Design, synthesis, and evaluation of 3,7-substituted coumarin derivatives as multifunctional Alzheimer’s disease agents

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Design, synthesis, and evaluation of 3,7-

substituted coumarin derivatives as

multifunctional Alzheimer’s disease agents

Sheunopa C. Mzezewa, Sylvester I. Omoruyi, Luke S. Zondagh, Sarel F. Malan,

Okobi E. Ekpo & Jacques Joubert

To cite this article: Sheunopa C. Mzezewa, Sylvester I. Omoruyi, Luke S. Zondagh, Sarel

F. Malan, Okobi E. Ekpo & Jacques Joubert (2021) Design, synthesis, and evaluation of 3,7-

substituted coumarin derivatives as multifunctional Alzheimer’s disease agents, Journal of Enzyme

Inhibition and Medicinal Chemistry, 36:1, 1607-1621, DOI: 10.1080/14756366.2021.1913137

To link to this article: https://doi.org/10.1080/14756366.2021.1913137

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2021, VOL. 36, NO. 1, 1607–1621

https://doi.org/10.1080/14756366.2021.1913137

BRIEF REPORT

Design, synthesis, and evaluation of 3,7-substituted coumarin derivatives as

multifunctional Alzheimer’s disease agents

Sheunopa C. Mzezewa^a, Sylvester I. Omoruyi^b, Luke S. Zondagh^a, Sarel F. Malan^a, Okobi E. Ekpo^band

Jacques Joubert^a

b

^aDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of the Western Cape, Bellville, South Africa; Department of Medical

Biosciences, University of the Western Cape, Bellville, South Africa

ABSTRACT

ARTICLE HISTORY

Received 7 February 2021

Revised 17 March 2021

Accepted 19 March 2021

Multitarget directed ligands (MTDLs) are emerging as promising treatment options for Alzheimer’s disease

(AD). Coumarin derivatives serve as a good starting point for designing MTDLs due to their inherent inhib-

ition of monoamine oxidase (MAO) and cholinesterase enzymes, which are complicit in AD’s complex

pathophysiology. A preliminary series of 3,7-substituted coumarin derivatives were synthesised and eval-

uated for enzyme inhibitory activity, cytotoxicity as well as neuroprotective ability. The results indicated

that the compounds are weak cholinesterase inhibitors with five compounds demonstrating relatively

potent inhibition and selectivity towards MAO-B with IC₅₀values between 0.014 and 0.498 hx00B5;mM.

Significant neuroprotective effects towards MPP^þ-compromised SH-SY5Y neuroblastoma cells were also

observed, with no inherent cytotoxicity at 10 mM for all compounds. The overall results demonstrated that

substitution of the phenylethyloxy moiety at the 7-position imparted superior general activity to the deriv-

atives, with the propargylamine substitution at the 3-position, in particular, displaying the best MAO-B

selectivity and neuroprotection.

KEYWORDS

Alzheimer’s disease;

coumarin; cholinesterase;

monoamine oxidase;

neuroprotection

1. Introduction

galantamine, and rivastigmine (Figure 1)^5,13. The clinical applica-

tion of these agents has demonstrated that cholinesterase inhib-

Neurodegenerative diseases (NDs) are a group of cognitive and

movement-related disorders resulting from neuronal loss in the

brain. Alzheimer’s disease (AD) is the most prevalent ND with over

30 million diagnosed patients worldwide^1,2. Its symptoms are pro-

gressive and irreversible, initially characterised by recurring short-

term memory loss which progresses to degeneration of higher

cognitive functions such as decision making and language. Due to

the nature of these symptoms, the disease comes at a great bur-

ition leads to modest improvement in cognitive functions^14–16

.

Monoamine oxidase (MAO) is a membrane-bound flavoenzyme

responsible for metabolising a variety of substrates throughout

the body such as serotonin, dopamine, and benzylamine. It exists

in two forms, namely MAO-A and MAO-B, with each isoform hav-

ing differing substrate selectivity and distribution throughout the

body. The MAO-B enzyme is the major isoform found in the brain,

den economically and socially^3,4. There is no singular cause or responsible for 80% of MAO activity in the brain. MAO-B has been

process that is responsible for this neurodegeneration, rather it is

the result of different interlinked mechanisms, underpinned by

genetic and environmental factors. This complex pathophysiology

has made treatment difficult and only symptomatic relief is pro-

implicated in the generation of neurotoxic free radicals and react-

ive oxygen species which trigger neuroinflammation and the

apoptotic cascade in neurons^17,18. Studies have found that MAO-B

enzyme activity increases with old age and correlates closely with

vided by the drugs currently available on the market^5–7

.

disease progression, making it a target for AD treatment^19–21

.

Of the multiple theories underlying AD’s pathophysiology, the

roles of oxidative stress, toxic amyloid-b (Ab) plaques, and the

cholinergic hypothesis have gained momentum in explaining

the disease’s pathology and in guiding treatment options^8–10. The

cholinergic hypothesis postulates that the symptoms present in

AD (particularly cognitive decline and amnesia) can be linked to

declining function in the cholinergic neurotransmitter system. The

cholinergic system is modulated by the neurotransmitter acetyl-

choline, metabolised by acetylcholinesterase (AChE) and butyryl-

cholinesterase (BuChE) in the brain^11,12. The primary therapeutic

approach has been to counter cholinergic depletion using acetyl-

cholinesterase inhibitors (AChEIs). This has thus far been done by

Dual inhibition towards the causative MAO and cholinesterase

enzymes has been demonstrated to not only improve cognitive

functions but also to achieve neuroprotection and subsequently

halt disease progression^19,21. Multitarget directed ligands (MTDLs)

provide researchers with a way to use a single drug agent to sim-

ultaneously target the mentioned set of enzymes. MTDLs are

made by combining pharmacophoric groups with differing

pharmacological activities into one molecule to achieve a syner-

gistic therapeutic effect, whilst avoiding the pitfalls of polyphar-

macy. MTDLs are being explored for use in a variety of complex

diseases such as cancer and malaria^7,22. Several promising MTDLs

using the second-generation AChEIs such as donepezil, have been explored for use in AD including ASS234 and ladostigil

CONTACT Jacques Joubert

jjoubert@uwc.ac.za

Department of Pharmaceutical Chemistry, School of Pharmacy, University of the Western Cape, Robert

Sobukwe Road, Private Bag X17, Bellville 7535, South Africa

ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

1608

S. C. MZEZEWA ET AL.

OH

H

O

H₃C

N

O

H₃C

CH₃

N

H₃C

CH₃

Rivastigmine

Galantamine

O

H₃C

N

O

Figure 2. Design strategy of the new coumarin derivatives in this study.

O

CH₃

Donepezil

Figure 1. Commercial AChEIs approved for the treatment of AD.

Analytical (RSA). All solvents used for reactions and chromatog-

raphy were purchased from various commercial sources and

unless mentioned used without further purification. All reactions

were monitored for impurities and completion using thin layer

chromatography performed on sheets of 0.20 mm thick aluminium

silica gel (TLC Silica gel 60 F245 Merck KGaA) with various mobile

phases using volume:volume ratios. Once the plates were devel-

oped, the spots were visualised using UV light (254 nm and

366 nm) or iodine vapour. Purification of the specified compounds

was performed in glass columns filled with appropriate amounts

of silica gel (0.063–0.200 mm/70–230 mesh ASTM, Macherey -

Nagel, Duren, Germany) as the stationary phase and with mobile

phases indicated for each compound. Nuclear magnetic resonance

(NMR) spectroscopy, Fourier Transform Infra-red (FT-IR) spectros-

copy, and High-resolution mass spectrometry (HR-MS) techniques

were used to elucidate the structures of the compounds. ¹H and

¹³C NMR spectra were obtained using a Bruker Avance III HD

Nanobay 400 MHz spectrometer (Rheinstetten, Germany) equipped

with a 5 mm BBO probe at 333 K using standard 1 D NMR pulse

sequence. Tetramethylsilane (TMS) was used as internal standard

and all chemical shifts are reported in parts per million (ppm) rela-

tive to its signal (d ¼ 0) in either deuterated dimethyl sulphoxide

(DMSO-d₆) or deuterated chloroform (CDCl₃). The following abbre-

viations are used to describe the multiplicity of signals: s – singlet;

d – doublet; dd – doublet of doublets; t – triplet; m – multiplet.

The IR data was obtained using a Perkin Elmer Spectrum 400

spectrometer (Waltham, USA) fitted with a diamond attenuated

total reflectance (ATR) attachment linked to a computer system.

HR-MS data were obtained using a Waters API Q-TOF Ultima spec-

trometer (Wilmslow, UK) using the electrospray ionisation tech-

which are based on the dual inhibition of MAO and

cholinesterases^23,24

.

The coumarin scaffold has many intrinsic pharmacological

properties and coumarin derivatives have shown promise as a

starting point for MTDL design strategies due to its ease of func-

tionalisation and inherent ChE and MAO inhibitory capacity^25–27

.

This study, therefore, aimed to further explore the potential of

coumarin derivatives as MTDLs by designing and synthesising a

small series of compounds that have substitutions of moieties

known to enhance inhibition of the MAO and cholinesterase

enzymes (Figure 2).

One such moiety is the benzyloxy moiety that has been found

to enhance the selectivity and potency of the coumarin scaffold

towards MAO-B^28–30. To optimise the coumarins’ activity towards

the target enzymes, a phenylethyl moiety instead of the benzyloxy

moiety was attached to the coumarin scaffold in this case. This

would ensure that the designed compounds are sufficiently elon-

gated to potentially interact with AChE’s peripheral anionic and

catalytic binding sites (also known as dual-site binding), whilst

preserving MAO-B binding interactions³¹.

Similarly, the incorporation of the carbamate moiety onto the

coumarin scaffold was investigated as well for this study.

Numerous studies have found that it confers significant ChE

inhibitory effect into various molecules which incorporate it,

including the promising MTDL ladostigil^24,32–34. These selected

moieties were to be incorporated at the 7-position of the scaffold,

as substitutions at this position are most beneficial for MAO-B

selectivity and inhibition^29,35,36

.

R

nique. Melting points were determined by using a Lasec^VMelting

Propargylamine, a moiety known for its capacity to confer neu-

roprotection and increase MAO inhibition was also selected to be

substituted at position 3 to enhance the pharmacological profiles

of the 7-substituted coumarins^37,38. Position 3 of the scaffold has

previously been described to be beneficial in achieving dual-site

binding in AChE’s active site, thus substitutions at this position

would serve an important role in producing dual ChE/MAO inhibi-

tors^38,39. These molecules (Figure 2) could have the potential to

relieve the symptoms of AD, slow down disease progression and

ultimately reduce the burden of the disease.

Point SMP 10 apparatus (Staffordshire, UK) and glass capillary

tubes. All experiments involving the use of microwave-assisted

methods were performed using a CEM Discover^TMclosed vessel

system (Buckingham, UK). The settings used were custom and spe-

cific for each experiment.

2.1.2. Synthesis of 7-hydroxy-3-methyl-2H-1-benzopyran-2-one (1)

A mixture of 2,4-dihydroxybenzaldehyde (7.2 mmol, 1.0 g), sodium

propionate (15.6 mmol, 1.5 g), propionic anhydride (19.4 mmol,

2.5 ml), and piperidine (1 mmol, 0.1 ml) was refluxed for 6 h and

then poured onto ice. The aqueous mixture was made acidic with

20 ml of a 0.1 N solution HCl and yielded a precipitate that was fil-

tered and treated under stirring with concentrated H₂SO₄(2 ml).

The resulting mixture was poured into ice again to afford a red-

brown precipitate of the desired product 1.

2. Materials and methods

2.1. Chemistry

2.1.1. General

All reagents used in the synthesis of the compounds were

Physical Properties: Yield: 60%. mp: 165 ^ꢀC. ¹H NMR (400 MHz,

R

obtained from Sigma-Aldrich^V(Steinheim, Germany) and Industrial DMSO-d₆): d 7.72 (s, 1H), 7.42–7.40 (d, 1H, J ¼ 8.5 Hz), 6.76–6.73

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1609

(dd, 1H, J ¼ 2.2, 8.4 Hz), 6.69–6.68 (d, 1H, J ¼ 2.1 Hz), 2.02 (s, 3H). temperature for 7 h and once the reaction was complete the suc-

¹³C NMR (100 MHz, DMSO-d₆): d 161.8, 160.2, 154.5, 140.1, 128.7, cinimide residue was filtered out. The solvent was removed in

120.0, 113.0, 111.8, 101.9, 16.5. HR-ESI [M þ H]^þ: calc. 175.0395, vacuo and the crude product was purified via flash column chro-

exp. 175.0398. FT–IR (ATR): ꢀ_max(cm^ꢁ1

1675, 1451.

)

3234, 2934, 1739, matography (mobile Phase; hexane:ethyl acetate:chloroform, 4:4:1).

An orange solid of compound 4 was obtained.

Physical Properties: Yield: 43%. mp: 101–105 ^ꢀC. ¹H NMR

(400 MHz, CDCl₃): d 7.70 (s, 1H), 7.43–7.27 (m, 6H), 6.86–6.83 (dd,

2.1.3. Synthesis of 3-methyl-7-(2-phenylethoxy)-2H-1-benzopyran-

2-one (2)

1H, J ¼ 2.3, 8.58 Hz), 6.81–6.80 (d, 1H, J ¼ 2.1 Hz), 4.41 (s, 2H),

4.25–4.21 (t, 2H, J ¼ 7.0), 3.14–3.11 (t, 2H, J ¼ 7.1). ¹³C NMR

(100 MHz, CDCl₃): 142.2, 129.0, 128.9, 128.9, 128.6, 128.6, 127.8,

126.8, 121.7, 113.4. 112.7, 101.3, 101.2, 69.3, 69.1, 35.5, 35.5, 28.2.

HR-ESI [M þ H]^þ: calc. 359.0283, exp. 359.0298. FT–IR (ATR): ꢀ_max

(cm^ꢁ1) 3029, 2918, 1707 1158, 698.

A microwave-compatible glass-vessel was charged with 200 mg

(0.71 mmol) of 7-hydroxy-3-methyl-2H-1-benzopyran-2-one (1),

68 mg (2.8 mmol) NaH (80% dispersion in oil), and dissolved in

10 ml of acetonitrile. To this mixture, 570 ml (4.2 mmol) of 2-bro-

moethylbenzene was added dropwise and subsequently stirred at

80 ^ꢀC for 5 h under microwave irradiation (maximum power ¼

150 W). The solvent was removed in vacuo and the resulting crude

mixture was washed with 30 ml ethyl acetate and 15 ml water.

The organic layer was washed with 15 ml of 1 M KOH and then

15 ml of brine, thereafter the solvent was removed in vacuo. The

2.1.6. Synthesis of 3-(bromomethyl)-2-oxo-2H-1-benzopyran-7-yl-

diethylcarbamate (5)

N-Bromosuccinimide (323 mg, 1.81 mmol) was added to a suspen-

sion of 3-methyl-2-oxo-2H-1-benzopyran-7-yl-diethylcarbamate

crude product was then purified using column chromatography (250 mg, 0.91 mmol) (3) in 9 ml CCl₄. Following this benzoyl perox-

ide (75%) (192 mg, 0.80 mmol) was added to the mixture. After

(mobile phase; hexane: ethyl acetate 3:1).

Physical Properties: Yield: 37%. mp: 89–92 ^ꢀC. ¹H NMR heating at reflux for 15 h, the hot reaction mixture was filtered to

(400 MHz, CDCl₃): d 7.39 (s, 1H), 7.31–7.22 (m, 6H), 6.78–6.59 (m, remove the succinimide by-product. The solvent was removed in

2H), 4.17 (t, 2H, J ¼ 7.0), 3.10–3.06 (t, 2H, J ¼ 7.1), 2.13 (s, 3H). ¹³C vacuo and the crude product was purified via flash column chro-

NMR (100 MHz, CDCl₃): 162.6, 160.9, 154.8, 139.3, 137.7, 128.9, matography (Mobile Phase; DCM: hexane: ethyl acetate, 4:3:1) to

128.6, 127.8, 126.7, 122.1, 113.2, 112.7, 101.2, 69.1, 35.5, 16.9. HR- obtain a white solid.

ESI [M þ H] ^þ: calc. 281.1178, exp. 281.1171. FT–IR (ATR): ꢀ_max

Physical properties: Yield: 15%. mp: 84–88 ^ꢀC. ¹H NMR

(400 MHz, CDCl₃): d 7.83 (s, 1H), 7.48–7.46 (d, 1H, J ¼ 8.6 Hz), 7.15

(d, 1H, J ¼ 1.9 Hz), 7.13–7.10 (dd, 1H, J ¼ 2.1, 8.5 Hz), 4.42 (s, 2H),

3.45–3.38 (m, 4H), 1.28–1.19 (m, 6H). ¹³C NMR (100 MHz, CDCl₃):

154.6, 154.4, 141.6, 128.5, 124.3, 118.9, 116.0, 110.2, 42.4, 42.1,

40.6, 35.1, 28.9, 27.7, 14.3, 13.3. HR-ESI [M þ H]^þ: calc. 354.0341,

(cm^ꢁ1) 3026, 2850, 1612, 1284, 1247.

2.1.4. Synthesis of 3-methyl-2-oxo-2H-1-benzopyran-7-yl-diethyl-

carbamate (3)

A microwave compatible glass-vessel was charged with 250 mg

(1.4 mmol) of 7-hydroxy-3-methyl-2H-1-benzopyran-2-one (1),

295 mg (2.1 mmol) of K₂CO_3,and a catalytic amount of TBAHSO₄

and dissolved in 10 ml of acetonitrile. Following this, 385 mg

(2.8 mmol) of diethyl carbamoyl chloride was added dropwise and

exp. 354.0344. FT–IR(ATR): ꢀ_max(cm^ꢁ1

) 2979, 2920, 1615,

1246, 1147.

2.1.7. Synthesis of 7-(2-phenylethoxy)-3-f[(prop-2-yn-1-yl) amino]

stirred at 100 ^ꢀC for 2.5 h under microwave irradiation (maximum methylg-2H-1-benzopyran-2-one (6)

power ¼ 150 W). Once the reaction was complete, K₂CO₃was fil- A mixture of 115 mg (0.32 mmol) of 3-(bromomethyl)-7-(2-phenyle-

tered out and the solvent was removed in vacuo. The crude mix- thoxy)-2H-1-benzopyran-2-one (5) and 441 mg (3.2 mmol) of K₂CO₃

ture was dissolved in 30 ml ethyl acetate and 15 ml water and was dissolved in 5 ml dried THF. Propargylamine (44 mg, 0.8 mmol)

transferred to a separatory funnel. The layers were separated and was added dropwise and the mixture was stirred at room tem-

the organic layer was washed with 15 ml of 1 M KOH and then perature for 48 h. The K₂CO₃was filtered off and the solvent was

15 ml of water. The combined aqueous layers were extracted with removed under reduced pressure. The resulting crude product

ethyl acetate (3 ꢂ 20 ml). The combined organic layers were then was purified using column chromatography (Mobile phase; hex-

washed with brine and concentrated under reduced pressure. The ane: ethyl acetate 2:1) and the product was obtained as a waxy

product was obtained as an amber coloured oil.

amber solid.

Physical Properties: Yield: 65%. mp: wax. ¹H NMR (400 MHz,

Physical Properties: Yield: 30%. mp: wax. ¹H NMR (400 MHz,

DMSO-d₆): d 7.85 (s, 1H), 7.62–7.60 (d, 1H, J ¼ 8.4 Hz), 7.20–7.19 (d, CDCl₃): d 7.70 (t, 1H, J ¼ 11.8 Hz), 7.36–7.27 (m, 6H), 6.84–6.81 (dd,

1H, J ¼ 2.1 Hz), 7.11–7.09 (dd, 1H, J ¼ 2.2, 8.5 Hz), 3.42–3.37 (m, 2H), 1H, J ¼ 2.4, 8.4 Hz), 6.80–6.78 (d, 1H, J ¼ 2.2H), 4.23– 4.20 (t, 2H,

3.36–3.28 (m, 2H), 2.08 (s, 3H), 1.21–1.10 (t, 6H, J ¼ 6.6). ¹³C NMR J ¼ 6.9 Hz), 3.82 (s, 2H), 3.52 (d, 2H, J ¼ 2.4), 3.14–3.1 (t, 2H,

(100 MHz, CDCl₃): 161.9, 153.5, 153.2, 153.0, 138.7, 127.2, J ¼ 7 Hz), 2.28 (t, 1H, J ¼ 2.4 Hz). ¹³C NMR (100 MHz, CDCl₃): 161.8,

124.5, 118.3, 116.6, 109.8, 42.3, 41.9, 29.6, 16.9, 14.1, 13.2. HR-ESI 161.7, 155.1, 140.5, 137.6, 128.9, 128.7, 128.6, 126.7, 122.2, 113.1,

112.7, 101.2, 80.8, 72.5, 69.2, 47.7, 3.4, 35.5. HR-ESI [M þ H]^þ: calc.

334.1443, exp. 334.1450. FT–IR(ATR): ꢀ_max(cm^ꢁ1) 3284, 3027, 2922,

2853, 1706, 1237.

þ:

[M þ H]

calc. 276.1236, exp. 276.1235. FT–IR (ATR): ꢀ_max(cm^ꢁ1

)

2920, 1708, 1471, 1316, 1240.

2.1.5. Synthesis of 3-(bromomethyl)-7-(2-phenylethoxy)-2H-1-ben-

zopyran-2-one (4)

A mixture of 114 mg (0.64 mmol) of N-bromosuccinimide, 150 mg 1-benzopyran-7-yl-diethylcarbamate (7)

2.1.8. Synthesis of 2-oxo-3-f[(prop-2-yn-1-yl) amino] methylg-2H-

(0.54 mmol) of 3-methyl-7-(2-phenylethoxy)-2H-1-benzopyran-2- 3-(Bromomethyl)-2-oxo-2H-1-benzopyran-7-yl-diethylcarbamate (4)

one (2) and 33 mg (0.14 mmol) of benzoyl peroxide (75%) were (120 mg, 0.34 mmol) was dissolved in 2.5 ml of THF before adding

dissolved in 7 ml CCl₄. The reaction vessel was stirred at room 480 mg (3.4 mmol) of K₂CO₃and 38 mg (0.68 mmol) of

1610

S. C. MZEZEWA ET AL.

propargylamine. The mixture was stirred at room temperature for NaCl) was prepared and refrigerated until further use. The enzyme

48 h following which the inorganic residue was filtered off after stock solutions were prepared by dissolving 2.5 mg of enzyme in

33.33 ml of phosphate buffer to produce stock solutions of

0.075 mg/ml which were stored in aliquots at ꢁ80 ^ꢀC until use.

Once in the final reaction mixture, this enzyme solution will have

a concentration of 0.0075 mg/ml. The substrate was prepared by

dissolving kynuramine in potassium buffer and was prepared as

two separate concentrations; 750 mM for MAO-A and

500 hx00B5;M for MAO-B. When placed in the reaction mixture,

these solutions ended up with final concentrations of 45 mM for

MAO-A and 30 mM for MAO-B respectively. The fresh substrate was

prepared on the day of the assay. The assay was carried out in

2 ml Eppendorf vials. To each vial, 207.5 hx00B5;l of phosphate

buffer was added followed by 2.5 hx00B5;l of the respective test

compound or control (positive control: clorgiline for MAO-A or

rasagiline for MAO-B; control: DMSO). Following this, 25 ml of the

enzyme stock were added to each vial in 10 s intervals and the

vials were incubated at 37 ^ꢀC for 10 min. After this time period

15 ml of kynuramine (750 mM for MAO-A and 500 mM for MAO-B)

was added at 10 s intervals and further incubated for 20 min. To

stop the reaction at the end of this time 150 hx00B5;l of 2 N NaOH

was added and the mixture was shaken. Using a micropipette

80 hx00B5;l of the mixture was transferred to a well in a black 96

well plate. The plate was placed in the fluorescent plate reader

and read at an excitation/emission wavelength of 310 nm/410 nm.

Each assay was run in triplicate and the data were analysed with

Graph Pad Prism 8 software (San Diego, USA).

washing with THF. The resulting solution was concentrated in

vacuo and purified using column chromatography (Mobile phase;

DCM: hexane: ethyl acetate in a 20:1:1 ratio) and an off-white solid

was obtained.

Physical Properties: Yield: 21%. mp: 124–126^ꢀC. ¹H NMR

(400 MHz, CDCl₃): d 7.72 (s, 1H), 7.46–7.44 (m, 1H), 7.13 (d, 1H,

J ¼ 2.0 Hz), 7.10–7.07 (dd, 1H, J ¼ 2.2, 8.4 Hz), 3.83 (s, 2H), 3.50 (s,

2H), 3.45–3.38 (m, 4H), 2.26–2.25 (t, 1H, J ¼ 2.1 Hz), 1.28–1.19 (m,

8H). ¹³C NMR (100 MHz, CDCl₃): 161.2, 153.9, 153.8, 153.2, 140.7,

128.4, 124.1, 118.7, 116.5, 109.9, 77.8, 74.5, 52.5, 42.9, 42.4, 42.0,

29.7, 14.3, 13.3. HR-ESI [M þ H]^þ: calc. 329.1496, exp. 329.1495.

FT–IR(ATR): ꢀ_max(cm^ꢁ1) 2921, 2851, 1634, 1616, 1245.

2.2. Biological studies

2.2.1. Cholinesterases assay

Cholinesterase activity is determined using a modified colorimetric

method first described by Ellman⁴⁰. The test compounds and posi-

tive control (donepezil) were dissolved in DMSO to prepare 10 mM

stock solutions. The stock was then further diluted by factors of

ten to produce solutions of 1 mM, 100 mM, 10 mM, 1 mM, and

0.1 mM which corresponds to concentrations of 100 mM, 10 mM,

1 mM, 0.1 mM, and 0.01 mM in the final reaction mixture. The test

concentrations were stored in the refrigerator until the day of the

assay. Trisma HCl buffer (50 mM, adjusted to pH 8 with 2 N NaOH)

was prepared as the buffer and used to prepare enzyme stock

solution (22 U/ml for Electrophorus electricus AChE and 12 U/ml

BuChE from equine serum), 15 mM acetylthiocholine iodide,

15 mM S-butyrylthiocholine iodide and 1.5 mM DTNB (Ellman’s

reagent). The enzyme stock solution was stabilised with 1%

bovine serum albumin and stored in aliquots at ꢁ80 ^ꢀC. A clear,

flat bottom 96 well plate was used to perform the experiments

with each concentration performed in triplicate. Prior to the assay

the background absorbance for each compound at the different

test concentrations was measured and accounted for when read-

ing the final absorbance. Before performing the assay, the enzyme

was diluted to 0.88 U/ml for AChE and 0.48 U/ml for BuChE. In

each well 148 ml DTNB, 50 ml of the enzyme and 2 ml of the test

compound or control (donepezil in the positive control and DMSO

in the negative control well) were added and incubated at 25 ^ꢀC

for 10 min. The concentration of DMSO was kept constant at less

than 1% in order not to influence the assay results. Following this,

30 ml of the substrate was added to each well simultaneously

2.2.3. MAO-B reversibility study

To investigate the reversibility of the observed MAO enzyme

inhibition, time-dependent inhibition studies were carried out on

compounds 6 and 7. Briefly, the recombinant human MAO-B

(0.0075 mg/ml) enzyme was preincubated with either 6 or 7 for

periods of 0, 15, 30, 60 min at 37 ^ꢀC. The concentration of inhibitor

in these incubations was approximately twofold the measured

IC₅₀value for the inhibition of the MAO-B enzyme. Potassium

phosphate buffer (100 mM, pH 7.4, made isotonic with NaCl) was

used as the incubation medium.

A final concentration of

30 hx00B5;M kynuramine for MAO-B was then added to the prein-

cubated enzyme preparations and the resulting 250 ml reactions

were incubated at 37 ^ꢀC for 15 min. The reactions were terminated

with the addition of 200 ml NaOH (2 N) and a volume of 600 ml dis-

tilled water was added to each reaction. The fluorescence of the

MAO-B generated 4-hydroxyquinoline in the supernatant fractions

were measured using a fluorescent microplate reader at an excita-

tion wavelength of 310 nm and an emission wavelength of

400 nm. Time-dependent graphs were plotted against the relative

fluorescent unit readings (RFU).

R

using a multi pipette. The plate was then placed in a Rayto^VRT-

2100C microplate reader and the absorbances were measured at

405 nm every 60 s over a 20-min period. The data obtained was

used to calculate the maximum inhibition over the 20-min period

and this was used to plot a dose-response graph, from which the

IC₅₀values of the compounds could be extrapolated. A one-sam-

ple t-test was performed on the data to test for statistical

significance.

2.2.4. Cell line and culture conditions

The human neuroblastoma SH-SY5Y cells were generously

donated by collaborators from the Blackburn Laboratory,

University of Cape Town. Cells were cultured in monolayer in

Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Life

Technologies Corporation UK), supplemented with 10% foetal

bovine serum (FBS, Gibco, Life Technologies Corporation, Paisley,

UK), 100 U/ml penicillin, and 100 mg/ml streptomycin (Lonza Group

Ltd., Verviers Belgium). Cultures were incubated at 37 ^ꢀC in a

humidified atmosphere with 5% CO₂. The growth medium was

replaced every 3 days and cells were sub-cultured when they

2.2.2. MAO assays

The test compounds and positive control (clorgiline for MAO-A

and selegiline for MAO-B) were dissolved in DMSO to prepare

10 mM stock solutions. The stock was then further diluted by fac-

tors of ten to produce solutions of 1 mM, 100 mM, 10 mM, 1 mM,

and 0.1 mM which would correspond to concentrations of 100 mM,

10 mM, 1 mM, 0.1 mM, and 0.01 mM in the final reaction mixture. A attained 70 to 80% confluency using a solution of 0.25% trypsin

potassium phosphate buffer (KH₂PO₃100 mM, pH 7.4, 0.9% w/v EDTA (Lonza Group Ltd., Verviers, Belgium).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1611

2.2.5. MTT assay

prepared enzyme was then saved in this state for use in the dock-

ing studies. The test ligands were drawn using ChemSketch 2016

SH-SY5Y cells were plated in growth medium in the flat bottom

96 well plates at a density of approximately 7500 cells/well. The software, saved as MDL files (V3000) and prepared for docking by

cells were allowed to adhere to the plate surface for 24 h and fol-

lowing this their media were replenished with fresh media con-

taining the test compounds at 10 mM, 50 mM and 100 mM

concentrations dissolved in DMSO. Vehicle control cells were

treated with DMSO, at a concentration similar to that of the high-

est concentration of the test compounds. Following an incubation

period of 48 h, 10 ml of MTT solution (5 mg/ml) was added to each

well. This was further incubated for 4 h and the formazan formed

was solubilised with 100 ml of DMSO. The plates were read using a

energy minimising them in MOE using the MMFF94 force field. A

database of these compounds was created and docking per-

formed. Poses were generated using Triangle Matcher placement

method and rescored using the ASE scoring function. The best

poses were visually inspected and binding interactions analysed.

To confirm the accuracy of the protocol it was repeated three

times and generated RMSD values <2 Å from the co-crystallised

ligand. This showed that the protocol could correctly predict the

binding orientations.

R

BMG Labtech Omega^VPOLARStar plate reader spectrophotometer

(Ortenberg, Germany) to determine the absorbance recorded at a

wavelength of 570 nm. The percentage cell viability was calculated 2.3.2. MAO docking studies

relative to the vehicle control using the following formula:

Docking of the ligands was performed with MOE 2018. The co-

crystallised MAO enzymes were obtained from the PSLIO/PDB

data bank with MAO-A co-crystallised with clorgiline (Code: 2BXS)

and MAO-B co-crystallised with safinamide (Code: 2V5Z). Prior to

the studies, the enzyme was first checked for missing atoms and

bonds. Partial charges, ionisation state, and hydrogens were

added to the macromolecular structure using the 3D protonation

function in the application. The co-crystallised ligand was then

selected to identify which binding site to use as the pocket for

docking. The prepared enzyme was then saved in this state for

use in docking studies. The test ligands were drawn using the

ChemSketch 2016 software, saved as MDL files (V3000) and pre-

pared for docking by energy minimising them in MOE using the

MMFF94 force field. A database of these compounds was created

and docking performed. Poses were generated using the Triangle

Matcher placement method and rescored using the ASE scoring

function. The best poses were visually inspected and binding

interactions analysed. To confirm the accuracy of the protocol it

was repeated three times and generated RMSD values < 1.5 Å

ꢀ

ꢁ

Absorbanceoftreatedwell

Absorbanceofuntreatedwell

Cellviability% ¼

ꢂ 100

2.2.6. Neuroprotection studies

2.2.6.1. MPP^þinduced neurotoxicity. SH-SY5Y cells were seeded

onto a 96-well plate in a growth medium at a density of 10,000

cells/well. Cells were pre-treated with test compounds at a con-

centration of 10 mM and thereafter 2000 mM MPP^þfollowing a 2-h

incubation period. Afterwards, cells were incubated for 24 h and

MTT colorimetric assay was used to measure cell viability relative

to untreated control. To determine the data’s statistical signifi-

cance, the One-way ANOVA Turkey’s multiple comparison test was

R

conducted on all experimental replicates using GraphPad Prism^V

(San Diego, USA).

8

2.2.6.2. Amyloid-beta 25–35 induced neurotoxicity. Human neuro- from the co-crystallised ligand. This showed that the protocol

blastoma cells (SH-SY5Y) were grown in

a culture medium

could correctly predict the binding orientations.

(DMEM/F-12) and supplemented with 10% foetal bovine serum.

Prophylactically, 1% v/v of penicillin/streptomycin was added to

the medium. Cells were maintained by incubation at 37 ^ꢀC with

5% carbon dioxide. All experiments were performed by seeding

the cells into 24-well plates. Once confluent, the cells were then

pre-treated with compounds 6 and 7 (10 mM) for 1 h, followed by

Ab 25–35 addition (20 mM) and incubated for 24 h. The untreated

control cells were treated with DMEM which contained DMSO and

were incubated in the same manner as the treated cells. After

incubation for the set period of time the MTT colorimetric assay,

as discussed under 2.2.5, was used to measure cell viability rela-

tive to untreated control. The level of significance was analysed

using One-way ANOVA statistical analysis and expressed using the

p-value.

2.4. In silico pharmacokinetic and drug-likeness evaluation

To evaluate the drug-likeness and pharmacokinetic profiles of

compounds 6 and 7, the SwissADME web tool (http://www.swis-

sadme.ch/) was utilised. The structures were drawn using

ChemSketch (2016) and saved as MDL molfiles. The files were con-

verted to SMILES (simplified molecular-input line-entry system) for-

mat which were then input into the web tool and the

pharmacokinetic parameters were generated.

3. Results and discussion

3.1. Chemistry

The synthesis of the compounds (Scheme 1) commenced with a

Pechmann condensation of dihydroxybenzaldehyde to obtain 7-

hydroxy-3-methyl-coumarin (1) which served as the base scaffold.

This scaffold was chosen due to the ease of substitution that the

–OH group at position 7 allows. Simultaneously, the poor reactiv-

ity of the 3-CH₃group protects the scaffold from unwanted substi-

tution reactions occurring during subsequent reactions.

Compound 1 was then functionalised at position 7 via either an

etherification reaction with 2-bromoethylbenzene (2) or carbamate

formation with diethyl carbamate (3). The synthesis of both 2 and

3 was optimised with the use of microwave (MW) irradiation to

2.3. Molecular modelling

2.3.1. AChE docking studies

Molecular Operating Environment (MOE) 2018 on a Windows plat-

form was used for the docking studies. The crystal structure of the

AChE enzyme was obtained from the PSLIO/PDB data bank co-

crystallised with donepezil (Code: 1EVE). Prior to the studies the

enzyme was first checked for missing atoms and bonds. Partial

charges, ionisation state and hydrogens were added to the macro-

molecular structure using the 3D protonation function in the MOE

application. The co-crystallised ligand was then selected to iden-

tify which binding site to use as the pocket for docking. The assist these S_N2 reactions, following the failure of traditional

1612

S. C. MZEZEWA ET AL.

Br

O

CH

O

3

(f)

(d)

N

H

CH

O

2

6

4

(b)

CH

O

3

(a)

HO

O

OH

HO

1

(c)

Br

O

(f)

O

NH

CH

O

3

(e)

CH

O

H C

N

H C

N

O

3

H C

N

O

3

H C

3

H C

5

3

7

Scheme 1. Synthesis pathway of the test compounds. Reagents and conditions: (a) Sodium propionate, propionic anhydride, piperidine, reflux, 6 h; (b) 2-bromoethyl-

benzene, NaH, acetonitrile, MW @ 150 W, 80 ^ꢀC, 5 h; (c) diethyl carbamoyl chloride, K₂CO₃, acetonitrile, MW @ 150W, 80 ^ꢀC, 2.5 h; (d) N-bromosuccinimide, benzoyl per-

oxide, CCl₄, stir at room temperature, 7 h; (e) N-bromosuccinimide, benzoyl peroxide, CCl₄, reflux, 15 h; (f) propargylamine, K₂CO₃, dry THF, stir for 48 h.

in compounds 6 and 7 was confirmed by the disappearance of

the singlet representing 3-CH₂Br present in the spectra of precur-

sor compounds 4 and 5. In its place, a doublet (for –CH₂) and trip-

let (for CH) appeared. ¹³C NMR data were used to confirm the

number of carbons in each structure and their respective environ-

ments corresponded with what was expected for the structures.

The FT-IR data were used to qualitatively identify the functional

groups present and compounds with similar substitutions had

spectra with similar signal patterns. HR-MS data confirmed the

molecular mass as well as molecular formulae of the compounds.

Table 1. Optimisation of reaction conditions using microwave-assisted methods

for compounds 2 and 3.

Compound

2

Conditions

Ethanol, NaH

Yield^a

0%

Stir at room temp., 11 h

Acetonitrile, NaH

37%

0%

MW @ 150 W, 80 ^ꢀC, 5 h

Dimethoxyethane, K₂CO₃

Stir at room temp., overnight

Dimethoxyethane, K₂CO₃

Reflux, 12 h

3

9%

Acetonitrile, K₂CO₃

27%

65%

MW @ 150 W, 100 ^ꢀC, 6 h

Acetonitrile, K₂CO₃, TBAHSO₄

MW @ 150 W, 100 ^ꢀC, 2.5 h

3.2. Biological evaluations

^aPercentage yield was calculated from the amount of 7-hydroxy-3-methyl-cou-

marin (1) used.

3.2.1. MAO inhibition studies

The MAO inhibitory activity of the compounds was assessed using

a fluorometric assay with recombinant human MAO-A and human

MAO-B enzymes. The compounds demonstrated good inhibition

of both MAO isoforms (Table 2). Against MAO-A, compounds 2, 4,

and 5 performed the best, displaying IC₅₀values between 0.36

and 0.66 mM. Compounds 4 and 5 demonstrate that replacing the

3-CH₃with 3-CH₂Br produced a significant increase in activity of

the compounds, with the lowest IC₅₀values of 0.48 mM and

0.36 mM for 4 and 5, respectively. Subsequent replacement of 3-

CH₂Br with the 3-propargylamine moiety led to a decrease in

activity towards MAO-A of several magnitudes as seen with com-

pounds 6 and 7.

Comparatively, the compounds performed significantly better

against MAO-B with five of the test compounds displaying nano-

molar IC₅₀values (Table 2). Compounds 2 and 6 performed best

in this assay with potent IC₅₀values of 14 nM and 29 nM, respect-

ively. The calculated selectivity index (SI) showed that the com-

pounds exhibit selectivity towards MAO-B, with the exception of

compound 4. This is quite important as this is the isoform found

predominantly in the brain and avoids the potential of a tyram-

ine-induced hypertensive crisis developing as seen with MAO-

A inhibitors.

synthetic methods (such as reflux) which took longer and pro-

duced inferior yields (Table 1). The 7-substituted compounds were

then a-brominated with N-bromosuccinimide to produce a -CH₂Br

group (compounds 4 and 5) at position 3, which further facilitated

the substitution of a propargylamine moiety to produce either

compound 6 or 7.

All of the synthesised compounds were characterised using

routine spectroscopic methods (NMR, IR, and MS). The ¹H NMR

spectra of all the synthesised compounds presented two singlets

which were paired with a set of two doublets between 7.1 ppm

and 7.8 ppm. This set of peaks is characteristic of the four aro-

matic protons found in the 3,7-substituted coumarin nucleus and

was largely conserved across all the compounds. The –N(CH₂CH₃)₂

chain present in the 7-carbamate derivatives (3, 5, and 7)

appeared as

a

pair of multiplets at 3.3–3.5 ppm and

1.20–1.28 ppm. These multiplets are representative of the chem-

ically equivalent –CH₂and –CH₃groups on this alkyl chain,

respectively. Likewise, the spectra for the 7-phenylethyloxy deriva-

tives (2, 4, and 6) had a characteristic multiplet representing the

five hydrogens of the phenyl moiety, which appeared in the aro-

matic region. The alkyl chain linking this phenyl ring to the cou-

marin nucleus was represented by two sets of triplets between

3.1 ppm and 4.2 ppm. This relative downfield shift is due to the

The results confirm that the initial substitutions to position 7

increased both the inhibitory capacity and MAO-B selectivity of

the coumarin scaffold. The phenylethyloxy moiety conferred

deshielding effect caused by the oxygen atom in the ether linkage superior inhibition and selectivity when compared to the carba-

with the coumarin nucleus. The presence of a 3-propargyl moiety mate moiety in this regard (see Table 2). Incorporation of the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1613

Table 2. In vitro biological activities of the synthesised and reference compounds.

Compound

AChE IC₅₀(mM)

BuChE IC₅₀(mM)

MAO-A IC₅₀(mM)

MAO-B IC₅₀(mM)

SI MAO-B^a(mM)

MPP^þneuroprotection^b(10 mM)

Donepezil

Clorgiline

Selegiline

1

2

3

4

5

6

7

0.007

n.d.

>100

108

>100

4.40

n.d.

0.001

n.d.

0.010

16.36

0.014

0.498

>100

0.333

0.029

0.101

20.25

>100

23.27

>100

33.40

0.658

2.822

0.476

0.355

3.86

2.04

47.7

5.67

n.d.

1.05

133.2

205.9

26.24%

24.85%

15.59%

22.11%

21.86%

35.96%

28.19%

20.80

^aMAO-B selectivity index ¼ IC₅₀(MAO-A)/IC₅₀(MAO-B). ^bPercentage neuroprotection values calculated as the difference between the final percentage cell viability of

the test compound treated cell line and that of the MPP^þonly treated cell line. n.d. ¼ not determined

Figure 3. (a) Diagrammatic representation of compound 1’s lack of binding interactions with amino acids in MAO-A’s active site. (b) 3D docking simulation showing 1

indicated in pink within the MAO-A active site.

progylamine moiety appears to not only increase the derivatives

potency towards MAO-B, but also has the greatest influence on

the compounds’ selectivity. The 3-propargylamine derivatives 6

and 7 displayed between 130 and 206 times greater selectivity to

MAO-B when compared to MAO-A. Together, these results show

that whereas, compounds 2 was more active in the inhibition of

MAO-B, compounds 6 and 7 showed better selectivity and this

can be attributed to the addition of the propargylamine moiety.

Figure 5 shows that the addition of the propargylamine scaf-

fold produced molecules that are unable to fit into MAO-As active

site. Docking conformations of compounds 6 and 7 demonstrate

that the molecules lack the flexibility to fit and subsequently inter-

act with amino acids in the cavity’s active site. This correlates with

the relatively poor MAO-A inhibitory capacity of these compounds

reported in the assays (Table 2).

3.2.2.2. MAO-B docking studies. Contrary to MAO-A, for MAO-B it

can be observed that the coumarin nucleus 1 fits well into the

entrance cavity, forming relatively stable p–H bonds between the

aromatic ring and the amino acids Ile 199 and Leu 171 (Figure 6).

This hydrogen bond with the Leu 171 residue on the fringe of the

substrate cavity was observed and conserved for the rest of the

compounds and may explain their selectivity towards MAO-B. The

7-phenylethyloxy derivatives’ (particularly compound 2) good

activity and selectivity can be attributed to the previously noted

interactions with Leu 171 coupled with new stable p–H bonds

which form between the phenylethyl moiety and the Ile 199 resi-

due. This interaction is important as the Ile 199 residue is critical

in forming part of the gating mechanism which allows access to

MAO-B’s substrate cavity⁴¹. The 7-carbamate compounds retain

the p–H interaction with Leu 171 which was first observed with 1,

however, they lack the hydrogen bond with Ile 199 and this may

account for their lower activity compared to their 7-phenylethy-

loxy counterparts.

3.2.2. MAO molecular modelling studies

3.2.2.1. MAO-A docking studies. Molecular Operating Environment

(MOE) was used to perform docking studies with the test com-

pounds in an attempt to shed light on the preliminary structure-

activity relationships responsible for the results obtained in the

assay. When the base scaffold, compound 1, was docked within

the MAO-A active site it was observed that the coumarin nucleus

simply enters the entrance cavity, with no significant binding

interactions taking place (Figure 3). This lack of binding correlates

to the compound’s low activity observed in the assay. The dock-

ing simulations also confirmed that the superior activity of the 7-

phenylethyloxy derivatives arose from the phenylethyloxy moiety

allowing compounds 2 and 4 to assume orientations that occupy

MAO-A’s rounder-shaped substrate cavity better than the other

compounds (Figure 4). By contrast, the 7-carbamate derivatives 3

and 5 are seen to only exhibit weak sidechain interactions with

the Cys 323 residue (near the entrance of the catalytic site) and

the oxygen atom on the carbamate moiety.

Similarly, in contrast to MAO-A, the elongated nature of the

MAO-B active site allows for better accommodation of the 3-

1614

S. C. MZEZEWA ET AL.

Figure 4. (a) 2D representation showing the 7-phenylethyl derivative 2’s orientation in MAO-A’s substrate cavity. (b) 3D docking simulation showing 2 indicated in

pink within the MAO-A active site.

Figure 5. (a) 2D interaction map showing compound 7’s orientation and binding in MAO-A’s substrate cavity. (b) 3D docking simulation showing 2 indicated in pink

within the MAO-A active site.

propargylamine derivatives 6 and 7. It was observed that both enzyme, a time-dependent reduction of enzyme activity would be

compounds were able to interact with the crucial flavin adenine expected. In this regard, the time-dependent inhibition of MAO-B

by the most promising and selective MAO-B inhibitors namely 6

and 7, were evaluated (Figure 8). Briefly, recombinant human

MAO-B was preincubated with the test compounds for periods of

0, 15, 30, and 60 min prior to starting the enzyme reaction and

the residual rates of the MAO-B catalysed oxidation of kynuramine

were measured. For this purpose, the concentrations of the test

compounds chosen were approximately twofold the measured

IC₅₀values for the inhibition of the respective enzymes. Selegiline,

an irreversible inhibitor of the MAO-B enzyme with a propargyl-

amine functional group within its structure, was used as a refer-

ence compound. As shown in Figure 8, MAO-B enzyme activity

was not reduced by the propargylamine containing coumarins 6

and 7 with increased preincubation time. However, selegiline

shows a clear time-dependent reduction in activity confirming its

irreversible mode of inhibition. This indicates that both com-

pounds 6 and 7 can be considered as reversible inhibitors of

MAO-B and that the propargylamine function does not bind to

dinucleotide (FAD) cofactor to varying extents due to the inclusion

of the propargylamine moiety. The FAD cofactor is essential for

substrate catalysis and thus compounds that can come into close

proximity or bind to this cofactor are known to inhibit the enzyme

function to a greater extent⁴². Compound 6 maintains the previ-

ously mentioned p–H bonding with Cys 172 as well as its propar-

gylamine moiety orienting in close proximity to FAD. Compound

7’s propargylamine moiety forms crucial p–H bonds with the

cofactor. From the low nanomolar IC₅₀and high SI values dis-

played by these compounds, the importance of the propargyl-

amine moiety can be seen in its ability to produce highly potent

and selective inhibitors of MAO-B (Figure 7) .

3.2.3. MAO-B reversibility studies

To determine the binding mode of the studied compounds on

MAO-B, the time dependency of enzyme inhibition was measured.

If the compounds form a covalent or irreversible adduct with the the FAD co-factor covalently as is the case in selegiline^43,44. A

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1615

Figure 6. Diagrammatic representation showing binding interactions of the (a) 7-phenylethyloxy compounds (represented by 2) and (b) 7-carbamate compounds (rep-

resented by 5) when docked with MAO-B. This compound’s lack of p–H interaction with Ile 199 may explain its lower activity.

Figure 7. (a) 2D interaction map showing compound 7’s orientation and p–H binding with FAD and Tyr 398 in MAO-B’s substrate cavity. (b) 3D docking simulation

showing 7 indicated in pink within the MAO-B active site.

compounds had low inhibitory activity against AChE up to a

100 mM concentration. The 3-bromoethyl derivative 5 demon-

strated the best activity exhibiting 49% enzyme inhibition at the

aforementioned concentration and an IC₅₀value of ꢃ108 mM. The

results illustrate that substitution with either the carbamate or

phenylethyl moieties at position 7 does not solely confer signifi-

cant AChE inhibitory activity to the scaffold, as compounds 2 and

3 have similar activity to 1. However, the a-bromination of the

methyl at position 3 to produce -3-CH₂Br demonstrated a signifi-

cant increase in the compound’s inhibitory capacity. As observed

with compounds 6 and 7, the 3-propargylamine compounds had

Figure 8. Time-dependent inhibition of the recombinant human MAO-B catalysed

oxidation of kynuramine by selegiline, 6 and 7. The enzyme was preincubated diminished activity compared to the 3-bromomethyl of compound

for various periods of time (0–60 min, x-axis). Data are expressed as the mean

RFU (relative fluorescent units) SEM of three independent experiments.

5. The increased length of these 3-propargylamine derivatives was

expected to correlate with an increase in inhibitory activity, how-

ever, their activity was only marginally better than the shorter 3-

methyl derivatives (2 and 3).

benefit of reversible MAO inhibitors is that they can possibly

reduce the toxicity of the molecules as the effect of the inhibitor

Interestingly, the test compounds were more active against

BuChE than AChE with higher percentage inhibitions at a 100 mM

concentration. Compounds 1 and 5 were the most active inhibi-

tors, with IC₅₀values of 20.25 mM and 23.27 mM respectively.

Compound 1, having the lowest IC₅₀value, demonstrates that nei-

can be reversed when the drug is withdrawn^45,46

.

3.2.4. Cholinesterase inhibition studies

The AChE and BuChE inhibitory capacity of the compounds was ther of the substitutions at the 7-position enhanced the latent

tested using a modified method of the assay developed by BuChE inhibitory capacity of the coumarin scaffold. Compound 5

Ellman⁴⁷. The results of the assay showed that generally, the was the exception having an IC₅₀close to that of 1, however,

1616

S. C. MZEZEWA ET AL.

Figure 9. (a) Diagrammatic representation of compound 3’s lack of binding interactions with AChE. (b) 3-Dimensional representation of docking simulation (bottom)

with 3 indicated in pink and AChE’s active site’s amino acids indicated in green. The carbamate portion of the molecule towards the left does not participate in any

interactions and lies towards the entrance of the active site.

there is no significant correlation between the nature of the sub- 3.2.6. Cytotoxicity studies

stitution and the compound’s resultant BuChE inhibitory activity. The

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) assay was used to assess the cytotoxicity of test com-

pounds following their exposure to the SH-SY5Y neuroblastoma

cells at concentrations of 10 hx00B5;M, 50 mM, and 100 mM using

neuroblastoma SH-SY5Y cells. The results show that in general,

the exposure to the test compounds led to a concentration-

dependent decrease in the SH-SY5Y cell viability (Figure 12). With

the majority of the compounds, this decrease in viability was not

statistically significant (p > 0.05) at the 50 mM and 10 mM concen-

trations. At 10 mM the compound’s cytotoxic effect is diminished

and can be seen to cause a slight favourable increase in cell via-

bility similar to the untreated control. The 3-bromomethyl deriva-

Based on their higher overall inhibition towards BuChE, the tested

compounds can be classified as weakly selective BuChE inhibitors.

The compounds’ selectivity may be ascribed to them having stron-

ger binding interactions with the higher density of hydrophobic

amino acids lining the active-site gorge of BuChE⁴⁰. The larger vol-

ume of BuChE’s active site may also allow the enzyme the ability

to accommodate the compounds better than in AChE⁴⁸.

3.2.5. Cholinesterase molecular modelling studies

The AChE enzyme active site is an ellipsoid-shaped gorge, consist-

ing of two main sites termed the peripheral anionic site (PAS) (at

the entrance of the active site) and the catalytic binding site (CAS)

containing the catalytic triad^49,50. The PAS is made up of largely

aromatic amino acids such as Trp 279 and is responsible for guid-

ing and stabilising substrates in the active site. The initial predic-

tions from literature anticipated that the coumarin nucleus will sit

in the PAS of the enzyme and the various substituents interact

with the CAS and catalytic triad^51,52. The best-ranked docking

results show that instead, the initial substitutions at position 7

performed in compounds 2 and 3 orients the coumarin nucleus

away from the PAS and towards the CAS (Figure 9). When the

molecules are oriented in this way, neither compound is able to

form favourable interactions with amino acids in either of

these regions.

The improved inhibitory capacity of compounds 4 and 5 can

be attributed to how the -CH₂Br group forms p–H interactions

with Trp 84 and the p–p stacking between the aromatic region of

the coumarin and Phe 330 (Figure 10). Both of these are key resi-

dues for substrate binding in the CAS. It can be hypothesised that

the electron-withdrawing effect caused by bromine may have led

to more stable interactions with the aromatic Trp 84 residue and

was responsible for these compounds’ superior inhibitory activ-

ities. Substitution with propargylamine in the same position in

derivatives 6 and 7 resulted in added length to the molecules,

however, this altered the conformation of the compounds in the

active site. As a result, the binding interactions which had been

demonstrated with 4 and 5 were disrupted (Figure 11).

tives

4

and

5

displayed the most concentration-related

cytotoxicity, with a significant reduction in cell viability at 50 mM

and 100 mM. Compound 4’s profile was significantly better with

65% and 7% survival at 50 mM and 100 mM, respectively, compared

to a 0% survival rate at the same concentrations for 5. The 3-bro-

momethyl substitution can be assumed to underpin this cytotox-

icity and this is in agreement with previous studies describing the

general cytotoxicity of halogen-containing coumarin deriva-

tives^53,54

. This cytotoxicity is mitigated at the lower 10 mM

concentration.

3.2.7. Neuroprotection studies

The neurotoxin 1-methyl-4-phenyl pyridinium (MPPþ) is widely

known to induce an apoptotic cascade in neurons and is thus

used to induce neurodegeneration in various in vitro and in vivo

models of NDs^55–58. In this study, the neuroprotective nature of

compounds was determined using an in vitro MTT assay based on

their ability to rescue SH-SY5Y neuroblastoma cells from this

MPP^þinduced in neurotoxicity.

The SH-SY5Y cells were pre-treated with 10 mM of the test com-

pounds for 2 h followed by the addition of 2000 mM of MPP^þ. The

10 mM concentration was selected as the compounds did not

cause any cytotoxicity at this concentration, thus it would be a

fair representation of their neuroprotective effect. The treated cells

displayed higher survival counts compared to the MPP^þ-treated

cells (Figure 13), which indicates that the compounds provided

neuroprotection to the cells. The cells exposed to the test

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1617

Figure 10. (a) Diagrammatic representation of compound 4’s binding interactions with Trp 84 and Phe 330 in the CAS of AChE. (b) 3-Dimensional representation of

docking simulation (bottom) with 4 indicated in pink and AChE’s active site’s amino acids indicated in green.

Figure 11. (a) Diagrammatic representation of 3-propargylamine derivatives’ lack of binding interactions with AChE. The p–p stacking with Phe 330 is diminished. (b)

3-Dimensional representation of docking simulation (bottom) with 6 indicated in pink and AChE’s active site’s amino acids indicated in green. The coumarin portion

now points away from the CAS and does not partake in significant interactions within the active site.

compounds increased significantly increased (p < 0.05) the survival

rate (15–36%) when compared to the MPP^þonly treated group.

The nature of the moiety at position 7 does not seem to influence

this neuroprotection greatly, as there was no significant trend

observed between the 7-benzeloxy and 7-carbamate derivatives.

100

100 µM

50 µM

10 µM

However, as expected, the propargylamine derivatives exhibited

the highest neuroprotective capability as reported in the litera-

ture^24,56,57. Cell lines treated with compounds 6 and 7 had 92%

and 85% survival rates respectively, further demonstrating the

moiety’s significance in neuroprotection.

The general neuroprotective profile of the compounds may

also suggest that the observed neuroprotection is independent of

their enzyme inhibitory capabilities, as all compounds performed

fairly similarly despite having different enzyme inhibitory profiles.

As described in the literature, coumarin derivatives may possess

inherent anti-inflammatory, anti-apoptotic and antioxidant effects

which, together with the MAO-inhibition effects of the test

50

0

Control 1

2

3

4

5

6

7

Figure 12. Percentage of cells viable following incubation with test compounds

relative to a control of untreated cells. Each bar represents the mean percentage

survival and SD (where n ¼ 3, three fields per repeat). The data was subjected to

ꢄ

an ANOVA statistical analysis and significance was defined as [ p < 0.05 and

ꢄꢄꢄꢄ

p < 0.0001] when comparing the compounds’ means to the nega-

tive control.

1618

S. C. MZEZEWA ET AL.

compounds may synergistically counter the MPP^þinduced apop- 25–35 peptide fragment was used to induce neurotoxicity in SH-

tosis^29,35,36,58. The results give an indication that the compounds

SY5Y cells. Ab 25–35 has been reported to be the actual biological

active site of Ab responsible for the peptide’s neurotoxic ability

would be tolerated well by neurons at or below the 10 mM con-

centration and most importantly, potentially ameliorate the neuro-

degeneration associated the with the progression of AD, and/or

other neurodegenerative disorders.

The accumulation of extracellular Ab plaques has been marked

as one of the pathological hallmarks of neurotoxicity associated

with AD^59–61and coumarin moieties have previously shown to

inhibit Ab plaque formation and Ab induced neurotoxicity^62,63. To

further explore the neuroprotective potential of the propargyl-

amine-containing coumarin derivatives 6 and 7, their ability to

curb neurotoxicity induced by the amyloid-b (Ab) protein was

thus assessed according to the method described in the experi-

mental section. In order to evaluate these compounds, the Ab

and has been used in similar models of neurodegeneration in pre-

vious studies^11–14

.

The exposure of Ab 25–35 on the SH-SY5Y cells for 24 h exhib-

ited significant (p < 0.01) detrimental effects, when compared to

the control, with a loss of cell viability of between 30% and 45%

in five independent experiments conducted in triplicate. This

reduction in cell viability in SH-SY5Y cells after Ab 25–35 treat-

ment was also reported in previous studies^64,65. As the addition of

Ab 25–35 in SH-SY5Y cells precipitates apoptosis^66,67, the neuro-

protective activity of 6 and 7 on mitigating cellular apoptosis was

examined at concentrations between 10 mM and 100 mM.

Unfortunately, the compounds, at all concentrations, did not show

any significant ability to mitigate the cytotoxic effects caused by

Ab 25–35. The significant neuroprotection observed in the MPP^þ

assay, however, highlights the need for further neuroprotective

and mechanistic studies. This will shed light on the potential of

these compounds as neuroprotective agents in not only AD but

other neurodegenerative disorders such as Parkinson’s disease

(PD) as well.

3.2.8. In silico pharmacokinetic and drug-likeness evaluation

In addition to displaying the relevant biological activities, it is

equally important for ideal drug candidates to have low toxicity

and access to their therapeutic target. Computer models used in

the discovery phase with online tools such as SWISSADME are

used to provide an early estimation of drug candidates’ ADME,

drug-likeness, and pharmacokinetic properties. This decreases the

fraction of pharmacokinetics-related failure in the clinical

phases^68,69. The most promising drug candidates from this study,

compounds 6 and 7, were assessed for their drug-likeness and

pharmacokinetic profiles using the SWISSADME web tool.

A bioavailability radar (Figure 14) was generated, which pro-

vides a graphical snapshot of the drug-likeness parameters of an

orally available bioactive drug. A range for each parameter was

defined and depicted as a pink area on the radar plot. The param-

eters are defined as lipophilicity (LIPO; ꢁ0.7 ꢅ XLOGP3 ꢅ þ5.0);

Figure 13. Bar graph comparing the effect of the test compounds (10mM) on

MPP^þ-induced toxicity in SH-SY5Y cells. The cell viability count of the untreated

control was defined as 100%. Error bars represent the standard deviation _þof the

ꢄ

mean data. Statistical significance ( p < 0.05) when compared to the MPP only

treated cells (purple bar) is indicated on the graph.

Figure 14. The simulated oral bioavailability of compounds 6 and 7 represented as a radar chart.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1619

Table 3. In silico drug-likeness properties of compounds 6 and 7

Drug-likeness

Compounds

BBB^a

GIA^b

P-gp^c

Lipinski

Ghose

Veber

Egan

Muegge

Bio. Score^d

6

7

Yes

High

No

Yes

0.55

^aBBB: Blood brain barrier permeant; ^bGIA: Gastrointestinal absorption; ^cP-gp: P-glycoprotein substrate; ^dBio. Score: Abbot bioavailability score (probability of >10%

bioavailability).

size (150 ꢅ MW ꢅ 500 g/mol); polarity (POLAR; 20 ꢅ TPSA ꢅ cytotoxicity and neuroprotection studies. S.C.M. and J.J. analysed

130 Å2); solubility (INSOLU; log S ꢅ 6); saturation (INSATU; fraction the NMR, IR, MS, and biological data; SCM and J.J. performed and

of sp3 hybridised carbons ꢆ 0.25) and flexibility (FLEX; ꢅ 9 rotat-

analysed the molecular modelling. O.E.E. provided the facilities

able bonds). For a molecule to be considered drug-like its radar

plot has to fall entirely within the defined range⁷⁰.

and consumables for the neuroprotection and cytotoxicity assays.

J.J. and S.F.M. provided supervisory and funding support. S.C.M.

prepared the paper and all authors edited the final draft. All

authors have read and agreed to the published version of

the manuscript.

The compounds were found to be within the desirable pink

area, barring compound 6’s high INSATU value. In spite of this,

further in silico simulations showed that both compounds have

high predicted intestinal absorption and BBB (blood–brain barrier)

permeability (Table 3)⁷¹. BBB permeability is of utmost importance

to the agents’ mechanism of action as they need to cross the bar-

rier to inhibit the action of enzymes in the brain as well as offer

neuroprotection to affected cells. The Abbot Bioavailability Score

predicted the compounds to have high oral bioavailability and

they are non-substrates of the permeability glycoprotein (P-gp). P-

gp is an efflux pump in the GIT which pumps out xenobiotics

(including drugs) away from the BBB and intestinal epithelium,

reducing their bioavailability⁷². Finally, the compounds were found

to comply with the computational filters developed for identifying

drug-like entities by pharmaceutical companies, Lipinski (Pfizer),

Ghose (Amgen), Egan (Pharmacia), Veber (GSK), and Muegee

(Bayer)^73,74. Based on these results the compounds could serve as

promising lead compounds for AD.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Funding

. This work was financially supported by the University of the

Western Cape as well as the National Research Foundation of

South Africa.

ORCID

Sarel F. Malan

Jacques Joubert

http://orcid.org/0000-0002-1640-6457

http://orcid.org/0000-0003-0378-7091

4. Conclusions

The objectives of this study were to synthesise a number of neu-

roprotective coumarin derivatives substituted with various privi-

leged moieties for potential cholinesterase and MAO inhibitory

activities. The compounds were synthesised and characterised and

biological evaluations showed that despite their lack of cholin-

esterase inhibitory activities, they are in general potent, selective

and reversible inhibitors of MAO-B and offer excellent in vitro neu-

roprotection in the MPP^þmodel with low cytotoxicity profiles.

Especially compounds 6 and 7 displayed the greatest potential as

MTDLs and this may be attributed to their inclusion of the propar-

gylamine functional group. In silico studies using the online

SwissADME tool predicted that the compounds should have

acceptable pharmacokinetic and drug-like properties. Additional

work is suggested for 6 and 7 to further explore their neuropro-

tective potential in AD and related neurodegenerative disorders,

such as PD.

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Article Doi

Design, synthesis, and evaluation of 3,7-substituted coumarin derivatives as multifunctional Alzheimer’s disease agents

DOI: 10.1080/14756366.2021.1913137

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