Synthesis and in vitro α-glucosidase and cholinesterases inhibitory actions of water-soluble metallophthalocyanines bearing ({6-[3-(diethylamino)phenoxy]hexyl}oxy groups

In this paper, we have prepared peripherally tetra-({6-[3-(diethylamino)phenoxy]hexyl}oxy substituted cobalt(II), copper(II), manganese(III) phthalocyanines (3, 4, 5) and their water-soluble derivatives (3a, 4a, 5a). Then, in vitro α-glucosidase and cholinesterases inhibitory actions of the water-soluble 3a, 4a, 5a were examined using spectrophotometric methods. 4a had the highest inhibitory effects among the tested compounds against α-glucosidase due to IC50 values. 4a and 5a had 40 fold higher inhibitory effects than the positive control. For cholinesterases, the compounds showed significant inhibitory actions that of galantamine which was used as a positive control. According to the SI value, 3a inhibited acetylcholinesterase enzyme selectively. In kinetic studies, 4a was a mixed inhibitor for α-glucosidase, 3a was a competitive inhibitor for AChE, and 4a was a mixed inhibitor for BuChE. The therapeutic potential of these compounds has been demonstrated by in vitro studies, but these data should be supported by further studies.


Experimental
The used materials, equipment, and in vitro inhibition assay on α-glucosidase, AChE, and BuChE are given in supplementary information.

Inhibition study of α-glucosidase
In this study, in vitro anti-α-glucosidase effects the compounds 3a, 4a, 5a were investigated by spectrophotometric methods.Acarbose was used as a positive control.The results were tabulated as IC 50 values in Table 1.All of the compounds showed higher inhibitory effects on α-glucosidase than that of acarbose (IC 50 = 60.28 ± 3.42 µM).4a has the best inhibitory actions with 1.36 ± 0.01 µM of IC 50 value among the tested compounds.4a and 5a had an inhibitory activity about 40 times higher inhibitory activity than that of acarbose.According to the literature, Güzel et al. investigated inhibitory effects of peripheral furan-2-ylmethoxy-substituted copper and manganese phthalocyanines on α-glucosidase [26].The IC 50 values of these compounds were determined as 911.20 µM and 695.37 µM, respectively.The peripherally tetra-({6-[3-(diethylamino)phenoxy]hexyl}oxy substituted cobalt(II), copper(II), manganese(III) phthalocyanines displayed a higher inhibitory effect on α-glucosidase than that of furan-2-ylmethoxy-substituted compounds on α-glucosidase, according to the IC 50 values.
In this work, Lineweaver-Burk and Dixon plots were investigated to evaluate the inhibitory type and inhibition constant (K i ) for 4a which had the best inhibitory actions on α-glucosidase.The results are given in Table 2 and Figure 4. On enhancing substrate and inhibitors concentrations against α-glucosidase, V max (maximum rate) value diminished and K m value increased.This result claimed that the compound inhibited the enzyme via mixed inhibition.

Inhibition studies of AChE and BuChE
The in vitro antiChEs actions of all synthesized compounds were investigated to determine their therapeutic potential in AD.Galantamine was used as a positive control.The results are shown in Table 3.The results showed that the compounds have higher inhibition efficiency on AChE and BuChE when compared to galantamine (IC 50 = 30.20 ± 0.58 µM for AChE and 52.04 ± 0.55 µM for BuChE).The IC 50 values of 3a, 4a, 5a were 0.65 ± 0.01 µM, 1.08 ± 0.03 µM, and 1.35 ± 0.01 µM,      [34].The results showed that copper, zinc, cadmium, and mercury inhibited on AChE [34].
In literature, Arslan reported novel peripherally tetra-chalcone substituted metal-free, manganese, cobalt and copper phthalocyanines and their inhibitory effects against AChE [34].These compounds had lower inhibitory effects than neostigmine (IC 50 = 0.136 ± 0.011 µM) which was used as a positive control but 3a showed higher inhibitory actions about 46 times than galantamine on AChE [35].In our previous study, the ChEs inhibitory effects of peripheral or nonperipheral tetra-[4-(9H-carbazol-9-yl)phenoxy] substituted cobalt, manganese phthalocyanines were investigated and IC 50 values of these compounds were determined ranging from 7.39 ± 0.25 µM to 58.02 ± 4.94 µM [36].The compounds used in this study were found to be more effective when compared to our previous study according to the IC 50 values [36].
In kinetic analysis, 3a showed competitive inhibition with V max unchanged and K m increased on AChE, according to the Lineweaver-Burk plot (Figure 5a).On the other hand, 3a was a mixed inhibitor due to the change of V max and K m values toward BuChE (Figure 6a).K i values of the compounds were determined as 0.51 ± 0.04 µM for 3a and 0.05 ± 0.01 µM for 4a, respectively (Figures 5b and 6b).

Conclusion
In this work, we have synthesized and characterized peripherally tetra-({6-[3-(diethylamino)phenoxy]hexyl}oxy) substituted metallophthalocyanines (3,4,5) and their water-soluble derivatives (3a, 4a, 5a).Also, we have reported α-glucosidase and ChEs inhibitory actions of 3a, 4a, 5a using spectrophotometric methods.All compounds had significant inhibitory properties on α-glucosidase and ChEs.According to the IC 50 values, 4a had the highest inhibitory effects among the tested compounds against α-glucosidase.4a and 5a showed 40 fold higher inhibitory effects than acarbose.In ChEs studies, the compounds had significant inhibitory actions when compared to galantamine (p < 0.0001).3a inhibited the AChE enzyme selectively, according to the SI value.In kinetic studies, 4a was a mixed inhibitor for α-glucosidase, 3a was a competitive inhibitor for AChE, and 4a was a mixed inhibitor for BuChE.Although it has been determined that these compounds have potential against DM and AD treatments in vitro, these data should be supported by further studies.

Acknowledgment
This study was not supported by any organization.

Table 1 .
IC 50 values of the compounds on α-glucosidase.

Table 2 .
Kinetic parameters of the compounds on AChE, BuChE, and α-glucosidase.

Table 3 .
IC 50 and SI values of the compounds on AChE, BuChE.In addition, the compounds displayed significant BuChE inhibitory properties that of galantamine (p < 0.0001).4a had the best BuChE inhibitory actions with 0.29 ± 0.01 µM of IC 50 value following 3a with 3.06 ± 0.02 µM of IC 50 value.According to the SI (selective index (IC 50 = BuChE/IC 50 = AChE)) values, 3a inhibited AChE selectively (SI value = 4.70).The difference in the results is thought to be due to the metal effect.Frasco et al. reported the metals inhibitory effects of different metals (copper, nickel, zinc, cadmium, and mercury) on AChE