2-(4-Hydroxyphenyl)benzothiazole Dicarboxylate Ester TACN Chelators for ⁶⁴Cu PET imaging in Alzheimer’s Disease

Abstract

Herein we report a new series of bifunctional chelators (BFCs) with high affinity for amyloid β aggregates, strong binding affinity towards Cu(II), and favorable lipophilicity for potential blood-brain barrier (BBB) penetration. The alkyl carboxylate ester pendant arms enable high binding affinity towards Cu(II). The BFCs form stable ⁶⁴Cu-radiolabeled complexes and exhibit favorable partition coefficient (log D) values of 0.75–0.95. Among the five compounds tested, the ⁶⁴Cu-YW-1 and ⁶⁴Cu-YW-13 complexes exhibit significant staining of amyloid plaques in ex vivo autoradiography studies.

Graphical Abstract

A series of bifunctional chelators (BFCs) with high affinity for amyloid β aggregates, strong Cu(II) chelating ability, and favorable lipophilicity for potential blood-brain barrier penetration are reported.

INTRODUCTION

According to the Alzheimer’s Association, Alzheimer’s disease (AD) is the 6^th leading cause of death in the United States, and most common cause of dementia worldwide.¹ AD is a neurodegenerative disease, as the disease progresses, symptoms include memory loss, synaptic dysfunction, and decline in learning and thinking abilities.^2–4 As the disease progresses, amyloid plaques formed by abnormal aggregation of amyloid β (Aβ) gradually build up in brains of AD patients, causing brain damage and neuron death. Aβ peptides are derived from the amyloid precursor protein (APP), and sequential cleavages of APP by β- and γ-secretases generates Aβ monomers with 39 to 43 amino acid residues in length. The two major isoforms of Aβ peptide are 40 and 42 amino acids long, named Aβ₄₀ and Aβ₄₂, respectively.^5–9

AD is currently incurable, and definitive diagnosis relies heavily on post-modern examination of brains of patients.^10–13 As researchers move to target early-stage of AD, determining the presence of amyloid deposits to confirm diagnosis becomes more and more important. Molecular imaging techniques, such as positron emission tomography (PET), have enabled non-invasive assessment of Aβ burden in brains of patients.^{14, 15} PET imaging utilizes radiotracers containing positron-emitting radionuclides, such as ¹¹C (t_1/2= 20.4 min) and ¹⁸F (t_1/2= 109.7 min), have been extensively studied in the last three decades. ¹¹C radiolabeled Pittsburgh Compound-B (¹¹C-PiB) is widely used for imaging fibrillar amyloid species by PET scanning.^{16, 17} Three ¹⁸F-labelled Aβ tracers, the benzothiazole derivative ¹⁸F-Flutemetamol (Vizamyl), the stilbene derivatives ¹⁸F-Florbetaben (Neuraceq) and ¹⁸F-Florbetapir (Amyvid) have been approved by the US Food and Drug Administration (FDA) for estimating Aβ plaque burdens in patients with cognitive impairment.^18–22

However, in clinical usage, both ¹¹C and ¹⁸F radiotracers suffer from the relatively short half-lives. In addition, these radioisotopes are covalently installed into the target molecules, requiring special synthetic approaches and equipment.²³ Therefore, long-lived radiometals, notably copper-64 (t_1/2 = 12.7 h, β⁺ = 17.8%; β⁻ = 38.4%), have gained considerable interest in PET imaging.²⁴ Compared with ¹¹C and ¹⁸F, the long half-life of ⁶⁴Cu allows for accumulation in the targeted region and broader clinical applications, especially for use in areas without access to on-site cyclotrons.^25–27 Moreover, ⁶⁴Cu could be easily incorporated into target bio-active agents via rapid chelation. The common oxidation state of ⁶⁴Cu in aqueous solution is ⁶⁴Cu(II), and its d⁹ electronic configuration enables formation of many coordination forms, varying from four-coordinate to six-coordinate using metal chelators containing nitrogen donors, anionic oxygen, or sulfur donors.²⁴

We have recently reported the development of bifunctional chelators (BFCs), which contain an Aβ-binding motif (2-phenylbenzothiazole derivative) and strong Cu(II)-chelating macrocyclic ligands, such as 1,4-dimethyl-1,4,7-triazacyclononane (TACN) and 2,11-diaza[3.3](2,6)pyridinophane (N4) macrocycles.^28–30 These BFCs exhibit high affinities for Aβ aggregates and Cu(II) ion, which could be used as ⁶⁴Cu amyloid PET imaging agents. The 2-phenylbenzothiazole derivative has shown appreciable Aβ-binding and fluorescent properties, therefore we continue to take advantage of this molecule as the amyloid binding motif and carry out the modification on the metal chelating fragment, by attaching two ester carboxylate pendant arms to the TACN backbone (Figure 1), in order to increase the lipophilicity of the bifunctional chelators (BFCs). Our purpose is to design diester-based ⁶⁴Cu PET imaging agents with favorable metal chelating ability and lipophilicity for in vivo amyloid PET imaging applications.

Figure 1.

Structures of the ligands investigated herein. The metal-binding and Aβ-interacting fragments are shown in blue and red, respectively.

RESULTS AND DISCUSSION

Design and synthesis of BFCs.

The bifunctional chelators (BFCs) discussed herein are created by linking the Aβ-binding fragment 2-(4-hydroxyphenyl)-benzothiazole and the metal-chelating TACN ligand via Mannich reaction (Scheme 1). The amyloid targeting motif was generated by the condensation of 2-aminothiophenol with vanillin, followed by oxidation with atmospheric oxygen (Scheme S1). To further enhance the metal chelation ability of BFCs, two pendant carboxylate arms were added to the TACN backbone.

Scheme 1.

Synthesis of BFCs.

Initially, the synthesis of final products was conducted by established procedures to generate the bi-substituted TACN ligands,³¹ followed by Mannich reaction with 2-(4-OH-phenyl)-benzothiazole and paraformaldehyde (Scheme S2). However, due to the lack of fluorescence emission for these precursors, we failed to detect the unreacted TACN ligands by either TLC or HPLC, and therefore unable to separate the impurities from the products. Moreover, the heating condition could lead to decomposition of the ester products. Therefore, we developed a new pathway (Scheme 1) by synthesizing YW-12 as the ligand precursor and then reacting YW-12 with a series of alkyl-bromoacetates in presence of base, generating the final products YW-1 to YW-5 that contain tert-butyl, iso-propyl, ethyl, and methyl ester groups, respectively. Hydrolysis of YW-1 in presence of concentrated hydrochloric acid generates YW-13.

UV-Vis absorption spectra were acquired to determine the maximum absorbance wavelength (λ_abs) of the ligands and their Cu(II) complexes in PBS (pH=7.4) buffer (Figure 2). Based on the UV-Vis absorbance spectra, excitation wavelengths (λ_ex) of 345–348 nm for BFCs and similar wavelengths for Cu(II) complexes were used to examine their fluorescent properties (Figure 3). Due to the quenching effect by chelation with Cu(II), the fluorescence intensities of ester BFCs decrease by 3~4 folds, while that of YW-13 decreases by 8 folds.

Figure 2.

UV-Vis spectra of a) YW-1 to YW-13 and b) their Cu(II) complexes in PBS.

Figure 3.

Emission spectra of a) YW-1 to YW-13 and b) their Cu(II) complexes in PBS.

Stability Constants with Metal Ions by pH-Spectrophotometric Titration.

Since all ligands contain several acidic and basic functional groups, their acidity constants (pKa) were determined by UV-Vis spectrophotometric titrations. In order to further investigate the metal chelation ability and to quantify it, we performed spectrophotometric titrations of ligands in presence of Cu(II) ion.

For YW-1, UV-Vis titrations from pH 3.0 to 11.0 reveal several changes in the spectra, such as the disappearance of the band at 325 nm and the increase of the band at 351 nm with an isosbestic point at 340 nm (Figure 4). The best fit to the data was obtained with four pKa values: 3.30(6), 5.75(5), 8.16(5) and 11.77(3) (Table 1). On the basis of previously reported acidity constants for phenols and amines,^{32, 33} we assigned the three lower pKa values to the deprotonation of the amine groups on TACN backbone, and the highest pKa value to the phenol deprotonation in ligand. The other ester containing ligands in the series show similar values to those obtained for YW-1 (Table 1).

Figure 4.

Variable pH (pH 3−11) UV-Vis spectra of YW-1 ([L] = 20 μM, 25 °C, I = 0.1 M NaCl) and species distribution plot.

Table 1.

Acidity constants (pK_a’s) of YW-1 to YW-13 determined by spectrophotometric titrations (errors are for the last digit).

Reaction	YW-1	YW-4	YW-2	YW-5	YW-13
[H4L]3+ = [H3L]2+ + H+ (pKa1)	3.30(6)	5.09(2)	4.78(3)	4.21(5)	3.83(5)
[H3L]2+ = [H2L]+ + H+ (pKa2)	5.75(5)	6.48(1)	7.41(2)	6.90(2)	6.44(3)
[H2L]+ = [HL] + H+ (pKa3)	8.16(5)	8.27(1)	9.42(2)	12.82(1)	7.92(1)
[HL]=[L]− + H+ (pKa4)	11.77(3)	11.98(1)	11.36(1)	11.30(4)	11.84(1)

Similar spectrophotometric titrations were performed to determine the stability constants and solution speciation of the Cu(II)-BFC complexes. The pKa values of the ligands and the deprotonation of metal-bound water molecules were included in the calculations. The stoichiometry of the Cu(II)-BFC complexes in solution was determined by Job’s plot analysis. Plot of Cu-YW-13 shows a break around 0.5 Cu mole fraction, suggesting the formation of a 1:1 complex (Figure S12).^28–30 The calculated stability constants show that YW-1 has larger binding constants (logK) with Cu(II) than YW-2, YW-4, and YW-5 (Table 2). In addition, YW-13, which contains two carboxylic groups instead of ester groups, exhibits the highest binding constant (logK) among all compounds, which is 2~3 orders of magnitude larger than those of ester BFCs.

Table 2.

Stability constants (logK) of the Cu(II) Complexes of YW-1 to YW-13.

Reaction	YW-1	YW-4	YW-2	YW-5	YW-13
M2+ + HL = [MHL]2+	5.61(13)	6.62(3)	6.89(9)	6.01(2)	3.89(3)
M2+ + L−1 = [ML]+	23.41(9)	23.27(2)	22.79(7)	22.36(1)	25.56(6)
[ML(H2O)]+ = [ML(OH)] + H+	15.27(9)	14.98(2)	15.36(8)	13.79(2)	17.70(7)

The species distribution plot of Cu-YW-1 was obtained based on the calculated stability constants (Figure 5), and the concentration of free Cu(II) with YW-1 is negligible above pH 4, as shown in the plot. The concentrations of free Cu(II) (pM =−log[M_unchelated]) at a specific pH value and total ion concentration can be calculated from the solution speciation diagrams, and the calculated pCu values for YW-1 and YW-13 are 11.3 and 12.4 at pH 7.4, respectively (Table 3), comparable to the value of 10.7 at pH 7.4 for the strong chelating agent DTPA (diethylenetriaminepentaacetic acid), indicating that our BFCs bind strongly to Cu(II) ions.^{32, 34, 35}

Figure 5.

Variable pH (pH 3−11) UV-Vis spectra of YW-1 and Cu(II) system ([L] = [Cu²⁺] =20 μM, 25 °C, I = 0.1 M NaCl) and species distribution plot.

Table 3.

Calculated pM (−log[M]_free; M = Cu²⁺) values for a solution containing a 1:1 Metal/Ligand Mixture ([M²⁺]_tot = [Ligand]_tot = 50 μM)

	YW1	YW4	YW2	YW5	YW13	DTPAa
pH 6.6	10.5	10.3	9.6	9.4	11.5	9.7
pH 7.4	11.3	11.1	10.6	10.3	12.4	10.7

^aDiethylenetriaminepentaacetic acid (DTPA).³⁴

EPR spectra of copper complexes.

To further characterize the Cu(II)-BFC complexes, their X-band CW EPR spectra were recorded in frozen glasses at 77 K. The Cu(II) complexes were prepared right before the EPR experiment by reacting the ligand with 0.8 equivalent of Cu(ClO₄)₂·6H₂O. The EPR spectrum of the Cu-YW-1 in a 1:3 (v:v) MeCN:PrCN glass solution reveals a pseudoaxial EPR pattern with three different g values: g_z = 2.258, A_z (Cu)=159 G, g_y = 2.060, and g_x = 2.045 (Figure 6). The EPR spectra of Cu-YW-4 and Cu-YW-5 were also obtained in the same way and exhibit a similar EPR pattern as Cu-YW-1’s with g_z = 2.255, A_z (Cu)=165 G, g_y = 2.055, g_x = 2.048, and g_z = 2.262, A_z (Cu) = 165 G, g_y = 2.041, g_x = 2.067, respectively (Figures 7 and 8). Overall, these EPR spectra are similar to the ones reported previously for related 2-(4-hydroxyphenyl)benzothiazole TACN derivatives,^28–30 and support the formation of mononuclear Cu complexes in solution.

Figure 6.

Experimental and simulated EPR spectra of the Cu-YW-1 complex in 1:3 MeCN:PrCN glass at 77 K. The following parameters were used for the simulation: g_z = 2.258, A_z (Cu) = 159 G, g_y = 2.060, and g_x = 2.045.

Figure 7.

Experimental and simulated EPR spectra of the Cu-YW-4 complex in 1:3 MeCN/PrCN glass at 77 K. The following parameters were used for the simulations: g_z = 2.255, A_z (Cu) = 165 G, g_y = 2.055, g_x = 2.048.

Figure 8.

Experimental and simulated EPR spectra of the Cu-YW-5 complex in 1:3 MeCN/PrCN glass at 77 K. The following parameters were used for the simulations: g_z = 2.262, A_z (Cu) = 165 G, g_y = 2.041, g_x = 2.067.

In general, the R parameter [R = (g_y − g_z)/(g_x − g_y) with g_x > g_y > g_z] can be indicative of the predominance of the d_z² or d_x²_−y² orbital in the ground state of the unpaired electron of the Cu²⁺ ion. When R > 1, the greater contribution to the ground state arises from the d_z² orbital, while when R < 1, the greater contribution to the ground state comes from the d_x²_−y² orbital. The R value of 0.076 determined for Cu-YW-1 is indicative of a predominantly d_x²_−y² ground state, which is characteristic for Cu(II) complexes with slightly rhombic symmetry and elongation of the axial bonds. Cu-YW-4 has a similar symmetry with an R value of 0.035.^36–38

Interaction of YW-13 with Aβ₄₂ fibrils.

BFCs investigated herein contain fragments derived from benzothiazole and o-vanillin, which bind tightly to Aβ species, while also exhibiting fluorescence emission that increases in the presence of amyloid fibrils.^{32, 39} When a solution of Aβ₄₂ fibrils was titrated with YW-13 and the emission intensity increase was corrected for the intrinsic fluorescence of YW-13, a saturation behavior is observed that is best fit with a one-site binding model to give a K_d = 121 ± 44 nM (Figure 9), indicating that this BFC can bind tightly to Aβ₄₂ species. We have also attempted to obtain K_d values for the ester BFCs or their Cu complexes, as performed previously,^28–30 yet the fluorescence intensities were low and negligible increases in intensity were observed upon increasing the concentration of BFCs or their Cu complexes.

Figure 9.

Direct binding constant measurements of YW-13 with Aβ₄₂ fibrils.

Fluorescence Imaging of Amyloid Plaques in 5xFAD Mouse Brain Sections.

Ex vivo mouse brain section staining was performed to evaluate each BFC’s affinity toward Aβ species. Brain sections were collected from 11-month-old 5xFAD transgenic mice, which overexpress mutant form of APP that can be cleaved by β- and γ-secretases to form Aβ peptides, and develop severe amyloid pathology found in AD.^{40, 41} An appreciable amount of fluorescence staining was observed upon incubation of the brain sections for 30 min with 50 μM solutions of BFCs, especially for YW-4 and YW-5 (Figure 10, left panels). The specific staining of amyloid plaques was confirmed by staining with Congo Red, another amyloid-binding fluorescent dye (Figure 10, middle panels).^{42, 43} The labeling ability of Cu(II) complexes was also probed using mouse brain sections of the same age. Compared to BFCs YW-4 and YW-5, the colocalization of their Cu(II) complexes signal with Congo Red signal is improved, as indicated by Pearson’s coefficients. The other BFCs and their Cu(II) complexes also show moderate staining of amyloid aggregates (Figures S1 and S2). Overall, these ex vivo amyloid binding studies suggest that these BFCs could be bind to Aβ species (see below).

Figure 10.

Fluorescence microscopy images of 5xFAD brain sections incubated with compound YW-4, YW-5, and their Cu(II) complexes (left panels), Congo Red (middle panels), and merged images (right panels). Magnification: 20x. Scale bar: 125 μm.

The amyloid binding affinity of the BFCs was further investigated with AF594-conjugated HJ3.4 antibody (AF594-HJ3.4) that binds to a wide range of Aβ species.^{30, 44–48} Using 6-month-old 5xFAD mouse brain sections, intense signal of fluorescence staining was observed with treatment of 50 μM BFCs or corresponding Cu(II) complexes solutions for 1 h. With less bulky substituents (from tert-butyl group to methyl group and hydrogen atom), there is an improvement of the colocalization between BFCs’ signal and antibody signal, as indicated by the calculated Pearson’s coefficients (Figure 11). The specific staining of Cu-YW-13 is further examined with the 40x lens of the fluorescence microscope (Figure S3), and it exhibits strong colocalization with the antibody stained regions.

Figure 11.

Fluorescence microscopy images of 5xFAD brain sections incubated with compound YW-1, YW-2, YW-13 and their Cu(II) complexes (left panels), AF594-HJ3.4 antibody (middle panels), and merged images (right panels). Concentrations used: [BFC] = [Cu²⁺] = 50 μM, [HJ3.4] = 1 μg/ml. Magnification: 20x. Scale bar: 125 μm.

Radiolabeling and Log D value determination.

The radiolabeling of compounds YW-1 to YW-13 was performed using ⁶⁴CuCl₂ and employing the conditions described in the experimental section.⁴⁹ Quality control assays were conducted using HPLC and/or TLC. All radiochemical yields were >95% within minutes at 45 °C, with specific activities of 100 Ci/mmol or greater. Therefore, all radiolabeled complexes were used directly without further purification.

For compounds to be used in neuroimaging applications, one critical factor is the penetration of blood brain barrier (BBB). Although the Lipinski’s rules could offer insights for designing molecules with favorable lipophilicity, it is necessary to obtain experimental results for evaluation of BBB permeability.⁵⁰ To determine the hydrophobicity of the radiolabeled compounds, the octanol/water partition coefficient values log D were determined for the ⁶⁴Cu complexes of YW-1 to YW-13 (Table 4). Gratifyingly, the obtained log D values for the ⁶⁴Cu-radiolabeled complexes YW-1 to YW-5 are in the range of 0.72 – 0.95, which suggests their potential ability to cross the BBB. By comparison, the ⁶⁴Cu complex of YW-13, which does not contain ester group on the acetate branch, exhibits a negative log D value of −0.68±0.10 and thus is not expected to cross the BBB. The radio-HPLC profiles suggested YW-13 chelate tightly with ⁶⁴Cu, while ⁶⁴Cu-YW-2 could undergo decomposition in the aqueous media, due to the potential hydrolysis of the ester groups (Figure S14).

Table 4.

Molecular weights (MWs) of ligands YW-1 to YW-13, measured log D values for the corresponding ⁶⁴Cu-radiolabeled complexes.

Ligand	MWs of ligands (g·mol−1)	log D (64Cu complexes)
YW-1	626.8	0.95 ± 0.07
YW-4	598.3	0.91 ± 0.07
YW-2	570.7	0.90 ± 0.03
YW-5	542.7	0.72 ± 0.02
YW-13	514.6	−0.68 ± 0.10

Autoradiography studies.

Ex vivo autoradiography studies using brain sections of 11-month-old 5xFAD and aged-matched WT mice were conducted to determine the specific binding of the ⁶⁴Cu-labeled BFCs to the amyloid plaques. The brain sections were stained, washed, and imaged as described in the experimental section.⁴⁹ Unfortunately, ⁶⁴Cu-YW-1 exhibits very low radioactivity intensity when staining the 5xFAD mouse brain sections (Figure 12, left column). Upon treatment with ⁶⁴Cu-YW-13 (Figure 12, right column), we observed an increased autoradiography intensity and ⁶⁴Cu-YW-13 exhibits a 2.26 intensity ratio of 5xFAD to WT staining (Figure 13). The specific binding to amyloid plaques of the radiolabeled BFCs was further confirmed by blocking the brain sections with the non-radioactive blocking agent (B₁, Figure S15).

Figure 12.

Autoradiography images of brain sections of WT and 5xFAD mice, in the absence and presence with a known Aβ specific blocking agent.

Figure 13.

Average radioactivity of the brain sections in the autoradiography images. The numbers in the bar graph are the intensity ratios of 5xFAD to WT in each group.

CONCLUSIONS

To summarize, we have designed and synthesized five BFCs YW-1 to YW-13 containing a 2-(4-hydroxyphenyl)benzothiazole fragment as the amyloid targeting motif and triazacyclononane-dicarboxylate ester metal chelators. Modification of the synthetic pathway allows for the generation of a series of BFCs in a direct approach. The evaluation of the Aβ-binding affinity for these BFCs and their ⁶⁴Cu complexes was probed by ex vivo AD mouse brain section fluorescence imaging and autoradiography studies. These results show that the Cu(II) complexes of the dicarboxylic acid YW-13 bind more selectively to the amyloid plaques than the Cu(II) complexes of the dicarboxylate esters YW-1 to YW-5. The ⁶⁴Cu-radiolabeled dicarboxylate ester BFCs exhibit moderate log D values around 0.9, suggesting they should be BBB permeable. Unfortunately, the ⁶⁴Cu complex of YW-13, which has favorable amyloid binding affinity and metal chelating ability, exhibits a negative log D value.

Overall, the resulting BFCs could be readily radiolabeled with ⁶⁴Cu for PET imaging purposes. More efforts need to be devoted for improving the lipophilicity of such BFCs for in vivo applications. These results further suggest that the presence of two carboxylic acid groups appended to the TACN macrocycle renders the corresponding chelators to hydrophilic for BBB permeability, and the related systems containing only one carboxylic acid or ester group should be more appropriate for in vivo applications.⁵¹