Synthesis of 9-Aminoacridine Derivatives as Anti-Alzheimer Agents

Abstract

In the present study, some 9-aminoacridine derivatives have been synthesized by condensation of 9-aminoacridine with substituted phenacyl, benzoyl, and benzyl halides (RM1-RM6). Compounds were investigated for acetylcholinesterase and butyrylcholinesterase inhibition potential, considering these enzymes playing a key role in Alzheimer’s disease. All derivatives showed better inhibition of enzymes than the standard galantamine, whereas except RM4, all exhibit better results than tacrine, a well-known acridine derivative used for the treatment of Alzheimer’s disease.

Introduction

Acridine-based pharmacophore ^1,2 has been found to be associated with a wide spectrum of biological activities, especially for the treatment of Alzheimer’s disease (AD) by inhibiting cholinesterases (ChE). ³ In symptomatic improvement of AD, acetylcholinesterase (AChE) has proven to be the most viable therapeutic goal because cholinergic deficit is a constant and early finding in this AD. There are 2 types of ChEs, AChE and butyrylcholinesterase (BuChE). Several available drugs target both AChE and BuChE in AD, but some are more selective. ⁴ 9-Aminoacridine (9AA) and its derivatives have long been known to be reversible inhibitors of acetyl cholinesterase, the most familiar of which is 9-amino-1,2,3,4-tetrahydroacridine, tacrine (Cognex, Pfizer Pharmaceuticals). ⁵ This was the first nonclassical inhibitor of AChE approved by the Food and Drug Administration (FDA) in 1993 ⁶ that binds to both AChE and BuChE. ⁷ However, widespread use of tacrine was limited as it caused a number of side effects including nausea, vomiting, dizziness, diarrhea, seizures, and liver toxicity. Short half-life of tacrine was another major problem. ⁸ Eventually, tacrine was discontinued due to hepatotoxicity. ⁹

Physostigmine, first-tested AChE inhibitor (AChEI), originally extracted from calabar beans. Physostigmine could improve memory in people with or without dementia, but this property has been limited by the very short half-life. ¹⁰ A number of physostigmine derivatives were synthesized, and some are failing in clinical trials due to severe side effects. ⁴ Rivastigmine (Exelon, Novartis Pharmaceuticals), a physostigmine derivative, is a centrally selective ChEI that was approved in 2000. Rivastigmine exhibits a low level of hepatotoxicity ^11,12 but frequently associated with side effects such as nausea, vomiting, anorexia, diarrhea, headache, fatigue, malaise, sweating, somnolence, dyspepsia, and sinusitis. ^13,14

Donepezil (Aricept, Pfizer Pharmaceuticals) is another nonclassic, centrally acting, reversible, noncompetitive AChEI that was approved in 1997 for mild to moderate AD and dementia and has little potential for hepatotoxicity, ^11,15 but on high dose, transient nausea, diarrhea, and insomnia were reported. ¹⁶

Galantamine (Razadyne, Global Pharmaceuticals), approved in 2000, an alkaloid found in plants of the family Amaryllidaceae, is a reversible inhibitor of AChE but does not inhibit BuChE and used for mild to moderate AD and dementia, and no hepatotoxicity is reported ^11,12 but associated with adverse events such as nausea, vomiting, diarrhea, anorexia, muscle cramps, headaches, dizziness, fatigue, and insomnia as a result of cholinergic stimulation. ^17,18

Naturally derived drugs such as huperzine A and huperzine B are potent ChEIs. The huperzine A had the least amount of activity against BuChE. ¹⁹ Huperzine A is considered to be the drug of choice in China for the treatment of memory disorders. Side effects would be expected to be similar in nature to other ChEIs. ⁴

Although available cholinesterase inhibitors (AChEIs) provide benefit in AD, all these drugs exhibit varied side effects and complications such as low bioavailability, shorter half-life, and hepatotoxicity. ^{20- 22}

The present study is an attempt to find better ChEI, a crucial target for the treatment of AD. All synthesized acridine derivatives showed promising AChE and BuChE inhibition. Pharmacophoric regions are also determined, highlighting the important structural features of ligands, which may be a great help and guidance for the designing of effective anti-Alzheimer drug. ²³

Chemicals and Instruments

All reagents were reagent grade purchased from Merck (Germany) and Sigma-Aldrich Company (Germany) and distilled twice. The monitoring of the reaction and purity of the final product were determined by thin-layer chromatography on silica gel 60 GF₂₅₄ (Merck). Thin-layer chromatography spot was visualized in UV light at 254 and 365 nm on HPUVIS Desaga (Heidelberg, Germany). Melting points were recorded on STUART (Bibby Sterlin Ltd, UK), SMP3 melting point apparatus, and were uncorrected. Spectroscopic data were recorded on ultraviolet spectrophotometer (CECIL 7200, USA), IR spectrophotometer (FTIR-8900; Shimadzu, Japan) using KBr disc, mass spectrometer (JEOL JMS-HX110, Japan), and ¹ H-NMR (Bruker Advance 300, France) in Dimethyl sulfoxide deprotonated (DMSO) at 300 MHz using tetramethylsilane as an internal standard.

Synthesis of Compounds

To a solution of 0.0025 mol/L 9AA in Acetone (15-20 mL), the substituted phenacyl, benzoyl, and benzyl halides (0.0025 mol/L) were added. Reaction mixture was stirred for 5 to 12 hours at room temperature and then refluxed for 15 to 78 hours at 50°C. After cooling, the solid precipitates were separated through vacuum filtration and washed with acetone. Purification was done through recrystallization using double solvent system (ethanol and ether). Pure compounds were dried in vacuum desiccators over silica beads. Melting point was recorded, and spectral studies were done for the confirmation of the resultant product (Figure 1).

Figure 1.

The synthetic scheme of compounds RM1, RM2, RM3, RM4, RM5, and RM6.

N-(3′-Bromophenyl-2″-oxoethyl)acridine-9-ammonium bromide (RM1)

Light yellow powder, yield: 71.25%. melting point decomposed (m.p. d) 215°C, UV(MeOH) ∊: 1817200. IR (KBr) νmax (cm⁻¹): 3107.1, 2993.3, 1595.0, 1479.3, EIMS (−HBr) [M]+: m/z 390. ¹H-NMR (DMSO, 300 MHz): δ 3.498 (s, 2H, H-11), 7.548-7.596 (t, J = 6.3 Hz, 3H, H-2, H-7, H-13), 7.949-8.301 (m, 6H, H-1, H-3, H-6, H-8, H-12, H-14), 8.723-8.752 (d, J = 8.7 Hz, 3H, H-4, H-5, H-15).

N-(Acridine-9-yl)-3′, 5′dinitrobenzamide (RM2)

Dark yellow powder, yield: 99.6%. m.p. d 277°C. UV (MeOH) ∊: 1115120. IR (KBr) νmax (cm⁻¹): 3342.4, 3139.9, 1658.7, 1502.4, EIMS (−HCl) [M]+: m/z 388. ¹ H-NMR (DMSO, 300 MHz): δ 7.503-7.552 (t, J = 7.2 Hz. 2H, H-2, H-7), 7.854-7.963 (m, 6H, H-1, H-3, H-4, H-5, H-6, H-8), 8.61-8.630 (d, J = 8.7 Hz, 3H, H-11, H-12, H-13).

N-(4′-Nitrophenyl-2″-oxoethyl)acridine-9-ammonium bromide (RM3)

Mustard yellow powder, yield: 99%. m.p. d 243°C. UV (MeOH) ∊: 1046820. IR (KBr) νmax (cm⁻¹): 3190.0, 3109.0, 1649.0, 1595.0, 1479.3, EIMS (−HBr) [M]+: m/z 357. ¹H-NMR (DMSO, 300 MHz): δ 3.352 (s, 2 H, H-11), 7.511-7.560 (d, J = 7.2 Hz, 4H, H-2, H-3, H-6, H-7), 7.847-7.966 (m, 6H, H-1, H-4, H-5, H-8, H-12, H-15), 8.587-8.615(d, J = 8.4 Hz, 2H, H-13, H-14).

N-(Acridine-9-yl)-4′-bromobenzamide (RM4)

Florescent yellow powder, yield: 92.2%. m.p. d 275°C. UV (MeOH) ∊: 792960. IR (KBr) νmax (cm⁻¹): 3340, 3138, 1589.2, 1479.3, EIMS (−HCl) [M]+: m/z 377. ¹H-NMR (DMSO, 300 MHz): δ 7.505-7.555 (d, J = 7.5 Hz, 2H, H-2, H-7), 7.852-7.964 (m, 6H, H-1, H-3, H-6, H-8, H-12, H-13), 8.597-8.06 (d, J = 8.7 Hz, 4H, H-4, H-5, H-11, H-14).

N-(4′-Bromobenyl)acridine-9-ammonium bromide (RM5)

Fluorescent yellow powder, yield: 39.9%. m.p. d 280°C. UV (MeOH) ∊: 310800. IR (KBr) νmax (cm⁻¹): 3307.7, 3109.0, 1595, 1477.4, EIMS (−HBr) [M]+: m/z 363. ¹H-NMR (DMSO, 300 MHz): δ 3.326 (s, 2H, H-11), 8.698-8.726 (d, J = 8.4 Hz, 2H, H-4, H-5), 7.183 (s, 2H, H-12, H-15), 7.559-7.688 (m, 4H, H-2, H-7, H-13, H-14), 7.835-8.039 (m, 4H, H-1, H-3, H-6, H-8).

N-(4′-Methylbenzyl)Acridine-9-Ammonium Chloride (RM6)

Yellow crystals, yield: 25.68%. m.p. 221°C, UV (MeOH) ∊: 858380. IR (KBr) νmax (cm⁻¹): 3182.3, 3049.2, 1485.1, 1392, EIMS (+HCl) [M]+: m/z 334. ¹H-NMR (DMSO, 300 MHz): δ 3.323-3.299 (d, J = 4.2 Hz, 5H, H-11 H-16), 7.272-7.322 (t, J = 7.5 Hz, 2H, H-12, H-15), 7.600-7.649 (t, J = 6.9 Hz, 4H, H-2, H-7, H-13, H-14), 7.73-7.821 (m, 4H, H-1, H-3, H-6, H-8), 8.354-8.382 (d, J = 8.4 Hz, 2H, H-4, H-5).

Pharmacology

All the compounds were screened for AChE and BChE inhibition activity. Electric eel AChE (EC 3.1.1.7), equine serum BuChE (EC 3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chloride, and 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) were purchased from Sigma (St Louis, Missouri). Buffers and other chemicals were of analytical grades.

Enzyme Inhibition Assay

All the assays were performed under 100 mmol/L sodium phosphate buffer, pH 8.0, using a 96-well microplate on SpectraMax340 (Molecular Device [U.S.A]). One hundred and fifty microliters of 100 mmol/L sodium phosphate buffer (pH = 8), 10 μL DTNB, 10 μL of test compound (9AA derivatives) solution, and 20 μL AChE or BuChE solution were mixed and incubated for 15 minutes at 25°C. The reaction was then initiated with the addition of 10 μL acetylthiocholine iodide or butyrylthiocholine chloride in that order. The activity was determined by measuring the increase in absorbance at 412 nm. The AChE and BuChE inhibiting activities were measured by slightly modifying the spectrophotometric method developed by Ellman. ¹²

Result and Discussion

All the newly synthesized molecules (RM1-RM6) were tested for their inhibition activities toward AChE and BuChE according to the modified Ellman method with parent molecule (9AA) and standards, galantamine and tacrine (Table 1). The ChE inhibition results were summarized in Table 1. The result showed that the target compounds possessed significantly even better ChE inhibition than galantamine and tacrine. In case of galantamine taken as standard, all derivatives showed better activity against AChE and BuChE. As compared to tacrine, compounds showed inhibitory potential against AChE in the order of RM2 > RM3 > RM6 > RM5 > RM1 > RM4. Only RM4 showed weak AChE inhibition than tacrine, whereas against BuChE, all derivatives showed better results than tacrine. All synthesized compounds presented more potent inhibitory activity against BuChE (IC₅₀ value 0.003-0.0715 nmol/L) as compared to AChE (IC₅₀ value 0.0004-0.006 nmol/L).

Table 1.

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Inhibiting Activity.

Compounds Codes	AChE Inhibition, Inhibitory Concentration 50% (IC50) ± SEM (nmol/L)	BuChE Inhibition, Inhibitory Concentration 50% (IC50) ± SEM (nmol/L)
RM1	0.022 ± 0.00046	0.0011 ± 0.00
RM2	0.0033 ± 0.0011	0.0011 ± 0.00
RM3	0.010 ± 0.00	0.00046 ± 0.00
RM4	0.0715 ± 0.00195	0.0062 ± 0.000208
RM5	0.015 ± 0.001	0.0004 ± 0.00
RM6	0.0127 ± 0.00	0.0004 ± 0.00
9AA (lead)	0.046 ± 0.00	0.0014 ± 0.00
Galantamine (standard)	1.6 ± 0.000167	6.6 ± 0.00038
Tacrine (standard)	0.021 ± 0.002	0.051 ± 0.005

Abbreviation: SEM, standard error of the mean.

Ligand-Based Pharmacophoric Study of Synthesized Compounds

Pharmacophoric study was carried out using the software LigandScout 3.02 [i1_10] with ChemDraw Ultra 8.0 for structure drawing. Important features were identified after analyzing the structures of the synthesized compounds along with the standards generated by LigandScout. The obtained main features of all the synthesized compounds were then superimposed into different standard ligands to get the shared features indicating the similar important binding regions, which may be involved in binding with the target.

Acetylcholinesterase and BuChE Inhibition

Pharmacophoric regions of the synthesized molecules determined with the help of the software “LigandScout” revealing the important structural features (Table 2). Donepezil, rivastigmine, galantamine, and tacrine approved by the United States Food and Drug Administration (FDA) and marketed for the treatment of AD. ^{24 -27}

Table 2.

Pharmacophoric Features of Parent, Synthesized Derivatives, and Standards.

Compounds	Aromatic Region(s)	Hydrophobic Region(s)	Hydrogen Bond Acceptor(s)	Hydrogen Bond Donor(s)	Ionic Interaction Positive/Negative
9AA	3	2	1	1	0
RM1	4	4	2	1	1/0
RM2	4	3	6	1	0
RM3	4	2	4	1	1/0
RM4	4	5	2	1	0
RM5	5	4	1	1	1/0
RM6	4	4	1	1	1/0
Galantamine	1	2	3	1	1/0
Physostigmine	1	2	2	1	1/0
Rivastigmine	1	4	2	0	1/0
Tacrine	2	1	1	1	1/0

Tacrine, physostigmine, rivastigmine, and galantamine taken as standard in this study are extensively investigated for the ChE inhibition as a major targets in AD. ⁶ In pharmacophoric generation, these standards displayed 5 to 9 pharmacophoric features (Figures 2 –5). 9-Aminoacridine has 7 pharmacophoric regions, whereas synthesized molecules (RM 1-6) comprised around 10 to 14 pharmacophoric regions (Figure 6) including hydrogen bond donor(s), hydrogen bond acceptor(s), ionic interaction, hydrophobic (HPB), and aromatic region(s).

Figure 2.

Two- and 3-dimensional picture of galantamine. Blue indicates aromatic region; yellow, hydrophobic region; red, hydrogen bond acceptor; green, hydrogen bond donor; blue star, positive ionic interaction. Note: Color version of the figure is available at http://aja.sagepub.com/

Figure 3.

Two- and 3-dimensional picture of physostigmine. Blue indicates aromatic region; yellow, hydrophobic region; red, hydrogen bond acceptor; green, hydrogen bond donor; blue star, positive ionic interaction. Note: Color version of the figure is available at http://aja.sagepub.com/

Figure 4.

Two- and 3-dimensional picture of rivastigmine. Blue indicates aromatic region; yellow, hydrophobic region; red, hydrogen bond acceptor; blue star, positive ionic interaction. Note: Color version of the figure is available at http://aja.sagepub.com/

Figure 5.

Two- and 3-dimensional picture of tacrine. Blue indicates aromatic region; yellow, hydrophobic region; red, hydrogen bond acceptor; green, hydrogen bond donor; blue star, positive ionic interaction. Note: Color version of the figure is available at http://aja.sagepub.com/

Figure 6.

Three-dimensional picture showing shared features of 9-aminoacridine (9AA), RM1, RM2, RM3, RM4, RM5, and RM6. Yellow, hydrophobic region; red, hydrogen bond acceptor; green, hydrogen bond donor. Note: Color version of the figure is available at http://aja.sagepub.com/

All synthesized molecules showed maximum shared features with tacrine as compared to other standards (Table 3). Tacrine shared 6 pharmacophoric regions with phenacyl, benzoyl, and benzyl derivatives (Figure 7) including 1 hydrogen bond acceptor, 1 hydrogen bond donor, 2 aromatic and 1 HPB region, and 1 ionic interaction. There is a possibility that all of these regions or some of them may be involved to interact with the active binding sites of enzymes.

Table 3.

Shared Pharmacophoric Features of Parent, Synthesized Derivatives, and Standards.

Compounds	Aromatic Region(s)	Hydrophobic Region(s)	Hydrogen Bond Acceptor(s)	Hydrogen Bond Donor(s)	Ionic Interaction Positive/Negative
9AA, RM1, RM2, RM3, RM4, RM5, RM6	0	2	1	1	0
Galantamine, RM1, RM3	1	2	1	0	1/0
Galantamine, RM2, RM4	0	1	1	0	0
Galantamine, RM5, RM6	1	2	1	0	1/0
Physostigmine, RM1, RM3	1	0	0	1	1/0
Physostigmine, RM2, RM4	0	2	1	0	0
Physostigmine, RM5, RM6	1	1	0	1	1/0
Rivastigmine, RM1, RM3	1	2	1	0	0
Rivastigmine, RM2, RM4	0	2	0	0	0
Rivastigmine, RM5, RM6	1	2	1	0	0
Tacrine RM1, RM3	2	1	1	1	1/0
Tacrine RM2, RM4	2	1	1	1	0
Tacrine RM5, RM6	2	1	1	1	1/0

Figure 7.

Three-dimensional picture showing shared features of tacrine, RM1, RM2, RM3, RM4, RM5, and RM. Blue indicates aromatic region; yellow, hydrophobic region; red, hydrogen bond acceptor; green, hydrogen bond donor; blue star, positive ionic interaction. Note: Color version of the figure is available at http://aja.sagepub.com/

Overall, this 3-dimensional structural investigation of synthesized compounds with different standards generates pharmacophoric regions showing good sharing of different important regions in structures. Several binding studies of the various cholinesterase inhibitory ligands with the target enzymes indicate the significance of hydrogen bonding (HB) and HPB interactions. ^28,29 In the present pharmacophoric investigation, all the synthesized compounds present greater number of hydrogen bondings (HBs 1-6) and HPB interactions (2-5) as compared to all standards (HB 1-3 and HPB 1-4). Presence of more HBs and HPB regions in the synthesized compounds provides possible justification of better enzymes inhibition as compared to standards.

Conclusion

In the given study, synthesized 9AA derivatives displayed highly significant potential for AChE and BuChE inhibition. As these enzymes have been studied and considered as a major target for the treatment of Alzheimer, better result than standards indicating the pronounced potential of these compounds to be used as anti-Alzheimer agents. These compounds have been selected to be investigated further for their toxicity profile as well as studied at molecular level for their binding mode with the target.