Bioorganic & Medicinal Chemistry 24 (2016) 5663–5684

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Review article

Cyclen-based Gd3+ complexes as MRI contrast agents: Relaxivity

enhancement and ligand design
Haroon Ur Rashid a,b,⇑, Marco Antonio Utrera Martines b, Juliana Jorge b, Paula Martin de Moraes b,
Muhammad Naveed Umar c, Kamin Khan a, Hanif Ur Rehman a
a
Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar, Khyber Pakhtunkhwa, Pakistan
b
Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil
c
Department of Chemistry, University of Malakand, Chakdara, Lower Dir, Khyber Pakhtunkhwa, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Magnetic Resonance Imaging (MRI) is a noninvasive radiology technique used to examine the internal
Received 31 August 2016 organs of human body. It is useful for the diagnosis of structural abnormalities in the body. Contrast
Revised 25 September 2016 agents are used to increase the sensitivity of this technique. 1,4,7,10-Tetraazacyclododecane (cyclen) is
Accepted 28 September 2016
a macrocyclic tetraamine. Its derivatives act as useful ligands to produce stable complexes with Gd3+
Available online 1 October 2016
ion. Such chelates are investigated as MRI contrast agents. Free Gd3+ ion is extremely toxic for in vivo
use. Upon complexation with a cyclen-based ligand, it is trapped in the preformed central cavity of the
Keywords:
ligand resulting in the formation of a highly stable Gd3+-chelate. Better kinetic and thermodynamic sta-
Cyclen
Chelate
bility of cyclen-based MRI contrast agents decrease their potential toxicity for in vivo use. Consequently,
Contrast agent such agents have proved to be safest for clinical applications. Relaxivity is the most important parameter
Relaxivity used to measure the effectiveness of a contrast agent. A number of factors influence this parameter. This
Octadentate article elucidates detailed strategies to increase relaxivity of cyclen-based MRI contrast agents. 1,4,7,
Gadolinium 10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7-
triacetic acid (DO3A) are two key ligands derived from cyclen. They also act as building blocks for the syn-
thesis of novel ligands. A few important methodologies for the synthesis of DOTA and DO3A derivatives
are described. Moreover, the coordination geometry of chelates formed by these ligands and their deriva-
tives is discussed as well. Novel ligands can be developed by the appropriate derivatization of DOTA and
DO3A. Gd3+-chelates of such ligands prove to be useful MRI contrast agents of enhanced relaxivity,
greater stability, better clearance, lesser toxicity and higher water solubility.
Ó 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5664
2. Cyclen, a building block for T1 contrast agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
3. Cyclen synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
3.1. Richman and Atkins procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
3.2. Weisman and Reed synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
3.3. Athey and Kiefer method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
3.4. Cyclen synthesis through the condensation of Triethylenetetraamine and Glyxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5665
4. Relaxivity enhancement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5666
4.1. Hydration number (q) and Gd-H distance (rGdH), . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5668
4.2. Rotational correlation time ðsR Þ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5669
4.3. Water residency time (sm ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5674
5. Cyclen derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5675
5.1. DO3A based ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5677

⇑ Corresponding author.
E-mail address: haroongold@gmail.com (H.U. Rashid).

http://dx.doi.org/10.1016/j.bmc.2016.09.069
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
5664 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

5.2. DOTA based ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5677

6. Summary and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5680
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5683
A. Supplementary data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5683
References and notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5683

1. Introduction widely used in hospitals around the world to diagnose malignant

Magnetic Resonance Imaging (MRI) is a noninvasive imaging
modality used in modern medicine to examine the detailed inter-
nal structure of human body. The technique is applied to diagnose
various human abnormalities. MRI scanners use magnetic field,
radio waves and a computer to produce images of various organs,
bones, soft tissues and almost all internal parts of body. The mech-
anism involves the detection of Nuclear Magnetic Resonance
(NMR) signals emitted by protons of water and fat molecules.
Human tissues contain hydrogen atoms of water molecules at
90 M. A strong magnetic field is applied to a specific region of
interest in the body which results in the alignment of a slight
excess of proton spins with the direction of the applied field (Lower
energy state). Radio waves of certain frequency are then projected
for a short period of time on the same area of a patient’s body.
Absorption of these waves causes ‘spin flip’ of excess protons. This
phenomenon brings about a change in the magnetic moment of the
protons which then opposes the applied magnetic field (Higher
energy state). When radio frequency source is turned off, the
absorbed frequencies are emitted by tissues and the excited pro-
tons in the higher energy state relax to the lower energy state caus-
ing the alignment of their spins again with the direction of the
applied magnetic field. As a result of this relaxation, the equilib-
rium is restored. Normal human tissues have a different rate of
re-establishment of equilibrium than the diseased tissues. Cross
sectional images are produced when an intensity plot of the
received signals on gray scale is recorded. In order to improve
the detection of emitted signals, receiver coils are used around
the region of interest in human body. Thus, the scanners (com-
monly between 0.5 and 1.5 T) produce signals proportional to the
number of spins in a volume element. Signals produced by the
emitted radio frequencies are processed by computers to create
digital images. Based on this phenomenon, a physician diagnoses
a human abnormality.1–6 MRI is a useful imaging modality as it
produces three-dimensional images of the body. Unlike Computed
Tomography (CT) and X-rays imaging techniques, it does not
involve the application of strong ionizing radiations to produce
images and is, therefore, safer for patients. Furthermore, a greater
contrast between normal and diseased tissues is observed in MRI
scans compared to both CT and X-rays scans.7–10 This modality is
tumors, infarcted artery, lesions, multiple sclerosis and necrotic
tissue. Paul C. Lauterbur and Sir Peter Mansfield were jointly
awarded the Nobel Prize in Physiology and Medicine in 2003 for
their work concerning MRI.4,11–14 However, key drawbacks of this
technique include (a) weak signal intensity and (b) longer time
to acquire a scan. In order to overcome these drawbacks, high
relaxivity contrast agents are used.4,15 These agents increase the
signal intensity in MRI images by altering the relaxation times of
water protons. They create contrast enhancement between dis-
eased and normal tissues (Fig. 1) by catalytically shortening the
relaxation times of bulk water protons. Most of the contrast agents
cause shortening of spin–lattice or longitudinal relaxation time (T1
relaxation time). It represents the rate of the transfer of energy
from nuclei in higher energy spin state to the surrounding (lattice).
In this relaxation phenomenon, nuclei in the less stable higher
energy spin state (spin aligned against the applied magnetic field)
return to the more stable lower energy spin state (spin aligned
with the applied field) by releasing their energy to the surrounding
in the form of heat. Initially, NMR was performed to investigate the
structure of solids (dissolved in some suitable solvent). Therefore,
the term ‘spin–lattice’ is commonly used to represent this process.
Such a relaxation occurs along z-direction and results in the
restoration of the Boltzmann equilibrium. Use of contrast agents
also shorten the time needed to acquire an MRI scan. It has been
reported that more than 35% of the MRI scans are performed with
contrast agents.16–21
Based on the relaxation process, MRI contrast agents are
broadly classified into two categories,
(a) T1 agents: T1 agents cause greater enhancement of the longi-
tudinal (spin–lattice) relaxation rates (1/T1) compared to the
transverse (spin–spin) relaxation rates (1/T2) of water pro-
tons present in the tissues. In T1 weighted images, the relax-
ation rate of protons is increased. Consequently, water
protons appear as bright spots of greater intensity. These
agents are also termed as positive contrast agents. Gadolin-
ium and Manganese chelates represent this class of contrast
agents. Commercially available T1 contrast agents (Gd3+-che-
lates) include Magnevist, Omniscan, Prohance, Gadovist and
Dotarem.

Figure 1. Transverse NMR images (0.6 T, 24 MHz) through the brain of a patient before (left) and 3 min later (right) intravenous injection of [Gd(DTPA)(H2O)]2
(dimeglumine salt; Schering/Berlex) at a dose of 0.1 mmol/kg. Characteristic ring enhancement of a tumor (high-grade astrocytoma) is seen in the post injection image. Pulse
sequence: spin echo TR = 500, TE = 20 ms. Reproduced with permission from Lauffer, R. B., Chem. Rev., 1987, 87, 901, Ó 1987 American chemical Society.1
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684
(b) T2 agents: T2 agents have a large transverse (spin–spin)
relaxations rates (1/T2) and cause a decrease in the signal
intensity. These agents produce darker images and, there-
fore, are also called negative contrast agents. Relaxation
phenomenon is caused by the interaction between excited
nuclei and those at lower energy through transfer of energy
(of radio frequency). This class is represented by siloxane or
dextran coated super paramagnetic iron oxide (SPIO)
nanoparticles (NPs). Commercially available T2 agents
include Resovist, Feridex, Gastromark.22–25
T1 agents are preferred over T2 agents because they produce
brighter images and are easily expelled from the body via renal
system thus causing lesser toxicity.
Paramagnetic complexes of gadolinium ion have the potential to
enhance the relaxation rates of water protons. Gadolinium is
located at the center of the lanthanide series. Its trivalent ion
(Gd3+) has the maximum number (seven) of unpaired electrons
among all the lanthanide ions. Furthermore, it possesses a long
electronic relaxation time (109 s) and a large magnetic moment
(7.9 BM). These qualities enable Gd3+ ion to produce the strongest
influence on the spin–lattice (T1) relaxation times of water protons.
Thus, Gd3+ ion acts as a best relaxation agent. However, in its free
state, Gd3+ ion is exceptionally toxic for living systems as its ionic
radius (1.05 Å) is almost equal to that of the Ca2+ ion (1.12 Å). Thus
in living systems, Gd3+ ion competes with Ca2+ ion and binds to tis-
sues instead of Ca2+ ion. In order to overcome this challenge and
render Gd3+ ion safe for use as T1 agent, it is complexed with organic
ligands. Gd3+ ion forms stable complexes with large molecular
weight octadentate organic ligands. It acts as an acceptor and form
electrostatic interactions with the coordinating atoms of organic
ligands. The chelate consists of two co-ordination spheres. The pri-
mary coordination sphere consists of Gd3+ ion directly attached to
the octadentate ligand by electrostatic interactions. As the coordi-
nation number of Gd3+ ion is nine, a water molecule in the primary
coordination sphere forms ninth bond with it. Water (solvent)
molecules reside in the secondary coordination sphere and estab-
lish weak interactions with the chelated Gd3+ ion. Such a combina-
tion of Gd3+ ion and ligand results in thermodynamically stable and
kinetically inert chelates which are safe for use as T1 contrast
agents. Figure 2 shows Gd3+-based contrast agents approved by
Food and Drug Administration (FDA) for clinical use.26–33
A few of these agents are formed by the chelation of Gd3+ ion with
acyclic ligands such as Diethylenetriaminepentaacetic acid (DTPA)
and its derivatives. Second class of ligands is represented by macro-
cyclic compounds. It consists of ligands obtained by the derivatiza-
tion of the macrocyclic tetraamine, 1,4,7,10-tetraazacyclododecnae
(cyclen).29 It has been reported that gadolinium chelates of multi-
dentate macrocyclic cyclen derived ligands exhibit greater thermo-
dynamic and kinetic stability as compared to the Gd3+-chelates of
acyclic ligands derived from DTPA. Therefore, cyclen-based contrast
agents are safer for clinical use due to their lesser toxicity.34
2. Cyclen, a building block for T1 contrast agents
Cyclen (Fig. 3) is the aza analogue of crown ether. It is an impor-
tant precursor for the preparation of MRI contrast agents. Different
types of contrast agents are synthesized by the appropriate substi-
tution at its four nitrogen as well as eight ring carbon atoms. In
stable Gd3+ chelates of cyclen-based ligands, either three or all four
of the ring nitrogen atoms are attached to pendant, ionizeable
groups. These groups are capable to coordinate with central Gd3+
ion. Phosphonic and carboxylic acid represent such groups. Since
Gd3+ ion is in +3 state, therefore, its complexation with ligands
having three such groups leads to the development of neutral
5665
chelates. This combination of metal ion and ligand results in the
neutralization of Gd3+ ion. Design and synthesis of such Gd3+-che-
lates is of great significance as the side effects of the resulting con-
trast agents depends on the hyper tonicity. Furthermore, such
chelates of Gd3+ ion have been reported to show less contribution
to the total osmolality of composition. Due to their low osmolality,
they minimize the pain and tissue sloughing when injected to the
patient. Owing to these key features, cyclen is considered an
important intermediate for the preparation of effective macro-
cyclic chelates.35–37 In modern medicine, a few cyclen-based MRI
contrast agents are used for imaging purpose. These chelates
ensure safe transport of Gd3+ ion to the targeted site in patient’s
body. Furthermore, they are eliminated in bound form (Gd3+ ion
attached to the ligands) via kidneys.38
3. Cyclen synthesis
A number of synthetic routes have been reported in literature
for the synthesis of cyclen. A few important procedures are
described below.
3.1. Richman and Atkins procedure
This is one of the oldest method commonly used for cyclen syn-
thesis (Scheme 1). It involves multistep protection–deprotection
approach. An important step in this procedure is the reaction
between two precursors. A preformed salt of a tritosylamide i.e.
[N,N0 ,N00 -tris(p-tolylsulfonyl)]diethylenetriamine disodium salt) is
treated with sulfonate esters i.e. [N,O,O0 -tris(p-tolylsulfonyl)]di-
ethanolamine in N,N-Dimethylformamide (DMF). In final step,
tosyl protecting groups are removed under severe conditions. This
step is performed with 97% H2SO4. This is a useful method since
cyclen is formed in high yield and purity. However, this procedure
demands absolutely dry and pure starting materials.39–41
3.2. Weisman and Reed synthesis
This is another convenient and efficient laboratory procedure
for cyclen synthesis (Scheme 2). However, a drawback of this
method is the use of expensive starting materials (Triethylenete-
traamine and Dithiooxamide). A key step in this method is double
reductive ring expansion of tricyclic bis-amidine with DIBALH
(Diisobutylaluminium hydride) in refluxing toluene for 15 h.
Cyclen is produced in 57% yield with a purity of more than 90%.
Ethanethiol is formed as a byproduct which must be flushed from
the reaction mixture.42
3.3. Athey and Kiefer method
Athey and Kiefer reported cyclen synthesis in three steps
(Scheme 3). In first step, bis-imidazoline is produced in more than
90% yield by the condensation of triethylene tetraamine (TETA)
with N,N-dimethylformamide dimethyl acetal. In second step, a
twelve-membered imidazolinium cyclized intermediate bromide
salt is produced in 70% yield by cyclization of bis-imidazoline with
1,2-dibromoethane. Final step involves the hydrolysis of interme-
diate bromide salt with hot aqueous caustic potash (pH >12) to
produce cyclen in more than 80% yield. It is a useful synthetic pro-
cedure in laboratory as well as for plant production quantities.43
3.4. Cyclen synthesis through the condensation of
Triethylenetetraamine and Glyxol
Herve et al. reported cyclen synthesis in three steps using tri-
ethylenetetraamine as starting material (Scheme 4). The synthesis
5666 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

O
O
O
N N N
N N
O Gd3+ O
O Gd3+ O
H
O N N O
O O O O
O
O O O O H
O
H H O
[Gd(DTPA)(H 2O)]2- (Magnevist TM) [Gd(DOTA)(H 2O)] - (Dotarem TM)
H H
O
H
H
Gd3+
O
O O O O
O o Gd3+
O O O O O O
O N N N N
O O N O
N N CH3
H3 C H O N O O
H O
O P
O O O
[Gd(C20H 32N5O 9) (H 2O)](OptiMark TM)

[Gd(DTPA-diphenylcyclohexyl-
N phosphate)(H2O)](Ablavar TM)
N O
N
O
O Gd3+ O
O
O
HN O O O NH N N
O
O O
O Gd3+
H H O
N N OH
[Gd(DTPA-BMA)(H 2O)] (Omniscan TM) O
OH
O
OH
H H

[Gd(DO3A-butrol)(H2O)] (Gadovist TM)

Figure 2. Structure of clinically approved G Gd3+-based MRI contrast agents.

NH HN

NH HN

Figure 3. Structure of 1,4,7,10-tetraazacyclododecane (cyclen).

involves the formation of bisaminal as the key intermediate. Gly-
oxal is used as carbon template source. In first step, the two reac-
tants are treated at 7 °C for 2 h in acetonitrile. Cyclization of the
bisaminal intermediate is performed by treating it with 1,2-dibro-
moethane in acetonitrile at 80 °C. Finally, the deprotection step is
performed by the treatment of the cyclic intermediate with hydra-
zine monohydrate for 20 h at 100 °C.32,44
4. Relaxivity enhancement
Relaxivity is an important parameter used to measure the effec-
tiveness of a contrast agent. It is the efficiency with which the
relaxation rate of water protons is increased per unit concentration
of the contrast agent. It must be sufficiently high to cause a pro-
nounced enhancement in the relaxation rate of protons at the tar-
get tissue. However, it is vital that the enhancement of proton
relaxation rate is caused by the administration of a low dose of
contrast agent to the patient in order to minimize the toxicity. High
relaxivity and stability are the two key features considered in the
design of a new contrast agent. Contrast agents of higher relaxivity
cause greater contrast at the same doses than the contrast agents
of lower relaxivity. In vivo stability of an intravenously adminis-
tered contrast agent is related to its acute and chronic toxicity.
The dose of Gd3+ ion needed to cause an increase in proton relax-
ation rates is toxic and, therefore, its complex must not dissociate
to a considerable extent. In case of dissociation, the toxicity of free
ligand also becomes a serious risk. Furthermore, these agents must
be excreted form patient’s body within a few hours after their
administration.1,45,46 Solomon-Bloembergen theory explains the
relaxation phenomenon of solvent nuclei. Gd3+ chelate causes
enhancement to both spin–lattice relaxation rates (1/T1) and
spin–spin relaxation rates (1/T2) of solvent nuclei (water protons).
Mathematically, the observed relaxation rate (1/Ti)obs of water pro-
tons (in the presence of Gd3+ ion) is obtained by the addition of
both the diamagnetic (1/Ti)d and paramagnetic (1/Ti)p relaxation
rates.
1 1 1
¼ þ i ¼ 1; 2 ð1Þ
T i;obs T i;d T i;p
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5667

TsCl EtONa Na Na
2HN N NH2 TsHN N NHTs TsN N NTs
H NaOH EtOH
Ts Ts
diethylenetriamine
[N, N', N''-tris(p-tolylsulfonyl)]- [N, N', N''-tris(p tolylsulfonyl)]-
diethylenetriamine diethylenetriaminedisodium salt

TsCl
HO N OH TsO N OTs
H Et3N Ts
diethanolamine
[N, O, O'-tris(p-tolylsulfonyl)]-
diethanolamine
Ts Ts
Na Na DMF N N
TsN N NTs + TsO N OTs
Ts Ts N N
Ts Ts
[N, N', N''-tris(p-tolylsulfonyl)]- [N, O, O'-tris(p-tolylsulfonyl)]-
diethylenetriaminedisodium salt diethanolamine 1, 4, 7, 10-tetrakis(p-tolylsulfonyl)
-1, 4, 7, 10-tetraazacyclododecane

Ts Ts
N N NH HN NH HN
H2SO4 base
4HCl
N N HCl NH HN NH HN
Ts Ts
1, 4, 7, 10-tetrakis(p-tolylsulfonyl)- 1, 4, 7, 10-tetraazacyclododecane 1, 4, 7, 10-Tetraazacyclododecane
1, 4, 7, 10-tetraazacyclododecane tetrahydrochloride

Scheme 1. Richman and Atkin’s method for cyclen synthesis.

NH 2 NH HN N N
+ (CH3)2NCH(OMe)2
NH
dimethylformamide
NH2 H2N dimethyl acetal N N
bis-imidazoline
S NH 2 NH Triethylene tetraamine
CH 3CH 2Br + Br
S NH 2 NH2
(TETA)
Br CO 3
NH2 S EtOH K2
triethylenetetraamine
NH2 S
Dithiooxamide +
2 Br- NH HN excess KOH
N N
+
>90 oC
N N
NH HN
NH NH Br
N N 1) DIBALH, CH3Ph
reflux, 15 h Cyclen 2,3,4,5,6,7,8,8c-Octahydro-1H-4a,6a,8a-triaza-2a-azo-
niacyclopent[fg]acenaphthylene Bromide salt
2) NaF, H 2O
N N NH NH
Scheme 3. Athey and Kiefer method for cyclen synthesis.

(2,3,5,6,8,9-Hexahydrodiimidazo-
[1,2-a:2',1'-c]pyrazine)
Scheme 2. Weisman and Reed method for cyclen synthesis.
Diamagnetic component (1/Ti)d represents the relaxation rates
of solvent (water) relaxation rate in the absence of Gd3+ ion.
The unpaired electron spins of the Gd3+ ion generate a fluctuating
local magnetic field which in turn interacts with the proton nuclear
spins. This kind of dipole–dipole interaction produces paramagnetic
relaxation of water protons. Therefore, the paramagnetic
Cyclen
contribution (1/Ti)p depends upon the Gd3+ ion concentration [Gd]
and the two are linearly proportional to each other. Therefore,
1 1
¼ þ r i ½Gd i ¼ 1; 2 ð2Þ
T i;obs T i;d
Relaxivity (ri ) is thus the slope of a plot between the observed
relaxation rate (1/Ti)obs and Gd3+ ion concentration. It is usually
measured in units of mM1 s1.15,17 The contrast enhancement
ability of an agent is directly related to the increase in the relax-
ation rate of water protons in its vicinity. It includes increase in

O H

H H O N NH
N NH2 glyoxal
H2N N R1 R2
H
Triethylentetraamine CH3CN, -7 oC,
N NH
2h

1) Heat = 80 oC
N NH 2) K2 CO3 ,
N N NH HN
Br(CH2 )2Br 1) Hydrazine monohydrate
R1 R2 R1 R2 . 4HCl
2) HCl
N NH N N NH HN

bisaminal intermediate cyclic intermediate 1,4,7,10-tetraazacyclododecane tetrahydrochloride

Scheme 4. Cyclen synthesis via the condensation of glyoxal and triethylenetetraamine.

5668 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

both longitudinal (1/T1) as well as transverse relaxation rates
(1/T2). The phenomenon of paramagnetic relaxation rate enhance-
ment arises as a result of dipole–dipole interaction between water
proton nuclear spins and fluctuating magnetic field produced by
the unpaired electrons of Gd3+ ion. It consists of inner sphere and
outer sphere contributions. Inner sphere relaxivity indicates the
proton relaxation rate enhancement produced by the interaction
of the unpaired electrons spin of Gd3+ ion and protons of water
molecules directly coordinated to the Gd3+ ion in first coordination
sphere. Outer sphere relaxivity is the proton relaxation rate
enhancement produced by the interaction between unpaired elec-
tron spins of Gd3+ ion and bulk water protons. Thus, the nature of
inner and outer sphere relaxations is ‘intra’ and ‘intermolecular’
interactions respectively. In case of clinical contrast agents, contri-
butions due to inner and outer sphere mechanisms towards the
total paramagnetic relaxation enhancement are almost of the same
order. Inner sphere relaxation can be increased by slowing down
the rotation of the complex. However, it is relatively difficult to
increase outer sphere relaxation. Therefore, inner sphere relaxivity
is of more importance. The total paramagnetic relaxation rate
enhancement caused by a Gd3+ based contrast agents is expressed
in Eq. 3.


IS
OS
1 1 1
¼ þ
T i;p T i;p T i;p
In terms of relaxivities, the total paramagnetic relaxation rate
enhancement is given in Eq. 4.
Eq. 7 is the sum of dipole–dipole (through space) and scalar or
contact (through bonds) components. In this equation, ‘!I’ shows
the proton gyro magnetic ratio, g is the electron g-factor, ‘b’ is Bohr
Magneton, ‘S’ is the total electron spin, ‘rGd-H’ is the distance
between the Gd3+ center and proton of coordinated water mole-
cule, ‘A/h’ represents the scalar coupling constant between the
electron of Gd3+ center and the proton of the coordinated water
molecule, ‘sc ’ and ‘se ’ are the correlation times for dipole–dipole
and scalar relaxation respectively. Whereas‘xI ’ and ‘xS ’ denote
the nuclear and electronic Larmor precession frequencies respec-
tively (x ¼ cB, ‘B’ is the magnetic field).1,47–49 Eq. 5 indicates that
a faster exchange rate between the coordinated water molecule
residing in the first coordination sphere and those in the bulk will
result in the dependency of relaxation rate enhancement (experi-
enced by these bulk water molecules) on the longitudinal relax-
ation rate (1=T 1m ) enhancement of the coordinated water
molecule. According to Solomon–Bloembergen theory, to enhance
the proton relaxivity of gadolinium based contrast agents, such
ligands should be designed which can allow the chelate to accom-
modate a greater number of water molecules ‘q’ in the first coordi-
nation sphere. Moreover, the complexes should have a long
rotational correlation time ‘sR ’ and optimum mean residence life-
time ‘sm ’ of coordinated water molecule (Fig. 4).50–54
ð3Þ Important parameters influencing the relaxivity are discussed
in detail.
4.1. Hydration number (q) and Gd-H distance (rGdH),

r i ¼ r IS
i þ ri
OS
ð4Þ From Eq. 5, it is also evident that increase in hydration number
In the above equations ‘IS’ and ‘OS’ represent Inner Sphere and (q) will result in significant enhancement of the inner sphere relax-
Outer Sphere respectively. ivity. However, ligands which provide space for more than one
In cyclen-based Gd3+ complexes, some water molecules (in sec- water molecules in the first coordination sphere, form Gd3+ com-
ond coordination sphere) also form weak hydrogen bonds with the plexes of decreased thermodynamic stability and kinetic inertness.
ligands (e.g. with their carboxylate or phosphates groups) or to the Consequently, such contrast agents will be more toxic. Therefore,
water molecules coordinated to the Gd3+ ion in the first coordina- this procedure of relaxivity enhancement is usually not recom-
tion sphere. This type of contribution to the relaxation is termed as mended. For the analysis of inner sphere proton relaxivity of a
second sphere relaxation. It also contributes to the total relaxivity given Gd3+ complex in solution, it is essential to get information
of a contrast agent. However, it is challenging to separate and mea- about its hydration number. X-ray analysis is usually used to deter-
sure second sphere contribution. Therefore, it is either neglected or mine the hydration number of solid Gd3+ complexes. However, it is
simply considered as an increase in the outer sphere contribution. imperative to determine the hydration number of a given Gd3+
The longitudinal (1/T1) and transverse (1/T2) relaxation rates of complex in solution form as well since its solid structure does
the bulk water protons are given in Eqs. 5 and 6 respectively. not always correlate with the species present in the solution form.
Different methods for the determination of hydration number

IS  
1 1 include NMR (Dysprosium Induced Shift, DIS),18,55–57 lumines-
¼ qP m ð5Þ
T1 ðT 1m þ sm Þ

IS
P m T 2
(τ R )
1 1
2m þ sm T 2m þ Dxm
2
1
¼ ð6Þ
T2 sm ðs1 þ T 1 Þ2 þ Dx2
g
in

m 2m m
bl
m

‘q’ stands for the number of water molecules coordinated to Gd3+

Tu
ar

Contrast Agent H
ion in first coordination sphere (Hydration Number), ‘Pm’ represents
l
cu

O O
o le

Bulk Water
the mole fraction of water molecule coordinated to Gd3+ ion, ‘sm ’ is
M

O HO H H
the mean residence life time of the coordinated water molecule in O O
N N H H
first coordination sphere (it is the reciprocal of water exchange rate, H H
Gd O 1st Sphere water molecule
kex), ‘Dxm ’ indicates chemical shift difference between bound and O q
⎛ 1 ⎞
H ⎜ ⎟
bulk water molecules, ‘1=T 1m ’ and ‘1=T 2m ’ are the longitudinal and O
N N
Water ⎝ τm ⎠ O
H H
transverse proton relaxation rates of the bound water respectively. O
Exchange Rate O
H
Mathematically, longitudinal proton relaxation rate of the bound O H
water is calculated by Solomon-Bloembergen equation (Eq. 7) as H O
under. H
2 2 2   2nd Sphere Water Molecule
1 2 !I g SðS þ 1Þb 7sc 3sc
¼ þ
T 1M 15 r 6Gd—H ð1 þ x2S s2c Þ ð1 þ x2I s2c Þ Figure 4. Parameters influencing the relaxivity of [Gd(HP-DO3A)(H2O)], a cyclen-

2   based contrast agent. ‘q’ is the number of water molecule(s) (Hydration number)
2 A se directly coordinated to Gd3+ in primary coordination sphere. ‘sR ’ represents
þ SðS þ 1Þ ð7Þ
3 h 1 þ x2S s2c rotational correlation time of the contrast agents. ‘1=sm ’ stands for water exchange
rate.
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684
cence58,59 or Electron Paramagnetic Resonance (EPR) Spec-
troscopy.60–62 Clinically approved cyclen-based contrast agents
i.e. Gd-DOTA (DotaremTM), Gd-HP-DO3A (ProHanceTM) and Gd-BT-
DO3A (GadovistTM) all contain one water molecule in their first
coordination sphere.55 Toth et al. for the first time studied the
behavior of [Gd(DO3A)(H2O)2], a cyclen-based contrast agent with
q ¼ 2. They observed that water exchange rate at [Gd(DO3A)
(H2O)2] was significantly slower than at Gd3+-aqua.63 Furthermore,
the relaxivity of [Gd(DO3A)(H2O)2] is easily suppressed when coor-
dinated water molecule(s) are displaced by small ligands like
endogenous phosphate, bicarbonate, amino- or hydroxyl car-
boxylic acid.64–67 Among all reported contrast agents with q = 1,
Gd3+ complexes of DOTA derivatives based on the Twisted Square
Ant prismatic (TSA) geometry offer a better choice. In such struc-
tures, the exchange rate of coordinated water molecules is rela-
tively fast and close to optimum values (predicted from
theory).68 Eq. 7 indicates that the inner

 sphere proton relaxivity
also depends on Gd-H distance (rGdH) 1
. Therefore, this param-
r 6GdH
eter is also vital to evaluate a contrast agent. From the equation
one can conclude that a noticeable relaxivity enhancement can
be achieved by shortening Gd-H distance. Its values have been
reported to be 2.7–3.5 Å in the literature. Experimentally, it is dif-
ficult to accurately measure Gd–H distance. However, it can be
indirectly calculated from fitting Nuclear Magnetic Resonance
Diffraction (NMRD) data. Neutron diffraction on single crystals or
isotopic exchange method (in very concentrated solution of the
contrast agents) is applied for the direct determination of Gd–H
distance. Electron Nuclear Double Resonance (ENDOR) spec-
troscopy is another useful technique to calculate Gd–H
distance.17,49
4.2. Rotational correlation time ðsR Þ
Relaxivity of a contrast agent also depends on its rotational cor-
relation time ðsR Þ. This parameter indicates the rotational tumbling
times of a contrast agent. It roughly reveals the mean time a mole-
cule requires to rotate one radian. It has been reported that slowly
rotating lanthanide ions cause relatively more relaxation enhance-
ment. Therefore, coupling of a Gd3+-chelate to macromolecules (via
covalent or non-covalent attachment) results in its slower rota-
tional tumbling. Long rotational correlation time results in relaxiv-
ity enhancement as it improves the nuclear and electron spins
coupling at magnetic field used typically in MRI (0.5–3 T). Useful
examples in the literature have been reported by the attachment
of Gd3+-chelates to bulky protein molecules. However, this type
of binding can also alter the water exchange time or mean resi-
dence lifetime of the coordinated water molecule by hydrogen
bonding of macromolecules with it. Due to increase in size of the
chelate, steric blocking of the water exchange path (to the bulk sol-
vent) can also occur. Therefore, it is imperative to optimize all the
parameters including rotational correlation time for relaxivity
enhancement.69–71 Furthermore, highest relaxivity can be achieved
at magnetic fields (0.24–1.65 T) conforming to the proton Larmor
frequency of 10–70 MHz. Majority of the clinical MRI examinations
are performed at about 1.5 T (64 MHz proton frequency) field
strength. A significant decrease in rotation of the Gd3+ based
contrast agents would result in large longitudinal relaxivity
enhancement at 1.5 T. However, at higher frequencies of about
100–400 MHz (2.35–9.4 T field strength), if the rotation of the con-
trast agent is decreased too much then x2 s2c > 1 (from Eq. 7)
applies and longitudinal relaxivity will decrease with increase in
rotational correlation time Therefore, it is worth mentioning that
increase in rotational correlation time causes relaxivity enhance-
ment at lower field strengths. At higher fields, rotational correla-
tion time should have an intermediate value. For the successful
5669
binding of Gd3+ complexes to macromolecules, there must be
reactive sites like isothiocyanato-, vinyl, –COOH, –SH, –HH2, –C
(O)CH2Br etc. in the macromolecules. A common method used
for binding of DOTA derivatives to a macromolecule is the forma-
tion of one amide bond in the acetate pendant of the parent
ligands.17,55,72,73 Covalent incorporation of DOTA based ligands
into natural and synthetic polymers can also lead to a significant
increase in their size. Gd3+-complexes of such large size polymeric
ligands have been reported to show high relaxivity due to their
decreased molecular tumbling. Ye et al. successfully synthesized
large molecular weight DOTA based ligand by introducing polysul-
fides into them. In the 1st step, they synthesized N1-lysylethylene-
diamine DOTA monoamide. (Scheme 5a) In order to increase the
size of this ligand, condensation copolymerization was performed
by slow dropwise addition of 3,30 -dithiobis[sulfosuccinimidylpro-
pionate] (DTTSP) to the concentrated aqueous solution of N1-
lysylethylenediamine DOTA monoamide while stirring. The mix-
ture was stirred for 4 h at room temperature. After purification
through dialysis against deionized water and freeze–drying, a
white color macromolecular polymeric ligand (OLS) was obtained.
Subsequently, the ligand was treated with twofold excess of
gadolinium acetate in deionized water for 48 h at room tempera-
ture and pH 6 to yield a neutral polydisulfide containing macro-
cyclic Gd3+-DOTA monoamide (GOLS) with apparent molecular
weight of 23.0 kDa at 1.5 T and 37 °C. (Scheme 5b) Longitudinal
and Transverse relaxivities of GOLS were measured to be
7.20 mM1 s1 and 9.70 mM1 s1 respectively. These values indi-
cate that the relaxivity of GOLS is almost two times greater than
the relaxivity of Gd(HP-DO3A) (4.1 mM1 s1 at 1.5 T, 37 °C).74
Synthesis of another similar macromolecular Gd3+-based con-
trast agent has also been reported by Ye and his coworkers. They
synthesized (N6-lysyl)lysine DOTA monoamide and 3-(2-car-
boxyethyldisulfanyl)propanoic acid copolymers (GODC) by copoly-
merization of (N6-lysyl)lysine DOTA monoamide and dithiobis
(succinimidyl propionate) followed by complexation with gadolin-
ium acetate. The apparent molecular weight of GODC was mea-
sured to be 26.4 kDa. The longitudinal relaxivity of his
polydisulfide containing Gd-DOTA monoamide complex was
recorded to be 8.25 mM1 s1 at 1.5 T and 37 °C. This value is
approximately two times greater than the relaxivity values of both
Gd-DOTA and Gd(HP-DO3A) at the same field strength and tem-
perature. Larger molecular size of GODC contributed to its
enhanced relaxivity.75 Casali and his coworkers investigated the
relaxivity of Gd complex of DO3A-monoamide coupled to a modi-
fied dextran polymer. The complex had an average molecular
weight of 52.1 kDa and its relaxivity (in water) was measured to
be 10.59 mM1 s1 (at pH 7.4, 37 °C, 20 MHz).76 In another attempt
Corsi et al. reported the synthesis of [Gd(APIDO3ASQ)] by binding
10-(2-Ethoxy-3,4-dioxo-1-cyclobutenyl)-1,4,7,10-tetraazacyclodo-
decan-1,4,7-triacetic acid (DO3ASQ) with O-(Aminopropyl)inulin.
Complexation of large molecular weight API-DO3ASQ conjugate
with Gd3+ ion was subsequently carried out. Due to large molecular
weight of the ligand, ‘sR ’ of the resulting complex was prolonged
and consequently a dramatic increase in the relaxivity was
observed.77
Aime et al. chose a different method to lengthen rotational cor-
relation time. They reported the entrapment of GdHPDO3A (Fig. 5a)
in Apoferritin. Ferritin is a ubiquitous protein which stores Iron and
releases it in a controlled way. It possesses a spherical shell (apo-
ferritin) which contains 24 sub units, each one having a central
cavity of 7–8 nm diameter. A single ferritin molecule can accom-
modate 4500 iron atoms (in the form of ferric oxyhydroxide crys-
tal) in its empty cavities. These cavities are also suitable sites for
the accommodation of contrast agents (Fig. 5b). Aime and his
coworkers carried out the dissociation of apoferritin into its sub-
units at pH 2. Then, they allowed these subunits to assemble in
5670 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

(a) H 2N NH 2
H
N H
N
+ Boc2O MeOH Boc Boc
NaHCO3, 88%
O O
O O

H
N H
N
H
N H ethylenediamine Boc
N Boc
Boc Boc anhydrous MeOH, stir in dark, 4 d H 2N
O
N
O H
O

H
N
Boc
H
N H
N O
Boc HN
Boc DOTA-tris(t-Bu)
N
Boc H
H 2N HOBT, PyBOP, DIPEA O
O HN
N
H
N N O t-Bu
O

t-Bu O N N O t-Bu

O O

H NH 2
N
Boc

O
O H 2N
HN
TFA N
N H
Boc H
O HN
HN COOH
O t-Bu N N
N N O
O

N N
t-Bu O N N O t-Bu
HOOC COOH

O O

Scheme 5a. Synthetic scheme of N1-lysylethylenediamine-DOTA monoamide.

the presence of GdHPDO3A molecules at pH 7. The resultant solu-
tion was analyzed through exhaustive dialysis. Apoferritin was
found to contain 10 molecules of GdHPDO3A trapped inside its cav-
ities. A large relaxivity enhancement (20 times higher) was
observed for GdHPDO3A trapped inside apoferritin (80 ± mM1
s1) as compared to free GdHPDO3A in water (4.2 mM1 s1) at
20 MHz and 25 °C. Increase in spherical size of the complex caused
its slow tumbling and longer ‘sR ’ which consequently lead to
remarkable relaxivity enhancement.78,79
The choice of a macromolecule used for binding purpose
depends upon the clinical efficacy of the contrast agent instead
of the macromolecule ability to enhance the relaxivity of that par-
ticular contrast agent. Blood Pool Agents (BPAs) represent an excel-
lent class of such contrast agents. These agents are designed for
prolonged vascular retention in order to detect abnormalities in
the vascular system via Molecular Resonance Angiography
(MRA). For a prolonged retention of these agents in the circulatory
system, they must possess larger molecular weight (>20 kDa).
Preferably, these agents should be larger in size than conventional
extracellular contrast agents. Thus covalent binding of small size
Gd3+ chelates to proteins, dendrimers or polymers result in the
increase of their size and molecular weight. Prolonged retention
of blood pool agents in intravascular system prevents their leakage
into the interstitium.66,80,81 Hydrophilic compound P760 is used as
a reference for all blood pool agents (Fig. 6). It has exhibited a sub-
stantial proton relaxivity enhancement (25 mM1 s1) at 37 °C and
20 mHz with respect to parent compound [GdDOTA(H2O)]. The
relaxivity enhancement is attributed to its longer rotational corre-
lation time (sR = 2 ns at 37 °C).82
Gadomer 17 (‘Digit 17’ denotes its molecular weight) is a poly-
lysine dendrimer which represent a class of Rapid Clearance Blood
Pool Agents (RCBPAs). It is a large molecule (MW = 17.5 kDa)
derived from 1,3,5-benzene tricarboxylic core bearing 24 [Gd
(DOTA-monoamide)(H2O)] chelates (Fig. 7). Large size of the agent
has contributed to its prolonged rotational correlation time. How-
ever, the relaxivity enhancement for this agent was lower than the
expected and its r1 value was reported to be only 11.9 mM1 s1 at
40 MHz. Internal flexibility of the molecule and slow water
exchange at the 24 Gd3+ centers are likely responsible for its fairly
low proton relaxivity.83,84 Kaneshiro and his coworkers have
reported the synthesis of three generations (G1, G2 and G3), large
size, rigid (Gd-DOTA-monoamide)-poly-L-lysine octasilsesquiox-
ane dendrimers as nanoglobular MRI contrast agents. These agents
were obtained by the reaction of globular lysine dendrimers with
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5671

(b) NH2
O
O- Na +
S
O O
H2N
N +Na -O
H
O O O
HN S N
COOH O O
N N S O
O
S

N N N
O
COOH O O
HOOC
O

pH=7~8

pH=6
Gd(CH3 COO)3

O
H H
N N
S
S
n O
O
N
H
HN
COO
N N
O 3+
Gd

N N
OOC COO

O
H
H

Scheme 5b. Synthetic scheme of N1-lysylethylenediamine Gd-DOTA monoamide and dithiobispropionic acid copolymers (GOLS).

O enhancement of the resulting Gd3+ chelate of DO3APABn bound to

OH
O the size of the complex–dendrimer adducts. A significant increase
N N
OH
HO
N N
O
HO CH3
Figure 5. Structures of (a) GdHPDO3A and (b) ferritin complex.
excess of DOTA-tris(t-Bu). They had compact structures with a
cubic octa (3-aminopropyl)silsesquioxane (OAS) core and their
relaxivity was found to be size-dependent. The size of the three
agents (G1, G2 and G3) was approximately 2.0, 2.4 and 3.2 nm
respectively. Their (G1, G2 and G3) relaxivity was reported to be
6.4, 7.2, and 10.0 mM1 s1 respectively at 3 T.85
An appropriate increase in rotational correlation time has been
reported for Gd3+ chelates by incorporating them at the surface of
polyamidoamine (PAMAM) dendrimers.86 This type of conjugation
of Gd3+ complexes with the surface of the dendrimers lead to size
increase of the resultant complex–dendrimer adducts. Rudovsky
et al. successfully attached monophosphinated DOTA like ligand,
DO3APABn at the peripheries of PAMAM. (Scheme 6) The relaxivity
PAMAM dendrimer showed ‘saturation effect’ due to increase in
in relaxivity of the complex was noted due to its stiff internal
frame and slower internal mobility. The relaxivity increased from
an initial value of 20.4 mM1 s1 to about 24.8 mM1 s1 (at
25 °C, 20 MHz and pH <6).87
Conjugation of a series of high-generation (G, where G = 5, 7, 9
and 10) ethylenediamine-core-PAMAM dendrimers with bifunc-
tional Gd3+ chelate of 2-(4-isothiocyanatobenzyl)-1,4,7,10-
tetraazacyclododecane-N,N0 ,N00 ,N000 -tetraacetate (p-SCN-Bz-DOTA)
has been reported. Results indicated that complex-dendrimers
adducts ranged from an average of 127 chelates and 96 Gd3+ ions
(per G = 5 dendrimer) to an average of 3727 chelates and 1860
Gd3+ ions (per G = 10 dendrimer). Relaxivity measurement
revealed that longitudinal proton relaxation value increased from
30 mM1 s1 for G = 5–35 mM1 s1 for G = 7 PAMAM-dendrimer
DOTA-Gd and exhibited a plateau of 36 mM1 s1 for G = 9 and
G = 10 dendrimersat 20 mHz and 23 °C.88
P792 is another high relaxivity macromolecular blood-pool
agent for Magnetic Resonance Imaging (Fig. 8). This agent is based
on Gd-DOTA structure substituted by hydrophilic arms. A signifi-
cant r1 relaxivity enhancement was noted for P792 (39 mM1 s1
in water) as compared to Gd-DOTA (3.5 nM1 s1 in water) at
20 mHz. This remarkable relaxivity enhancement is attributed to
increase in molecular weight of P792 (6.47 kDa) compared to the
5672 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

COR ROC

COOH N [CH 2(CHOH)4CH 2OH]2

HOOC N N Br

R= NH HN Br

N N
Br N [CH 2(CHOH)4CH2OH]2
COOH
HOOC

COR ROC

P760

Figure 6. Structure of hydrophilic compound P760.

GlyGdDOTA
HN
GlyGdDOTA
NH
O GlyGdDOTA
HN HN GlyGdDOTA

GlyGdDOTA HN

HN GlyGdDOTA GlyGdDOTA
NH H NH
N H
N GlyGdDOTA
O O O
NH NH O
NH O
O H
O O N GlyGdDOTA
GlyGdDOTA N N
O H C NH H
N O N
HN
N
NH H

GlyGdDOTA N O HN O
GlyGdDOTA HN GlyGdDOTA
O HN O
H HN
N O NH N H
HN O N GlyGdDOTA
O H
HN HN
GlyGdDOTA NH
O GlyGdDOTA
O
NH
GlyGdDOTA HN GlyGdDOTA
GlyGdDOTA HN HN GlyGdDOTA
GlyGdDOTA
HN
NH
N O GlyGdDOTA
HN H NH
GlyGdDOTA

HN
GlyGdDOTA
Gadomer 17

Figure 7. Chemical structure of Gadomer 17.

NH 2 NH2 NH HN
H2N NH 2 HN NH
O
NH 2 HN N N NH
H2N N N HOOC P
N N
NH 2 DO3APABn OH N N N N
COOH HN
N N N N N N NH
HOOC N
H2N N
NH2 HN
N NH
N
N NH 2 1. H2O/CCl4, CSCl2, pH 2-3 (HCl), 12 h N N N
H 2N N 2. H2O, pH 9-10 (KOH), 12 h
N N N
HN NH
NH 2 N N
H2N N N
NH 2 HN NH
NH HN
H 2N NH 2
NH2

Scheme 6. Simplified representation of the reaction sequence leading to G2-PAMAM dendrimeric conjugate.
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5673

low molecular weight of Gd-DOTA (0.56 kDa). Increase in molecu- sR ¼ 4pa3 g=3kT ð8Þ
lar weight of P792 caused its slow rotation in solution. Thus, ‘sR ’ of
The above equation indicates that ‘sR ’ is directly proportional to
P792 was prolonged as a result of its slow rotation.89–91
the microviscosity of the medium ‘g’ and third power of the radius
Helbich et al. reported covalent binding of polysaccharide with
‘a’ of the spherical molecule. Whereas ‘k’ refers to Boltzmann con-
multiple Gd3+-chelates. They successfully synthesized a new con-
stant and ‘T’ shows absolute temperature. The above equation is of
trast agent, carboxymethyl hydroxyethyl starch-(Gd-DO3A)35
less importance as the exact values of ‘a’ and ‘g’ are difficult to
[CMHES-(Gd-DO3A)35]. The agent consists of a polysaccharide
determine. However, it still holds good to compare the ‘sR ’ values
backbone covalently derivatized with 35 macrocyclic chelating units
of two different Gd3+ complexes (having different molecular
and has an average molecular weight of 72 kDa. T1 relaxivity per
weights) if their densities and microviscosities are assumed to be
Gd3+ ion of this agent was reported to be 14.1 ± 0.1 L mmol1 s1
the same. ‘sR ’ values can also be determined by applying the
(at 39 °C and 20 mHz). This value is more than 4 times as compared
nuclear relaxation measurements of nuclei other than proton.
to the T1 relaxivity value measured for reference agent
Merbach et al. applied longitudinal 17O relaxation rates mea-
Magnevist92. Non-covalent interaction between Human Serum
surement (from H217O) to estimate ‘sR ’ values. The T1 relaxation
Albumin (HSA) and suitably functionalized Gd-DOTA complexes
of 17O follows both quadrupolar (1/T1q) and dipolar (1/T1d) mecha-
can also slow down molecular tumbling. HSA is the most abundant
nisms. Both these mechanisms depend on rotation.
protein in human blood plasma and it has a number of possible
binding spots for hydrophobic functional groups (long aromatic
or aliphatic substituents). Thus, it acts as a slow tumbling host
for Gd3+ complexes.93,94 Aime et al. examined such interaction
R// R/
between DOTA like Gd3+ complexes containing hydrophobic ben-
zyloxymethyl (BOM) substituents (Fig. 9) and HSA. The non-
covalent interaction between hydrophobic moieties of Gd-DOTA OOC COO

like complexes and appropriate binding sites of HSA molecules

N N
caused size increase of the resultant complexes. Slower rotational
tumbling resulted in significant proton relaxivity enhancement of Gd 3+

macromolecular Gd3+-adducts.95
N N
Recently Schmitt et al. have reported the synthesis of a poten-
tial theranostic agent, [DPP-ZnP-GdDOTA] by coupling a dike-
COO
OOC
topyrrolopyrrole–porphyrin component DPP–ZnP with Gd-DOTA
complex. They observed a considerably high longitudinal relaxivity R

(19.94 mM1 s1 measured at 20 MHz and 25 °C) for the resultant R = R/ = R// = H Gd DOTA

complex. Slow rotation of the complex due to the extended and

R= O R/ = R// = H Gd DOTA (BOM)
stiff aromatic entities incorporated in the ligand molecule resulted
in its high longitudinal relaxivity.96
R = R/ = O R// = H cis - Gd DOTA (BOM)2
The value of ‘sR ’ can be determined in a number of ways. Major-
ity of the ‘sR ’ values for Gd3+ chelates reported in the literature O R/ = H trans - Gd DOTA (BOM)2
R = R// =
have been estimated by proton relaxation measurements. How-
ever, this way of calculating ‘sR ’ value is uncertain as proton relax- R = R/ = R// = O Gd DOTA (BOM)3
ivity itself depends on several factors. Therefore, independent
determination of ‘sR ’ value is preferred. Debye–Stokes equation is Figure 9. Structures of Gd-DOTA derivatives containing benzyloxymethyl (BOM)
used to determine ‘sR ’ for spherical molecule of radius ‘a’. substituents.

O O O O
R HN NH R

N N
O O
Gd
O
N N O
HO
R HN OH
NH R
O O O O HO
OH
Na +
OH
O OH OH OH
N
H H H O Br
N O N N
R= OH
O O N Br OH
OH OH
H
Br
N
O OH OH OH
HO
OH
HO
OH
HO

Figure 8. Structure of P-792, VistaremÒ.

5674 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

1 3p2 2I þ 3 the Gd3+ center, then it unnecessarily occupies the first coordina-
¼ v2 ð1
T 1q 10 I2 ð2I  1Þ tion sphere thus delays the protons relaxation of water molecules
  in the second coordination sphere as well as in the bulk solvent.
sR sR
þ g2 =3Þ 0:2 þ 0:8 ð9Þ Majority of the Gd3+-based contrast agents reported so far have
1 þ x2I s2R 1 þ 4x2I s2R
exhibited very slow water exchange and is far from optimum
 l 2   value.69,98–102
1 2 c2I g 2 lB2 sc2 sc1 At current clinical magnetic field (60 MHz/1.5 T), short ‘sm ’ val-
¼ SðS þ 1Þ o
7 þ 3 ð10Þ
T 1d 15 r 6GdO 4p 1 þ x2s s2c2 1 þ x2I s2c1 ues (10–30 ns) of the coordinated water molecule are desirable for
large molecular weight contrast agents. This will cause consider-
where ‘I’ represents the nuclear spin,: v2 ’ is the quadrupolar cou-
able relaxivity enhancement.18,55,103 However, at higher fields,
pling constant, ‘g’ is the asymmetry parameter, ‘rGdO’ is the Gd3+ -
even shorter ‘sm ’ values (1–10 ns) are needed for such agents.104,105
O distance, ‘sc ’ is the correlation time of dipole–dipole
The potential relaxivity enhancement will be even more significant
relaxation. However, a limitation of this procedure is that the values
if ‘sm ’ values for such agents are close to that of the coordinated
of ‘v2 ’ and ‘rGdO’ cannot be determined accurately. Nevertheless, this
water molecules of [Gd(H2O)8]3+ complex (1.2 ns).106 A number
is still a useful technique for the comparison of ‘sR ’ values of anal-
of parameters influence ‘sm ’ values. These include complex charge,
ogous Gd3+ chelates. Another advantage is that the ‘sR ’ value deter-
solvent accessibility, steric hindrance in the vicinity of water bind-
mined by this method represents the rotation of the Gd3+-
ing site and water exchange mechanism. For all the ligands derived
coordinated water oxygen (Gd3+ OH2) in the first coordination
from DOTA, ‘sm ’ values also depend on the abundance of TSA
sphere of the complex. This rotation of Gd3+ ion coordinated to
(Twisted Square Antiprismatic)/SP (square Antiprismatic) iso-
water oxygen (Gd3+ OH2) is similar to the rotation of Gd3+ ion
mers.55 An effective strategy to speed up the water exchange at
coordinated to water proton which itself shows inner sphere proton
Gd3+ center is to increase electronic cloud around it. This will
relaxivity.
reduce the demand of electron density from lone pair of water oxy-
Direct determination of ‘sR ’ value is possible by 13C or deu-
gen. This effect will then result in the weakening of the electro-
terium relaxation measurements on a diamagnetic substance sim-
static attraction between Gd3+ center and coordinated water
ilar to Gd3+ chelate (Y3+, La3+ or Lu3+). However, there are two
molecule. Consequently, the dissociation of bound water molecule
major limitations of this technique. (1) It is less sensitive due to
will speed up. Therefore, water exchange will possibly be the fast-
low natural abundance of 13C and 2D and, therefore, high concen-
est for ligands containing anionic donor atoms or groups. Based on
trations of 13C and 2D labeling are needed. (2) It does not measure
this concept, it is suggested that ligands containing carboxylate as
the rotation of Gd3+ ion coordinated to water molecule. Conse-
electron donating groups will show faster water exchange than
quently, this technique is practically inappropriate for the determi-
neutral ligands such as amides or alcohols. Thus, water exchange
nation of ‘sR ’ values of large size chelates.1,18,49,97
at the Gd3+ center can be accelerated by introducing stronger elec-
In brief, rotational correlation time represents the molecular
tron donating groups in the ligand. It has been reported for Gd3+
tumbling time of Gd3+-chelates. It is considered the most impor-
complexes of DOTA and its amides that the negatively charged spe-
tant parameter in the design of new MRI contrast agents. Large size
cies exhibit much shorter water residence time (50–300 ns) than
Gd3+-chelates rotate slowly which results in their prolonged rota-
the analogous neutral (e.g. complexes of DOTA monoamides) and
tional correlation time. Consequently, such agents show a signifi-
positively charged (e.g. complexes of DOTA tetraamides) species
cant relaxivity enhancement. Based on this idea, low molecular
whose ‘sm ’ values have been reported in milliseconds. Sherry and
weight cyclen-based Gd3+-chelates are linked to macromolecules
his coworkers showed that increasing the number of ionic groups
in order to increase the relaxivity of the resultant adducts. How-
in a complex significantly shortened the mean residency time of
ever, relaxivity enhancement due to prolonged rotational correla-
water molecule. They substituted neutral amide ligating donors
tion time is effective only if all other parameters are
by anionic acetates donors (Fig. 10). Their results are shown in
simultaneously optimized as well.49,55,69 Different techniques like
Table 1.18,55,99
proton relaxation measurements, longitudinal 17O relaxation rates
measurement and deuterium relaxation measurements are used to
determine the ‘sR ’ value18,49
PO 32-
R1
4.3. Water residency time (sm ) NH
  N N
Water residency time, sm kex ¼ s1m is the third key parameter O

which influences the relaxivity. It represents the life time of water N N

molecule coordinated to Gd3+ ion in the first coordination sphere of R2 R1
the chelate. The relaxation effect is transmitted to the bulk water 1. GdDOTA-4AmP5- : R1 = R2 = CH2CONHCH2PO 32-, 2. GdDOTA-2AmP3-: R1
molecule when the coordinated water molecule is in rapid = CH2CO2- R2 = CH2CONHCH2PO 32-, 3. GdDOTA-1AmP 2- , R1 = R2 = CH2CO 2-
exchange with them. It also influences the overall ‘sR ’ which gov-
erns the nuclear and electron spins coupling. Longer ‘sR ’ values Figure 10. Structure shows the replacement of neutral amide donors by the anionic
acetates donors.
are effective and cause significant relaxivity enhancement only
when water residence lifetime of the coordinated water molecule
is optimized (sm ’ 10  40 ns). This kind of exchange at Gd3+ cen- Table 1
Increasing the number of acetates in the complex shortened the water residence life
ter occurs by the detachment of coordinated water molecule leav-
time. Reproduced with permission from Rashid, H. Ur.; Yu, K.; Zhou, J. J. Struct. Chem.
ing the first coordination sphere, changing the complex to 2013, 54 223, Ó 2013 Springer4,99
octadentate. Another water molecule from the second coordination
S. no Complex # of acetates s298
m /ls
sphere gets attached to the Gd3+ ion and thus restores its coordina-
tion number back to nine. If the coordinated water molecule does 1 GdDOTA-4AmP5 0 26
not spend enough time at the Gd3+ center, then its protons will not 2 GdDOTA-2AmP3 2 6.2
3 GdDOTA-1AmP2 3 1.3
be efficiently relaxed before the water molecule dissociates. On the
4 GdDOTA 4 0.24
contrary, if the coordinated water molecule spend longer time at
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5675

" #
Another approach to optimize mean residency time is to 1 1 ð1=sm þ 1=T 2m Þ=T 2m þ ðDxm Þ2 1
increase steric bulk in DOTA complexes around Gd3+ center. ¼ þ ð12Þ
T 2r sm ð1=sm þ 1=T 2m Þ2 þ ðDxm Þ2 T 2OS
Research suggest that substitution of acetate arms in DOTA with
more bulky groups such as phosphonate/phosphinate or introduc- Terms (1/T2) and (1/T2A) show 17O NMR relaxation rates of the
tion of a –CH2 unit in macrocyclic backbone will result in consider- paramagnetic solution and of an external reference respectively.
able shortening of mean residency lifetime of the coordinated External reference is a solution of a similar diamagnetic Gd3+ com-
water molecule. Due to the resultant steric crowding, a water plex having same concentration and pH as the sample solution of
molecule will not be able to approach the Gd3+ center close Gd3+ complex. It has been reported that in case of 17O NMR relax-
enough. Furthermore, the steric crowding will cause a decrease ation rates measurements of Gd3+ complexes containing inner
in the electrostatic interaction between Gd3+ ion and the coordi- sphere water molecules, the outer sphere contributions are negli-
nated water molecule. Consequently, the water molecule is held gible. The term ‘Dx2m ’ in Eq. 12 is also negligible compared to other
far from Gd3+ center and thus exchanges rapidly.87,107–109 quantities. Therefore, Eq. 12 on simplification can be written as,
It is important to mention that mean residency lifetime
1 1
becomes a key factor to limit or enhance relaxivity after slowing ¼ ð13Þ
T 2r T 2m þ sm
down the rotational correlation time by the covalent or non-cova-
lent binding of small Gd3+-chelates to macromolecules. The effect Since the oxygen atom is directly coordinated to Gd3+ ion,
of water exchange on relaxivity is more prominent in slow tum- therefore, the term ‘1/T2m’ is governed by scalar mechanism whose
bling large chelates as compared to fast tumbling small complexes. rate at high fields is given as,
A considerable change in ‘sm ’ values (three orders of magnitude)
2

1 1 SðS þ 1Þ A sE2
can be brought by the alteration of only one donor group of poly- ffi ¼ sE1 þ ð14Þ
amino-carboxylate Gd3+ chelates. Carvan and his coworkers inves- T 2m T 2sc 3 h 1 þ x2s s2E2
tigated the effect of single donor atom substitution on water where ‘A/h’ is the Gd–17O scalar coupling constant (3.8  106
exchange of serum-albumin bound gadolinium-1,4,7,10-tetraaza- rad s1 for the polyaminocarboxylate Gd3+ chelate having just one
cyclododecane-N,N0 ,N00 ,N000 -tetraacetato derivatives. They prepared metal coordinated water molecule). The value of Scalar coupling
a total of 38 such chelates with the same HSA-binding group and constant (A/h) in the above equation does not exhibit much varia-
measured their relaxivity at different temperature and magnetic tion for different Gd3+ chelates. However, it is suggested that its
field in the presence and absence of HSA. They further explored value should separately be calculated from chemical shift measure-
the effect of substitution of acetate with a different donor group ments for each chelate in order to ensure accurate determination of
on water exchange rate. Based on their results, they determined exchange rates. ‘xs ’ is the Larmor frequency, ‘S’ is the electron spin
an increasing order of water exchange rate for these donor groups quantum number (7/2 for Gd3+ ion) and ‘sE ’ represents the correla-
as under, tion time of the process modulating the scalar interaction.
Phosphonate  phenolate < a-substituted acetate < acetate < Water residency time (sm = 1/kex) is given by Eyring equation,
hydroxamate  sulfonamide < acetamide  pyridyl  imidazole.110,111 !
In another study, they examined the effect of different donor atoms 1 kb T DSy DHy
¼ kex ¼ exp  ð15Þ
on the water exchange rate of Gd3+ complexes with cyclen-based sm h R RT
ligands. They prepared 20 Gd-DOTA like chelates and measured
their relaxivity in the presence and absence of HSA at variable tem- where (DSy ) and (DHy ) are the activation entropy and the activation
perature and magnetic field. It was reported that changing two enthalpy respectively. Table 2 shows ‘sm ’ values of cyclen based
acetate groups to acetamides decreased the water exchange rate Gd3+-chelates.18,49,50,103,113–115
while changing third acetate group to phosphonate resulted in
the increase of water exchange rate. It was concluded that water 5. Cyclen derivatives
exchange at the Gd3+ center can be predictably tuned by the selec-
tion of donor groups and hence relaxivity can be enhanced.69 Siri- Contrast agents of high thermodynamic stability and kinetic
wardena-Mahanama and Allen have written a comprehensive inertness are desirable in order to minimize the potential in vivo
review on strategies to optimize the water exchange rates of lan- toxicity. Cyclen-based chelates have exhibited better thermody-
thanides based chelates. Based on the coordination chemistry of namic stability and kinetic inertness. Therefore, such chelates are
lanthanide chelates, they concluded that certain conditions are less toxic for in vivo use. However, continuous research is needed
necessary for optimum water exchange rates of such species. These
include, the chelates should (a) undergo associative water
exchange (b) have steric crowding around paramagnetic center
Table 2
(c) be negatively charged (d) have side chains with non-polar sub- Water exchange lifetime at 25 °C for cyclen-based Gd3+ chelates as determined from
stituents (e) have large TSA/SP isomers ratio for DOTA-type the analysis of the temperature dependence of water 17O transverse relaxation rate.
complexes.112 Reproduced with permission from S. Aime, M. Botta, M. Fasano, E. Terreno, Chem. Soc.
The water exchange rate at the Gd3+ center can be estimated Rev. 27 (1998) 19–29, Ó 1998 American chemical Society [Ref. 103]
through 17O NMR by the measurement of transverse relaxation Ligand Q Charge sm /ns
rate of coordinated water molecule in the presence and absence
DOTAa 1 1 244
of Gd3+ complex. Swift and Connick equation is used to describe HP-DO3Ab 1 0 350
relationship of paramagnetic Transverse 17O relaxation rate DOTMAc 1 1 68
enhancement (1/T2r) with ‘sm ’ and relaxation rate of the coordi- DTMAd 1 3+ 19,000
DOTA(BOM)3e 1 1 77
nated water molecule (1/T2m).
a
1,4,7,10-Tetraazacyclododecan-1,4,7,10-tetraacetic acid.
" # b
10-(2-Hydroxypropyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid.
2
1 1 Pm 1 ð1=sm þ 1=T 2m Þ=T 2m þ ðDxm Þ 1 c
(a,a00 ,a00 ,a000 )-Tetramethyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic
 ¼ ¼ Pm þ acid.
T 2 T 2A T 2r sm ð1=sm þ 1=T 2m Þ2 þ ðDxm Þ2 T 2OS d
1,4,7,10-Tetrakis-[(N-methylcarbamoyl)methyl]-1,4,7,10-
ð11Þ tetraazacyclododecane.
e
1,4,7,10-Tetraazacyclododecan-1,4,7,10-tetraacetic acid-tris-benzyloxymethyl.
5676 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

H
COOH
COOH COOH

N N N N

N N N N

COOH COOH COOH COOH

DO3A DOTA

Figure 11. Structures of DO3A and DOTA.

COOH COOH COOH

HOOC COOH
H3 C CH3 N N
N N HOOC
N N
N N O
N N HOOC OH
H3C H DO3A-L2 HN N N
(2) OH
COOH DO3MA O HOOC COOH
(1) DO3ACE
O N (3)
O N
PO(OH)2
HOOC HOOC
N N N N HOOC
COOH N N

HOOC N N COOH N N
N N
HOOC COOH HOOC COOH
DO3AP
DO3ApyNO-C (5) DO3ApyNO
(4) (6)

HOOC COOH HOOC COOH

N N HOOC COOH
N N
N N

N N O O
HOOC N N N N
P S P
COOH HOOC N
OH HO
N N
H H HO
N
(DO3APABn )2CS DO3APhenOH
(7) (8)

HOOC COOH
HOOC COOH HOOC COOH
N N
N N N N
OH
N N N N N N
HOOC HOOC HOOC
OH OH
HO
HP-DO3A DO3ABn
DO3A-butrol
(9) (10) (11)
HOOC COOH
HOOC COOH COOH
HOOC N N
N N N N
COO O
PO3 H2
N N
N N NH2 N
N N N HOOC H
HOOC PO3 H2
HOOC COO BPAMD
DO3A-pic
(12) α
DO3A-N- -aminopropionate
(14)

(13)

HOOC COOH
HOOC COOH
N N
N N
PO3H2
N O
N N N
HOOC
HOOC R
ODOTRA
DO3A-L1 O
(15)
R= H, CH2 PO3H2 or CH2 COOH
(16)

Figure 12. DO3A like ligands.

 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684
to design new contrast agents of improved properties due to their
growing demand in the field of MRI. Improvements in the specific
biological and physiochemical properties of these agents through
synthetic modifications will enable them to bind to a particular
macromolecule or localize at a specific tissue. This idea is useful
to extend the scope of their future applications.116–118
For the synthesis of novel ligands, cyclen is considered an
attractive precursor due to its cavity size, facile synthetic methods
and a range of methodologies for functionalization. Incorporation
of acetate pendant arms into cyclen ring makes it a strong binding
ligand which is then capable to coordinate effectively with Gd3+
ion. DO3A and DOTA are the two important ligands which serve
as a base for MRI contrast agents (Fig. 11).117 A number of other
cyclen based ligands with different functional groups such as alco-
hols, amides, phosphinic or phosphonic acids were also reported
and their complexes with Ln3+ were investigated.55
5.1. DO3A based ligands
1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid (DO3A) is a
tri-N-alkylated cyclen based heptadentate ligand which is of enor-
mous importance in the design and synthesis of MRI contrast
agents. It forms a chelate containing two water molecules coordi-
nated to Gd3+ center. In DO3A, three carboxylates occupy three
N-positions of cyclen ring. These groups are used for coordination
with Gd3+ ion. The remaining secondary amine in the ring is free
and is derivatized through a variety of synthetic modifications to
prepare a range of octadentate ligands for bifunctional, smart
(responsive) and targeted contrast agents. Thus, a wide range of
contrast agents of improved lipophilicity, hydrophilicity, biodistri-
bution and tissue selectivity can be prepared.55,119–121 Figure 12
shows DO3A like ligands.
1,4,7-Tris-(tert-butoxycarbonylmethyl)-1,4,7,10-Tetraazacyclodo-
decane, hydrogen bromide (DO3A-tri-t-butyl ester) is a protected
form of DO3A related compounds (Fig. 13). Besides a ligand itself,
it also serves as a starting material for the synthesis of a wide range
of DO3A based ligands.38
Majority of the research groups apply protection-deprotection
strategy to synthesize novel DO3A based ligands.122–132 They treat
cyclen with tert-butyl bromoacetate to produce DO3A-tri-t-butyl
ester. In this protected form of DO3A, three secondary amine posi-
tions are occupied by tert-butoxycarbonyl methyl moieties and
thus are not available for substitution. Numerous functional groups
are incorporated at the free macrocyclic nitrogen. Electrophiles
derived from halides (preferably bromide and chloride) are more
appropriate for substitution at fourth NH position of DO3A-tri-t-
butyl ester. It has been reported that three tert-butyl protective
moieties (tert-butoxycarbonylmethyl arms) are deprotected to car-
boxymethyl groups by the treatment of the ligands with trifluo-
roacetic acid (TFA) in dichloromethane at room temperature.
Ligands synthesized via this methodology are then complexed with
Gd3+ ion to obtain their chelates.133–137
Li and Wong have described a convenient and straightforward
methodology for the synthesis of DO3A derivatives. The synthesis
O with Gd3+ ion coordinated to the eight donors of the ligand (includ-
H
N N
O
.HBr
N N
O O
O DOTA is another important ligand derived from cyclen. It is syn-
O
Figure 13. Structure of 1,4,7-Tris-(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-
cyclododecane, hydrogen bromide.
5677
is performed in two steps, (a) First step involves direct mono alky-
lation of cyclen under mild reaction conditions resulting in the for-
mation of a variety of mono-N-alkylated cyclen derivatives. (b) In
2nd step, mono-N-alkylated cyclen derivatives were subsequently
derivatized at their remaining three secondary amine positions
through tert-butyl bromoacetate.138 Massue et al. have also
described selective N-alkylation of cyclen in a single step. They
used a stoichiometric ratio of 1:4 (Alkyl halide/cyclen). Excess
cyclen was successfully recovered via aqueous extraction and
reused. Selective mono-N-alkylation of cyclen followed by deriva-
tization of remaining three secondary amines using tert-butyl bro-
moacetate appears to be a simple and convenient method to
produce DO3A derivatives. However, the major limitation of this
methodology is the formation of different byproducts which then
require tedious and time consuming purification procedures.139 A
few other synthetic strategies to prepare DO3A derivatives have
been described in a recent review article by Cakic et al.140
Gd3+ complexes of DO3A derivatives commonly exist as capped
SA isomers in their solid state. The distances between the planes of
four ring nitrogen and three carboxylate oxygen are about 2.35 Å
while all coordinate bond lengths are in normal range. Chang
et al. isolated Gd (DO3A) complex as a hydrated trimer in solid
state. X-ray single-crystal analysis indicated that the complex
exists as [(Gd(DO3A)}3Na2CO3]17H2O. Each Gd atom in the trimer
is coordinated to four ring nitrogen, three oxygen of the carboxyl
moieties and two oxygen of the carbonate ion. Thus, none of the
three Gd atoms is coordinated to any water molecule in the solid
trimer of Gd(DO3A).141 X-ray structure analysis of Gd3+ chelate of
ligand DO3MA (ligand 1, Fig. 12) has shown that it exists as dimer
[(DO3MA)Gd] [(DO3MA)Gd(H2O)2] in solid state. Both the gadolin-
ium atoms form nine coordinate bonds. It was further reported
that the dimer is a mixture of capped SA and capped TSA isomers.
Capped SA isomer has two water coordinated water molecules
while in capped TSA isomer, a directly coordinated water molecule
is absent since bidentately coordinated acetate moieties (from the
SA unit) reside at the two free coordination spots.142 The crystal
structure analysis of Gd(HP-DO3A) indicates that it exists in both
capped SA and TSA geometries and Gd3+ ion forms nine coordinate
bonds with different donating groups of the ligand. Apart from
three carboxylate oxygen and four ring nitrogen, oxygen of the
hydoxylalkyl group and a water molecule are also coordinated to
Gd3+ ion.143 Another similar complex Gd(DO3A-butrol) exists only
in capped TSA geometry having Gd3+ ion coordinated to eight
donating groups of the ligand and an oxygen of the bridging car-
boxylate group of the adjacent complex.144 ODOTRA(1-oxa-
4,7,10-triazacyclododecane-4,7,10-triacetate) is a ligand similar
to DO3A. (Ligand 15 in Fig. 12) Its structure shows that one NH
group in the macrocyclic ring has been replaced by ether oxygen.
X-ray crystal analysis of Gd(ODOTRA) reveals that it exists in
capped SA geometry and Gd3+ ion is nine coordinate. It is coordi-
nated to oxygen and three nitrogen of the macrocyclic ring, three
oxygen of the carboxylate groups and a water molecule. The ninth
coordinate bond is formed by oxygen of the bridging carboxylate
from a neighboring molecule.145 In DO3A-L2, one of the DOTA acet-
ate moieties has been substituted with an amide hydroxyl group
and its a-carbon is derivatized with benzyloxy functionality. Its
complex Gd(DO3A-L2) is reported to be in capped TSA geometry
ing oxygen atom of the amide group) and a water molecule.146
5.2. DOTA based ligands
thesized by the substitution of four secondary amine hydrogen
(–N–H) of the cyclen ring with acetic acid (–CH2COOH) moieties.
DOTA is an octadentate ligand which utilizes its four ring nitrogen
5678 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

atoms and four carboxylate groups for attachment with Gd3+ ion.
DOTA and its derivatives form gadolinium complexes of high
kinetic inertness and thermodynamic stability as all their donor
atoms effectively enclose the Gd3+ ion. Thus, Gd3+ chelates of DOTA
and its derivatives find useful applications as MRI contrast
agents.147–149 Stetter and Frank for the first time reported the syn-
thesis of DOTA by carrying out tetra N-alkylation of cyclen.
(Scheme 7) They treated cyclen with chloroacetic acid in an aque-
ous alkaline medium. This is still a widely applied methodology for
the synthesis of DOTA. Other haloacetic acid (like bromo and iodo)
have also been reported to react with cyclen to yield DOTA.150
In recent past, research was mainly focused on the modification
in the pendant arms of DOTA to produce next generation contrast
agents. Consequently, a large number of functional MRI contrast
agents comprising targeted, pH sensors, metal sensors and multi-
merics were reported. A range of functionalities such as triazoles,
pyridyl, amides and malimides have been used as side arms for
the synthesis of DOTA derivatives. Research has shown that high
thermodynamic stability and kinetic inertness are maintained by
the DOTA derivatives synthesized by the incorporation of various
functional groups as side arms in parent DOTA ligand.151 Figure 14
shows DOTA based ligands.
HOOC
NH HN N N
XCH2COOH, X=Cl, Br, I
K 2CO3, MeCN/DMF
NH HN N N
Based on the methodology used for the preparation of DOTA
derivatives, they are classified into two categories: (1) N-function-
alized DOTA derivatives (2) C-functionalized DOTA derivatives.
N-Functionalized DOTA derivatives are prepared via mono alky-
lation of cyclen with a particular group. Subsequently, another
group is used for the alkylation of the remaining three secondary
amine groups. Another strategy used to prepare N-functionalized
DOTA type ligands include the derivatization of the only free sec-
ondary amino group on the protected DO3A-tri-t-butyl ester.140
Suchy and Hudson have written a comprehensive review article
on the preparation of N-functionalized DOTA derivatives using
acyclic precursors via 2+2, 3+1 and 4+1 cyclization processes. How-
ever, these traditional methodologies are not preferred anymore as
cyclen is now commercially available. Intermolecular or
intramolecular cyclization of suitable precursors is a useful strat-
egy for the preparation of C-functionalized DOTA derivatives.152
For example, Moi and Meares reported the synthesis of C-function-
alized cyclen by intramolecular ring closure of tosylamide at high
dilution, using concentrated sulfuric acid for subsequent detosyla-
tion. (Tosylamide was prepared by the reflux of tetrapeptide with
BH3.THF followed by tosylation with p-Toluenesulfonyl chloride).
C-Functionalized DOTA derivative was finally obtained by the alky-
lation of the C-functionalized cyclen with bromoacetic acid in
water (Scheme 8).153
COOH The above synthetic methodology was further improved by car-
rying out the ring closure of diamine with suitable protected amino
disuccinimido ester at 90 °C in dioxane (Scheme 9).154
Generally, N-functionalized DOTA derivatives are chosen for

HOOC
their role as MRI contrast agents due to their more convenient syn-
COOH
thesis as compared to the C-functionalized DOTA derivatives. PA-
Scheme 7. Synthesis of DOTA. DOTA is one of the first bifunctional chelating agents developed

O O
O O
(HO)2OP PO(OH)2
HO P P OH
N N H2N N N NH2 N N R
R
N N N NH 2 R
H 2N N R
(HO)2OP PO(OH) 2 N N
HO P P OH
DOTP O
DOTAM O O DOTPR O
(1) (2) (3)

HOOC COOH COOH

HOOC COOH HOOC
N N
N N N N

N N
HOOC N N N N
COOH
HOOC COOH HOOC COOH
DOTA-pNB
(4) TRITA TETA
(5) (6)

NO2

HOOC HOOC COOH

HOOC COOH HOOC
N N N N
HOOC COOH
N N
N N NO2
N N
COOH HOOC COOH
N N HOOC
HOOC COOH (TE)2DOTA NO2BnDOTMA
(8)
(9)
HOOC HOOC
TCE-DOTA HOOC COOH
N N
(7)

N N COOH
HOOC

DOTASA
HOOC (10)

Figure 14. Structures of DOTA like ligands.

 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5679

O O O

OH N N OH
N N BH3.THF NH
H H H
O NH H
Reflux, 31 h
NH2
R
R NH2 .TFA (2)
(3)

Ts Ts
N N OTs N N
NTs Cs2C03 in DMF
TsCl, CH3CN/Et 3N Ts Ts
8 h, Room Temp. 5 h, 60oC
NHTs
R (4) R N N
Ts Ts
(5)

H H HOOC COOH
N N N N
96% H 2S0 4, 16 equiv of Phenol BrCH 2COOH

48 h, 100oC 3 h, 70 oC, pH 10
R N N R N N
H H COOH
HOOC
(6) (1)

Scheme 8. Synthetic strategy for the preparation of C-functionalized DOTA derivative.

O O
O O
O N HN R-
O H2N R/
N OR
Et 3N
+
Dioxane, 90oC, High Dilution N HN
N H2N O
OR
O
(3) O O
O (2) O (4)
O
NO2

Where R = N and R- =

O
HOOC COOH
NH HN R- N N R-
(1) HCl/dioxane(2) BF3. THF BrCH 2COOH

NH HN N N

COOH
HOOC
(5)
(1)

Scheme 9. Synthetic route for C-functionalized DOTA derivative by the cyclization of a diamine and BOC-protected amino disuccinimido ester.

by Chappell et al. The preparation of the ligand is initiated by

monoalkylation of cyclen with an alkyl halide of the N-func-
HOOC COOH
HOOC COOH N N tionalization pendent group (methyl(D,L)-2-bromo-4-(4-nitro-
N N
phenyl)butanoate). The remaining three secondary amine groups
are subsequently alkylated with ethyl bromoacetate. The interme-
N N N N diate thus formed contains a p-nitrophenyl moiety which is then
COOH
HOOC COOH
HOOC modified to isothiocyanate through a series of reactions. The isoth-
NH iocyanate functionality of PA-DOTA enables it to undergo further
O synthetic modifications and couple to primary amines (‘ligand a’
O in Fig. 16).155 A number of N-functionalized DOTA based ligands
having self-immolative arms have been reported through a similar
SCN methodology. Ln3+ Complexes of these ligands were evaluated as
PA-DOTA potential enzyme responsive MRI contrast agents (‘ligand b’ in
X
Fig. 15).156
(a) X-Y = ß-D-galactopyranoside Y Lately, a bifunctional chelating agent, DOTAGA-Anhydride
(b)
(GA = Glutaric Acid) has been reported to be a useful building block
Figure 15. Examples of N-functionalized DOTA ligands. for the preparation of several DOTA like ligands carrying various
5680 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

m better choice than their respective SA type isomers. Another

M prominent aspect of Gd(DOTA)-like chelates is their ability to exist
SA Δ(λλλλ) TSA Δ(δδδδ)
in several dynamic isomeric forms in aqueous solution. These iso-
O
O mers may exchange/interconvert on the NMR time scale produced
either by the rotation of the acetate moieties or by the conforma-
O N N
tional interconversion of the –CH2 groups belonging to cyclen ring.
N N Ring Inversion It is important to mention that long electronic relaxation time of
Ln
O Ln O Gd3+ ion inhibits the NMR spectra of its complexes to be recorded.
N However, 13C and 1H NMR spectra of other related Ln3+ chelates are
N O
N N used to determine the solution structures of Gd(DOTA)-like
chelates.160,161 At room temperature, the 13C and 1H NMR spectra
O of [Ln(DOTA)] complexes consist of two sets of peaks confirming
O
the presence of two interchanging structural isomers. In the spec-
Arm Rotation tra of Ln3+ ion complexes, the Lanthanide Induced Shifts (LIS) val-
ues of one isomer are greater than the other. The correlation of
Arm rotation
temperature with NMR spectral features further reveals the rigid-
O ity of the macrocyclic ring in the Ln(DOTA)-like complexes. Due
to large size of Ln3+ ion, it cannot fit into the internal cavity of
O
the ring and, therefore, resides out of it. In this case, Ln3+ ion is
coordinated to four ring nitrogen and four oxygen of the carboxy-
O
Ring Inversion N N late moieties. All the –CH2 groups of the macrocyclic ring adopt
N N
O Ln O same gauche conformations having either ‘k’ or ‘d’ configuration.
Ln
Consequently, two possible square conformations [3,3,3,3] of the
N
O N macrocyclic ring (kkkk and dddd) arise. Two possible alignments
N N
(D or K) also arise for the pendant acetate arms. As a result, in
the solutions of Ln(DOTA)-like complexes, four stereo isomers
O
O can form either through cooperative ring inversion (k
d) or via
concerted rotation of the pendant acetate arms (D
K). These
TSA Λ (λλλλ) SA Λ(δδδδ) stereoisomers exist in two pairs of enantiomers i.e. Ddddd=Kkkkk
m M and Dkkkk=Kdddd.
It is evident from Figure 16 that exchange of SA and TSA isomers
Figure 16. Possible stereo isomers of [Ln(DOTA)] complexes. The coordinated occurs when either of the two phenomena (ring inversion or pen-
water molecule is omitted for simplicity (Readapted with permission from dant acetate arm rotation) is carried out alone. However, combined
Hermann, P.; Kotek, J.; Kubíček, V.; Lukes, I. Dalton Trans., 20N-func08, 3027, Ó
execution of the two processes (either in succession or concur-
2008 Royal Society of Chemistry).
rently) causes exchange of the enantiomeric pairs (Fig. 16).
2D NMR studies of the two enantiomers in solution indicate a
functionalities such as thiol, esters, isothiocyanate and amino resemblance in the vicinal couplings in the ethene bridges. Based
acids. A number of nucleophiles can selectively open the precursor on this finding, it was deduced that the two isomers have the same
(DOTAGA-anhydride) to produce a variety of N-functionalized macrocyclic ring and their pendant acetate moieties have different
DOTA derivatives (Scheme 10). Such DOTA based ligands are useful orientations. The angle (x) formed by the mutual rotation of N4
as their reactive sites can link to macromolecules resulting in their and O4 planes is different for the two isomers. SA isomer is formed
size increase. Thus, contrast agents of high relaxivity can be in the Dkkkk=Kdddd pair by an approximate rotation of 40°. [Gd
obtained on the complexation of the resulting adducts with Ln3+ (DOTA)(H2O)] solution contains a higher percentage of this iso-
ion.157 mer and, therefore, it is termed as ‘MAJOR’ (M) isomer. In
C-Functionalized DOTA derivatives containing isothiocynate Ddddd=Kkkkk enantiomeric pair, TSA or ‘minor’ (m) isomer is pro-
pendant moiety also couple to macromolecules. Gd3+-chelates of duced by a rotation of about 24° (Fig. 17).55,162
such ligands link to the terminal –NH2 moiety of the dendrimer
producing large size contrast agents (Scheme 11).158 6. Summary and future prospects
X-ray solid structure of [Gd(DOTA)] reveals that the arrange-
ment of macrocyclic ligand around central Gd3+ ion results in SA MRI is a noninvasive investigation used in medicine for the
geometry. Four nitrogen of aza crown and four carboxylate oxygen diagnosis of various human abnormalities. It is based on the detec-
form N4 and O4 parallel planes respectively. The twist angle (h) tion of NMR signals emitted by the water protons in human body
between two square planes is approximately 39°. The coordinating placed in a strong magnetic field. Contrast agents are used to
water molecule resides in a capping position above the O4 plane of increase the sensitivity of MRI technique. With the availability of
the four carboxylate oxygen atoms.159 Crystal analysis of Gd3+-che- scanners of high field strengths, use of MRI contrast agents has
lates of DOTA derivatives further indicated that diethylenetriamine become an essential component of a comprehensive scanning pro-
groups (bound to Gd3+ ion) exist either in kk or dd conformations. tocol. Majority of these agents are stable Gd3+-complexes of
(Scheme 12) Such chelates have minimum steric interactions. organic ligands. Cyclen is a macrocyclic tetra amine which acts
In solution state, [Gd(DOTA)] can exist in either of the two as a fundamental precursor for the synthesis of MRI contrast
coordination geometries i.e. SA or TSA. The two structures differ agents. Cyclen-based macrocyclic ligands form chelates of high
in the twist angle between the square planes of the antiprism. thermodynamic and kinetic stability with Gd3+ ion. Therefore,
Coordination geometry of Gd(DOTA)-like chelates also influence use of such chelates as MRI contrast agents is preferred. A number
the water exchange at Gd3+ center. It has been reported to be much of strategies have been reported for the synthesis of cyclen. Rich-
faster for TSA structure than for SA geometry. Therefore, for relax- man and Atkins methodology is widely applied due to its cheaper
ivity enhancement, TSA isomers of Gd(DOTA)-like chelates offer a starting materials and high product yield. The effectiveness of a
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684 5681

O COOH

N N N
H COOH

N N COOH
O2N HOOC
Cl
-
O NH2 H
N+
NH2 HCl
O
O

COOH O O
HOOC O HOOC COOH
N N O
N N
HOOC
N N Cl HS
N N NO2 H NH2 N N
HOOC COOH COOH
HOOC
N N
HOOC COOH
O DOTAGA–anhydride O
NH NH
O HO
O HS

O2N HOOC COOH

N N

N N
HOOC COOH

O
O

Scheme 10. Reaction of DOTAGA-anhydride with various nucleophiles to produce N-functionalized DOTA ligands.

OOC
COO OOC
COO

N N N N
PAMAM
Gd3+ Gd 3+
N N
N N S
COO
OOC
COO N N PAMAM
OOC

NCS n

Scheme 11. Gd3+ chelate of C-functionalized DOTA derived ligand to coupling to PAMAM.

contrast agent is measured in terms of its relaxivity. Contrast
agents with enhanced relaxivity are highly desirable since they
increase the sensitivity of MRI by causing greater contrast between
normal and diseased tissues. The use of high relaxivity contrast
agents also ensures lesser toxicity to the patients due to their smal-
ler dose. Thus, continuous efforts are being made to synthesize
contrast agents with enhanced relaxivity. Increasing the hydration
number (q) of the Gd3+ complexes results in significant relaxivity
enhancement. However, complexes with more than one coordi-
nated water molecule (q > 1) show a significant decrease in stabil-
ity. Research is being conducted to prepare stable cyclen-based
Gd3+ complexes with q = 2. However, it is necessary to carefully
test their in vivo stability to ensure their future use in the field
of MRI. Another approach towards relaxivity enhancement is to
slow down the rotation of the Gd3+ ion. Covalent or non-covalent
coupling of cyclen-based Gd3+ chelates to macromolecules slow
down their rotational tumbling and consequently lead to relaxivity
enhancement. Optimization of water exchange rate at Gd3+ center
causes relaxivity enhancement. Most of the reported Gd3+ based
agents have a slower water exchange rate. However, structural
modifications in the ligand architecture (like incorporation of
stronger electron donating groups in them) accelerate water
exchange rate. Studies further indicate that increase in steric hin-
drance around Gd3+ center by the substitution of acetate arms of
DOTA with bulkier groups will speed up the water exchange
rate. However, simultaneous control and optimization of all the
5682 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684

Ha R

R N
Ha N

Gd
Gd

Hb N
H N

Hb
H

λλ δδ
Scheme 12. Equilibrium between two gauche conformations of the ethylenediamine moieties in polyaminocarboylate.

Figure 17. Possible stereoisomers of [Ln(DOTA)] complexes. The coordinated water molecules above the O4 plane are not shown for simplicity. Pink colored balls denote
oxygen atoms, aqua colored balls show nitrogen atoms while yellow colored balls at the center represent lanthanide ions. Symbols ‘x’ and ‘d’ represent average angle of
rotation and distance between O4 and N4 planes respectively. ‘W’ is the minimum value of O–Ln–O opening angles. (Readapted with permission from Hermann, P.; Kotek, J.;
Kubíček, V.; Lukes, I. Dalton Trans., 2008, 3027, Ó 2008 Royal Society of Chemistry).55

parameters is needed to bring about substantial relaxivity
enhancement. Preferably, cyclen-based Gd3+-chelates should have
a high hydration number with a prolonged rotational correlation
time and high water exchange rate. For safety purpose, they must
exhibit increased thermodynamic stability and kinetic inertness.
DO3A and DOTA are the two most important ligands derived from
cyclen. They not only form stable complexes with Gd3+ ion but also
act as precursors for the synthesis of novel ligands through their
derivatization. The synthesis of DO3A based ligands is initiated
by treatment of cyclen with tert-butyl bromoacetate to yield
DO3A-tri-t-butyl ester (protected form of DO3A). A variety of
octadentate ligands are synthesized by the introduction of various
electrophiles at the reaming secondary ring amine. Alternatively,
DO3A based ligands are also synthesized by direct mono alkylation
of cyclen followed by the introduction of tert-butoxycarbonyl
methyl moieties at the remaining three secondary ring amines. In
both methodologies, tert-butoxycarbonylmethyl groups are depro-
tected to carboxymethyl groups by treating the protected ligands
with TFA in dichloromethane at room temperature. Majority of
the Gd3+ chelates of DO3A derivatives exist as capped SA isomer
in their solid state. However, a few complexes of DO3A derivatives
with Gd3+ ion are found to possess both capped SA and TSA geome-
tries. DOTA is another important octadentate ligand derived from
cyclen by the substitution of four secondary amine hydrogen
atoms of the macrocyclic ring with acetic acid moieties. DOTA like
ligands form stable chelates with Gd3+ ion. Useful ligands are syn-
thesized for next generation MRI contrast agents by modifying the
pendant arms of parent DOTA using a range of functionalities.
N-Functionalized DOTA derivatives are synthesized more conve-
niently and are, therefore, preferred over C-functionalized
derivatives for their role as ligands in the development of new
MRI contrast agents. In solid state, DOTA arrangement around
Gd3+ ion results in SA coordination geometry. In solution state,
its arrangement around Gd3+ produces both SA and TSA geome-
tries. Current research around the world has been focused on the
preparation of target specific MRI contrast agents of enhanced
relaxivity. The scope of the research can further be extended to
the synthesis of contrast agents with greater stability, better clear-
ance, lesser toxicity, higher water solubility and larger relaxivity as
compared to the currently available contrast agents. Higher relax-
ivity of these agents will increase the signal intensity of MRI tech-
nique. These agents will cause greater contrast between the
diseased and normal tissues as compared to the existing agents
of lower relaxivity. Consequently, such contrast agents will prove
 H. U. Rashid et al. / Bioorg. Med. Chem. 24 (2016) 5663–5684
more useful for in vivo use. For this purpose, new types of cyclen-
based ligand architecture need to be explored. Furthermore,
cyclen-based ligands (e.g. DO3A derivatives) with suitable func-
tionalities on coupling to macromolecules such as solid nanoparti-
cles, polymers, proteins, dendrimers and plasma proteins will
result in their size increase. Gd3+-chelates of such large sized con-
jugates are expected to possess enhanced relaxivity. They will be
retained in the vascular system for a prolonged period of time
and will not leak into the interstitium. Thus, such agents can be
evaluated as high relaxivity blood pool contrast agents. These
agents are expected to be useful in Magnetic Resonance angiogra-
phy and will provide a non-invasive substitute to X-ray angiogra-
phy, a potentially dangerous procedure for acquiring images of
arterial blockages in the body. Since nanoparticle-based MRI con-
trast agents exhibit enhanced vascular permeability. Therefore,
another future prospect can be the development of high relaxivity
tumor specific MRI contrast agents by the conjugation of nanopar-
ticles with cyclen-based Gd3+-chelates. Such agents will tend to be
localized in tumor cells and will, consequently, show remarkable
tumor-specific targeting ability. Gadolinium also strongly absorbs
X-rays due to its heavy nature. Therefore, gadolinium containing
nanoparticle-based agents can also be applied as multimodal con-
trast agents (for X-ray tomography, Photoluminescence and MRI).
Acknowledgment
The authors are thankful to Brazilian Foundation Agency (CNPq)
for providing financial support to execute this project
(400662/2013-2).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.09.069.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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