method22 and was con-
jugated to BTA in the presence of triphenyl phosphite to form
a white solid 2. tert-Butyl was cleaved using a mixture of
trifluoroacetic acid (TFA) and dichloromethane to yield a pale-
yellow product 3 after precipitation. Alkylation with tert-butyl
bromoacetate under conditions reported previously23 resulted
in a high yield of the protected EDTA-BTA 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 Mn-EDTA-BTA was
prepared by reaction of L with a stoichiometric equivalent of
MnCl2·4H2O at pH 6 followed by removal of inorganic
impurities by reverse-phase chromatography. The pure chelate Figure 1. Transmetalation of 1 mM MnDPDP (△), Mn-EDTA (▽),
was isolated as a sodium salt in moderate yield by Gd-DTPA (□), and Mn-EDTA-BTA (red circle) by 10 mM Zn2+
lyophilization. The formation of the ligand and its Mn complex plotted by ΔR2 as a function of time at 3 T and 293 K (ΔR2(t) = R2(t)
was confirmed by microanalysis and spectroscopic techniques − R2(0)).

2994 DOI: 10.1021/acs.jmedchem.6b01799

J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

Table 1. Relaxivity and Octanol−Water Partition Coefficients Data of Mn-EDTA-BTA, Mn-EDTA, MnDPDP, and Gd-DTPA in
Water (64 MHz, 297 K)
r1 (mM−1 s−1) r2 (mM−1 s−1)
c
water HSA water HSAc log Poct/wat
Mn-EDTA-BTA 3.5 ± 0.1 15.1 ± 1.9 4.9 ± 0.1 34.5 ± 3.9 −1.84
Mn-EDTA 1.9 ± 0.1 3.7 ± 0.1 −2.72
MnDPDPa 2.8 3.7 −3.07
Gd-DTPAb 3.3 4.3 3.9 4.4 −3.16
a b c
Data obtained from ref 7. Data obtained from ref 31. [HSA] = 0.67 mM in water.

evaluate transmetalation of MnDPDP, Mn-EDTA, and Gd- obtained by measuring the proton longitudinal relaxation rate
DTPA. The ΔR2(t) of MnDPDP rapidly increased and reached as a function of the concentration of Mn-EDTA-BTA, at a fixed
saturation. In contrast, the ΔR2(t) of Mn-EDTA-BTA rapidly concentration of HSA. To estimate lipophilicity, we determined
increased at first, but subsequently slowed to reach saturation at the octanol−water partitioning coefficient (log P). The log P
a much lower ΔR2(t) value compared to that of MnDPDP. value for Mn-EDTA-BTA (log P = −1.84) was higher than
Therefore, compared to MnDPDP, Mn-EDTA-BTA is those of MnDPDP (log P = −3.07), Mn-EDTA (log P =
significantly more inert to Mn2+ transmetalation. Figure 1 −2.72), and Gd-DTPA (log P = −3.16), demonstrating the
also shows that Mn-EDTA-BTA is kinetically more stable than higher lipophilicity of Mn-EDTA-BTA (Table 1).
Mn-EDTA is, suggesting the possible role of the BTA moiety in In Vitro Cytotoxicity. Tests of Mn-EDTA-BTA and Gd-
stabilizing Mn chelation. Furthermore, the pattern of ΔR2(t) DTPA cytotoxicity were performed on the human prostate
for Mn-EDTA-BTA is almost identical to that for Gd-DTPA, cancer cell line DU 145 and the mouse liver cell line NCTC
the widely used clinical MRI CA, indicating that Mn-EDTA- 1469. Figure 3 shows that viability of DU 145 cells was above
BTA is as stable as Gd-DTPA is. 88% and that of NCTC 1469 cells was above 86% when
Relaxivity and Lipophilicity. The relaxivity values of Mn- incubated with various concentrations of Mn-EDTA-BTA up to
EDTA-BTA are summarized in Table 1 along with those of 50 μM. As shown in Figure 3, Mn-EDTA-BTA showed lesser
MnDPDP, Mn-EDTA, and Gd-DTPA for comparison. Of the cytotoxicity than Gd-DTPA did at all concentrations. These
observations indicate that Mn-EDTA-BTA has negligible
three complexes, Mn-EDTA-BTA had the highest relaxivity
cytotoxicity in the concentration range needed to sufficiently
(Table 1). Although Mn2+ (S = 5/2) has fewer unpaired
enhance signal intensity in MRI.
electrons than Gd3+ (S = 7/2) does, the lipophilic moiety BTA
In Vivo MRI and in Vivo Biodistribution. In vivo MRI
has a slower tumbling rate and might contribute to high using Mn-EDTA-BTA was performed by obtaining T1-
relaxivity relative to that reported for Gd-DTPA or Mn-EDTA. weighted MR images of 6-week-old male mice of the Institute
In addition, the shorter distance between Mn2+ and water may for Cancer Research strain (ICR mice) after a bolus injection of
contribute to the increased relaxivity of Mn-EDTA-BTA.21 We Mn-EDTA-BTA through the tail vein (Figure 4). The most
also measured relaxivity in an aqueous solution of human serum characteristic MR feature of Mn-EDTA-BTA is that it shows
albumin (HSA). In this solution as well, the relaxivity values of strong contrast enhancement in the liver and kidney after
Mn-EDTA-BTA were higher than those of Gd-DTPA and injection and subsequently in the gallbladder and intestine as
indicated an interaction between HSA and Mn-EDTA-BTA. well. In vivo MRI therefore indicates that Mn-EDTA-BTA is
The binding constant (Ka), reflecting the interaction of Mn- eliminated via hepatobiliary and renal pathways. In the
EDTA-BTA with HSA was determined following a previously hepatobiliary pathway, Mn-EDTA-BTA is eliminated via the
published method.26 The Ka of Mn-EDTA-BTA binding to bile duct following hepatobiliary uptake. Interestingly, this dual
HSA (95 M−1) was higher than that of Gd-DOTA binding to elimination property of Mn-EDTA-BTA is similar to that of the
HSA (21 M−1).27 Figure 2 shows the result of fitting data clinically approved Gd-based liver-specific agent Gd-DTPA-
EOB.21 In vivo biodistribution of Mn-EDTA-BTA was
quantitatively measured with an inductively coupled plasma
(ICP) spectrophotometer (Figure 5). The data show the
highest Mn(II) accumulation in the liver and intestine,
indicating Mn-EDTA-BTA 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 Mn-EDTA-BTA is
eliminated via dual pathways (renal and hepatobiliary).
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 Mn-EDTA-BTA. These experiments
provide direct evidence that the liver MR signal enhancement is
Figure 2. Proton longitudinal paramagnetic relaxation rates of Mn- a result of hepatocyte targeting by Mn-EDTA-BTA. However, a
EDTA-BTA as a function of [Mn] in aqueous solution of HSA (0.67 further study is warranted to investigate the detailed
mM) at 64 MHz and 293 K, fitted using eq 1. mechanism of hepatocyte targeting of Mn-EDTA-BTA,
2995 DOI: 10.1021/acs.jmedchem.6b01799
J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

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 Mn-EDTA-BTA (black bars) or Gd-DTPA (gray bars).

Figure 4. In vivo T1-weighted spin echo (SE) MR images of normal ICR mice obtained after tail vein injection of Mn-EDTA-BTA (0.05 mmol/kg).

Figure 5. Biodistribution of Mn-EDTA-BTA (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.

although its lipophilicity may be partly responsible for this. In Tumor Imaging: Distinction between Normal Liver
the case of Gd-DTPA-EOB, the active transport system on the and Tumor Tissue. Because Mn-EDTA-BTA behaves as a
hepatocyte membrane plays an important role. Specifically, Gd- liver-specific agent, we performed an in vivo test to determine
whether it can distinguish between normal and tumor liver
DTPA-EOB enters hepatocytes through two different organic
tissue. Figure 7a and Figure 7b show T2-weighted and T1-
anion transport systems.21 In addition, in future studies, it will weighted MR images from a HepG2 xenograft mouse model
be important to identify more efficient alternatives to BTA for before and after injection of Mn-EDTA-BTA or MnDPDP. T2-
hepatocyte targeting. weighted images without CA injection clearly showed the
2996 DOI: 10.1021/acs.jmedchem.6b01799
J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

Gd-DTPA. Furthermore, its R1 relaxivity (3.47 mM−1 s−1) was

higher than those of MnDPDP (R1 = 2.8 mM−1 s−1) and Gd-
DTPA (R1 = 3.3 mM−1 s−1). In vivo biodistribution and in vivo
MRI patterns effectively demonstrated that Mn-EDTA-BTA is
a liver-specific MRI CA that utilizes both renal and
hepatobiliary elimination pathways. This elimination pattern
is similar to that of Gd-based hepatobiliary agents such as Gd-
Figure 6. T1-weighted MR images of NCTC-1469 cell fractions DTPA-EOB and Gd-BOTPA. Finally, in an animal model of
incubated with Mn-EDTA-BTA (100 μM) for 12 h. liver cancer, Mn-EDTA-BTA significantly improved tumor
detection and characterization compared to that observed with
tumor location and size. In the case of Mn-EDTA-BTA, greater MnDPDP, suggesting that Mn-EDTA-BTA may be a good
signal enhancement is seen in normal liver tissue than in tumor diagnostic MRI agent for liver cancer.

■
tissue. However, in the case of MnDPDP, positive signal
enhancement is seen in both normal liver and tumor tissue, EXPERIMENTAL SECTION
suggesting that normal hepatocytes and tumor cells efficiently
General Remarks. D,L-2,3-Diaminopropionic acid monohydrobro-
take up MnDPDP. Mn-EDTA-BTA thus showed a higher mide, di-tert-butyl dicarbonate, triphenyl phosphite, N,N-diisopropyl-
difference in contrast-to-noise ratio (ΔCNR) between tumor ethylamine, and ethylenediaminetetraacetic acid manganese disodium
and normal liver tissue (Figure 7c) than MnDPDP did, salt hydrate (Mn-EDTA) were purchased from Tokyo Chemical
suggesting a significant improvement in tumor detection and Industry (Tokyo, Japan). tert-Butyl bromoacetate was obtained from
characterization. From the kinetic stability data (Figure 1), one Alfa-Aesar (Ward Hill, MA, USA). Sodium bicarbonate (NaHCO3)
possible mechanism for a significant improvement in contrast was purchased from Daejung Chem. (Korea). Potassium iodide (KI)
between tumor and normal liver tissue is that Mn-EDTA-BTA and sodium sulfate (Na2SO4) anhydrous were purchased from Duksan
remains as a stable complex and thus is taken up only by Scientific Corp. (Korea). All other commercial reagents were
normal liver cells. However, MnDPDP is dissociated into free purchased from Sigma-Aldrich (St. Louis, MO, USA) and used
without further purification unless otherwise stated. Solvents were
Mn2+ ions and DPDP, and free Mn2+ ions enter all cells with purified and dried using standard procedures. Deionized water was
equal efficiency, enhancing both tumor and normal liver tissue used for all experiments. 1H NMR spectra was recorded on a Bruker
equally. Advance 500 MHz spectrometer at the Center for Instrumental
In summary, Mn-EDTA-BTA shows good potential as a Analysis, Kyungpook National University (KNU). Chemical shifts are
diagnostic agent for liver cancer. It should be mentioned that given as δ values with reference to tetramethylsilane (TMS) as the
although the injection dose of MnDPDP used in the current internal standard. Coupling constants are in Hz. Elemental analyses
study was lower than that of Mn-EDTA-BTA, based on the (EA) were performed by the Center for Instrumental Analysis, KNU.
suggested clinical dose of MnDPDP,28 the main reason for the High-resolution fast atom bombardment mass spectra (HR-FAB-MS)
low ΔCNR of MnDPDP between tumor and normal liver were obtained using a JMS-700 model (Jeol, Japan) mass
spectrophotometer at the Korea Basic Science Institute (KBSI). An
tissue was not the low dose but the uptake of MnDPDP in both HPLC (high-pressure liquid chromatography, LC-Forte/R, YMC,
tumor and normal liver tissue. Furthermore, the injection dose Japan) system equipped with a Luna C18 column (250 mm × 21.2
of MnDPDP could not be increased because of possible toxicity mm, Phenomenex Inc. USA) was used for the purification and purity
in vivo, especially due to neurotoxicity of Mn2+ released from tests at room temperature. The methods used in the purity test were as
DPDP. follows: (method A) eluent A, 0.1% TFA in water; B, 0.1% TFA in

■ CONCLUSIONS
In the current study, we synthesized and characterized an Mn-
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.
EDTA complex incorporating a BTA functionality and The purity of all products was determined using elemental analysis or a
investigated its possible diagnostic utility in liver MRI. The reverse-phase HPLC with UV−vis detection at 320 nm.
kinetic inertness of this complex was much higher than those of Synthesis and Characterization. 2,3-Bis-tert-butoxy-
MnDPDP and Mn-EDTA, and was comparable to that of stable carbonylaminopropionic Acid (1). The title compound was

Figure 7. Axial T2-weighted and T1-weighted MR images of HepG2 xenograft mice (a) before injection and 30 min after injection with Mn-EDTA-
BTA (0.05 mmol/kg) and (b) before injection and 30 min after injection with MnDPDP (0.01 mmol/kg). The white arrows show the liver tumor
lesion. (C) Difference of CNR between normal and tumor liver tissue as a function of time. CNR was measured in T1-weighted images. Mn-EDTA-
BTA (filled squares) and MnDPDP (filled circles).

2997 DOI: 10.1021/acs.jmedchem.6b01799

J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

prepared according to the literature method with little modification.22 (Figure S10). Yield: 0.89 g (83%). 1H NMR (CDCl3): δ = 8.06−8.01
Yield: 7.8 g (95%). 1H NMR (CDCl3): δ = 1.45 (s, 18H, CH3), 3.55 (m, 2H, BTA), 7.99−7.96 (d, 2H, BTA), 7.85−7.83 (d, 2H, BTA),
(m, 2H, CH2), 4.12 (s, 1H, CH), 4.27−5.17 (m, 1H, NH), 5.85 (s, 7.52−7.49 (t, 1H, BTA), 7.42−7.39 (t, 1H, BTA), 5.50−5.48 (s, 2H,
1H, OH), 2.05 (s, 1H, NH). Anal. Calcd for C13H24O6N2·2EtOAc: C, OH), 4.02−3.98 (d, 1H, CH), 3.96−3.76 (m, 8H, CH2), 3.57−3.41
51.71; H, 8.04; N, 8.10. Found: C, 51.87; H, 8.15; N, 7.75. (m, 2H, CH2). Anal. Calcd for C24H24N4O9S·H2O: C, 51.24; H, 4.66;
[2-(4-Benzothiazol-2-ylphenylcarbamoyl)-2-tert- N, 9.96; S, 5.70. Found: C, 51.02; H, 4.65; N, 9.64; S, 5.54. HR-FAB-
butoxycarbonylaminoethyl]carbamic Acid tert-Butyl Ester (2). MS (m/z) for C24H25N4O9S: calcd, 545.1342 [M + H]+; found,
2,3-Bis-tert-butoxycarbonylaminopropionic acid (7.00 g, 23.01 mmol) 545.1340 [M + H]+.
was dissolved in pyridine (40 mL), and benzothiazole aniline (BTA) [Mn(EDTA-BTA)(H2O)]2−. Compound L (1 g, 1.8 mmol) was
(5.20 g, 23.01 mmol) in pyridine (20 mL) was added slowly. Resulting dissolved in MeOH (30 mL) and the pH of the solution adjusted to
mixture was stirred for 30 min, and triphenyl phosphite (7.13 mL, pH 6 with NaOH (1.0 M), then the mixture changed to suspension
23.01 mmol) was added dropwise. The solution was stirred for 3 h at including the off-white precipitation. To this mixture, MnCl2·4H2O
80 °C and then overnight at rt. Solid obtained was filtered and washed (0.36 g, 1.8 mmol) in MeOH (3 mL) was slowly added and stirred at
with DI water and acetone. Crude product was recrystallized in rt for overnight. During this time the mixture was readjusted to pH 6.
absolute acetonitrile. The desired product was obtained as white The precipitate was filtered, washed with cold methanol, and dried
powder. Yield: 9.6 g (81.4%). 1H NMR (CDCl3): δ = 9.28 (s, 1H, under vacuum and then was dissolved in a minimum amount of water
NH), 8.06−8.01 (m, 3H, BTA), 7.89−7.85 (d, 1H, BTA), 7.68−7.64 and more purified by flash column chromatography (C18, 95:5 to
(d, 2H, BTA), 7.49−7.44 (t, 1H, BTA), 7.38−7.33 (t, 1H, BTA), 85:15, water/MeOH) to yield a white solid. Further purification was
5.97−5.93 (s, 2H, NH), 4.02−3.98 (d, 1H, CH), 3.57−3.41 (m, 2H, carried out using preparative HPLC (method B), and also purity was
CH2), 1.50−1.40 (d, 18H,CH3). Anal. Calcd for C26H32N4O5S: C, confirmed (Figure S11). Yield: 0.35g (32%). HR-FAB-MS (m/z) for
60.92; H, 6.29; N, 10.93; S, 6.26. Found: C, 60.55; H, 6.36; N, 10.60; C24H20MnN4O9S: calcd, 595.0331 [M]+; found, 595.0327 [M]+.
S, 5.98. HR-FAB-MS (m/z) for C26H33N4O5S): calcd, 513.2172 [M + Transmetalation Kinetics. This experiment was performed
H]+; found, 513.2170 [M + H]+. following published literature with a slight modification.18 About 20
2,3-Diamoniumtrifluoroacetate-N-(4-benzothiazol-2-yl- μL of a 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffered
phenyl)propionamide (3). TFA (10 mL) was added dropwise into solution (pH 6.0) of ZnCl2 was added to 1 mL of a buffered solution
solution of 2 (1.5 g, 2.92 mmol) in CH2Cl2 at 0 °C. Resulting yellow of 1 mM metal complex. The mixture was shaken briefly and
solution was stirred until starting material was consumed. Solvent was immediately used for measuring solvent T2 as a function of time.
removed and Et2O was added to obtain precipitate. The solid was Control studies were also conducted with MnDPDP (Teslascan), Gd-
filtered and washed three times with Et2O and then dried to give the DTPA (Magnevist), and Mn-EDTA for comparison. The measure-
title compound as a pale yellow solid in quantitative yield. Yield: 0.82 g ments were performed on a 3 T whole body system (Discovery
(90%). 1H NMR (MeOH-d4): δ = 8.75 (s, 1H, NH), 8.01−7.99 (d, MR750w 3.0T, GE healthcare) at room temperature. The graph was
2H, BTA), 7.9−7.89 (d, 2H, BTA), 7.82−7.78 (d, 2H, BTA), 7.45 (t, plotted using the equation ΔR2(t) = R2(t) − R2(0) as a function of
1H, BTA), 7.35 (t, 1H, BTA), 4.43 (t, 1H, CH), 3.62−3.55 (dd, 1H, time.
CH2), 3.51−3.44 (dd, 2H, CH2), 1.94 (s, 2H, NH). Anal. Calcd for Relaxivity. Using an inversion recovery method, T1 measurements
C16H16N4OS·3CF3COOH: C, 40.37; H, 2.93; N, 8.56; S, 4.90. Found: were made with a variable inversion time (TI) at 1.5 T (64 MHz, GE
C, 40.74; H, 2.68; N, 8.86; S, 5.26. HR-FAB-MS (m/z) for
Healthcare Milwaukee, WI, USA). Magnetic resonance (MR) images
C16H17N4OS: calcd, 313.1123 [M + H]+; found, 313.1121 [M + H]+.
were acquired at 35 different TI values over the frequency range 50−
{[1-(4-Benzothiazol-2-yl-phenylcarbamoyl)-2-(bis-tert-
butoxycarbonylmethylamino)ethyl]-tert-butoxycarbonyl- 1750 ms. T1 relaxation times were achieved from the nonlinear least-
methylamino}acetic Acid tert-Butyl Ester (4). Compound 3 (2 g, square fit of the signal intensity measured at each TI value. In place of
3.70 mmol), N,N-diisopropylethylamine (7.66 mL, 59.2 mmol), and T2 measurements, the CPMG (Carr−Purcell−Meiboon−Gill) pulse
KI (1.84 g, 11.1 mmol) were dissolved in DMF (20 mL), and the sequence was adapted for multiple spin−echo measurements. Thirty-
solution was warmed to 45 °C. tert-Butyl bromoacetate (5.77 mL, 29.6 four images were achieved with 34 different echo time (TE) values
mmol) was added dropwise into this solution over 30 min. Resulting ranging from 10 to 1900 ms. T2 relaxation times were attained from
solution was stirred for 4 h at 110 °C and cooled to rt. The solvent was the nonlinear least-squares fit of the mean pixel values of multiple
removed and the residue was partitioned between 10% NaHCO3 and spin−echo measurements at each echo time. Relaxivities (R1 and R2)
ethyl acetate. The aqueous layer was extracted three more times with were then measured as an inverse of relaxation time per mM. The
EtOAc. Combined organic layer was then washed with brine and dried determined relaxation times (T1 and T2) and relaxivities (R1 and R2)
over Na2SO4. Evaporation gave a dark brown oil that was purified by were finally image-processed to provide the relaxation time map and
column chromatography (silica, hexanes/EtOAc, 9:1). The desired relaxivity map, respectively.
product 4 was obtained as a light brown oily solid. Yield: 3.37 g (82%). Octanol−Water Partition Coefficients. This experiment was
1
H NMR (CDCl3): δ = 10.85 (s, 1H, NH), 8.08−8.01 (m, 3H, BTA), performed following published literature.29 Manganese(II) complex (1
7.91−7.82 (dd, 3H, BTA), 7.47 (t, 1H, BTA), 7.36 (t, 1H, BTA), 3.75 mg) was dissolved in 2 mL of a 1:1 mixture of water and 1-octanol.
(t, 1H, CH), 3.62−3.40 (m, 8H, CH2-tBu), 3.04−2.96 (dd, 2H, CH2), The solution was shaken for 30 s, then the vial containing the mixture
1.46 (s, 36H, tBu). Anal. Calcd for C40H56N4O9S·3H2O: C, 58.37; H, was placed on a rotator for gentle mixing to equilibrate for 48 h. The
7.59; N, 6.81; S, 3.90. Found: C, 58.61; H, 7.31; N, 6.46; S, 3.58. HR- sample was then allowed to settle at room temperature for 24 h.
FAB-MS (m/z) for C40H57N4O9S: calcd, 769.3846 [M + H]+; found, Mn(II) concentrations of each layer were determined by ICP-MS
769.3842 [M + H]+. (inductively coupled plasma mass spectrometry). Partition coefficients
[{1-(4-Benzothiazol-2-yl-p henylcarbamoyl)-2-(bis- were calculated from the equation log P = log(Co/Cw), where log P is
carboxymethylamino)ethyl]carboxymethylamino}acetic Acid] the logarithm of the partition coefficient, Co is the concentration of
(L). Compound 4 (1.5 g, 1.95 mmol) was dissolved in ACN (30 mL), Mn in the 1-octanol layer, and Cw is the concentration of Mn in the
and conc HCl (15 mL) was added at 0 °C. Resulting solution was water layer.
stirred for 20 h. An additional portion of HCl (5 mL) was added, and Determination of Binding Constants. The binding constants of
the solution was stirred for one more hour. The volume was reduced several CAs with HSA were measured following published
by approximately 75% by rotary evaporation. The residue was diluted literature.26,27 The nonlinear increase of the proton paramagnetic
with water (30 mL), and the pH was adjusted to 2 by the addition of relaxation rates measured at 64 MHz in solutions containing 0.67 mM
sodium hydroxide. The white precipitate appears which was collected HSA was fitted using eq 1, where Ka is the binding constant of the
by filtration and washed with DI water (pH 2.0), ethyl acetate (30 interaction with HSA, p0 is the concentration of the HSA, s0 is the
mL), and ether (30 mL). The desired product L was obtained as a paramagnetic complex concentration, N is the number of independent
white powder. The purity also confirmed using HPLC (method A) interaction sites (N was set to 1), and r1c and r1f are the relaxivities of

2998 DOI: 10.1021/acs.jmedchem.6b01799

J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry Article

the complex HSA-contrast agent and of the free contrast agent, before and after tail vein injection of paramagnetic complexes. After
respectively. each measurement, the mice were revived from anesthesia and placed
in cages with free access to food and water. During these
R1p
obs
= 1000 ×{(r s ) + 12 (r − r )⎡⎣(Np ) + s + K
f 0
1
c
1
f
1
0 0
a
−1 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
− ((Np ) + s + K ) − 4Ns p ⎥}
0 0 −1 2 0 0
was of receiver type bird cage with an inner diameter of 50 mm. The
a ⎦ (1)
imaging parameters for spin echo (SE) T1-weighted images were as
Cell Culture. The culture medium consisted of Dulbecco’s follows: repetition time (TR) = 300 ms; echo time (TE) = 12 ms; 11
modified Eagle medium (DMEM, Gibco Invitrogen, Carlsbad, CA) mm field of view (FOV); 192 × 128 matrix size; 1.2 mm slice
or Roswell Park Memorial Institute (RPMI, Gibco Invitrogen, thickness; number of acquisition (NEX) = 8. The imaging parameters
Carlsbad, CA), 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) for fast spin echo (FSE) T2-weighted images were as follows: TR =
penicillin−streptomycin. Cells were plated at a density of 2 × 105 2000 ms; TE = 40 ms; 11 mm FOV; 192 × 128 matrix size; 1.2 mm
cells/35 mm dish, incubated overnight for stabilization, and then slice thickness; NEX = 8.
treated with Mn for 24 h in serum-depleted media. Image Analysis. Anatomical positions with enhanced contrast
Liver Tumor Model. An orthotopic xenograft mouse liver tumor were identified with respect to heart, liver, gallbladder, kidney, and
model was approved by the Institutional Animal Care and Use of bladder on postcontrast MR images. For quantitative measurement,
Committee of Daegu-Gyeongbuk Medical Innovation Foundation signal intensities in specific regions of interest (ROI) were measured
(DGMIF). Five-week-old male nude mice (BALB/c nu/nu) were using the image processing program ImageJ (National Institutes of
purchased from Orient Bio (Seongnam, Korea) and housed in a Health, USA). The CNR (contrast-to-noise ratio) was calculated using
specific pathogen-free facility at the Laboratory Animal Center of eq 2, where SNR is the signal-to-noise ratio.
DGMIF before use. The mice were inoculated with HepG2-luc2 cells CNR = SNR post − SNR pre (2)
(1 × 106 cells in 50 μL of HBSS) in the subcapsular parenchyma of the
left liver lobe. The HepG2-luc2 cell line was purchased from Biodistribution. Mn-EDTA-BTA was administered intravenously
PerkinElmer Inc. Four weeks after inoculation, mice were imaged by as a bolus (0.05 mmol/kg) via tail veins of four normal male mice
the IVIS system to check tumor induction (Figure S12 in Supporting (ICR mice; 25−30 g) for each time point. The mice were anesthetized
Information). and killed by exsanguination from the vena cava at each time point
Bioluminescence Imaging. Bioluminescence images were (after 30 min, 1, 6, 12, and 24 h injection time). The Mn
acquired using the IVIS Lumina system (PerkinElmer). Mice were concentration was measured in various tissues (brain, heart, liver,
intraperitoneally administered firefly D-luciferin potassium salt gallbladder, spleen, intestine, bladder, kidney, blood, and lung) by
(PerkinElmer) at a dose of 150 mg/kg body weight in Dulbecco’s digesting the tissues with HNO3 (70%) and H2O2 (30%) at 180 °C for
phosphate-buffered saline. During image acquisition, anesthesia was 3 h and measuring the concentration in the clear diluted solution with
maintained with 2% isoflurane. Analysis was performed with Living an inductively coupled plasma spectrophotometer (ICP spectropho-
Image software by measuring the photon flux (measured in photons/ tometer, Optima 7300DV, PerkinElmer, USA). The detection limit of
[s·cm2·sr]) using a region of interest manually drawn over the body of this method is 0.01 ppm.30
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 96-well plates. The DMEM or
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
RPMI growth medium was removed, and the cells were incubated with
Gd-DTPA or Mn-EDTA-BTA in DMEM or RPMI serum-depleted ACS Publications website at DOI: 10.1021/acs.jmed-
medium for 24 h. Cell viability was assessed using the CCK-8 kit chem.6b01799.
(Dojindo, Sunnyvale, CA) according to the manufacturer’s protocol. 1
H NMR and HR-FAB-MS data of synthesized
In brief, 10 μL of CCK-8 solution was added to each well and the complexes, HPLC spectra of L and Mn-EDTA-BTA,
samples were incubated for 4 h before the absorbance was measured at and bioimage of liver tumor model (PDF)
450 nm. Molecular formula strings and some data (CSV)

■
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 Mn-EDTA-BTA (100 AUTHOR INFORMATION
μM) in DMEM serum-depleted media for 12 h. The cells were washed Corresponding Authors
with phosphate buffered saline (PBS) and harvested. For cell *T.-J.K.: phone, (+)82-53-950-4127; e-mail, [email protected].
fractionation, cancer cells were lysed with three cycles of freezing *Y.C.: phone, (+)82-53-420-5471; e-mail, [email protected].
and thawing in PBS, and lysates were centrifuged at 1000 rpm for 5
min at 4 °C. The supernatant was the cytosolic fraction, and the first ORCID
pellet was resuspended in radioimmunoprecipitation assay (RIPA) Yongmin Chang: 0000-0002-0585-8714
buffer for 1 h at 4 °C and centrifuged at 12 000 rpm for 10 min at 4 Author Contributions
°C. The supernatant corresponded to RIPA buffer-soluble membrane ◆
Md.K.I. and S.K. contributed equally to the work. The
fraction, and the final pellet contained nuclei and cell organelles.
Collected cells were subject to MRI using a 1.5 T MRI scanner.
manuscript was written through contributions of all authors. All
In Vitro MRI. In vitro MR images were obtained with a 1.5 T MR authors have given approval to the final version of the
unit (GE Healthcare, Milwaukee, WI, USA) equipped with a manuscript.
homemade small animal rf coil. The imaging parameters for SE Notes
(spin echo) were as follows: repetition time (TR) = 500 ms; echo time The authors declare no competing financial interest.

■
(TE) = 13.6 ms; 10 mm field of view (FOV); 192 × 128 matrix size;
1.0 mm slice thickness; number of acquisition (NEX) = 15. ACKNOWLEDGMENTS
In Vivo MRI. All animal experiments were approved by and
performed in accordance with the rules of Kyungpook National This work was supported by the Basic Research Laboratory
University Animal Care Committee. Six-week-old male ICR mice (BRL) Program (Grant 2013R1A4A1069507) through the
weighing 25−30 g were used for the MRI study. The mice were National Research Foundation funded by the Ministry of
anesthetized with 1.5% isoflurane in oxygen. Measurements were made Science, ICT and Future Planning.
2999 DOI: 10.1021/acs.jmedchem.6b01799
J. Med. Chem. 2017, 60, 2993−3001
Journal of Medicinal Chemistry

■
Article

ABBREVIATIONS USED (13) Bock, N. A.; Silva, A. C. Manganese: a unique neuroimaging

contrast agent. Future Neurol. 2007, 2, 297−305.
EDTA, ethylenediaminetetraacetic acid; BTA, benzothiazole (14) Ni, Y.; Marchal, G. Clinical implications of studies with
aniline; MRI, magnetic resonance imaging; MnDPDP, MnDPDP in animal models of hepatic abnormalities. Acta Radiol.
mangafodipir trisodium; NSF, nephrogenic systemic fibrosis; 1997, 38, 724−731.
CA, contrast agent; Gd-DTPA, gadopentetic acid; HSA, human (15) Karlsson, J. O. G.; Ignarro, L. J.; Lundström, I.; Jynge, P.;
serum albumin; ICR, Institute of Cancer Research; CNR, Almén , T. Calmangafodipir [Ca 4 Mn(DPDP) 5 ], mangafodipir
contrast to noise ratio; Gd-EOB-DTPA, gadoxetic acid; Gd- (MnDPDP) and MnPLED with special reference to their SOD
BOPTA, gadobenic acid; rt, room temperature; HPLC, high mimetic and therapeutic properties. Drug Discovery Today 2015, 20,
pressure liquid chromatography; MES, (2-(N-morpholino)- 411−421.
ethanesulfonic acid); Mn-EDTA, ethylenediaminetetraacetic (16) Rocklage, S. M.; Cacheris, W. P.; Quay, S. C.; Hahn, F. E.;
acid manganese disodium salt hydrate; TI, inversion time; Raymond, K. N. Manganese(II) N,N′-dipyridoxylethylenediamine-
CPMG, Carr−Purcell−Meiboon−Gill pulse sequence; TE, N,N′-diacetate 5,5′-bis(phosphate). Synthesis and characterization of a
paramagnetic chelate for magnetic resonance imaging enhancement.
echo time; TR, repetition time; SE, spin echo; FSE, fast spin
Inorg. Chem. 1989, 28, 477−485.
echo; FOV, field of view; NEX, number of acquisition; ROI, (17) O’Neal, S. L.; Zheng, W. Manganese toxicity upon over-
regions of interest; DMEM, Dulbecco’s modified Eagle exposure: a decade in review. Curr. Environ. Health Rep. 2015, 2, 315−
medium; RPMI, Roswell Park Memorial Institute; FBS, fetal 328.
bovine serum; HSA, human serum albumin; PBS, phosphate (18) Gale, E. M.; Atanasova, I. P.; Blasi, F.; Ay, I.; Caravan, P. A
buffered saline; HBSS, Hank’s balanced salt solution; IVIS, in manganese alternative to gadolinium for MRI contrast. J. Am. Chem.
vivo imaging instruments; RIPA, radioimmunoprecipitation Soc. 2015, 137, 15548.
assay; CCK-8, cell counting kit-8 (19) Baroni, S.; Serra, S. C.; Mingo, A. F.; Lux, G.; Giovenzana, G. B.;

■
Lattuada, L. Synthesis and relaxometric characterization of a new
Mn(II)-EDTA-deoxycholic acid conjugate complex as a potential MRI
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3001 DOI: 10.1021/acs.jmedchem.6b01799

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