The Antioxidant Additive Approach for Alzheimer's Disease Therapy

Brief Article pubs.acs.org/jmc

The Antioxidant Additive Approach for Alzheimer’s Disease Therapy: New Ferulic (Lipoic) Acid Plus Melatonin Modified Tacrines as Cholinesterases Inhibitors, Direct Antioxidants, and Nuclear Factor (Erythroid-Derived 2)-Like 2 Activators Mohamed Benchekroun,† Alejandro Romero,‡ Javier Egea,§,∥ Rafael León,§,∥ Patrycja Michalska,§,∥ Izaskun Buendía,§,∥ María Luisa Jimeno,⊥ Daniel Jun,# Jana Janockova,∇ Vendula Sepsova,# Ondrej Soukup,∇ Oscar M. Bautista-Aguilera,† Bernard Refouvelet,† Olivier Ouari,○ José Marco-Contelles,◆ and Lhassane Ismaili*,† †

Neurosciences Intégratives et Cliniques EA 481, Laboratoire de Chimie Organique et Thérapeutique, UFR SMP, Université Bourgogne Franche-Comté, 19 rue Ambroise Paré, CS 25000 Besançon, France ‡ Department of Toxicology & Pharmacology, Faculty of Veterinary Medicine, Complutense University of Madrid, E-28040 Madrid, Spain § Instituto de Investigación Sanitaria, Servicio de Farmacología Clínica, Hospital Universitario La Princesa, C/Diego de León 62, E-28006 Madrid, Spain ∥ Instituto de I+D del Medicamento Teófilo Hernando (ITH), Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, E-28029 Madrid, Spain ⊥ Centro Química Orgánica “Lora-Tamayo” (CENQUIOR), CSIC, C/Juan de la Cierva 3, E-28006 Madrid, Spain # Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, CZ-500 01 Hradec Kralove, Czech Republic ∇ Biomedical Research Center, University Hospital Hradec Kralove, CZ-500 05 Hradec Kralove, Czech Republic ○ ICR UMR 7273, Aix Marseille University, CNRS, 13013 Marseille, France ◆ Laboratory of Medicinal Chemistry, IQOG, CSIC, C/Juan de la Cierva 3, E-28006 Madrid, Spain S Supporting Information *

ABSTRACT: Novel multifunctional tacrines for Alzheimer’s disease were obtained by Ugi-reaction between ferulic (or lipoic acid), a melatonin-like isocyanide, formaldehyde, and tacrine derivatives, according to the antioxidant additive approach in order to modulate the oxidative stress as therapeutic strategy. Compound 5c has been identified as a promising permeable agent showing excellent antioxidant properties, strong cholinesterase inhibitory activity, less hepatotoxicity than tacrine, and the best neuroprotective capacity, being able to significantly activate the Nrf2 transcriptional pathway.

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progress and development of AD pathogenesis.3 Indeed, it has been evidenced that the accumulation of senile plaques release reactive oxygen species (ROS), which result in severe damages to the nucleus, mitochondrial membranes, and cytoplasmic proteins of neurons. Consequently, the development of antioxidants for aging diseases has been of paramount importance in the last decades.4 In this context, melatonin5 has been shown to protect neurons and glia from OS.6 This role is due to its ability to scavenge different types of ROS in cells7 and its powerful indirect

INTRODUCTION Among aging-diseases, Alzheimer’s disease (AD) remains the most common cause of memory impairment and dementia in elderly people.1 AD exhibits highly interconnected physiopathological processes leading to the accumulation of abnormal extracellular deposits of β-amyloid peptide (Aβ) and intracellular neurofibrillary tangles, along with dramatic neuronal death and decreased levels of choline acetyltransferase associated with worsened scores in mental status examinations.2 In the search of understanding the mechanisms leading to neurodegeneration, and to find new and more efficient therapies, oxidative stress (OS) have emerged as crucial point in the © 2016 American Chemical Society

Received: August 5, 2016 Published: October 13, 2016 9967

DOI: 10.1021/acs.jmedchem.6b01178 J. Med. Chem. 2016, 59, 9967−9973

Journal of Medicinal Chemistry

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antioxidant functions.8 Melatonin plays likewise a neuroprotective role against Aβ9 and easily crosses the blood−brain barrier (BBB). Recently, melatonin has been suggested as a modulator of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ARE pathway,10 an intrinsic mechanism of defense that, under neuropathological conditions, reduces OS and inflammation by triggering the endogenous expression of detoxifying enzymes and leads to the downregulation of iNOS and COX-2 enzymes. On the other hand, tacrine, one of the most potent known ChEIs to date, was the first FDA-approved drug for the treatment of AD.11 However, because of its dose-dependent hepatotoxicity, it was discontinued shortly after its approval.12 Even so, many medicinal chemistry programs were launched, aiming to design safer tacrine derivatives. One good example is 9-amino-7methoxy-1,2,3,4-tetrahydroacridine (7-MEOTA), which showed reduced hepatotoxic side effects when compared to tacrine.13 In the wake of the repeated failure of the traditional “magic bullet” approach to treat AD, new therapeutic strategies based on the multitarget-directed ligands able to bind simultaneously at the diverse enzymatic systems or receptors involved in the progress and development of the disease have been advanced.14−17 With these precedents in mind, we propose here the antioxidant additive approach as a new strategic approach in order to design new and more potent antioxidant drugs based on the simple concept “two better than one”, which regarding the previously reported tacrine−melatonin hybrids, means to incorporate, in addition to the melatonin, a second one to improve and/or reinforce it. For this purpose, we and others have selected ferulic acid (FA),18 lipoic acid (LA),4 and thio derivatives19,20 to prepare multifunctional tacrines for AD. FA is one of the dominating plant phenolic acids and a potent antioxidant that can greatly attenuate neuronal cell death caused by ROS and protect the brain from Aβ neurotoxicity, possessing hepatoprotective effects.18 LA is a natural antioxidant that protects neurons against cytotoxicity induced by Aβ.4 Thus, we designed the new ferulic acid−tacrine−melatonin hybrids (FATMHs) and lipoic acid−tacrine−melatonin hybrids (LATMHs) (Figure 1), whose

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RESULTS AND DISCUSSION

The synthesis of the FATMHs 5a−d was carried out as shown in Scheme 1 from ferulic acid (1), 3-(2-isocyanoethyl)-5-methoxy1H-indole (2),21 formaldehyde (3), and tacrines 4a−d,22 under Scheme 1a

a

Reagents and conditions: (a) MeOH/CH2Cl2 (3:1, v/v), rt, 24 h (31−61%).

typical U-4CR conditions, from modest to good overall yields. Similarly, but starting from lipoic acid (6), we obtained the LATMH (5e−h) hybrids (Scheme 1). Structural characterization of compounds 5a−h was carried out by the combined use of 1D and 2D [1H,1H] and [1H−13C] NMR experiments (gCOSY, TOCSY, NOESY, multiplicity-edited gHSQC and gHMBC). Compounds 5a−h exist as a mixture of two nonseparable E/Z rotamers, which showed the corresponding separate signals in the 1H and 13C NMR spectra. Conformational equilibrium is due to the hindered rotation around the (O)C−N bonds, being the barrier of rotation in tertiary amides (ca. 15−20 kcal/mol).23,24 In a recently published paper, we have described the dynamic behavior of related derivatives.18 Full NMR study for the major and minor rotamers of selected 5a,d,e,h molecules are gathered in the Supporting Information (SI). These results indicated that at ambient temperature they show a slow exchange process, the Z-rotamer being the major for all the compounds. The novel hybrids 5a−h were analyzed for their anti-ChE activity following the Ellman protocol25 and their hepatotoxicity profile. Thus, on the basis of the IC50 values shown in Table 1, we conclude that FATMHs 5a−d and LATMHs 5e−h are potent, in the nanomolar range, ChEIs. The most effective eqBuChEI was LATMH 5e (IC50= 1.25 nM), showing also the best EeAChE inhibition activity (IC50 = 3.62 nM), which means that 5e is 4- and 12.2-fold more potent than tacrine for the inhibition of eqBuChE and EeAChE, respectively, and 18.9% less hepatotoxic at 300 μM than tacrine. Regarding the eqBuChE inhibition by the methoxy-substituted FATMHs 5b−d, the inhibition power remains quite similar, around 3 nM, on going from 5b (n = 6) to 5c (n = 7), but strongly diminishes on going from hybrids 5b/5c to 5d (n = 8), 4-fold less potent than 5b or 5c and the poorest eqBuChEI analyzed in this work. For the same length in the linker (n = 8), FATMH 5a (R1 = H) is significantly 5.3-fold more potent for the inhibition of eqBuChE, but not suprisingly, 4.7% more hepatotoxic than 5d (R2 = OMe) at 1000 μM.

Figure 1. Antioxidant additive approach for new multifunctional FATMHs and LATMHs.

biological activities [inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), in vitro toxicity in HepG2 cells, neuroprotection in SH-SY5Y cells, Nrf2 pathway induction in AREc32 cells, BBB permeability] as well as their antioxidant capacity through oxygen radical absorbance capacity fluorescein (ORAC) assay have been assessed. From these hybrids, compound 5c (Scheme 1) has been identified as a strong direct antioxidant, potent BuChE inhibitor (BuChEI), less hepatotoxic than tacrine, showing good neuroprotective activity and performing as an activator of the Nrf2 signaling pathway. 9968



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Table 1. Inhibition (IC50, nM) of ChEs, ORAC, BBB Prediction, and HepG2 Cells Viability by Hybrids 5a−h, Tacrine (Ta), Ferulic Acid (FA), Lipoic Acid (LA), and Melatonin (Mel)

HepG2 cell viability (μM)e

compd

EeAChEa

eqBuChEa

hAChEa

hBuChEa

SRb

ORACc

BBB prediction (PAMPA)d

5a 5b 5c 5d 5e 5f 5g 5h Ta Mel FA LA

13.94 ± 1.40 17.80 ± 2.25 8.37 ± 0.08 29.66 ± 5.92 3.62 ± 0.30 16.02 ± 1.14 7.56 ± 0.25 14.84 ± 0.61 44.30 ± 1.54 nd nd nd

2.26 ± 0.19 2.97 ± 0.27 2.91 ± 0.35 12.00 ± 2.15 1.25 ± 0.10 3.92 ± 0.10 5.38 ± 0.62 8.22 ± 1.10 5.07 ± 0.19 nd nd nd

43.7 ± 0.9 >10000 1290 ± 70 >10000 180 ± 4 >10000 >10000 >10000 420 ± 2 nd nd nd

23.5 ± 1.2 358 ± 10 234 ± 8 336 ± 12 211 ± 8 1880 ± 20 2650 ± 100 1730 ± 20 25 ± 1 nd nd nd

1.86

7.98 ± 0.19 8.51 ± 0.19 9.11 ± 0.21 7.27 ± 0.17 1.46 ± 0.34 1.79 ± 0.15 1.80 ± 0.08 3.68 ± 0.15 60

a

AREc32 cells were treated with increasing concentrations of the corresponding compound (1, 3, 10, and 30 μM) for 24 h and, thereafter, luciferase reporter activity was measured as relative luminescence units and normalized respect to untreated cells. Data was represented as concentration−response curves and fitted to nonlinear equation. Data are expressed as the concentration required to double the specific luciferase reporter activity (CD). The CD value was used to quantify and compare the induction potency.

thus, this mechanism must be relevant at the concentrations used.33 Taking into account the use of Aβ1−42 as toxic, 5b,c showed similar potencies at the concentration of 3 μM (85.1% and 81.4% protection, respectively). Nevertheless, 5d showed a very close value of 78.9% protection at the concentration of 1 μM. Compound 5d showed the most potent Nrf2 induction properties; however, hybrids 5b,c showed better scavenger abilities. Consequently, the combination of both effects might explain these neuroprotective trends; at low concentration, the Nrf2 induction properties are the driving force for neuroprotection, and at higher concentrations, the scavenger ability becomes more important. This hypothesis might also explain the neuroprotection against H2O2 because the higher protection value was obtained for 5a,b at 3 μM. Instead, when R/O was used as toxic, hybrid 5d showed the best neuroprotective effect

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EXPERIMENTAL SECTION

General Procedure for the Synthesis of Tacrines 4a−d. A solution of chlorotacrines22,39−41 (1.0 mmol, 1 equiv), and the corresponding alkylenediamine (3.0 mmol, 3 equiv) in pentan-1-ol (3 mL) 9971



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was refluxed for 18 h. The reaction was cooled to rt, diluted with CH2Cl2 (50 mL), and washed with a 10% (w/v) aqueous KOH solution (2 × 50 mL) and water (2 × 50 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford the crude product that was purified by flash column chromatography CH2Cl2/ MeOH/aqueous 30% NH3 (7:3:0.1, v/v/v) to afford the products 4a-d, whose spectroscopic and analytical data were in good agreement with previously described data.22 General Procedure for the U-4CR Synthesis of α-Acylaminocarboxamides 5a−h. A solution of the corresponding N1-(1,2,3,4tetrahydroacridin-9-yl)alkane-1,n-diamine (4a) or N1-(7-methoxy1,2,3,4-tetrahydroacridin-9-yl)alkane-1,n-diamine (4b−d) (1.0 equiv) and formaldehyde (1.0 equiv) in MeOH/CH2Cl2 (7 mL, 3:1, v/v) was stirred for 1 h at rt. The corresponding acid (1.0 equiv) and 3-(2-isocyanoethyl)-5-methoxy-1H-indole (2) (1.0 equiv) were then added, and the reaction was stirred 24 h at rt. The mixture was concentrated under reduced pressure to dryness, and the crude product was purified by flash column chromatography to afford the corresponding α-acylaminocarboxamides 5a−h. The purity of the new compounds was checked by elemental analyses, conducted on a Carlo Erba EA 1108 apparatus, and confirmed to be ≥95%. In addition, new compounds were found to be ≥95% pure by HPLC analysis using a Hitachi Lachrom Elite series instrument equipped with a L2400 Lachrom Elite DAD detector and a Uptisphere ODB column (4.6 mm × 100 mm, Ø = 3 μm). Peaks were detected at 210 nm and the system was operated at 25 °C with a flow rate of 2 mL/min. The mobile phase was an isocratic mixture of acetonitrile and water (1:1, v/v) containing 0.1% (w/v) monopotassium phosphate.

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CZ-DRO (UHHK, 00179906) and by Long Term Development Plan-1011 of Faculty of Military Health Sciences, University of Defence (Czech Republic). R.L. thanks ISCIII (Programa Miguel Servet, CP11/00165; grant PI14/00372), Bayer AG “From targets to novel drugs” (grant 2015-03-1282) and Fundación FIPSE (grant 12-00001344-15). P.M. thanks MECD for a FPU fellowship (FPU13/03737).

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ABBREVIATIONS USED Aβ, β-amyloid; ARE, antioxidant response element; COX-2, cyclooxygenase 2; ChEs, cholinesterases; HepG2, human liver hepatocellular carcinoma cell line; iNOS, nitric oxide synthase; Nrf2, nuclear factor (erythroid-derived 2)-like 2; SH-SY5Y, human dopaminergic neuroblastoma cell line; U-4CR, Ugi fourcomponent reaction

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01178. Experimental procedures covering the synthesis, spectral characterization of 2 and 4a−d and hybrids 5a−h as well as the pharmacological methods and docking studies (PDF) Molecular formula strings (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33381665543. Fax: +33363082319. E-mail: lhassane. [email protected]. Author Contributions

The manuscript was written through the contribution of all the authors. M.B., O.M.B.A., B.R., and J.M.C. contributed to the chemistry, ChEs inhibition, and ORAC-FL assays, L.I. contributed to the chemistry, ChEs inhibition, and ORAC-FL assays and conceived the project. M.L.J. contributed to the NMR analysis. J.E., P.M., I.B., R.L., and A.R. contributed to the in vitro cell studies. R.L. contributed to docking studies. D.J., J.J., V.S., and O.S. contributed to human ChEs inhibition assays, ChEs kinetics, and PAMPA-BBB assay. O.O. has carried out the EPR analysis. J.E. and R.L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS B.R. and L.I. thank the Regional Council of Franche-Comté (France) for financial support, and M.-J. Henriot (PHV Pharma) for support in the HPLC analyses. M.B. thanks the Regional Council of Franche-Comté (France) for a Ph.D. grant. J.E. has a grant from ISCIII (Programa Miguel Servet, CP14/00008). D.J., J.J., V.S., and O.S. thank Jitka Pichova for a skillful technical assistance. This work was supported by the project MH 9972



Brief Article

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The Antioxidant Additive Approach for Alzheimer's Disease Therapy

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