Synthesis and Evaluation of Diaza-Crown Ether-Backboned Chelator Containing Hydroxamate Groups for Zr-89 Chelation Chemistry

Abstract

Zirconium-89 (⁸⁹Zr) has been explored for molecularly targeted positron emission tomography (PET) imaging of various diseases. We synthesized and evaluated a novel chelator (DA-18C6-BHA) for ⁸⁹Zr. The new chelator is structured on a macrocyclic backbone (1,10-diaza-18-crown-6) and contains hydroxamates as acyclic donor groups. The new chelator ((DA-18C6-BHA) was rapidly labeled with ⁸⁹Zr under mild conditions. The ⁸⁹Zr-labeled DA-18C6-BHA complex remained stable in human serum and apotransferrin for 7 days. When challenged with excess EDTA solution, ⁸⁹Zr-labeled DA-18C6-BHA was shown to hold ⁸⁹Zr without losing considerable radioactivity to EDTA. The ⁸⁹Zr-labeled DA-18C6-BHA complex displayed high complex stability in normal mice as evidenced by low bone uptake.

Positron-emitting radionuclides used for positron emission tomography (PET) imaging include ¹⁸F (t_1/2 = 1.83 h, E_β+ = 0.63 MeV), ⁶⁴Cu (t_1/2 = 12.7 h, E_β+ = 0.66 MeV, E_γ = 1.35 MeV), ⁶⁸Ga (t_1/2 = 1.12 h, E_β+ = 1.88 MeV, E_γ = 0.51 MeV), and ⁸⁹Zr (t_1/2 = 78.4 h, E_β+ = 0.90 MeV, E_γ = 0.91 MeV).^1–5 Research efforts have been made on discovery of PET imaging agents with high binding affinity and selectivity to specific biomarkers on tumor cells.^1–3 A number of target-specific biomolecules including peptides and antibodies have been labeled with radionuclides for preclinical and clinical evaluations of targeted PET imaging of cancers.^5–7

Among the positron-emitting radionuclides in clinical use, a long-lived ⁸⁹Zr is well suited for PET imaging of molecular targets using an antibody with a long biological half-life.^1,5–8 In particular, ⁸⁹Zr-based PET tracers have a clinical value in pre- and post-treatment imaging and monitoring tumor response in antibody therapy.^{5–6 89}Zr(IV) is a highly charged cation with strong binding with anionic oxygens such as hydroxyl group in bone mineral hydroxylapatite (HA).^1,5,9

DFO (desferrioxamine) is a hydroxamate-based chelator in clinical use for the treatment of iron overload diseases including β-thalassemia.¹⁰ Given the high binding avidity of the hydroxamates for a hard oxophilic cation, DFO has been also extensively explored for labeling of various biomolecules with ⁸⁹Zr.^8,11 DFO was shown to rapidly form a 6-coordinate complex with Zr(IV), and ⁸⁹Zr-DFO complex remained intact in human serum.^12,13 However, DFO-antibody conjugate labeled with ⁸⁹Zr has a limited in vivo stability in mice and displayed high bone uptake.^12,13 The elevated bone uptake of ⁸⁹Zr-DFO can be rationalized by high affinity of ⁸⁹Zr(IV) released from the complex for bone mineral, hydroxylapatite (HA). While research efforts have been made to develop improved chelation chemistry that can sequester the bone-seeking ⁸⁹Zr with high in vivo stability, DFO remains the standard chelator for clinical use.^1,12

In this paper, we report synthesis and evaluation of a novel chelating agent (DA-18C6-BHA) that is built on a macrocyclic backbone (1,10-diaza-18-crown-6) tethered with hydroxamate as an acyclic donor group. The new chelator and DFO were comparatively studied for radiolabeling with ⁸⁹Zr. The ⁸⁹Zr-radiolabeled new chelator (⁸⁹Zr-DA-18C6-BHA) and ⁸⁹Zr-DFO were comparatively evaluated for complex stability in human serum and apotransferrin and excess EDTA solution and biodistribution profile in normal mice.

The novel chelator (DA-18C6-BHA) contains diaza-18-crown-6 as a macrocyclic backbone and hydroxamates as tetradentate binding moieties. The four oxygens in the macrocyclic backbone are expected to participate in complexation with ⁸⁹Zr(IV) and contribute to the formation of an eight-coordinate Zr complex. Synthesis of the new chelator (DA-18C6-BHA) includes reaction of compound 2¹⁴ as a key precursor molecule with 1,10-diaza-18-crown-6 (3, Scheme 1). Compound 2 was prepared from O-paramethoxybenzyl (PMB)-protected N-methyl hydroxylamine (1)¹⁵ by a modified procedure of the reported method.¹⁴ Compound 1 was reacted with bromoacetyl bromide in CH₃CN at 0 ^oC to afford compound 2 in 77% isolated yield. Compound 2 was further reacted with 1,10-diaza-18-crown-6 (3) to produce O-PMB protected chelating agent 4 in 41% isolated yield. Deprotection of PMB in 4 was accomplished by reaction of 4 with TFA and triethylsilane to produce the desired chelating agent 5 in nearly quantitative yield.

Scheme 1.

Synthesis of chelator DA-18C6-BHA (5) for Zr(IV)-89

The new chelator (DA-18C6-BHA) and DFO were comparatively evaluated for radiolabeling with ⁸⁹Zr at room temperature (Supporting Information). The new chelator (DA-18C6-BHA) and DFO rapidly bound to ⁸⁹Zr and were shown to be effective in binding ⁸⁹Zr at pH 7 and room temperature. Radiolabeling of DA-18C6-BHA or DFO with ⁸⁹Zr was nearly complete in 10 min (>98% radiolabeling efficiency). Stability of ⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO in human serum was determined (Table 1 and Supporting Information). Dissociation of ⁸⁹Zr from ⁸⁹Zr-DA-18C6-BHA or ⁸⁹Zr-DFO in human serum (pH 7.0 and 37 ^oC) was measured over 7 days. The serum stability data suggest that DA-18C6-BHA and DFO remained inert in serum for 7 days. A minimal amount of ⁸⁹Zr from ⁸⁹Zr-DA-18C6-BHA or ⁸⁹Zr-DFO was released to serum (<1% at 168 h time point).

Table 1.

Complex Stability of ⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO in human serum (pH 7.0, 37°C, ITLC, duplicate).

	89Zr-Chelator Complex (%)
Time	DA-18C6-BHA	DFO
0 h	99.4 ± 0.1	99.5 ± 0.5
24 h	99.1 ± 0.0	99.2 ± 0.1
48 h	98.8 ± 0.7	99.3 ± 0.3
72 h	98.7 ± 0.2	98.7 ± 0.8
168 h	99.0 ± 0.3	99.0 ± 0.5

⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO were evaluated for complex stability in the presence of EDTA (Table 2 and Supporting Information). ⁸⁹Zr-DA-18C6-BHA or ⁸⁹Zr-DFO was incubated with a 100-fold molar excess of EDTA. ⁸⁹Zr-DA-18C6-BHA was shown to have high complex stability in EDTA solution, and only a trace amount of ⁸⁹Zr (<1%) was transchelated to EDTA. In contrast, a significant amount of ⁸⁹Zr was dissociated from ⁸⁹Zr-DFO complex at 168 h-post incubation (>12%).

Table 2.

Complex Stability of ⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO in EDTA solution (pH 7.0, 37°C, ITLC, duplicate).

	89Zr-Chelator Complex (%)
Time	DA-18C6-BHA	DFO
0 h	99.3 ± 0.1	99.4 ± 0.1
1 h	99.6 ± 0.1	98.1 ± 0.3
24 h	99.0 ± 0.2	92.5 ± 0.4
48 h	99.0 ± 0.2	89.1 ± 0.2
168 h	99.1 ± 0.3	87.3 ±1.8

⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO were further evaluated for complex stability in the presence of apotransferrin (Table 3 and Supporting Information). ⁸⁹Zr-DA-18C6-BHA or ⁸⁹Zr-DFO was incubated with a 5-fold molar excess of apotransferrin. Both ⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO complex remained stable in apotransferrin solution (PBS, pH 7.0), and no considerable amount of ⁸⁹Zr was released to the apotransferrin solution over 7 days.

Table 3.

Complex Stability of ⁸⁹Zr-DA-18C6-BHA and ⁸⁹Zr-DFO in apotransferrin solution (pH 7.0, 37°C, ITLC, duplicate).

	89Zr-Chelator Complex (%)
Time	DA-18C6-BHA	DFO
0 h	98.8 ± 0.5	99.0 ± 0.1
24 h	98.3 ± 0.4	98.8 ± 0.2
48 h	98.0 ± 0.2	97.8 ± 0.6
72 h	97.9 ± 0.4	98.0 ± 0.3
168 h	98.8 ± 0.3	98.4 ± 0.4

We performed biodistribution studies in CD-1 normal mice (intravenous injection, n = 4) to evaluate in vivo stability of ⁸⁹Zr-DFO and ⁸⁹Zr-DA-18C6-BHA. The mice were euthanized at 1 h, 4 h, and 24 h. The selected organs (liver, kidney, muscle, bone) and the blood were harvested, wet weighed, and the radioactivity was measured in a γ-counter (Figures 1 and 2 and Supporting Information). Both ⁸⁹Zr-DA-18C6-BHA (Figure 1) and ⁸⁹Zr-DFO (Figure 2) showed rapid blood clearance and low uptake in normal organs (<0.44% ID/g) over 24 hours. ⁸⁹Zr-DA-18C6-BHA exhibited a negligible level of radioactivity in blood at all time points (≤0.04 %ID/g). The bone and muscle uptake of ⁸⁹Zr- DA-18C6-BHA remained minimal at all time points (≤0.11% ID/g). ⁸⁹Zr-DA-18C6-BHA exhibited the highest retention in liver (0.44 %ID/g at 4 h). Renal uptake of ⁸⁹Zr-DA-18C6-BHA was decreased from 0.21 %ID/g (1 h) to 0.12 %ID/g (24 h). ⁸⁹Zr-DFO showed a minimal uptake in blood, liver, and muscle at 24 hours (0.02 %ID/g, 0.01 %ID/g, and 0.03 %ID/g, respectively). ⁸⁹Zr-DFO showed relatively higher accumulation of radioactivity in the kidney at all time points (≤0.25 %ID/g) when compared to other organs. ⁸⁹Zr-DFO displayed minimal bone uptake which was decreased from 0.09 %ID/g (4 h) to 0.04 %ID/g (24 h). The in vivo data suggest that both ⁸⁹Zr-DFO and ⁸⁹Zr-DA-18C6-BHA presented an excellent biodistribution profile in normal mice as evidenced by low uptake in blood and normal organs including bone.

Figure 1.

In Vivo Biodistribution of ⁸⁹Zr-DA-18C6-BHA in CD-1 mice (n = 4, intravenous injection)

Figure 2.

In Vivo Biodistribution of ⁸⁹Zr-DFO in CD-1 mice (n = 4, intravenous injection)

In summary, the new chelator (DA-18C6-BHA) built on diaza-18-crown-6 containing two hydroxamate donor groups was synthesized and evaluated. The new chelator rapidly bound to ⁸⁹Zr, and the corresponding ⁸⁹Zr-labeled DA-18C6-BHA was shown to display high complex stability in human serum and apotransferrin for 7 days. When challenged by excess EDTA solution, ⁸⁹Zr-DA-18C6-BHA remained inert and was favorably compared to ⁸⁹Zr-DFO for complex stability. Both ⁸⁹Zr–DA-18C6-BHA and ⁸⁹Zr–DFO were shown to be stable in mice and have favorable biodistribution profiles with low bone uptake. The radiolabeling and in vitro and in vivo complex stability data clearly demonstrate that DA-18C6-BHA is an efficient chelator for ⁸⁹Zr radiolabeling.