Mn(II) Complex of Lipophilic Group-Modified Ethylenediaminetetraacetic Acid (EDTA) as a New Hepatobiliary MRI Contrast Agent

Article abstract of DOI:10.1021/acs.jmedchem.1c00393

Liver-specific contrast agents (CAs) can improve the Magnetic resonance imaging (MRI) detection of focal and diffuse liver lesions by increasing the lesion-to-liver contrast. A novel Mn(II) complex, Mn-BnO-TyrEDTA, with a lipophilic group-modified ethylenediaminetetraacetic acid (EDTA) structure as a ligand to regulate its behavior in vivo, is superior to Gd-EOB-DTPA in terms of a liver-specific MRI contrast agent. An MRI study on mice demonstrated that Mn-BnO-TyrEDTA can be rapidly taken up by hepatocytes with a combination of hepatobiliary and renal clearance pathways. Bromosulfophthalein (BSP) inhibition imaging, biodistribution, and cellular uptake studies confirmed that the mechanism of hepatic targeting of Mn-BnO-TyrEDTA is the hepatic uptake of the amphiphilic anion contrast agent mediated by organic anion transporting polypeptides (OATPs) expressed by functional hepatocytes.

Full text of DOI:10.1021/acs.jmedchem.1c00393

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Article
Mn(II) Complex of Lipophilic Group-Modified
Ethylenediaminetetraacetic Acid (EDTA) as a New Hepatobiliary MRI
Contrast Agent
Keyu Chen, Pan Li, Chunrong Zhu, Zhiyang Xia, Qian Xia, Lei Zhong, Bin Xiao, Tao Cheng,
Changqiang Wu, Chengyi Shen, Xiaoming Zhang,* and Jiang Zhu*
Cite This: J. Med. Chem. 2021, 64, 9182−9192
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Supporting Information
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ABSTRACT: Liver-specific contrast agents (CAs) can improve
the Magnetic resonance imaging (MRI) detection of focal and
diffuse liver lesions by increasing the lesion-to-liver contrast. A
novel Mn(II) complex, Mn-BnO-TyrEDTA, with a lipophilic
group-modified ethylenediaminetetraacetic acid (EDTA) structure
as a ligand to regulate its behavior in vivo, is superior to Gd-EOB-
DTPA in terms of a liver-specific MRI contrast agent. An MRI
study on mice demonstrated that Mn-BnO-TyrEDTA can be
rapidly taken up by hepatocytes with a combination of
hepatobiliary and renal clearance pathways. Bromosulfophthalein
(BSP) inhibition imaging, biodistribution, and cellular uptake
studies confirmed that the mechanism of hepatic targeting of Mn-
BnO-TyrEDTA is the hepatic uptake of the amphiphilic anion contrast agent mediated by organic anion transporting polypeptides
(OATPs) expressed by functional hepatocytes.
hydrophilic, do not bind to proteins or receptors, and are
rapidly excreted unmetabolized in urine.2,6−8
INTRODUCTION
■
Magnetic resonance imaging (MRI) is one of the most
powerful noninvasive diagnostic tools in medical imaging and
biomedical research. Without using ionizing radiation, this
technique can generate superb tissue contrast imaging with
high spatial resolution.1 Due to its relatively low sensitivity,
more than one-third of clinical MR scans are being contrast-
enhanced by the use of MRI contrast agents (CAs), which can
catalytically shorten the relaxation times (T1 and T2) of bulk
water protons to increase signal intensity. Compared with
unenhanced MR images, contrast-enhanced MRI can produce
more morphologic and functional information, which results in
more accurate diagnosis of diseases. The most widely used MR
CAs are dominated by the paramagnetic chelates of
gadolinium-based contrast agents (GBCAs), owing to
gadolinium(III)’s high effective magnetic moment and
relatively slow electronic spin relaxation.2
Usually, an amphiphilic structure formed by introducing
lipophilic groups (e.g., aromatic or steroid structures) to the
ligand of a highly hydrophilic gadolinium chelate will promote
hepatic uptake. For instance, Gd-EOB-DTPA and Gd-BOPTA,
two Gd-DTPA-derived chelates that are composed of an
ethoxybenzyl moiety and a benzoylmethyl moiety, respectively,
are the most wildly used contrast agents in clinic hepatic MR
imaging. Liver-specific CA-enhanced liver MRI can provide
valuable anatomic and functional diagnosis information in
numerous focal and diffuse liver lesions,9 such as facilitating
the differentiation of focal nodular hyperplasia (FNH) from
hepatocellular carcinoma (HCA),10,11 quantitatively assessing
regarding liver perfusion and hepatocyte function.
Recent studies revealed that highly expressed organic anion
transporting polypeptides (OATPs; OATP1B1 and
OATP1B3) on hepatocyte membranes are the main trans-
porters of amphiphilic structural small-molecule liver-specific
To reduce the toxicity of free Gd3+ ion, the gadolinium ion is
chelated with multidentate ligands (chelators), either macro-
cyclic (H4DOTA) or linear (H5DTPA), to form high stability
(thermodynamic and kinetic) charged or charge-neutral
chelates (Chart 1). It is well known that the chemical structure
of ligand determined the overall biology behavior of these Gd3+
chelates in vivo (biodistribution, pharmacokinetics, pharmaco-
dynamics, and safety profiles).3−5 In general, most clinically
approved small molecular gadolinium chelates are called
nonspecific, extracellular fluid (ECF) agents, as they are highly
Received: March 5, 2021
Published: June 21, 2021
https://doi.org/10.1021/acs.jmedchem.1c00393
© 2021 American Chemical Society
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Chart 1. Ligand and Chelate Structures Discussed in This Paper
a
Scheme 1. Synthesis Procedure of the Mn-BnO-TyrEDTA Complex
a
Reagents and conditions. a. (i) K2CO3, KI, BnCl, 90 °C, 6.0 h; (ii) NaOH, H2O, THF, 50 °C,1.0 h, and then acidified with diluted HCl; b. (i)
isobutyl chloroformate, DIPEA, THF, rt, 1.0 h, and then NH3(g), 24.0 h, rt; (ii) TFA, CH2Cl2, rt, 12.0 h; c. BH3·THF, THF, refluxed, 24.0 h; d.
tert-butyl bromoacetate, DIPEA, KI, acetonitrile (ACN), 50 °C, 12.0 h; e. TFA, dichloromethane (DCM), 12.0 h; and f. MnCl2·4H2O, NaOH (1.0
M), pH 7.4.
contrast agents (e.g., Gd-EOB-DTPA).12−16 High-spin
manganese(II) (5/2) with five unpaired electrons and
relatively slow electron relaxation has lower intrinsic toxicity
and a faster water-exchange rate than gadolinium(III).
Recently, the identification of nephrogenic systemic fibrosis
(NSF)17−20 and brain gadolinium deposition,21−24 a rare but
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Table 1. r1 and r2 in pH 7.4 Tris Buffer and 4.5% w/v Bovine Serum Albumin at 0.47 T and 32 °C
chelate
Mn-EDTA
Mn-BnO-TyrEDTA
Gd-EOB-DTPA
r1 pH 7.4 tris (mM−1 s−1
)
r2 pH 7.4 tris (mM−1 s−1
)
r1 BSA (mM−1 s−1
)
r2 BSA (mM−1 s−1
)
2.83 0.06
4.34 0.08
6.15 0.11
3.90 0.06
6.21 0.17
6.99 0.12
3.46 0.07
15.81 1.72
8.36 0.23
4.57 0.06
20.70 2.36
9.41 0.38
serious disorder associated with adventurous Gd(III), has led
to manganese(II) as an attractive alternative to gadolinium-
(III) for MRI application. For example, excellent kinetically
inert Mn-based MR CAs such as Mn-PyC3A,25 Mn-bispidine
chelate,26 and liver-specific Mn(II) CAs have been reported.27
It is well known that the structure of ethylenediaminetetra-
acetic acid (EDTA) and its derivates have been widely used as
ligands for chelating Mn2+ in the research of manganese-based
contrast agents.28,29 Previously, we reported a hexameric
Mn(II) dendrimer that was synthesized from a tyrosine-
derived bifunctional precursor featuring a backbone-substi-
tuted EDTA structure (named TyrEDTA)30 (Chart 1, Mn-
TyrEDTA). The corresponding moderate molecular weight
Mn(II) dendrimer exhibited high relaxivity at different fields
and mixed renal and hepatobiliary clearance in vivo.31 In this
work, we are pursuing the further development of this
bifunctional structure to develop a new T1 liver-specific Mn2+
contrast agent ([Mn(TyrEDTA)]2−) by introducing a lip-
ophilic group on the TyrEDTA ligand (BnO-TyrEDTA; Chart
1). The synthesis, characterization, and MRI liver enhance-
ment patterns of this new amphiphilic, anionic Mn(II)
complex in mice were demonstrated. In addition, the hepatic
uptake mechanism was further investigated by OATP
inhibition imaging and cell uptake study.
measured the relaxivities (r1 and r2) of Mn-EDTA, Mn-BnO-
TyrEDTA, and Gd-EOB-DTPA (Table 1). At 0.47 T, the r1
value of Mn-BnO-TyrEDTA was 4.34 mM−1 s−1 (in Tris buffer
(50 mM), pH = 7.4) and increased to 15.81 mM−1 s−1 (in
bovine serum albumin (BSA), 4.5%), higher than those of Gd-
EOB-DTPA and Mn-EDTA (8.36 and 3.46 mM−1 s−1 in BSA).
The lipophilic group in Mn-BnO-TyrEDTA promotes its
strong binding to albumin, as evidenced by the significant
increase of relaxivity in the BSA solution since the tumbling
rate slows down after macromolecule complex formation.
log P. Due to the negatively charged exterior of the cell,
most anionic GBCAs are usually not internalized by the cells
and considered as extracellular fluid (ECF) agents. Liver-
specific contrast agents such as Gd-EOB-DTPA could be
structurally considered as amphiphilic derivates of highly polar
hydrophilic anionic Gd-DTPA covalently linked to a lipophilic
p-ethoxybenzyl (EOB) moiety. Caravan et al. found that the
liver uptake and the rate of blood clearance are directly
correlated with log P values through the study of the
structure−activity relationship (SAR) of a series of small
molecular Mn(II) complexes.27 In our work, we measured the
estimated log P values of Mn-BnO-TyrEDTA and Mn-EDTA
using the reverse-phase high-performance liquid chromatog-
raphy (HPLC) method. Mn-BnO-TyrEDTA (log P = 0.18)
shows higher lipophilicity than Mn-EDTA (log P = − 2.27)
based on the estimated log P values (Table S1 in the
Supporting Information).
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The ligand (BnO-
TyrEDTA, compound 6) was synthesized via a multiple-step
reaction with Boc-L-tyrosine methyl ester (compound 1) as the
starting material (Scheme 1). In brief, the phenol group of
compound 1 was protected by the benzyl group and then the
methyl ester group was hydrolyzed to give compound 2. The
acid group of compound 2 was activated with isobutyl
chloroformate and treated with ammonia gas, and then the
Boc group was removed to give compound 3. The amide
carbonyl of compound 3 was reduced by the borane−
tetrahydrofuran (THF) complex to afford the key intermediate
diamine (compound 4). Exhaustive N-alkylation of compound
4 with tert-butyl bromoacetate and then acid cleavage of the
tert-butyl ester yielded the ligand compound (compound 6).
To ensure the preparation of the Mn(II) complex of 1:1 Mn/
ligand, the precise ligand concentration was determined by
titration of an aliquot ligand solution with Mn(II).31 Finally,
the corresponding Mn( II) complex (abbreviated as Mn-BnO-
TyrEDTA) was prepared by mixing the ligand and MnCl2·
4H2O at pH 7.4 proportionally based on the titration data
(Figure S24 in the Supporting Information). All of the
intermediate compounds and the final product were
characterized by spectroscopic techniques, such as NMR and
mass spectrometry (Figures S1−S22 in the Supporting
Information).
Cell Viability Assay. The toxicity of Mn-BnO-TyrEDTA
was evaluated by the cell viability assay with commercially
available Gd-EOB-DTPA as a comparison (Figure 1). Raw
Figure 1. Cell cytotoxicity assay via CCK-8 against RAW 264.7 cells
with different concentrations of Mn-BnO-TyrEDTA and Gd-EOB-
DTPA at 37 °C for 24 h. Error bars represent the standard deviation
( SD) on a triplicate analysis.
264.7 cells were incubated with different concentrations of
CAs for 24 h at 37 °C, and the cytotoxicity was estimated using
the Cell Counting Kit-8. At low drug loading concentrations
(<125 μM), it was found that Mn-BnO-TyrEDTA did not
show appreciable cytotoxicity, and cells were significantly
viable with viability over 80% after incubation for 24 h. At high
concentrations (>250 μM), the cytotoxicity of Mn-BnO-
TyrEDTA was higher and the cell viability percentage dropped
Relaxivity. The efficiency of a contrast agent is expressed
by its relaxivity (r1 or r2, in mM−1 s−1), which is defined as the
change of the relaxation rate (R1,2 = 1/T1,2 in units of s−1) of
the water protons upon addition of a normalized concentration
of the contrast agent (in units of mmol/L, mM).32 We
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Figure 2. Spoiled gradient recalled echo T1-weighted images (SPGR-T1WI) of normal Swiss mice recorded at 3.0 T capturing the liver (A) and
kidneys (B) obtained prior, 1, 5, 10, and 20 min after tail vein injection of Mn-BnO-TyrEDTA (a), Gd-EOB-DTPA (b), and Mn-EDTA (c) at a
dose of 0.1 mmol/kg, respectively. BL, bladder. Graphs show the normalized signal-to-noise ratio (nSNR) time course in the liver (C) and kidneys
(D) after injection of CAs. (E) ΔCNR (liver vs muscle) recorded 5 min postinjection demonstrates that the liver enhancement is the greatest for
Mn-BnO-TyrEDTA. Data are reported as mean standard deviation (n = 3; **P < 0.01 and ***P < 0.001).
to 75% (500 μM, 90% for Gd-EOB-DTPA). It is noteworthy
that the cytotoxicity could be negligible, as found from the
rapid clearance of Mn-BnO-TyrEDTA in the pharmacokinetic
study on rats.
TyrEDTA and Gd-EOB-DTPA produced a significantly
greater and more prolonged liver signal enhancement than
Mn-EDTA, especially between 5 and 20 min at the same dose
(Figure 2A). Among the three complexes, Mn-BnO-TyrEDTA
produced the overwhelmingly highest signal-to-noise ratio
(SNR) in the liver within 20 min postinjection (Figure 2C)
and the strongest liver-to-muscle contrast-to-noise ratio
(ΔCNR) at 5 min postinjection (Figure 2E). The relaxivity
in the liver tissue was increased probably due to the increased
microviscosity inside the hepatocyte or the transient
interaction with intracellular proteins.36
All three CAs showed renal excretion revealed by the images
of the kidneys and bladder with the initial strong kidney
enhancement at 1 min and the subsequent enhancement of the
urinary bladder between 5 and 20 min (Figure 2B). The renal
excretion of Mn-EDTA was much slower than those of Mn-
BnO-TyrEDTA and Gd-EOB-DTPA evidenced by an increase
in the kidney signal after 8 min (Figure 2D). The structure of
Mn-EDTA is kinetically less inert than Mn-BnO-TyrEDTA,
which was the backbone-substituted structure of EDTA. Both
slow renal excretion and liver enhancement of Mn-EDTA
could be attributed to the free Mn2+ ion dissociated from Mn-
EDTA. Overall, Mn-BnO-TyrEDTA with an amphiphilic and
In Vivo MR Images of Mice. Gd-EOB-DTPA-enhanced
MRI is now the gold standard for the diagnosis of liver-specific
diseases33 with approximately 50% of the administered dose
being taken up into hepatocytes and excreted via the biliary
system.34,35 To investigate the hepatobiliary imaging capacity
and pharmacokinetics of Mn-BnO-TyrEDTA, we performed
dynamic contrast-enhanced (DCE) MRI studies in normal
mice at 3.0 T using a clinical whole-body scanner with Gd-
EOB-DTPA and Mn-EDTA as comparisons. After the tail was
intravenously administrated with the same dose of contrast
agents, mice were scanned starting from preinjection to 20 min
postinjection using a spoiled gradient recalled echo T1-
weighted image (SPGR-T1WI) sequence.
Figure 2A,B showed the coronal T1-weighted images
recorded at baseline, 1, 5, 10, and 20 min postinjection. The
signal intensity of the liver and kidneys were measured, and the
normalized signal-to-noise ratio (nSNR) was calculated as a
function of time to analyze the contrast enhancement behavior
in vivo of three contrast agents (Figure 2C,D). Both Mn-BnO-
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Figure 3. In vivo spoiled gradient recalled echo T1-weighted imaging (SPGR-T1WI) capturing the liver (A) and kidneys (B) after injection of Mn-
BnO-TyrEDTA (0.1 mmol/kg) in the absence and presence of bromosulfophthalein (BSP). Normalized SNR (nSNR) time courses in the liver (C)
and kidneys (D) that were recorded out to 20 min from the MR images. BL, bladder. Data are reported as mean SD (n = 3).
negatively charged structure shows excellent liver-specific
contrast enhancement and a combination of hepatobiliary
and renal clearance in our imaging study in vivo.
contrast agent. The mechanism of liver-targeting of Mn-BnO-
TyrEDTA can be attributed to the hepatic uptake mediated by
OATPs expressed by hepatocytes based on the above BSP
inhibition imaging study.
Bromosulfophthalein (BSP) Inhibition Images of
Mice. Organic anion transporting polypeptides (OATPs) are
the key transporters for the disposition of various drugs and
endogenous substrates, influencing the pharmacokinetics of
multiple classes of drugs and participating in many clinical
drug−drug interactions.37 The hepatic uptake of Gd-EOB-
DTPA is also mediated by OATPs (OATP1B1 and
OATP1B3) and known to be inhibited by bromosulfoph-
thalein (BSP)38 and rifampicin.39
We performed the DCE-MR study to explore the liver-
targeting mechanism of Mn-BnO-TyrEDTA using BSP as a
competitive inhibitor. BSP was administered intravenously
with a blood concentration of 0.1 mM maintained before bolus
injection of Mn-BnO-TyrEDTA. Compared with the BSP (-)
group, the BSP (+) group had less pronounced liver
enhancement and maintained lower signal intensity (nSNR)
during the experiment (Figure 3). In contrast, the BSP (+)
group showed better renal enhancement and remained stable
until 20 min. The pronounced decrease in hepatic enhance-
ment after the preadministration of BSP can be explained by
the competitive inhibition of the hepatocellular uptake of Mn-
BnO-TyrEDTA. As a surrogate, the decrease of hepatic
excretion results in the enhanced renal clearance of the
Cell Uptake Study. We further performed the cell uptake
study to verify that OATPs (OATP1B1 and OATP1B3) are
master transporters in the hepatic uptake of Mn-BnO-
TyrEDTA based on the results of BSP inhibition imaging.
Herein, we performed the cell uptake study for Mn-BnO-
TyrEDTA in the OATP1B1-transfected HEK-293 cell line
(HEK-OATP1B1). Both real-time quantitative polymerase
chain reaction (PCR) analysis and immunofluorescence
staining indicated high expression of OATP1B1 in HEK-
OATP1B1 cells. It should be noted that low expression of
OATP1B1 in HEK-293 cells could be observed in the
immunofluorescence assay (Figure S27 in the Supporting
Information). As illustrated in Figure 4A, the uptake rate of
Mn-BnO-TyrEDTA by HEK-OATP1B1 cells was significantly
higher than HEK-293 cells (approximately 2-fold higher after
incubation for 30 min with 0.1 mM drug loading). Competitive
inhibition studies using BSP as an inhibitor of OATP1B1 could
inhibit nearly 50% of the hepatic uptake of Mn-BnO-
TyrEDTA, further confirming the importance of OATP1B1
in the hepatic uptake of the contrast agent (Figure 4B).
Biodistribution and Biliary Excretion. The in vivo
biodistribution in nine tissues (heart, brain, liver, spleen, lungs,
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plasma at different time points was quantitatively analyzed by
ICP-MS after single-dose intravenous injection (0.1 mmol/kg;
Figure 6). Table 2 shows the pharmacokinetic parameters
Figure 4. (A) Uptake of Mn-BnO-TyrEDTA (0.1 mM) in HEK cells
transfected with human OATP1B1 (HEK-OATP1B1) and normal
control cells (HEK-293) after incubation for 5−30 min. (B) Uptake
of Mn-BnO-TyrEDTA (100 μM) in HEK-OATP1B1 cells was
measured at 10 min in the absence (control) and presence of BSP
(100 μM). Mean SD is given for n = 3 experiments. ***P < 0.001.
Figure 6. Plasma Mn content−time curve of Mn-BnO-TyrEDTA in
rats (n = 6). Data are reported as mean SD.
kidneys, bone, muscle, and small intestine) of the Mn content
was quantitatively measured by inductively coupled plasma
mass spectrometry (ICP-MS). The bile was collected (up to
about 80 min postinjection) and the Mn content was
quantified to evaluate the biliary excretion of Mn-BnO-
TyrEDTA. The ICP-MS data show that about 44 and 7% of
the accumulated administrated dose was in the liver at 5 min
and 30 min, respectively (Figure 5A). The change of the
accumulated dose in the liver at the two different time points
(5 vs 30 min) was roughly in agreement with the dose
recovered in the bile over this period (5−30 min). During this
period, a rapid washout of the contrast agent in the liver could
be estimated (approximately 37%), which was roughly
consistent with the biliary excretion of the contrast agent
(approximately 25%; Figure 5B). Biliary clearance data indicate
that approximately 50% of the administered dose is excreted
within 80 min.
In addition to the liver, the kidneys showed relatively high
Mn accumulation, resulting from glomerular excretion via the
renal pathway and Mn levels returned to the baseline within 24
h. Therefore, the in vivo biodistribution data strongly suggest
that Mn-BnO-TyrEDTA is excreted by both renal and
hepatobiliary routes. This dual excretion pattern of Mn-BnO-
TyrEDTA is very similar to the excretion pattern of liver-
specific Gd-EOB-DTPA.
Table 2. Pharmacokinetic Parameters of Mn-BnO-
TyrEDTA
a
parameter
[Mn]-time
unit
t
t
1/2α
1/2β
0.83 0.24
9.33 1.92
min
min
AUC0‑t
Vd
CL
5.28 1.50
0.078 0.013
0.019 0.006
mmol/L·min
(mmol/kg)/(mmol/L)
L/kg/min
a
t
1/2α and t1/2β indicate distribution and elimination half-lives,
respectively. AUC0‑t indicates the area under the drug−time curve
from the start of the administration to the last observation time point.
Vd, apparent volume of distribution; CL, clearance rate.
estimated from the Mn-BnO-TyrEDTA plasma clearance data.
Mn-BnO-TyrEDTA exhibits rapid biexponential plasma
clearance, which is the typical behavior of extracellular contrast
agents, with a half-life of distribution (t1/2α) of 0.83 0.24
min and a half-life of elimination (t1/2β) of 9.33 1.92 min.
CONCLUSIONS
■
In summary, we have designed, synthesized, and characterized
a novel Mn(II) complex with a lipophilic group-modified
EDTA structure as a ligand and investigated its diagnostic
ability as a hepatobiliary specific MRI contrast agent. The in
vivo MRI study on mice demonstrated that this Mn(II)
Pharmacokinetic Study. To study the pharmacokinetic
behavior of Mn-BnO-TyrEDTA, the manganese content in
Figure 5. (A) Biodistribution study of Mn-BnO-TyrEDTA (0.1 mmol/kg) in normal mice represented by the Mn percentage in each tissue (n =
6). (B) Biliary excretion of Mn-BnO-TyrEDTA in rats after intravenous (IV) administration of Mn-BnO-TyrEDTA at 0.1 mmol/kg (n = 6). Data
are reported as mean SD.
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After removing the solvent, the product was collected as a white solid.
Yield: 23.5 g (93%). H NMR (400 MHz, CDCl3) δ 7.39 (m, J =
complex, Mn-BnO-TyrEDTA, can produce superb liver
enhancement with a combination of hepatobiliary and renal
clearance pathways, which is similar to that of the clinically
used liver-specific agent Gd-EOB-DTPA. In vivo biodistribu-
tion further confirmed its hepatic specificity, and plasma
pharmacokinetic studies revealed its rapid plasma distribution
and elimination rate, which is typical for extracellular fluid
contrast agents. Finally, BSP inhibition imaging and cellular
uptake studies confirmed that the mechanism of hepatic
targeting of Mn-BnO-TyrEDTA is the hepatic uptake of the
amphiphilic anion contrast agent mediated by OATPs
expressed by functional hepatocytes. Mn-BnO-TyrEDTA,
which showed excellent liver specificity with an OATP-
transport mechanism in our study, may contribute to the
MRI diagnosis of cancer with altered OATP expression (e.g.,
liver, breast, and prostate cancer).40
1
22.2, 13.5, 7.0 Hz, 5H), 7.13 (d, J = 7.5 Hz, 2H), 6.94 (d, J = 8.0 Hz,
2H), 5.09 (s, 2H), 4.59 (d, J = 6.5 Hz, 1H), 3.11 (ddd, J = 43.3, 13.8,
5.7 Hz, 2H), 1.40 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 176.18,
157.93, 155.45, 137.02, 130.48, 128.59, 127.97, 127.50, 114.95, 80.20,
70.01, 60.55, 37.00, 28.32. (m/z) for C21H25NO5: calcd, 394.16 [M +
Na]+, 370.16 [M − H]−; found, 394.1 [M + Na]+; 370.2 [M − H]−.
2-Amino-3-(4-benzyloxy-phenyl)-propionamide (3). (i) Com-
pound 2 (12 g, 32.31 mmol) and N,N-diisopropylethylamine (8.35
g, 64.62 mmol) were dissolved in THF. Isobutyl chloroformate (4.85
g, 35.54 mmol) was slowly added and stirred for 1 h at room
temperature. The reaction was confirmed by TLC. Then, ammonia
was injected for 10 min and stirred overnight at room temperature.
The reaction was confirmed by TLC. After the solvent was removed
by rotary evaporation, washed with water, and extracted with ethyl
acetate, an organic layer was collected, which then was washed with
brine and dried over Na2SO4. After drying, the crude compound was
recrystallized with ethanol, and the pure product was collected as a
1
EXPERIMENTAL SECTION
white solid. Yield: 10.0 g (84%). H NMR (400 MHz, CDCl3) δ
■
7.57−7.31 (m, 5H), 7.18 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 8.0 Hz,
2H), 5.73 (s, 1H), 5.31 (s, 1H), 5.06 (s, 2H), 4.33 (s, 1H), 3.04 (dd, J
= 17.7, 6.8 Hz, 2H), 1.44 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
174.11, 174.11, 157.88, 157.88, 157.59, 147.27, 136.95, 130.40,
130.40, 128.60, 128.60, 128.00, 128.00, 127.49, 127.49, 115.06,
115.06, 77.25, 69.99, 55.36, 37.56, 28.21. (m/z) for C21H26N2O4:
calcd, 393.18 [M + Na]+; found, 393.2 [M + Na]+.
General Remarks. All reagents were purchased from Aladdin
Industrial Corporation (Shanghai, China), such as Boc-tyr-OMe/
THF/TFA/N, N-diisopropylethylamine/isobutyl chloroformate/tert-
butyl bromoacetate, etc., MnCl2·4H2O was purchased from Sigma-
Aldrich (St. Louis, MO), and anhydrous acetonitrile/benzyl
bromoacetate was purchased from Alfa-Aesar (Ward Hill, MA).
Other common reagents are domestic analytical pure (methanol,
ethanol, petroleum ether, acetonitrile, dichloromethane, concentrated
hydrochloric acid, dichloro-sulfoxide, sodium bicarbonate, sodium
chloride, anhydrous sodium sulfate, etc.). All reagents were purchased
commercially and used without further purification. The purity of all
compounds by biological evaluation was ≥95%. Purity was evaluated
by 1H NMR and the purity of the final complex compound was
evaluated by HPLC using the method described in HPLC for
estimating log P. Ultrapure water (UP) was used for all experimental
procedures. Mass (electrospray ionization mass spectrometry (ESI-
MS)) spectra and nuclear magnetic resonance (1H NMR 400 MHz,
Bruker Advance) hydrogen spectra were commonly used to identify
the structures. Tetramethyl silane (TMS) was used as an interior
standard for chemical shifts. No unexpected or unusual high safety
hazards were encountered during experimental procedures.
(ii) [2-(4-Benzyloxy-phenyl)-1-carbamoyl-ethyl]-carbamic acid
tert-butyl ester (20 g, 53.99 mmol) was dissolved in dichloromethane
(200 mL), and trifluoroacetic acid (20 mL) was carefully added and
stirred for 12 h at room temperature. The reaction was confirmed by
TLC. The solvent was removed and a small amount of ethyl acetate
was added to dissolve the crude product, and then sedimentation with
hexane was performed. After precipitation, the substance was dried
1
and collected as a white solid. Yield: 10.0 g (66%). H NMR (400
MHz, DMSO) δ 7.39 (m, 5H), 7.17 (d, J = 8.0 Hz, 2H), 6.99 (d, J =
8.1 Hz, 2H), 5.08 (s, 2H), 3.87 (t, J = 6.4 Hz, 1H), 2.95 (dddd, J =
21.7, 14.0, 6.7 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 170.28,
158.00, 137.54, 131.06, 128.92, 128.34, 128.17, 127.46, 115.24, 69.58;
54.01; 36.46. (m/z) for C16H18N2O2: calcd, 371.15 [M + H]+; found,
371.1 [M + H]+.
3-(4-Benzyloxy-phenyl)-propane-1,2-diamine (4). Compound 3
(10 g, 36.99 mmol) was dissolved in THF, followed by the addition of
the boron hydrogen reagent (222 mL, 222 mmol); then the mixture
was heated to reflux and reacted for 24 h. The reaction was confirmed
by TLC. Twenty milliliters of methanol was added slowly under an ice
bath, and then the mixture was heated to reflux for 30 min. After the
solvent was removed, the pH of the solution was adjusted to 2 by
careful addition of concentrated hydrochloric acid. Then, after
washing with ethyl ether and extracting with water, a water layer
was collected and NaOH was added to adjust the pH to 8−9. After
extracting with DCM, an organic layer was collected, washed with
brine, and dried over Na2SO4. After drying, the pure product was
All experiments were executed in accordance with the Guidelines
for Care and Use of Laboratory Animals and were approved by the
Ethics Committee of North Sichuan Medical College. All animals
were fasted 12 h prior to drug administration and had access to water
ad libitum and were fed 4 h postdose.
Synthesis and Characterization. 3-(4-Benzyloxy-phenyl)-2-
tert-butoxycarbonylamino-propionic Acid (2). (i) Compound 1
(20 g, 67.72 mmol), K2CO3 (20.59 g, 148.59 mmol), and KI (1.12 g,
6.77 mmol) were dissolved in ACN (120 mL), and Bn−Cl (9.43 g,
74.49 mmol) was slowly added and the mixture was stirred for 6 h in a
90 °C oil bath. The reaction was monitored and confirmed by thin-
layer chromatography (TLC). Inorganic salts were removed using a
filter and the filtrate was rotated to evaporate the solvent. The crude
compound was recrystallized with ethanol, and the pure product was
1
collected as a white solid. Yield: 6.0 g (63%). H NMR (400 MHz,
CDCl3) δ 7.59−7.28 (m, 5H), 7.11 (d, 2H), 6.94 (d, 2H), 5.04 (s,
2H), 2.92 (s, 1H), 2.87−2.61 (m, 2H), 2.49 (dd, J = 22.2, 10.4 Hz,
2H). 13C NMR (100 MHz, DMSO) δ 157.37, 137.03, 131.41, 130.29,
128.62, 128.01, 127.53, 114.88, 70.01, 55.13, 47.97, 41.34. (m/z) for
C16H20N2O: calcd, 257.17 [M + H]+; found, 257.2 [M + H]+.
{[3-(4-Benzyloxy-phenyl)-2-(bis-tert-butoxycarbonylmethyl-
amino)-propyl]-tert-butoxycarbonylmethyl-amino}-acetic Acid
tert-Butyl Ester (5). Compound 4 (5 g, 19.5 mmol), N,N-
diisopropylethylamine (15.13 g, 117.03 mmol) and KI (0.32 g, 1.95
mmol) were dissolved in ACN (60 mL). Then, tert-butyl
bromoacetate (22.82 g,117.03 mmol) was added and stirred for 4 h
in an 60 °C oil bath. The reaction was confirmed by TLC. Inorganic
salts were filtered using a filter, and after the filtrate was washed with
water and extracted with ethyl acetate, an organic layer was collected,
washed with brine, and dried over Na2SO4. After evaporation, the
crude product was collected and purified by column chromatography
1
collected as a white solid. Yield: 25.0 g (96%). H NMR (400 MHz,
DMSO) δ 7.46−7.26 (m, 5H), 7.14 (d, J = 8.0 Hz, 2H), 6.92 (d, J =
8.0 Hz, 2H), 5.06 (s, 2H), 4.19−3.98 (m, 1H), 3.59 (s, 3H), 2.99−
2.68 (m,2H), 1.29 (s, 9H). 13C NMR (100 MHz, DMSO) δ 173.18,
157.47, 155.90, 137.61, 130.57, 130.08, 128.88, 128.24, 128.09,
114.96, 78.78, 69.55, 55.92, 52.22, 36.05, 28.56. (m/z) for
+
C22H27NO5: calcd, 408.18 [M + Na] , 384.18 [M − H]−; found,
408.1 [M + Na]+. 384.2 [M − H]−.
(ii) 3-(4-Benzyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic
acid (25 g, 67.72 mmol) and NaOH (6.75 g, 135.44 mmol) was
dissolved in THF (150 mL) and a small amount of water (50 mL).
The solution was stirred for 1 h at 50 °C. The reaction was confirmed
by TLC. The pH of the solution was adjusted to 2 by careful addition
of HCl (37%, w/w). After extracting with ethyl acetate, an organic
layer was collected and washed with brine and dried over Na2SO4.
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(silica, hexanes/EtOAc, 6:1). Yield: 9.0 g (65%). 1H NMR (400
MHz, CDCl3) δ 7.58−7.31 (m, 5H), 7.16 (d, J = 8.1 Hz, 2H), 6.88
(d, J = 8.1 Hz, 2H), 5.03 (s, 2H), 3.49 (d, J = 25.3 Hz, 8H), 3.10 (t, J
= 5.5 Hz, 1H), 2.89 (m, 2H), 2.60 (m, 2H), 1.55−1.42 (d, 36H). 13C
NMR (100 MHz, CDCl3) δ 171.44, 170.98, 157.01, 137.22, 132.69,
130.22, 128.58, 127.92, 127.53, 114.58, 77.25, 69.99, 56.25, 55.75,
54.98, 53.55, 35.94, 28.15. (m/z) for C40H60N2O9: calcd,713.44 [M +
H]+; 735.42 [M + Na]+; found, 713.5 [M + H]+; 735.6 [M + Na]+.
{[3-(4-Benzyloxy-phenyl)-2-(bis-carboxymethyl-amino)-propyl]-
carboxymethyl-amino}-acetic Acid (6). Compound 5 (5 g, 7.02
mmol) was dissolved in dichloromethane (80 mL), and trifluoroacetic
acid (16 mL) was carefully added and stirred for 12 h at room
Wuhan, China) and then incubated at 37°C for 1 h; the absorbance of
the samples was measured at 450 nm using a microplate reader
(Thermo Scientific). Cell viability was calculated according to the
manufacturer’s protocol.
MR Image. Swiss mice (20 5 g; Laboratory Animal Center of
NSMC, Sichuan, China) were imaged with a 3.0 T MR scanner
(Discovery MR750, GE Medical System, Milwaukee, WI) with a
custom-made mice coil. Anesthesia was maintained by inhaling
isoflurane (RWD Life Science, Shenzhen, China) through a face mask
(Anesthetic Conc.: 0.8−1.5%; 20−30 mL O2/kg). After baseline
imaging, contrast agents (0.1 mmol/kg Mn or Gd) were administered,
separately, and imaging was repeated in a dynamic fashion.
Bromosulfophthalein (BSP) as a competitive inhibitor was injected
30, 20, and 15 min before the injection of Mn. The mouse was imaged
immediately after the injection of the contrast agents using gradient
recalled echo T1-weighted imaging sequences for 20 min. MR images
were obtained in the coronal plane, and MRI imaging parameters
were TE = 2.7 ms, TR = 9.5 ms, FA = 30°, FOV = 80 mm × 64 mm,
matrix size = 320 × 192, 44 slices, slice thickness = 1.0 mm, NEX =
1.0, and bandwidth = 62.50 kHz. Images were analyzed using a
RadiAnt DICOM viewer by drawing regions of interest (ROIs) on the
liver and kidneys. The normalized signal-to-noise ratio (nSNR) was
used with nSNR = SNRpost/SNRpre and SNR = SI/SDair, where SI is
the signal intensity and SDair is the standard deviation of the signal
intensity in the ROI outside the animal. The contrast-to-noise ratio
(CNR) values were calculated as CNR = (SIliver − SImuscle)/SDair, and
ΔCNR values were calculated by subtracting the preinjection CNR
map from the CNR map after contrast agent administration.
Real-Time Quantitative PCR Analysis and Immunofluor-
escence Staining. Human embryonic kidney (HEK-293) cells were
provided by the Chinese Academy of Sciences Cell Bank (Shanghai,
China). Human embryonic kidney cells stably overexpressing
OATP1B1 (HEK-OATP1B1) were purchased from Genechem Co.,
Ltd (Shanghai, China). The cells were tested for the OATP1B1
receptor by real-time fluorescent quantitative PCR and immuno-
fluorescence. The RNA was extracted from HEK-293 cells and HEK-
OATP1B1 cells using the TRIzol method, and the mRNA expression
levels of OATP1B1 were detected by SYBR green gene expression
assays (primer OATP1B1: forward primer, 5′-AACTCCTACT-
GATTCTCGATGGG-3′ and reverse primer, 5′-GTTTCCAGCA-
CATGCAAAGAC-3′. Primer GAPDH: forward primer, 5′-
TGACTTCAACAGCGACACC-CA-3′ and reverse primer, 5′-
CACCCTGTTGCTGTAGCCA-AA-3′). Immunofluorescence im-
ages were recorded using an Olympus laser scanning confocal
microscope. The cells were fixed with PFA, permeabilized in 0.1%
Triton X-100, and then blocked in 5% BSA for 1 h at 25 °C. The
samples were incubated with the SLCO1B1 antibody (Affinity
Biosciences) at 4 °C overnight. The expression of the transporter
protein was assessed using the Alexa Fluor 594 conjugated goat anti-
rabbit antibody (Affinity Biosciences).
1
temperature. The reaction was confirmed by H NMR. The solvent
was removed and an appropriate amount of methanol was added. The
solvent was swirl dried and repeated three times. After drying, the
pure product was collected as a primrose yellow solid. Yield: 3.0 g
(87%). 1H NMR (400 MHz, D2O) δ 7.54−7.22 (m, 5H), 7.08 (d, J =
7.8 Hz, 2H), 6.98−6.85 (d, 2H), 5.05 (s, 2H), 3.28−2.77 (m, 8H),
2.74−2.50 (m, 3H), 2.37 (t, J = 12.6 Hz, 1H), 2.11 (t, J = 12.2 Hz,
2H). 13C NMR (100 MHz, D2O) δ 172.91, 170.91, 170.29, 169.89,
157.06, 136.68, 130.05, 129.82, 128.04, 115.18, 114.75, 69.51, 60.5,
57.01, 56.04, 48.71. (m/z) for C24H28N2O9: 489.19 [M + H]+; 487.17
[M − H]−; found, 489.2 [M + H]+. 487.2 [M − H]−.
Mn-BnO-TyrEDTA. The ligand (compound 6) (0.620 g, 1.19
mmol) was dissolved in ultrapure water, and the pH of the solution
was adjusted to 7.4 by careful addition of NaOH (1.0 M). Based on
the NMR titration results, MnCl2·4H2O (0.217 g, 1.10 mmol) was
slowly added and the resulting solution was stirred. Finally, the pH of
the solution was readjusted to 7.4. After lyophilization, the product
was collected as a solid. ESI-MS (m/z) for C24H24MnN2O9: calcd,
608.6 [M + 3Na]+; 562.08 [M + Na]−; found, 608.1 [M + 3Na]+;
562.18 [M + Na]−.
Relaxivity. Relaxivities were measured with a 0.5 T NMR contrast
agent relaxation rate analyzer (PQ001, Shanghai Newmag Electronic
Technology Co., Ltd.). Longitudinal (T1) relaxation was acquired via
an IR (inversion recovery) sequence using 10 inversion times of
duration ranging between 0.05 × T1 and 5 × T1. For transverse (T2)
relaxation, the CPMG (Carr−Purcell−Meiboon−Gill) pulse sequence
was used. The nonlinear least-squares fit of the average pixel was used
to estimate T1 and T2 relaxation times. Then, r1 and r2 relaxivities
were computed by the linear fit of T1 and T2 relaxation times for each
concentration (pick five concentrations by half dilution, such as
0.03125, 0.0625, 0.125, 0.25, and 0.5 mM).
Estimates of 1-Octanol: the Water Partition Coefficient
(Estimated log P). Log P values were estimated using high-
performance liquid chromatography (HPLC 1640, Agilent) as
follows: a Kromasil C4 column (150 mm × 4.6 mm 100 Å); eluent
A, ultrapure water; eluent B, 100% MeCN; gradient, 5% B from 0 to 1
min, 5−50% B from 1 to 10 min, 50−95% B from 10 to 11 min, 95%
B from 11 to 12 min, 95 to 5% B from 12 to 13 min, and 5% B from
13 to 15 min; and flow rate at 0.7 mL/min (eluent ultrasonography 1
h in advance). A linear calibration curve correlating log P of four
standards (DMSO, nitrobenzene, phenol, and pyridine) to HPLC
retention time (tR) under the above method conditions was
generated. The estimated log P values of complexes Mn-EDTA and
Mn-BnO-TyrEDTA were estimated from their corresponding tR using
this calibration curve.
Cell Uptake Assay. HEK-OATP1B1 cells and HEK-293 cells
were seeded in 12-well plates at 10 000 cells per well. After 24 h of
incubation, the cells were incubated with 0.1 mM Mn-BnO-TyrEDTA
at 37 °C for 0, 5, 10, 20, and 30 min, washed with ice-cold phosphate-
buffered saline, and then disintegrated using 1% HNO3. In inhibition
assays, the uptake of Mn-BnO-TyrEDTA (0.1 mM) was measured at
10 min in the absence (control) and presence of 0.1 mM BSP.
Prewarmed (37 °C) uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM
K2HPO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5
mM HEPES, pH 7.3−7.4) was used as a cell culture medium.41 The
concentration of Mn was quantified in cell lysates by inductively
coupled plasma mass spectrometry (ICP-MS), and the protein
concentration of each sample was determined using the BCA protein
concentration determination kit (Boster Biological Technology,
Wuhan, China). The Michaelis−Menten constant (Km) and the
maximum transport velocity (Vmax) were calculated using the
following equation: y = Vmax × x/(Km + x).12
Cytotoxicity Assay. Mn-BnO-TyrEDTA and Gd-EOB-DTPA
(Primovist) were tested for cytotoxicity on mouse macrophages
(RAW 264.7) cells. RAW 264.7 cells were provided by the Chinese
Academy of Sciences Cell Bank (Shanghai, China). The cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high
glucose (4.5 g/L glucose) containing 10% (v/v) fetal bovine serum
(Biological Industries, Israel) and 1% (v/v) penicillin−streptomycin
(Hyclone, Logan, UT) at 37 °C, 5% CO2, and saturated humidity.
RAW 264.7 cells were plated in 96-well plates at 1 × 104 cells/well in
100 μL of complete media. Following incubation for 24 h, the cells
were added with varying concentrations (31.25, 62.5, 125, 250, and
500 μM) of contrast agents. Each well was added with a 10 μL Cell
Counting Kit-8 (CCK-8) solution (Boster Biological Technology,
Biodistribution and Biliary Excretion Studies. Swiss mice
were used in the biodistribution study. After intravenous injection of
Mn-BnO-TyrEDTA (0.1 mmol/kg) to normal male and female mice
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(n = 6; 20
5 g) through tail veins, the mice were euthanized
Phone: (+)86-817-2242635; Email: zhangxm@
nsmc.edu.cn
postinjection at 4 different time points (5 min, 30 min, 1 h, and 24 h).
Various tissues (brain, heart, liver, spleen, lung, kidneys, bone, muscle,
and small intestine) were weighed and then digested in concentrated
nitric acid for 48 h. Samples for analysis were prepared by diluting
them in an aqueous solution of 0.1% Triton X and 5% HNO3. The
manganese concentration of the sample was quantitatively analyzed
by ICP-MS. The total amount of bile excretion was calculated based
on the test results. An arterial cannula (i.d., 0.6 mm; Linhwa, Suzhou,
China) was introduced into the ductus choledochal of six SD rats
(180−220 g) while the animals were kept under anesthesia. Rats were
anesthetized with isoflurane and administered 0.1 mmol/kg Mn-BnO-
TyrEDTA injection into the tail vein. Bile was collected quantitatively
before and 5, 10, 15, 20, 30, 40, 50, 60, 70, and 80 min after contrast
medium administration. The concentration of the contrast medium in
the bile was determined by measuring [Mn] using ICP-MS.
Pharmacokinetic Study. Sprague-Dawley (SD) rats (200 20 g;
Laboratory Animal Center of North Sichuan Medical College
(NSMC), Sichuan, China) were used in the pharmacokinetic study.
The rats (n = 6) were anesthetized by isoflurane (Anesthetic Conc.:
2−2.5%; 25−35 mL O2/kg). The tail vein cannula was used for
administration at a dose of 0.1 mmol/kg, and the carotid artery
catheter was used for blood sampling. Approximately 100 μL of blood
was collected at 0 (predose to serve as a blank), 0.33, 0.67, 1, 1.5, 2,
2.5, 3, 4, 5, 7, 10, 15, 20, 25, and 30 min postdose from the carotid
artery via a catheter after IV administration of Mn-BnO-TyrEDTA.
The samples were diluted with 0.1% Triton X-100 (Inno-chem
Science & Technology, Beijing, China) in 5% nitric acid.27
Manganese samples were quantified by elemental analysis by ICP-
MS.42 Quantitative analysis was performed using a linear calibration
curve ranging from 0.1 to 200 ppb Mn calibration standard
manufactured under ISO 9001 quality assurance system by
PerkinElmer. The blood pharmacokinetic data were fit to the
following biexponential model C(t) = A exp(−αt) + B exp(−βt),
where C(t) is the manganese metal ion concentration at time t, α and
β are the distribution and elimination rates, respectively, and A and B
are the distribution and elimination coefficients, respectively.25 A two-
compartment pharmacokinetic model was used to analyze the data
and calculate the pharmacokinetic parameters with PKSolver software
(China Pharmaceutical University).43 The compartment model fitting
evaluation standard is based on the akaike information criterion
(AIC) and R-squared (R2).
Jiang Zhu − Sichuan Key Laboratory of Medical Imaging,
School of Pharmacy, and Nanchong Key laboratory of MRI
Contrast Agent, North Sichuan Medical College, Nanchong
637000, China; Department of Oncology, Affiliated Hospital
of North Sichuan Medical College, Nanchong 637000,
China; orcid.org/0000-0001-7437-2956; Phone: (+)86-
181-81085956; Email: zhujiang@nsmc.edu.cn
Authors
Keyu Chen − Sichuan Key Laboratory of Medical Imaging
and School of Pharmacy, North Sichuan Medical College,
Nanchong 637000, China
Pan Li − Department of Radiotherapy, Sichuan Cancer
Hospital & Institute, Chengdu 610041, China
Chunrong Zhu − Sichuan Key Laboratory of Medical Imaging
and College of Preclinical Medicine, North Sichuan Medical
College, Nanchong 637000, China
Zhiyang Xia − Sichuan Key Laboratory of Medical Imaging
and College of Preclinical Medicine, North Sichuan Medical
College, Nanchong 637000, China
Qian Xia − Sichuan Key Laboratory of Medical Imaging and
Nanchong Key laboratory of MRI Contrast Agent, North
Sichuan Medical College, Nanchong 637000, China
Lei Zhong − Sichuan Key Laboratory of Medical Imaging and
Nanchong Key laboratory of MRI Contrast Agent, North
Sichuan Medical College, Nanchong 637000, China
Bin Xiao − Sichuan Key Laboratory of Medical Imaging,
North Sichuan Medical College, Nanchong 637000, China
Tao Cheng − Sichuan Key Laboratory of Medical Imaging,
North Sichuan Medical College, Nanchong 637000, China
Changqiang Wu − Sichuan Key Laboratory of Medical
Imaging and Nanchong Key laboratory of MRI Contrast
Agent, North Sichuan Medical College, Nanchong 637000,
China; orcid.org/0000-0002-6363-2721
Chengyi Shen − College of Preclinical Medicine and Nanchong
Key laboratory of MRI Contrast Agent, North Sichuan
Medical College, Nanchong 637000, China
Data Analysis. Data analysis was performed using Graphpad
Prism 8 software. Statistical analysis was performed using unpaired
Student’s t-test. P values less than 0.05 were considered to indicate
statistical significance.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00393
Author Contributions
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00393.
■
K.C. and P.L. contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript.
sı
Notes
ESI-MS and NMR spectrum (1H and 13C NMR spectra)
of compounds 2−6 and Mn-BnO-TyrEDTA; HPLC
trace of Mn-BnO-TyrEDTA; NMR titration for
determining the precise Mn/L ratio; HPLC trace for
determining the estimated Log P; and liquid chromatog-
raphy-mass spectrometry (LC-MS) traces analysis of
urine and feces (PDF)
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge financial support from the National
Nature Science Foundation of China (81671675, 21172025,
and 81601490) and Science and Technology Project of
Municipal School Strategic Cooperation, Nanchong
(NSMC20170100, 18SXHZ0091, 18SXHZ0379, and
NCKL201704).
Compounds 1−6 and Mn-BnO-TyrEDTA as molecular
formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
Xiaoming Zhang − Sichuan Key Laboratory of Medical
Imaging, North Sichuan Medical College, Nanchong 637000,
China; Department of Radiology, Affiliated Hospital of North
Sichuan Medical College, Nanchong 637000, China;
■
ABBREVIATIONS USED
■
BSP, bromosulfophthalein; DCE, dynamic contrast-enhanced;
EDTA, ethylenediaminetetraacetic acid; GBCA, gadolinium-
based MRI contrast agent; ICP-MS, inductively coupled
plasma mass spectrometry; MRI, magnetic resonance imaging;
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fibrosis: risk factors and incidence estimation. Radiology 2007, 243,
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Steever, A.; Bluemke, D. A. MR imaging in patients at risk for
developing nephrogenic systemic fibrosis: protocols, practices, and
imaging techniques to maximize patient safety. Radiographics 2009,
29, 9−22.
OATPs, organic anion transporting peptides; SPGR-T1WI,
spoiled gradient recalled echo T1-weighted images
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