Inorganic Chemistry
Article
1H NMR (300 MHz, CDCl3, RT): δ 10.23 (s, 1H), 8.32 (d, J = 8.5,
1H), 8.17 (s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.63 (t, J = 8.5 Hz, 1H),
7.45 (dd, J = 8.4 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H). 13C NMR (75
MHz, CDCl3, RT): δ 192.8, 153.1, 150.3, 137.9, 137.6, 131.1, 130.5,
118.2, 118.1, 111.4. LR-ESI-MS: [C10H7NO2 + Na]+ calcd., 196.0;
[M + Na]+ found, 196.3.
with Sc3+, two in-batch methods were used to determine the stability
constants: UV spectrophotometric and fluorometric titrations. The
metal−ligand concentration ([ML] = 6.51 × 10−6 M) was lower than
that in the other experiments due to the lower solubility of the neutral
Sc(glyox) species. A Ross combination pH electrode was calibrated
for hydrogen ion concentration using HCl as described before,37 and
the results were analyzed by the Gran72 procedure. The pH of the
samples containing ligand and the metal−ligand complex was
measured between the pH range of 1.8 and 11.5. For the most
acidic samples, the equilibrium H+ concentration was calculated from
solution stoichiometry and was not measured with the glass electrode.
For the ligand protonation equilibria study, the samples were left for 2
min to achieve equilibrium before measuring the pH and the UV
absorption spectrum. For the samples containing the metal
complexes, the measurements were performed only when equilibrium
was achieved. The protonation constants of H3glyox and the metal-
complex stability constants were calculated from the experimental
data using the HypSpec201449 program. Proton dissociation
constants corresponding to hydrolysis of Ga3+, In3+, and Sc3+ aqueous
ions included in the calculations were taken from Baes and Mesmer.73
The species formed in the studied systems are characterized by the
general equilibrium: pM + qH + rL = MpHqLr (charges omitted).
For convention, a complex containing a metal ion M, proton H, and
ligand L has the general formula MpHqLr. The stoichiometric indices
p might also be 0 in the case of ligand protonation equilibria, and
negative values of q refer to proton removal or hydroxide ion addition
during formation of the complex. The overall equilibrium constant for
the formation of the complexes MpHqLr from its components is
designated as log β. Stepwise equilibrium constants log K correspond
to the difference in log units between the overall constants of
sequentially protonated (or hydroxide) species. pM value is defined as
−log[Mn+]free calculated at specific conditions ([Mn+] = 10 μM, [Lx−]
= 1 μM, pH 7.4).52
Radiolabeling Experiments. A stock solution of the ligand,
H3glyox, was prepared (7.64 × 10−5 M) in MQ water. For
concentration dependent experiments, different ligand solutions
were prepared by diluting the stock solution using a corresponding
buffer solution such that the total volume per reaction was 500 μL
after the addition of [111In]InCl3, [68Ga]GaCl3, or [44Sc]ScCl3.
Concentration Dependent [111In]In(glyox) Radiolabeling
and Complex Stability Studies in Human Serum. All labeling
reactions were done over a time period of 15 min at room
temperature and pH 7. The total volume of the reaction mixture
was 500 μL after the addition of ∼900 kBq of [111In]InCl3 using 0.1
M NaOAc/HOAc buffer (pH = 7) and an appropriate amount of the
ligand stock solution. The RCYs were obtained by analyzing the
reaction mixture by spotting an aliquot on an aluminum-backed silica
TLC plate and developed using a 10 mM EDTA solution (pH = 7) as
a mobile phase. The [111In]In(glyox) complex stays at the baseline (Rf
= 0) while the uncomplexed [111In]In3+ migrates up the plate as the
EDTA complex (Rf > 0.7). Developed plates were counted
immediately, and radiolabeling yields were calculated by integrating
the peaks in the radio-chromatogram, consistent with the well-
separated radio peaks of the “free” [111In]In3+ metal ion and the
Ethyl Bis((8-hydroxyquinolin-2-yl)methyl)glycinate (2). Com-
pound 1 (1 g, 5.7 mmol, 2.1 equiv) was dissolved in 50 mL of 1,2-
dichloroethane, and to this solution was added glycine ethyl ester,
HCl (0.8 g, 2.7 mmol, 1 equiv), and NaBH(OAc)3 (2.8 g, 13.5 mmol,
5 equiv). The mixture was refluxed overnight at 50 °C, then quenched
with saturated aqueous Na2CO3 (20 mL) and extracted with
dichloromethane (3 × 20 mL). The combined organic layers were
dried over anhydrous MgSO4. The solvent was removed under a
vacuum to give crude product 2, which was then purified by column
chromatography (eluted with a gradient of 100% hexane to 100%
ethyl acetate) to afford 2 as an off-white solid (0.7 g, 1.6 mmol, 61%,
1
Rf = 0.50 in 20% EtOAc in hexane). H NMR (300 MHz, CDCl3,
RT): δ 8.11 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.41 (m,
2H), 7.31 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 7.5 Hz, 2H), 4.19 (m, 6H),
2.03 (s, 2H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3,
RT): δ 171.3, 160.6, 157.1, 145.3, 140.8, 136.5, 136.4, 134.1, 128.9,
128.8, 127.0, 125.8, 122.9, 122.2, 60.7, 60.3, 55.4, 14.4.
Bis((8-hydroxyquinolin-2-yl)methyl)glycine, H3glyox (3). A por-
tion of 2 was dissolved in HCl (10 mL, 6 M) and refluxed overnight.
The solution was cooled, and the solvent was evaporated under
reduced pressure to give a yellow colored solid product 3 (0.5 g, 1.3
mmol, 76%). 1H NMR (300 MHz, RT, D2O): δ 8.54 (d, J = 7.7 Hz,
2H), 7.72 (d, J = 7.9 Hz, 2H), 7.45 (t, J = 7.2 Hz, 2H), 7.31 (d, J = 7.6
Hz, 2H), 7.15 (d, J = 6.7, 2H), 4.52 (s, 4H), 4.04 (s, 2H). 13C NMR
(75 MHz, RT, D2O): δ 176.1, 155.1, 150.4, 146.9, 146.6, 130.9, 128.8,
128.4, 122.3, 119.2, 117.0, 58.5, 57.3. HR-ESI-MS calcd for
C22H19N3O4: 389.1400. Found: 390.1451 [M + H]+. Anal. calcd.
for [H3glyox·2.8 HCl·1.3 CH3OH)]: C, 52.49; H, 5.1; N, 7.88.
Found: C, 52.48; H, 5.11; N, 7.93.
In(glyox). A portion of H3glyox (10 mg, 0.02 mmol) was dissolved
in 1 mL of distilled water, and In(ClO4)3 (11.7 mg, 0.02 mmol) was
added to it. Using 0.1 M NaOH, the pH was adjusted to ∼7, and the
solution was left to stir for 1 h. The solvent was evaporated under
reduced pressure to give yellow colored metal complex as a hydrate.
HR-ESI-MS calcd for C22H16InN3NaO4: 524.0077. Found: 524.0076
[M + Na]+.
Ga(glyox). To a solution of H3glyox (10 mg, 0.02 mmol) dissolved
in 1 mL of distilled water was added Ga(NO3)3 (7.23 mg, 0.02
mmol), and the pH was adjusted to ∼7 using 0.1 M NaOH. The
solution was left to stir overnight, and the solvent was evaporated
under reduced pressure to give a pale yellow colored metal complex as
a hydrate. 1H NMR (300 MHz, RT, D2O): δ 7.69 (d, J = 8.4 Hz, 2H),
7.11 (t, J = 7.8 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.6 Hz,
2H), 6.59 (d, J = 7.7 Hz, 2H), 3.76 (s, 4H), 3.05 (s, 2H). 13C NMR
(75 MHz, CDCl3, RT): δ 179.8, 164.1, 155.4, 143.0, 136.9, 129.4,
128.5, 121.7, 114.0, 111.3, 60.7, 59.3. HR-ESI-Ms calcd for
C22H16GaN3NaO4: 478.0294. Found: 478.0296 [M + Na]+.
Sc(glyox). H3glyox (10 mg, 0.02 mmol) was dissolved in 1 mL of
distilled water in a scintillation vial, and ScCl3 (5.18 mg, 0.02 mmol)
was added, followed by pH adjustment to ∼6 using 0.1 M NaOH.
The solution was left to stir for 1 h, and formation of the metal
complex was confirmed using LR-ESI MS as well as fluorescence
under a UV lamp. HR-ESI-MS calcd for C22H17N3O4Sc: 432.0778.
Found: 432.0777 [M + H]+.
Solution Thermodynamics. Protonation constants and metal
stability constants were calculated from UV spectrophotometric
titration data obtained using a Cary 60 UV−vis spectrophotometer in
the spectral range of 200−450 nm. The path length was 1 cm for all
samples. Individual samples (5 mL) containing the ligand (H3glyox,
[L] = 2.5 × 10−5 M) and the corresponding metal complexes (M =
Ga3+, In3+; [ML] = 2.5 × 10−5 M) were prepared in pure water by
adjusting the pH using standardized HCl or NaOH solutions, and
NaCl was added to maintain a constant 0.16 M ionic strength over the
pH range ∼0.64−11.64. In the complex formation equilibria studies
[
111In]In(glyox) complex on the HPLC radiotraces (tR = 4.9 min for
“free” 111In and 12.2 min for [111In]In(glyox) complex). For the
human serum challenge, 500 μL of human serum was added to a
quantitative radiolabeled complex solution (500 μL). The mixture was
then incubated at 37 °C. An aliquot of the reaction mixture was
spotted on silica plates at desired time points to determine the
amount of intact complex (RCY%).
pH and Concentration Dependent [68Ga]Ga(glyox) Radio-
labeling Procedure. All pH dependent radiolabeling reactions were
done at room temperature over a 5 min period, and the total volume
of the reaction mixture was kept to 500 μL after addition of 5 MBq
[68Ga]GaCl3 and the corresponding NaOAc/HOAc buffer (0.1 M,
pH = 5.5, 6.5, 7.3) such that the final ligand concentration was 10−4
M. For the concentration dependent radiolabeling, reaction solutions
with varying ligand concentrations (10−4 M to 10−9 M) were prepared
by diluting the stock solution of the ligand (∼10−3 M) using 0.1 M
J
Inorg. Chem. XXXX, XXX, XXX−XXX
Choudhary, Neha
