Synthesis, conjugation and relaxation studies of gadolinium(iii)-4- benzothiazol-2-yl-phenylamine as a potential brain specific MR contrast agent

Article abstract of DOI:10.1039/c2dt32391e

Magnetic resonance (MR) imaging is widely used in clinical research to map the structural and functional organization of the brain. We have designed and synthesized a Gd-based specific MR contrast agent that binds to regions in the brain. The presented co

Full text of DOI:10.1039/c2dt32391e

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Synthesis, conjugation and relaxation studies of
gadolinium(III)-4-benzothiazol-2-yl-phenylamine as a
Cite this: Dalton Trans., 2013, 42, 4994
potential brain specific MR contrast agent
Nisha Saini,a,b Raunak Varshney,a Anjani K. Tiwari,*a Ankur Kaul,a Michele Allard,c
M. P. S. Isharb and Anil K. Mishra*a
Magnetic resonance (MR) imaging is widely used in clinical research to map the structural and functional
organization of the brain. We have designed and synthesized a Gd-based specific MR contrast agent that
binds to regions in the brain. The presented compound {4-[(4-benzothiazol-2-yl-phenylcarbamoyl)-
methyl]-7,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl} acetic acid (DO3A-BT) was synthesized
by conjugating the chloroacetylated product of 4-benzothiazol-2-yl-phenylamine with a trisubstituted
cyclen. The lanthanide complex (Ln–DO3A-BT) was evaluated in vitro for both MR (Gd–DO3A-BT) and
optical (Eu–DO3A-BT) imaging applications. The complex Gd–DO3A-BT displays a relaxivity of r1
=
4.18 mM−1 s−1 at 4.7 T which is 1.2 times greater than Dotarem and significantly higher than the brain
specific MR contrast agent Luxol Fast Blue (LFB). The protonation constant of the ligand (pKa1 = 9.91,
pKa2 = 8.22, pKa3 = 5.01) and the stability constant of the complex formed between Gd(III), Eu(III) and
Ca(II) and ligand DO3A-BT (log βGdL = 18.4, log βEuL = 18.3, log βZn2L = 7.1, log βCa2L = 6.3) were recorded
by potentiometric titration. The constants reflect the high stability of the ligand with lanthanides com-
pared with endogenous metal ions. The transmetalation stability of Gd–DO3A-BT toward Zn proved to
be excellent with a rate constant of 3.07 × 10−5 s−1 which is in line with other tetraazatetraacetic acid
(DOTA)–monoamide complexes. The hydration number (q) was found to be 0.92, and is calculated from
the difference in the luminescence lifetime of Eu–DO3A-BT in H2O and D2O solutions to determine the
coordination state of this complex. The in vivo biodistribution of 99mTc–DO3A-BT in BALB/c mice showed
a brain uptake of 1.2% ID g−1 at 2 min post injection when injected with mannitol which disrupts the
blood–brain-barrier (BBB) due to osmotic shock. In vitro binding on the brain homogenate revealed a
high uptake by the neuronal/glial cells for in vivo applications.
Received 1st June 2012,
Accepted 7th January 2013
DOI: 10.1039/c2dt32391e
www.rsc.org/dalton
The advantage of using MRI for neuroscience is to perform
imaging in conjunction with contrast agents. Polyaminopoly-
Introduction
Magnetic resonance imaging (MRI) is emerging as a powerful carboxylic acid complexes of lanthanides have widespread
tool for studying the microstructure of many tissues, including application as contrast agents in MRI imaging. Thus the
the architecture of the human brain. There is an important coordination chemistry of lanthanide complexes of polyamino-
need for increased MR research for contrast agents with carboxylates has become important in biomedical science for
improved soft-tissue contrast which will enhance the capacity MR contrast agents3–6 and as luminescent probes.7,8 The MR
of MRI to provide functional information of different images are achieved by changing the magnetic relaxation
regions.1,2 The longitudinal MRI studies can provide a non- times of the protons in tissue-contained water. These images
invasive tool to understand brain functions, as well as are enhanced by the use of contrast agents as they increase the
measure the effects of drug and other therapeutic interven- longitudinal and transverse relaxation rate of the water
tions with a high degree of specificity.
protons which contribute to the contrast of the image.9 Gadoli-
nium(III) ions are an ideal choice due to their high spin state
(s = 7/2), high magnetic moment, slow electronic relaxation rate
and labile hydration sphere for water exchange.10–16 According
to the Soloman–Bloembergen–Morgan equation theory high
relaxivity can be obtained in the presence of a greater number
of inner sphere water molecules q; an optimally short water
aDivision of Cyclotron and Radio-Pharmaceutical Sciences, Institute of Nuclear
Medicine and Allied Sciences, Brig S. K. Majumdar Road, Delhi-54, India.
E-mail: akmishra63@gmail.com, anjani7797@rediffmail.com;
Tel: +91-11-2390511709968314130
bDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-005, India
cUniversite de Bordeaux, UMR-CNRS 5287-EPHE, Bordeaux, France
residence time τm; and
a slow tumbling rate τr while
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maintaining sufficient thermodynamic stability.17–19 The DO3A-BT has lower brain permeability than [125I]IMPY and
complexation with numerous ions such as Ca2+, Zn2+, Cu2+ there is no exposure to ionizing radiation which makes our
and precipitation of Gd3+ salts may contribute to the in vivo compound a more promising candidate. Hence the encour-
dissociation of the Gd3+ complexes. Gd forms thermodynami- aging in vitro and in vivo studies of DO3A-BT supports the
cally and kinetically stable complexes with polyaminopolycarbox- choice of this derivative for further evaluation in brain
ylate-type ligands such as the tetraazatetraacetic acid macrocycle research. Thus with the aim to synthesize a bifunctional probe
(DOTA) and diethylenetetraamine pentaacetic acid (DTPA).20,21
which can be applied in brain targeted magnetic resonance
The action of MRI contrast agents requires one or more water imaging (MRI) and optical imaging. To achieve this, we conju-
molecules to be coordinated to the paramagnetic metal ion, and gated an arylbenzothiazole moiety with a macrocyclic ring.
these are important in determining the molar relaxivity of the The compounds were characterized by NMR and ESI-MS
agent. Thus the structural dynamics of MR contrast agents are spectroscopy and their relaxivity, luminescence lifetime and
very important for clinical aspects and can be measured by the biological applications were evaluated.
luminescence decay rate and the high resolution absorption
spectra of the Eu3+ complex.22–24 More precise determination of
Results and discussion
Design and synthesis of the contrast agent
the complex hydration state (q) gives unique information on the
local symmetry and specification of 7F0 → 5D0 band.
The selected benzothiazole moiety is present in various 4-Benzothiazol-2-yl-phenylamine
3 was synthesized from
dyes such as primuline and thioflavin which possess the 2-amino-benzenethiol and 4-aminobenzoic acid using polyphos-
potential to fluorescently stain diseased brain tissues.25,26 phoric acid. It was chloroacetylated to obtain N-(4-benzothi-
Recently, an attempt has been made to use Luxol Fast Blue azol-2-yl-phenyl)-2-chloroacetamide 4. The chloroacetylated
(LFB), a histological stain for brain tissue with a paramagnetic product was conjugated to a trisubstituted cyclen to yield the
copper core, as a MR probe. LFB shows a low relaxivity (r1
=
desired compound i.e. 10-[N-(4-benzothiazol-2-yl-phenylcarba-
0.09 mM−1 s−1 at 4.7 T) and a modest solubility27 but the moyl)-methyl]-1,4,7-tri(carbobutoxymethane)-1,4,7,10-tetraaza-
longitudinal relaxivity of DO3A-BT is ∼34 times more than that cyclododecane 6. Compound 6 was deprotected to remove the
of LFB. On the other hand, biodistribution studies of the thio- tert-butyl group using trifluoroacetic acid at 0 °C, and, on
flavin analogue i.e. [125I]IMPY28 in normal mice exhibited a further complexation with GdCl3 and EuCl3, gave Gd–DO3A-BT
brain uptake of 2.2% ID g−1 with a fast washout. However, Gd– 8 and Eu–DO3A-BT 9 respectively (Scheme 1). The above
Scheme 1 Synthesis of Ln–DO3A-BT (Gd, Eu). Reagents and conditions: (a) polyphosphoric acid, 120 °C; (b) Cl–CH2–CO–Cl, triethylamine (TEA), 0 °C–rt; (c) K2CO3,
70 °C, 12 h; (d) trifluoroacetic acid (TFA); (e) LnCl3 (Ln = Gd, Eu), 70 °C, 14 h.
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complexes were lyophilized and white solids were obtained in
90% and 93% yields, respectively. After the completion of both
reactions, the reaction mixture was passed through chelex-100
at room temperature to trap the free Ln(III), and the absence of
free Gd(III) was checked using a xylenol orange indicator.29
Gd–DO3A-BT and Eu–DO3A-BT were characterized by mass
spectrometry and the isotopic pattern of seven peaks and five
peaks confirms the complexation of the ligand DO3A-BT with
Gd and Eu, respectively.
Fig. 1 (a) Longitudinal and (b) transverse relaxivity of Gd–DO3A-BTA.
Relaxometric studies
The presence of paramagnetic Gd(III) in the complexes resulted
in an enhancement of the longitudinal relaxation rate of water
protons (1/T1). To express the proficiency of the Gd(III) com-
plexes, relaxivity (r1, mM−1 s−1) values are used; relaxivity is the
increase in the longitudinal proton relaxation rate and is
measured using the Gd3+ complex at 1 mM concentration.
Longitudinal and transverse 1H spin relaxation rates have been
measured for Gd–DO3A-BT in aqueous solution (pH = 7) at
6 different concentrations (0.625, 1.25, 2.5, 5, 10, and 20 mM).
MR studies in the concentration range of 0.625–20 mM of Fig. 2 Experimental UV-visible spectra of Eu(III)–DO3A-BT (25 °C, pH 7).
Gd–DO3A-BT exhibited a contrast enhancement in T1-weighted
images with the effect being more pronounced at lower con-
provide an efficient non-radiative pathway for the de-excitation
centrations. Enhanced contrast was obtained at concentrations
of the emissive state of certain lanthanide ions. On the other
as low as 0.625 mM whereas a darker image intensity was
hand, OD oscillators of coordinated D2O molecules are quite
obtained using higher concentrations of the complex. At low
inefficient in accomplishing this de-excitation. Thus, the
concentrations, an increase in the contrast results in an
different quenching frequencies of the O–H and O–D
increase in signal intensity due to the effect on T1 until the
bonds give an estimation of the number of coordinated water
optimal concentration is reached. A further increase in concen-
molecules by using the equation developed by Supkowski
tration reduces the signal because of the effect on T2. Longi-
and Horrocks.30 The values for the luminescence lifetimes
tudinal (T1) and transverse (T2) relaxation times of solvent-
of Eu–DO3A-BT at pH 7.4 and 25 °C for the 7F0
→
5D0
water protons as
(0.625–20 mM) were measured to investigate their relaxation
behaviour.
a function of DO3A-BT concentration
transition in H2O and D2O solutions are 638 μs and
2430 μs and the hydration number (q) is calculated to be
0.92, which clearly indicates a monoaqua Eu-coordinated
system.
The longitudinal relaxivity r1, and transverse relaxivity r2,
was determined to be 4.18 mM−1 s−1 and 4.54 mM−1 s−1
respectively at 4.7 T and 37 °C. A plot of relaxivities as a func-
tion of the concentration of this complex is shown in Fig. 1
and a linear enhancement in the relaxivity with the concen-
tration of the Gd complex is shown. The measured relaxivities
arise from the exchange between the coordinated water mole-
cules and the surrounding water molecules in solution. The
DO3A-BT longitudinal relaxivity is approximately 35 times
greater than that of LFB (r1 = 0.09 mM−1 s−1 at 4.7 T and
25 °C), which is an MR probe for differentiating myelinated
regions.27 It makes our compound superior to LFB as an MR
contrast agent for imaging brain regions.
An interesting aspect of Eu(III) luminescence involves the
7F0
→
5D0 excitation spectrum. Since the excited state and
ground state are both nondegenerate and cannot be split
further by the ligand field, each peak in the spectrum must
correspond to a distinct Eu(III) environment. The absorption
spectrum (Fig. 2) of Eu–DO3A-BT consists of a single peak due
to the 7F0
→
5D0 transition around 570 nm confirming the
presence of a monoaqua complex.
Determination of the protonation and thermodynamic
stability constants
We have summarized the protonation constant of DO3A-BT
and the stability constant of the complex DO3A-BT with
Inner sphere hydration number (q) determination
The number of water molecules directly coordinated to Gd3+ is Gd3+, Eu3+ and biologically active divalent metal cations
one of the fundamental parameters influencing the relaxivity. obtained by potentiometric titration in Table
To determine the hydration number, the luminescence life- respectively.
1 and 2
time decay of the 5D0 excited state of the Eu(III) complex in
A pH-potentiometric titration of the ligand DO3A-BT over
H2O and D2O is measured. The luminescence lifetime studies the pH range 2–12 gave three protonation constants which are
established that OH oscillators of coordinated H2O molecules likely to be associated with the three carboxylic acid groups.
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Table 1 Protonation constants (log K
SD, n = 3) of the metal complexes
Table 3 Pseudo first order rate constants characterizing the metal exchange
reactions of Gd–DO3A-BT with Eu3+ and Zn2+ (25 °C, 0.1 M KCl)
DO3A-BT, DOTA and DTPA in NMe4Cl (0.1 M) at 298 K with SD
DOTA31
DTPA31
DO3A-BT
kobs (s−1
)
log KH1
log KH2
log KH3
11.14 0.02
9.69 0.01
4.85 0.07
10.49 0.03
8.37 0.05
4.09 0.04
9.91 0.06
8.22 0.02
Ligand
Eu3+
Zn2+
5.01 0.05 Gd–DO3A-BT
3.0 0.7 × 10−5
Gd–DOTA33
2.1 0.2 × 10−5
1.2 0.1 × 10−3
2.3 0.4 × 10−3
Gd–DTPA34
1.1 0.1 × 10−5
1.4 0.3 × 10−2
Gd–DO3A35
Table 2 Stability constants (log
β
SD, n = 3) of the metal complexes
DO3A-BTA, DOTA and DTPA (25 °C, μ = 0.1 M NMe4Cl)
required for conversion. Pseudo first order rate constants of
the metal exchange reactions of Gd–DO3A-BT along with other
Gd complexes with Zn2+ and Eu2+ are shown in Table 3. Replace-
ment of one carboxylate group with an amide group in Gd–
DO3A-BT results in a slight decrease in the stability constant
but has practically no effect on the kinetic stability of the Gd–
DOTA32
DTPA32
DO3A-BT
log βGdL
log βEuL
23.2 0.01
24.5 0.06
18.1 0.06
22.2 0.03
22.2 0.04
20.0 0.03
16.5 0.07
22.4 0.01
18.4 0.06
18.3 0.02
7.1 0.04
6.3 0.04
log βZn L
2
log βCu L
2
DO3A-BT complex, which has a rate constant of 3.0 × 10−5 s−1
.
This indicates that compared with the carboxylate group, the
amide group has a lower reactivity with Zn2+ and thus it will
not undergo transmetallation with endogenous metal ions.
The suitability of Gd3+ chelates for in vivo application is deter-
mined by their thermodynamic parameters. Most of the poly
(aminocarboxylate) ligands form complexes with increasing
stability across the lanthanide series. Here, the stability con-
stant of DO3A-BT with Gd3+ falls almost in the same range as
that of DOTA and DTPA but it is very low for Zn2+ and Cu2+.
These findings conclude that DO3A-BT forms exclusively stable
complexes with Gd and does not undergo transmetallation
with endogenous metal ions.
99mTc radiocomplexation of DO3A-BT
The Rf of the labelled complex and free technetium was esti-
mated using an instant thin layer chromatography method
and was found to be 0.0 and 1.0, respectively, using acetone as
a mobile phase. The optimum pH for radiolabelling was
6.0–7.5, and from the mole calculation, it was calculated that
52 μg of stannous chloride resulted in a maximum labelling
efficiency with a minimum amount of 99mTcO4−. The labelling
efficiency for 99mTc–DO3A-BT was calculated to be >98%, and
was determined in a different solvent system chromatographi-
cally. The in vitro stability of 99mTc–DO3A-BT in the PBS buffer
at pH 7.0 was checked at different time intervals: 4, 6, 24 and
48 h, and the percentage labelling efficiency at 48 h was found
to be 95%, implying that the labelled conjugate was stable up
to 48 h post labelling.
Kinetic stability
The aminopolycarboxylate complexes of Gd3+ are used as MR
contrast agents and are kinetically inert compounds. However,
a number of animal and human experiments indicate that the
excretion of Gd3+ from the body is not complete, which is
explained by assuming that a small amount of dissociation of
Gd3+ from the Gd3+ complexes occurs in vivo. To understand
the in vivo fate of the Gd3+ containing contrast agents, we have
to know the rate of dissociation of the complex, which presum-
ably takes place in the exchange reactions with endogenous
metal ions. Thus sufficiently high thermodynamic stability of
the Gd(III) complex and good selectivity of the chelating ligand
for Gd(III) over other endogenous metal ions such as Cu2+ and
Zn2+ are prerequisites for all biomedical applications. In order
to obtain information on the rate of dissociation of the
complex Gd–DO3A-BT, the kinetics of the exchange reactions
were studied in 0.1 M KCl at 25 °C,
Blood kinetic study
The blood kinetics of 99mTc–DO3A-BT fits in to a two compart-
ment model by regression analysis. The blood-activity curve
studied in rabbits reveals a biphasic pattern of clearance with
t1/2 fast and t1/2 slow. Initially the clearance of the agent exhib-
ited a slow trend, but after 10 h the activity of 99mTc–DO3A-BT
was greatly reduced. The biological half life was found to be
t1/2(F) = 2 h and t1/2(S) = 13 h (Fig. 3). After 1 h, the 99mTc–
DO3A-BT has approximately 20% activity remaining whereas
only 3.1% remained after 24 h, which accounts for the excel-
lent in vivo stability of the radioconjugate. This data correlates
with the hydrophilic behaviour of the DO3A-like ligand as they
are cleared from blood circulation mainly through the renal
pathway.
½Gd–DO3A ꢀ BTꢁ þ Mnþ Ð ½M–DO3A ꢀ BTꢁ þ Gd3þ
where Mn+ = Zn2+ or Eu2+. The rate of the exchange reactions
were investigated in the presence of excess exchanging metal
ions. The stability of Eu–DO3A-BT is similar to that of Gd–
DO3A-BT and conversion occurs with 5–15 fold excess of Eu3+.
The log KML value (where KML is the stability constant of the
metal–ligand complex) of the Zn(II) complex is lower than that
Serum stability assay
of Ln–DO3A-BT (Ln = Eu, Gd) and despite the high stability of In vivo kinetic inertness plays a critical role in determining
the Zn2L complex, a large excess (50–80 fold) of metal ions is competition experiments using native biological chelators
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having pdb id-1E78 which gave the following docking
parameters:
Docking score
Glide evdw
Ecoul
Glide energy
Glide H-bond
−6.655
−30.946
−8.704
−39.651
−2.226
It is clear from the docking score that the DO3A-BT
complex has considerably poor HSA binding affinity, which
confirms the high serum stability of the complex. More nega-
tive docking scores indicate that the complex has a higher
affinity for human serum albumin and a lower stability.
Fig. 3 Blood kinetics of 99mTc–DO3A-BT administered through the ear vein in a
normal rabbit.
Binding affinity for neuronal cells
To determine the binding affinity of 99mTc–DO3A-BT to neuro-
nal cells, a saturation binding assay was performed using
increasing concentrations (10–0.001 mM) of the radioligand
on brain homogenates. The percent specific binding of 99mTc–
DO3A-BT was then measured as a function of the concen-
tration of the radiolabeled complex. The data (Fig. 5) shows
that when the concentration of 99mTc–DO3A-BT is increased
there is an increase in the percentage uptake, which became
saturated above 1 mM. The maximum percentage uptake for
the brain homogenate was 78% with a 1 mM concentration of
DO3A-BT. This study was performed to analyze the in vitro
uptake of 99mTc–DO3A-BT which is further evaluated in in vivo
biodistribution studies.
Fig. 4 In vitro serum stability of 99mTc–DO3A-BT at 37 °C (n = 3).
Biodistribution studies
The results of in vivo biodistribution studies in BALB/c mice
showed that 99mTc–DO3A-BT is excreted through the renal
pathway. It penetrates well through the intact blood–brain-
barrier (BBB) when mannitol36 is injected prior to injection of
drug. The brain has a maximum uptake (1.2%) 15 min after
the injection of the radiolabelled complex, but only 0.51%
penetrates the brain when administered without mannitol as
shown in Table 4 and 5. 99mTc–DO3A-BT displayed an initial
brain penetration and a fast washout from the brain with only
0.06% ID g−1 remaining 1 h after injection. There was a signifi-
cant correlation between log P and clearance as expressed by
the 2 min to 30 min ratio. The least lipophilic compound
cleared faster from the brain and the most lipophilic
such as those contained in blood serum (e.g. apo-transferrin,
albumin). In vitro assay is useful for predicting the in vivo
stability and kinetic inertness of a radiometal ion complex.
Lanthanides are trivalent metal ions, and behave similarly to
Fe3+ cations that are present in blood such as transferrin,
which is an iron transport protein in human serum. In
addition to binding to iron, transferrin also forms com-
plexes with lanthanides, and therefore the serum stability of
these complexes is of major concern under physiological
conditions.
99mTc–DO3A-BT demonstrated high serum stability as
shown in Fig. 4 with approximately 95% of the radioactivity
remaining after 24 h. The decomposition of 99mTc–DO3A-BT is
slightly greater than DOTA and DTPA. The lower serum stabi-
lity of the complex is due to a weaker octadentate structure for
the DO3A-BT complex as compared to the octadentate struc-
tures of DTPA and DOTA. Lanthanide complexes generally
require at least eight donor atoms to fully saturate their inner
coordination sphere. DTPA and DOTA have eight potential
atoms for metal coordination, whereas the eighth coordinating
amide group of DO3A-BT is not as strong as the DTPA and
DOTA carboxylate group, which explains the dissociation of
the complex.
The human serum albumin (HSA) stability of the complex
is further verified from a docking study of DO3A-BT with HSA Fig. 5 The saturation binding assay of 99mTc–DO3A-BT in brain homogenate.
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Table 4 The biodistribution profile of 99mTc–DO3A-BT in normal mice without
mannitol (%ID g−1 SD, n = 3)
of 4-aminobenzoic acid (3 g, 0.02 mmol) in polyphosphoric
acid (30 ml). The reaction mixture was heated to 130 °C for
5 h. The reaction mixture was cooled to room temperature and
neutralized with 10% Na2CO3 (300 ml). A yellow solid precipi-
tated from the reaction mixture and was filtered under
reduced pressure to give the desired product (3). Yield (5.50 g,
91.6%). Rf: 0.8 (9 : 1; DCM–MeOH). Elemental analysis: found
C, 68.01; H, 4.21; N, 12.99; S, 14.35. Calc. for (C13H10N2S):
C, 69.00; H, 4.45; N, 12.38; S, 14.17. 1H NMR (400 MHz,
DMSO-d6, Me4Si): δH = 6.75 (d, 2H, J = 8.7 Hz, CH), 7.32 (t, 1H,
CH), 7.44 (t, 1H, CH), 7.78 (d, 2H, J = 8.4 Hz, CH), 7.89 (d, 1H,
J = 8.0 Hz, CH), 7.97 (d, 1H, J = 7.6 Hz, CH). 13C NMR
(100 MHz, DMSO-d6, Me4Si): δC = 154.21, 151.42, 134.18,
129.22, 126.72, 124.88, 122.39, 122.26, 114.80, 68.46 (CvN).
ESI-MS: m/z found 227 [M + H]+ (C13H10N2S), calculated 226.
Synthesis of N-(4-benzothiazol-2-yl-phenyl)-2-chloroaceta-
mide (4). To a stirred solution of 3 (1 g, 4.42 mmol) in dried
CH2Cl2 (20 ml), Et3N (0.92 ml, 6.64 mmol) and chloroace-
tylchloride (0.42 ml, 5.30 mmol) in CH2Cl2 (5 ml) were added
drop wise to the reaction mixture at 0 °C. The reaction mixture
was stirred for 1 h at 0 °C and then was stirred further for 24 h
at room temperature. The completion of the reaction was moni-
tored by TLC and on completion, the reaction mixture was fil-
tered and evaporated under reduced pressure to give the crude
product. The trituration of the crude product gave a pure white
powder. Yield (1.43 g, 80%). Rf: 0.66 (8 : 2; DCM–MeOH).
Elemental analysis: found C, 59.22; H, 3.99; N, 8.99; S, 11.06.
Calc. for (C15H11ClN2OS): C, 59.50; H, 3.66; N, 9.25; S, 10.59.
1H NMR (400 MHz, DMSO-d6, Me4Si): δH = 4.34 (s, 2H, CH2),
7.44 (t, 1H, CH), 7.53 (t, 1H, CH), 7.82 (d, 2H, J = 8.8 Hz, CH),
8.02 (d, 2H, J = 6.4 Hz, CH), 8.1 (d, 2H, J = 7.6 Hz, CH).
13C NMR (100 MHz, DMSO-d6, Me4Si): δC = 167.33 (CvN),
165.59 (CvO), 154.01, 141.87, 134.74, 128.53, 127.06, 125.77,
123.05, 122.75, 120.00, 44.03. ESI-MS: m/z found 338
[M + K − 2H]− (C15H11ClN2OS), calculated 302.
Organs
2 min
15 min
30 min
1 h
Blood
Brain
Heart
Liver
Spleen
Kidney
Stomach
Intestine
17.53 0.93
0.51 0.03
3.17 0.71
14.18 2.30
9.15 0.95
63.84 1.14
0.87 0.12
1.86 0.51
12.42 0.89
0.12 0.02
6.81 0.41
12.13 1.41
7.41 0.54
15.11 0.94
0.40 0.17
0.89 0.06
8.54 0.71
0.09 0.01
2.13 0.29
9.14 0.93
5.61 0.34
13.44 0.73
0.20 0.04
0.51 0.04
2.60 0.51
0.02 0.00
0.94 0.07
7.15 0.53
4.50 0.24
5.50 0.51
0.04 0.01
0.04 0.00
Table 5 The biodistribution profile of 99mTc–DO3A-BT in normal mice with
mannitol (%ID g−1 SD, n = 3)
Organs
2 min
15 min
30 min
1 h
Blood
Brain
Heart
Liver
Spleen
Kidney
Stomach
Intestine
19.87 0.91
0.87 0.17
8.60 1.23
13.19 1.14
5.06 1.13
43.59 2.10
0.73 0.05
2.50 0.41
15.13 1.01
1.20 0.51
9.45 1.09
10.31 1.31
2.89 0.90
19.39 2.91
0.35 0.02
2.33 0.23
9.84 1.51
0.54 0.07
3.76 0.91
11.71 1.02
2.33 0.13
15.26 2.63
0.87 0.06
1.91 0.45
2.46 0.99
0.06 0.01
0.68 0.08
8.10 0.06
1.37 0.09
7.53 1.92
0.61 0.08
0.59 0.08
compound accumulated in the brain over 30 min. The log P
value of DO3A-BT–Gd is 2.27 whereas for 99mTc–DO3A-BT
log P = −1.189, which indicates that there is no need to inject
mannitol when using DO3A-BT–Gd, but due to the hydrophilic
nature of 99mTc–DO3A-BT, mannitol is required for the disrup-
tion of the BBB.
Experimental
Materials
Synthesis of 1,4,7-tri(tert-butoxymethane)-1,4,7,10-tetraaza-
cyclododecane (t-Bu-DO3A) (5). NaHCO3 (1.3 g, 15.5 mmol)
was added to a solution of 1,4,7,10-tetraazacyclododecane (1 g,
5.18 mmol) in dry acetonitrile (30 ml) at 0 °C under a nitrogen
atmosphere and stirred for 0.5 h. tert-Butylbromoacetate
(2.29 ml, 15.5 mmol) in acetonitrile (5 ml) was added slowly to
the above reaction mixture at 0 °C and stirred for 40 h at room
temperature. The progress of the reaction was monitored by
TLC (9 : 1; DCM–methanol). After the completion of the reac-
tion, the reaction mixture was filtered and the filtrate was evapo-
rated to dryness. The crude compound was purified by column
chromatography (silica gel, 10% methanol in dichloro-
methane) to give a white powder of t-Bu-DO3A (2.30 g, 80%).
Rf: 0.65 (9 : 1; DCM–MeOH). Elemental analysis: found C,
All reagents and solvents were used in the purest grade that
was available commercially without further purification.
1,4,7,10-Tetraazacyclododecane was purchased from Strem,
France. 2-Aminothiophenol, tert-butylbromoacetate, 4-amino-
benzoic acid, polyphosphoric acid, anhydrous gadolinium
trichloride, 2-chloroacetylchloride, potassium carbonate,
acetonitrile, methanol, trifluoroacetic acid, HPLC water,
dichloromethane, chloroform and triethylamine were pur-
chased from Aldrich, Germany. Column chromatography was
carried out using silica MN60 (60–200 μm), TLC on aluminium
plates coated with silica gel 1160, F254 (Merck).
Instrumentation
1H and 13C NMR spectra were recorded using a Bruker 60.33; H, 9.05; N, 10.56. Calc. for (C26H50N4O6): C, 60.67; H,
1
Avance II 400 MHz. Chemical shifts are reported relative to 9.79; N, 10.89. H NMR (400 MHz, CDCl3, Me4Si): δH = 1.45 (s,
TMS. ESI-MS was performed using the Agilent 6310 system, 27H, C(CH3)3), 2.85 (d, J = 10 Hz, 12H, 6 × CH2), 3.03 (s, 4H,
Germany, with the ion trap detection in the positive and nega- 2 × CH2), 3.36 (s, 6H, 3 × CH2). 13C NMR (100 MHz, CDCl3,
tive mode.
Me4Si): δC = 170.54, 169.65 (CvO), 81.86 (C(CH3)3), 47.48,
Synthesis of 4-benzothiazol-2-yl-phenylamine (3). 2-Amino- 48.81, 49.17, 51.30 (CH2), 28.21 (CH3). ESI-MS: m/z found 515
benzenethiol (2.34 ml, 0.02 mmol) was added to the solution [M + H]+ (C26H50N4O6), calculated 514.
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Synthesis of 10-[N-(4-benzothiazol-2-yl-phenylcarbamoyl)- S, 4.21. ESI-MS: m/z (Eu–DO3A-BT) found 763 [M]+,
methyl]-1,4,7-tri(carbobutoxy-methane)-1,4,7,10-tetraaza- 785 [M + Na]+, calculated 763.
cyclododecane (6). 1,4,7-Tris(carbobutoxymethane)-1,4,7,10-
tetraazacyclododecane (1 g, 1.94 mmol) was dissolved in dry
acetonitrile (20 ml). K2CO3 (2.15 g, 15.5 mmol) was added
to the reaction mixture and stirred at room temperature for
0.5 h. N-4-Benzothiazol-2-yl-phenyl-2-chloroacetamide (0.58 g,
1.94 mmol) was added to the reaction mixture and heated to
70 °C. After 14 h the reaction mixture was filtered and evapo-
rated under reduced pressure to give a crude oily residue. The
compound was purified by column chromatography to give a
yellow oil (yield 0.83 g, 60%). Rf: 0.34 (8 : 2; DCM–MeOH).
Elemental analysis: found C, 62.97; H, 7.65; N, 10.98; S, 4.24.
Calc. for (C41H60N6O7S): C, 63.05; H, 7.74; N, 10.76; S, 4.11.
1H NMR (400 MHz, MeOD, Me4Si): δH = 1.52 (s, 9H, C(CH3)3),
2.09 (b, 8H, CH2), 2.79 (b, 4H, CH2), 3.24 (s, 6H, CH2),
3.14 (s, 4H, CH2), 3.31 (s, 2H, CH2), 8.08 (d, J = 8.2 Hz, 3H),
In vitro relaxivity measurement
Experiments were carried out using a 4.7 Tesla horizontal
Bruker Biospect 47/50 (Bruker, Ettlingen, Germany). The
system was equipped with a 12 cm BG12 gradient system
capable of 193 mT m−1. Measurements were performed with a
birdcage resonator (60 mm in diameter and 120 mm long)
tuned to 20 MHz. T1 measurements were performed at 37 °C
using an Inversion-Recovery scheme (increment of inversion
delay: 10 ms with 456 increments) followed by a RARE imaging
sequence (TR/TEeff: 5000/2.6 ms; FOV (field of view): 30 ×
30 mm; matrix: 128 × 128; slice thickness: 1 mm). T2 measure-
ments were performed with
a Carr–Purcell–Meiboon–Gill
imaging sequence (TR: 2500 ms; inter-echo time: 11.6 ms;
number of echoes: 64; FOV: 30 × 30 mm; matrix: 128 × 128;
slice thickness: 1 mm). Relaxation data were analyzed with
home-made software developed on Igor Pro (Wave metrics,
Lake Oswego, OR, USA).
To measure the changes in the longitudinal relaxivity (r1),
MRI experiments were performed at six different concen-
trations (0.625–20 mM) of [GdL] prepared in phosphate
buffered saline in 1.5 mL Eppendorf tubes at physiological pH.
Each tube was filled with 400 μL of the contrast agent solution.
Relaxivity at different concentration was then calculated using
eqn (1):
7.84 (d,
J = 7.8 Hz, 1H), 7.54 (t, 1H), 7.45 (t, 1H).
13C NMR (100 MHz, MeOD, Me4Si): δC = 173.12, 171.54,
168.14, 153.63, 141.70, 134.44, 128.19, 127.57, 126.34, 125.15,
122.08, 121.57, 119.62, 56.32, 55.35, 55.26, 26.99. ESI-MS:
m/z found 782 [M + 2H]+ and 804 [M + Na]+ (C41H60N6O7S),
calculated 780.
Synthesis
of
{4-[(4-benzothiazol-2-yl-phenylcarbamoyl)-
methyl]-7,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl}-
acetic acid (DO3A-BT) (7). Compound 6 (400 mg, 0.51 mmol)
was dissolved in 5 ml of neat trifluoroacetic acid at 0 °C and
stirred for 5 h. The solvent was evaporated under reduced
pressure. After trituration with cold ether, the compound pre-
cipitated as a white powder The compound was dried, dis-
solved in water and neutralized to pH 7 by the addition of
1 M NaOH. The product was obtained as brown solid. Yield
(283 mg, 75%). Elemental analysis: found C, 57.09; H, 6.11;
N, 13.35; S, 5.55. Calc. for (C30H37N5O7S): C, 56.85; H, 5.92;
N, 13.72; S, 5.23. 1H NMR (400 MHz, D2O, Me4Si): δH = 2.59–3.85
r1;obs ¼ ð1=T1;obs ꢀ 1=T1;dÞ=½GdLꢁ
ð1Þ
where T1,obs is the measured T1; T1,d is the diamagnetic contri-
bution of the solvent and [GdL] is the concentration in mmol
of the appropriate Gd(III) complex.
Equilibrium measurements
(m, 26H, CH2), 7.02 (s, 1H, CH), 7.16–7.26 (m, 6H, CH), 7.56 Potentiometric titrations for the determination of the ligand
(s, 1H, CH). 13C NMR (100 MHz, D2O, Me4Si): δC = 38.69, protonation constant and stability constant of the complexes
42.24, 47.72, 49.00, 50.08, 51.68, 53.09, 54.83, 117.43, 119.69, with Gd(III), Eu(III), Cu(II) and Zn(II) were measured with an
127.09, 163.07, 174.37, 178.61. ESI-MS: m/z found 614 [M + H]+ automatic titration system consisting of a Metrohm 713 pH
(C30H37N5O7S), calculated 613.
meter equipped with a Metrohm A.60262.100 glass electrode,
Synthesis of lanthanide(III) complexes with DO3A-BT (Ln– 800 Dosino autoburet. It has a precision of 0.002 pH unit and
DO3A-BTA) (8). The pH of the ligand DO3A-BTA (500 mg, is thermostated (25 °C) with a glass-jacketed titration cell
0.81 mmol) was adjusted to 7.0 by the drop wise addition of fitted with a combination glass electrode and a Metrohm
1 M NaOH. Anhydrous LnCl3 (GdCl3 and EuCl3) (257 mg, piston buret with a capillary tip placed below the surface of the
0.98 mmol) was added to the above reaction mixture and sample. This avoids absorption of CO2 by the base solution.
stirred for 18 h at 70 °C and pH kept at 7.0. After 18 h, the reac- The pH meter–electrode system was calibrated with standard
tion mixture was cooled and passed through chelex-100 at buffers. The protonation constants of DO3A-BT were deter-
room temperature to trap the free lanthanide. The absence of mined potentiometrically by titrating 1 mM of DO3A-BT with
free Gd(III) was checked by using xylenol orange as an indi- 10 mM tetramethylammoniumhydroxide (TMAOH). Potentio-
cator. The crude product was filtered, evaporated and lyophi- metric titrations were carried out with 0.1 M ionic strength of
lized to get the desired product as a white solid. Elemental tetramethylammoniumchloride (TMACl) at 25 °C. Titrations
analysis: found C, 43.09; H, 4.02; N, 9.99; S, 4.76. Calc. for were performed in the pH range of 2–12 for protonation
(C29H33GdN6O7S): C, 45.42; H, 4.34; N, 10.96; S, 4.18. ESI-MS: constants.
m/z (Gd–DO3A-BT) found 768 [M]+, 790 [M + Na]+, calculated
The stability constants of DO3A-BT with Gd(III), Eu(III),
768. Elemental analysis: found C, 46.22; H, 5.12; N, 12.04; Cu(II) and Zn(II) were determined by direct pH potentiometric
S, 4.56. Calc. for (C29H33EuN6O7S): C, 45.73; H, 4.37; N, 11.03; titration (0.002–0.004 M Mn+ and 0.002 M ligand solutions),
5000 | Dalton Trans., 2013, 42, 4994–5003
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I = 0.1 M using TMACl. Solutions of Gd(III), Eu(III) and Cu(II) integration time = 1 s, flash count = 8 and proper slit widths
were prepared by dissolving the appropriate amounts of their were used for no saturation of the signal by using a different
salts in milliQ water. For titrations of the complexes, an delay times. Solutions of the Eu–DO3A-BT complex were pre-
appropriate volume of the previously prepared metal solution pared in situ by mixing its appropriate volume in milliQ water
was added via a micropipette to reach the 1 : 1 ligand to or in D2O with the ligand in Tris buffer 0.1 M (pH = 7.4) or in
metal ratio. A minimum of two duplicate measurements D2O followed by the addition of NaOD until pH = 7. The exci-
were taken. The protonation and stability constants were tation source was a hydrogen gas filled nanosecond flash lamp
evaluated using titration data using the program with a low hydrogen gas pressure of 0.4 bar operating at a fre-
Tiamo 2.0. The protonation constant of ligand is expressed by quency of 40 kHz. The slit widths for both the excitation and
eqn (2) and the stability constant of the metal complex is given emission monochromators are kept open. The intensity decay
by eqn (3):
curves were obtained at emission maximum and fitted as sum
of exponentials as:
½HiLꢁ
X
Ki ¼
ð2Þ
ð3Þ
½Hiꢀ1Lꢁ½Hþꢁ
It ¼ I0
Aiexpðꢀt=τiÞ
ð5Þ
½MHiLꢁ
where, τi and Ai represent the fluorescence lifetime and pre
exponential factor for ith component. The number of coordi-
nated water molecules (q) present in the inner sphere of the
complex is calculated from the luminescence rate constants of
Eu–DO3A-BT in light and heavy water using equation deve-
loped by Supkowski and Horrocks for Eu(III):30
KMH L
¼
i ¼ 0; 1; 2; 3
i
½MHiꢀ1Lꢁ½Hþꢁ
Kinetic measurement
High kinetic stability is an important requirement for Gd3+
complexes that are used as contrast enhancement agents in
MRI. The extent of the dissociation depends on the selectivity
of the ligand for Gd3+ over the endogenous metal ions present
in body fluids. For example, in vivo, endogenous cations
(Fe3+, Ca2+, Zn2+ and Cu2+) can react with Gd chelates by
displacing Gd3+ in a metal–metal transmetallation exchange.
Thus to understand the in vivo fate of the Gd3+ containing
contrast agent, we have to know the rate of the exchange
reaction of Gd–DO3A-BT with Eu3+ and Zn2+. The rate of the
metal exchange reactions of Gd–DO3A-BT with Eu3+ and Zn2+
were studied using a UV-visible spectrophotometer, following
the formation of the Eu3+ and Zn2+ complexes at 280 and
317 nm, respectively. The concentration of the complex
Gd–DO3A-BT was 6.2 × 10−5 M, whereas the concentration of
the metal ion was 30–40 times higher, respectively, guarantee-
ing pseudo-first-order conditions. The temperature was main-
tained at 25 °C and the ionic strength of the system was kept
constant with 0.1 M KCl at pH 7.4. In the presence of an
excess of the exchanging ion, the transmetallation process may
be assumed to be a pseudo-first-order process and the rate of
reaction can be expressed with eqn (4),
q ¼ AEuð1=τH O ꢀ 1=τD O ꢀ aEu
Þ
ð6Þ
2
2
where τH O and τD O are the luminescence lifetimes in H2O
2
2
and D2O,37 giving an inner sphere hydration number. The
value of AEu = 1.11 ms and aEu = 0.31 ms−1; aEu is used for the
correction of error which accounts for closely diffusing OH
oscillators.
Biological studies
99mTc radiocomplexation of DO3A-BT
DO3A-BT (2 μmol) was dissolved in 1 ml of doubly distilled
water. 100 μl of this solution was taken in a shielded glass
vial and stannous chloride (1 μmol, dissolved in 1 ml of 10%
acetic acid purged with N2) was added to it, followed by the
addition of 100 μl of 74 MBq of freshly eluted (<1 h) 99mtech-
necium pertechnetate saline solution. The pH of the reaction
mixture was adjusted to 7 using 0.1 M sodium carbonate solu-
tion. All contents were thoroughly mixed and incubated for
30 min at room temperature to achieve an optimal labelling
yield.
At ¼ ðAo ꢀ ApÞeꢀk
ð4Þ
obst
where kobs is a pseudo-first-order rate constant and At, Ao, and
Ap are the absorbance values at time t, and at the starting and
equilibrium points of the reaction, respectively.
Radiochemical purity of 99mTc–DO3A-BT complex
The radiolabelling efficiency of the above complex was deter-
mined by using ascending instant thin layer chromatography
ITLC-SG (Paul Gelman, USA) using 100% acetone simul-
UV-visible absorption and luminescence measurement
The inner sphere hydration number of Eu–DO3A-BT was taneously with pyridine–acetic acid–water (PAW) (3 : 5 : 1.5) and
recorded on a Cary Varian double beam UV/visible spectro- saline. TLC strips were cut into 0.5 cm fragments and counts
photometer with Perkin-Elmer Luminescence Cells with a path of each fragment were taken. The percentage of free Na
length of 1 cm at 293 K. The luminescence lifetime τL was cal- 99mTcO4−, reduced 99mTc and complexed 99mTc could be calcu-
culated by measuring the decay at the maximum of emission lated from this method. Complexed 99mTc remained at the
spectra and the signals were analysed as single exponential origin whereas uncomplexed 99mTc moved with the solvent
decays. The instrument settings were: gate time = 10 ms, front.
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Blood kinetics
A biodistribution study of 99mTc–DO3A-BT was performed
on BALB/c mice (25–30 g) with and without mannitol, as man-
nitol disrupts the BBB due to osmotic shock. MR contrast
agents (Gd chelates) in combination with mannitol allow the
contrast agent to cross the BBB. 30% of mannitol was injected
5 min before the injection of the drug. An aliquot of 2 mCi of
99mTc–DO3A-BT was injected in each mouse intravenously
through the tail vein and the animals were humanely sacri-
ficed at 5, 15, 30, 60 min post injection. Tissue samples includ-
ing the lung, liver, spleen, kidney, stomach, blood, heart,
intestine and brain were removed and weighed. Uptake of
99mTc radioactivity in each sample was measured as %ID g−1
of the tissue with an automated gamma scintillation counter
and was normalized using the 99mTc-decay correction.
Blood clearance studies were carried out using normal rabbits
and 0.1 ml of 99mTc–DO3A-BT was administered intravenously
through the dorsal ear vein of the animal. Blood samples
(10 μl) were collected at different time intervals and the radio-
activity was counted using a NaI well type gamma counter. The
percentage radioactivity in the whole blood was calculated
assuming that the whole blood volume is 7% of the body
weight.
Human serum stability
Human serum was prepared by allowing blood collected from
healthy volunteers to clot for 1 h at 37 °C in a humidified incu-
bator maintained at 5% carbon dioxide/95% air. Then, the
samples were centrifuged at 400 g, and the serum was filtered
through a 0.22 μm syringe filter into sterile plastic culture
tubes. The radio labelled DO3A-BT was immediately placed in
Summary
a CO2 chamber, incubated at 37 °C and then analyzed to check We have designed a biocompatible probe which can investigate
for any dissociation of the complex. The percentage of free per- the brain non-invasively, and represents a platform for the
technetate at a particular time point was estimated using development of multimodal imaging tools for neuroscience.
acetone and pyridine, acetic acid, and water (PAW) (3 : 5 : 1.5) We have presented the synthesis and characterization of a dual
as the mobile phase which represent the percentage dis- modality imaging agent Ln–DO3A-BT (Ln = Gd or Eu), which
sociation of the complex at that particular time point in was proved to be an excellent MR contrast and optical imaging
serum. The HSA binding of the complex is also determined agent. Its longitudinal relaxivity is ∼35 times higher than that
from docking experiments carried out by using GLIDE (grid of LFB which is a selective MR contrast agent for the brain
based ligand docking with energetic) and a ligand docking commonly found in the literature. The described macrocyclic
programme in extra precision (XP) mode using HSA pdb complex fulfils the requirements of a luminescent probe as an
id-1E78.
optical agent: it has a good hydrophilicity and a high kinetic
inertness in aqueous solutions. Moreover the pharmacokinetic
profile of 99mTc–DO3A-BT showed a good serum stability,
a fast clearance through the renal route and a substantial
brain uptake when injected along with 30% mannitol. The
highest in vitro binding affinity for neuronal cells makes it
highly specific for mapping brain regions.
Radioligand binding assay for neuronal cells
BALB/c mice were sacrificed, and their brains were excised and
homogenized in 0.1 M phosphate buffer (pH = 8). An 0.4 ml
aliquot of the brain homogenate was added to Eppendorf
tubes containing different concentrations of 99mTc–DO3A-BT
(10–0.001 mM) and incubated for 30 min at 37 °C. After incu-
bation, 0.1 ml of 10% trichloroacetic acid was added to each
Eppendorf tube which caused the protein to precipitate out.
Then samples were centrifuged and washed twice with PBS to
obtain a pellet of protein which was then resuspended in PBS.
The uptake of 99mTc radioactivity in each sample was
measured with an automated gamma scintillation counter and
was normalized with 99mTc-decay correction as well as for
diluted homogenates. Percentage uptake in the brain was
plotted against the different concentrations of 99mTc–DO3A-BT
used in the binding assay.
Acknowledgements
We are highly thankful to Dr R. P. Tripathi, Director INMAS,
for providing the necessary facilities. This work was supported
by the Council of Scientific and Industrial research (CSIR) and
the Defence Research and Development Organization, Ministry
of Defence, under R&D project INM-311(3.1).
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