Cyclen-based chelators for the inhibition of Aβ aggregation: Synthesis, anti- oxidant and aggregation evaluation

The aggregation of the protein amyloid-β in the brain has been associated with neurodegenerative diseases such as Alzheimer’s disease. Such aggregation of amyloid-β can be induced by misregulated metal ions such as Cu and Zn. Here we present a series of four metal chelating compounds based on the cyclen macrocycle that bears pendant arms to provide additional anti-oxidant activity. The corresponding Cu, Zn and Ni complexes have been synthesized and characterized to examine the ability of the chelators to bind to the metal centres. Aβ40 de-aggregation by the cyclen compounds was assessed using turbidometry and the re-solubilization of the Aβ40 was also examined. Our results show that the cyclen compounds have the ability to effectively chelate Cu and Zn metal ions and thus de-aggregate Aβ40 that has been aggregated due to the presence of these ions. The antioxidant properties of the cyclen compounds was tested by using the DPPH scavenging assay and the results show that some of the compounds can decrease oxidative stress. Introduction Alzheimer’s disease (AD) is a progressive illness that is often characterized by deposits of the peptide amyloid-β (Aβ), oxidative stress, and elevated levels of particular metal ions within regions of the brain [1-4]. Amyloid-β peptides are generally derived from amyloid precursor protein (APP) [5, 6], which is located on the cell membrane [7]. APP can be cleaved either by a non-amyloidogenic pathway yielding the soluble APPα, or by the amyloidogenic pathway, where β-secretase cleaves APP to give APPβ and the c99 terminal. The c99 is further cleaved to give amyloid β[5, 8, 9], which is normally degraded and escorted from the brain[10]. However, mis-regulated metal ions (zinc, copper, nickel, iron) can prevent the clearance of amyloid β proteins by blocking their passage through the blood brain barrier (BBB)[11]. At the same time, accumulated free metal ions can stimulate the production of extracellular amyloid β by binding the active site of α-secretase in APP thus favouring the amyloid pathway over the non-amyloid pathway. Subsequently, amyloid β may aggregate in the brain to form plaques [10]. Misregulated metal ions can also catalyze the production of reactive oxygen species (ROS) that induce oxidative stress in the brain [12]. The metals copper, zinc, and iron have been implicated in this regard and copper in particular has been proposed to have a cumulative effect that impairs processes that remove Aβ from the brain [10]. Currently, the role of Aβ and indeed that of the various metal ions and their interactions appears to be far from fully understood. Other factors such as neuroinflammation are currently receiving intense investigation [13] and quite recent work points to a connection between copper ions and the inflammatory response [14]. The binding and removal of excess metal species using metal chelating compounds, coupled with antioxidant activity to remove ROS, has been identified as a promising strategy to treat AD [15]. A number of metal chelators have been investigated as metal ion chelation therapy agents [16, 17]. For example, desferrioxamine has a high affinity towards free iron [18] as well as Cu and Zn, with which it forms stable complexes that slow down the development of Alzheimer’s disease [19]. 8-Hydroxyquinoline derivatives such as clioquinol have been tested. Clioquinol has a hydrophobic character and readily crosses the blood brain barrier (BBB) and can bind to Zn and Cu that are attached to Aβ. On the other hand, clioquinol-copper complexes can increase the level of biological copper in the brain and has been shown to redistribute copper ions [16, 20]. Macrocyclic polyamine compounds have a strong affinity for metals ions [21] and have found applications in diagnostic imaging, as therapeutic agents [22-24] and in targeting Aβ amyloid aggregation [25]. These compounds have important physical properties such as low molecular weights, neutral charge, amphiphilic solubility and low toxicities [22]. Fig. 1. Chemical structures of the chelators investigated in the current work. In this work, cyclen-based chelators have been synthesized (Fig. 1) that bear pendant arms with redox properties that increase the lipophilicity of the resultant compounds and that may reduce the oxidative stress that is caused by particular metals ions. In compound 1, the phenolic hydroxyl moiety of the pendant 8-hydroxyquinoline group act as an antioxidant [26] while the quinolinyl moiety has been shown to decrease levels of Aβ in the brain of AD patients without affecting serum metal ion levels (Zn and Cu) [27]. The pendant arm of 2 (Fig. 1) bears a 2-(pyridin-2-yl)ethylacetamide group that may also increase lipophilicity and decrease oxidative stress. For comparison, compound 3, which bears a propylpyridin-4-yl pendant arm should increase lipophilicity but have limited effect on oxidative stress. We predicted that the anti-oxidant activity of 4 would be similar to that of 2 but allows the effect of more polar substituents to be examined. We report here our findings describing the synthesis of compounds 1-4 and some X-ray structural determinations of complexes with Cu, Zn, and Ni. Results and Discussion Scheme 1. Synthesis of 1-4 where R = pendant arm (see Figure 1). The chelators, 1-4, were synthesized by the reaction of 1,4,7,10-tetrazacyclododecane (cyclen) with the corresponding alkylbromide in a 2:1 (cyclen:RBr) molar ratio. Using this ratio, reasonable yields were obtained and the formation of diand tri-substituted cyclen was minimized. The monosubstituted cyclen is significantly less reactive than unsubstituted cyclen and so only small amounts of diand trisubstituted cyclen (~10% and ~3%, respectively) were obtained. The resultant mixtures could be readily separated using column chromatography. Attempts to protect three of the cyclen nitrogen atoms using P(NMe2)3 (and thus avoid multiple substitution products) [28] resulted in only very low yields and so this method was not pursued further. The synthesis and characterization of compounds 1, 3 and 4 has not been previously reported while 2 has been reported elsewhere [29]. Scheme 2: Synthesis of 2-bromomethyl-8-hydroxyquinoline The syntheses of the R-Br groups that subsequently form the pendant arms are shown in Schemes 2 and 3. 2-Bromomethyl-8-hydroxyquinoline (Scheme 2) was synthesized in two steps from 2-formyl-8-hydroxyquinoline by first reducing the aldehyde to the corresponding alcohol followed by bromination of the alcohol using hydrobromic acid. 2-Bromo-N-(2(pyridin-2-yl)ethyl)acetamide and bis(2-hydroxyethyl)carbamic bromide were prepared by reacting the commercially available amines with bromoacetyl bromide. Scheme 3: Synthesis of 2-bromo-N-(2-(pyridin-2-yl)ethyl) acetamide and bis(2hydroxyethyl)carbamic bromide Metal complexes (Cu, Zn, and Ni) of 1-4 were synthesized by reaction of the substituted cyclen compounds with the corresponding metal salts (either sulfate or chloride) in methanol or water. The resultant complexes could be readily purified by recrystallization, which also gave some crystals suitable for structure determination by X-ray diffraction. X-ray Crystallography Fig. 2. ORTEP diagram showing the structure of the Zn-1 unit.Main Text Paragraph. X-ray crystal structures were obtained for the complexes Cu-2, Cu-3, Zn-1, and Ni-3. An ORTEP diagram for Zn-1 is shown in Figure 2. Table 1 shows selected bond lengths and angles for Zn-1, Cu-2, Cu-3, and Ni-3. Other crystal data are collected in the supporting information. Table 1: Selected bond lengths and angles for Zn-1, Cu-2, Cu-3, and Ni-3. Zn-1 Cu-2 Cu-3 Ni-3 M-N1 2.304 (5) 2.055 (13) 2.047 (4) 2.143 (6) M-N2 2.120 (5) 2.016 (12) 2.019 (4) 2.044 (7) M-N3 2.211 (5) 2.024 (13) 2.022 (4) 2.113 (7) M-N4 2.111 (5) 2.058 (11) 2.020 (4) 2.071 (7) M-N5 2.094 (4) M-O 2.340 (4) 2.108 (9) 2.138 (3) 2.099(5) M-N1-C9 109.4 (3) 112.9 (9) 113.2 (3) 112.6 (5) N1-C9-C10 112.3 (5) 116.4 (13) 115.3 (5) 118.0 (6) N1-M-N2 80.26 (17) 85.6 (5) 86.61 (18) 84.9 (3) N2-M-N3 79.87 (19) 86.6 (5) 85.91 (18) 84.3 (3) N3-M-N4 80.4 (2) 86.7 (5) 85.99 (18) 81.4 (3) N4-M-N1 79.78 (18) 86.4 (5) 86.58 (18) 82.0 (3) In each of the structures, the cyclen macrocycle is bound to the metal centre in the expected tetradentate fashion. In the structure of Zn-1, the remaining two coordination sites of the distorted octahedral geometry are occupied by the donor atoms (N and O) of the 8hydroxyquinolinyl pendant arm. Thus, this ligand binds in a hexadentate fashion. The charge of the Zn-1 cation is balanced by a tetrahedral ZnCl4 2anion (not shown). The metal-nitrogen bond distances for Zn-1 (Table 1) are generally longer than the metalnitrogen distances for the other complexes. Similarly, the N-M-N angles for Zn-1 are smaller than those the other complexes as are the M-N1-C9 and N1-C9-C10 angles. These geometries allow for accommodation of the bidentate quinolinyl group in the coordination sphere of the metal. In the structures of Cu-2, Cu-3 and Ni-3, the pendant arms of 2 and 3 are not coordinated to the metal centre. In the cases of Cu-2 and Cu-3, an O atom of the sulfate anions occupies an apical position to give square pyramidal coordination geometry. In the case of Ni-3, there are two molecules in the asymmetric unit. Each complex has two water molecules coordinated to the Ni centre resulting in a distorted octahedral geometry and no coordination of the accompanying sulfate anions. A turbidimetric assay [30] was used to investigate the effect of 1-4 on the Cu-induced aggregation of Aβ40. Aggregation of Aβ40 was induced by the addition of Cu 2+ ions. This was accompanied by a significant increase in optical absorbance, which was monitored at 405 nm. Upon addition of 1-4, disaggregation of Aβ40 occurred with a corresponding decrease in optical absorbance as the turbid suspension of the Aβ40 aggregates re-dissolved due to the withdrawal and binding of the Cu ions by the chelators. Experimental data are presented in Figure 3. Each of the chelators was effective at decreasing turbidity to levels only marginally above that of un-aggregated Aβ40. Within error, each of 1-4 exhibited the same effect on turbidity. 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16


Introduction
Alzheimer's disease (AD) is a progressive illness that is often characterized by deposits of the peptide amyloid-β (Aβ), oxidative stress, and elevated levels of particular metal ions within regions of the brain [1][2][3][4]. Amyloid-β peptides are generally derived from amyloid precursor protein (APP) [5,6], which is located on the cell membrane [7]. APP can be cleaved either by a non-amyloidogenic pathway yielding the soluble APPα, or by the amyloidogenic pathway, where β-secretase cleaves APP to give APPβ and the c99 terminal.
The c99 is further cleaved to give amyloid β [5,8,9], which is normally degraded and escorted from the brain [10]. However, mis-regulated metal ions (zinc, copper, nickel, iron) can prevent the clearance of amyloid β proteins by blocking their passage through the blood brain barrier (BBB) [11]. At the same time, accumulated free metal ions can stimulate the production of extracellular amyloid β by binding the active site of α-secretase in APP thus favouring the amyloid pathway over the non-amyloid pathway. Subsequently, amyloid β may aggregate in the brain to form plaques [10].
Misregulated metal ions can also catalyze the production of reactive oxygen species (ROS) that induce oxidative stress in the brain [12]. The metals copper, zinc, and iron have been implicated in this regard and copper in particular has been proposed to have a cumulative effect that impairs processes that remove Aβ from the brain [10]. Currently, the role of Aβ and indeed that of the various metal ions and their interactions appears to be far from fully understood. Other factors such as neuroinflammation are currently receiving intense investigation [13] and quite recent work points to a connection between copper ions and the inflammatory response [14].
The binding and removal of excess metal species using metal chelating compounds, coupled with antioxidant activity to remove ROS, has been identified as a promising strategy to treat AD [15]. A number of metal chelators have been investigated as metal ion chelation therapy agents [16,17]. For example, desferrioxamine has a high affinity towards free iron [18] as well as Cu and Zn, with which it forms stable complexes that slow down the development of Alzheimer's disease [19]. . 8-Hydroxyquinoline derivatives such as clioquinol have been tested. Clioquinol has a hydrophobic character and readily crosses the blood brain barrier (BBB) and can bind to Zn and Cu that are attached to Aβ. On the other hand, clioquinol-copper complexes can increase the level of biological copper in the brain and has been shown to redistribute copper ions [16,20].
Macrocyclic polyamine compounds have a strong affinity for metals ions [21] and have found applications in diagnostic imaging, as therapeutic agents [22][23][24] and in targeting Aβ amyloid aggregation [25]. These compounds have important physical properties such as low molecular weights, neutral charge, amphiphilic solubility and low toxicities [22]. In this work, cyclen-based chelators have been synthesized (Fig. 1) that bear pendant arms with redox properties that increase the lipophilicity of the resultant compounds and that may reduce the oxidative stress that is caused by particular metals ions. In compound 1, the phenolic hydroxyl moiety of the pendant 8-hydroxyquinoline group act as an antioxidant [26] while the quinolinyl moiety has been shown to decrease levels of Aβ in the brain of AD patients without affecting serum metal ion levels (Zn 2+ and Cu 2+ ) [27]. The pendant arm of 2 ( Fig. 1) bears a 2-(pyridin-2-yl)ethylacetamide group that may also increase lipophilicity and decrease oxidative stress. For comparison, compound 3, which bears a propylpyridin-4-yl pendant arm should increase lipophilicity but have limited effect on oxidative stress. We predicted that the anti-oxidant activity of 4 would be similar to that of 2 but allows the effect of more polar substituents to be examined. We report here our findings describing the synthesis of compounds 1-4 and some X-ray structural determinations of complexes with Cu 2+ , Zn 2+ , and Ni 2+ .

Results and Discussion
Scheme 1. Synthesis of 1-4 where R = pendant arm (see Figure 1).
The chelators, 1-4, were synthesized by the reaction of 1,4,7,10-tetrazacyclododecane (cyclen) with the corresponding alkylbromide in a 2:1 (cyclen:RBr) molar ratio. Using this ratio, reasonable yields were obtained and the formation of di-and tri-substituted cyclen was minimized. The monosubstituted cyclen is significantly less reactive than unsubstituted cyclen and so only small amounts of di-and tri-substituted cyclen (~10% and ~3%, respectively) were obtained. The resultant mixtures could be readily separated using column chromatography. Attempts to protect three of the cyclen nitrogen atoms using P(NMe2)3 (and thus avoid multiple substitution products) [28] resulted in only very low yields and so this method was not pursued further. The synthesis and characterization of compounds 1, 3 and 4 has not been previously reported while 2 has been reported elsewhere [29].  Metal complexes (Cu, Zn, and Ni) of 1-4 were synthesized by reaction of the substituted cyclen compounds with the corresponding metal salts (either sulfate or chloride) in methanol or water. The resultant complexes could be readily purified by recrystallization, which also gave some crystals suitable for structure determination by X-ray diffraction. X-ray crystal structures were obtained for the complexes Cu-2, Cu-3, Zn-1, and Ni-3. An ORTEP diagram for Zn-1 is shown in Figure 2. Table 1 shows selected bond lengths and angles for Zn-1, Cu-2, Cu-3, and Ni-3. Other crystal data are collected in the supporting information. In each of the structures, the cyclen macrocycle is bound to the metal centre in the expected tetradentate fashion. In the structure of Zn-1, the remaining two coordination sites of the distorted octahedral geometry are occupied by the donor atoms (N and O) of the 8hydroxyquinolinyl pendant arm. Thus, this ligand binds in a hexadentate fashion. The charge of the Zn-1 cation is balanced by a tetrahedral ZnCl4 2anion (not shown).

X-ray Crystallography
The metal-nitrogen bond distances for Zn-1 (Table 1)  A turbidimetric assay [30] was used to investigate the effect of 1-4 on the Cu 2+ -induced aggregation of Aβ40. Aggregation of Aβ40 was induced by the addition of Cu 2+ ions. This was accompanied by a significant increase in optical absorbance, which was monitored at 405 nm.
Upon addition of 1-4, disaggregation of Aβ40 occurred with a corresponding decrease in optical absorbance as the turbid suspension of the Aβ40 aggregates re-dissolved due to the withdrawal and binding of the Cu 2+ ions by the chelators. Experimental data are presented in  The effect of 1-4 on Aβ40 aggregation was further evaluated using the BCA assay [31,32], which assesses the concentration of Aβ40 in solution. Figure 4 and Table S9 show solubility data for Aβ40 before and after treatment with Cu 2+ ions and then with 1-4. After incubation for 48 hours with Cu 2+ ions, the solubility of Aβ40 decreased to ~25 % of the initial concentration due to Cu 2+ -induced aggregation. Upon addition of 1-4, the solubility increased significantly after incubation for 4 h. The re-solubilisation of the Aβ40 is ascribed to copper sequestration by 1-4 to disassemble the aggregates and thus return the Aβ40 to solution.
Comparison of the four chelators reveals that treatment with 1 returns considerably more Aβ40 to solution (~83% of the original material) than does 2-4 (58%, 59% and 57%, respectively). We propose that the enhanced chelating ability of 1 may be due to its hexadentate binding compared to the tetradentate nature of 2-4. The UV-visible spectrum of Cu-1 ( Figure S13) exhibits a maximum absorbance of 646 nm in the visible region, similar to the value of 648 nm reported for a six-coordinate Cu II cyclen complex bearing a picolinate pendant group [33]. Similarly, the complexes bearing the tetradentate ligands 2-4 show absorption bands in the range 603-621 nm ( Figure S13), indicative of five-coordinate Cu II species [33].
To confirm that the Cu 2+ ions responsible for Aβ40 aggregation were subsequently chelated upon introduction of the cyclen compounds 1-4, and that unbound Aβ40 was returned to solution, aliquots of the supernatant from centrifuged Aβ40/Cu 2+ /cyclen mixtures were examined using ESI-MS ( The antioxidant activities of 1-4 were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay [34] using ascorbic acid as a reference material. DPPH is a stable free radical that can be scavenged by efficient antioxidants. Thus, the decrease in optical absorbance (at a wavelength corresponding to that of DPPH absorbance) upon introduction of an antioxidant provides a measure of the free radical scavenging activity.   Table 3. All of the tested compounds exhibit activities lower than that of ascorbic acid.  Pharmacokinetic properties of 1-4 were calculated and the data are collected in Table S10.
Compounds 1-3 have predicted physicochemical properties that would provide adequate blood-brain barrier (BBB) permeability, while 4 is predicted not to penetrate the BBB to any significant degree. Each compound is predicted to be a non-inhibitor for CYP2D6 and wellmetabolized in the first phase metabolism. All of the compounds have polar surface area (PSA) < 140 Å 2 and AlogP98 (logarithm of the partition coefficient between n-octanol and water at the 98% confidence level) <5 with the exception of 4. A plot of these two parameters ( Figure S12) shows that 1, 2 and 3 are highly likely to have adequate intestinal absorption and BBB permeability while 4 is less likely to possess to do so.

Conclusions
Compounds based on the cyclen macrocycle that bear pendant groups have been synthesized and characterized. Each of the compounds 1-4 has been shown to coordinate Cu 2+ , Zn 2+ and Ni 2+ metal ions in aqueous solution. X-ray crystal structures revealed that 1 binds in a hexadentate fashion while 2 and 3 are tetradentate. Importantly, each of the compounds can remove Cu 2+ ions from A β40 to inhibit aggregation. Of the four chelating compounds, 1 was superior in returning aggregated Aβ40 to solution compared to 2-4. In terms of anti-oxidant properties, 1 showed the greatest free radical scavenging activity although none were greater than Vitamin C.

Experimental Section
General: Reagents and analytical grade solvents were purchased from commercial sources. The Aβ40 peptide (purity >95%) was purchased from GL Biochem Ltd (Shanghai).
Anhydrous dichloromethane, triethylamine and acetonitrile were prepared by refluxing over calcium hydride for several hours. Anhydrous toluene was prepared by standing over sodium wire for 2 days. 1,4,7,10-tetrazacyclododecane was synthesized according to the procedure of Reed and Weisman [35]. Pierce BCA protein assay kit was obtained from Thermo Scientific.  [36] were applied and the data were corrected for Lorentz and polarisation effects using Bruker APEX2 software [37]. The structure was solved by Direct methods and the full-matrix least-square refinement was carried out using Shelxl [38] in Olex2 [39]. The non-hydrogen atoms were refined anisotropically. The molecular graphic was generated using program Olex2 [39].
Pharmacokinetics parameters were calculated using Discovery Studio 4.5 (Accelrys, San Diego, CA, USA). Aqueous sodium carbonate (2 M) was added until pH 7. The mixture was extracted with dichloromethane (3 x 50 mL) and the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated using a rotary evaporator. The crude product was purified by silica gel column chromatography eluting with hexane: ethyl acetate (1:1) to give pure 2- where Absblank is the absorption of pure methanol. The IC50 ascorbic acid (vitamin C) and

Synthesis
1-4 is the samples concentration at which 50% of the DPPH free radical was scavenged.

Assays to assess interactions with Aβ40
Turbidity assay: This assay was used to assess the Cu 2+ -induced aggregation and was performed according a modified published method [30]. Briefly, aqueous copper(II) sulfate The colour change monitored by UV-visible absorbance using a microplate reader at 540 nm.
Mass spectrometric analysis of chelated species: Samples prepared using the procedure outlined in 4.4.1 (before the addition of 1-4) were centrifuged (12000 rpm) for 30 minutes to precipitate any undissolved material. The supernatant was decanted, the pellet was washed with water, centrifuged a second time (12000 rpm) for 30 minutes, and the supernatant decanted. Aqueous solutions of 1-4 (5 μL, 4mM) were added followed by incubation at 37 ºC for 4 hours. The pH was adjusted at 7.4 (aq. NaOH (0.5M) or aq. HCl (0.5 M)) and the mixture was centrifuged (12000 rpm) for 30 minutes. Aliquots of the supernatant were examined using ESI-MS.