Bioorganic & Medicinal Chemistry Letters 22 (2012) 2885–2888

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Prospective acetylcholinesterase inhibitory activity of indole and its analogs

Nantaka Khorana a,⇑, Kanokwan Changwichit a, Kornkanok Ingkaninan a, Maleeruk Utsintong b

a Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences and Center for Innovation in Chemistry, Naresuan University,Phitsanulok 65000, Thailandb School of Pharmaceutical Sciences, University of Phayao, Phayao 56000, Thailand

a r t i c l e i n f o

Article history:Received 14 September 2011Revised 26 January 2012Accepted 20 February 2012Available online 27 February 2012

Keywords:Indolesb-CarbolinesQuinolinesAcetylcholinesterase inhibitory activityAlzheimer’s disease

0960-894X/$ - see front matter � 2012 Published bydoi:10.1016/j.bmcl.2012.02.057

⇑ Corresponding author. Tel.: +66 55 961 862; fax:E-mail addresses: nantakak@nu.ac.th, nantaka@ho

a b s t r a c t

Acetylcholinesterase (AChE) inhibitory activity is one of the proposed targets for indole analogs. Simpleindoles with substitution of methoxy, carboxy or hydroxy at the benzene ring showed a low percent ofinhibitory activity in eel-AChE. Adding a side chain at the pyrrole ring, such as serotonin, b-carbolinesand quinolines (the bioisostere of indole), improved the inhibitory activity significantly. However, propersubstitution and conformation of the ring were required for good binding. The result of inhibition inhuman-AChE of serotonin, b-carbolines and quinolines showed similar profile as eel-AChE with lowermagnitude. The data from molecular docking showed that they shared the same binding site asgalantamine.

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Indole is a bicyclic structure of benzene fused to a five-mem-bered pyrrole ring. Indole is a part of the structure in the neuro-transmitter serotonin and melatonin which are derived from theamino acid tryptophan. Various chemicals in our daily life; suchas pharmaceutical products, fragrances and some natural occurringcompounds (indole alkaloids), also contain the indole ring in theirstructures. Indole-containing compounds play a role in diversepharmacological actions; for example oncolytic activity of Vincris-tine and Vinblastine1 from Catharanthus roseus, antinociceptiveactivity of 7-hydroxymitragynine2 from Mitragyna speciosa, neuro-leptic activity of synthetic 4-aryltetrahydropyrrolo[3,4-b]indoles3

and acetylcholinesterase (AChE) inhibitory activity of b-carbolinederivatives from Haplophyton crooksii4 and Tabernaemontanaaustralis5, vobasinyl-iboga bisindole alkaloids6 from Tabernaemon-tana divaricata and ibogaine derivatives from Tabernaemontanaaustralis5 and Ervatamia hainanensis.7

AChE inhibition is one of the proposed mechanisms for treat-ment of Alzheimer’s disease for which the incidence rate of diseaseis predicted to increase in every year.8 Hence, prevention and treat-ment methods are required to slow down the prevalence of the dis-ease. Plant extracts are one of the major sources for discovery ofnew compounds with high AChE inhibitory activity. Various indolestructures were discovered from plant extracts in both monomericand bis-indole derivatives. From T. divaricata extract, compoundswith bis-indole structure (Fig. 1) were isolated and showed goodAChE inhibitory activity. 19,20-Dihydrotabernamine (1) and

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+66 55 963 731.tmail.com (N. Khorana).

19,20-dihydroervahanine A (2) from Ingkaninan et al.6 displayedsignificantly high IC50 for AChE, 227 ± 154 and 71 ± 13 nM, respec-tively. However, some indole-structures, tabernaelegantine A (3),displayed much lower inhibition. From Figure 1, compound 1, 2and 3 showed similar structure in monomer-1 which might be pos-sible to conclude that only monomer-2 responsible for the AChEinhibition. The appropriate substitution on the indole ring waslikely to be required for improving the inhibitory activity, but thesubstitution at the R group in structure 1 and 2 might play someminor cause for activity. In this study, simple indole derivativesand b-carboline derivatives were used to investigate the AChEinhibitory activity, to simplify and to understand the structure-activity relationship of indole structure.

The commercially available compounds of simple indoles withmethoxy (4–7), carboxy (8–11) or hydroxyl (12,13) substitutionat position 4–7 were examined for % inhibition on AChE at concen-tration 10�4 M (Table 1). The assay for eel-AChE9 inhibitory activitywas performed according to the methods developed by Ellmanet al.10 and Ingkaninan et al.11, using galantamine as a referencestandard. It appeared that the indole ring with substitutions eitherelectron donating or electron withdrawing group only on the ben-zene ring showed extremely low % inhibition of less than 20% atconcentration 10�4 M for indole with electron donating groupand less than 40% at concentration 6.2 � 10�4 M for indole withelectron withdrawing group. It implied that the structures mightbe too small and they were not appropriate to fit within the AChEbinding pocket. Comparison of the influence between electrondonating (methoxy and hydroxy substitution) and electron with-drawing (carboxy) substitution of indole ring on inhibitory activity

NH

NCH3

H

H3COOC

NH

N

RH

Monomeric-1

Monomeric-2

1.R=H2.R=COOCH3

NH

NCH3

H

H3COOC

NH

N

H3COOC H

H3CO

3

Figure 1. Chemical structures of 19,20-dihydrotabernamine (1), 19,20-dihydroervahanine A (2) and Tabernaelegantine A (3) from Tabernaemontana divaricata.

Table 1AChE inhibitory activity of simple indole ring derivatives on eel-AChE at concentra-tion 10�4 M (N = 3)

Indole ringNH1

2

3

45

6

7

R

Compound R % Inhibition (±SD)

4 4-OCH3 9.24 ± 4.035 5-OCH3 4.26 ± 2.416 6-OCH3 11.41 ± 2.207 7-OCH3 8.17 ± 3.078 4-COOH 36.82 ± 5.59⁄

9 5-COOH 29.05 ± 3.86⁄

10 6-COOH 28.50 ± 4.42⁄

11 7-COOH 23.35 ± 7.62⁄

12 4-OH 19.04 ± 5.8713 5-OH 7.33 ± 4.1014 2-CH3 17.97 ± 6.1015 5-OCH3, 3-CH2-CH2-NH2 7.19 ± 3.0216 5-OH, 3-CH2-CH2-NH2 62.59 ± 3.74Ganlantamine 100.00 ± 0.00⁄⁄

Remark: ⁄ = % Inhibition at concentration 6.2 � 10�4 M.⁄⁄ = % Inhibition at concentration 3.4 � 10�4 M; IC50 = 0.60 ± 0.01 lM.

2886 N. Khorana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2885–2888

could not be concluded by this data because both substitution typeat the indole ring exhibited fairly low activity. Moving the substi-tution to the pyrrole ring at position 2 also showed low activityas in 2-methylindole (14). Adding a longer substitution, ethyla-mine, to pyrrole and 5-methoxy at the benzene ring as in 15 didnot improve any AChE inhibition comparing to the 5-methoxy in-dole (5). However, serotonin (16) which had the hydroxyl group atposition-5 showed significant improvement in% inhibition com-pared to 4–15. It was likely that more sterical structure at the pyr-role ring and specific type of substitution on the benzene ring werepreferred for increasing the inhibition. Hence, the more rigid struc-tures of ethylamine side chain reconnect to indole ring with onecarbon linker, b-carboline derivatives, were investigated withexpectation to improve the AChE inhibitory activity.

Mostly of the reported active indole derivatives found fromplants contained electron donating group on the benzene ring ofindole, so we would like to concentrate only the molecule withelectron donating group in this study. Harmane (17), a b-carbolinestructure with 1-methyl substituted, displayed a good % inhibitoryactivity on eel-AChE with % inhibition more than 80% at concentra-tion 10�4 M and IC50 20.91 ± 1.64 lM (Table 2). Substitution of

harmane at position-7 with methoxy (18) did not harm the AChEinhibitory activity but replacement of methoxy to hydroxyl group(19) improved the activity by 2 to 3-fold. Reduction of one doublebond in pyridine ring of 18 increased the activity approximatelyfivefold as shown in 20. It indicated that flexibility of the moleculein the appropriate orientation was required for binding at the ac-tive site. In contrast, compound 21, the reduced form of 19 didnot affect the inhibitory activity. Substitution at 7-position mightbe both methoxy and hydroxy depending on the conformation ofthe ring. The tetrahydro-b-carboline analog (22) showed a ten-dency to reduce the inhibitory activity compared to the other lessflexible b-carboline. However, it cannot be concluded at this pointbecause the tested compounds showed more than one change inthe molecule from 17–21 (Table 2). 6-Methoxy and 1-carboxylicsubstitution at tetrahydro-b-carboline ring might not favor forthe activity as in 23 and 24. Moreover, the quinoline ring whichwas considered as the bioisostere of the indole ring, was investi-gated in another study.12 The commercially available 6-methoxy-quinoline (25) did not show any inhibitory activity similar to itsbioisostere 5. From Markmee et al.13 and Khorana et al.14, the iso-quinoline derivatives have shown some activity by adding thesubstituted group at position 1. Hence, N-alkyl substitutions ofquinoline were synthesized by interaction of 6-methoxy quinoline25 with alkyl halide in acetonitrile without base in the reaction. 6-Methoxy-1-methyl quinolinium iodide (26) and 1-benzyl-6-meth-oxy quinolinium iodide (27) were synthesized and examined forthe AChEI activity. They drastically improved the AChE inhibitoryactivity better than the 6-methoxy quinoline (25) with IC50

7.67 ± 1.71 lM and 2.46 ± 0.83 lM, respectively.Compounds 15–27 were chosen to examine for human-AChE

inhibitory activity.15 % Inhibition from human-AChE of all testedcompounds (Table 3) showed a similar profile as the result fromeel-AChE with reduction approximately 2 to 3-fold of % inhibitionat the same concentration. With exception, compound 20, 26 and27 gave comparable % inhibition to eel-species and they gave IC50

as 23.70 ± 6.26, 49.73 ± 9.96 and 5.29 ± 1.99 lM, respectively.The most active compounds of each ring system, compound 20

and 27, were chosen to perform the docking study using Auto-Dock16 to determine possible binding modes. Torpedo califonicaacetylcholinesterase complexed with galantamine (PDB 1DX6)was selected as the AChE template for the validation study. Thedockings were done with the following parameters: grid spacingwas set to 0.375 Å with a grid box of 40 � 40 � 40 points, initialpopulation was set to 150, and the number of evaluations wasset to 2,500,000. The dockings were run 100 times each and theresults clustered at 2 Å RMSD. The docking runs were prepared

Table 2AChE inhibitory activity of b-carboline and quinoline derivatives on eel-AChE (N = 3)

NH

N

CH3

R1

2

345

6

78

9

Compound R % Inhibition (±SD)⁄ IC50 (lM)

17 H 83.19 ± 3.58 20.91 ± 1.6418 OCH3 74.10 ± 2.27 22.89 ± 8.2419 OH⁄⁄⁄ 87.07 ± 2.50 8.02 ± 1.93

NH

N

CH3

R

Compound R % Inhibition (±SD)⁄ IC50 (lM)

20 OCH3 85.52 ± 5.15 4.90 ± 2.1521 OH 88.61 ± 1.20 11.28 ± 0.36

NH

NH

R'

R

Compound R R0 % Inhibition (±SD)⁄ IC50 (lM)

22 H H 60.56 ± 5.48 97.08 ± 7.7023 OCH3 H 43.07 ± 3.24 227.03 ± 47.8524 OCH3 COOH 14.29 ± 5.91 ND⁄⁄

N

H3CO

R

Compound R % Inhibition (±SD)⁄ IC50 (lM)

25 — 0 ND⁄⁄

26 CH3 87.15 ± 1.36 7.67 ± 1.7127 CH2-Ph 99.68 ± 0.55 2.46 ± 0.83

Remark: ⁄ = % Inhibition at concentration 10�4 M.⁄⁄ = Not determine (ND).⁄⁄⁄ = HCl�2H2O salt.

Table 3AChE inhibitory activity of b-carboline and quinoline derivatives on human-AChE(N = 3)

Compound % Inhibition (±SD)⁄ IC50 (lM)

15 14.68 ± 1.19 ND⁄⁄

16 28.97 ± 2.73 ND17 43.54 ± 4.99 ND18 23.75 ± 4.08 ND19 41.86 ± 4.22 ND20 65.89 ± 2.78 23.70 ± 6.2621 49.66 ± 3.10 ND22 46.71 ± 2.41 ND23 13.69 ± 8.64 ND24 13.67 ± 9.45 ND25 4.33 ± 7.51 ND26 61.67 ± 9.61 49.73 ± 9.9627 94.33 ± 1.53 5.29 ± 1.99Galantamine 0.30 ± 0.07

Remark: ⁄ = % Inhibition at concentration 10�4 M.⁄⁄ = Not determine (ND).

Figure 2. The ribbon model and binding site of AChE (PDB 1DX6); (A) Shows thebackbone of AChE catalytic domain with ligand in magenta, (B) Possible bindingmode of compound 20, and (C) Possible binding mode of compound 27. Atom colorsused for studied compounds were demonstrated as follows: red for Oxygen, blue forNitrogen, and grey for Carbon. Figures were generated using AutoDockTools.17

N. Khorana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2885–2888 2887

using AutoDockTools.17 The enzyme model used was prepared andvalidated by redocking with galantamine. The result showed 100%of the docked conformations grouped into a single cluster using anRMSD clustering tolerance of 2.0 Å, and the docked orientation was

close to that of the crystal structure with an RMSD of 0.74 Å. Theredocking result indicated that the prepared AChE protein was agood model for docking studies of the compounds. Figure 2 showedthe best possible binding mode of each compound generated bythe docking. Figure 2A demonstrated the ligand (galantamine)which was colored in magenta bound in the binding pocket ofAChE. The best possible binding mode predicted for compound20 was shown in Figure 2B with a docking energy of �6.94 kcal/mol and a cluster size of 69 out of 100. In this mode, 20 made ahydrogen bond with the backbone CO of Trp84 of AChE. The possi-ble binding between amino acids in AChE crystal structure and 20were shown in Figure 3A.

The best binding mode of 27 was shown in Figure 2C which hada docking energy of �7.51 kcal/mol with a clustering of 44 out of100. In this mode, the quinoline ring of 27 formed a hydrophobicbond with the backbone of Phe330 of AChE. The amino acids ofthe AChE from the crystal structure that binded with 27 were

Figure 4. The superimposed model of galantamine (magenta), compound 20 (blue)and compound 27 (red) in the active site of AChE.

Figure 3. The bound conformation of compound 20 (A) and 27 (B) in the active siteof AChE. All interacting amino acid residues shown as stick and ball models in atomcolors. H-bonding and hydrophobic bond are shown in green line and dash box,respectively.

2888 N. Khorana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2885–2888

shown in Figure 3B. The docking energy from the modeling studygave the corresponding data to the in vitro binding data whichshowed the superior in binding of 27 over 20. Compound 20 and27 were likely to bind in the similar pocket with ganlantamine.Hence, all three compounds were docked with the same enzyme.It was found that there were some overlapped portions at bindingsite among ganlanthamine, 20 and 27 as shown in Figure 4.

A number of commercially available indoles, b-carbolines andsynthetic quinolines were tested in this study. It could be con-cluded that indole rings with substitution only on the benzenering were likely to be too small to fit in the active site of AChE.Adding more bulk to the molecule, as in serotonin or b-carbolineanalogs, could dramatically increase the inhibitory activity onAChE. In the b-carboline structure, reduction of the pyridine ringaffected the conformational change in the molecule which mightbe another factor for the difference of percent inhibition. It alsoinfluenced the difference of the preference of the substitutionon the benzene ring. The quinoline compounds, the bioisotereto that of indole ring, also gave good inhibitory activity for AChE.Both b-carboline and quinoline analogs bound in the same bind-ing site with some slightly overlapped structure. From the dock-ing model, it showed the corresponding information to thein vitro enzyme assay that the tested compounds showed lowerinhibitory activity than galantamine and compound 27 coulddock to enzyme better than 20. Nevertheless, some compoundsof b-carboline and quinoline showed modest binding which mightimply that both ring types could be used for developing AChEinhibitors in the future.

Acknowledgments

The study was supported by National Research Council of Thai-land (NRCT) 2007 and Center of Excellence for Innovation in Chem-istry (PERCH-CIC), Commission on Higher Education, Ministry ofEducation. The authors would like to thank Dr. Rodney Harris formodeling facility.

References and notes

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