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2021 Investigating Bismuth complexes with benzoazacrown tri- and tetra-acetates

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Investigating the Bismuth Complexes with Benzoazacrown
Tri- and Tetra-Acetates
Bayirta V. Egorova,*[a] Ekaterina V. Matazova,[a] Gleb Yu. Aleshin,[a] Anastasia D. Zubenko,[b]
Anna V. Pashanova,[b, c] Ekaterina A. Konopkina,[a] Artem A. Mitrofanov,[a]
Anastasia A. Smirnova,[a] Alexander L. Trigub,[d] Valentina A. Karnoukhova,[b]
Olga A. Fedorova,[b, c] and Stepan N. Kalmykov[a, d]
The complexation properties towards Bi3 + of benzoazacrown
ligands with five (H3BA3A) and six (H4BATA) heteroatoms and
corresponding number of acetic arms have been investigated.
Complexation constants were determined via potentiometric
titration for BA3ABi complex (logβ = 26.2) and refined for
BATABi complex (logβ = 31.7). According to NMR, X-ray and
EXAFS results, chelation of cation by H3BA3A appears to be
outside the macrocyclic cavity both in the solid state and in the
aqueous solution. This out-cage location despite the high
stability constant causes serum and in vivo instability of
complex of smaller azacrown H3BA3A in contrast to H4BATA.
Whereas the larger macrocycle and four pendant arms of
H4BATA provide long-range coordination effectively shielding
the cation thereby ensuring high inertness in vitro, low
accumulation of the radionuclide in the organs, and fast
oxygen ligating atoms. Hence, the most effective chelators used
with this cation in radiotherapeutics are polyaminopolyacetates,
such as H4DOTA and CHX-DTPA.[6,7] The latter represents
rigidified H5DTPA and is commonly used for labeling of heatsensitive molecules to improve the kinetic inertness of the
formed complex compared with H5DTPA. The higher kinetic
inertness is accompanied by longer radiolabeling,[8] which is
crucial for short-lived Bismuth radioisotopes. Recently, it was
shown that azacrown ethers with a cavity of 6 heteroatoms and
picolinate groups – macropa and derivative ligands – are
promising for use with Ac3 + and rare earth elements (REE).[9,10]
Additionally, we determined that the aza-18-crown-6 tetraacetate (H4BATA) with the same macrocycle as macropa is a
candidate for the Bi3 + cation.[11] We suggested that its high
efficiency is associated with the H4DOTA structure: H4BATA
seems to be an opened version of H4DOTA, resulting in
decreased restriction inside the macrocycle compared with
H4DOTA. Additionally, the presence of the rigid benzyl moiety
in the macrocycle provides a way for bifunctionalization
distanced from the cation-coordinating part. H3NOTA analogs
have also been considered for Bismuth cations.[12,13] Based on
this, in this work, we studied the complexation features of the
aryl-containing azacrowns H4BATA and H3BA3A (as opened
H3NOTA) (Scheme 1) with Bi3 +, including the structural features
Alpha particles have a high linear energy transfer that causes
more efficient DNA breakage of double-stranded cancer cells
compared with β particles.[1] Additionally, the short range of
the α-particle leads to low toxicity to the surrounding healthy
tissues.[2] Bismuth isotopes 212,213Bi are particularly attractive
because of their suitable radiation characteristics and the
availability of methods to produce these isotopes, such as using
convenient radionuclide generator systems in particular.[3,4]
Radiopharmaceuticals based on peptides and antibodies labeled with 213Bi isotopes have already demonstrated their
potency in clinical trials.[5] One of the challenges for targeted
alpha therapy (TAT) using Bi radioisotopes is the design of
chelators that are appropriate for forming complexes of high
thermodynamic stability and kinetic inertness to avoid possible
transchelation and transmetallation in biological media. Bismuth, as an intermediate cation according to the HSAB theory,
can be coordinated via combination of basic nitrogen and
[a] Dr. B. V. Egorova, E. V. Matazova, Dr. G. Y. Aleshin, E. A. Konopkina,
Dr. A. A. Mitrofanov, A. A. Smirnova, Dr. S. N. Kalmykov
Chemistry Department
Lomonosov Moscow state university
119991 Leninskie Gory, 1/3, Moscow, Russian Federation
E-mail: bayirta.egorova@gmail.com
[b] Dr. A. D. Zubenko, A. V. Pashanova, V. A. Karnoukhova, Dr. O. A. Fedorova
A. N. Nesmeyanov Institute of Organoelement Compounds of Russian
Academy of Sciences
119991 Vavilova, 28, GSP-1, Moscow, Russian Federation
[c] A. V. Pashanova, Dr. O. A. Fedorova
D. Mendeleev University of Chemical Technology of Russia
125047, Miusskaya sqr. 9, Moscow, Russian Federation
[d] Dr. A. L. Trigub, Dr. S. N. Kalmykov
National Research Center “Kurchatov Institute”
123098 Akademika Kurchatova sqr., 1, Moscow, Russian Federation
Supporting information for this article is available on the WWW under
Eur. J. Inorg. Chem. 2021, 3344 – 3354
Scheme 1. Ligands H3BA3A and H4BATA.
© 2021 Wiley-VCH GmbH
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of the complexes in aqueous solution via X-ray absorption
spectroscopy, 1H NMR, and single crystal X-ray diffraction. The
stability constants of the bismuth(III) complexes were determined, and in vivo experiments were conducted. Additionally,
full labeling and serum stability studies of the complexes are
also described.
Results and Discussion
Studies of the Bismuth(III) complexes H3BA3A and H4BATA
In general, the thermodynamic stability of bismuth(III) complexes of macrocyclic ligands like H4DOTA[14] cannot be
determined by potentiometric titrations because of the rapid
formation of the hydroxide species even at very low pH.
However, in the present research this method was applicable
for the determination of the stability constants. An excess of
chloride-ion along with fast binding of Bismuth(III) by considered ligands caused an effective hampering of cation’s
hydrolysis upon pH increasing and allowed to collect data for
constants calculation (Table 1). The stability constants of
bismuth(III) complexes with H4BATA were previously reported in
a brief communication[11] and have been further clarified in this
work, and refined values are presented in Table 1.
Table 1. Stepwise (KBiHnL) complexation constants (in log units) of H4BATA
(refined for wider pH range vs[11]), H3BA3A, and H4DOTA at 25.0 °C in 0.1 M
KNO3[a] and the corresponding pBi values
BiHL + H + ⇄BiH2L
BiL + H + ⇄BiHL
Bi3 + + L⇄BiL
BiLOH + H + ⇄BiL
log KBiHnL
H4DOTA[b] [14,15]
[a] L denotes the ligand; charges of ligand and complex species were
omitted for simplicity. [b] I = 1.0 M NaBr
The equilibrium state was reached quickly during the
titration experiments. The H3BA3A bismuth(III) complex began
to dissociate at high pH, forming a hydroxide chelate species
which was easily distinguishable in the species distribution
diagram (Figure 1). Further pH increase caused visually observable formation of the insoluble hydroxide species in the range
of pH 9–10 resulting in the titration experiments with Bi3 + and
H3BA3A being terminated. The value of the BA3ABi stability
constant was lower than that for DOTABi , however, it was still
sufficiently high to form a stable complex that can be defined
by potentiometric titration. In the neutral pH range, the main
form of the complex in both cases was LBi (Figure 1). The HLBi
complex forms in acidic media, and the formation of LBiOH was
observed at pH values > 8.
To unify logβ for the bismuth complexes with both ligands,
we repeatedly titrated solutions of Bi3 + with H4BATA over a
wider pH range starting from pH 1.6 (compared to the data
presented in[11]). This experiment allowed us to refine logβ
values for protonated forms of complex that affected value for
BATABi as well, which turned out to be higher by 1.4 orders
than that of logβ(DOTABi ).
It should be noted that the high basicity of H4BATA would
not allow chelation at the initial low pH value, leading to the
presence of the BiCln species up to pH 2. Whereas, H3BA3A with
lower protonation constants readily binds the cation at the start
of the titration, even in the monoprotonated form (Figure 1).
For correct comparison of the complexation ability of the
different ligands, the different basicity of the ligands should be
considered. Thus, pM values (pM = log[Metal]free), calculated
for a certain pH by taking into account the known values of the
stability constants are often applied for better estimation of the
comparative complex stability. Hence, pBi values for complexes
with H4BATA, H3BA3A, and the well-known H4DOTA were
calculated at pH 7.4 using stability constant values for an excess
of ligand: [Bi3 + ]tot = 1.0 μM and [L]tot = 9.0 μM (Table 1). Without
the impact of protonation, the obtained pBi values of both the
H3BA3A and H4BATA were of the same order of magnitude,
while the value of DOTABi complex was higher. These results
Figure 1. Species distribution diagram of bismuth(III) in the presence of H3BA3A (a) and H4BATA (b) in an aqueous solution at [Bi3 + ]total = [L]total = 1.0 mM,
[Cl ]total = 23 mM.
Eur. J. Inorg. Chem. 2021, 3344 – 3354
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demonstrate that both the studied ligands have a good ability
to maintain bismuth cation binding despite the different levels
of logβ. Although pBi had a lower value than for H4DOTA,
dissociation of complexes with H3BA3A and H4BATA was still
expected to be extremely low, which is promising for complexation of bismuth radioisotopes.
Structural characterization of the complexes
X-ray Crystal Structure of [BA3A·Bi]
The structure of complex [BA3A·Bi] was established via singlecrystal X-ray diffraction analysis (Figure 2, S1). The complex
crystallizes as a hydrate with three water molecules in the
symmetrically independent part of the unit cell. Bond lengths
and angles in the macrocyclic ligand were within normal
ranges, as confirmed by a Mogul geometry check.[16] The
alicyclic crown fragment of the macrocycle was significantly
distorted from the idealized planar conformation to adopt the
geometry required for effective coordination with a large
bismuth(III) cation; for this, the alicyclic part bent out from the
average plane of the rigid part. The cation was located outside
the cavity of the macrocycle, and was coordinated by three
oxygen atoms of the carboxyl groups and three nitrogen atoms
of the macrocycle, forming a distorted pentagonal pyramid
with an O(6) atom on the vertex and N(1), N(2), N(3), O(4), and
O(8) atoms at the base with bond lengths in the range
2.1757(16)-2.5745(18) Å (Table 2). The cation was located outside the cavity of the macrocycle, and was coordinated by three
oxygen atoms of the carboxyl groups and three nitrogen atoms
of the macrocycle, forming a distorted pentagonal pyramid
with an O(6) atom on the vertex and N(1), N(2), N(3), O(4), and
O(8) atoms at the base (Table S2). Besides, two shortened
distances to oxygen atoms of the macrocycle O(1) and O(2)
(2.9010(15) and 2.9832(15) Å, respectively) could be observed.
To ensure formation of bonds between Bi and the mentioned
atoms we performed QTAIM[17] analysis implemented into
multiwfn package[18] (Fig S2, Table S1).
Two carboxyl groups of the macrocycle participated in
hydrogen bonding with three water molecules. The set of
hydrogen bonds connected two complexes into the centrosymmetric dimeric-like structure, with six water molecules located
between the complex moieties (Figure S1).
NMR study of the complex formation
Figure 2. General view of the [BA3ABi] complex in crystal. Anisotropic
displacement parameters are drawn at the 50 % probability level, hydrogen
atoms were omitted for clarity.
The complexation of the ligands H3BA3A and H4BATA with the
bismuth cation in aqueous solutions was studied using 1H NMR
spectroscopy. The 1H NMR spectra of the free ligands, the
spectra after the addition of bismuth nitrate and adjusting the
pD of the solution to a certain value were recorded. The spectra
in the presence and absence of the Bi3 + ion were analyzed to
assess the interactions between the cation and the ligands
H3BA3A and H4BATA.
Table 2. Selected bond lengths [Å] and angles [°] of the metal coordination environment in [BA3ABi] complex.
Bond lengths [Å]
Bond angles [°]
Eur. J. Inorg. Chem. 2021, 3344 – 3354
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Figure 3 shows the 1H NMR spectra of the deprotonated
form of the ligand BA3A3 and the complex BA3ABi. Changes in
the 1H NMR spectrum observed after the addition of the Bi3 +
ion to the D2O solution of H3BA3A demonstrate geminal
coupling of the methylene protons belonging to the macrocyclic fragment as well as pendant side-arms. As a result, the
seven proton signals of the free ligand BA3A3 transform to
twelve proton signals (Figure 3).
This behavior is consistent with the presence of a single
complex BA3ABi with a relatively rigid structure in solution;
fluxional interconversion at ambient temperatures was not
observed (on the NMR time scale). Additionally, no change in
the number of peaks in the 13C NMR spectrum of the ligand
H3BA3A was found upon Bi3 + coordination, suggesting no
chemically distinct isomers were formed (Figure S3).
In the BA3ABi complex spectrum, the resonances of the
protons of the macrocycle, benzene, and pendant arms downfield change because of the polarization effect of the metal ion
(Table S2). It can be seen that the H1, H2, and H4 protons are
characterized by smaller shifts compared with the H5, H6, and H7
protons, which may indicate weaker binding of the Bi3 + cation
by the oxygen atoms of the crown ring. Additionally, the
methylene protons of the side arms exhibit the largest signal
shifts. In this regard, it can be assumed that all heteroatoms of
the macrocycle as well as all three acetate groups of the ligand
participate in complexation with the bismuth cation.
Interestingly, a splitting of the signals belonging to the
geminal H8 protons of two opposite arms can be observed,
while methylene H10 protons of the central arm appear as a
singlet. This can be explained by the fact that the central
acetate group is in the plane of symmetry, which is located
perpendicular to the macrocycle, and therefore, the geminal
protons H10 are magnetically equivalent. In contrast, the two
opposite acetate groups are located above the macrocyclic
cavity, coordinating the metal cation.
Figure S6 shows the 1H NMR spectra of the free ligand
H4BATA, as well as in the presence of the Bi3 + cation (pD = 6.3).
The resulting spectrum of the complex is much more
complicated than the initial ligand. Analysis of the aromatic
region of the spectrum using 2D COSY spectroscopy revealed
two sets of signals corresponding to the protons of the benzene
ring (Figure S7). Using 2D NOESY spectroscopy, it was possible
to identify the H4 protons of the macrocycle closest to benzene
(Figure S8). Further complete correlation of signals was complicated by the proximity of a large number of different signals to
each other. Based on the potentiometric titration data, the
aqueous solution contains only LBi- particles at pH 6.3 (Figure 1b). In this regard, it can be assumed that the obtained
NMR spectrum corresponds to complexes with the same
composition, but with different structures, i. e., two conformers
of the H4BATA complex with Bi3 + cation in a molar ratio of 4 : 1
are simultaneously present in the solution (based on the
integral intensity of the protons of the benzene ring). On the
2D NOESY spectrum, in addition to negative NOE’s correlations,
positive off-diagonal peaks can be seen. These peaks are
correlations from conformational exchange, so the BATABi
complex has two different conformations in slow exchange
with each other on the NMR time scale and gives the proton
spectrum with a doubling of each resonance. Compared to the
spectrum of the free H4BATA ligand, the signals of the benzene
ring with different values are shifted to the high-field region.
This effect can be caused by the shielding of benzene protons
by closely spaced carbonyl groups. Thus, it can be assumed that
the two conformers of the BATABi complex formed in solution
have different positions of the chelating groups relative to the
macrocyclic plane. In addition, all four acetate groups are
involved in the coordination of the Bi3 + cation. Analysis of the
Figure 3. 1H NMR spectra of the free ligand BA3A3 (CL = 8 mM, pD = 12.4) and its complex with Bi3 + (CL = 10 mM, pD = 5.3) in D2O.
Eur. J. Inorg. Chem. 2021, 3344 – 3354
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integral intensities of signals in the spectrum led to the
conclusion that each proton appears as an individual signal,
thus both conformers of the complex have C1 symmetry. This
assumption is in good agreement with the 13C NMR spectrum of
the BATABi complex (Figure S9), in which an increased set of
signals can be seen compared to the spectrum of the free
ligand, namely: 20 signals in the aromatic region instead of 5
signals, and 28 signals in the aliphatic region instead of 7
signals, respectively.
X-ray absorption spectroscopy and DFT calculations of bismuth
The formation and coordination environment of the bismuth
cation in the presence of H3BA3A and H4BATA were further
confirmed by EXAFS studies. The nearly similar atomic scattering factors of C, N and O do not allow the atoms to be
distinguished in the local surrounding of the Bi atom. First, the
absence of Bi Bi interactions indirectly proved complete
complexation of Bi3 + by both ligands at pH 5.0–5.5; otherwise,
at millimolar concentrations, bismuth forms polynuclear
hydroxyl species at pH > 2 or precipitates.[19] Secondly, distances
of 2.2–2.5 Å (Figure 4, Table 3) agree well with the X-ray single
crystal data for other complexes of bismuth with
polyaminopolycarboxylates[20] including crystal structure of
[BA3A·Bi] (Table 2). The closest distances in BA3ABi 2.2–2.4 Å
correspond to acetic oxygens as O(4), O(6), O(8) while longer
~ 3 Å can be associated with macrocyclic atoms of C, N, O.
Furthermore, the obtained structural data concerning the first
coordination sphere for both complexes were similar to each
other and the total coordination numbers can formally be
evaluated as 8 for BATABi and 9 for BA3ABi. Noteworthy, the
coordination sphere that is further away is more precisely fitted
for the BATABi complex (Table 3, Figure 4), which means
higher structural organization in the outer coordination shells
(on the long range). This is why the CNs at larger distances are
much higher for BA3ABi than for BATABi .
To model coordination of Bi3 + in complexes we performed
DFT calculations of optimized geometry for both BA3ABi and
BATABi (Figure 5, Table 4). Firstly, arrangement of coordinating
atoms in BA3ABi agrees with crystallographic data: cavity needs
to be bent out to chelate the cation by macrocyclic nitrogens
and carboxylic oxygens. This causes one side location of all the
pendant arms relative to macrocycle. Additionally, order of
distances with coordinating groups is the same as in the solid
Figure 4. The fitted spectra for X-ray absorption of·Bismuth complexes with H3BA3A (a) and H4BATA (b) in the R- and k –spaces.
Table 3. Structural parameters refined from fitting the EXAFS Bi L3-edge spectra for Bi-BA3A and Bi-BATA at pH 5–5.5.
Coordination shell
Bi-BA3A (pH 5–5.5)
R, Å
σ, Å2
2.23 � 0.01
2.40 � 0.02
3.03 � 0.02
3.24 � 0.02
3.72 � 0.02
4.41 � 0.04
4.97 � 0.07
0.007 � 0.003
0.007 � 0.003
0.007 � 0.003
0.005 � 0.003
0.005 � 0.007
0.005 � 0.007
0.005 � 0.007
Eur. J. Inorg. Chem. 2021, 3344 – 3354
Bi-BATA (pH 5–5.5)
R, Å
σ, Å2
2.17 � 0.01
2.47 � 0.02
3.15 � 0.02
3.45 � 0.06
4.31 � 0.03
4.51 � 0.09
0.0045 � 0.0006
0.007 � 0.003
0.005 � 0.003
0.005 � 0.003
0.005 � 0.007
0.004 � 0.025
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Figure 5. Optimized geometries of the complexes: BA3ABi (a), BATABi conformation 1 (b) and BATABi conformation 2 (c).
Table 4. Interatomic distances [Å] of the metal coordination environment
in BA3ABi and BATABi complexes according to optimized geometry.
Conformation 1
Conformation 2
state: acetic oxygens are the closest, then aminogroups and
macrocyclic oxygens at the distance almost 3 Å (Table 2,
Table 4). Furthermore such environment of bismuth is consistent with fitting of EXAFS spectrum and light C/N/O atoms are
found at corresponding distances (Table 3). In general, the
structure of complex in crystal form is more “compact” than the
calculated gazeous one, resulting in smaller M-ligand distance.
For the BATABi complex two conformations were established (Figure 5, Table 4). With regard to larger size of macrocycle cation encapsulates inside the cavity in both geometries.
It is in line with earlier published bismuth complex of triacetate
derivative of aza-18-crown-6[21] where cation was also embedded into the cavity and chelated by three acetates from
both sides of macrocycle. The obtained structures of BATABi
differ in an arrangement of pendant arms relative to azacrown
cavity. The first one is characterized by three arms (O(4), O(6),
O(8)) from one side plus capped by the fourth (O(10)) from
another (Figure 5b). In the second conformation arms are
altering to chelate cation from both sides with respect to
macrocycle: two (O(4), O(8)) from above and two (O(6), O(10))
from below (Figure 5c). Apparently such surrounding on all
sides causes elongation of distances with chelating atoms
compared to BA3ABi. As soon as coordination sphere is
saturated by four aminogroups and four acetates macrocyclic
oxygens O(1) and O(2) are distanced from Bi3 + as far as it is
possible in the cavity - 4–5 Å. In addition QTAIM[17] analysis
implemented into multiwfn package[18] for BATABi complex (Fig
S2, Table S1) has shown absence of interaction between cation
and macrocyclic oxygens in contrast to BA3ABi.
Furthermore regarding effectiveness of BATABi complex[11] we
attempted to evaluate the changing of coordination environments
of Bi3 + in the BATABi complex at different pH by EXAFS
measurements of the solutions at pH 1, 3 and 11 (Figure 6,
Table 5) in addition to pH 5-5.5 discussed above. Sample at pH 1
was used as reference to show the spectrum and obtained
environment of free cation in presence of Cl- and H4BATA. The
obtained distance 2.68 Å corresponds to Bi Cl bond in analogous
compounds.[22] Light atoms at 2.33 Å can be attributed to the
hydration shell. According to obtained distances decreasing and
increasing of pH in relation to coordination at pH 5–5.5 causes
extension of the number of the closest neighbors. In the case of
pH 3 high CN at 2–3 Å is caused by an incomplete chelation of
cation by donor atoms of ligand due to the protonation as it is
evident from the speciation diagram (Figure 1b). Cation is partially
surrounded by solvent molecules and reflections of plenty light
Table 5. Structural parameters refined from fitting the EXAFS Bi L3-edge spectra for Bi-BATA solutions at varied pH.
R, Å
σ, Å2
R, Å
σ, Å2
Bi C/N/O
Bi C/N/O
Bi C/N/O
Bi C/N/O
Bi C/N/O
Bi C/N/O
2.33 � 0.01
3.18 � 0.03
4.19 � 0.07
4.54 � 0.06
0.005 � 0.002
0.006 � 0.004
0.005 � 0.005
0.003 � 0.0005
2.32 � 0.02
2.53 � 0.02
3.20 � 0.03
4.78 � 0.03
4.64 � 0.07
0.006 � 0.002
0.004 � 0.001
0.007 � 0.003
0.002 � 0.004
0.005 � 0.011
2.68 � 0.03
0.009 � 0.006
Eur. J. Inorg. Chem. 2021, 3344 – 3354
R, Å
σ, Å2
2.17 � 0.01
2.47 � 0.02
3.15 � 0.02
3.45 � 0.06
4.31 � 0.03
4.51 � 0.09
0.0045 � 0.0006
0.007 � 0.003
0.005 � 0.003
0.005 � 0.003
0.005 � 0.007
0.004 � 0.025
R, Å
σ, Å2
2.36 � 0.03
2.61 � 0.03
3.29 � 0.03
4.64 � 0.06
0.005 � 0.003
0.004 � 0.003
0.005 � 0.002
0.007 � 0.007
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Figure 6. The fitted spectra for X-ray absorption of Bismuth complexes with H4BATA in the R- (a) and k –spaces (b).
atoms are observed in the spectra. Upon pH increase at pH 5–5.5
both monoprotonated and deprotonated complexes exist with
prevalence of the latter and cation introduces into the cavity in
agreement with DFT calculated structure. Finally at highest pH 11
bismuth cation starts to hydrolyze and external OH groups
facilitate gradual escape of the cation from surrounding by
ligands’ donor atoms enhancing number of closest light atoms as
in the case of lower pH 3.This approach experimentally confirmed
structural reorganizations of the BATABi complex upon pH
variation in agreement with speciation diagram (Figure 1)
obtained by potentiometric studies: deprotonation of the ligand
causes encapsulation of the cation while protonated forms as well
as hydroxide specie force its escape out of the cage. This way is
similar to H4DOTA complexation of cations[23,24] but significantly
larger macrocycle of H4BATA provides much less restrictions for
deprotonation and consequent embedment of bismuth inside the
ligand’s cavity.
Because the pH value could influence complexation, we
evaluated labeling yields for several concentrations at different
pH values, and no significant differences were observed. The
labeling yield reached > 96 % at pH 6 and 77–100 μM of H4BATA
and > 98 % at pH 7 and 60–100 μM of H3BA3A (Table 6).
In vitro stability studies
As a preliminary estimation before in vivo behavior analysis,
challenge of the labeled compounds [207Bi]LBi with fetal bovine
serum was carried out. Serum stability was studied using [207Bi]
BATABi and [207Bi]BA3ABi radiolabeled solutions (radiochemical
purity 97 %, 1 kBq of 207Bi) mixed with a fetal bovine serum. Serum
stabilities were controlled after several time periods at 37 °C.
Figure 7 shows the comparative protein binding of radionuclide
upon challenging solutions with [207Bi]BA3ABi and [207Bi]BATABi.
After 1 h of incubation (~ 1 decay period of 212,213Bi), the [207Bi]
The labeling efficiency of each studied ligand was determined
using the TLC technique. The radiolabeling yields of H4BATA
with 207Bi have previously been determined in a number of
buffer systems[11] and are described here for comparative
purposes, and these values as well as the radiolabeling yields of
H3BA3A are presented in Table 6.
Table 6. Percentage of incorporation of
mean values � SD (n = 3).
Bi by H4BATA and H3BA3A:
c(L), [μM]
H3BA3A, pH 6.0
H3BA3A, pH 7.0
H4BATA, pH 6.1
H4BATA, pH 8.0
49 � 5 %
80 � 2 %
88 � 5 %
70 � 5 %
77 � 4 %
89 � 3 %
82 � 3 %
97 � 3 %
91 � 4 %
98 � 3 %
95 � 5 %
78 � 6 %
95 � 5 %
98 � 6 %
Eur. J. Inorg. Chem. 2021, 3344 – 3354
Figure 7. Transchelation of [207Bi]Bi3 + from complexes with H4EDTA,[21]
H3BA3A, and H4BATA[11] by serum proteins.
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BA3ABi complex demonstrated 23 % 207Bi3+ protein binding and it
was 38 % after 2 h (~ 2–3 decay periods of 212,213Bi). Additionally,
there was no any significant 207Bi3 + protein binding for the case of
[207Bi]BATABi even after 2 days, which indicated remarkable
complex stability. This indicates that, in solution, it is more likely
that the cation is not included in the cavity of H3BA3A, which is
similar to its crystalline structure. For the BATABi complex, with
regards to the higher organization on the outer coordination
shells revealed by EXAFS, it is reasonable to suggest that inclusion
of Bi3 + inside the macrocycle provides effective shielding of the
cation from competing agents.
Interestingly that despite much higher stability constant
complex BA3ABi demonstrates completely the same trend for
dissociation in 100-fold excess of serum proteins as complex with
pyridine bisamide aza-18-crown-6 triacetate – L6 (logβ = 21.3).[21]
This fact might reflect the moderate ability of “opened NOTA”
backbone to hold Bismuth cation.
In vivo studies
Studying the behavior of a complex without a biological vector is
an important step for understanding the stability of the complex
inside the body. For the case of a stable complex, we expect to
observe fast clearance of radioactive substances from the body
without significant accumulation in normal tissues. When instability of the complexes is demonstarted in the body, their further
conjugation with a biological vector is not reasonable. A total of 6
normal male BALB/c mice were used for the experiment with the
[207Bi]BA3ABi complex. The radiolabeled ligand in 100 μL (pH 6.8,
0.01 M MES) was injected intraperitoneally. The mice were then
euthanized at 1 and 6 h, and the blood and major organs were
harvested, wet weighed, and the radioactivity was measured via
gamma-spectrometry. The percent injected dose per gram (% ID/
g) was determined for each tissue (Table S3).
All the obtained results are summarized in Figure 8 (a, b). The
profile of the biodistribution of complex [207Bi]BA3ABi is similar to
that of [207Bi]BATABi and [207Bi]DOTABi. The clearance was also
carried out by the kidneys, and it is the organ with the highest
radioactivity accumulation compared with other tissues for all
complexes. However, much higher accumulation in the kidneys
could be observed for the [207Bi]BA3ABi complex: 23 � 9 % of ID/g
after 1 h, decreasing to 14 � 3 % of ID/g after 6 h. There was also
some accumulation of the H3BA3A complex with 207Bi3 + in the liver
5.0 � 1.1 % of ID/g, and it decreased to 2.7 �0.5 % of ID/g after 6 h.
Additionally, the biodistribution profile of the [207Bi]BA3ABi
complex was also very similar to those of the [207Bi]EDTABi
complex (Figure 8), which is known to be unstable in vivo despite
its high stability constant. The accumulation of 207Bi was more
pronounced at 6 h post injection (p.i.) of [207Bi]BA3Abi, which
indicated slower clearance, and it was also at the same percentage
as the complex with H4EDTA. This indicated that the [207Bi]BA3ABi
complex released radionuclide in vivo similarly to [207Bi]EDTABi.
It is important to notice that according to serum studies and
neutral charge of LBi species we could expect the similar
biodistribution of [207Bi]L6Bi[21] and [207Bi]BA3ABi. However comparison of biodistribution profiles of these complexes especially at 6 h
Eur. J. Inorg. Chem. 2021, 3344 – 3354
Figure 8. Biodistribution of 207Bi-labeled compounds: H4EDTA,[21] H3BA3A,
H4BATA,[11] and H4DOTA[11] in normal BALB/c Mice at 1 h (a) and 6 h (b) after
p.i. clearly shows definitely less stability of [207Bi]BA3ABi in vivo
revealing the important role of macrocyclic cavity for Bismuth
The stability of [207Bi]BATABi differed significantly from [207Bi]
BA3ABi. A [207Bi]BATABi complex like [207Bi]DOTABi is rapidly
excreted from the body, and no significant accumulation of
radioactivity in any healthy tissues was observed. The major
radioactivity was found in the kidneys (~ 3 % ID/g), which reduced
even further after 6 h. Based on this we can conclude that the Bi3 +
complex of H4BATA has considerably higher stability than H3BA3A,
and it has almost the same in vivo inertness as that of H4DOTA.
After summarizing the serum and in vivo behavior of all the
considered complexes, the aza-18-crown-6 cavity seems to be
more appropriate for Bi3+ particularly in H4BATA, providing
markedly better biodistribution of the formed chelate compared
to [207Bi]BA3ABi regardless of the nearly similar pBi values.
Furthermore, the tetraacetate ligands H4BATA and H4DOTA form
complexes of nearly similar in vivo stability, and this demonstrates
the effectiveness of negatively charged complex compounds such
as BATABi and DOTABi . Additionally, based on the EXAFS data,
H4BATA provides Bi3 + a stable long-range environment that allows
this complex not only to be stable in the presence of external
competitors, such as a 100-fold excess of serum protein, but also
© 2021 Wiley-VCH GmbH
Full Papers
ensures fast in vivo clearance and avoids absorption of radionuclides in the organs.
Taking into account the nearly similar ionic radii of Na +
(1.02 Å) and Bi3+ (1.03 Å) CN6,[25] it is reasonable to suggest
inclusion of the Bi3+ cation inside the cavity of the aza-15-crown-5
in H3BA3A. However, we do not observe such inclusion in the
crystal structure of BA3ABi and clearly observed that the out-cage
location of Bi3 + enables its transchelation, which provides
moderate serum stability. It is likely that despite visual suitability of
the cavity of the aza-15-crown-5 for Bi3+, additional factors such as
the conformation of the azacrown-ligand and the 6 s2 lone pair of
Bi3 + influence the resulting out-cage structure of the H3BA3A
complex. In addition, by considering all the data,including
described NETA and NPTA complexes[12] with Bi3 +, we can assume
that 6 donor groups (3 amines and 3 acetates) in H3BA3A is not
sufficient for formation of a highly stable complex with the
bismuth cation in a competing medium.
In this work, we studied the complexation of two ligands H4BATA
and H3BA3A with bismuth(III). The thermodynamic stability of
bismuth(III) complexes of H4BATA and H3BA3A and their pH
speciation were analyzed via a potentiometric method. The
stability of the studied complexes was reasonably high based on
the thermodynamic stability constants and pBi values. However,
the results of the serum challenge and in vivo biodistribution show
that the structural peculiarities significantly impacted the inertness
of complexes in a competing medium. The macrocyclic cavity of
aza-18-crown-6 appeared to be the most appropriate for the
bismuth cation; in particular, it has four acetate arms and forms a
negatively charged complex, which leads H4BATA to be the ideal
ligand for Bi3 +. Based on EXAFS measurements in aqueous
solutions, the bismuth cation chelated by H4BATA is effectively
shielded from the surrounding competitors even in a living
organism. The difference in the long-range neighbors of complexes with H3BA3A and H4BATA reflects the ability of the cation
to be released from the complex in vitro and in vivo. The H4BATA
ligand can be bifunctionalized via the benzene group, providing
sufficient distancing of the biomolecule from the cation-coordinating sites to eliminate possible effects on labeling of the
bioconjugate. Therefore, H4BATA can be considered as a promising
candidate for conjugation with biological vectors (such as peptides
and antibodies) for TAT with 212,213Bi radioisotopes.
Experimental Section
Synthesis of ligands
All commercially available reagents were used without further
purification. The ligands H3BA3A and H4BATA were prepared as
described earlier.[11,26]
Eur. J. Inorg. Chem. 2021, 3344 – 3354
NMR study
H NMR spectra were recorded at 25 °C on Varian Inova 400
spectrometers. Chemical shifts are reported in parts per million (δ)
relative to the deuterated solvent used as the internal reference
(D2O δ = 4.75). The coupling constant J is given in Hertz. Spectral
assignments were based in part on two-dimensional NMR experiments, COSY and NOESY (Figure S3, Figure S4). Samples of the Bi3 +
complexes for the NMR measurements were prepared by dissolving
the corresponding ligand and 1.5 eq. Bi(NO3)3 in D2O, followed by
adjustment of the desired pD with small volumes of concentrated
HClO4 or NaOH. Accurate pD measurements in D2O were obtained
by direct reading in a D2O solution using a combined glass/AgCl
electrode after appropriate calibration procedures using standard
buffers. Numbering of the hydrogen and carbon nuclei used to
describe the 1H NMR spectra is given in Figure 2.
Synthesis of [Bi(BA3A)]: Bi(NO3)3 · 5H2O (5.3 mg, 0.011 mmol) was
added to solution of H3BA3A (4.4 mg, 0.010 mmol) in H2O (1 mL),
and the pH value of the solution was increased to 7–8. The solution
was heated to 90°C with vigorous stirring for 10 min. After cooling
to room temperature, the formed precipitate was filtered off. Slow
evaporation of the mother solution led to the formation of single
crystals suitable for X-ray analysis (5 mg, 71 % yield). 1H NMR (D2O,
400 MHz): 2.52 (t, 2H, H(7e), J = 13.3 Hz), 3.00 (t, 2H, H(6e), J =
13.3 Hz), 3.20 (d, 2H, H(5a), J = 12.9 Hz), 3.41 (t, 2H, H(5e), J =
13.3 Hz), 3.41 (d, 2H, H(8y), J = 16.4 Hz), 3.54 (d, 2H, H(7a), J =
13.3 Hz), 3.65 (d, 2H, H(6a), J = 15.2 Hz), 4.16 (t, 2H, H(4e), J =
10.9 Hz), 4.20 (s, 2H, H(10)), 4.49 (d, 2H, H(4a), J = 10.9 Hz), 6.64 (d,
2H, H(8x), J = 16.8 Hz), 7.05 (m, 4H, H(1,2)). 13C NMR (D2O, 400 MHz):
51.31 (C-7), 54.77 (C-10), 55.09 (C-6), 59.86 (C-5), 64.27 (C-8), 64.82
(C-4), 112.50 (C-2), 122.62 (C-1), 144.28 (C-3), 179.37 (C-9), 185.95 (C11).
Synthesis of [Bi(BATA)]: Bi(NO3)3 · 5H2O (5.3 mg, 0.011 mmol) was
added to solution of H4BATA (5.4 mg, 0.010 mmol) in H2O (1 mL),
and the pH value of the solution was increased to 7–8. The solution
was heated to 90°C with vigorous stirring for 10 min. After cooling
to room temperature, the formed precipitate was filtered off. Slow
evaporation of the mother solution did not lead to the formation of
single crystals suitable for X-ray analysis. The product was isolated
as a white powder (7.1 mg, 96 % yield). The 1H and 13C NMR
spectrum is shown in Figures S6 and S7.
Crystal data for [BA3ABi]
Single crystals of the bismuth(III) complex BA3A3 were obtained
from an aqueous solution via the slow evaporation method. Data
collection for [BA3ABi] was performed on a Bruker SMART APEX II
diffractometer (λ(MoKα) = 0.71073 Å, ω-scans, 2θ < 61.3°). The
colorless crystals of C20H32BiN3O11 (M = 699.46) at 120 K were
monoclinic (space group P21/c; a = 10.3041(4) Å; b = 14.4533(5) Å;
c = 15.8501(6) Å; β = 103.9340(10)°; V = 2291.07(15) Å3; Z = 4 (Z’ = 1);
dcalc = 2.028 g cm 3; μ = 7.763 mm 1; F(000) = 1376). Frames were
integrated using the Bruker SAINT software package[27] with a
narrow-frame algorithm. A semi-empirical absorption correction
was applied with the SADABS[28] program using the intensity data
of equivalent reflections. The intensities of 7,047 independent
reflections (Rint = 0.0341) out of 30,487 collected were used for
structure solution and refinement. The structure was solved using
the dual-space approach with the SHELXT program,[29] and then
refined via the full-matrix least-squares technique against F2hkl in an
anisotropic approximation for non-hydrogen atoms with the
SHELXL[30] program. The hydrogen atoms of water molecules were
found from difference Fourier synthesis and refined in an isotropic
approximation. All other hydrogen atoms were placed in the
calculated positions and refined in the riding model with Uiso(H)
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parameters equal to 1.2 Ueq of the connected carbon atoms. The
refinement converged to R1 = 0.0195 (calculated for 6,099 observed
reflections with I > 2σ(I)), wR2 = 0.0404, and GOF = 1.024 for all
independent reflections.
Thermodynamic Stability Studies
The setup for potentiometric titrations has been described
before.[31] The titrant was a carbonate-free solution of NaOH
analytical grade at ca. approximately 0.10 M. The exact concentration of NaOH solution was obtained by application of the Gran’s
method upon titration of a previously standardized amounts of HCl
and determining the equivalent point by the Gran’s method using
the GLEE software.[32] The ionic product of water pKw = 13.78 at
25.0 °C and ionic strength of 0.10 � 0.01 M using KNO3 as a
background electrolyte was kept constant. A stock solution of
H4BATA and H3BA3A was prepared at ca. 0.01 M. An analytical
solution of Bi(NO3)3 was prepared at 0.025 M in 0.7 M aqueous HCl
to avoid metal hydrolysis. Potentiometric titrations were run with
ca. 0.016 mmol of ligand in a total volume of 16.00 mL at 25.0 �
0.5 °C. Data were collected in the pH range 1.5–9.5/11.0. The [H + ] of
the solutions was determined by measurement of the electromotive force of the cell, E = E0 + Q log[H + ] + Ej. The terms E0 and Q
were determined by titration of a solution of known hydrogen-ion
concentration at the same ionic strength. Each titration consisted of
80–100 equilibrium points and a minimum of two replicates were
performed. The stability constants of the complexes were calculated from the electromotive force titration data with the Hyperquad software[32] and speciation diagrams were plotted using the
Hyss software.[33] Experimental curves and other details are given in
the ESI (Table S4).
The overall equilibrium (formation) constants βHhL and βMmHhLl are
defined by βHhL = [HhLl]/[H]h[L]l and βMmHhLl = [MmHhLl]/[M]m[H]h[L]l,
while stepwise equilibrium constants are given by KMmHhLl =
[MmHhLl]/[MmHh–1Ll][H] and correspond to the difference in log units
between overall constants of sequentially protonated (or
hydroxide) species.
EXAFS measurement and data treatment
The Bi L3-edge X-ray spectra for sample of aqueous solution
containing Bi3 + + H3BA3A and Bi3 + + H4BATA were collected at the
beamline “Structural Materials Science”[34] using the equipment of
“Kurchatov Synchrotron Radiation Source” (Moscow, Russia). The
storage ring with electron beam energy of 2.5 GeV and a current of
80–100 mA was used as the source of radiation. All the spectra
were collected in the transmission mode using a Si (111) channelcut monochromator. EXAFS data (χexp(k)) were analyzed using the
IFEFFIT data analysis package.[35] EXAFS data reduction used
standard procedures for the pre-edge subtraction and spline
background removal. The Fourier transformation (FT) of the k2weighted EXAFS functions χexp(k) was calculated over the range of
photoelectron wave numbers k = 2–10.0 Å 1. The structural parameters, including interatomic distances (Ri), coordination numbers
(Ni) and Debye-Waller factors (σ2), were found by the non-linear fit
of theoretical spectra (eq.) to experimental ones.
Ni Fi ðkÞ
R2i k
e lðkÞ e
2s2i k2
sinð2kRi þFi ðkÞÞ
The theoretical data were simulated using the photoelectron mean
free path λ(k), amplitude Fi(k), and phase shift Fi(k) calculated ab
initio using the program FEFF6.[36]
Eur. J. Inorg. Chem. 2021, 3344 – 3354
Computational details
All the calculations were performed at the DFT[37,38] level of theory with
hybrid density functional PBE0[39] and dispersion correction D3.[40] The
atomic orbitals were described with def2-svp[41] basis set (in a case of
Bi ion, the inner orbitals were included together with the core into an
effective core potential def2-SD[42]). We also used ‘chain-of-spheres’
resolution of identity approximation (RIJCOSX[43–45]) for calculation of
the electron repulsion integrals. The preliminary scan of potential
energy surface and search of a global energy minimum was
performed with a stochastic algorithm described in details in our
previous work.[46] All the calculations were done in the orca[47] package.
Protocol for radiolabeling with 207Bi
All reagents and solvents were purchased from commercially available
sources and used as received. The initial concentration of 207Bi was
determined by radioactivity counting. To study the labeling efficiency,
30 μL containing 300 Bq [207Bi]BiCl3 in 0.1 M HCl was equilibrated with
1–1000 μM of the ligand in a 0.01 M MES buffer solution at room
temperature in a plastic Eppendorf tube with a total volume of
300 μL. The average 207Bi concentration was 0.2 nM. After preparation,
the mixtures were analyzed by thin layer chromatography (TLC). The
TLC plates (cellulose on Al support, Sigma) were developed in a
mixture of 0.9 % NaCl/10 mM NaOH. The plates were cut in half and
the radioactivity on each part was measured via gamma spectrometry.
The activity was quantified by the 570 keV gamma emission of 207Bi.
Autoradiography was performed with a Perkin Elmer Cyclone Plus
Storage Phosphor System and the associated software. Percentages of
Bi incorporation were deduced from the ratio of the radioactivity
intensities at Rf = 0.9 � 0.1 (which corresponds to [207Bi]BA3ABi or [207Bi]
BATABi) and Rf ~ 0 (which corresponds to the remaining 207Bi salts).
The average values of at least two experiments were used (error
estimated: � 6%).
In vitro stability study of [207Bi]BA3ABi
A solution of the radiolabeled complex [207Bi]BA3ABi with ca. 0.6 mM
of ligand was buffered at pH 6.8 with a 0.01 M MES solution at room
temperature (RT) (radiochemical purity: 97 %). Fetal bovine serum
(triple 0.12 μm sterile filtered) was purchased from HyClone (South
Logan, Utah), and all required storage measures were followed. The
ratio of the complex [207Bi]BA3ABi solution and serum volume was
established at 1 :100 at 37 °C. After 1, 5, 15, 30, 60, 120, 240 min, 1 day,
and 2 days an aliquot of each sample was taken; after protein
precipitation via ethanol radioactivity of each supernatant, an aliquot
of the initial sample with the same volume and geometry was
measured by gamma-spectrometry and the percentage of the
complex was determined. Additionally, aliquots of the supernatants
were analyzed by TLC on cellulose plates with an Al support.
In vivo biodistribution study of [207Bi]BA3ABi
All in vivo experiments were performed in accordance with EU
Directive 2010/63/EU for animal experiments and approved by The
Bioethics Commission of Lomonosov Moscow State University (meeting number 125-d held on 28. 01. 2021, protocol No122-a). For the
in vivo experiments a total of 12 normal male BALB/c mice were used.
The mice were housed with 12-h light/dark cycles with access to water
and food ad libitum. A solution of 1 mM H3BA3A and 207Bi3+ (2.0–2.5
kBq) was buffered at pH 6.8 with 0.01 M MES at RT and diluted in a
sterile isotonic solution. The radiochemical purity according to TLC
reached 97 %. Then, 100 μl of solution was administered to the mice
(weight 28–37 g) via intraperitoneal injection as previously
described.[48] The mice (3 per data point) were euthanized at 1 and 6 h
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Full Papers
by cervical dislocation. Blood was collected right after euthanasia and
mixed with 100 μl of heparin solution. The major organs were
harvested and the blood and peritoneal liquid was washed off with
0.9 % NaCl solution, the organs were then wet-weighed and the
radioactivity of each was measured using a γ-scintillation counter
(with a HPGe-detector GR3818 Canberra Ind.). The percent injected
dose per gram (% ID/g) was determined for each tissue, and the
values presented are the mean and standard deviation.
Deposition Number 2058062 (for [BA3ABi]) contains the supplementary crystallographic data for this paper. These data are
provided free of charge by the joint Cambridge Crystallographic
Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.
The reported study was funded by the Russian Science Foundation
project no. 18-73-10035 (synthesis of ligands and complexation
study). E.A.K., A.L.T. and S.N.K. acknowledge support by the
Russian Ministry of Science and Education under grant No 075-152019-1891 (EXAFS measurements). X-ray diffraction experiment
was performed with financial support from the Ministry of Science
and Higher Education of the Russian Federation using equipment
at the Center for Molecular Composition Studies of INEOS RAS.
The research was carried out using the equipment of the shared
research facilities of HPC computing resources at Lomonosov
Moscow State University (DFT calculations).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: Benzoazacrown · Bismuth · complexation · EXAFS ·
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Manuscript received: March 29, 2021
Revised manuscript received: July 26, 2021
Accepted manuscript online: July 27, 2021
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