Design and synthesis of calcium responsive magnetic resonance imaging agent: Its relaxation and luminescence studies

Article abstract of DOI:10.1016/j.ejmech.2014.05.046

Calcium concentration modulation both inside and outside cell is of considerable interest for nervous system function in normal and pathological conditions. MRI has potential for very high spatial resolution at molecular/cellular level. Design, synthesis and evaluation of Gd-DO3A-AME-NPHE, a calcium responsive MRI contrast agent is presented. The probe is comprised of a Gd³⁺-DO3A core coupled to iminoacetate coordinating groups for calcium induced relaxivity switching. In the absence of Ca²⁺ ions, inner sphere water binding to the Gd-DO3A-AME-NPHE is restricted with longitudinal relaxivity, r₁ = 4.37 mM^-1 s^-1 at 4.7 T. However, addition of Ca²⁺ triggers a marked enhancement in r₁ = 6.99 mM^-1 s^-1 at 4.7 T (60% increase). The construct is highly selective for Ca²⁺ over competitive metal ions at extracellular concentration. The r₁ is modulated by changes in the hydration number (0.2 to 1.05), which was confirmed by luminescence emission lifetimes of the analogous Eu³⁺ complex. T₁ phantom images establish the capability of complex of visualizing changes in [Ca²⁺] by MRI.

Full text of DOI:10.1016/j.ejmech.2014.05.046

European Journal of Medicinal Chemistry 82 (2014) 225e232
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of calcium responsive magnetic resonance
imaging agent: Its relaxation and luminescence studies
,
,
Jyoti Tanwar a b, Anupama Datta a *, Kanchan Chauhan a, S. Senthil Kumaran c,
Anjani K. Tiwari a, K. Ganesh Kadiyala a b, Sunil Pal a, M. Thirumal b, Anil K. Mishra a
,
, *
a Institute of Nuclear Medicine and Allied Sciences, Brig S. K. Mazumdar Road, Delhi 110054, India
b Department of Chemistry, University of Delhi, Delhi 110054, India
c Department of N.M.R. and MRI, All India Institute of Medical Sciences, New Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Calcium concentration modulation both inside and outside cell is of considerable interest for nervous
system function in normal and pathological conditions. MRI has potential for very high spatial resolution at
molecular/cellular level. Design, synthesis and evaluation of Gd-DO3A-AME-NPHE, a calcium responsive
MRI contrast agent is presented. The probe is comprised of a Gd3þ-DO3A core coupled to iminoacetate
coordinating groups for calcium induced relaxivity switching. In the absence of Ca2þ ions, inner sphere
water binding to the Gd-DO3A-AME-NPHE is restricted with longitudinal relaxivity, r1 ¼ 4.37 mMꢀ1 sꢀ1 at
4.7 T. However, addition of Ca2þ triggers a marked enhancement in r1 ¼ 6.99 mMꢀ1 sꢀ1 at 4.7 T (60% in-
crease). The construct is highly selective for Ca2þ over competitive metal ions at extracellular concen-
tration. The r1 is modulated by changes in the hydration number (0.2 to 1.05), which was confirmed by
luminescence emission lifetimes of the analogous Eu3þ complex. T1 phantom images establish the capa-
bility of complex of visualizing changes in [Ca2þ] by MRI.
Received 28 September 2013
Received in revised form
15 May 2014
Accepted 20 May 2014
Available online 20 May 2014
Keywords:
Magnetic resonance imaging
Calcium sensing
1,4,7,10-Tetraaacyclododecane-1,4,7-
tris(methylenecarboxylic acid)
Gadolinium
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
abnormalities in diseased states [5]. The multiple roles of calcium
are being majorly understood in greater details with the help of
Calcium ion, Ca(II), is one of the most versatile and universal
signalling agent in the biological system. Around 1% of calcium in
the body is present in solution form in the intra and extracellular
fluids at varying concentrations (100 nM‒2 mM) [1,2]. Its essenti-
ality can be well understood from the multitude of cellular process
controlled by it right from the cell birth, throughout life and unto
death [3]. A number of intracellular processes in the brain are
dependent on Ca2þ, both pre synaptic and post synaptic [4] which
include its essential presence for neurotransmitter release, as a
mediator of synaptic plasticity and in cellular processes supporting
learning and memory. The modulation in Ca2þ concentration both
inside and outside the cell is a significant factor in determining the
nervous system function in both the normal as well as diseased
state. Therefore, tracking the fluctuations in the systematic con-
centration of Ca2þ can well serve as a Ca2þ signalling “tool kit” for
understanding its participation in neuronal regulation and
selective fluorescent reporters [6]. However, intrinsic depth limi-
tations associated with optical imaging and production of toxic
photobleaching byproducts of fluorescent dyes restricts these ap-
plications to superficial regions [7]. With the development of metal
selective MRI contrast agents, MRI has evolved as one of the most
promising alternative for tracking [Ca2þ] changes for biomedical
research where depth penetration is not a restriction [8]. With the
better understanding of BloembergeneSolomoneMorgan theory
[9e11], significant progress has been made in the past two decades,
towards the development of contrast agents and their applications
to provide insight into the relationship between the structure and
efficacy of a contrast agent [12e14]. For the detection of functional
activity by MRI, the main challenge lies in translating the activity at
molecular level into changes in the MR image contrast [15,16]. In
pursuit of this, conventional linear polyamine or polyaza macro-
cycle based gadolinium complexes of diethylenetriaminepenta-
acetic acid, [Gd(DTPA)(H2O)] and 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid, [Gd(DOTA)(H2O)], respectively with coor-
dinating acetate arms, have been commercially employed as first
generation approved contrast agents. These complexes are suffi-
ciently stable with one coordination site left available for
* Corresponding authors.
E-mail addresses: anupama@inmas.drdo.in (A. Datta), akmishra@inmas.drdo.in
(A.K. Mishra).
http://dx.doi.org/10.1016/j.ejmech.2014.05.046
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

226
J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
surrounding water molecules [17e19]. However, their non-specific
nature has directed the focus in designing new contrast agents with
improved magnetic properties as well as specificity. Thus the quest
for novel complexes that are able to detect particular changes in
their environment by altering relaxivity thereby generating a MRI
signal has led to the development of various MR contrast agents
that provide information about biochemical events and physio-
logical signals [20].
two nucleophilic reactive centres which were poised for intro-
duction of carboxylates moieties to bind strongly with Ca2þ metal
ion and another moiety for alkylation of secondary amine of
DO3A.
2.1. Synthesis and characterization
A synthetic strategy for calcium specific magnetic resonance
Smart contrast agents responsive to the essential metal ions of
life viz-a-viz. Ca2þ [21e23], Zn2þ [24,25], Cu2þ [26e29] and Mg2þ
[30e32] are of paramount importance for biomedical research
prompting development of many metal sensitive contrast probes.
Towards this goal, Meade and co-workers have developed gado-
linium (Gd) based smart MRI contrast agents responsive towards
calcium leading to changes in the longitudinal relaxation time of
water protons (T1) in its presence [15,33,34]. Similarly, Logothetis,
Toth and co-workers have developed agents for studying fluctua-
tion in extracellular Ca2þ levels using BAPTA (1,2-bis(o-amino-
phenoxy)ethane-N,N,N0,N0-tetraacetic acid bisamide, EDTA
(ethylenediamine tetraacetic acid), DTPA and EGTA (ethyl-
eneglycoltetraacetic acid) linked DO3A cores which show 10e40%
increase in relaxivity in response to excess Ca2þ [21,22,35]. DO3A
system conjugated with metal-selective molecular recognition el-
ements provides a promising new class of chemosensors for mo-
lecular imaging of biological systems. However, EDTA, EGTA and
their derivatives exhibit strong chelation for Ca2þ and form com-
plexes with log kCaL ~10 [36]. Consequently, for the development of
Ca2þ chelator for extracellular calcium concentration ranges,
modifications in their structure is required without compromising
the selectivity towards Ca2þ over other ions. Encouraged by the
successful development of a Gd based bis-polyazamacrocyclic non
ionic MRI contrast agent, DO3A-AME-DO3A, exhibiting enhanced r1
[37], an attempt has been made to develop a metal ion specific T1
agent derived from Gd3þ-DO3A linked to chelating iminoacetate
groups. Towards this objective, we have synthesized Gd-DO3A-
AME-NPHE which is selective towards calcium and is showing
potential influence of variation in physiological Ca2þ concentration
by reflecting changes in T1 relaxivity.
contrast agent was designed based on a framework derived from
DO3A derivative and L-phenylalanine. The synthesis of DO3A-AME-
NPHE, 2,20,200-(10-(2-(4-(2-(bis(carboxymethyl)amino)-2-carbox-
yethyl) phenylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl) triacetic acid, was achieved in multi-step reactions.
Amino bis (methylene acetates) are known to exhibit efficient
chelating properties for biologically relevant ions like Ca2þ, Mg2þ or
Zn2þ. But the studies on these type of functionalities are more
focussed for Ca2þ due to its selective binding with desirable for-
mation constants in the concentration region suitable for tracking
changes of Ca2þ [41e43]. Thus the properties of the probe were
tuned for Ca2þ sensing by appending its functionalities with
carboxylate groups. L-phenylalanine was nitrated at the para posi-
tion by carrying out electrophilic nitration with concentrated H2SO4
and concentrated HNO3 giving compound 1 in 81.0% yield. The
amino group was derivatized to introduce bis (methylene acetate)
coordinating groups which was followed by the reduction of nitro
group to the amino group using standard procedure to give com-
pound 3. Finally, reaction of compound 3 with 2-chloroacetyl
chloride gave compound 4, (2-(bis-(2-tert-butoxy-2-oxoethyl)
amino)-3-(4-(2-chloroacetamido)phenyl) propanoic acid). Among
the variety of linkers available 2-chloroacetyl chloride was used as
the source for linkage as its synthetic procedure is straightforward
and simple [44]. DFT studies [45] on DO3A-AME-NPHE were
performed to calculate the binding proximity on the basis of their
chemical potentials and its chemical hardness based on HOMO
and LUMO energy parameters with Ca2þ. It was found that with
2-chloroacetyl chloride as a linker there was an energy gap of
0.21 between these energy parameters along with a chemical
potential of ꢀ5.423 eV which suggests that 2-chloroacetyl chlo-
ride provides suitable proximity consequently augmenting the
binding properties of acetates. It also possessed desirable rigidity
and two nucleophilic reactive centres which were poised for
introduction of carboxylates moieties to bind strongly with Ca2þ
metal ion and another moiety for alkylation of secondary amine
of DO3A.
Compound 4 was then conjugated with t-Bu-DO3A yielding
compound 5 which on treatment with trifluoroacetic acid gave the
title compound 6, DO3A-AME-NPHE with a yield of 82% (Scheme 1).
Column chromatography was used for the purification of com-
pounds. Structure elucidation was done using 1H NMR, 13C NMR,
HETCOR, DEPT and ESI-MS spectrometric techniques. Finally, the
complexation of the ligand DO3A-AME-NPHE with the lanthanide
ions (Ln ¼ Gd3þ, Eu3þ) was carried out at a pH 6.5e7.0 at a tem-
perature of 70 ꢁC. After the complexation reaction of DO3A-AME-
NPHE with the lanthanide ion, the reaction mixture was passed
through chelex-100 (widely used for binding of lanthanide metals)
at room temperature to trap the free lanthanide ion and lanthanide
complex of DO3A-AME-NPHE was collected as an eluent. Absence
of free Ln3þ was checked by xylenol orange indicator [46,47]. The
lanthanide complexes of DO3A-AME-NPHE were characterized by
MS spectrometry. Characteristic isotopic pattern of peaks in the
ESI-MS for the complexes confirmed the presence of respective
lanthanide ions. The relaxometric, UV and luminescence studies
and spectrophotometric studies were carried out with metal-ligand
complex to check the potential of the developed compound as a
calcium sensing agent.
As the MRI probes must be compatible with biological systems
and their practical properties includes water-soluble, non-toxic,
and able to image extracellular and/or intracellular spaces, thus the
MRI probe discussed here has been synthesized from a biomole-
cule, an amino acid L-phenylalanine which was derivatized to
introduce iminoacetate groups, the site of recognition for Ca2þ. It
was finally linked to DO3A via a linker. The r1 was modulated by the
presence and absence of calcium ion thus inducing changes in
hydration number due to the alteration in the structure of the
agent. The selectivity and sensitivity of the system was studied in
the presence of biologically relevant alkali, alkaline earth and d-
block metal ions. Luminescence and UVeVis absorbance mea-
surements on the corresponding Eu-complex were carried out to
assess the hydration state and its variation on Ca2þ addition.
2. Results and discussion
Molecular imaging techniques are being utilized for the diag-
nosis of different diseases and identification of specific markers
upregulation during various physiological processes [38e40].
With the aim to develop a calcium specific contrast agent for
magnetic resonance imaging, a synthetic strategy was designed
based on a framework derived from an optically active amino acid,
L
-phenylalanine and DO3A derivative. L-phenylalanine being an
essential amino acid is expected to exhibit less toxicity in-vivo and
was further derivatized to a reactive group to serve the role of
bifunctional chelating agent. It possessed desirable rigidity and

J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
227
Scheme 1. Synthetic scheme for 2,20,200-(10-(2-(4-(2-(bis(carboxymethyl)amino)-2-carboxyethyl) phenylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic
acid (DO3A-AME-NPHE) and Ln-DO3A-AME-NPHE.
2.2. Relaxometric Ca2þ titration of Gd-DO3A-AME-NPHE
maximum of 60% to 6.99
.0.16 mMꢀ1 sꢀ1 at 1.0 equivalent of
calcium metal ion and levelled off with further addition. This sig-
nificant enhancement in investigations thus confirmed our pre-
sumption that in the absence of Ca2þ, the amino bis(methylene
acetate) groups exhibit weak interaction ~3e4 Å with Gd3þ
through ionic attraction leaving no space for water to interact.
However, in the presence of Ca2þ, they rearranged to bind Ca2þ thus
increasing the probability of water to bind directly to Gd3þ leading
to enhancement of r1. The sensitivity of the Gd3þ complex at
different concentration levels was also investigated for which the
titration curves were drawn by plotting the relaxivity as a function
of the Ca2þ/Gd- molar ratio. Upon addition of equimolar amount of
EDTA after titration of Gd-DO3A-AME-NPHE with Ca2þ, the relax-
Gd-DO3A-AME-NPHE was evaluated as a Ca2þ sensing MRI
agent by studying its relaxation behaviour in the absence and
presence of Ca2þ. The longitudinal and transverse relaxation rates
of the Gd-DO3A-AME-NPHE protons were measured. Gd-DO3A-
AME-NPHE
displays
a
relaxivity
(Fig.
1)
of
r1 ¼ 4.37
0.12 mMꢀ1 sꢀ1 and r2 ¼ 3.13
0.25 mMꢀ1
s
ꢀ1. On
addition of Ca2þ the relaxivity r1 gradually enhanced reaching to a
ivity of the complex returned to the initial value observed in Ca2þ
free solution, indicating the reversible nature of r1 and its exclusive
dependence on Ca2þ. Further, to check the contribution of the
COOH of -phenylalanine on the relaxometric response, methyl
-
a
-
L
ester derivative of phenylalanine was used for the preparation of
the ligand and relaxation studies were performed on its gadolinium
complex. The initial r1 and r2 values in the presence and absence of
Ca2þ ion were found to be similar with the methyl ester derivatized
ligand 7 suggestive of participation of a-COOH in the interaction/
coordination with either Gd3þ or Ca2þ was negligible and did not
contribute in relaxivity.
The relaxivity results of Gd-DO3A-AME-NPHE are found to be
better than the relaxivities of MRI contrast agents prepared from
Gd-DOPTA (500 MHz, 298 K, 3.26 to 5.76 mMꢀ1 sꢀ1), bismacrocyclic
Gd3þ chelate with a BAPTA-bisamide conjugated Gd-DO3A core
(500 MHz, 298 K, 3.35 to 3.85 mMꢀ1
s
ꢀ1), DTPA-Gd-derivative
ꢀ1). EDTA based Gd-derivative
Fig. 1. Dependence of T1 on the concentration of Gd-DO3A-AME-NPHE at pH 7.4, 4.7 T,
(500 MHz, 298 K, 4.7 to 5.4 mMꢀ1
s
27 ꢁC.

228
J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
(500 MHz, 298 K, 3.44 to 6.29 mMꢀ1 sꢀ1) exhibited 32% increase in
relaxivity in response to Ca2þ [23,24,38]. Gd-DO3A-AME-NPHE
displayed longitudinal relaxivity, r1, higher than that of
[Gd(DOTA)(H2O)]ꢀ (3.38 mMꢀ1 sꢀ1, 37 ꢁC) which can be attributed
to the introduction of rigidity due to the phenyl group in the
molecule. However it was found to be quite close to the r1
measured in the calcium sensing MRI probes (GdL1-GdL4) wherein
variation in the r1 was in response to the structure [23].
2.4. Luminescence studies
To assess the complex hydration, measurement of the radiative
rate constants for the decay of Eu3þ or Tb3þ excited state in H2O and
D2O are useful. Therefore for examining the behaviour of Gd-DO3A-
AME-NPHE, it was both prudent and informative to study the
photophysical properties of europium analogues. Eu3þ complex of
DO3A-AME-NPHE was prepared and it was assumed that the
number of inner sphere water molecules estimated for Eu3þ should
be close to the corresponding Gd3þ complexes. Luminescence
lifetime measurements on Eu-DO3A-AME-NPHE showed specific
2.3. Selectivity studies
transition of Eu3þ complex (7F0
/
5D0) at 578e582 nm. Upon
The selectivity of the MRI contrast agent is influenced by the
architecture of the molecule including the type of donor sites used
for recognition (hard/soft, neutral/charged, etc.), number of do-
nors, cavity size, and geometry which can all be tailored to match
a metal ion of choice. With no vector conjugated to the developed
agent which could aid its internalization of the cell, so with the
objective to track the changes in extracellular Ca2þ concentration,
studies were performed to confirm that the amino bis(methylene
acetates) would afford higher selectivity for Ca2þ over other
abundant extracellular metal ions by measuring its relaxivity
values in the presence of other competing, biologically relevant
metal ions with added calcium at physiological condition. The
selectivity experiments were conducted with alkali metal ions,
Naþ (10.0 mM) and Kþ (2.0 mM), alkaline, Mg2þ(2.0 mM) and d-
orbital, Zn2þ (2.0 mM) metal ions were used to check the selec-
tivity of the probe (Fig. 2). The addition the metal ions, Naþ and Kþ
led to a slight increase in the r1 from the original (<3%), however
interference of Zn2þ ion upon addition brought 6 and 8% variation
in r1 calculated in the absence and presence of the calcium. The
variation was <2 and 6% in the response with Mg2þ. Selectivity
experiments conducted proved that the ions triggered little vari-
ation in the relaxivity of Gd-DO3A-AME-NPHE and their presence
led to slight relaxivity enhancements or interfered with calcium
ion turn-on responses.
addition of calcium to the solution of Eu-DO3A-AME-NPHE
(0.04 mM) the emission band did not produce any change in its
pattern but the intensity of the band increased by 25% at pH 7.4
maintained with HEPES buffer.
The hydration number was determined in the first coordination
sphere before and after Ca2þ is added by measuring the lumines-
cence lifetimes of lanthanide complex. The hydration numbers
were calculated according to the Equation (3) [48].
ꢀ ꢁ
ꢁ
ꢂ
q ¼ A0 1 tH O ꢀ 1 tD O
(3)
2
2
where, A0 is 1.05 ms.
In the absence of Ca2þ, the luminescence
t
values were found to
be, tH O ¼ 0.618 ms and tD O ¼ 0.713 ms for 10ꢀ4 M solution of the
2
2
complex (Table 1). Luminescence lifetime remained constant over
the pH range 7.0e8.0, consistent with a single species in solution
under these conditions. The experiment was carried out in a set of
six. The time resolved luminescence experiment for the complex,
Eu-DO3A-AME-NPHE recorded in H2O and D2O shows a hydration
number (q) of 0.2. Luminescence decay constant was recorded in
presence of calcium metal ion an increase in the hydration number
(q) to 1.05 was observed with tH O ¼ 0.495 ms and tD O ¼ 0.984 ms.
2
2
The pattern of the band did not show any variation in the presence
of Kþ and Naþ ions while the intensity decreased by ~3e4% (Kþ; 3%,
Naþ;4%) in their presence.
The potential of the title compound to selectively target Ca2þ
was further demonstrated by the competitive co-binding experi-
ment involving the addition of Ca2þ to the Gd3þ complex contain-
ing Zn2þ/Mg2þ metal ion solutions. Impressively, 55% increase in
relaxivity was observed upon the addition of calcium to the solu-
tion. The selectivity studies imply that the developed MRI sensors
respond strongly to calcium metal ion thus reducing the probability
of false signals and contrast will not be altered by the high back-
ground of cations.
2.5. Kd measurements
The absorption spectrum of a solution of Eu-DO3A-AME-
NPHE shows an absorbance band at 277 nm changing linearly
with an increase in the concentration of Ca2þ up to molar ratio of
1:1 Ca2þ/Eu-DO3A-AME-NPHE and remained as a plateau upon
adding more Ca2þ (Fig. 3). Binding analyses using the method of
continuous variations obtained through UVeVis absorption
measurements, showed inflection points at a mole fraction of 0.5
for both the Gd-DO3A-AME-NPHE and calcium ion. Furthermore,
plots of relaxivity versus [Ca2þ] revealed that the observed
relaxivity reached a maximum at 1.0 equiv of Ca2þ and levelled
off at higher concentrations of added Ca2þ. These data are
consistent with a 1:1 Ca2þ/Gd-DO3A-AME-NPHE binding stoi-
chiometry, which was then assumed in all of the Kd calculations.
The dissociation constant (Kd) for 1:1 complex was estimated to
be 0.027 ꢂ 10ꢀ3 M.
Table 1
Luminescence lifetime measurements of Eu-DO3A-AME-NPHE at pH 7 and 25 ꢁC.
Ca2þ (mM)
tH ðmsÞ
2 O
tD ðmsÞ
2O
tH O=tD
q
2 O
2
Fig. 2. Selectivity studies of Gd-DO3A-AME-NPHE (0.5 mM) towards calcium in
presence of Naþ, Kþ, Mg2þ and Zn2þ (10.0, 2.0, 2.0 and 2.0 mM respectively) before and
after the addition of Ca2þ (pH 7.4, 4.7 T, 27 ꢁC).
0.0 mM
1.0 mM
0.618
0.495
0.713
0.984
0.866
0.503
0.22
1.05

J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
229
3. Conclusion
A calcium specific magnetic resonance contrast agent was
developed based on a framework derived from an optically active
amino acid, L-phenylalanine and DO3A derivative. The preliminary
results demonstrate the capability of the probe, Gd-DO3A-AME-
NPHE as a new Ca2þ responsive MRI contrast agent. The probe
exhibits a distinct Ca2þ triggered enhancement (ca. 60%) in relax-
ivity with specific selectivity towards Ca2þ over alkali (Kþ and Naþ),
alkaline earth (Mg2þ) and d-block (Zn2þ) metal cations. The hy-
dration number of analogous Eu-DO3A-AME-NPHE was deter-
mined from luminescence lifetime measurements in the absence
(q ¼ 0.2) and presence of Ca2þ (q ¼ 1.05). Visual changes in T1
phantom images with Ca2þ demonstrate the potential feasibility of
the MR sensor for molecular imaging applications.
Fig. 3. UVeVis titration of Eu-DO3A-AME-NPHE with at pH 7.2 in presence and
absence of various concentrations of Ca2þ ions (0e1.0 equivalent). On addition of Ca2þ
the relative intensity of the band increases but the spectrum hardly changed
(A ¼ Absorbance).
4. Experimental section
4.1. Chemistry
4.1.1. General
2.6. T1 weighted imaging studies
1,4,7,10-Tetraazacyclododecane was procured from Strem
Chemicals,
France.
2-Chloroacetyl
chloride,
tert-butyl-
With spectroscopic data exhibiting the turn on relaxivity re-
sponses, binding properties, metal ion selectivity and calcium
induced change in q value for DO3A-AME-NPHE, the ability of
probe was tested to detect the change in aqueous calcium using
MRI. T1 weighted images were acquired in the presence and
absence of Ca2þ at different concentration. Images were taken in
absence and presence of (1.0, 0.5, 0.2, 0.1 mM) solution of Ca2þ ion
to the solution of complex (Fig. 4). T1 weighted images acquired got
brighter upon addition of Ca2þ ions and a linear expression was
observed between the concentration of Ca2þ and brightness. The
phantom MR depicts that the title compound can detect contrast
between samples with and without added Ca2þ and readily visu-
alizes difference in Ca2þ level.
bromoacetate, trifluoroacetic acid, L-phenylalanine, nitric acid,
sulphuric acid, hydrochloric acid, triethylamine, ammonium hy-
droxide, sodium hydroxide, chelex-100, xylenol orange, palladium
over activated charcoal, celite, acetonitrile, chloroform, gadolinium
trichloride hexahydrate, europium trichloride hexahydrate, zinc
chloride, calcium chloride, potassium carbonate, sodium carbonate,
were purchased from Aldrich, Germany. Column chromatography
was carried out using silica MN60 (60e200 mm), TLC on aluminium
plates coated with silica gel 1160, F254 (Merck, Germany). HEPES
buffer (0.01 M) of pH 7.4 was used in relaxation and luminescence
studies.
NMR spectra were recorded on a Bruker Avance II 400 MHz
spectrometer. Electrospray ionization mass spectrometry (ESI-MS)
was performed on 6310 system (Agilent, Germany) with ion trap
detection in the positive and negative modes. HPLC analyses were
performed on Agilent 1200 LC coupled to UV detector (
The C-18 RP Agilent column (5
applying elution system as described. The flow rate was 3 mL/min
with mobile phase being isocratic with 20% 10 mM ammonium
acetate and 80% acetonitrile.
l
¼ 214 nm).
m
, 1 mm ꢂ 125 mm) were used
For DFT methodology, all the calculations were performed with
€
the Jaguar 7.9 quantum chemistry module of Schrodinger software.
The calculated stationary points were characterized by calculating
vibrational frequencies with the Hessian obtained during the ge-
ometry optimization. Real frequencies were obtained for optimized
structures.
4.1.2. Synthesis of 4-nitrophenylalanine (1)
In a round bottom flask, conc. H2SO4 (0.183 mol, 10.0 mL) was
taken and L-phenylalanine (0.030 mol, 5.0 g) was added slowly to
dissolve completely. The reaction mixture was cooled down to 0 ꢁC
and conc. HNO3 (0.019 mol, 1.70 mL) was added dropwise to the
reaction mixture. The reaction was stirred for 1.0 h at 0 ꢁC. The
reaction mixture was then poured into ice-cooled water (50.0 mL)
and stirred for 15 min, heated to boil and neutralized by the
addition of ammonium hydroxide solution after bringing down to
room temperature. The reaction mixture was concentrated under
vacuum and kept overnight for crystallization. The crystals formed
were filtered, washed with water and recrystallized in water-
ethanol solvent system to give product 1 (5.10 g, 81.0%) as white
crystalline solid. dH (400 MHz, D2O, Me4Si): 3.16e2.96 (2H, m, CH2),
3.52 (1H, t, CH), 7.34 (2H, d, CH), 8.17 (2H, d, CH); dC (100 MHz, D2O,
Fig. 4. T1-weighted phantom MR images of a 0.5 mM solution of Gd-DO3A-AME-NPHE
in phosphate buffered saline at various concentrations of Ca2þ. A: 0.0 mM Ca2þ, B:
0.10 mM Ca2þ, C: 0.20 mM Ca2þ, D: 0.50 mM Ca2þ, E: 1.0 mM Ca2þ
.

230
J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
Me4Si): 36.54 (CH2), 55.61 (CH), 1230.42, 130.26 (CH aromatic),
143.87,147.36 (C aromatic), 171.70 (COOH); m/z (ESI) 211.2 (MþþH
C9H10N2O4 requires 210.0). Elemental analysis calcd for C9H10N2O4:
C, 51.43; H, 4.80; N, 13.33. Found: C, 51.38; H, 4.71; N, 13.40.
CDCl3, Me4Si): 1.46 (18H, s, C(CH3)3), 2.96e3.14 (2H, dd, CH2),
3.23e3.40 (2H, q, CH2), 3.61 (1H, t, CH), 4.20 (2H, s, CH2), 4.50 (2H, s,
CH2), 7.27 (2H, d, CH aromatic), 7.49 (2H, d, CH aromatic); dC
(100 MHz, CDCl3, Me4Si): 27.93 (CH3), 38.76, 42.86, 49.12, 61.32,
61.75 (CH2), 81.30, 82.62 (C(CH3)3), 120.17, 129.43, 130.06, 135.43
(CH aromatic), 163.67, 166.47, 170.72, 172.96 (CO); m/z (ESI) 485.0
(MþþH C21H32N2O6 requires 484.2). Elemental analysis calcd for
4.1.3. Synthesis of 2-(bis-tert-butoxycarbonylmethyl-amino)-3-(4-
nitrophenyl)-propionic acid (2)
Under an atmosphere of nitrogen, 4-nitrophenylalanine (1)
(0.007 mol, 1.50 g) was dissolved in acetonitrile (15.0 mL), potas-
sium carbonate (0.014 mol, 1.95 g) was added to the solution and
stirred for 30.0 min. To the stirred solution tert-butylbromoacetate
(0.014 mol, 2.09 mL) in acetonitrile (10.0 mL) was added dropwise.
After the complete addition, the reaction mixture was refluxed for
18.0 h. The reaction mixture was cooled down to room tempera-
ture, filtered and the filtrate was evaporated under reduced pres-
sure. The compound was purified by column chromatography
(silica gel, 30% ethyl acetate in petroleum ether, Rf ¼ 0.55) to give
the final compound 2 (2.66 g, 85.0%) as yellow solid. dH (400 MHz,
CDCl3, Me4Si): 1.45 (18H, s, C(CH3)3), 3.05e3.20 (2H, dd, CH2),
3.27e3.36 (2H, q, CH2), 3.69 (1H, t, CH), 4.57 (2H, q, CH2), 7.45 (2H,
d, CH aromatic), 8.11 (2H, d, CH aromatic); dC (100 MHz, CDCl3,
Me4Si): 27.30 (CH3), 38.95, 49.46, 61.19 (CH2), 81.37, 82.40
(C(CH3)3), 123.73, 130.55 (CH aromatic), 141.32, 146.93 (C aromatic),
166.63, 170.63, 172.57 (CO); m/z (ESI) 439.6 (MþþH C21H30N2O8
requires 438.2). Elemental analysis calcd for C21H30N2O8: C, 57.52;
H, 6.90; N, 6.39. Found C, 57.48; H, 6.88; N, 6.43.
C23H33ClN2O7: C, 56.96; H, 6.86; N, 5.78. Found C, 57.06; H, 6.77; N,
5.88.
4.1.6. Synthesis of 2-(bis(2-tert-butoxy-2-oxoethyl)amino)-3-(4-(2-
(4,7,10-tris(2-tert-butoxy-2-oxoethyl) 1,4,7,10-
tetraazacyclododecane-1-yl)acetamido) phenyl) propanoic acid (5)
Under an inert atmosphere t-Bu-DO3A22 (0.001 mol, 0.50 g) was
taken in a round bottom flask and dissolved in acetonitrile
(10.0 mL). Potassium carbonate (0.0019 mol, 0.27 g) was added and
stirred for 30.0 min. To the stirred solution of reaction mixture
compound 4 (0.0015 mol, 0.71 g) dissolved in acetonitrile was
added dropwise. After the complete addition of compound 4, the
reaction was refluxed for 18.0 h. The progress of the reaction was
checked by TLC (dichloromethane: methanol/9:1). The reaction was
cooled down to room temperature and filtered. The filtrate was
evaporated under reduced pressure and the obtained residue was
purified by column chromatography (silica gel, 5.0% methanol in
dichloromethane, Rf ¼ 0.51) to give the white compound 5 (0.79 g,
85.0%). dH (400 MHz, CDCl3, Me4Si): 1.43 (54H, s, C(CH3)3),
2.77e1.73 (18H, br, CH2), 3.10e2.90 (2H, dd, CH2), 3.43e3.21 (2H, q,
CH2), 3.63 (1H, t, CH), 4.49 (2H, s, CH2), 7.06 (2H, d, CH aromatic),
7.71 (2H, d, CH aromatic); dC (100 MHz, CDCl3, Me4Si): 27.95 (CH3),
39.00, 49.04, 53.41, 55.66, 61.32, 61.75 (CH2), 81.04, 82.12 (C(CH3)3),
120.40, 129.01, 131.26, 138.13 (CH aromatic), 166.58, 167.41, 170.97,
172.95 (CO); m/z (ESI) 963.7 (MþþH C21H32N2O6 requires 962.59).
Elemental analysis calcd for C49H82N6O13: C, 61.10; H, 8.58; N, 8.73.
Found C, 61.07; H, 8.47; N, 8.79.
4.1.4. Synthesis of 3-(4-amino-phenyl)-2-(bis-tert-
butoxycarbonylmethyl-amino)-propionic acid (3)
In a three necked round bottom flask under an inert atmosphere
of nitrogen 2-(bis-tert-butoxycarbonylmethyl-amino)-3-(4-nitro-
phenyl)-propionic acid 2 (4.60 mol, 2.0 g), was taken and dissolved
in absolute ethanol (20.0 mL) with stirring. Palladium over acti-
vated carbon (10.0%, 2.30 mol, 4.87 g) was added to the stirred
solution. The hydrogen gas was purged into the solution till the
completion of reaction. The progress of the reaction was monitored
by TLC, ethyl acetate: petroleum ether; 1:1. The solution was
filtered through celite, filtrate was collected and evaporated under
reduced pressure. The compound was purified by column chro-
matography (silica gel, ethyl acetate/petroleum ether 1:1, Rf ¼ 0.50)
to give the final compound 3 (1.67 g, 90.0%) as a pale yellow solid. dH
(400 MHz, CDCl3, Me4Si): 1.44 (18H, s, C(CH3)3), 2.87e3.10 (2H, dd,
CH2), 3.26e3.42 (2H, q, CH2), 3.63 (1H, t, CH), 4.59 (2H, s, CH2), 6.62
(2H, d, CH aromatic), 7.03 (2H, d, CH aromatic); dC (100 MHz, CDCl3,
Me4Si): 28.69 (CH3), 38.33, 49.71, 61.20, 62.40 (CH2), 81.19, 82.15
(C(CH3)3), 126.09, 130.14 (CH aromatic), 145.13 (C aromatic), 166.54,
170.73, 173.21 (CO); m/z (ESI) 409.6 (MþþH C21H32N2O6 requires
408.2). Elemental analysis calcd for C21H32N2O6: C, 61.75; H, 7.90; N,
6.86. Found C, 61.72; H, 7.76; N, 6.92.
4.1.7. Synthesis of 2,20,200-(10-(2-(4-(2-(bis(carboxymethyl)amino)-
2-carboxyethyl) phenylamino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triaceticacid(DO3A-AME-NPHE,6)
The ligand 5 (0.0033 mol, 1.0 g) was dissolved in 3.0 mL of neat
trifluoroacetic acid at 0 ꢁC and stirred for 4.0 h. After that the re-
action mixture was stirred at room temperature for additional
10.0 h. The solvent was evaporated under reduced pressure and the
residue was dissolved in 1.0 mL of MeOH, followed by addition of
30.0 mL of diethyl ether dropwise at 0e5 ꢁC. The reaction solution
was stirred for 1.0 h at room temperature. The compound was
dried, dissolved in water and neutralized to pH 7.0 by the addition
of 1.0 M aqueous NaOH. The crude product was purified by pre-
parative HPLC to give compound 6 (0.58 g, 82.0%). dH (400 MHz,
D2O, Me4Si): 4.35e2.75 (18H, br, CH2), 4.51 (1H, t, CH), 7.21 (2H, d,
CH aromatic), 7.39 (2H, d, CH aromatic); dC (100 MHz, D2O, Me4Si):
34.49, 48.25, 60.34, 62.37, 65.95 (CH2), 120.60, 121.09, 130.05 (CH
aromatic), 162.31, 162.66, 163.37, 167.85, 168.53, 170.71 (CO); m/z
(ESI) 681.2 (MþꢀH C29H42N6O13 requires 682.2). Elemental analysis
calcd for C29H42N6O13: C, 51.02; H, 6.20; N, 12.31. Found C, 51.01; H,
6.12; N, 12.35.
4.1.5. Synthesis of 2-(bis-(2-tert-butoxy-2-oxoethyl)amino)-3-(4-
(2-chloroacetamido) phenyl) propanoic acid (4)
In a three necked round bottom flask, under inert atmosphere,
compound 3 (0.0012 mol, 0.50 g) was taken and dissolved in dry
acetone (15.0 mL). The reaction mixture was brought to 0 ꢁC.
Triethylamine (0.0015 mol, 205.0
mixture was stirred for 15.0 min. To the reaction flask 2-
chloroacetyl chloride (0.0015 mol, 118.0 L) dissolved in dry
mL) was added and the reaction
4.1.8. Synthesis of 2,20,200-(10-(2-(4-(2-(bis(carboxymethyl)amino)-
3-methoxy-3-oxopropyl)phenylamino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid (DO3A-AME-
NPHME, 7)
The synthetic procedures followed were similar to the pro-
cedures used in the synthesis of compound 1e6 wherein instead of
m
acetone (10.0 mL) was added dropwise at 0 ꢁC and stirred over-
night. The solvent was evaporated under reduced pressure and the
residue obtained was extracted in dichloromethane/water mixture.
The organic solvent was collected and evaporated under reduced
pressure. The compound 4 was purified by column chromatography
(silica gel, 50.0% ethyl acetate in petroleum ether, Rf ¼ 0.65) and
obtained (0.539 g, 91.0%) as a cream coloured solid. dH (400 MHz,
L-phenylalanine, methyl ester of L-phenylalanine was taken as one
of the precursors. The yield of the final purified compound 7 was
85.2%. dH (400 MHz, D2O, Me4Si): 4.32e2.81 (18H, br, CH2), 3.74(3H,

J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
231
s, COOCH3) 4.52 (1H, t, CH), 7.21 (2H, d, CH aromatic), 7.39 (2H, d,
CH aromatic); dC (100 MHz, D2O, Me4Si): 34.45, 48.25, 60.40, 62.41,
65.91 (CH2),42.01(CH3) 120.60, 121.12, 130.00 (CH aromatic), 162.41,
162.60, 163.29, 167.05, 168.33, 170.71 (CO); m/z (ESI) 696.2 (MþꢀH
substance, and [Gd] is the concentration of Gd3þ complex. Relax-
ivities, r1 and r2 were calculated from the slope of linear regression
fits of 1/T1 and 1/T2 against the gadolinium concentration.
For metal ion selectivity experiments, stock solution (1.0 M) of
NaCl, KCl, ZnCl2 and MgCl2 were prepared and appropriate con-
centrations (NaCl: 10.0 mM, KCl: 2.0 mM, MgCl2: 2.0 mM, ZnCl2:
2.0 mM) were prepared by dilution method. Solutions of Gd-DO3A-
AME-NPHE of concentration, 0.5 and 0.75 mM were used for the
selectivity studies. The titrations were performed at 4.7 T, 25 ꢁC and
pH 7.0e7.4. Metal ion solutions of known concentration were
added in stepwise portions to the complex solution and the lon-
gitudinal proton relaxation time (T1) was measured after each
addition of the analyte. The solution of Ca2þ was added in the 1:1 M
ratio and the relaxation time, T1 was again measured. The relaxivity
(r1) was calculated using the actual Gd3þ concentration each point
of the titrations.
C
30H45N6O13 requires 697.2).
4.1.9. Synthesis of lanthanide (III) complexes of DO3A-AME-NPHE
The introduction of lanthanide ion into the macrocyclic frame-
work was done at pH 7.0. To a stirred aqueous solution of ligand
DO3A-AME-NPHE (10ꢀ4 mol), a solution of 1.0 M NaOH was added
until pH 7.0 was attained. A solution of LnCl3 was prepared in water
and was added dropwise to the ligand solution in 1:1 M ratios. The
reaction mixture was heated at 65e70 ꢁC for 18.0 h and the pH of the
solution was periodically adjusted to 7.0 by addition of aliquots of
NaOH solution. After 18.0 h, the reaction mixture was cooled to room
temperature and then passed through an ion-exchange column
packed with chelex-100 resin at room temperature. The absence of
any free lanthanide (III) ion was checked by xylenol orange indicator.
The compound was lyophilized, and white solid was obtained. The
4.3. UVeVis and luminescence studies
formation of the metal complex Ln-DO3A-AME-NPHE (Ln ¼ Gd3þ
,
UVevis spectrum of the Eu-DO3A-AME-NPHE complex was
obtained on a Cary Varian double beam spectrophotometer (Cary
BIO 100, Australia at 450e600 nm). A stock solution of Eu-DO3A-
AME-NPHE of concentration 0.1 M was prepared and diluted as
per requirement. The emission wavelength for ligand was found at
348 nm while for specific transition of Eu(III) complex (7F0 / 5D0)
was found 578e582 nm.
The lifetime experiment was carried out in D2O and H2O solu-
tions in the absence and presence of Ca2þ metal ions. Luminescence
decay was recorded in the short phosphorescence lifetime mode
and was repeated 6 times under each condition. Luminescence
lifetime was calculated from the mono exponential fitting of the
average decaying data. A stock solution (0.10 M) of Eu-DO3A-AME-
NPHE complex was prepared and diluted as per requirement. The
pH of the solution was adjusted to 7.0 and the solution was stirred
overnight at 25 ꢁC.
Eu3þ) was confirmed by mass spectrometry. Calculated mass [Mþ]
834, found m/z (ESI) 833.0 (MþꢀH Gd-DO3A-AME-NPHE,
C29H37GdN6O13 requires 834), Elemental analysis calcd for
29H37GdN6O13: C, 41.77; H, 4.35; N, 10.08. Found C, 41.90; H, 4.28; N,
C
10.21. m/z (ESI) 826.0 (MþꢀH Eu-DO3A-AME-NPHE, C29H37EuN6O13
requires 827). Elemental analysis calcd for C29H37EuN6O13: C, 42.04;
H, 4.38; N, 10.14. Found C, 41.95; H, 4.28; N, 10.27.
4.2. Relaxation studies
Experiments were carried out on a 4.7 T horizontal Bruker
Biospec 47/50 (Bruker, Ettlingen, Germany) at 27 ꢁC. The mea-
surement of longitudinal and transverse relaxation rates (r1 and r2)
of the complex Gd-DO3A-AME-NPHE were carried out at different
concentrations. A set of Gd-complex solution of different concen-
tration (0.06e6.0 mM) were prepared. The total volume of the
solution was kept constant (5.0 mL) and the sampling was done by
avoiding air gap in the tube. The solutions were prepared in HEPES
buffered saline of physiological pH. A stock solution of calcium
chloride of concentration (10.0 mM) was prepared and its appro-
priate concentration was added to the Gd-DO3A-AME-NPHE solu-
tions. An incubation time of 30 min was given to the solutions prior
to the experiments. Relaxation rate measurements were performed
at 27 ꢁC on a 4.7 T MRI machine.
Longitudinal (T1) measurements were performed with an
inversion-recovery pulse sequence (increment of inversion delay:
10 ms with 456 increments) followed by a RARE imaging sequence
(RARE Factor: 16; TR/TEeff: 5000/7.7 ms; FOV: 25 ꢂ 25 mm; Matrix:
128 ꢂ 128; slice thickness: 1 mm). T1 values were measured from
six data points generated by inversion recovery pulse sequence.
Fitting of T1 value was done voxel wise on selected ROIs using
IGORpro (Wavemetric).
The pseudo-first-order kinetics were measured by putting the
conditions M >> L. Thermodynamic parameters were calculated by
using standard least-square procedures to fit the data to the
expression.
Luminescence measurements were carried out on spectrofluo-
rimeter FS920 (Edinburgh Instruments, UK) equipped with Xenon
arc lamp. The temperature of the sample holder was regulated with
a peltier cooled thermostat. Luminescence lifetime measurements
were calculated by customized integrated steady state spectroflu-
orimeter and fluorescence lifetime instrument FL900CDT (Edin-
burgh Analytical Instruments, UK). The excitation source was
hydrogen gas filled nanosecond flash lamp nF900 filled with low
hydrogen gas pressure of 0.4 bar operating at frequency of 40 KHz.
The intensity decay curves were obtained at emission maximum
and fitted as sum of exponentials as:
X
It ¼ I0
Aiexpðꢀt=tiÞ
(2)
Transverse (T2) measurements were performed with a Carr-
Purcell-Meiboon-Gill imaging sequence (TR: 5000, inter-echo
time: 5 ms, number of echoes: 128; FOV: 25 ꢂ 25 mm; Matrix:
128 ꢂ 128; slice thickness: 1 mm). The longitudinal relaxivity (r1)
and transverse relaxivity (r2) was measured from concentration
dependent relaxation times (T1 and T2) of the Gd-complex. The
effective relaxivity was determined from the equation [9e11]:
where, ti and Ai represent the fluorescence lifetime and pre expo-
nential factor for ith decay component. The luminescence decay
was noted in a short phosphorescence lifetime mode and repeated
at least five times under each condition. The luminescence lifetime
was determined from the mono exponential fitting of the average
decay data.
.
.
1
Tobs;ð1;2Þ ¼ 1 Td;ð1;2Þ þ r
½Gdꢃ
(1)
ð1;2Þ
Acknowledgements
Where, Tobs,(1,2) is the observed longitudinal relaxation time, Td,(1,2)
is the diamagnetic contribution in the absence of the paramagnetic
We are thankful to Dr. R. P. Tripathi, Director, Institute of Nuclear
Medicine and Allied Sciences, Delhi for providing the necessary

232
J. Tanwar et al. / European Journal of Medicinal Chemistry 82 (2014) 225e232
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of interest.
Appendix A. Supplementary data
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