5-HT1A targeting PARCEST agent DO3AM-MPP with potential for receptor imaging: Synthesis, physico-chemical and MR studies

Article abstract of DOI:10.1016/j.bioorg.2020.104487

Contrast enhancement in MRI using magnetization or saturation transfer techniques promises better sensitivity, and faster acquisition compared to T₁ or T₂ contrast. This work reports the synthesis and evaluation of 5-HT_1A

Full text of DOI:10.1016/j.bioorg.2020.104487

Bioorganic Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
5
-HT1A targeting PARCEST agent DO3AM-MPP with potential for receptor
imaging: Synthesis, physico-chemical and MR studies
a
,
b
b
,
*
a
,
b
c
d
Anju , Shubhra Chaturvedi , Vishakha Chaudhary , Pradeep Pant , Preeti Jha ,
e
a
b,*
Senthil S. Kumaran , Firasat Hussain , Anil Kumar Mishra
a
Department of Chemistry, University of Delhi, North Campus, Delhi 110007, India
b
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, Brig. S. K
Mazumdar Road, Timarpur, Delhi 110054, India
c
Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India
d
Department of Immunology, Genetics and Pathology, Uppsala University, 75185 Uppsala, Sweden
e
All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Contrast enhancement in MRI using magnetization or saturation transfer techniques promises better sensitivity,
Chemical Exchange Saturation Transfer
Computational Studies
1 2
and faster acquisition compared to T or T contrast. This work reports the synthesis and evaluation of 5-HT1A
targeted PARACEST MRI contrast agent using 1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide (DO3AM) as
the bifunctional chelator, and 5-HT1A-antagonist methoxyphenyl piperazine (MPP) as a targeting unit. The multi-
step synthesis led to the MPP conjugated DO3AM with 60% yield. CEST-related physicochemical parameters
were evaluated after loading DO3AM-MPP with paramagnetic MRI active lanthanides: Gadolinium (Gd-DO3AM-
MPP) and Europium (Eu-DO3AM-MPP). Luminescence lifetime measurements with Eu-DO3AM-MPP and
computational DFT studies using Gd-DO3AM-MPP revealed the coordination of one water molecule (q = 1.43)
Density Functional Theory
Water Residence Lifetime
Longitudinal and Transverse relaxivity
Fluorescence quenching
with metal-water distance (r
of K
62 ± 0.02 pM as evaluated from fluorescence quenching of 5-HT1A (protein) and docking score of ꢀ 4.81 in
theoretical evaluation reflect the binding potential of the complex Gd-DO3AM-MPP with the receptor 5-HT1A
Insights of the docked pose reflect the importance of NH (amide) and aromatic ring in Gd-DO3AM-MPP while
interacting with Ser 374 and Phe 370 in the antagonist binding pocket of 5-HT1A. Gd-DO3AM-MPP shows
M
-H
2
O) of 2.7 Å and water residence time (
τ
m
) of 0.23 ms. The dissociation constant
d
.
2
ꢀ
1
ꢀ 1
longitudinal relaxivity 5.85 mM
s
with a water residence lifetime of 0.93 ms in hippocampal homogenate
3
+
containing 5-HT1A. The potentiometric titration of DO3AM-MPP showed strong selectivity for Gd over phys-
iological metal ions such as Zn2 and Cu . The in vitro and in vivo studies confirmed the minimal cytotoxicity
and presential binding of Gd-DO3AM-MPP with 5-HT1A receptor in the hippocampus region of the mice. Sum-
marizing, the complex Gd-DO3AM-MPP can have a potential for CEST imaging of 5-HT1A receptors.
+
2+
1
. Introduction
Magnetic Resonance Imaging (MRI) is among the most sought-after
pulse sequences or by introducing certain chemical entities. The chem-
ical entities, popularly referred to as contrast reagents in the jargon of
imaging sciences, have been widely applied and researched extensively.
Mechanistically, the contrast agents alter the relaxation rate of water
molecular imaging techniques mainly due to low invasiveness, excel-
lent soft-tissue contrast, no ionizing radiation, and good temporal-
spatial resolution. [1] The variation in the concentration and the
bound state of water molecules in different tissues results in contrast in
MRI. Further, image contrast in MRI can be altered either by variation in
protons in their vicinity resulting in contrast. [2] Both longitudinal (T
or (and) transverse (T ) relaxations can be affected by contrast agents.
For instance, lanthanide-based contrast agents affect mainly T , whereas
superoxide or the iron nanoparticles alter T . [3,4] The lanthanide-
1
)
2
1
2
Abbreviations: 5-HT1A, 5-hydroxy tryptamine Receptor (Class-1A); MRI, Magnetic Resonance Imaging; CEST, Chemical Exchange Saturation Transfer; MPP,
′
′
Methoxyphenyl piperazine; Gd-DO3AM, Gadolinium 2, 2 , 2 ’-(1,4,7,10 -tetraazacyclododecane-1, 4, 7triyl) triacetamide; 2D, Two Dimensional; 3D, Three
Dimensional.
*
Corresponding authors.
E-mail addresses: shubhra@inmas.drdo.in (S. Chaturvedi), akmishra@inmas.drdo.in (A. Kumar Mishra).
https://doi.org/10.1016/j.bioorg.2020.104487
Received 4 November 2020; Accepted 17 November 2020
Available online 24 November 2020
0
045-2068/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Anju, Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2020.104487

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
based contrast agents, particularly gadolinium complexes, hold a
considerable share in MRI and account for 30% of MRI scans. The
complexes are tuned to study fluctuations in the cellular micro-
environment by being responsive or are targeted -gadolinium com-
G6530AA (LC-HRMS-Q-TOF) at Delhi University. Ultra-Pure Liquid
Chromatography (ESI-MS) was performed at Jubilant Pvt. Ltd., Noida,
India. In vitro and in vivo MR studies were performed using 7 T Bio-Spec
USR 70/20 animal MRI Scanner at All India Institute of Medical Sci-
ences, Delhi India. Computational studies were performed using
1
plexes as T agents have spawned interest in receptor imaging. [5,6]
¨
However, the sensitivity required to detect the low concentration and
perturbation in receptor expression has remained a challenge for an
effective MRI contrast agent, even though few examples have been re-
ported for ex-vivo targeting of neuro-receptors (Fig. 1 [7–10].
Parametric analysis for MRI based receptor imaging reveals the
importance of increasing the relaxation rate while designing sensitive
gadolinium complexes. Relaxation rate or relaxivity is a complex
SchrOdinger Software LLC, New York, 2017 Maestro.
2
2
.1. Synthesis and characterization
.1.1. 1-(3-Chloropropyl)-4-(2-methoxy-phenyl)-piperazine (1)
A solution of 1, 3-bromochloropropane (1.22 g, 7.80 mmol) dis-
solved in acetonitrile was added to a stirring solution of 1-(2-Methoxy-
parameter relying on the variables: (i) rotational correlation time (
τ
R
phenyl)-piperazine (1 g, 5.20 mmol) and potassium carbonate (2.1 g,
0
.2–0.5 ns) (ii) rate of water exchange ( < 100 ns) and, (iii) contri-
τ
M
◦
1
5.60 mmol) in 20 ml acetonitrile at 70 C. The mixture was refluxed for
4 h. The progress of reaction was monitored by TLC (DCM:MeOH 9:1).
bution from water molecules in the second sphere of hydration. [9]
While tuning the three variables, the physical and stability constraints
limit the increase in relaxivity much lower than the theoretically
possible values and restrict the sensitivity of the gadolinium complexes.
The mechanistically alternate method that is chemical exchange
saturation transfer, to achieve contrast, relies on magnetization transfer
instead of relaxivity. The contrast produced is a darker image instead of
2
After the completion of reaction, inorganic salts were separated by fil-
trations and solution of reaction mixture was evaporated to dryness
under vacuum to get yellow-green liquid oil. The compound was puri-
fied by column chromatography using (60–120) mesh size silica, the
desired product was eluted at 1% MeOH in DCM (R
f
= 0.75) yield (75%)
which was evaporated to get light blue liquid oil.
1
brighter contrast as provided by T modulating contrast agents. CEST
1
H NMR (400MHz,CDCl
3
) (δ in ppm) δ = 6.91–6.89 (m, 4H, ArH, J =
), δ = 3.11 (piperazine
), δ = 2.69 (br, s, 4H, –CH ) (piperazine
ring hydrogens), δ = 2.49 (t, J = 10.8 Hz, 2H) δ = 1.84 (quintet, J = 10.8
contrast agents have around two order higher sensitivity, fast acquisi-
tion, and better spatial resolution. [11,12,1]
2
Hz) (aromatic region), δ = 3.83 (s, 3H, OCH
3
ring hydrogens) (br, s, 4H, –CH
2
2
In order to further explore the utility of CEST contrast agents, we
have attempted to design a CEST agent with Gadolinium and Europium
and evaluate for serotonin receptors imaging. Targeting towards 5-HT1A
receptors was accomplished by well-exploited antagonist pharmaco-
phore, methoxyphenyl piperazine (MPP). MPP as targeting moiety has
been reported for a gamut of PET/ SPECT imaging (nuclear imaging)
1
3
◦
3
) 25 C,
Hz, 2H), δ = 1.22 (t, J = 10.8 Hz, 2H), C NMR (100 MHz CDCl
1
5
52.1, 141.0, 122.9, 120.9, 111.0 (aromatic carbons), 56.0, 55.2, 53.3,
+
0.3, 30.8, 23.7; MS(ESI ) m/z calculated for C14
H
21ClN
2
O is 268.1342
+
found (M + H) 269.1407.
1
and MRI-T contrast agents. [13,14] The complex was studied for
2
.1.2. 1, 4, 7, 10 tetraaza-Cyclododecane-1,4,7-tricarboxylic acid tri-tert-
physical parameters that affect CEST viz., coordinating water molecules
and water exchange through luminescence lifetime measurements and
butyl ester (2)
To a solution of cyclen (1 g, 2.314 mmol) and triethylamine (710 mg,
1
theoretical calculations, alteration in T and relaxivity through MRI and
7
.021 mmol) in chloroform, solution of di-tert- butyl dicarbonate (3.396
targeting ability through evaluation of binding parameters using fluo-
rescence quenching and theoretical predictions.
g, 17.414 mmol) in chloroform was added dropwise over a course of 2 h
◦
at 0 C. Then resulting solution was allowed to warm to room temper-
ature and stir for additional 18 h. The reaction mixture was filtered, and
solvents were removed under vacuum. The dried mixture was then
washed with water (2 × 100 ml) and the organic layer was dried over
2
. Materials and methods
1
-(2-methoxyphenylpiprazine),
cyclen), 1-bromo-3-chloropropane, potassium carbonate, sodium bi-
carbonate, trifluoroacetic acid, di-tert-butyl-dicarbonate and sodium
sulphate were purchased from Sigma-Aldrich. Metal salts (GdCl O,
⋅6H
EuCl O) were purchased from Aldrich with 99.9% purity. Aceto-
⋅6H
nitrile (HPLC Grade), Dichloromethane (HPLC Grade), Chloroform
HPLC Grade) were obtained from Merck Ltd., India, and used as such
1,4,7,10-tetraazacyclododecane
2 4
Na SO , and the solvents were removed under vacuum. Further the
(
residue was purified by column chromatography over a silica gel using
mesh size (60–120) R = 0.5 (50% EtOAc: PET Ether) and the product
was eluted at 60% P-ET Ether in ethylacetate with yield 70%. H NMR
f
3
2
1
3
2
1
(
400 MHz, CDCl
3
) (in ppm) H NMR (400 MHz, CDCl
3
) δ = 3.57, (s, 4H,
br), □=3.35 (s, 8H), δ = 2.80 (s, 4H, br), δ = 1.42 (doublet, J = 9.6 Hz,
(
–
2
2
7H).13C NMR C
δ = 49.5 (ring CH
m/z calculated for C33
O δ = 151.9, (C(CH
3
)
3
), δ = 81.2, δ = 51.0 (ring CH ),
2
without any purification. Compounds Purifications carried out using
column chromatography using silica gel having mesh size (60–120 µm).
TLC was run on silica gel coated aluminum sheets (silica gel 60, F254,
Merck) and analyzed under UV detection set at fixed wavelength 254
), δ = 28.7, (ring CH
2
), δ = 27.5 (C(CH . MS (ESI + )
3 3
)
+
44 4 6
H N O
is 472.3263 found (M + H) 473.3335.
1
13
2.1.3. 10-(3-(4-(2-Methoxy-phenyl)-piperazin-1-yl)-propyl)-1, 4, 7, 10
teraaza-cyclododecane-1,4,7tricarboxylic acid tri-tert-butyl ester (3)
nm. H and C on the Jeol Model JNM-EXCP 400 MHz system at Delhi
University. Chemical Shift (ꢀ) represented in ppm with respect to TMS.
High-Resolution mass spectroscopy (ESI-MS) was performed on Agilent
To a solution of 2 (500 mg, 1.05 mmol) and K
2 3
CO (438.94 mg,
3
.177 mmol) in acetonitrile, 1 was dissolved in acetonitrile (1.05 mmol,
Fig. 1. Gadolinium based T
1
contrast agents for receptor imaging.
2

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
2
83.89 mg) was added dropwise and this mixture was allowed to stir for
2.1.6. Preparation of (lanthanide) Europium (III) complex of DO3AM-
MPP (6)
◦
4
8 h at 70 C. The progress of reaction of mixture was monitored by
running TLC (40% PET Ether in ethyl-acetate). Further inorganic salts
were filtered, and solution of reaction mixture was evaporated to dry-
ness under dryness to get brownish liquid oil. The compound was pu-
rified by column chromatography using (60–120) mesh and the desired
To a solution of DO3AM-MPP (50 mg, 0.0869 mmol) (4) in water was
added a solution of Europium Chloride (31.86 mg, 0.0869 mmol) in
aqueous solution. The pH of solution was maintained between 4 and 5
by addition of 1 M aqueous solution of KOH. The reaction was heated at
◦
product was eluted at 50% PET Ether in ethylacetate with yield 45% (R
0.32). 1H NMR (400 MHz, CDCl
) (δ in ppm) δ = 7.007–6.835
Aromatic-H (4H, m J = 2 Hz), δ = 4.12 (2H, triplet, J = 7.6 Hz CH
f
60 C for 24 h and solvent was evaporated under vacuum to obtain
1
=
3
Europium complex as cream white solid. H NMR (400 MHz, D
2
O) (δ in
2
)
ppm) δ = 7.91(–NH
J = 6 Hz), δ = 5.15 (quintet, piperazine ring, 8H, J = 4.4 Hz), δ = 5.0 (s,
16H, cyclen ring) δ = 4.17 (t, 2H, linker J = 6.4 Hz), δ = 3.9 (–OCH
methoxy group) δ = 2.52 (2H, t, J = 6.4 Hz), δ = 2.23 (quintet, 2H, J =
2
amide, 6H), δ = 7.31–7.23 (m, aromatic hydrogens,
linker, δ = 3.84 (OCH
3
, 3H, s), δ = 3.60 (cyclen ring CH
, 8H, s) δ = 3.08 (CH Piperazine ring, 4H, s br), δ = 2.82
, 4H, s), δ = 2.65 (4H, s br) piperazine ring, δ = 2.48 (triplet, J
7.6 Hz, CH linker), δ =
linker) δ = 1.87 (2H, quintet, J = 7.6 Hz, CH
.44 (27H, doublet, J = 8 Hz). C NMR δ = 171.5 aromatic C, δ = 151.9
2
4H, s), δ = 3.26
(
cyclen ring CH
ring CH
2
2
3
,
(
2
3
+
3+
O)/3)
=
2
2
6.4 Hz); HRMS-ESI-MS calculated for C28
H
51GdN
9
O
5
((M + H
2
1
3
+
1
m/z = 248.7736 and found ((M + H
2
O)/3) + 3H) =251.1822
–
(
C
–
O), δ = 141, 123.1, 122, 120, 118.2, 111 δ = 81.6 (quarternary
carbon), δ = 62.8 (methoxy OCH ) aromatic C, δ = 55.1 piperazine
ring, δ = 53.4 Cyclen ring Carbon, δ = 53.4 piperazine ring, δ = 50.6
alkyl group linker, Cyclen ring carbon δ = 28.2 alkyl carbon CH carbon
linker (C(CH is
–
3
2.2. CEST parameters
2
2.2.1. Luminescence lifetime measurement
3
)
3
), δ = 26.1. MS (ESI + ) m/z calculated for C37
H
64
N
6
O
7
The luminescence lifetime (
τ
m) and q value measurements were
+
+
7
04.483 found (M + H) is 705.4908 and (M + K) is 743.4422.
recorded on HIDEX Instrument.
τ
for Eu-DO3AM was calculated by
m
measuring the variation in luminescence count with a change in decay
time. The measurements were carried in water and deuterated water (pH
2
.1.4. 2-(4,7-Bis-carbamoylmethyl-10-(3-(4-(2-methoxy-phenyl)-
7
.4). The source used for analysis was a hydrogen flash lamp. Slit widths
piperazin-1-yl)-propyl)-1,4,7,10teraaza-cyclododec-1-yl)-acetamide (4)
for emission and excitation were kept open. The intensity decay curves
were obtained at emission maximum and fitted as the sum of
exponential
Compound 3 was dissolved in dichloromethane and 1 ml of tri-
floroacetic acid was added and reaction mixture was allowed to stir at
room temperature for 24 h. Then the solvent was evaporated, and res-
idue was re-dissolved in DCM and washed with 10% aqueous solution of
NaOH to make the pH 9 of reaction mixture. Further solution was
evaporated and resulting product was used as such without any purifi-
cation. To a solution of resulted product (100 mg, 0.247 mmol) and
∑
t
τ
I
t
= I
o
A
i
eꢀ
Where
τ
and A represent the fluorescence lifetime and pre-
exponential factor, respectively.
2 3
K CO (102.56 mg, 0.742 mmol) in acetonitrile, bromo-acetamide
The number of water molecules coordinated to the inner sphere (q)
of the complex is calculated from luminescence rate constants in water
and heavy water using the equation given by Supkowski and Horrocks:
(
101.84 mg, 0.247 mmol) dissolved in acetonitrile was added drop-
◦
wise. The reaction mixture was refluxed at 65 C and the reaction
mixture was allowed to stir for additional 24 h. Then reaction mixture
was filtered, and inorganic salts were filtered, and solution was evapo-
rated to dryness under vacuum. The residue was dissolved in water and
1
H2O
1
D2O
q = AEu(τ
ꢀ τ
ꢀ aEu)
1
diethyl ether was added to remove nonpolar impurities. H NMR was
recorded in deuterated DMSO where as 13C NMR in deuterated H
Where
τ
H2O
&
τ
D2O represent luminescence lifetime of Eu-DO3AM-
2
O
ꢀ 1
MPP in H
2
O and D
2
O. The value of AEu = 1.1 ms and aEu is 0.31
because amide hydrogens are superimposed (broad spectra obtained) in
deuterated water (individual peaks are not clearly visible) and in 13
ꢀ 1
ms . aEu is used for the correction of an error, which accounts for
closely diffusing OH oscillators.
C
NMR due to presence of residual water in deuterated DMSO peaks in-
1
tensity is very low. So, H NMR(400 MHz, DMSO) (δ in ppm) δ = 8.00
2
.2.2. Relaxometric Studies: In vitro studies
(
4H, s) –NH
2
amide, δ = 7.94 (2H, s) –NH amide, δ = 7.14 (4H, s, br)
2
MR Imaging was done using a 7 T Bruker Biospec USR 20/70 animal
aromatic H, δ = 5.37 (2H, triplet, J = 5.6 Hz), δ = 3.7 (17H s, br) ((3H,
OCH , 8H piperazine ring , 6H CH adjacent to NH
MRI scanner at AIIMS, Delhi. Rare T
1
+ T
2
sequence was used to obtain
3
2
2
)), δ = 3.3 (DMSO
relaxivity. In vitro and in vivo T and T
1
2
measurements were performed
residual water), δ = 2.50 (quintet 3H, DMSO), δ = 2.04 (18H, s) (16H
Cyclen ring Hydrogens, 2H alkyl linker), δ = 1.18 (2H, J = 5.6 Hz,
triplet). 13C NMR (400 MHz, deuterated water) δ = 173.9 (Carbonyl
oxygen of amide pendant arm), δ = 169.2 (aromatic carbon), δ = 134.9,
at 37℃ with eight different TR repetition time (8 ms, 24 ms, 40 ms, 56
ms, 72 ms, 88 ms, 104 ms, 120 ms) and TE echo time (5376 ms, 2876 ms,
1
376 ms, 676 ms, 376 ms, 176 ms, 26 ms, 11.59 ms). MRI experiments to
measure the change in longitudinal relaxivity (r ) were performed with
1
1
34.0, 128.7, 128.5, 128.3 (five aromatic carbons), δ = 68.1 (amide
six different concentrations of Gd-DO3AM-MPP (0.625–8 mM, pH-7).
Agar (1% in water) was added to all solutions for maintaining homo-
geneity. The relaxivity at different concentration was calculated using
the equation:
pendant arm), δ = 67.02 (methoxy carbon), δ = 51.2 (piperazine ring
carbon), δ = 48.1 (Cyclen ring), δ = 30.1, 28.9, 24.5 (linker carbon). MS
+
(
ESI + ) m/z calculated for C28
H
49
N
9
O
4
is 575.3921 and found (M + H)
+
+
is 576.3990, (M + Na) 598.3814 and (M + K) 614.3540.
(
)
1
1,obs
1
1,d
r
1,obs
=
ꢀ
[GdL]
T
T
2
.1.5. Preparation of (lanthanide) Gadolinium (III) complex of DO3AM-
MPP (5)
To a solution of DO3AM-MPP (50 mg, 0.0869 mmol) (4) in water was
where 1/T
1
is the relaxation rate and 1/T
1
, d is the solvent’s
diamagnetic contribution, and (GdL) is the concentration of complex in
mM. Relaxation data were analyzed with Topspin 2.0 Bruker Software,
added a solution of Gadolinium nitrate (32.32 mg, 0.0869 mmol)
aqueous solution. The pH of solution was maintained between 4 and 5
1 2
and the relaxivity (r & r ) measured by regression analysis.
by addition of 1 M aqueous solution of KOH. The reaction was heated at
◦
6
0
C for 24 h and solvent was evaporated under vacuum to obtain
2.3. Physiochemical characterization
Gadolinium-complex as pure white solid. HRMS-ESI-MS calculated for
3
5
3
+
+
3+
C
28
H
51GdN
9
O
((M + H
2
O)/3) m/z = 250.4413 and found ((M +
2.3.1. Equilibrium measurements
H
2
O)/3) + H) =251.2000
Potentiometric titrations were used to determine protonation con-
3

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
stant (pka) of DO3AM and stability constant (β) of the complexes with
from phosphate buffer and Milli-Q water was subtracted from the sample
emission. The data reflected a change in fluorescent emission with
quencher concentration and was plotted following the Stern-Volmer
Equation.
3
+
3+
2+
2+
Gd , Eu , Cu , and Zn using Metrohm potentiometer. The pro-
tonation constants were measured by titrating 0.5 mM ligand with 0.01
M tetramethylammonium hydroxide (TBAOH) at 25℃ in the pH range
2
–14. The protonation constant was calculated according to the
equation
Mechanism of quenching. To establish the quenching mechanism of the
ligands on serotonin, the Stern-Volmer equation was used for analysis.
[
H L]
i
i
K =
+
[H
iꢀ 1L][H ]
F
O
F
=
1 + KSV[q]
where i = 1, 2, 3
where: F
O
= Fluorescence intensity of pure Serotonin (negative
Stability constants of DO3AM-MPP were determined by potentio-
metric titration of 1:1 Metal: Ligand solutions. Solutions of metal salts
were prepared by dissolving the appropriate amount in Milli-Q water
and titrated with tetra-butyl ammonium hydroxide. Stability constants
of the metal complex were evaluated as per the equation:
control) F = Fluorescence intensity of Serotonin + analog: KSV = bio-
molecular quenching constant: [q] = molar concentration of quencher
(
ligand Eu-DO3AM)
2
.4.3. 5-HT1A receptor binding assay through fluorescence studies
[
MH
i
L]
The hippocampus of the Sprague Dawley rat brain was homogenized
β =
+
[
MHiꢀ 1L][H ]
in 10 volumes of ice-cold PBS buffer (50 mM, pH 7.4) using an Ultra
Truax T10 (IKA) and centrifuged at 3000 rpm for 20 min. The resulting
pellet was suspended using an Ultra Truax and centrifuged again at
Where i = 1, 2, 3
3
000 rpm for 20 min. A similar procedure was repeated. The obtained
2
.3.2. Kinetic stability
pellet was resuspended in 10 volumes of buffer and stored at ꢀ 80℃ until
used in receptor binding assays. The purity of extracted serotonin pro-
tein was confirmed by measuring absorbance ratio at 260, and 280 nm,
which conforms to the protein obtained from Westar rats (1.86 mg/ml
protein), is more than 95% pure. The binding of protein with synthe-
sized ligands was studied through fluorescence quenching of the protein.
The free amount of ligand which did not bind to the serotonin was
calculated using the following equation:
Kinetic stability is an important parameter for Gadolinium com-
plexes as these are used in MRI as contrast enhancement. The extent of
dissociation of gadolinium complexes depends on the selectivity of li-
gands with gadolinium ion over endogenous metal ion present in body
fluids. As in vivo, endogenous cations (Fe3 , Ca , Zn +, and Cu ) may
+
2+
2
2+
3
+
react with Gadolinium chelates by displacing Gd in a metal–metal
trans-metalation exchange. Thus, to understand the in vivo fate of the
3
+
Gd containing contrast agent, we must know the rate of the exchange
(
Free Ligand = Total Conc of ligand used – ((Q)* (Conc of serotonin))
3
+
2+
3+
reaction of Gd–DO3M-MPP with Eu and Zn . The formation of Eu
Scatchard plot is plotted using following equation:
2
+
and Zn complexes at 276 and 332 nm were used to study the metal
exchange rate in Gd–DO3AM-MPP. The concentration of the complex
Gd-DO3AM-MPP taken was 1 mM, whereas the Europium concentration
was used 10-15fold excess and that of Zinc metal ion was 30–40 times
higher, respectively, confirming pseudo-first-order conditions. The
Q
1
(Q)
+ kn
=
ꢀ k
LigandFree
d
d
Non-specific binding was performed using 100fold excess of seroto-
nin and MPP.
◦
temperature was kept constant at 25 C, and the ionic strength main-
tained with 2 M KCl at pH 7.4. In the presence of an excess of the
exchanging ion, the trans-metalation process may be assumed to be a
pseudo-first-order process, and the rate of reaction can be expressed
with the equation:
2
.4.4. Characterization of Gd-DO3AM-MPP using power X-ray diffraction
Powder X-ray diffraction analysis was performed using the Bruker D8
Discover instrument at the University of Delhi. The Cu-K radiation was
α
used in the source, and the instrument was operated at 40 kV and 40 mA.
ꢀ
ꢁ
◦
A
t
= A
0
ꢀ A
p
eꢀ kobs
t
The XRD patterns were recorded within 10-50 range of 2θ as a function
of intensity with a scanning rate of 2 /min. The miller indices (hkl) were
◦
where kobs is a pseudo-first-order rate constant, A
t
is the absorbance
is the absorbance values at time t = 0, and A is the
absorbance values at time t = equilibrium point.
calculated using Bragg’s Equation and XRD pattern was plotted using
Origin7.
values at time t, A
o
p
2
.5. Computational modeling
2
2
.4. Fluorescence studies
2
The geometry of Gd-DO3AM-MPP and Gd-DO3AM-MPP-H O in
.4.1. Fluorescent emission of Eu-DO3AM-MPP
aqueous solution (including water molecule in the inner sphere) were
optimized ab initio with DFT calculations on Gaussian 09 software. [15]
Hybrid functional B3LYP and MWB53 [16] basis sets for Gadolinium
and 6-31G* for C, H, N, and O were chosen to compute the energy dif-
ference between two conformations. After optimization (cartesian input
coordinates of Gd-DO3AM-MPP in ESI Table S1), the complexes were
obtained in a square ant prismatic geometry. Energies of optimized
complexes were calculated after convergence. The PDB files generated
were further used for docking studies. Docking studies to visualize the
interaction between the complex (Gd-DO3AM-MPP) and receptor 5-
HT1A were accomplished on the Glide module of Schrodinger molecular
modeling software.
Fluorescence emission spectra of varying concentrations of Eu-
DO3AM (10 µm ꢀ 100 µm) were recorded using 300 to 700 nm on
Synergy 2 multi-reader (M/S Biotech Instruments, USA) with excitation
at 276 nm.
2
.4.2. Fluorescence quenching
The experiment was performed as reported previously. Briefly, the
concentration of serotonin (=fluorophore, 0.1 µM in Phosphate Saline
buffer) was fixed, and dilutions of Eu-DO3AM-MPP (10 µM-100 µM)
(quencher) were prepared in Milli-Q water. In a 96 well black opaque
plate, 10 µl of serotonin was incubated with different ligands concentra-
tions. The final volume in each well was 200 µl. The solutions were mixed
thoroughly and allowed to incubate for 30 min. The fluorescence emission
was recorded from 300 nm to 700 nm using Synergy 2 Multi-Mode reader
2.6. MTT assay
(
M/S Biotech Instrument USA) with excitation at 260 nm. The experi-
MTT assay was conducted to analyze the cytotoxicity of the Gd-
DO3AM-MPP complex on HEK (Human Embryonic kidney) cell lines.
ments were carried out at room temperature. The background emission
4

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 2. PARACEST Agents with different application.
In a 96 well plate, growing cells were seed at a density of 4000 cells/
wall. Cells were treated with varying concentrations of the Gd-DO3AM-
MPP complex ranging from 100 mM to 1pM. At predefined intervals of
MPP), after rupturing the Blood-Brain-Barrier with 25% w/v of
mannitol. MR Imaging was performed in the mice using T
Rare sequence with a scanning time of 54 min.
1
+ T
2
map
2
4 h, 48 h and 72 h the cells were washed and replenished with fresh
media to ascertain the cytotoxicity of the Gd-DO3AM-MPP complex.
Further cells were treated with MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2Htetrazolium bromide (concentration 0.05 mg/ml) for two
hours so that cells convert the MTT (blue colored) into Formazan crys-
tals (purple). Addition of DMSO led to solubilization of formazan crystal
and optical density of these crystals were measured at 570 nm. HBBS
3. Results and discussion
3.1. Design consideration
Gadolinium complexed in the macrocyclic cage of DOTA
′
′
′
(2,2 ,2 ’,2 ’’-(1,4,7,10-teraazacyclododecane-1,4,7,10-tetraethyl) tetra-
′
′
(
Hank’s Balances salt solution) were used as negative control. The
acetic acid) or DO3A (2,2 ,2 ’-(1,4,7,10-teraazocyclododecane-1,4,7-
triyl) tri-acetic acid) has been the preferred strategy to develop para-
magnetic contrast agents due to the complex’s excellent kinetic inertness
and thermodynamic stability, making it suitable for in vivo applications.
Retaining the same scaffold of Gd-DOTA, researchers have developed
paramagnetic CEST agents. The modification focuses on introducing
labile protons (–NH, –OH) that can be exchanged with bulk water pro-
tons in tissues. For example, Markwood and co-workers synthesized pH-
responsive gadolinium-cyclen contrast agents with carboxylate and
amide side chains as CEST agents. [17] Other examples are Ln-DOTAM-
Gly [18] and Yb-DOTAM [19] as a pH-responsive probe, Tm-DOTAM-
βAla-py [20] for imaging of endogenous zinc and copper and Eu-
DOTAM-Glu-Lys-OH and Tm-DOTAM-Glu-Lys-OH[21] to study their
magnetic properties (Fig. 2).
experiment was performed in triplicate.
2
.7. In vivo MR imaging
In vivo MR imaging was performed in All India Institute of Medical
Sciences Delhi, India using Biospec USR 70/20 7 T animal MRI Instru-
ment on two mice (male sex and age 8 weeks, breed BALB/C). One mice
was considered as control which was injected with saline. The second
mouse was sedated with ketamine/xylazine with a concentration of
1
7.5 mg/ml and 2.5 mg/ml respectively. Respiration pulse was moni-
tored during the whole experiment and maintained at 90 beats/min. In
addition, for the estimation of 5-HT1A binding affinity of Gd-DO3AM-
MPP in the hippocampus region of the brain, the mice was injected
with 0.625 mM concentration of Gd-DO3AM-MPP intravenously (as
brighter contrast obtained at 0.625 mM concentration of Gd-DO3AM-
The replacement of carboxylates with amides provides reduced
electron density at the metal centre, leading to a slow exchange of
3
+
3+
Scheme 1. Where M = Gd , Eu . (a) Br-CH
BrCH CONH , acetonitrile, 70℃ (e) GdCl
⋅6H
2
-CH
2
-Cl, K
2
CO
3
, acetonitrile, 70℃ (b) ((CH
3
)
3
CO)
2 3 2 3
O, Et N, chloroform, 0℃, (c) K CO , acetonitrile. (d) TFA/DCM,
2
2
3
2
O, water, 0.1 M NaOH 70℃ (f) EuCl
3
⋅6H
2
O, water, 0.1 M NaOH, 70℃.
5

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
exchangeable protons at the metal centre to the tune of 1–3 ms [22,23].
This variation resulted in compromised thermodynamic stability.
However, it was reasonably compensated by the high kinetic inertness
the metal complex for optimization also determines the complex’s
relaxivity and stability.
Luminescence lifetime measurements of water molecules bonded to
the metal ion can provide information about the hydration state. In
contrast to Gadolinium, Europium and terbium are most studied
lanthanide ions due to their longer excited-state lifetime. Europium and
Gadolinium are the neighboring elements in the periodic table and share
many fundamental properties like ionic radius, chemical reactivity, and
valency. However, Europium possesses one extra physical property
luminescence under UV light, which is not shown by Gadolinium. Due to
resemblance in fundamental properties, Europium analog Eu-DO3AM-
MPP can be used in place of Gadolinium Gd-DO3AM-MPP to monitor
the physical processes Luminescence and tissue distribution through
fluorescence. Among all the lanthanides, Europium has found to have a
slower rate of water exchange and low paramagnetic relaxation
[
24].
As the fourth arm of DOTA remains free, targeting was achieved by
appending the serotonin 5-HT1A receptor known antagonist pharmaco-
phore, MPP. Scheme 1 includes the synthesis and structure of the Para-
CEST agent.
The first step involved MPP functionalization through 1-bromo, 3-
chloro propane linker (1) synthesized in more than 75% yield. The
formation of the compound was asserted by the appearance of two
triplets and quintet in 1H NMR. Compound (1) was appended with
triprotectedboc-cyclen (2). After TFA/DCM deprotection, the compound
(
3) was alkylated with bromo-acetamide with an overall yield of 60 ±
% for the purified product. Loading of Gadolinium and Europium was
5
accomplished by complexing metal chloride (metal = Gadolinium or
Europium) with ligand DO3AM-MPP. Mass spectrometry confirmed the
formation of Gd-DO3AM-MPP and Eu-DO3AM-MPP (Isotopic Pattern)
enhancement. (maximum T
1 2
and T shortening effect). These properties
of Europium are favorable in the design of the PARCEST agent. [27] The
decay of excited Eu3 -DO3AM-MPP complex was measured in H
O as a concentration function. As well documented, the O-H
oscillator (in H O) is an effective quencher for the excited lanthanide ion
compared to the O-D oscillator of D O.
Thus, there is a difference in quenching frequencies of O
bonds that can estimate the number of coordinated water molecules. The
+
O and
2
(
ESI-MS).
D
2
–
2
3
.2. CEST parameters
2
–
H and O-D
The parameters governing CEST contrast are similar to those gov-
erning T
1
contrast and includes (i) water residence lifetime (
τ
m
) (ii) no of
and T
luminescence lifetime of Eu-DO3AM (pH 7.4 and 7F
was found to be 0.23 ms in H O and 0.38 ms in D
0
to 5D
0
transition)
◦
water molecules coordinated to the inner sphere (q) (iii) T
1
2
2
2
O at 25 C. Using the
relaxivity of surrounding water protons and (iv) rGd-H distance between
Gadolinium and a coordinated water molecule. [25]
Supkowski and Horrocks equation. [28] The hydration number of Eu-
DO3AM was calculated to be 1.43, which confirms mono aqua com-
plex formation.
3
.2.1. Luminescence based evaluation of (i) water residence time and (ii)
number of water molecules
3.2.2. MRI based evaluation of (iii) NMR relaxivity: In vitro studies
The exchange rate of water protons with bulk water protons of tis-
sues depends on the metal ion apart from other physical parameters viz.,
pH, temperature, and ionic environment. Ideally, for a CEST agent, the
The longitudinal (T
DO3AM-MPP were measured as a function of concentration to deter-
mine the longitudinal relaxivity (r : r ) and transverse relaxivity
(r : r ). Linear enhancement was obtained for 1/T
= 1/T
the [Gd-DO3AM] (r = 0.999 for 1/T and r = 0.985 for 1/T
1 2
) and transverse (T ) relaxation time of Gd-
1
1
= 1/T
1
metal-bound water residence lifetime (
2
τ
m
) should range from 10-5 to 10-
2
2
2
1
and 1/T
2
with
), as
s. In the case of cyclen derived contrast agents, the change in the rate
can be introduced by variation of side pendant arms design. The
replacement of acetate in DOTA/DO3A with the amide in DO3AM re-
sults in the slow exchange of metal-bound water molecules, presumably
because the pendant arms of DOTA or DO3A contain acetate groups,
which more basic as compared to DO3AM amide arms. Thus, the metal
center interacts strongly with DO3A/DOTA in comparison to the water
molecule. On the other hand, in DO3AM, the metal center interacts
strongly with water molecules leading to higher residence time/ slower
exchange.
1
2
depicted in Fig. 3(b) & (c) respectively. The longitudinal relaxivity r
1
-
1 ꢀ 1
-1 ꢀ 1
and transverse relaxivity r
2
were 5.85 mM s
and 6.41 mM s ,
◦
respectively, on 7 T at 37 C (see Fig. 3).
The Gadolinium loaded complex showed shortening in relaxation
time with respect to water (T1 pure-water = 2916.79 ms and T = 305.703
ms and T2 pure-water = 112.643 ms and T = 42.241 ms at a lower con-
1
2
centration. . At [Gd-DO3AM-MPP] = 0.625 mM, an increase in
enhancement and brightest contrast was recorded; however, increasing
[Gd-DO3AM-MPP] lead to the formation of a darker image. The linear
enhancement with an increase in the ligand concentration, followed by
darker contrast at [Gd-DO3AM-MPP] = 8 mM, can be attributed to the
saturation achieved between the energy levels. [30] (see Fig. 3).
The nature of the metal ion also influences the water exchange rate.
Gadolinium-DOTA complexes have a faster exchange rate than the
Europium-DOTA complexes, which are of the order of »100-300 ns. [26]
The number of water molecules (q) in the first coordination sphere of
3
+
Fig. 3. (a) T Weighed MRI Contrast Images of Gd -DO3AM-MPP at a different concentration ranging from 0.625 mM to 8 mM, and 3(b) show the linear regression
1
3
+
1 2
analysis of relaxivity of Gd -DO3AM-MPP complex with concentration to determine longitudinal relaxivity (r ) and 3(c) transverse relaxivity (r ) respectively.
6

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 1
exchange reactions. The exchange rate constant of Gd-DO3AM-MPP was
investigated in the presence of Eu3 , and Zn metal ion and value of
+
2+
pka values of ligands.
proton assisted exchange rate constants are summarised in Table 3.
pka
DOTA[33]
DO3A-MPP
DO3AM-MPP
Gd -DO3AM-Propyl-MPP) + M (M +-DO3AM-Propyl-MPP) +
3
+
n+
n
(
pka1
pka2
pka3
11.14 ± 0.02
9.69 ± 0.01
4.85 ± 0.07
9.93 ± 0.02
6.72 ± 0.06
4.65 ± 0.01
9.12 ± 0.02
6.09 ± 0.03
4.02 ± 0.02
Gd3 where M =Cu , Eu
+
n+
2+
3+
)
The exchange rate of Gd-DO3AM-MPP with Eu-DO3AM-MPP and Zn-
DO3AM-MPP is very low compared to DO3A or DOTA derivatives due to
lower basicity of amide oxygen atom which conforms higher kinetic
inertness to Gd-DO3AM-MPP over Gd-DOTA, making it suitable for in
vivo application. The plausible explanation lies in already existing
mechanistic data. [38] According to which acid-catalyzed dissociation
of the Ln-DO3AM complex occurs by a different mechanism as compared
to Ln-DOTA. In Ln-DOTA, the first step is the protonation of the unco-
ordinated carboxylate oxygen atoms followed by the proton transfer to
ring nitrogen. The electrostatic repulsion between now positively
charged nitrogen and Ln (III) metal ion facilitates the expulsion of Ln
Table 2
Stability Constant of metal complexes.
Stability Constant
DOTA[34]]
DO3A-Propyl-MPP
DO3AM-Propyl-MPP
LogβGdL
LogβEuL
23.2 ± 0.01
24.5 ± 0.06
18.1 ± 0.06
22.2 ± 0.03
17.6963 ± 0.03
17.3245 ± 0.02
7.1879 ± 0.05
6.0102 ± 0.03
13.7439 ± 0.04
13.5486 + 0.06
11.3860 ± 0.08
18.0548 ± 0.01
2
LogβZn L
2
LogβCu L
(
III) metal ion from the cage. In Ln-DO3AM, due to weaker electrostatic
3
+
interaction between Ln ion and the neutral ligand DO3AM, it is much
challenging to protonate neutral arm as compared to negatively charged
acetate arm which ultimately results in limiting their rate of acid-
catalyzed dissociation. Although stability constant values of Gd-
DO3AM is much less than Gd-DOTA or Gd-DO3A, high comparable ki-
netic inertness of Gd-DO3AM with Gd-DOTA makes it suitable for CEST
Imaging.
Table 3
Pseudo First order rate constants characterizing the metal exchange reactions of
3
+
3+
2+.
Gd -DO3AM-MPP with Eu and Zn
Metal-Ligand Complex
3
+
2+
Zn
Eu
ꢀ
ꢀ
ꢀ
5
3
6
Gd-DOTA[36]
Gd-DO3A[37]
Gd-DO3AM-MPP
2.1 × 10
2.3 × 10
3.5 × 10
Not observed
ꢀ 2
1.4 × 10
ꢀ 5
3.4 × 10
3
.3.3. rGd-H distance between gadolinium and coordinated water
3
3
.3. Physicochemical characterization
The distance between the water molecule and Gadolinium was
evaluated theoretically using the Gaussian software Fig. 4. The unsol-
vated Gd-DO3AM-MPP was found to have lower energy than the sol-
vated Gd-DO3AM-MPP with a difference in energy (DE = E[Gd-DO3AM-
.3.1. Equillibrium Measurements
The complexation properties of DO3AM-MPP are different from
DOTA and DO3A due to three amide groups. The differences in the
stability constants of complexes formed between DO3AM and metal ions
were determined using potentiometric titration. The metal selected were
MPP]-
-E[Gd-DO3AM-MPP]-
Unsolv
) of 7.31 kJ/mol. The un-
Solv
stabilization of solvated Gd-DO3AM-MPP is due to the presence of a
water molecule in the inner coordination that weakens the coordination
3
+
3+
lanthanide trivalent ions (Eu ) and biologically significant divalent
bonds of Gd with other pendant arms. The distance of the coordinated
metals ions (Zn2 , Cu ). The pKa’s are summarized in Table 1 .
It was observed that the pka values of DO3AM are lower than cor-
responding DOTA and DO3A due to the difference in amide and acetate
arms basicity. Similarly, stability constant (Logβ, Table 2) of DO3AM
+
2+
3+
water molecule from Gd is 2.7 Å, which is in fair agreement for the
NMR relaxometry experiment. [32]
3.4. Targeting capability
3
+
complexes are lower than DOTA due to weaker Ln -amide oxygen bond
in DO3AM than Ln3 -acetate oxygen bond in DOTA or DO3EA. [35] The
stability constant of Gd-DO3AM-MPP is slightly higher than Eu-DO3AM-
MPP due to lanthanide contraction, which leads to an increase in elec-
tron density on the metal and consequently, a weak coordinate bond
between amide oxygen and metal ion.
+
3.4.1. & 3.4.2 Binding constant through fluorescence quenching studies
Fluorescence provides information about the molecular environment
in the vicinity of the chromophore molecule. Fluorescence quenching
studies were performed to evaluate the binding constant between the
ligand (quencher:(Eu-DO3AM-MPP)) and protein (5-HT1A receptor).
The incremental addition of Eu-DO3AM-MPP (UV plot of Eu-DO3AM-
3
.3.2. Kinetic stability
MPP λ
max
276 nm, ESI Figure 16) resulted in the reduction of fluores-
To avoid the toxic effects of free Gadolinium, trans-metalation of
cence intensity for 5-HT1 ) Fig. 5. Under the experimental conditions,
A
Gadolinium with endogenous metal cation should not occur. The extent
of dissociation of Gd-DO3AM-MPP was estimated from the kinetics of
Eu-DO3AM-MPP illustrated no fluorescence emission in the range
300–400 nm and did not affect serotonin intrinsic fluorescence. As
Fig. 4. DFT minimized the structure of (a) Unsolvated Gd-DO3AM-MPP (b) solvated Gd-DO3AM-MPP. The presence of water molecules in the inner coordination
sphere destabilizes the coordination bonds reflected in increases in distances, thereby giving an energy difference of E [Gd-DO3AM-MPP]-Solv -E[Gd-DO3AM-
MPP]-Unslov of 7.31 kJ/mol. The rGd-H is 2.7 Å.
7

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 5. (a) Emission Spectra of Eu-DO3AM-MPP with Excitation at wavelength 276 nm (b) Fluorescence spectra of serotonin (λemission 330 nm) with varying con-
centration of Eu-DO3AM-MPP (c) Stern-Volmer Plot of Serotonin-Eu-DO3AM at 25℃ (d) Scatchard plot for Serotonin-Eu-DO3AM-MPP at 25℃.
deduced by the Stern Volmer plot and equation [39]) (Fig. 5c), the as-
3.4.4. Characterization of Gd-DO3AM-MPP using power X-ray diffraction
The obtained powder XRD pattern for Gd-DO3AM-MPP resembles
well with monoclinic symmetry with space group P12/c1 (ICSD Number
98–010-9886), which confirm the presence of Gadolinium and other
chemical constituents and, therefore, supports the chelating affinity of
the synthesized complex. The crystal parameters and pattern for the Gd-
DO3AM-MPP are depicted in Table S1 and Fig. 7, respectively. It was
analysed that due to the presence of organic moiety in Gd-DO3AM-MPP
XRD pattern is amorphous.
-
1
sociation constant, KSV was found to be 0.0051 (micro M) .
3
.4.3. Binding affinity assay
The specific binding of Eu-DO3AM-Propyl-MPP with 5-HT1A recep-
tor was estimated through the saturation binding assay. 5-HT1A re-
ceptors isolated in murine brain hippocampal homogenate were
incubated with the complex. [40] Specific binding was calculated by
Scatchard plot analysis (Fig. 5d), which revealed the high affinity of Eu-
DO3AM-MPP towards neuronal hippocampus receptor with k
0.02 pM. Non-specific binding was calculated by using a 100 fold
excess of serotonin and methoxyphenyl piperazine.
d
value 62
±
3
.5. Theoretical docking studies
Theoretical docking studies gave an insight into the interactions
between ligand Gd-DO3AM- MPP and 5-HT1A receptors. The ligand
Fig. 6. (a) and (b) 3D and 2D Ligand Interaction Diagram of Gd-DO3AM-MPP with 5-HT1A receptor.
8

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
Gd3 -DO3AM-MPP was found to dock on a 5-HT1A model with a G-Score
of ꢀ 4.81 and binding energy ꢀ 57.29 kcal/mol. Way-100635, the well-
known non-metal antagonist-ligand for the 5-HT1A receptor had a
docking score was ꢀ 7.7 (2D and 3D Ligand Interaction Diagram in ESI)
under similar docking set up. The lowering of G-score may be attributed
to the presence of metal and steric hindrance in Gd-DO3AM-MPP. In 2D
and 3D ligand interaction diagram (Fig. 6 and Table 4), it was observed
that residues Ser 182, Thr 188, Ser 190, and Ser 374 constituted the
polar groups of ligand-binding pocket. One of the amide arms in DO3AM
interacted through H-bonding with Arg 181. Another amide arm showed
polar and covalent interaction with Lys 191 and Ser 190. The MPP
piperazine was involved in polar covalent interaction with Asp116 and
retained its antagonist mode of binding with the receptor.
+
Fig. 7. Powder X-ray Diffraction Pattern for Gd-DO3AM-MPP.
Table 4
GScore and Ligand Interaction of DO3AM-MPP and DFT optimized Gd-DO3AM-MPP and antagonist WAY-1OO635 with 5-HT1A receptor system.
Residual Interactions
Ligand
Non-Polar Residues
Polar Residues
GScore (Kcal/
mol)
Gd-DO3AM-
MPP
Leu 366, Val 367, Phe370, Cys 371, Cys 375, Pro 378, Leu 380, Ala 383, Ile 189
Ile 189, Cys 187, Leu 380, Ile 384, Trp 387, Leu 388, Tyr 96, Phe 370, Val 367, Phe
Hip 193, Asp 192, Lys 191, Ser 190, Thr 188, Thr 379,
Ser 374, Asp 180, Glu 179
ꢀ 4.81
ꢀ 7.7
WAY-100635
Lys 191, Thr 188, Thr 379, Thr 200, Asp 116
1
12, Val 364, Ile 363, Val 117, Cys 120
Fig. 8. MTT assay to analyse the cytotoxicity of Gd-DO3AM-MPP complex on HEK cell line.
Fig. 9. In vivo MR Imaging (a) Control mice (b) showing preferential binding of Gd-DO3AM-MPP in 5-HT1A receptor post injection of Gd-DO3AM-MPP contrast
agent after BBB disruption with mannitol.
9

Anju et al.
Bioorganic Chemistry xxx (xxxx) xxx
3
.6. Cytotoxicity studies
[2] A.C. Sedgwick, J.T. Brewster, P. Harvey, D.A. Iovan, G. Smith, X.-P. He, H.e. Tian,
J.L. Sessler, T.D. James, Metal-based imaging agents: progress towards
interrogating neurodegenerative disease, Chem. Soc. Rev. 49 (10) (2020)
Cytotoxicity assay was performed on HEK cell line to analyse the
biological assessment of the resulting complex Gd-DO3AM-MPP using
MTT as a biomarker. The toxicity of Gd-DO3AM-MPP was evaluated in
the concentration range from 100 µM to 1 pM. In between concentration
range 100 µm to 1 pM, maximum cells death of 9.6% was noticed after
completion of 72 h (Fig. 8) which indicates the good percentage cell
viability even after the prolonged time treatment. These results suggest
that the Gd-DO3AM-MPP complex has minimum cytotoxicity, making it
suitable for in vivo medical imaging applications.
2
886–2915, https://doi.org/10.1039/C8CS00986D.
[3] T. Kanda, M. Matsuda, H. Oba, K. Toyoda, S. Furui, Gadolinium Deposition after
Contrast-enhanced MR Imaging, Radiology 277 (3) (2015) 924–925, https://doi.
org/10.1148/radiol.2015150697.
[
4] L.M. De Le o´ n-Rodríguez, A.F. Martins, M.C. Pinho, N.M. Rofsky, A.D. Sherry, Basic
MR relaxation mechanisms and contrast agent design: MR Relaxation Mechanisms
and Contrast Agents, J. Magn. Reson. Imaging 42 (3) (2015) 545–565, https://doi.
org/10.1002/jmri.24787.
[
[
5] S. Chaturvedi, A. Kaul, P.P. Hazari, A.K. Mishra, Mapping neuroreceptors with
metal-labeled radiopharmaceuticals, Med. Chem. Commun. 8 (5) (2017) 855–870,
https://doi.org/10.1039/C6MD00610H.
6] G.R. Naumiec, G. Lincourt, J.P. Clever, M.A. McGregor, A. Kovoor, B. DeBoef,
Synthesis of a β-CCT-lanthanide conjugate for binding the dopamine transporter,
Org. Biomol. Chem. 13 (9) (2015) 2537–2540, https://doi.org/10.1039/
C4OB02165G.
3
.7. In vivo CEST MR imaging
[
[
[
7] I. Zigelboim, A. Weissberg, Y. Cohen, Target-Specific Ligands and Gadolinium-
Based Complexes for Imaging of Dopamine Receptors: Synthesis, Binding Affinity,
and Relaxivity, J. Org. Chem. 78 (14) (2013) 7001–7012, https://doi.org/
The longitudinal and relaxation time and binding of metal complex
Gd-DO3AM-MPP in mouse was analyzed using MR Imaging technique.
The Gd-DO3AM-MPP complex shows preferential uptake in the hippo-
campus region of the brain Fig. 9 which confirms the applicability of the
complex for medical imaging purpose.
1
0.1021/jo400646k.
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All animal experiments were performed following the guidelines of
the CPCSEA, Government of India, New Delhi. Animal handling and
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
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DRDO, Ministry of Defense and Department of Science and Tech-
nology (DST) jointly supported this research under the project. The work
was carried at the Institute of Nuclear Medicine and Allied Sciences
(
INMAS)-Delhi, All India Institute of Medical Sciences-Delhi, and the
Supercomputing Facility for Bioinformatics and Computational Biology
SCF-BIO)-IIT Delhi. The authors gratefully acknowledge the Institutes
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for providing facilities and support. The authors also thank the Chem-
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