Probing Unexpected Reactivity in Radiometal Chemistry: Indium-111-Mediated Hydrolysis of Hybrid Cyclen-Hydroxypyridinone Ligands

Chelators based on hydroxypyridinones have utility in incorporating radioactive metal ions into diagnostic and therapeutic agents used in nuclear medicine. Over the course of our hydroxypyridinone studies, we have prepared two novel chelators, consisting of a cyclen (1,4,7,10-tetraazacyclododecane) ring bearing two pendant hydroxypyridinone groups, appended via methylene acetamide motifs at either the 1,4-positions (L1) or 1,7-positions (L2) of the cyclen ring. In radiolabeling reactions of L1 or L2 with the γ-emitting radioisotope, [111In]In3+, we have observed radiometal-mediated hydrolysis of a single amide group of either L1 or L2. The reaction of either [111In]In3+ or [natIn]In3+ with either L1 or L2, in aqueous alkaline solutions at 80 °C, initially results in formation of [In(L1)]+ or [In(L2)]+, respectively. Over time, each of these species undergoes In3+-mediated hydrolysis of a single amide group to yield species in which In3+ remains coordinated to the resultant chelator, which consists of a cyclen ring bearing a single hydroxypyridinone group and a single carboxylate group. The reactivity toward hydrolysis is higher for the L1 complex compared to that for the L2 complex. Density functional theory calculations corroborate these experimental findings and importantly indicate that the activation energy required for the hydrolysis of L1 is significantly lower than that required for L2. This is the first reported example of a chelator undergoing radiometal-mediated hydrolysis to form a radiometalated complex. It is possible that metal-mediated amide bond cleavage is a source of instability in other radiotracers, particularly those in which radiometal complexation occurs in aqueous, basic solutions at high temperatures. This study highlights the importance of appropriate characterization of radiolabeled products.


X-ray Crystallography
The The N9-H hydrogen atom in the structure of 2 was located from a ΔF map and refined freely subject to an N-H distance constraint of 0.90 Å. Figure S19. Representation of 2 (50% probability ellipsoids). S16 Figure S20. Proposed intramolecular amide hydrolysis mechanism.

Gallium Complexation
We elected to further probe the reactivity of L 1 with Ga 3+ , which has exceptionally high affinity for hydroxypyridinone ligands. in HPLC experiments ( Figure S22). Based on this data, we hypothesise that cyclen coordination to a metal ion is important for hydrolytic activity of this ligand system, although extensive further experiments are required to assess reactivity of these compounds with other metal ions.

Kinetic analysis
We have qualitatively shown that both In 3+ coordination and In 3+ -mediated hydrolysis is dependent on the concentration of OH -. The kinetics of these consecutive reactions are complicated: the rate laws are likely of a high order, complexation will be dependent on [OH - In combination, these factors mean that determining experimental rate constants and activation energies for each process is difficult. However, in radiochemical reactions, due to the large excess of ligand, OHand solvent ions relative to 111 In 3+ , pseudo-first order reactions with respect to 111 In 3+ can be approximated.

S23
BS2. NBO calculations were performed at this level. Higher level single point calculations were performed on each B97X-D optimised structure using the larger basis set, BS3.
BS1 was built as follows. The SDD basis set and corresponding effective core potential was used for In. The split-valence 6-31(d) basis set was used for C and H atoms. The triple-ξ 6-311+G(d) basis set was used for N and O.
BS2 was built as follows. The SDD basis set and corresponding effective core potential was used for In. The split-valence 6-31+G(d,p) basis set was used for C and H atoms. The triple-ξ 6-311+G(d) basis set was used for N and O.
BS3 was built as follows. The SDD basis set and corresponding effective core potential was used for In. All other elements (C, H, N and O) were described using Ahlrichs quadruple-ξ basis set def2-QZVPP. 14 Full coordinates for all the calculated stationary points are included as part of the supplementary information (.xyz).
For simplicity, the coordination environment of L 1B with In 3+ was chosen as a starting point.
These structures were modelled using the B97X functional and BS1 with no solvent corrections. The input geometries were pre-selected based on the possible coordination groups of each ligand i.e. each carboxylic acid on one arm, and amide or hydroxypyridinone (hopo) on the other. Due to the basic conditions of the reaction being modelled, both the carboxylic acid and hopo moieties were deprotonated. This led to five possible permutations.
All five permutations were explored, with and without an additional bound hydroxide to In (Tables S1 and S2). Note that several structures were calculated where the amide proton had migrated to the hopo-O -, or where the exposed hopo-Owas bound to the electrophilic amide carbon.   For In-L 1B , in each case the output geometry showed the carboxylate arm was coordinated, and stationary points were found with either amide or hopo coordination from the other arm.

Input Geometry Output Geometry G (hartrees) G (kcal mol -1 )
In all cases, coordination of hydroxide was favoured over a cationic complex by > 100 kcal  (Table S4). Similar to L 1B , hydroxide coordination and the hopo-carb geometry is favoured.   Table S8. Calculated free energies for the coordination and hydrolysis of L 1  Finally, each optimised structure at the B97X-D/BS2 level was single point corrected at BS3.
The tabulated data for the correction are presented in Table S9.