Design, synthesis and evaluation of 3,4-dihydroxybenzoic acid derivatives as antioxidants, bio-metal chelating agents and acetylcholinesterase inhibitors

Abstract

Metal ions, especially copper, zinc and iron, play an important role in the neurodegeneration process because they can affect protein misfolding, leading to the formation of the amyloid deposits and oxidative stress leading to reactive oxygen species (ROS). Here we report the synthesis and evaluation as antioxidant and metal chelating agents of 3,4-dihydroxybenzoic acid derivatives. Synthesized compounds were tested by the 2,2-diphenyl-2-picrylhydrazyl (DPPH) method showing a radical scavenging ability (EC₅₀ = 0.093–0.118 μM) higher than Trolox used as reference. Furthermore, these compounds were able to bind both iron and copper, especially the iron (III), by the formation of hexa-coordinated complexes. Synthesized compounds were tested to evaluate their ability to inhibit acetyl- and butyryl-cholinesterase; the obtained results have demonstrated that they are selective inhibitors of AChE (K_i = 1.5–18.9 μM) and result weakly active versus butyrylcholinesterase (BChE).

• Antioxidant activity
• bio-metal chelation
• cholinesterase inhibitors
• DPPH method
• Job’s method

Introduction

The events leading to various neurodegenerative diseases, e.g. Alzheimer’s (AD) or Parkinson’s (PD) disease, are still unknown. However, metal ions have long been suspected to play a key role in the pathogenesis of neurodegenerative disorders. The relationship between the development of diseases and metals is very complex, but it has become clear that alterations in metal homeostasis and their interactions with biomolecules can drive the progression of neurological diseases1.

In the central nervous system (CNS), iron, copper and zinc are required for many enzymatic activities, mitochondrial function, neurotransmission, learning and memory. The concentration of these vital metals needs to be finely tuned and any dysregulation may lead to severe disease as a consequence of cell death in specific brain areas2.

High levels of Cu and Zn ions are found in amyloid plaques, commonly seen in AD brains, and in the cerebrospinal fluid of patients affected by AD or PD. High Fe ions levels are also frequently observed in PD and AD. Several transition metal ions also bind proteins associated with neurodegenerative diseases, such as β-amyloid (Aβ) and α-synuclein, and affect their aggregation process2.

Moreover, it is well known that iron and copper play a well-established role as catalysts for the production of reactive oxygen species (ROS) via the Fenton reactions, so, they are a major cause of the oxidative stress3. Thus, the modulation of bio-metals in the brain has been proposed as a potential therapeutic strategy for the treatment of neurodegenerative diseases, especially for AD4^,5.

In view of the multifactorial nature of AD, more efforts are directed to develop new drugs that could be beneficial to the development and progression of symptoms by acting at different levels on the involved mechanisms. Recently, many published papers report the synthesis and the study of cholinesterase inhibitors that are at the same time anti-oxidants and / or metal chelating and / or inhibitors of amyloid aggregation6–8, and the great amount of work on this topic is widely reported in numerous reviews9–11.

Protocatechuic acid (3,4-dihydroxybenzoic acid) possesses well known antioxidant and metal chelating properties12–14 and it appears to be an interesting starting molecule which can be structurally modified by addition of further functional groups that can provide additional biological activity. In particular, keeping in mind the cholinergic hypothesis of AD15 and the key role played by metal cations and ROS in the neurodegeneration correlated to AD, we designed derivatives 8–11, joining antioxidant and chelating properties of 3,4-dihydroxybenzoic acid with anticholinesterase activity due to the presence of a carbamic moiety typical of many acetylcholinesterase (AChE) inhibitors16.

Methods

Chemical synthesis

All reagents, solvents and deuterated were purchased from Sigma-Aldrich (Milano, Italia). Benzene was dried just before use by mean of Dean Stark apparatus. Acetonitrile was freshly purified by distillation over calcium hydride.

IR spectra were recorded on a FT-IR Perkin-Elmer SpectrumOne equipped with ATR system; signals are given as ν (cm⁻¹). ¹H and 13C NMR spectra were recorded on AVANCE400 Bruker spectrometer operating at 400 and 100 MHz, respectively; chemical shifts are given in ppm (δ) with respect to TMS as internal reference, coupling constant are given in Hz. Melting points were determined on Tottoli apparatus (Buchi) and are uncorrected. Elemental analyses were obtained by a PE 2400 (PerkinElmer, Waltham, MA) analyzer.

Synthesis of carbamates 4--7

4-Aminobutyl-diethylcarbamate (4)

4-Aminobutan-1-ol (1.0 g, 11.2 mmol) was added with 1.5 mL of aqueous 6 M HCl and 30 mL of benzene and was refluxed for 3 h in a Dean-Stark apparatus. After this time the benzene solution was cooled at room temperature (RT) and then the solvent was removed under reduced pressure to give a crude residue (4-aminobutan-1-ol hydrochloride) that was used without further purification. To the obtained residue 15.0 mL of anhydrous CH₃CN and 4.6 g of diethylcarbamoyl chloride (33.7 mmol) were added and the suspension was refluxed until complete dissolution (approximately 12 h).

After this time, the cooled solution was added with 6.0 mL of water and 0.1 M HCl until pH 4, stirred for 15 min, added with 0.1 M Na₂CO₃ until pH 8 and extracted with chloroform (5 × 15.0 mL). Combined organic layers were dried over Na₂SO₄, then the solvent was removed under reduced pressure to give rise to 1.55 g of a colorless oily product (73.5% yield). I.R. (neat, ν, cm⁻¹): 2934, 1685, 1273, 1172, 1071, 1004, 748. ¹H NMR (CDCl₃): δ ppm 3.97 (t, 2 H, J = 6.5 Hz, -CH₂-O), 3.19–3.11 (m, 4 H, CH₃-CH₂-N), 2.65 (t, 2 H, J = 7.1 Hz, -CH₂-CH₂-N), 1.58–1.47 (m, 4 H, J = 7.3 Hz, -CH₂-CH₂-), 1.09–0.98 (m, 6 H, CH₃). 13C NMR (CDCl₃): δ ppm 155.9, 64.5, 41.5, 41.2, 40.6, 27.9, 26.4, 13.9, 13.4. Elemental analysis: Calcd for C₉H₂₀N₂O₂ C 57.42%, H 10.71%, N 14.88%; Found C 57.21% H 10.74%, N 14.82%.

5-aminopentyl-dimethylcarbamate (5)

Compound 5 was prepared with the same procedure described for 4 using 5-aminopentan-1-ol and dimethylcarbamoyl chloride. Pure 5 was obtained as a pale yellow waxy solid (yield 70%). I.R. (neat, ν, cm⁻¹): 2933, 1688, 1186, 1059, 954, 770. ¹H NMR (CDCl₃): δ ppm 4.00 (t, 4 H, J = 6.5 Hz,-CH₂-O), 2.82 (s, 6 H, -CH₃), 2.57 (t, 2 H, J = 7.1 Hz, -CH₂-N), 1.56–1.60 (m, 2 H, -CH₂-CH₂-O), 1.36–1.41 (m, 4 H, -CH₂-CH₂-). 13C NMR (CDCl₃): δ ppm 156.8, 65.2, 41.9, 36.3, 35.8, 32.9, 28.9, 23.3. Elemental analysis: Calcd for C₈H₁₈N₂O₂ C 55.15%, H 10.41%, N 16.08%; Found C 54.99%, H 10.45%, N 16.03%.

5-aminopentyl-diethylcarbamate (6)

Compound 6 was prepared with the same procedure described for 4 using 5-aminopentan-1-ol and diethylcarbamoyl chloride. Pure 6 was obtained as a pale yellow viscous oil (yield 60%). I.R. (neat, ν, cm⁻¹): 2935, 1676, 1275, 1174, 1072, 770. ¹H NMR (CDCl₃): δ ppm 3.95 (t, 2 H, J = 6.6 Hz, -CH₂-O), 3.15 (s, 4 H, CH₃-CH₂-N), 2.75 (t, 2 H, J = 7.4 Hz, -CH₂-NH₂), 1.55–1.59 (m, 4 H, -CH₂-CH₂-O +-CH₂-CH₂-NH₂), 1.31–1.38 (m, 2 H, -CH₂-CH₂-CH₂-), 1.00 (t, 6 H, J = 7.1 Hz, CH₃). 13C NMR (CDCl₃): δ ppm 155.8, 64.4, 41.3, 40.1, 29.0, 28.4, 22.8, 13.4. Elemental analysis: Calcd for C₁₀H₂₂N₂O₂ C 59.37%, H 10.96%, N 13.85%; Found C 59.15%, H 10.99%, N 13.81%.

6-aminohexyl-diethylcarbamate (7)

Compound 7 was prepared with the same procedure described for 4 using 6-aminopentan-1-ol and diethylcarbamoyl chloride. Pure 7 was obtained as a pale yellow viscous oil (yield 86%). I.R. (neat, ν, cm⁻¹): 2934, 1690, 1273, 1172, 1071, 1004, 748. ¹H NMR (dimethyl sulfoxide (DMSO) + 1% C₆D₆): δ ppm 4.00 (t, 2 H, J = 6.5 Hz, -CH₂-O), 3.23 (q, 4 H, J = 7.1 Hz, CH₃-CH₂-N), 2.62 (t, 2 H, J = 7.0 Hz, -CH₂-NH₂), 1.53–1.60 (m, 2 H, -CH₂-CH₂-O), 1.41–1.47 (m, 2 H, -CH₂-CH₂-NH₂), 1.30–1.36 (m, 4 H, C-CH₂-CH₂-C), 1.07 (t, 6 H, J = 7.1 Hz, CH₃). 13C NMR (CD₃OD): δ ppm 156.0, 64.8, 41.5, 41.2, 40.9, 30.8, 28.9, 26.3, 25.6, 13.9. Elemental analysis: Calc for C₁₁H₂₄N₂O₂ C 61.07%, H 11.18%, N 12.95%; Found C 60.88%, H 11.19%, N 12.90%.

Synthesis of carbamates 8--11

4-[(3,4-Dihydroxybenzoyl)amino]butyl-diethylcarbamate (8)

3,4-Dihydroxybenzoic acid (0.2 g, 1.29 mmol) 15.0 mL of anhydrous CH₃CN was heated at 40 °C until complete dissolution. N,N'-Bis(4-methylphenyl)carbodiimide (0.29 g, 1.30 mmol) was added and the suspension was stirred 24 h at 40 °C. After this time, 5.0 mL of anhydrous CH₃CN, containing 0.25 g (1.33 mmol) of carbamate 4, were added and the suspension was stirred for additional 24 h at 40 °C.

The suspension was cooled, the white precipitate was collected by centrifugation and washed with cold MeOH (4 × 1.5 mL); the obtained white precipitate was discarded and the combined methanolic solutions were evaporated under reduced pressure. The obtained crude residue was further washed with cold methanol (4 × 1.0 mL), each time discarding the precipitate, and the combined methanolic solutions was then evaporated under reduced pressure to provide a white residue that was crystallized from 2-propanol to give pure 8 as a pale yellow hygroscopic solid (yield 48%). Melting point (mp) 56–60 °C. I.R. (neat, ν, cm⁻¹): 2971, 1666, 1600, 1276, 1174, 1007, 947, 770. ¹H NMR (CD₃OD): δ ppm 7.48 (d, 1 H, J = 2.0 Hz, Ar), 7.41 (dd, 1 H, J = 2.0 and 8.2 Hz, Ar), 6.78 (d, 1 H, J = 8.2 Hz, Ar), 4.09–4.14 (m, 2 H, -CH₂-O), 3.29 (q, 4 H, J = 7.1 Hz, CH₃-CH₂-N), 2.90–2.96 (m, 2 H, -CH₂-NH-), 1.69–1.75 (m, 4 H, -CH₂-CH₂-), 1.13 (t, 6 H, J = 7.1 Hz, CH₃-CH₂-). 13C NMR (CD₃OD): δ ppm 174.2, 156.3, 147.8, 144.1, 128.8, 121.6, 116.4, 113.9, 64.3, 41.6, 41.2, 39.1, 25.8; 24.3, 12.9, 12.4. Elemental analysis: Calcd for C₁₆H₂₄N₂O₅ C 59.24%, H 7.46%, N 8.64%; Found C 59.01%, H 7.49%, N 8.60%.

5-[(3,4-Dihydroxybenzoyl)amino]pentyl dimethylcarbamate (9)

Compound 9 was prepared with the same procedure described for 8; carbamate 5 was used as reactant and the reaction mixture was heated 72 h at 40°C. Pure 9 was obtained as a light yellow hygroscopic solid (yield 60%). I.R. (neat, ν, cm⁻¹): 2936, 1672, 1600, 946, 893, 769. ¹H NMR (CD₃OD): δ ppm 7.70 (s, 1 H, Ar), 7.39 (d, 1 H, J = 8.2 Hz, Ar), 6.79 (d, 1 H, J = 8.2 Hz, Ar), 4.03 (t, 2 H, J = 6.4 Hz, -CH₂-O), 2.92 (t, 2 H, J = 7.6 Hz, -CH₂-NH-), 2.88 (s, 6 H, CH₃), 1.60–1.67 (m, 4 H, -CH₂-CH₂-), 1.38–1.44 (m, 2 H, -CH₂-CH₂-CH₂-). 13C NMR (CD₃OD): δ ppm 176.1, 159.3, 146.4, 123.8, 118.7, 116.3, 67.1, 41.4, 37.0, 30.4, 29.1, 24.8. Elemental analysis: Calcd for C₁₅H₂₂N₂O₅ C 58.05%, H 7.15%, N 9.03%; Found C 57.81%, H 7.19%, N 9.01%.

5-[(3,4-Dihydroxybenzoyl)amino]pentyl diethylcarbamate (10)

Compound 10 was prepared with the same procedure described for 8; carbamate 6 was used as reactant and the reaction mixture was heated 72 h at 40°C. Pure 10 was obtained as a white hygroscopic solid (yield 45%). I.R. (neat, ν, cm⁻¹): 2937, 1666, 1640, 1280, 1181, 1093, 771. ¹H NMR (DMSO): δ ppm 7.36 (d, 1 H, J = 2.0 Hz, Ar), 7.29 (dd, 1 H, J = 8.2 and 2.0 Hz, Ar), 6.77 (d, 1 H, J = 8.2 Hz, Ar), 4.00 (t, 2 H, J = 6.6 Hz, -CH₂-O), 3.23 (q, 4 H, J = 7.1 Hz, CH₃-CH₂-N), 2.76 (t, 2 H, J = 7.2 Hz, -CH₂-NH-), 1.53–1.61 (m, 4 H, -CH₂-CH₂-O +-CH₂-CH₂-NH-), 1.32–1.40 (m, 2 H, CH₂-CH₂-CH₂), 1.08 (t, 6 H, J = 7.1 Hz, CH₃-CH₂-). 13C NMR (CD₃OD): δ ppm 169.4, 154.9, 147.7, 127.4, 114.9, 112.7, 63.0, 39.8, 37.8, 26.8, 25.4, 21.1, 11.0. Elemental analysis: Calcd for C₁₇H₂₆N₂O₅ C 60.34%, H 7.74%, N 8.28%; Found C 60.13%, H 7.76%, N 8.26%.

5-[(3,4-Dihydroxybenzoyl)amino]hexyl diethylcarbamate (11)

Compound 11 was prepared with the same procedure described for 8; carbamate 7 was used as reactant and the reaction mixture was heated 24 h at 40 °C. Pure 11 was obtained as a white solid (yield 62%). mp 155–159 °C. I.R. (neat, ν, cm⁻¹): 3038, 2936, 1665, 1603 1278, 1172, 1071, 1004, 748. ¹H NMR (CD₃OD): δ ppm 7.46 (d, 1 H, J = 2.1 Hz, Ar), 7.39 (dd, 1 H, J = 8.2 and 2.1 Hz, Ar), 6.78 (d, 1 H, J = 8.2 Hz, Ar), 4.10 (t, 2 H, J = 7.2 Hz, -CH₂-O), 3.30 (q, 4 H, J = 7.5 Hz, CH₃-CH₂-N), 2.93 (t, 2 H, J = 6.6 Hz, -CH₂-NH-), 1.65–1.77 (m, 4 H, -CH₂-CH₂-O +-CH₂-CH₂-NH-), 1.40–1.51 (m, 4 H, CH₂-CH₂-CH₂-CH₂), 1.13 (t, 6 H, J = 7.5 Hz, CH₃-CH₂-N). 13C-NMR (CD₃OD): δ ppm 173.5, 156.5, 147.9, 144.1, 128.1, 121.7, 116.4, 113.9, 64.8, 41.3, 39.3, 28.5, 27.2, 25.6, 25.1, 12.7. Elemental analysis: Calcd for C₁₈H₂₈N₂O₅ C 61.34%, H 8.01%, N 7.95%; Found C 61.13%, H 8.03%, N 7.93%.

Antioxidant activity tests

The antioxidant activity was evaluated using the spectrophotometric DPPH method according to Brand-Williams et al.^.based on the ability of antioxidant compounds to react with the stable radical 2,2-diphenyl-2-picrylhydrazyl (DPPH^·) characterized by an absorption maxima at 490 nm. An UV-Vis lambda 40 Perkin Elmer spectrophotometer, optical polystyrene cuvettes and methanol as solvent were used; each measure was repeated at least in triplicate.

A calibration equation was obtained (r²= 0.996) by measuring the absorbance of DPPH solutions at concentrations ranging between 25 and 150 µM. To assess the antioxidant activity of compounds (8–11) 3.0 mL of 100 µM DPPH were placed in a cuvette and the absorbance at 490 nm was read in order to determine the initial concentration of DPPH radical; therefore, the opportune amount of methanolic solution of each carbamate was added to obtain cuvette concentrations ranging from 5 to 30 µM. For each cuvette the absorbance was read each 20 min until a plateau was reached. For each carbamate concentration, the residual plateau percentage of DPPH radical was calculated from residual absorbance using the calibration equation and the obtained values were plotted versus the molar ratio [carbamate]/[DPPH] to graphically extrapolate the EC₅₀ values (efficient concentration = EC₅₀ expressed as the moles of antioxidant per moles of DPPH needed to reduce to 50% the initial DPPH).

Complexation studies

The ability of carbamates 8–11 to complex the metal ions Fe⁺³, Fe⁺², Cu⁺² and Zn⁺² was achieved using the continuous variation method of Job, based on the UV-Vis absorption maxima intensity changes due to the complex formation. This method requires to mix, in adequate proportions, equimolar solutions of metal ion and ligand, recording the UV spectra and plotting the difference in the maximum absorbance versus the molar ratio (X) of the ligand; in this way the stoichiometric coefficient of the complex can be obtained.

FeCl₃·6H₂O, FeSO₄·7H₂O, CuSO₄·6H₂O and ZnSO₄·7H₂O were purchased from CARLO ERBA REAGENT (Milano, Italia); UV-Vis lambda 40 Perkin Elmer spectrophotometer and quartz HELLMA 10 mm cuvette were used. Each measure was triplicate and data were elaborated using UV-WIN Lab Version 2.0, Perkin Elmer Corporation and Microsoft Excel 2010.

The same procedure was followed for all carbamates towards each metal cations. Briefly, 4 × 10⁻⁴ M solutions of each carbamate in 1:1 MeOH/water and inorganic salts 4 × 10⁻⁴ M in water were prepared. The solutions of each carbamate and each metal ion were mixed in order to obtain an increasing molar ration of carbamate (from 0.1 to 0.95) and maintaining unchanged the total concentration of carbamate + metal ion (4 × 10⁻⁴ M). Each solution was placed in cuvette and the UV-Vis spectra were recorded between 200 and 400 nm using an equimolar carbamate solution as blank; from these spectra was algebraically subtracted that of an equimolar solution of the same cation in order to obtain the exclusive spectral changes due to the complex formation.

The molar ratio (X) that causes the maximum ΔA can be obtained plotting the differences in absorbance measured at two wavelength (usually absorption maxima) versus molar ratio of ligand. The stoichiometry of the complex can be easily calculated with the following equation: where n is the number of ligand molecules per cation present in the complex.

Enzymatic inhibition studies

Electric eel AChE (EC 3.1.1.7), equine BChE (EC 3.1.1.8), acetylthiocholine iodide, butyrylthiocholine iodide and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (Milano, Italy). All other chemical and biological reagents and solvents used were of the highest analytical, commercially available grade. Enzymatic inhibition of carbamates 8–11 were assessed by means of Ellman method, as previously described25, using a double beam UV-Vis lambda 40 Perkin Elmer spectrophotometer and optical polystyrene cuvettes (10 × 10 × 45 mm, 340–800 nm optical transparency); each measure was repeated at least in triplicate. AChE and BChE were daily tested to evaluate the effective enzymatic activity.

Enzymatic inhibition tests were carried out by mixing in a polystyrene cuvette 3.0 mL of 0.1 M (pH 7.4) phosphate buffer containing DTNB (0.25 mM) and AChE (0.028 U mL⁻¹) or BChE (0.08 U mL⁻¹) with each carbamate derivatives 8–11 (final concentration range 1.0–10.0 µM). The reaction was started by the addition of acetylthiocholine iodide or butyrylthiocholine iodide (200–600 μM) and the changes in the absorbance at 412 nm were recorded at 25 °C between 0.5 and 1.5 min after reagent addition. The data were analyzed with the enzyme kinetic module of SigmaPlot, version 8.02 a, in order to find the best fitting model of inhibition, as indicated by calculated linear regression coefficient r². K_i values were obtained according to Dixon’s method, reporting the reciprocal of the hydrolysis rate versus the carbamate concentrations.

Molecular docking studies

Molecular docking study was performed in order to suggest a possible binding mode of all synthesized compounds with AChE and BChE.

The crystal complex (code: 1EVE, resolution of 2.50 Å) of Torpedo californica AChE (TcAChE) with donepezil and the crystal (code: 1P0I, resolution of 2.00 Å) of hBChE were downloaded from PDB (http://www.rcsb.org).

Prior to performing molecular docking, the protein PDB file was prepared using the Dock Prep tool available in the free software package UCSF Chimera 1.7. This involved the addition of hydrogen atoms, removal of solvent (water) molecules and assigning partial charges (using the AMBER99 force field).

Docking calculations were carried out using SwissDock, an online server that docks ligand to protein using EADock DSS software. Docking runs were performed using the “Accurate” parameters option, which is the most exhaustive in terms of the number of binding modes sampled.

Output clusters were obtained after each docking run and were ranked according to the FullFitness (FF) scoring function specified by the SwissDock algorithm (cluster 0 being the cluster with the best FF score). A greater negative FF score indicates a more favorable binding mode with a better fit. Within each cluster, the individual binding poses were further arranged and ranked based on their FF score.

The EADock DSS software was able to identify the native binding mode of donepezil as the best FF score binding conformation.

Results and discussion

Chemistry

The general synthesis of 8–11 is illustrated in Scheme 1. The amino-alcohols 1–3 were treated with aqueous 6 M HCl and benzene and dried by Dean-Stark distillation; the corresponding hydrochlorides formed were subsequently suspended in dry acetonitrile and treated with dimethylcarbamic or diethylcarbamic chloride to furnish the aminocarbamates 4–7. Afterwards, intermediates 4–7 were added to the 3,4-dihydroxybenzoic acid, pre-activated by 12 h treatment with N,N′-bis(4-methylphenyl)carbodiimide in dry acetonitrile at 40 °C; the suspension was stirred at 40 °C for additional 24–72 h. The solvent was then removed and the residue was extracted with 3–4 small portions of cold methanol to furnish pure carbamates 8–11 as solids. The IR, ¹H and 13C NMR spectroscopic data of compounds 4–11 are in agreement with proposed structures.

Scheme 1. Reagents and conditions: (a): 6 M HCl, benzene, Dean-Stark distillation; (b): R₂NCOCl, CH₃CN, reflux, 12 h; (c): N,N′-bis(4-methylphenyl)carbodiimide, CH₃CN, 40 °C, 12 h; (d): 4–7, CH₃CN, 40 °C, 24–72 h.

The antioxidant activity of carbamates 8–11 was evaluated using the DPPH method that allows to assess the ability of the compounds to scavenge the stable DPPH radical17; this capability is expressed as EC₅₀ (moles of antioxidant per mole of DPPH needed to reduce to 50% the initial DPPH); lower EC₅₀ values indicate better antioxidant properties. The obtained data are summarized in Table 1.

Table 1. Antioxidant EC₅₀ values of compounds 8--11 obtained with DPPH method.

Compound	EC50^a
8	0.093
9	0.110
10	0.112
11	0.118
Trolox	0.150

^aEC₅₀: efficient concentration, moles of antioxidant per mole of DPPH needed to reduce to 50% the initial DPPH.

The obtained EC₅₀ values range from 0.093 for compound 8 to 0.118 for compound 11 and are lower than the hydrophylic tocopherol derivative Trolox (EC₅₀ = 0.150), thus indicating the good radical scavenging and antioxidant properties of catecholic derivatives 8–11.

The chelation abilities of 8–11 towards bio-metal cations such as Fe⁺³, Fe⁺², Cu⁺² and Zn⁺² were investigated by Job’s method18^,19 based on the changes in the UV-Vis absorption spectra due to the complex formation. The results of complexation studies are summarized in Table 2 and indicate that all compounds are able to form metal complex with Fe⁺³, Fe⁺² and Cu⁺² (Figures S1–S12 Supplementary material). The major changes in the UV-Vis spectra of carbamates 8–11 were observed in presence of Fe³⁺, which leads to the highest values. By the Job’s plot (ΔA versus molar ratio of ligand) the stoichiometry of the complex can be derived (Figure 1); in some cases three molecules of catechol derivative are needed per each Fe³⁺ cation, thus indicating that a single molecule of carbamate can form a bidentate coordination with the iron (III). Otherwise, reduced changes were recorded for Fe²⁺ and Cu²⁺ complex and some differences in complex stoichiometry were also observed. Compound 10 produced a noticeable turbidity in presence of Fe⁺³, Fe⁺² and Cu⁺², probably due to the formation of insoluble complexes, that could explain the lower ΔA values observed in the complexation studies of this compound. Negligible or no changes in the absorption spectra were recorded in the UV-Vis spectra of compounds 8–11 in presence of Zn²⁺.

Figure 1. (a) UV-Vis spectral changes obtained for Fe³⁺ in presence of increasing amounts of carbamate 11; (b) Job’s plot of ΔA observed at 269 and 309 nm versus the molar ratio of the carbamate.

Table 2. Complexation of Fe⁺³, Fe⁺², Cu⁺² and Zn⁺²with carbamates 8--11.

	Fe^+3			Fe^+2			Cu^+2			Zn^+2
Compound	ΔAmax^a	X^b	n^c	ΔAmax^a	X^b	n^c	ΔAmax^a	X^b	n^c	ΔAmax^a	X^b	n^c
8	1.48	0.75	3	0.63	0.75	3	0.62	0.7	2	^d
9	1.63	0.75	3	0.88	0.8	4	0.55	0.3	0.5	0.2	0.7	2
10	1.02^e	0.75	3	0.38^e	0.75	3	0.26^e	0.75	3	0.2	0.7	2
11	1.22	0.75	3	0.41	0.75	3	0.61	0.75	3	^d

^aMaximum absorbance changes at absorption maxima.

^bMolar ratio of ligand needed to obtain ΔA_max.

^cLigand stoichiometry – molecules of ligand per metal cation.

^dNo changes observed.

^eTurbidity was observed, probably due to the formation of insoluble complexes.

Ellman spectrophotometric method20 was used to assess the ability of carbamates 8–11 to inhibit AChE and butyrylcholinesterase (BChE).

All synthesized compounds were found to inhibit AChE at low μM concentration (Table 3, Figures S13–S16 Supplementary material), in particular carbamate 9–11 showed mixed inhibition with K_i values, respectively, of 6.6, 18.9 and 1.5 μM, suggesting that these compounds could interact with peripheral anionic site (PAS) and the deepest catalytic site. Differently, the carbamate 8, endowed with an alkyl linker of four methylene units, showed a non-competitive inhibition with a K_i value of 5.8 μM suggesting that it could interact exclusively with PAS. All the studied compounds resulted weak inhibitors of BChE, underlining their high selectivity towards the AChE.

Table 3. Anticholinesterase activity of carbamates 8–11.

	AChE			BChE
Compd	Ki, μM	SD, μM	r2	% inh	Conc, μM
8	5.8^a	0.4	0.945	13	100
9	6.6^b	2.4	0.983	41	100
10	18.9^b	17.0	0.986	0	100
11	1.5^b	1.1	0.958	23	100

^aNon-competitive inhibition.

^bMixed inhibition.

To support the experimental results obtained by enzymatic inhibition, we carried out a molecular docking study using SwissDock freely available on Swiss Institute of Bioinformatics Website21^,22. Three-dimensional crystal structures of Torpedo californica AChE (TcAChE) complexed with donepezil and of human BChE (hBChE) were obtained by RCSB Protein Data Bank (PDB codes: 1EVE for AChE; 1P0I for BChE). The protein preparation and the molecular docking results visualization were carried out using the UCSF Chimera package23.

The complex 1EVE has been chosen for the high degree of homology with Electric eel AChE used in the enzymatic inhibition tests. The results of the TcAChE docking study led to the hypothesis that the carbamates 8–11 could bind the enzyme active site; the binding conformations resulted within the two highest FF score clusters. The binding poses were found very similar for all compounds (Figure 2, Figures S18–S21 Supplementary material): the catechol moieties were found in the enzyme active site, interacting mainly with Trp84 and with Ser200 or Glu199; the 4--6 methylene alkyl chains are close to aromatic aminoacid side chains of the enzyme gorge (Phe330 and 331); finally, the carbamic function was found in proximity of the PAS aminoacids (Trp279, Tyr70 and Tyr121).

Figure 2. Docking conformations of carbamates 8 (yellow), 9 (blue), 10 (red) and 11 (green) in the active site of TcAChE (1EVE). The selected aminoacids are dark cyan colored.

In the case of BChE, the docking study is more difficult, indeed most of the crystal structures available in the PDB were complexes with irreversible inhibitors, such as organophosphorus compounds. Then, we decided to use the crystal 1P0I on the basis of informations from the literature24. All compounds formed π–π interactions with Trp82 and/or hydrogen bond with Glu197. The conformations of 8–11 are slightly bent, not linear, and the fit of these molecules is not satisfying as in the case of AChE, confirming the high selectivity against AChE demonstrated by the enzymatic assay. In particular, all compounds do not interact with the PAS residues (Trp 231, Ala 277, Gln 119, Gln 71 -- Figures S22--S25 Supplementary material).

In conclusion, we have designed and synthesized the carbamates 8–11 possessing good antioxidant and radical scavenging properties, as showed by EC₅₀ values lower than trolox. These compounds are also able to chelate the bio-metal cations Cu (II), Fe (II) and especially Fe (III). Interestingly, 8–11 also resulted highly selective non-competitive or mixed inhibitors of AChE, characterized by low μM K_i values. Based on the molecular docking study, the carbamates 8–11 could inhibit the AChE by interaction with active catalytic site, access gorge and PAS. Moreover, the reduced activity towards BChE could be explained by the weak interactions with PAS aminoacid residues.

For these reasons, carbamates 8–11 can be considered interesting lead compounds in the development of new multifactorial agents potentially useful in the treatment of neurodegenerative disorders that typically possess a complex multifactorial etiology.

Supplementary material available online

Supplementary Figures S1–S25.

Declaration of interest

The authors report no declarations of interest.

This research was financially supported by grants from Sapienza, Università di Roma – Progetti di Ricerca di Ateneo, and by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR).