Copper(II) complexation of tacrine hybrids with potential anti-neurodegenerative roles.

JIB-09738; No of Pages 9 Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio

Copper(II) complexation of tacrine hybrids with potential anti-neurodegenerative roles Catarina Quintanova a, Rangappa S. Keri a,b, Sílvia Chaves a,⁎, M. Amélia Santos a a b

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal Centre for Nano and Material Sciences, Jain University, Jain Global Campus, Bangalore, Karnataka 562112, India

a r t i c l e

i n f o

Article history: Received 2 February 2015 Received in revised form 5 June 2015 Accepted 6 June 2015 Available online xxxx Keywords: Tacrine S-allylcysteine S-propargylcysteine Anti-neurodegeneratives Copper chelation

a b s t r a c t The complexity and multifactorial nature of neurodegenerative diseases turn quite difficult the development of adequate drugs for their treatment. Multi-target analogues, in conjugation with natural moieties, have been developed in order to combine acetylcholinesterase (AChE) inhibition with antioxidant properties, metal-binding capacity and inhibition of amyloid-β (Aβ) aggregation. Due to the recent interest on natural-based drugs and also the importance of studying the role of transition metal ions in the disease process, we herein evaluate the copper chelating capacity and inhibitory ability for self- and Cu-induced Aβ1–42 aggregation of two nature-base hybrid model compounds obtained from conjugation of a tacrine moiety with a S-allylcystein (1) or Spropargylcystein (2) moiety. Both compounds show a moderate chelating power towards Cu(II) (pCu 7.13– 7.51, CL/CCu = 10, CCu = 10−6 M, pH 7.4), with predominant formation of 1:1 complex species (CuL, CuH−1 L) for which the coordination sphere involves the N-amide and the NH2 amine of the cysteine derivative as well as the NH of tacrine. The compounds are able to improve the inhibition of Aβ aggregation in the presence of Cu(II) and this is slightly more relevant for the allyl derivative (1), a stronger copper chelator, than for the propargyl (2). Moreover, the presence of a chloro atom in the tacrine moiety and the size of the chain length between the two NH groups appeared also to improve the inhibition capacity for Aβ aggregation. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative agelinked disease associated to dementia, cognitive deficit and memory loss, which can lead to incapacitation and death [1]. The AD brain shows several typical pathological features, such as accumulation of misfolded amyloid-β (Aβ), metal ion (Cu, Zn, Fe) dyshomeostasis and elevated oxidative stress [2]. Moreover, metal ions, such as Cu and Zn, bind to Aβ peptides enabling their aggregation, while deregulated redox active metal ions, Cu(I/II) and Fe(II/III), promote overproduction of reactive oxygen species (ROS) with consequent disruption of biological molecules such as proteins, DNA and lipids. Due to the multifactorial nature of AD and to the potential interconnection of various factors in its pathogenesis, there is still absence of a drug for AD cure. Tacrine (TAC, see Fig. 1) was the first drug approved by the U.S. FDA for the palliative treatment of AD. It demonstrated improvement on the cholinergic system and a modest reduction in the decline of cognitive performance, due to inhibition of acetylcholine esterase (AChE) [3], but it has some limitations due to hepatotoxicity and it is therefore seldom used nowadays in clinical application. ⁎ Corresponding author. E-mail address: [email protected] (S. Chaves).

To overcome TAC drawbacks, this compound has been used as an inspiration for the development of multi-target analogues [4] in conjugation with natural moieties such as melatonin [5], hydroxyquinoline [6] or thioflavine [7], in order to combine the AChE inhibition with antioxidant properties, metal-binding capacity and/or inhibition of Aβ aggregation. Continuing our research on the development of multifunctional heterocyclic compounds with potential application in AD [7–9], and taking also in account the recent interest on the neuro-protective role of some natural products, hybrid compounds containing TAC and also S-allyl-cysteine (SAC, garlic constituent) or S-propargyl-cysteine (SPRC, attenuator of spatial learning and memory impairment [10]) moieties were designed and developed [11]. Based on the hypothesis that AD brains suffer from metallostasis, it has been world widely accepted that metal chelators can interfere in metal-induced Aβ aggregation and neurotoxicity. This hypothesis has been mostly focused on three transition elements (iron, copper, zinc) [12] and research on the role of these metal ions on neurodegenerative diseases has also been receiving particular attention after failures on large-scale clinical trials based on Aβ-targeting drugs [13]. In fact, there is pathological, biochemical, pharmacological and genetic evidence for the role of metal fatigue in AD pathogenesis. Recently, degenerative disease-related proteins such as amyloid precursor protein (APP) [14], tau [15] and presenilin [16] have been attributed with

http://dx.doi.org/10.1016/j.jinorgbio.2015.06.008 0162-0134/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: C. Quintanova, et al., Copper(II) complexation of tacrine hybrids with potential anti-neurodegenerative roles, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.06.008

2

C. Quintanova et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

Fig. 1. Molecular structure of tacrine (TAC) and tacrine derivatives under study.

important roles in metal homeostasis, their malfunction being associated to changed metal compartmentalization in the brain. As a continuation of our study on a set of tacrine hybrids, respectively TAC–SAC and TAC–SPRC, with previously demonstrated biological activity towards a series of pathological AD pathways (e.g. inhibition of AChE, Aβ aggregation and anti-oxidant activity [11]), we have decided to gain some insight on a further potential metal-related role of these compounds, since they enclose a cysteine derivative in their structure. Therefore, two hybrid compounds, 1 and 2 (see Fig. 1), from each of the two series (TAC–SAC and TAC–SPRC) of tacrine derivatives, were selected and are herein studied in terms of their chelating capacity towards Cu(II), involving different techniques such as UV-Visible spectrophotometry (UV–vis), 1H NMR, pH-potentiometry and electrospray ionization–mass spectrometry (ESI–MS), as well as their inhibitory ability for Cu(II)-induced Aβ1–42 aggregation. The study of this two model compounds will allow one to evaluate the effect of S-allyl, S-propargyl and chloride atom on Cu(II) chelation and Cu(II)-induced Aβ1–42 aggregation. Some studies on the Zn(II)/1 system were also performed, since they can represent a contribution to the understanding of the metal coordination modes, even though some limitations could arise from the fact that the zinc complexes are expected to have lower stability than the copper ones. 2. Experimental section 2.1. Materials and methods The compounds (1,2) were synthesized according to previously reported strategies. The general methodology involves the derivatization of the two main molecular moieties, namely the 9-aminoalkylenetacrine and the S-allyl- or S-propargyl-cysteine by standard nucleophilic substitutions [7], followed by condensation of both units with formation of amide linkages, using T3P as carboxylic activating agent [8]. Specific experimental details are described elsewhere (R.S. Keri, C. Quintanova, S. Chaves, D.F. Silva, S.M. Cardoso, M.A. Santos, manuscript submitted). General analytical grade reagents were purchased from current suppliers and they were used without further purification. Acetonitrile (CH3CN, p.a. (p.a. = pro analysi, reagent grade)) was purchased from Carlo Erba, potassium dihydrogen orthophosphate (KH2PO4, p.a.) from BDH and sodium hydroxide (NaOH, p.a.) from Eka Chemicals. Dimethyl sulfoxide (DMSO, dried, p.a), potassium chloride (KCl, p.a.), sodium carbonate (Na2CO3, p.a.), deuterium chloride (DCl, 99%), potassium deuterium oxide (KOD, 98%), thioflavin T (ThT, p.a.) and glycine (N 98.5%)

were obtained from Sigma-Aldrich. Deuterium oxide (D2O, 99.9%) and deuterated dimethylsulfoxide (d6-DMSO, 99.9%) were acquired from Cambridge Isotope Laboratories Inc. 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP, 99%) was purchased from TCI and Aβ-peptide (1–42) (Aβ1–42) was obtained from Biopeptide Co. Inc. as a lyophilized powder and stored at −20 °C. The aqueous copper (0.015 M) and zinc (0.0156 M) stock solutions were prepared from 1000 ppm standards (Titrisol) and their metal content was evaluated by atomic absorption. The 0.1 M HCl solution used in calibration of the glass electrode was prepared from a Titrisol ampoule. The titrant used in pH–potentiometric and spectrophotometric titrations was prepared from carbonate free commercial concentrate (Titrisol, KOH 0.1 M ampoules). The KOH solution was standardized by titration with a solution of potassium hydrogen phthalate and was discarded whenever the percentage of carbonate, determined by Gran's method [17], was greater than 0.5% of the total amount of base. The electronic spectra were recorded with a Perkin Elmer Lambda 35 spectrophotometer equipped with a temperature programmer PTP 1 + 1 Peltier System, using thermostated (T = 25.0 ± 0.1 °C) 0.4 and 1 cm path length cells. The 1H NMR spectra were recorded on Bruker AVANCE III spectrometers (300 or 400 MHz) and chemical shifts (δ) are reported in ppm from the standard internal reference DSS (sodium 3-trimethylsilyl-d4-propionate). Mass spectra (ESI–MS) were performed on a Bruker HCT quadrupole ion trap equipped with an electrospray ion source. Fluorescence spectra were obtained from a Varian Cary Eclipse spectrophotometer.

2.2. Potentiometric and spectrophotometric studies The metal-complexation studies were performed by pH– potentiometric and UV–vis spectroscopic titration methods, using experimental conditions analogous to those previously reported [18]. Titrations of 1 and 2, alone or in the presence of copper, were accomplished in a 30% w/w DMSO/H2O medium, at T = 25.0 ± 0.1 °C and ionic strength (I) 0.1 M KCl, by using 0.1 M KOH as titrant. The glass and Ag/AgCl reference electrodes were previously conditioned for some days in several DMSO/H2O mixtures of increasing DMSO % composition and the response of the glass electrode was checked by strong acid–strong base (HCl/KOH) calibrations and analysis of the respective Nernst parameters by Gran's method [17]. For the pH–potentiometric and spectrophotometric titrations (total volume 20 mL), the ligand concentrations (CL) were, respectively, 1 × 10−3 M and (5 − 8) × 10−5 M, but keeping the same CCu/CL ratios (0:1, 1:1 and 1:2). The spectrophotometric measurements were carried out in a 250–400 nm wavelength range at pH ca 2–10. Potentiometric titrations were also performed for the systems Zn(II)/1 (1:1 and 1:2 Zn/L stoichiometries) under the same conditions used for copper. The value determined for the water ionization constant (pKw) was 14.4. The stepwise protonation constants of both ligands, Ki = [HiL] / [Hi–1 L][H] (i = 1 − 2), and the overall metal(II)–complex stability constants, βMm Hh Ll = [MmHhLl] / [M]m[H]h[L]l, were calculated by fitting the pH–potentiometric and spectrophotometric data with, respectively, Hyperquad 2008 [19] and PSEQUAD programs [20]. The Cu(II) and Zn(II) hydrolysis models were determined under the defined experimental conditions (I = 0.1 M KCl, 30% w/w DMSO/H2O, T = 25.0 ± 0.1 °C) and the following values of stability constants were included in the fitting of experimental data towards the equilibrium models related to the Cu(II)/L and Zn(II)/L systems: log βCu2 H−2 = -9.94; log βZnH−2 = -14.7, log βZnH−3 = -22.08. The species distribution curves were obtained with the Hyss program [19]. Electronic spectra of the ligands and copper complexes (0:1, 1:1 or 1:2 metal to ligand stoichiometries) in 30% w/w DMSO/H2O medium were recorded in the ranges 250–400 nm (CL = 8 × 10−5 M) and 400–1000 nm (CL = 5 mM) for selected pH values (ca 5.5–9), according to the data provided by previously obtained species distribution diagrams in order to guarantee major formation of complex species.



3

2.3. 1H NMR studies

3. Results and discussion

Proton NMR titrations of solutions of the ligands (CL = 10 mM) alone or in the presence of Cu(II) or Zn(II) (1:1 M/L stoichiometry) in 50% w/w d6-DMSO/D2O (1) or 30% w/w d6-DMSO/D2O (2) were performed using DCl or CO2 free KOD solutions and an Orion Star Thermo Scientific instrument fitted with a combined Mettler Toledo U402-M3S7/200 microelectrode. Although, for solubility reasons, the 1H NMR studies for 1 have been performed in a different medium (50% w/w d6-DMSO/D2O), no problems arose from this fact since the NMR studies were only used to evaluate the protonation sequence of the ligands or to try to identify chemical shift deviations in the M(II)/L vs L systems and no calculations were performed based on this data in order to obtain stability constants. The microelectrode was calibrated with standard buffered aqueous solutions (pH 4 and 7) and pH* corresponds to the reading of the pH meter previously calibrated with aqueous buffers. 1 H–1H COSY spectra in d6-DMSO were also recorded for the systems L, Cu(II)/L 1:1 and Zn(II)/L 1:1 (CL = 10 mM) in the 300 MHz spectrometer.

It has been proven that the use of metal chelators, namely metalprotein attenuating compounds, results in the regulation of metalinduced Aβ aggregation and neurotoxicity. Thus, after assessing the biological properties of a series of tacrine-conjugates [11], we chose two compounds (1 and 2) as models of anti-neurodegenerative drugs and decided to conduct studies in order to evaluate their acid–base behavior, chelating capacity towards Cu(II) and respective coordination modes, as well as to analyze their effect on the Cu(II)-induced Aβ1–42 aggregation.

2.4. ESI–MS spectra The pH of the Cu(II)/L systems in aqueous solutions was previously ascertained to ca 6 (CL / CCu = 1) or ca 7.5–8 (CL / CCu = 2, CL = 6 × 10−4 M,) by using KOH solution, and the respective mass spectra were obtained from a Bruker HCT quadrupole ion trap equipped with an electrospray ion source, operated in the positive ion mode. Mass spectra were acquired using the following typical instrumental parameters: solution flow rate, 2.5 μL/min; ESI needle spray voltage, +4 kV; capillary exit voltage, − 129 V; nebulizer gas pressure, 8 psi; dry gas flow rate, 4 L/min; dry gas temperature, 250 °C; octopole RF amplitude, 187 Vpp. The spectra were recorded in the range 100–2000 Da and typically correspond to the average of 20–35 scans.

3.1. Acid–base properties of the compounds In order to evaluate the copper(II) chelating ability of compounds 1 and 2, their acid–base behavior was firstly studied. The protonation constants of both ligands were determined by pH–potentiometric and UV– vis spectrophotometric techniques (see Fig. 2, in which a represents moles of added base per mole of ligand), both procedures giving mostly similar values for the constants determined except for the log K2 values of 1 (see Table 1). The compounds were isolated in the dihydrochloride form, which means that they were in the fully protonated form (H2L2 +). Accordingly, two sets of protonation constants were determined, as depicted on Table 1: log K1 (7.9–8.7) and log K2 (6.2–6.5). 1H NMR titration curves were further used to aid the identification of the protonation sequence. For the subsequent calculations on the copper/ligand systems, the protonation values of the ligands obtained from the pH–potentiometric

2.5. Self-induced and Cu(II)-induced Aβ1–42 aggregation by the compounds Following a reported protocol [7,21], samples were treated with HFIP to avoid self-aggregation and reserved. HFIP pre-treated Aβ42 samples were re-solubilized with a CH3CN/Na2CO3 (300 μM)/NaOH (250 mM) (48.3: 48.3: 3.4, v/v/v) solvent mixture in order to have a stable stock solution. This Aβ42 alkaline solution (500 μM) was diluted in phosphate buffer (0.215 M, pH 8.0) to obtain a 40 μM solution. Due to the hydrophobic nature of the compounds under study, they were firstly solubilized in methanol (1 mg/mL), and then further diluted in phosphate buffer to a final concentration of 80 μM. To study the effects of the compounds on copper-induced Aβ1–42 aggregation, solutions of Cu(II) (200 μM) in phosphate buffer (0.2 M, pH 8.0) were prepared from a CuCl2 stock solution (0.015 M). The assay consisted on the incubation of Aβ1–42 (40 μM) with or without Cu(II) (40 μM) in phosphate buffer with or without the ligands (80 μM). The incubation was performed at 37 ° C, for 24 h, and afterwards the samples were diluted with 180 μL of glycine-NaOH buffer (50 mM, pH 8.5) in the presence of ThT (5 μM). Fluorescence intensities were measured at 490 nm (λem) with λexc = 446 nm [22]. The percentage inhibition of the self-induced aggregation due to the presence of the test compound was calculated using Eq. (1), where IFi and IF0 correspond to the fluorescence intensities in the presence and absence of the test compound, respectively, minus the fluorescence intensities due to the respective blanks. %I ¼ 100−

I Fi 100 I F0

ð1Þ

The reported values were obtained as the mean ± SEM (standard error of the mean) from two different experiments, each one made in duplicate.

Fig. 2. a) pH–potentiometric titration curves of 2 (CL = 1 × 10−3 M; a represents moles of added base per mole of ligand); b) spectrophotometric absorption spectra of 1 recorded for 2.59 b pH b 11.08 (CL = 5 × 10−5 M); inset contains the individual calculated spectra by PSEQUAD.


4


Table 1 Stepwise protonation constants of the compounds and global formation constants of their Cu(II) complexes (T = 25.0 ± 0.1 °C, I = 0.1 M KCl, 30% DMSO). Ligand 1

2

Method Pot Spect Pot Spect Pot Spect Pot Spect

Log Ki

CumHhLl

log β Mm Hh Ll

pCu⁎

7.99(1) 7.93(3) 6.20(2) 6.50(9) 8.69(4) 8.61(1) 6.29(7) 6.29(3)

(101) (1–11) (1–22)

6.62(3) −0.27(6) −5.2(1)

7.51

(101) (1–11) (1–22)

7.21(2) −0.26(6) −4.0(1)

7.13

⁎ pCu = −log [Cu(II)] with CL / CCu = 10 and CCu = 10−6 M at pH = 7.4.

titration were adopted since the involved errors in the spectrophotometric titrations are higher, in particular for compound 1. Analysis of the inset of Fig. 2b and of Fig. 3, enclosing the calculated spectra by PSEQUAD of the different protonated species for 1, as well as the respective species distribution curves merged with the molar extinction coefficients at the maximum absorption wavelengths, evidences that the absorptions at 339 nm and 351 nm correspond to both protonated species (H2L2+ and HL+) and that the deprotonated L species has a quite different spectra with maximum absorption at ca 328 nm. Moreover, at pH 7.4 (CL = 10− 5 M), as an attempt to mimic the physiological conditions, the predominant species is the monoprotonated one (HL+) for both ligands (75.8% for 1 and 88.6% for 2) with some expected hydrophilic contribution to these compounds, even though the lipohydrophilic character is not only determined by the molecular charge but also by the ability to establish solute-solvent interactions. The 1H NMR titration curves for both ligands showed that the first center to be protonated is associated to the tacrine NH group, while the second one is the amine NH2 group of the SAC or SPRC moiety. In fact, Fig. 4 evidences for 2 downfield shifts of peaks 1, 2, 5 and 7 for 8.0 b pH ∗ b 10.5 and afterwards downfield shifts of peaks 6 and 11 in the pH* range 5.3–7.6, which is according to the above mentioned sequence of protonation. Even though the first center to be protonated is attributed to the tacrine NH group, it must be reminded that an amino/imino tautomeric equilibrium occurs in the tacrine moiety, involving both N atoms of the amino-pyridine derivative [23]. Also, both log K1 (ca 8–8.7) and log K2 (6.2–6.5) values herein determined are significantly lower than, respectively, the protonation constants reported in the literature for the NH2 group of tacrine (10.44 [24] or 9.94 [25] in water) and of 4-aminopyridine (9.14 [26]) or for the NH2 group of cysteine (10.2 [27]). This simultaneous decrease in both values of the protonation constants can be explained on the basis

Fig. 3. Species distribution curves for 1 with molar extinction coefficients at the maximum absorption wavelengths (CL = 5 × 10−5 M).

Fig. 4. 1H NMR titration curves of 2 in 30% d6-DMSO/70% D2O medium.

of the stabilization of the deprotonated forms by formation of bifurcated hydrogen bonding involving the vicinal amide nitrogen atom as well as the tacrine nitrogen and the amine NH2. In the case of 1, there is also a cumulative effect resulting in an even lower log K1 value (ca 8) due to the electron-withdrawing effect of the chloro-substituent in the tacrine moiety. Of course that the differences in the solution media when comparison to literature is made account as well for changes in the values of the constants and that the log K2 values herein determined reflect also the influence of the substitution on the carbonyl CO group of the ligands that does not occur with the carboxylic COOH group of cysteine. 3.2. Studies on copper complexation The chelating ability of the compounds towards Cu(II) was estimated on the basis of the global formation constants of their complexes, determined mainly by UV–vis spectrophotometric (PSEQUAD program [20]) and also pH–potentiometric (Hyperquad 2008 program [19]) techniques. Fig. 2a shows representative pH–potentiometric curves for ligand 2 alone and in the presence of Cu(II) in a 1:1 and 1:2 metal ion to ligand molar ratio. It is possible to observe that, for both Cu(II)/L ratios, the formation of the copper complexes only occurs above pH ca 4 and that the presence of the metal ion in solution induces large changes in the deprotonation profile of the ligand. In fact, for 0 b a b 2, the curves corresponding to the copper-ligand systems lye below that of the ligand, which indicates that the completely deprotonated ligand (L) forms complexes with the copper ion. For a N 2, where virtually all the acidic protons of H2L2+ are neutralized, the copper titration curves are still under the ligand curve which may be due to an additional acid release from hydrolysis and/or from the deprotonation of the amide nitrogen that only occurs in the presence of copper. For the 1:1 systems, above pH ca 7–7.5 precipitation occurred during the pH–potentiometric titration, which may be attributed to the formation of hydroxo complexes. The 1:2 pH– potentiometric titration curves gave less indications about the speciation in solution, due to the presence of somehow larger buffer regions than those in the 1:1 Cu(II)/L curve, but the model suggested the formation of a 1:2 Cu(II)/L complex species. Spectroscopic UV–vis titrations were also carried out for both ligands at the 1:1 and 1:2 stoichiometries, in order to improve the understanding of the complexation process (see Fig. S1 in Supplementary Material for the 1:1 Cu(II)/1 system).



The pH–potentiometric and spectrophotometric titration curves of the systems Cu(II)/1 and Cu(II)/2 lead to the best complexation model shown in Table 1. In Fig. 5, the species distribution curves in the concentration conditions of the spectrophotometric titrations are shown for the systems Cu(II)/1 1:1 and Cu(II)/2 1:2. At these experimental conditions, it is possible to see that copper complexation begins above pH 5 with the formation of CuL, the species CuH− 1 L appears for pH ca 6 and CuH−2 L2 forms above pH 7.5–8.0. At pH 7.4 and micromolar metal concentration (CL = 10 × CM), CuH−1 L is the predominant complex species for the Cu/1 system, while both CuL and CuH−1 L coexist in case of the Cu/2 system. Analysis of the pCu values (pCu = −log [Cu(II)], CL/CCu = 10, CCu = 10−6 M, at pH 7.4) depicted in Table 1 (7.51 for 1 and 7.13 for 2) evidences that the chelating capacity of these two cysteine derivatives towards Cu(II) is similar and somehow higher than that of cysteine (log βCuL2 = 16 in 0.17 M phosphate buffer [28], pCu = 6.3), though discrepancies can be due to the quite different redox and complexation behavior of thiol- and thioether-moieties, as well as to the different media used in the equilibrium studies. To get some insight on the metal coordination mode adopted in the Cu(II) complex species, some further considerations and studies were performed. In fact, reported infrared and X-ray absorption spectroscopies confirmed that the Cu(II) binding in pale pink complexes of cysteine (1:2, 1:4 and 1:6 Cu/L stoichiometries) occurs via the thiol group, although some type of weak electrostatic interaction seems to happen between copper and the carboxylate group [29]. On the other hand, the copper chelating ability of ethylenediamine is higher (log βCuL = 10.54, log βCuL2 = 19.6 [26], pCu = 10.1) than those of the herein studied complexes, both ethylenediamine N atoms being involved in the coordination to the copper ion. In order to get some understanding into the nature of the bluegreenish Cu(II) complexes with the two ligands, the spectroscopic characterization of the complexes in solution was performed through a combination of different techniques: UV–vis, ESI–MS and 1H NMR studies.

a)

% formation relative to Cu

100 Cu

80

CuL

CuH-2L2

5

The UV–vis spectroscopic study was carried out taking advantage of the data provided by the species distribution diagrams formerly achieved and therefore selecting the most significant pH regions corresponding to major complex formation. A representative selection of UV–vis absorption spectra obtained for the Cu/1 systems is presented in Fig. 6, suggesting their attribution to mainly d–d bands although with some overlapping with charge-transfer (CT) bands. The 1:1 CuL copper complexes display λmax corresponding to d–d bands within a small range of 709 to 735 nm in the high wavelength region (spectra at pH ca 5.6), therefore indicating an electronic environment created by the ligands around the copper ion, involving the NH2 amine, the NH amide and eventually the NH tacrine. For pH around 7.6, it is possible to observe a blue shift of that band (ca 620 nm), corresponding to the species CuH− 1 L in the case of the Cu/1 (1:1) System. This last value is somehow near that one presented by square planar copper complexes of L-hystidine containing peptides (590 nm [30]), involving three nitrogen atoms (N imidazole, N amine, N peptide deprotonated) in the coordination sphere. For the Cu/1 system herein studied, the stronger ligand field effect seems to result from the deprotonation of the NH amide group. For higher pH values (9), the major species in solution is CuH− 2 L2 for both 1:2 systems and the shift of the d–d band (overlapped also by CT bands) for 565–601 nm seems to be according to the formation of a complex that involves four nitrogen atoms (two NH2 amine and two deprotonated N-amide) in the coordination shell, with eventually a square pyramidal environment for the copper. In fact, that coordination geometry was already observed for copper complexes of bis (amino amide) ligands (580 nm [31]) as well as of tetramethylcyclam (Me4-CYCLAM) macrocycle (583 nm [32]) in which the macrocyclic effect is lost upon methyl substitution of CYCLAM with concomitant changes from four to penta-coordination. ESI–MS measurements, performed for solutions of Cu(II)/1 and Cu(II)/2 in 1:1 and 1:2 stoichiometric conditions and adequate pH values confirmed the existence of 1:1 copper complexes as the species [CuLCl]+, [Cu(L + H)Cl2]+ and[Cu(L − H)Cl2]- for both systems (see Table 2 and Fig. 7). Under the experimental conditions of ESI/MS it was not possible to observe the formation of the 1:2 CuH−2 L2 complexes (dominant species above pH 8.6 for the 1:2 Cu(II)/L systems) probably due to some precipitation of hydroxo species. In order to try to further understand the metal coordination mode in the complexes formed with the studied ligands, some further studies were implemented, namely a 1H NMR titration of 1 in the presence of Zn(II). However, prior to these studies, pH–potentiometric titrations of the systems Zn(II)/1 1:1 and 1:2 were performed. The pH–potentiometric studies clearly indicate the zinc complex formation, namely based on the observed change in the shape of the titration curves in the presence of

b)

CuH-1L

60 40 20 0 4

6

8

10

pH Fig. 5. Species distribution curves for the systems a) Cu(II)/1 1:1 (CL = 5 × 10−5 M) and b) Cu(II)/2, 1:2 (CL = 8 × 10−5 M).

Fig. 6. Representative UV–vis absorption spectra for 1:1 and 1:2 Cu(II)/1 systems (CL = 5.7 mM).


6


Table 2 ESI/MS ions for the copper complexes in aqueous solution (1:1 Cu(II)/L, CL = 6 × 10−4 M). Species

1 (m/z)

2 (m/z)

[CuLCl]+ [Cu(L + H)Cl2]+ [Cu(L − H)Cl2]−

516/518 552/554 550/552

480/482 516/518 514/516

zinc, when compared to that of the ligand, particularly in the zone corresponding to the deprotonation of the NH+ 3 group. As it was observed for the copper systems, the zinc complexes are not formed at the beginning of the titration, the species ZnHL occurring at pH above 4 (log βZnHL = 11.71(7)). Due to the formation of a precipitate at pH ca 7, and therefore an insufficient number of experimental points, the 1:2 species could not be found. Nevertheless, from the profile of the pH–potentiometric titration curves and calculated value for log βZnHL (11.71), it is possible to infer that 1 forms a weaker complex with zinc than with copper (log βCuL = 6.62 and log K2 = 6.2 for ligand 1). Some further 1H NMR studies were performed aimed at getting some insight on the hetero atoms involved in the coordination to the metal ion. Firstly, proton NMR titrations were performed with ligand solutions (CL = 10 mM) in the presence of Cu(II) or Zn(II) (1:1 M/L stoichiometry) in 50% w/w d6-DMSO/D2O (1) or 30% w/w d6-DMSO/ D2O (2), using DCl or CO2 free KOD solutions to ascertain the pH values, but the results were not conclusive because, for the copper complexes the peaks became too much broad above pH 3.6, while for the zinc complexes precipitation occurred for pH ca 6. Therefore, COSY 1H–1H spectra were performed in d6-DMSO, as illustrated for the systems 1, 1:1 Cu(II)/1 and 1:1 Zn(II)/1 in Fig. 8. In this solvent, it was possible to identify the proton peaks corresponding to the two N-amine and one N-amide groups; furthermore, Fig. 8 shows that the NH amide (NH*) proton presents significant upfield shifts upon complexation either with the copper or the zinc ion while smaller shifts were observed for

the NH tacrine proton peak. This effect can also be observed in Fig. 9, which evidences the chemical shift deviations for the protons associated to the above referred three nitrogen groups of the ligand and metal complexes. In particular, such deviations are more prominent for the proton of NH* (see Figs. 8 and 9), which seems to leave no doubts about its presence in the coordination sphere of the metal ion. Furthermore, since no chemical shift deviation was observed for the protons in the vicinity of the sulfur atom, it is possible to conclude that the coordination sphere of the metal ion involves mostly two nitrogen atoms (NH2 and N amide) but also the NH tacrine group in the 1:1 M/L systems. 3.3. Molecular modeling of the copper complexes In the absence of X-ray structure, we have performed a brief molecular modeling study aimed at providing some further inside into the 1:1 copper complex structures. These studies were carried out with full geometry optimization of the Cu(II) complexes by quantum mechanical calculations based on density functional theory (DFT) methods [33] included in the Gaussian 03 program software [34] with the B3PW91 functional [35]. Geometry optimizations were obtained by using 321G basis set [36] for all elements, first, and afterwards re-optimized using 6-31G** [37] for the main group elements and the Stuttgart– Dresden pseudopotential (SDD) and associated basis set for Cu [38]. No symmetry constrains were enforced during geometry optimizations. Both energy-minimized structures of complexes CuL and CuH−1 L are similar in terms of the Cu(II) coordination, presenting an approximately square planar four-coordinate configuration (see Fig. 10), in agreement with our expectations and solution spectroscopic results. Nevertheless, this modeling simulations suggest that the Cu(II) coordination sphere should have a higher distortion from planarity in CuL than in CuH−1 L; although both complexes present some identical coordination bond distances, such as Cu–Cl (2.16–2.17 Å) and Cu–NH2 (2.064–2.069), that one involving the N amide atom is particularly

a)

b)

Fig. 7. ESI(+) and ESI(−) MS spectra for the complexes (1:1 Cu(II)/L, CL = 6 × 10−4 M): a) Cu-1 (pH = 5.84); b) Cu-2 (pH = 6.08).



7

Fig. 8. Superposition of 1H–1H COSY spectra for the systems 1 (blue), 1:1 Cu(II)/1 (purple, marked with an asterisk mark) and 1:1 Zn(II)/1 (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

shortened (from 2.099 to 1.897 Å) when the respective proton is removed, and it is accompanied by a slight increase in the Cu(II)–NH tacrine distance (from 2.075 to 2.132 Å). Therefore, it seems that CuH−1 L has a stronger coordination to Cu(II) then CuL, in accordance with the previous copper complexation studies, accompanied by a less distorted Cu(II) coordination shell (e.g. NH tacrine–Cu(II)–NH2 bond angle is 151.70 for CuL and 165.30 for CuH−1 L). Since these two complexes do not have exactly the same number of atoms, and charge, comparison of complex stability based on the calculated minimum energies cannot be done.

resulting in higher inhibition percentages. So, even though the copper complexes with the studied ligands are not very strong, the moderate chelating capacity of the ligands towards copper is enough to explain the metal mediation. The effect of the compounds on the inhibition of Cu-induced Aβ aggregation seems slightly more relevant for the allyl derivative (1), a stronger chelator for Cu(II), than for the propargyl (2). Moreover, the presence of the chloro atom in the tacrine moiety and a chain length corresponding to n = 2 appeared also to improve the inhibition capacity for Aβ aggregation.

3.4. Cu(II)-free and Cu(II)-induced Aβ1–42 aggregation by the compounds

4. Conclusions

To evaluate the ability of compounds 1 and 2 to inhibit Aβ42 self- and copper-induced aggregation, an in vitro assay was performed based on the quantification of the amyloid fibril formation by the thioflavin T fluorescence method [7,21]. Five other compounds from previously developed series — 3, 4, 5, 6 and 7 (see Table 3) — were also assayed in order to compare results. All the compounds were incubated for 24 h, at 37 °C, with the Aβ42 peptide (40 μM) and with or without a CuCl2 solution (40 μM). After incubation, a ThT solution (5 μM) was added and fluorescence measurements were carried out. Analysis of the results contained in Table 3 show that all the compounds studied reveal inhibitory capacity for the self-induced aggregation of the same order of magnitude (8.5–14.7%). Nevertheless, the ability of the compounds to inhibit copper-induced aggregation seems to be higher than that in the absence of copper. This may indicate that the compounds are able to modulate the metal-mediated aggregation of the Aβ42 peptide. The low ability of these tacrine hybrids to inhibit the Abeta selfaggregation can be explained by the linear structure of the SAC and SPRC moieties and the concomitant low binding interaction with the Aβ fibers. However, when Aβ is treated with copper, the properties of the synthesized compounds as metal chelators may compete with the amyloid for the copper and decrease their aggregation ability therefore

Due to the interest in modulating the brain metal dyshomeostasis in patients with AD, a series of cysteinyl-tacrine hybrid derivatives with anti-neurodegenerative properties has been reevaluated for their potential extra-role as modulators of metal concentrations and associated pathways such as the amyloid peptide aggregation. Thus, two model compounds (1 and 2) were selected from previously studied series (TAC–SAC and TAC–SPRC) and herein studied in terms of their chelating capacity towards copper, as well as their ability for inhibition of self- or Cu-induced Aβ1–42 aggregation. Both compounds showed a moderate chelating ability towards Cu(II) (pCu 7.13–7.51, CL / CCu = 10, CCu = 10−6 M, at pH 7.4) and also that, at the conditions of pCu determination, CuH−1 L is the predominant complex species for the Cu(II)/1 system while both CuL and CuH−1 L coexist in case of the Cu(II)/2 system. Several spectroscopic techniques, such as UV–vis, ESI–MS and 1H NMR, were used to determine the metal coordination modes in the 1:1 and 1:2 Cu(II)/L complexes formed. It was concluded that the coordination sphere of the 1:1 complex species (CuL and CuH− 1 L) include mainly two N atoms of the cysteine derivative (N-amide and NH2) and also the NH tacrine group; the amide group is protonated in the CuL species and deprotonated in the CuH−1 L species. Molecular modeling of these 1:1


8


Fig. 10. DFT-minimized structures of copper complexes with 1 (L): CuL (top) and CuH−1 L (bottom). Color of atoms: Cu (light blue), N (blue), Cl (green), S (yellow), O (red), C (gray) and H (white). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

corresponding to n = 2 appeared also to improve the inhibition capacity for Aβ aggregation. Abbreviations Aβ amyloid-β AChE acetylcholinesterase AD Alzheimer's disease APP amyloid precursor protein DFT density functional theory ESI–MS electrospray ionization–mass spectrometry HFIP 1,1,1,3,3,3-hexafluoropropan-2-ol Me4-CYCLAM 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane Table 3 Inhibition of Aβ1–42 aggregation in the absence and in the presence of copper. Fig. 9. Superposition of 1H NMR spectra for the systems 1 (blue), 1:1 Cu(II)/1 (purple) and 1:1 Zn(II)/1 (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

copper complexes gave support to the proposed coordination modes for the copper ion. For higher pH values, a 1:2 Cu(II)/L species is also formed, CuH−2 L2, with a coordination sphere involving probably two NH2 amine and two deprotonated N-amide groups. The self- and Cu-induced Aβ aggregation was analyzed for both compounds and the obtained results compared with those found for other compounds of the same series, with different number of carbon atoms (n = 2–4) in the alkylic linker and different tacrine R1 groups (H, Cl). It can be concluded that, although the compounds are only moderate copper chelators, they are able to affect the Aβ aggregation in the presence of Cu(II) and this inhibitory capacity is enhanced when compared to the values obtained in the absence of the metal ion. The percent inhibition is slightly more relevant for the allyl derivative (1), a slightly stronger copper chelator, than for the propargyl (2). Moreover, the presence of the chloro atom in the tacrine moiety and a chain length

Compounds

3 R1 = H, R2 = allyl, n = 2 1 4 R1 = Cl, R2 = allyl, n = 3 5 R1 = Cl, R2 = allyl, n = 4 2 6 R1 = Cl, R2 = propargyl, n = 2 7 R1 = Cl, R2 = propargyl, n = 3

Inhibition of Aβ aggregation (%)a Aβb

Aβ + Cub

8.5 10.9 14.7 10.3 13.0 11.4 12.1

30.9 50.3 35.1 32.1 44.4 49.3 34.3

a

The standard deviation is within 10% of the values. Inhibition of self-mediated Aβ (1–42) aggregation (40 μM) with or without copper (40 μM). The thioflavin-T fluorescence method was used and the measurements were carried out in the presence of an inhibitor (80 μM). b



ROS reactive oxygen species SAC S-allyl-cysteine SEM standard error of the mean SPRC S-propargyl-cysteine TAC tacrine ThT thioflavin T UV–vis ultraviolet–visible spectrophotometry Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2015.06.008. Acknowledgments The authors thank the Portuguese NMR (IST-UL Center) and Mass Spectrometry Networks (Node IST-CTN) for providing access to their facilities. Financial support is acknowledged to the Portuguese Fundação para a Ciência e Tecnologia (FCT) with the project UID/QUI/00100/2013 and the postdoctoral fellowship SFRH/BPD/75490/2010 (R.S.K.).

[15]

[16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]

References [1] [2] [3] [4] [5]

[6] [7] [8] [9]

[10] [11] [12] [13] [14]

R. Jakob-Roene, H. Jacobsen, Angew. Chem. Int. Ed. 48 (2009) 3030–3059. L.M. Sayre, G. Perry, M.A. Smith, Chem. Res. Toxicol. 21 (2008) 172–188. E. Giacobini, Neurochem. Int. 32 (1998) 413–419. C. Rodríguez-Rodríguez, M. Telpoukhovskaia, C. Orvig, Coord. Chem. Rev. 256 (2012) 2308–2332. M.I. Fernández-Bachiller, C. Pérez, N.E. Campillo, J.A. Páez, G.C. González-Muñoz, P. Usán, E. García-Palomero, M.G. López, M. Villarroya, A.G. García, A. Martínez, M.I. Rodríguez-Franco, ChemMedChem 4 (2009) 828–841. M.I. Fernández-Bachiller, C. Pérez, G.C. González-Muñoz, S. Conde, M.G. López, M. Villarroya, A.G. García, M.I. Rodríguez-Franco, J. Med. Chem. 53 (2010) 4927–4937. R.S. Keri, C. Quintanova, S.M. Marques, A.R. Esteves, S.M. Cardoso, M.A. Santos, Bioorg. Med. Chem. 21 (2013) 4559–4569. A. Nunes, S.M. Marques, C. Quintanova, D.F. Silva, S.M. Cardoso, S. Chaves, M.A. Santos, Dalton Trans. 42 (2013) 6058–6073. J. Sebestík, S.M. Marques, P.L. Falé, S. Santos, D.M. Arduíno, S.M. Cardoso, C.R. Oliveira, M.L. Serralheiro, M.A. Santos, J. Enzyme Inhib. Med. Chem. 26 (2011) 485–497. Q.H. Gong, Q. Wang, L.L. Pan, X.H. Liu, H. Xin, Y.Z. Zhu, Brain Behav. Immun. 25 (2011) 110–119. C. Quintanova, R.S. Keri, S. Chaves, M.A. Santos, ISMEC Acta 4 (2014) 74–75 (ISSN 2239–2459, OC-29). H. Kozlowski, M. Luczkowski, M. Remelli, D. Valensin, Coord. Chem. Rev. 256 (2012) 2129–2141. S. Ayton, P. Lei, A.I. Bush, Free Radic. Biol. Med. 62 (2013) 76–89. J.A. Duce, A. Tsatsanis, M.A. Cater, S.A. James, E. Robb, K. Wikhe, S.L. Leong, K.A. Perez, T.J. Johanssen, M.A. Greenough, H.-H. Cho, D. Galatis, R.D. Moir, C.L. Masters, C.

[28] [29] [30] [31] [32] [33] [34]

[35] [36] [37] [38]

9

Mclean, R.E. Tanzi, R. Cappai, K.J. Barnham, G.D. Ciccotosto, J.T. Rogers, A.I. Bush, Cell 142 (2010) 857–867. P. Lei, S. Ayton, D.I. Finkelstein, L. Spoerri, G.D. Ciccotosto, D.K. Wright, B.X. Wong, P.A. Adlard, R.A. Cherny, L.Q. Lam, B.R. Roberts, I. Volitakis, G.F. Egan, C.A. Mclean, R. Cappai, J.A. Duce, A.I. Bush, Nat. Med. 18 (2012) 291–295. M.A. Greenough, I. Volitakis, Q.X. Li, K. Laughton, G. Evin, M. Ho, A.H. Dalziel, J. Camakaris, A.I. Bush, J. Biol. Chem. 286 (2011) 9776–9786. F.J.C. Rossotti, H. Rossotti, J. Chem. Educ. 42 (1965) 375–378. S. Chaves, A. Capelo, L. Areias, S. Marques, L. Gano, M.A. Esteves, M.A. Santos, Dalton Trans. 42 (2013) 6033–6045. A. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739–1753. L. Zékány, I. Nagypál, G. Peintler, PSEQUAD version 5.01, 2001. M. Bartolini, C. Bertucci, M.L. Bolognesi, A. Cavalli, C. Melchiorre, V. Andrisano, ChemBioChem 8 (2007) 2152–2161. X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J. Yu, Y. Tan, Y. Huang, J. Qin, S. Rapposelli, Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502. S.J. Angyal, C.L. Angyal, J. Chem. Soc. (1952) 1461–1466. S. Bollo, L. Munoz, L.J. Núnez-Vergara, J.A. Squella, Electroanalysis 12 (2000) 376–382. H. Yu, W.-M. Li, K.K.W. Kan, J.M.K. Ho, P.R. Carlier, Y.-P. Pang, Z.-M. Gu, Z. Zhong, K. Chan, Y.-T. Wang, Y.-F. Han, J. Pharm. Biomed. Anal. 46 (2008) 75–81. R.M. Smith, A.E. Martell, Critical Stability Constants, vol. 2, Plenum Press, New York, 1975. V.K. Sharma, F. Casteran, F.J. Millero, C. de Stefano, J. Solut. Chem. 31 (2002) 783–792. G. Berthon, Pure Appl. Chem. 67 (1995) 1117–1240. K.M. Dokken, J.G. Parsons, J. McClure, J.L. Gardea-Torresdey, Inorg. Chim. Acta 362 (2009) 395–401. G.F. Bryce, F.R.N. Gurd, J. Biol. Chem. 241 (1966) 122–129. I. Martí, A. Ferrer, J. Escorihuela, M.I. Burguete, S.V. Luis, Dalton Trans. 41 (2012) 6764–6776. L. Hertli, T.A. Kaden, Helv. Chim. Acta 57 (1974) 1328–1333. R.G. Parr, W. Yang, Density Functional Theory of Atoms and Molecules, Oxford University Press, New York, 1989. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford CT, 2004. J.P. Perdew, K. Burke, Y. Wang, Phys. Rev. B 54 (1996) 16533–16539. K.D. Dobbs, W.J. Hehre, J. Comput. Chem. 8 (1987) 861–879. P.C. Hariharan, J.A. Pople, Theor. Chim. Acta 28 (1973) 213–222. T. Leininger, A. Nicklass, H. Stoll, M. Dolg, P. Schwerdtfeger, J. Chem. Phys. 105 (1996) 1052–1059.


Synthesis and biological evaluation of novel tacrine derivatives and tacrine-coumarin hybrids as cholinesterase inhibitors.

Novel tacrine-grafted Ugi adducts as multipotent anti-Alzheimer drugs: a synthetic renewal in tacrine-ferulic acid hybrids.

Tuning electron donor-acceptor hybrids by alkali metal complexation.

Molecular Docking and Pharmacological Investigations of Rivastigmine-Fluoxetine and Coumarin-Tacrine hybrids against Acetyl Choline Esterase.

Tacrine-flurbiprofen hybrids as multifunctional drug candidates for the treatment of Alzheimer's disease.

Discovery of 3-Arylcoumarin-tetracyclic Tacrine Hybrids as Multifunctional Agents against Parkinson's Disease.

Inhibition of cholinesterase and monoamine oxidase-B activity by Tacrine-Homoisoflavonoid hybrids.

Multi-target tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties against Alzheimer's disease.

Novel tacrine analogs as potential cholinesterase inhibitors in Alzheimer's disease.

Tacrine: a pharmacological review.

Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase.

Tacrine in Alzheimer's disease.

Tacrine in Alzheimer's disease.

Tacrine in Alzheimer's disease.

Identification of quinoline-chalcone hybrids as potential antiulcer agents.

Complexation of DNA with ruthenium organometallic compounds: the high complexation ratio limit.

Potential roles of BMP9 in liver fibrosis.

Transport properties of hybrids with ferromagnetic MnAs nanoclusters and their potential for new magnetoelectronic devices.

Multisubstituted indole-acrylonitrile hybrids as potential cytotoxic agents.

Tacrine and lecithin in Alzheimer's disease.

Extracellular vesicles: potential roles in regenerative medicine.

A double-blind, placebo-controlled multicenter study of tacrine for Alzheimer's disease. The Tacrine Collaborative Study Group.

Molecular perception of interactions between bis(7)tacrine and cystamine-tacrine dimer with cholinesterases as the promising proposed agents for the treatment of Alzheimer's disease.

Mapping with B-cell hybrids.