Synthesis and evaluation of chromone derivatives as inhibitors of monoamine oxidase

Molecular Diversity

https://doi.org/10.1007/s11030-019-09917-8

ORIGINAL ARTICLE

Synthesis and evaluation of chromone derivatives as inhibitors

of monoamine oxidase

Annah N. Mpitimpiti¹· Jacobus P. Petzer^1,2· Anél Petzer^1,2· Johannes H. L. Jordaan³· Anna C. U. Lourens^1,2

Received: 14 November 2018 / Accepted: 6 January 2019

Abstract

Based on reports that chromone compounds are good potency inhibitors of monoamine oxidase (MAO), the present study

evaluates the efect of substitution with fexible side chains on the 3 position on MAO inhibition potency. Fifteen chromone

derivatives were synthesised by reacting aromatic and aliphatic amines and alcohols with chromone 3-carboxylic acid in

the presence of carbonyldiimidazole (CDI). This yielded chromane-2,4-dione and ester chromone derivatives. Generally,

the esters exhibited weak MAO inhibition, while the chromane-2,4-dione derivatives showed promise as selective MAO-B

inhibitors with IC₅₀values in the micromolar range. Compound 14b, 3-[(benzylamino)methylidene]-3,4-dihydro-2H-1-ben-

zopyran-2,4-dione, was the most potent MAO-B inhibitor with an IC₅₀value of 638 µM. This compound was shown to be a

reversible and competitive MAO-B inhibitor with a K_iof 94 µM. In conclusion, the efect of chain elongation and introduction

of fexible substituents on position 3 of chromone were explored and the results showed that aminomethylidene substitution

is preferable over ester substitution. Good potency MAO-B inhibitors may act as leads for the design and development of

therapy for Parkinson’s disease where these agents reduce the central metabolism of dopamine.

Graphical abstract

Chromane-2,4-dione derivaꢀves show promise as selecꢀve MAO-B inhibitors.

Keywords Chromone · Chromandione · Monoamine oxidase · MAO · Inhibitor · Competitive · Parkinson’s disease

Introduction

Electronic supplementary material The online version of this

Parkinson’s disease is the second most common neurodegen-

erative disorder afecting the brain and is the most common

progressive neurodegenerative movement disorder [1–3].

Pathologically, Parkinson’s disease is characterised by the

death of the neurons in the substantia nigra and the presence

article (https://doi.org/10.1007/s11030-019-09917-8) contains

supplementary material, which is available to authorized users.

* Jacobus P. Petzer

jacques.petzer@nwu.ac.za

Extended author information available on the last page of the article

Vol.:(0123456789)

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Molecular Diversity

of proteinaceous deposits known as Lewy bodies, resulting

in the characteristic motor symptoms [4]. Due to its distinc-

tive pathology, Parkinson’s disease is primarily character-

ised by motor symptoms, although non-motor symptoms

frequently appear especially in later stages of the disease [5].

Current treatment strategies are mainly aimed at restoring

striatal dopamine activity with levodopa as the gold standard

of therapy. Levodopa is administered in combination with

carbidopa, an aromatic l-amino acid decarboxylase (AADC)

inhibitor which prevents peripheral levodopa metabolism [1,

6]. Other dopaminergic therapies include dopamine agonists,

which stimulate dopamine receptors, monoamine oxidase

B (MAO-B) inhibitors and catechol-O-methyltransferase

(COMT) inhibitors which reduce dopamine degradation

[6]. Several potential neuroprotective or disease-modifying

agents have been evaluated in clinical trials, but none have

been conclusivel y proven to be neuroprotective in Parkin-

son’s disease [7, 8]. As discussed below, however, a mecha-

nistic rationale and preclinical evidence exist for a potential

neuroprotective efect by MAO-B inhibitors.

increase with age in humans, thus providing an additional

rationale for the use of MAO-B inhibitors in Parkinson’s

disease with the aim of reducing oxidative neuronal dam-

age [18]. Three MAO-B inhibitors are currently approved

for use in Parkinson’s disease. These are the mechanism-

based inhibitors, rasagiline and selegiline, and the revers-

ible inhibitor safnamide [9, 19]. These inhibitors are spe-

cifc for the MAO-B isoform since MAO-A inhibition may

result a potentiall y fatal hypertensive crisis when taken with

certain food [20, 21]. For the design and development of

new MAO-B inhibit ors, isoform specifcity is an important

consideration.

Several heterocyclic scafolds have been considered for

the design of MAO inhibitors. These include coumarins,

chalcones as well as chromones [22, 23]. Chromone

(4H-1-benzopyran-4-one, 1) is an isomer of coumarin (2)

and forms part of the favone backbone (Fig. 1). Chromone

derivatives occur in nature, and a large range of pharmaco-

logical activities suc h as immune s timulation [24, 25 ], anti-

oxidant [26 ], anti-HIV [27], anticancer [28 ], biocidal [29],

w ound healing [30 ], antibacter ial and antifung al activities

[31] have been reported for these compounds. Due to their

presence in plants, chromones are part of the human diet

and generally exhibit low mammalian toxicity [28]. The chr-

omone ring system is therefore considered to be a privileged

scafold due to its range of pharmacological and biological

efects as well as the low risk of toxicity associated with

chromone compounds [32].

MAO is an enzyme distributed extensively in higher

eukaryotes and in mammals and is present in two isoforms,

MAO-A and MAO-B [9]. The MAOs contain favin adenine

dinucleotide (FAD) as a cofactor and are bound to the outer

mitochondrial membrane. The MAOs catalyse the oxidative

deamination of various biogenic and dietary monoamines,

and abnormal MAO levels may be associated with several

disease states such as depression and Parkinson’s disease

[9]. Since MAO-A and MAO-B are products of distinctive

genes, they difer with respect to their amino acid sequence,

three-dimensional structure, tissue distribution as well as

substrate and inhibitor specifcity [10–12]. MAO-A prefer-

entially deaminates serotonin, while MAO-B shows pref-

erence for benzylamine and phenethylamine as substrates.

Both MAO isoforms deaminate dopamine equally well [9].

Since inhibition of the MAOs can potentially modulate neu-

rotransmitter levels (for example dopamine) in the brain that

are activel y involved in the pathogenesis of some disease

states [13, 14], MAO inhibitors have found application in the

therapy of disorders such as Parkinson’s disease [15, 16]. In

this respect, MAO-B inhibitors are efective as symptomatic

treatment by compensating for diminished striatal dopamine

levels; however, arresting disease progression remains the

ultimate goal [8, 14].

The MAO inhibitory potential of chromone deriva-

tives has been illustrated. For example, series of C6- and

C7-substituted chromone derivatives were found to be

potent, reversible MAO-B inhibitors. Compounds 3–6 are

examples of high potency MAO-B inhibitors from these

studies [22, 33, 34]. Although some of these chromones

also exhibited IC₅₀values in the nM range for the inhibition

of MAO-A, these compounds are specifc inhibitors of the

MAO-B isoform.

Interestingly, C5 substitution of chromone yields poor

MAO-B inhibition when compared to the C6- and C7-sub-

stituted derivatives (e.g. 7) [35]. Also of interest is the fnd-

ing that chromone 3-carboxylic acid (8) is a potent and

specific MAO-B inhibitor (IC₅₀= 0.048 µM), while the

presence of the carboxylic acid group in position 2 of t he

4-pyrone nucleus (9) results in a loss of activity [36–38].

Similarly, phenylcarboxamide substitution on position 3 of

the 4-pyrone nucleus results in potent MAO-B inhibition,

with compounds 10 and 11 exhibiting IC₅₀values of 0.40

and 0.063 µM, respectively, while related 2-phenylcarbo xa-

mide substitution generally results in poor activity [37–39].

From the above discussion it is thus clear that c hr-

omones are suitable for the design of MAO-B-specifc

inhibitors. The MAO inhibitory activity of the chromones

As mentioned above, a mechanistic rationale exists for a

potential neuroprotective efect by MAO-B inhibitors. The

MAO catalytic cycle produces hydrogen peroxide as a by-

product which, via the Fenton reaction, is a precursor to

harmful reactive oxygen species that may lead to oxidative

damage and exacerbate neurodegeneration [14]. Thus, the

inhibition of the MAO enzymes may reduce the formation

of hydrogen peroxide and act as potential neuroprotectants

[17]. Furthermore, MAO-B levels in several brain regions

1 3

Molecular Diversity

Fig. 1 The structures of chromone (1), coumarin (2) and chromone derivatives 3–11 referred to in the text

has been validated, and this study will contribute by exam-

ining the MAO inhibition potential of C3-substituted chr-

omones. Although the MAO inhibitory activity of rigid

C3-substituted phenylcarboxamide derivatives has previ-

ously been investigated, the efect of chain elongation and

the introduction of a more fexible substituent in this posi-

tion have not been explored [37–39]. As discussed above,

fexible substituents on positions 6 and 7, and rigid amide

functions on position 3 have resulted in potent MAO inhib-

itors [33, 34, 37–39]. It is thus postulated that the introduc-

tion of a f e xible ester- or amide-containing substituents

on position 3 of chromone will result in potent, specifc

MAO inhibition. This study therefore attempted to synthe-

sise chromone derivatives (12 and 13) where fexible sub-

stituents will be attached via ester and amide functions on

position 3 of chromone (Fig. 2). The chromone derivatives

were synthesised by reacting aromatic and aliphatic alco-

hols and amines with chromone 3-carboxylic acid (8). As

will be shown, this yielded the anticipated ester chromone

derivatives (12), while the amide derivatives 13 were not

Fig. 2 The general structures of chromone derivatives 12–14 that

obtained. Instead, the reactions of amines with chromone

were considered for this study, and the synthesis of ester chromone

derivatives (12a–e) and 3-aminomethylidene-2,4-chromandiones

(14a–j). Reagents and conditions a CDI, DMF, 60 °C, 2 h; b R–OH,

DMF, rt, overnight; c R–NH₂, DMF, rt, overnight

3-carboxylic acid yielded chromane-2,4-diones (14).

1 3

Molecular Diversity

experimental conditions, and activation of the carboxylic

acid probably precedes nucleophilic attack by the amine on

C2. Careful consideration of the NMR data revealed that

the desired amide derivatives were in fact not synthesised,

but that the products that were obtained were the structur-

ally related chromane-2,4-diones 14. In the ¹H NMR spectra

for example, the presence of a methine proton (H-9) that

occurs as a doublet coupling to the adjacent imino proton

provided evidence that the keto-enamines were obtained and

not the desired amides (where both H-2 and the NH signals

are expected to appear as singlets). A crystal structure was

obtained for compound 14j (Fig. 3) and provided further

confrmation that this was indeed the case.

Results

Chemistry

The aim of this study was to synthesise ester (12) and amide

(13) derivatives of chromone. The synthesis of these com-

pounds was attempted using standard literature procedures

with the synthetic strategy employed for both series involv-

ing a coupling reaction of chromone 3-carboxylic acid (8)

with an equimolar quantity of an alcohol or amide in the

presence of carbonyldiimidazole (CDI) as coupling reagent

(Fig. 2) [40]. In total, 15 compounds were synthesised in

poor to moderate yields (13–70%). All synthesised com-

pounds were characterised by NMR and infrared spec-

troscopy and mass spectrometry. For all compounds, the

mass data correlated well with the calculated mass values.

Purity of the compounds was determined by HPLC and was

between 79 and 100%. Decomposition of the ester deriva-

tives was observed over time. Melting points generally cor-

related with reported literature values; however, diferences

in the melting points of the chromane-2,4-diones (14) when

compared to the literature values are most likely due to dif-

ferences in the ratios of the E/Z-isomers [41].

A reaction mechanism for the formation of the chromane-

2,4-dione derivatives, based on liter ature reports for related

systems, is illustrated in Fig. 4 [39, 43]. It is postulated that

the carboxylic acid is activated b y CDI as expected. Reaction

of the amine however does not result in the displacement

of imidazole, but leads to ring opening of the pyrone ring,

followed by ring closure through lactonisation with loss of

imidazole.

NMR data further revealed that the chromane-2,4-diones

exist as mixtures of isomers. In the proton spectra there are

two distinct NH signals as well as two signals for H-9 and

generally also for H-5. Double signals of almost all carbons

also occur, but are especially clear for C-4, C-2 and C-9.

A literature survey revealed that these compounds exist as

mixtures of E- and Z-isomers, with one isomer predomi-

NMR assignments were based on interpretation of both

1D NMR (¹³C, H, DEPT) and 2D NMR (COSY, HSQC,

1

HMBC) spectra, while NMR data of known compounds

were also considered. The results of the attempted syn-

thesis of amide derivatives 13 were initially surprising, as

analogous amide derivatives were obtained using related

methodology [38, 42 ]. However, in a study done by Cag-

ide and co-workers [39], similar results to this study were

obtained using PyBOP as coupling reagent in the presence

of N,N-diisopropylethylamine (DIPEA), while Gaspar and

co-workers used PyBOP or BOP as coupling agent, also in

the presence of DIPEA [38]. Under acidic conditions (POCl₃

in DMF), however, the target carboxamide derivatives could

be obtained [39]. The formation of the chromane-2,4-diones

versus the carboxamide derivatives is thus dependant on the

1

nating (Fig. 5) [41, 44–47]. In the H NMR spectrum of

compound 14i for exam ple, the two NH signals are present

at 13.57 and 11.91 ppm for the major and minor products,

respectively. It is postulated that the more downfeld signal

corresponds to the E-isomer, where the NH forms a short,

strong intramolecular hydrogen bond with the C(4)=O

group. The NH signal for the Z-isomer (thus at 11.91 ppm)

also forms a hydrogen bond with the ester carbonyl (C2), but

it has been reported that this bond is signifcantly weaker

[41, 44]. On the other hand, the H-9 proton of the minor

Fig. 3 ORTEP drawing repre-

senting the crystal structure of

the E-isomer of compound 14j

1 3

Molecular Diversity

Fig. 4 Proposed mechanism for aminomethylidene-2,4-chromandione formation (adapted from the literature) [39, 43]

HMBC, HSQC and COSY spectra as well as DEPT data.

The assignments and observed HMBC correlations of the

major isomer of compound 14i are shown in Table S1 (sup-

plementary) as an example. The most characteristic dif-

1

ference between the H NMR spectra of these chromane-

2,4-diones (14) and those of the ester derivatives (12) is

the presence of H-9 which appears as a doublet coupling

to the adjacent imino proton. The NH signals further also

occur quite far downfeld due to intramolecular hydrogen

bonding with C(4)=O (major isomer) or C(2)=O (minor

isomer). The shifts of the aromatic protons of ring A of

these derivatives were similar to those of the ester deriva-

tives and were assigned as follows for compound 14i: δ_H:

8.05 (H-5), 7.63–7.56 (H-7), 7.32–7.23 (H-6 and H-8). The

¹³C NMR spectrum for compound 14i showed the presence

of 30 signals, thus providing further evidence of the pres-

ence of two isomers. Analysis of the ¹³C spectrum together

with the DEPT 135 spectra indicated the presence of six

quaternary carbons and nine CH carbons (for each isomer).

Chemical shifts and observed 2D correlations were used to

further assign carbon signals (Table S1, supplementary).

Similar correlations were observed in all cases for the minor

Z-isomer.

Fig. 5 The E- and Z-isomers of the chromane-2,4-diones obtained in

this study

isomer (presumably the Z-isomer) is found more downfeld

(9.71 ppm) than that of the major E-isomer (9.61 ppm).

In the ¹³C NMR spectrum of 14i, the signal for the C-4

carbonyl at 182.4 ppm for the major (E-isomer) is shifted

downfeld compared to the signal of the minor Z-isomer at

178.8 ppm. The opposite is observed for the C-2 carbonyl,

where the signal for the minor Z-isomer is more downfeld

(165.3 ppm) compared to the signal for the major E-isomer

which is present at 163.3 ppm. This is due to the possi-

bility that C-4 carbonyl oxygen is hydrogen bonded to the

NH proton in the E-isomer, whereas C-2 carbonyl oxygen

is hydrogen bonded to the NH for the minor Z-isomer. The

carbonyl that undergoes hydrogen bonding therefore occurs

further do wnfeld than its non-hydrogen-bonded counter-

part [41, 44]. Since similar observations were made for all

the 3-chr omane-2,4-diones synthesised in this study, it is

proposed that the E-isomer is the major isomer. Accord-

ing to the literature further evidence for the E-isomer as

major product is provided by molecular calculations where

the energy of formation of the E-keto-enamine is lower than

that of the Z-keto-enamine form [41].

Ratios of the E:Z mixtures may be calculated using the

integral intensities of the H-9 signals. Interestingly, Traven

and co-workers noted that the rate of equilibration between

these isomers strongly depends on solvent polarity, where

for 3-(p-tolyliminomethyl)chromane-2,4-dione equilibrium

is almost immediately established after dissolution in metha-

nol, 4 h after dissolution in DMSO and 24 h after dissolution

in chloroform [41]. Thus, if a diferent solvent was used to

dissolve these compounds and if they were observed over

time, the observed ratios of these isomers could change. In

order to investigate the efect of temperature on the ratios

NMR assignments were based on chemical shifts, inte-

gration, multiplicities, coupling constants, correlations in

1 3

Molecular Diversity

Table 1 IC₅₀values for the inhibition of MAO-A and MAO-B by

ester chromone derivatives (12)

of isomers, variable temperature NMR experiments were

performed. The results with compound 14b are shown in

Fig. S1 (supplementary). Based on these results it appears

that changes in temperature from −10 to 45 °C do not alter

the observed ratios of the E- and Z-isomers.

O

R

O

Monoamine oxidase inhibition: IC₅₀values

O

The compounds synthesised in this study were evaluated

as inhibitors of MAO-A and MAO-B. Recombinant human

MAO-A and MAO-B enzymes were used for this purpose.

MAO activity was measured by making use of a fuoro-

metric assay with kynuramine as the enzyme–substrate.

Kynuramine is a non-selective substrate for both MAO-A

and MAO-B and is oxidised to yield 4-hydroxyquinoline,

a metabolite that fuoresces in alkaline media [48, 49]. By

measuring MAO activity in the absence and presence of

diferent concentrations of the test inhibitors, sigmoidal

plots of enzyme activity versus the logarithm of inhibitor

concentration were constructed from which the IC₅₀values

were estimated. Figure 6 gives examples of representative

sigmoidal plots.

R

IC₅₀(µM)^a

MAO-A

SI^b

MAO-B

12a

12b

66.7 2.06

27.3 0.946

14.7 0.416

2.4

1.8

26.8 0.560

12c

12d

21.6 8.72

18.5 8.85

14.8 1.09

1.5

Cl

12.0 0.400

12e

18.6 3.27

9.74 0.123

1.9

Cl

The r esults of the MAO inhibition studies are given in

Tables 1 and 2. Selectivity index (SI) values provide an indi-

cation of t he selectivity for the inhibition of MAO-B over

the MAO-A isoform. From Table 1 it is apparent that the

ester derivatives are generally weak MAO-A and MAO-B

inhibitors with IC₅₀values >9 µM. Comparing the MAO-A

and MAO-B potencies of compound 12a (phenyl substi-

tuted) and 12b (benzyl substituted), it is evident that the

benzyl derivative is more potent than the phenyl derivative.

Thus, chain elongation from phenyl to benzyl improved

MAO inhibition activity. A similar trend is observed when

the activity of compound 12c is compared to that of 12a.

^aAll values are expressed as the mean SD of triplicate determina-

tions

^bSelectivity index (SI)=IC₅₀(MAO-A)/IC₅₀(MAO-B)

4-Chlorophenyl substitution (12c) has improved MAO

inhibition activity compared to unsubstituted phenyl side

chain (12a). This trend is also observed when comparing

compound 12e, a 4-chlorobenzyl-substituted derivative, to

compound 12b. Comparing the 4-chlorophenyl (12c) with

3-chlorophenyl substitution (12d), it is clear that the position

of the chlorine does not signifcantly afect MAO inhibition

activity. It may therefore be concluded that elongation of

the side chain from phenyl to benzyl and substituting with

a chlorine on the 3ʹ- or 4ʹ-position enhance MAO inhibition

activity of the ester derivatives.

100

75

50

25

0

From Table 2 it may be concluded that the chromane-

2,4-diones are selective inhibitors of MAO-B with IC₅₀

values in the micromolar range. Generally, the chromane-

2,4-diones are more potent MAO-B inhibitors than the ester

derivatives. (They are however weaker inhibitors of MAO-

A.) Compound 14b is the most potent MAO-B inhibitor with

an IC₅₀value of 0.638 µM, while 14d is the most selective

MAO-B inhibitor with a SI value of 218. MAO-B inhibition

increases with side chain elongation from compound 14a to

14b (phenyl to benzyl) and is similar for compounds 14a

and 14c, but from compounds 14d to 14e, as further chain

elongation occurs, MAO-B inhibitory activity decreases.

Comparing the MAO-A and MAO-B inhibition potencies

of compounds 14f, 14g and 14h, it is evident that chain

-3

-2

-1

0

1

2

3

Log[I]

Fig. 6 Sigmoidal plots for the inhibition of MAO-B by 14a (open cir-

cles), 14b (flled circles) and 14c (triangles). Each data point repre-

sents a mean SD of triplicate determinations

1 3

Molecular Diversity

Table 2 IC₅₀values for the inhibition of MAO-A and MAO-B by

3-aminomethylidene-2,4-chromandiones (14)

MAO-B inhibitors that the ester chromone derivatives 12.

For example, the phenyl-substituted compound, 14a, is

approximately 28-fold more potent than 12a. Similarly,

the chromane-2,4-diones 14b and 14f are also signifcantly

more potent MAO-B inhibitors than their corresponding

ester derivatives 12b and 12c. It should be noted that the

3-aminomethylidene-2,4-chromandiones are represented by

inseparable mixtures of E- and Z-isomers, and the MAO

inhibition potencies recorded are that of the mixtures. The

observation that chromane-2,4-diones are weak inhibitors

of MAO-A is in agreement with a recent report of the MAO

inhibition properties of four 3-[(phenylamino)methylidene]

chromane-2,4-dione derivatives [39]. These compounds did

not inhibit human MAO-A at a maximal tested concentra-

tion of 10 µM. The IC₅₀values reported for the inhibition

of human MAO-B by 14a (0.268 µM) and 14f (0.065 µM),

however, difer signifcantly from the values recorded in the

present study. The reason for this discrepancy is unclear,

but may be related to diferent experimental approaches to

measuring MAO activity.

O

R

N

H

O

R

IC₅₀(µM)^a

MAO-A

SI^b

MAO-B

14a

14b

79.6 30.2

77.9 25.5

0.947 0.125

84

0.638 0.287

0.897 0.166

122

14c

101 3.49

113

14d

14e

14f

14g

312 89.1

155 19.1

72.1 6.53

288 61.8

1.43 0.103

142 47.2

3.08 0.279

NI^c

218

1.1

23

–

Monoamine oxidase inhibition: reversibility

Cl

The reversibility of the MAO-B inhibition by compound

14b was examined using dialysis [49]. Compound 14b

was selected since it is the most potent MAO-B inhibitor

of this study. None of the study compounds were potent

MAO-A inhibitors, and therefore reversibility of MAO-A

inhibition was not examined. For these studies MAO-B

and compound 14b (at 4×IC₅₀) were combined and prein-

cubated for 15 min. As controls, the MAO-B enzyme was

similarly preincubated in the absence of inhibitor (negative

control) and presence of the irreversible MAO-B inhibitor,

(R)-deprenyl (positive control). The preincubated mixtures

were subsequently dialysed for 24 h and diluted twofold to

yield a fnal inhibitor concentration of 2×IC₅₀. The residual

MAO-B activity of the diluted samples was measured and

is shown in Fig. 7. Non-dialysed mixtures of MAO-B and

the test inhibitor were maintained over the same time period

(24 h) for comparison. The results show that the inhibition

of MAO-B by compound 14b is completely reversed after

24 h of dialysis. After dialysis of mixtures containing 14b

and MAO-B, the MAO-B catalytic activity was recovered

to 115% of the negative control value (100%). This sug-

gests that compound 14b is a reversible inhibitor of MAO-

B. After dialysis of the positive control samples containing

(R)-deprenyl and MAO-B, catalytic activity is not recovered

with only 3% activity remaining. As anticipated, inhibition

of MAO-B by 14b persists in non-dialysed samples with the

residual activity at 41%.

Cl

14h

NI^c

–

14i

14j

73.1 20.4

41.6 2.29

38.7 1.73

16.6 1.04

1.9

2.5

N

^aAll values are expressed as the mean SD of triplicate determina-

tions

^bSelectivity index (SI)=IC₅₀(MAO-A)/IC₅₀(MAO-B)

^cNo inhibition at a maximum tested concentration of 100 µM

elongation results in loss of activity. Interestingly, com-

pounds 14g and 14h do not inhibit MAO-B. This may be

due to lengthening of the side chain in combination with

the addition of a sterically large chlorine. Comparing the

MAO inhibition activities of compounds 14a–c with those of

compounds 14f–h, it may be concluded that chlorine substi-

tution reduces MAO-B inhibition potency. The compounds

with pyridine-containing side chains (compounds 14i and

14j) both show poor MAO inhibition; however, with chain

elongation from pyridyl (14i) to ethylpyridyl (14j) a twofold

increase in MAO inhibition is observed. The results fur-

ther show that the chromane-2,4-diones 14 are more potent

A set of Lineweaver–Burk plots were also constructed

to determine the mode of MAO-B inhibition of 14b. Four

Lineweaver–Burk plots were constructed with the frst plot

1 3

Molecular Diversity

that the lines are linear and intersect at a single point just

to the left of the y-axis. This suggests that 14b interacts

competitively with the MAO-B enzyme. From a replot of

the slopes of the Lineweaver–Burk plots versus inhibitor

concentration, the K_ivalue is estimated at 0.94 µM.

125

100

75

50

25

0

Discussion and conclusion

This study attempted to synthesise ester (12a–e) and amide

(13) derivatives of chromone by reacting chromone 3-car-

boxylic acid with aromatic and aliphatic alcohols and amines

in the presence of CDI. While the target esters were suc-

cessfully synthesised, the reaction of chromone 3-carboxylic

acid with amine compounds yielded the chromane-2,4-di-

ones, which was not entirely unexpected (14a–j). Qualitative

analysis using 1D and 2D NMR and X-ray crystallography

assisted in structure elucidation of the chromane-2,4-diones.

The results of the MAO inhibition studies showed that the

ester derivatives are weak MAO inhibitors, while several of

the chromane-2,4-diones exhibited good MAO-B inhibition

potencies. The most potent MAO-B inhibitor is compound

14b with an IC₅₀value of 0.638 µM. This compound is much

less potent as an MAO-A inhibitor with an IC₅₀of 77.9 µM.

It was further established that 14b is a reversible MAO-B

inhibitor. Compared to the reversible MAO-B selective

inhibitor, lazabemide (IC₅₀=0.091 µM), which was evalu-

ated under identical experimental conditions, compound 14b

is however a lower potency MAO inhibitor [50]. Similarly,

the MAO-B inhibition potency of 14b is signi f cantly lower

compared to pr eviously reported C6- and C7-substituted

chromones [22, 33, 34]. This is exemplifed by chromones

3–5, which are at leas t an order of magnitude more potent

MAO-B inhibitors compared to 14b.

NI

14b depr

14b

dialysed

not dialysed

Fig. 7 The reversibility of inhibition of MAO-B by compound 14b.

MAO-B was preincubated and dialysed in the absence of inhibitor

(NI—dialysed) and the presence of inhibitor 14b (14b—dialysed) and

(R)-deprenyl (depr—dialysed). The residual MAO-B activities were

recorded and are given as the mean standard deviation. The resid-

ual activities of non-dialysed mixtures of MAO-B and 14b were also

recorded (14b—not dialysed)

[I],

M

Molecular docking is considered to be a useful tool for

predicting potential binding orient ations and interactions of

ligands in the MAO active site [51–53]. Based on the crystal

structures of MAO-B in complex wit h reversible inhibitors,

it may be expected that chromane-2,4-diones will bind to the

active site with the chromane-2,4-dione moiety in proxim-

ity to the FAD, the most polar region. Here the chromane-

2,4-dione moiety can establish hydrogen bonding with

amino acid residues and water molecules. The 3-aminometh-

ylidene side chain is expected to extend into the entrance

cavity, which is reported to be lined by mostly non-polar

residues. To illustrate this, the trans and cis isomers of 14b

were docked into the active site of MAO-B (PDB: 2Z5X)

[54] with the CDOCKER module of Discovery Studio 3.1

(Accelrys) according to the reported protocol [49]. The

results are given in Fig. 9 and show that both isomers indeed

bind with the chromane-2,4-dione moiety placed in the sub-

strate cavity. Interestingly, the binding orientations of the

trans and cis isomers are very similar. While conventional

1/[S]

Fig. 8 Lineweaver–Burk plots of the inhibition of MAO-B by 14b.

The inset is a plot of the slopes of the Lineweaver–Burk plots versus

inhibitor concentration. Key: no inhibitor (flled squares); inhibitor

concentration=½×IC₅₀(circles), 1×IC₅₀(triangles), 1¼×IC₅₀(dia-

monds)

prepared in the absence of inhibitor, while the remaining

three plots were prepared in the presence of diferent con-

centrations of 14b (½ × IC₅₀, 1 × IC₅₀; 1¼ × IC₅₀). Kynu-

ramine at eight diferent concentrations (15–250 µM) was

used as substrate. The results are given in Fig. 8 and show

1 3

Molecular Diversity

Fig. 9 Overlay of the predicted

binding orientations of the

trans (purple) and cis (yellow)

isomers of 14b in the MAO-B

active site

hydrogen bonding is not observed, a pi–pi stacking interac-

tion is seen with Tyr-398, as well as a pi-sulphur interaction

with Cys-172 (cis isomer). Pi-alkyl interactions between the

chromane-2,4-dione and Leu-171 (both isomers) and Ile-

198 and Cys-172 (trans isomer) are formed, while the side

chain phenyl ring is stabilised by pi–alkyl interactions with

Ile-199 and Ile-316 (both isomers). Ile-199 is of further sig-

nifcance by establishing carbon-hydrogen bonding with a

benzylic H of the side chain and the C2 carbonyl oxygen of

the chromane-2,4-dione (both isomers). The phenolic oxy-

gen of Tyr-326 also forms a carbon-hydrogen bond with the

benzylic H (both isomers). This analysis suggests that both

isomers of 14b are able to bind and interact with MAO-B,

with the binding modes and interactions being very simi-

lar. It may be concluded that both isomers contribute to the

observed MAO-B inhibition.

elongation occurs to yield 14e. Considering the results of

the molecular docking study, it may be concluded that steric

conficts may occur between the 3-aminomethylidene side

chain and the MAO-B entrance cavity, as the length of the

side chain increases. A side chain linker length of 3 atoms

(e.g. 14b) seems to be optimal for avoiding steric conficts

and/or maximising productive interactions with the entrance

cavity. The placement of the side chain in the entrance cav-

ity thus appears to be an important determining factor for

MAO-B inhibition potency, and further enhancement of

activity should focus on modifcation of the 3-aminometh-

ylidene side chain and binding interactions with the entrance

cavity. As outlined in the introduction, MAO-B inhibitors

are useful agents in the treatment of Parkinson’s disease.

The chromane-2,4-diones may be particularly suitable for

further development since, as exemplifed by 14b, they are

fully reversible MAO-B inhibitors with comparatively weak

MAO-A inhibition potencies. This will greatly reduce the

liability for hypertensive crisis which is associated with irre-

versible MAO-A inhibition.

An analysis of the structure–activity relationships (SARs)

for MAO-B inhibition revealed some interesting trends.

While the ester derivatives are weak MAO-B inhibitors, for

the chromane-2,4-diones substitution with the benzyl group

appears to be appropriate for MAO-B inhibition among the

compounds evaluated. In this regard, larger substituents or

further substitution with chlorine on the side chain phenyl

results in decreased activity. It is noteworthy that substitu-

tion with the benzyl moiety (e.g. 14b) yielded the highest

potency MAO-B inhibition among the chromane-2,4-di-

ones. This structural element is present in many experimen-

tal MAO inhibitors as well as in the clinically used agent,

safnamide. Since MAO inhibition of these compounds is

modest, further structural modifcations will be required

to optimise these compounds for further development as

potent, reversible inhibitors of MAO-B. As mentioned, an

interesting observation is that MAO-B inhibition decreases

with side chain elongation from compound 14b to 14c

and 14d, with a large decrease in potency as further chain

Experimental section

Chemicals and instrumentation

All reagents and chemicals were purchased from Sigma-

Aldrich and were used without further purifcation. All

solvents used were obtained from ACE, while deuterated

solvents used for NMR spectroscopy were purchased from

Merck. Routine reaction monitoring was performed by thin-

layer chromatography (TLC). Silica gel 60 F254 sheets

(Merck) were used for TLC with mobile phases consisting

of ethyl acetate: methanol (8:2) and ethyl acetate: petroleum

ether (8:2). Carbon (¹³C) and proton (¹H) NMR spectra were

1 3

Molecular Diversity

recorded on a Bruker Avance III 600 spectrometer at fre-

quencies of 150 MHz and 600 MHz, respectively. All NMR

samples were dissolved in either deuterated chloroform

(CDCl₃) or deuterated dimethyl sulfoxide (DMSO-d₆). The

chemical shifts (δ) are reported in parts per million (ppm)

and coupling constants (J) in Hz. The spin multiplicities are

given in abbreviated format: s (singlet), br s (broad singlet),

d (doublet), br d (broad doublet), dd (doublet of doublets),

ddd (doublet of doublet of doublets), t (triplet), br t (broad

triplet), q (quartet), p (pentet/quintet), s (septet) or m (mul-

tiplet). Chemical shifts are referenced to the residual solvent

signal, at 7.26 ppm for ¹H and 77.0 ppm for ¹³C in CDCl₃

and at 2.50 ppm for ¹H and 39.5 ppm for ¹³C in DMSO-d₆.

High-resolution mass spectra (HRMS) were recorded on a

Bruker micrOTOF-Q II mass spectrometer in atmospheric-

pressure chemical ionisation (APCI) mode, in positive ioni-

sation mode. Melting points were determined with a Büchi

B-545 melting point apparatus and are uncorrected. An

Alpha FTIR spectrometer (Bruker) was used to record IR

spectra. The OPUS/Mentor software interface was used to

process the spectrometer readings. Fluorescence spectropho-

tometry was carried out using a Varian Cary Eclipse fuo-

rescence spectrophotometer. Microsomes from insect cells

containing recombinant human MAO-A and MAO-B (5 mg

protein/mL) and kynuramine dihydrobromide were obtained

from Sigma-Aldrich.

silicon crystal) Mo-K_α-radiation (0.71073 Å) from a sealed

microfocus tube. Generator settings were 50 kV, 1 mA.

APEX2 software was used for preliminary determination of

the unit cell and determination of integrated intensities, and

unit cell refnement was performed using Bruker SAINT.

The data collection temperature was –73 °C. Data were cor-

rected for absorption efects with SADABS using the multi-

scan technique. The structure was solved with XS, and sub-

sequent structure refnements were performed with XL. The

structure was refned by anisotropic full-matrix least-squares

refnement on F²: C₁₇H₁₄N₂O₃, M=294.30 g mol⁻¹, crys-

tal size: 0.20 × 0.20 × 0.60 mm³, monoclinic, space group

C 1 2/c 1, a=33.050(5) Å, b=4.9754(7) Å, c=21.666(3)

Å, α = γ=90°, β = 128.152(9)°, V = 2801.6(8) Å³, Z = 8,

ρ_calcd= 1.395 g cm⁻³, µ = 0.096 mm⁻¹, λ = 0.71073 Å,

T = 200(2) K, θ_range= 1.56–25.04°, refections collected:

13307, independent: 2474 (R_int=0.0338), 203 parameters,

fnal R indices [I>2σ(I)]: R₁=0.0584; wR₂=0.1593, max/

min residual electron density: 0.468/− 0.368 e⁻/Å³, good-

ness of ft on F²1.061, F(000)=1232. CCDC 1859532 con-

tains the supplementary crystallographic data for this paper.

The data can be obtained free of charge from The Cambridge

Crystallographic Data Centre via www.ccdc.cam.ac.uk/struc

tures.

Procedure for synthesis of ester derivatives 12a–e

Purity determination by HPLC

1,1′-Carbonyldiimidazole (CDI) (5.26 mmol, 1 equiv.) was

added to a stirred suspension of chromone 3-carboxylic acid

(5.26 mmol, 1 equiv.) in N,N-dimethylformamide (DMF)

(20 mL). This mixture was stirred for 2–3 h at 60 °C under

nitrogen, after which the reaction was cooled to 0 °C and the

alcohol (5.26 mmol, 1 equiv.) was added. The mixture was

allowed to return to room temperature and stirred overnight.

On completion, 40 mL of distilled water was added and the

aqueous phase was extracted to ethyl acetate (3 × 20 mL).

This organic fraction was washed with distilled water

(5×40 mL) and brine, dried over magnesium sulphate and

concentrated in vacuo. The resulting crude product was puri-

fed by recrystallisation from methanol.

Purity of the synthesised compounds was determined by

HPLC using an Agilent 1200 HPLC system equipped with

a quaternary pump and an Agilent 1200 series diode array

detector. HPLC grade acetonitrile (Merck) and Milli-Q water

(Millipore) were used for the chromatography. A Venusil

XBP C18 column (4.60×150 mm, 5 µm) was used with ace-

tonitrile (30%) and Milli-Q water (70%) as the initial mobile

phase at a fow rate of 1 mL/min. At the start of each injec-

tion, a solvent gradient programme was initiated by linearly

increasing the composition of the acetonitrile in the mobile

phase to 85% acetonitrile over a period of 5 min. Each run

lasted 15 min and a time period of 5 min was allowed for

equilibration between runs. A volume of 20 µL of solutions

of the test compounds in acetonitrile (1 mM) was injected

into the HPLC system, and the eluent was monitored at

wavelengths of 210, 254 and 300 nm.

Phenyl 4‑oxo‑4H‑chromene‑3‑carboxylate (12a) The title

compound was prepared from chromone-3-carboxylic acid

and phenol in a yield of 22%: mp. 154.7–155.5 °C (metha-

nol), pale yellow crystals. ¹H NMR (600 MHz, DMSO-d₆)

δ 9.26 (s, 1H, H-2), 8.13 (dd, J=7.9, 1.7 Hz, 1H, H-5), 7.88

(ddd, J=8.7, 7.2, 1.7 Hz, 1H, H-7), 7.76 (dd, J=8.5, 1.0 Hz,

1H, H-8), 7.58 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H, H-6), 7.50–

7.43 (m, 2H, H-3ʹ/5ʹ), 7.34–7.27 (m, 1H, H-4ʹ), 7.28–7.22

(m, 2H, H-2ʹ/6ʹ). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.5

(C-4), 164.0 (C-2), 161.1 (C-9), 155.2 (C-8a), 150.2 (C-1ʹ),

135.0 (C-7), 129.6 (C-3ʹ/5ʹ), 126.7 (C-6), 126.1 (C-4ʹ),

125.5 (C-5), 124.3 (C-4a), 121.9 (C-2ʹ/6ʹ), 118.7 (C-8),

X‑ray crystallography

X-ray crystallographic studies were carried out with a

Bruker SMART X2S benchtop crystallographic system. For

this purpose a single colourless crystal of compound 14j

was mounted on a MiTeGen MicroMount. Intensity meas-

urements were taken using monochromated (doubly curved

1 3

Molecular Diversity

114.8 (C-3). IR ν_max(cm⁻¹) 3057, 1746, 1645, 1614, 1486,

1094, 750. APCI-HRMS m/z calcd. for C₁₆H₁₁O₄, 267.0652,

[M+H]⁺found 267.0658. Purity (HPLC): 81%.

sample decomposed upon standing and purity was thus

deemed acceptable based on NMR.

(4‑Chlorophenyl)methyl 4‑oxo‑4H‑chromene‑3‑carboxylate

(12e) The title compound was prepared from chromone-

3-carboxylic acid and 4-chlorobenzyl-alcohol in a yield of

59%: mp. 116.6–117.7 °C (methanol), yellow crystals. ¹H

NMR (600 MHz, DMSO-d₆) δ 9.00 (s, 1H, H-2), 8.09 (dd,

J = 8.0, 1.7 Hz, 1H, H-5), 7.83 (ddd, J = 8.6, 7.1, 1.7 Hz,

1H, H-7), 7.70 (br d, J=8.4, 1H, H-8), 7.57–7.49 (m, 3H,

H-6, H-2′/6′ or H-3′/5′), 7.45 (d, J=8.4 Hz, 2H, H-2′/6′ or

H-3′/5′), 5.29 (s, 2H, H-10). ¹³C NMR (151 MHz, DMSO-

d₆) δ 172.5 (C-4), 162.9 (C-2), 162.6 (C-9), 155.2 (C-8a),

135.0 (C-1′), 134.8 (C-7), 132.6 (C-4′), 129.6 (C-2′/6′ or

C-3′/5′), 128.4 (C-2′/6′ or C-3′/5′), 126.5 (C-6), 125.5 (C-5),

124.4 (C-4a), 118.6 (C-8), 115.5 (C-3), 65.2 (C-10). IR ν_max

(cm⁻¹): 3080, 1732, 1647, 1614, 1461, 1083, 766. APCI-

HRMS m/z calcd. for C₁₇H₁₂ClO₄, 315.0419 [M+H]⁺found

315.0388. Purity (HPLC): 97%.

Benzyl 4‑oxo‑4H‑chromene‑3‑carboxylate (12b) The title

compound was prepared from chromone-3-carboxylic acid

and benzyl alcohol in a yield of 16%: mp. 107.3–108.3 °C

1

(methanol), pale brown crystals. H NMR (600 MHz,

CDCl₃) δ 8.65 (s, 1H, H-2), 8.27 (dd, J=8.0, 1.7 Hz, 1H,

H-5), 7.67 (ddd, J=8.7, 7.1, 1.7 Hz, 1H, H-7), 7.48–7.40

(m, 4H, H-6, H-8, H-2ʹ/6ʹ or H-3ʹ/5ʹ)*, 7.39–7.33 (m, 2H,

H-2ʹ/6ʹ or H-3ʹ/5ʹ)*, 7.34–7.28 (m, 1H, H-4ʹ), 5.35 (s, 2H,

H-10). ¹³C NMR (151 MHz, CDCl₃) δ 173.3 (C-4), 163.1

(C-9), 161.9 (C-2), 155.5 (C-8a), 135.5 (C-1ʹ), 134.2 (C-7),

128.6 (C-2ʹ/6ʹ or C-3ʹ/5ʹ), 128.24 (C-4ʹ), 128.16 (C-2ʹ/6ʹ

or C-3ʹ/5ʹ), 126.5 (C-5 or C-6), 126.2 (C-5 or C-6), 125.1

(C-4a), 118.1 (C-8), 116.0 (C-3), 66.9 (C-10). IR ν_max(cm⁻¹)

3087, 1727, 1650, 1613, 1462, 1079, 754. APCI-HRMS m/z

calcd. for C₁₇H₁₃O₄, 281.0808, [M+H]⁺found 281.0809.

Purity (HPLC): 97%. *In no particular order.

Procedure for synthesis

of 3‑aminomethylidene‑2,4‑chromandiones 14a–j

4‑Chlorophenyl 4‑oxo‑4H‑chromene‑3‑carboxylate (12c) The

title compound was prepared from chromone-3-carboxylic

acid and 4-chlorophenol in a yield of 21%: mp. 181.9–

CDI (5.26 mmol, 1 equiv.) was added to a stirred suspen-

sion of chromone 3-carboxylic acid (5.26 mmol, 1 equiv.)

in 20 mL DMF. This mixture was stirred for 2–3 h at 60 °C

under nitrogen, after which the reaction was cooled to 0 °C

and the amine (5.26 mmol, 1 equiv.) was added. The mix-

ture was allowed to return to room temperature and stirred

overnight. On completion of the reaction, 40 mL of distilled

water was added and the aqueous phase was extracted to

ethyl acetate (3×20 mL). This organic fraction was washed

with distilled water (5×40 mL) and brine, dried over mag-

nesium sulphate and concentrated in vacuo. The result-

ing crude product was purifed by recrystallisation from

methanol.

1

182.3 °C (methanol), pale yellow crystals. H NMR

(600 MHz, CDCl₃) δ 8.83 (s, 1H, H-2), 8.29 (dd, J= 8.0,

1.7 Hz, 1H, H-5), 7.72 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H,

H-7), 7.54–7.45 (m, 2H, H-6, H-8)*, 7.35 (d, J = 8.8 Hz,

2H, H-3ʹ/5ʹ), 7.16 (d, J = 8.8 Hz, 2H, H-2ʹ/6ʹ). ¹³C NMR

(151 MHz, CDCl₃) δ 173.0 (C-4), 162.8 (C-2), 161.6 (C-9),

155.5 (C-8a), 148.7 (C-1ʹ), 134.5 (C-7), 131.5 (C-4ʹ), 129.5

(C-3ʹ/5ʹ), 126.61, 126.57 (C-5, C-6)*, 125.0 (C-4a), 123.1

(C-2ʹ/6ʹ), 118.2 (C-8), 115.3 (C-3). IR ν_max(cm⁻¹) 3056,

1752, 1643, 1615, 1461, 1070, 758. APCI-HRMS m/z calcd.

for C₁₆H₁₀ClO₄, 301.0262, [M+H]⁺found 301.0268. Purity

(HPLC): 83%. *In no particular order.

3‑Chlorophenyl 4‑oxo‑4H‑chromene‑3‑carboxylate (12d) The

title compound was prepared from chromone-3-carboxylic

acid and 3-chlorophenol in a yield of 19%: mp. 151.2–

(3E/Z)‑3‑[(Phenylamino)methylidene]‑3,4‑dihydro‑2H‑1‑ben-

zopyran‑2,4‑dione (14a) The title compound was prepared

from chromone-3-carboxylic acid and aniline in a yield of

62%: m p. 206.7–209.5 °C (methanol), (lit. 208–210 °C,

[41]; 204–208 °C; [55], 201–202 °C (DMSO), [42]; 194–

236 °C (CHCl₃—petroleum ether), [45]), yellow f u fy crys-

tals. Ratio of major isomer: minor isomer 1:0.9. ¹H NMR

(600 MHz, CDCl₃) δ Major isomer: 13.68 (d, J=13.5 Hz,

1H, NH), 8.88 (d, J=13.6 Hz, 1H, H-9), 8.05 (dd, J=7.8,

1.7 Hz, 1H, H-5), 7.62–7.55 (m, 1H, H-7), 7.49–7.42 (m,

2H, H-2ʹ/6ʹ or H-3ʹ/5ʹ), 7.39–7.22 (m, 5H, H-2ʹ/6ʹ or H-3ʹ/5ʹ,

H-4′, H-6, H-8). Minor isomer: 11.92 (d, J =14.4 Hz, 1H,

NH), 9.02 (d, J = 14.5 Hz, 1H, H-9), 8.12 (dd, J = 7.8,

1.7 Hz, 1H, H-5), 7.62–7.55 (m, 1H, H-7), 7.49–7.42

(m, 2H, H-2ʹ/6ʹ or H-3ʹ/5ʹ), 7.39–7.22 (m, 5H, H-2ʹ/6ʹ or

H-3ʹ/5ʹ, H-4′, H-6, H-8). ¹³C NMR (151 MHz, CDCl₃) δ

1

155 °C (methanol), yellow crystals. H NMR (600 MHz,

DMSO-d₆) δ 9.27 (s, 1H, H-2), 8.13 (dd, J = 7.9, 1.7 Hz,

1H, H-5), 7.88 (ddd, J=8.7, 7.2, 1.7 Hz, 1H, H-7), 7.76 (dd,

J=8.5, 1.0 Hz, 1H, H-8), 7.58 (ddd, J=8.1, 7.2, 1.1 Hz, 1H,

H-6), 7.50 (t, J=8.1 Hz, 1H, H-5ʹ), 7.44 (t, J=2.1 Hz, 1H,

H-2ʹ), 7.39 (ddd, J=8.1, 2.1, 0.9 Hz, 1H, H-4ʹ), 7.26 (ddd,

J=8.2, 2.3, 1.0 Hz, 1H, H-6ʹ). ¹³C NMR (151 MHz, DMSO-

d₆) δ 172.5 (C-4), 164.4 (C-2), 160.7 (C-9), 155.2 (C-8a),

150.9 (C-1ʹ), 135.1 (C-7), 133.4 (C-3ʹ), 131.1 (C-5ʹ), 126.8

(C-6), 126.3 (C-4ʹ), 125.6 (C-5), 124.3 (C-4a), 122.4 (C-2ʹ),

121.0 (C-6ʹ), 118.8 (C-8), 114.4 (C-3). IR ν_max(cm⁻¹) 3072,

1738, 1642, 1615, 1462, 1088, 755. APCI-HRMS m/z calcd.

for C₁₆H₁₀ClO₄, 301.0262, [M+H]⁺found 301.0272. Purity:

1 3

Molecular Diversity

Major isomer: 181.8 (C-4), 163.6 (C-2), 154.95 (C-8a),

154.92 (C-9), 137.7 (C-1ʹ), 134.8 (C-7), 130.18 (C-3′/5′),

127.5 (C-4ʹ), 126.6 (C-5), 124.2 (C-6), 120.3 (C-4a), 118.45

(C-2′/6′), 117.43 (C-8), 98.7 (C-3). Minor isomer: 178.6

(C-4), 165.2 (C-2), 154.7 (C-8a), 153.4 (C-9), 137.7 (C-1ʹ),

134.7 (C-7), 130.16 (C-3′/5′), 127.4 (C-4ʹ), 125.8 (C-5),

124.4 (C-6), 120.6 (C-4a), 118.51 (C-2′/6′), 117.37 (C-8),

98.8 (C-3). IR ν_max(cm⁻¹) 3180, 1684, 1594, 1567, 755.

APCI-HRMS m/z calcd. for C₁₆H₁₂NO₃, 266.0812, [M+H]⁺

found 266.0807. Purity (HPLC): 90%.

C-3′/5′)*, 126.6 (C-4′), 125.3 (C-5), 123.95 (C-6), 120.3

(C-4a), 116.9 (C-8), 95.6 (C-3), 51.6 (C-10), 35.8 (C-11).

Minor isomer: 177.0 (C-4), 163.2 (C-2), 160.6 (C-9), 154.1

(C-8a), 137.9 (C-1′), 134.4 (C-7), 128.9, 128.5 (C-2′/6′,

C-3′/5′)*, 126.6 (C-4′), 125.8 (C-5), 124.04 (C-6), 120.4

(C-4a), 117.0 (C-8), 95.3 (C-3), 51.7 (C-10), 35.9 (C-11). IR

ν_max(cm⁻¹) 3063, 1708, 1590, 1463, 741. APCI-HRMS m/z

calcd. for C₁₈H₁₆NO₃, 294.1125, [M+H]⁺found 294.1129.

Purity (HPLC): 100%. *In no particular order.

(3E/Z)‑3‑{[(3‑Phenylpropyl)amino]methylidene}‑3,4‑dihy-

dro‑2H‑1‑benzopyran‑2,4‑dione (14d) The title compound

was prepared from chromone-3-carboxylic acid and 3-phe-

nyl-1-propylamine in a yield of 58%: mp. 171.3–171.6 °C

(methanol), white crystals. Ratio of major isomer: minor

isomer 1:0.7. ¹H NMR (600 MHz, DMSO-d₆) δ Major iso-

mer: 11.76–11.57 (m, 1H, NH), 8.44 (d, J= 14.7 Hz, 1H,

H-9), 7.96–7.89 (m, 1H, H-5), 7.68–7.61 (m, 1H, H-7),

7.40–7.08 (m, 7H, H-6, H-8, H-2′–6′)*, 3.60 (q, J=6.8 Hz,

2H, H-10), 2.64–2.57 (m, 2H, H-12), 1.95 (p, J = 7.3 Hz,

2H, H-11). Minor isomer: 10.52–10.21 (m, 1H, NH), 8.55

(d, J=15.5 Hz, 1H, H-9), 7.96–7.89 (m, 1H, H-5), 7.68–7.61

(m, 1H, H-7), 7.40–7.08 (m, 7H, H-6, H-8, H-2′–6′), 3.60

(q, J=6.8 Hz, 2H, H-10), 2.64–2.57 (m, 2H, H-12), 1.95 (p,

J=7.3 Hz, 2H, H-11). ¹³C NMR (151 MHz, DMSO-d₆) δ

Major isomer: 179.3 (C-4), 162.7 (C-2), 162.2 (C-9), 154.2

(C-8a), 140.9 (C-1′), 134.28 (C-7), 128.4, 128.3 (C-2′/6′,

C-3′/5′)*, 125.9 (C-4ʹ), 125.3 (C-5), 123.9 (C-6), 120.3

(C-4a), 116.9 (C-8), 95.7 (C-3), 50.0 (C-10), 31.9 (C-12),

31.15 (C-11). Minor isomer: 177.1 (C-4)**, 163.5 (C-2),

160.6 (C-9), 154.2 (C-8a), 140.9 (C-1′), 134.33 (C-7), 128.4,

128.3 (C-2′/6′, C-3′/5′)*, 125.7 (C-4′), 125.3 (C-5), 124.0

(C-6), 120.5 (C-4a), 117.0 (C-8), 95.7 (C-3), 50.2 (C-10),

31.9 (C-12), 31.2 (C-11). IR ν_max(cm⁻¹) 3023, 1711,

1603, 1462, 752. APCI-HRMS m/z calcd. for C₁₉H₁₈NO₃,

308.1281, [M+H]⁺found 308.1280. Purity (HPLC): 100%.*

In no particular order. **Shift based on HMBC correlation,

signal not visible on ¹³C NMR.

(3E/Z)‑3‑[(Benzylamino)methylidene]‑3,4‑dihydro‑2H‑1‑ben-

zopyran‑2,4‑dione (14b) The title compound was prepared

from chromone-3-carboxylic acid and benzylamine in a

yield of 58%: mp. 163.8–164.6 °C (methanol), (lit. 165–

167 °C, [41]), pink crystals. Ratio of major isomer: minor

isomer 1:0.3. ¹H NMR (600 MHz, DMSO-d₆) δ Major iso-

mer: 11.96–11.88 (m, 1H, NH), 8.62 (d, J= 14.6 Hz, 1H,

H-9), 7.89 (dd, J = 7.8, 1.4 Hz, 1H, H-5), 7.67–7.58 (m,

1H, H-7), 7.44–7.23 (m, 7H, H-6, H-8, H-2′–6′)*, 4.82 (d,

J=6.2 Hz, 2H, H-10). Minor isomer: 10.71–10.65 (m, 1H,

NH), 8.72 (d, J=15.4 Hz, 1H, H-9), 7.93 (br d, J=7.7 Hz,

1H, H-5), 7.67–7.58 (m, 1H, H-7), 7.44–7.23 (m, 7H,

H-6, H-8, H-2′–6′)*, 4.82 (d, J = 6.2 Hz, 2H, H-10). ¹³C

NMR (151 MHz, DMSO-d₆) δ Major isomer: 179.4 (C-4),

162.7 (C-2), 162.2 (C-9), 154.2 (C-8a), 136.7 (C-1′), 134.3

(C-7), 128.8 (C-2′/C-6′ or C-3′/C-5′), 128.0 (C-2′/C-6′ or

C-3′/C-5′), 127.98 (C-4′), 125.3 (C-5), 123.9 (C-6), 120.2

(C-4a), 116.9 (C-8), 96.0 (C-3), 53.2 (C-10). Minor isomer:

177.1 (C-4), 163.2 (C-2), 160.6 (C-9), 154.1 (C-8a), 137.0

(C-1′), 134.4 (C-7), 128.8 (C-2′/C-6′ or C-3′/C-5′), 128.0

(C-2′/C-6′ or C-3′/C-5′), 127.9 (C-4′), 125.8 (C-5), 124.0

(C-6), 120.4 (C-4a), 117.0 (C-8), 95.9 (C-3), 53.1 (C-10). IR

ν_max(cm⁻¹) 3033, 1708, 1592, 1463, 746. APCI-HRMS m/z

calcd. for C₁₇H₁₄NO₃, 280.0968, [M+H]⁺found 280.0972.

Purity (HPLC): 100%. *In no particular order.

(3E/Z)‑3‑{[(2‑Phenylethyl)amino]methylidene}‑3,4‑dihy-

dro‑2H‑1‑benzopyran‑2,4‑dione (14c) The title compound

was prepared from chromone-3-carboxylic acid and pheneth-

ylamine in a yield of 59%: mp. 178.2–178.7 °C (methanol),

white crystals. Ratio of major isomer: minor isomer 1:0.5.

¹H NMR (600 MHz, DMSO-d₆) δ Major isomer: 11.68–

11.62 (m, 1H, NH), 8.34 (d, J=14.3 Hz, 1H, H-9), 7.90 (dd,

J=7.7, 1.7 Hz, 1H, H-5), 7.66–7.59 (m, 1H, H-7), 7.33–7.17

(m, 7H, H-2ʹ–6ʹ, H-8, H-6)*, 3.83 (q, J=6.7 Hz, 2H, H-10),

2.96 (t, J = 7.2 Hz, 2H, H-11). Minor isomer: 10.40 (br s,

1H, NH), 8.45 (d, J=14.8 Hz, 1H, H-9), 7.90 (dd, J=7.7,

1.7 Hz, 1H, H-5), 7.66–7.59 (m, 1H, H-7), 7.33–7.17 (m,

7H, H-2ʹ–6ʹ, H-8, H-6)*, 3.83 (q, J=6.7 Hz, 2H, H-10), 2.96

(t, J=7.2 Hz, 2H, H-11). ¹³C NMR (151 MHz, DMSO-d₆) δ

Major isomer: 179.3 (C-4), 162.7 (C-2), 162.2 (C-9), 154.2

(C-8a), 137.9 (C-1′), 134.3 (C-7), 128.9, 128.5 (C-2′/6′,

(3E/Z)‑3‑{[(4‑Phenylbutyl)amino]methylidene}‑3,4‑dihy-

dro‑2H‑1‑benzopyran‑2,4‑dione (14e) The title compound

was prepared from chromone-3-carboxylic acid and 4-phe-

nylbutylamine in a yield of 13%: mp. 142.4–143.3 °C (meth-

anol), white crystals. Ratio of major isomer: minor isomer

1:0.4. ¹H NMR (600 MHz, CDCl₃) δ Major isomer: 11.86

(br s, 1H, NH), 8.34 (d, J = 14.1 Hz, 1H, H-9), 7.99 (dd,

J = 7.8, 1.7 Hz, 1H, H-5), 7.54 (ddd, J = 8.4, 7.4, 1.7 Hz,

1H, H-7), 7.30–7.12 (m, 7H, H-6, H-8, H-2ʹ–H-6ʹ)*, 3.50

(q, J = 6.4 Hz, 2H, H-10), 2.65 (t, J = 6.8 Hz, 2H, H-13),

1.79–1.66 (m, 4H, H-11, H-12)*. Minor isomer: 10.21 (br

s, 1H, NH), 8.50 (d, J=14.9 Hz, 1H, H-9), 8.07 (dd, J=7.8,

1.6 Hz, 1H, H-5), 7.57–7.53 (m, 1H, H-7), 7.30–7.12 (m,

7H, H-6, H-8, H-2ʹ–H-6ʹ)*, 3.52 (q, J=6.4 Hz, 2H, H-10),

1 3

Molecular Diversity

2.65 (t, J = 6.8 Hz, 2H, H-13), 1.79–1.66 (m, 4H, H-11,

H-12)*. ¹³C NMR (151 MHz, CDCl₃) δ Major isomer: 181.3

(C-4), 163.9 (C-2), 162.2 (C-9), 154.8 (C-8a), 141.08 (C-1ʹ),

134.2 (C-7), 128.5, 128.3 (C-2ʹ/6ʹ, C-3ʹ/5ʹ)* 126.1 (C-5),

125.6 (C-4ʹ), 123.9 (C-6), 120.5 (C-4a), 117.3 (C-8), 96.83

(C-3), 50.9 (C-10), 35.2 (C-13), 29.57, 28.0 (C-11, C-12)*.

Minor isomer: 178.4 (C-4), 165.2 (C-2), 160.6 (C-9), 154.6

(C-8a), 141.10 (C-1ʹ), 134.3 (C-7), 128.5, 128.3 (C-2ʹ/6ʹ,

C-3ʹ/5ʹ)*, 126.3 (C-5), 125.6 (C-4ʹ), 124.1 (C-6), 120.7

(C-4a), 117.2 (C-8), 96.76 (C-3), 50.8 (C-10), 35.2 (C-13),

29.64, 28.0 (C-11, C-12)*. IR ν_max(cm⁻¹) 3195, 1687,

1606, 1463, 754. APCI-HRMS m/z calcd. for C₂₀H₂₀NO₃,

322.1438, [M+H]⁺found 322.1445. Purity (HPLC): 100%.

*In no particular order.

(C-2), 162.3 (C-9), 154.3 (C-8a), 135.9 (C-1′), 134.5 (C-7),

132.7 (C-4′), 130.0, 128.8 (C-2′/6′, C-3′/5′)*, 125.3 (C-5),

124.0 (C-6), 120.3 (C-4a), 117.0 (C-8), 96.2 (C-3), 52.5

(C-10). Minor isomer: 177.3 (C-4), 163.2 (C-2), 160.8 (C-9),

154.3 (C-8a), 136.1 (C-1′), 134.5 (C-7), 132.7 (C-4′), 130.0,

128.8 (C-2′/6′, C-3′/5′)*, 125.8 (C-5), 124.1 (C-6), 120.4

(C-4a), 117.1 (C-8), 96.0 (C-3), 52.5 (C-10). IR ν_max(cm⁻¹)

3183, 1698, 1617, 1464, 753. APCI-HRMS m/z calcd. for

C₁₇H₁₃ClNO₃, 314.0578, [M+H]⁺found 314.0557. Purity

(HPLC): 100%.*In no particular order.

( 3 E / Z ) ‑ 3 ‑ ( { [ 2 ‑ ( 3 ‑ C h l o r o p h e n y l ) e t h y l ] a m i n o }

methylidene)‑3,4‑dihydro‑2H‑1‑benzopyran‑2,4‑dione

(14h) The title compound was prepared from chromone-

3-carboxylic acid and 2-(3-chlorophenyl)ethan-1-amine in a

yield of 70%: mp. 216.3–217.6 °C (methanol), white fufy

crystals. The ratio of major isomer: minor isomer 1:0.5. ¹H

NMR (600 MHz, DMSO-d₆) δ Major isomer: 10.45–10.35

(m, 1H, NH), 8.37 (d, J = 14.6 Hz, 1H, H-9), 7.94–7.88

(m, 1H, H-5), 7.68–7.60 (m, 1H, H-7), 7.39–7.18 (m, 6H,

H-2′,H-4′, H-5′, H-6′, H-6, H-8), 3.83 (q, J = 7.0 Hz, 2H,

H-10), 3.01–2.94 (m, 2H, H-11). Minor isomer: 11.66–11.61

(m, 1H, NH), 8.48 (d, J = 15.5 Hz, 1H, H-9), 7.94–7.88

(m, 1H, H-5), 7.68–7.60 (m, 1H, H-7), 7.39–7.18 (m, 6H,

H-2′,H-4′, H-5′, H-6′, H-6, H-8), 3.83 (q, J = 7.0 Hz, 2H,

H-10), 3.01–2.94 (m, 2H, H-11). ¹³C NMR (151 MHz,

DMSO-d₆) δ Major isomer: 179.3 (C-4), 162.7 (C-2),

162.3 (C-9), 154.2 (C-8a), 140.5 (C-1′), 134.3 (C-7), 133.1

(C-3′), 130.3, 128.9, 127.7, 126.6 (C-2′, C-4′, C-5′, C-6′)*,

125.3 (C-5), 124.0 (C-6) 120.3 (C-4a), 116.9 (C-8), 95.6

(C-3), 51.2 (C-10), 35.3 (C-11). Minor isomer: 177.0 (C-4),

163.2 (C-2), 160.7 (C-9), 154.1 (C-8a), 140.5 (C-1′), 134.4

(C-7), 133.1 (C-3′), 130.3, 128.9, 127.7, 126.5 (C-2′, C-4′,

C-5′, C-6′)*, 125.8 (C-5), 124.1 (C-6), 120.3 (C-4a), 117.0

(C-8), 95.4 (C-3), 51.3 (C-10), 35.3 (C-11). IR ν_max(cm⁻¹)

3140, 1705, 1623, 1464, 752. APCI-HRMS m/z calcd. for

C₁₈H₁₅ClNO₃, 328.0735, [M+H]⁺found 328.0734. Purity

(HPLC): 98%. *In no particular order.

(3E/Z)‑3‑{[(4‑Chlorophenyl)amino]methylidene}‑3,4‑dihy-

dro‑2H‑1‑benzopyran‑2,4‑dione (14f) The title compound

was prepared from chromone-3-carboxylic acid and 4-chlo-

roaniline in a yield of 57%: mp. 251.7–252.7 °C (metha-

nol), (lit. 250 °C, [56]), yellow solid. Ratio of major isomer:

minor isomer 1:0.4. ¹H NMR (600 MHz, DMSO-d₆) δ Major

isomer: 13.37 (d, J=13.8 Hz, 1H, NH), 8.84 (d, J=13.8 Hz,

1H, H-9), 8.00–7.95 (m, 1H, H-5), 7.74–7.67 (m, 3H, H-7,

H-2′/6′), 7.56–7.49 (m, 2H, H-3′/5′), 7.39–7.30 (m, 2H, H-6,

H-8)*. Minor isomer: 11.83 (d, J=14.5 Hz, 1H, NH), 8.86

(d, J=14.8 Hz, 1H, H-9), 8.00–7.95 (m, 1H, H-5), 7.74–7.67

(m, 3H, H-7, H-2′/6′), 7.56–7.49 (m, 2H, H-3′/5′), 7.39–

7.30 (m, 2H, H-6, H-8)*. ¹³C NMR (151 MHz, DMSO-d₆)

δ Major isomer: 180.3 (C-4), 162.3 (C-2), 155.8 (C-9),

154.4 (C-8a), 137.1 (C-1′), 135.1 (C-7), 131.2 (C-4′), 129.6

(C-3′/5′), 125.5 (C-5), 124.4 (C-6), 121.3 (C-2′/6′), 119.9

(C-4a), 117.2 (C-8), 98.3 (C-3). Minor isomer: 177.5 (C-4),

163.1 (C-2), 154.5 (C-9), 154.2 (C-8a), 137.6 (C-1′), 135.0

(C-7), 131.1 (C-4′), 129.5 (C-3′/5′), 125.9 (C-5), 124.5

(C-6), 121.7 (C-2′/6′), 120.2 (C-4a), 117.3 (C-8), 98.2 (C-3).

IR ν_max(cm⁻¹) 3076, 1711, 1614, 1463, 750. APCI-HRMS

m/z calcd. for C₁₆H₁₁ClNO₃, 300.0422, [M + H]⁺found

300.0439. Purity (HPLC): 79%. *In no particular order.

( 3 E / Z ) ‑ 3 ‑ ( { [ ( 4 ‑ C h l o r o p h e n y l ) m e t h y l ] a m i n o }

methylidene)‑3,4‑dihydro‑2H‑1‑benzopyran‑2,4‑dione

(14g) The title compound was prepared from chromone-

3-carboxylic acid and 4-chlorobenzylamine in a yield of

37%: mp. 182.7–183.1 °C (methanol), white crystals. Ratio

of major isomer: minor isomer 1:0.5. ¹H NMR (600 MHz,

DMSO-d₆) δ Major isomer: 11.87 (br s, 1H, NH), 8.61 (br s,

1H, H-9), 7.95–7.86 (m, 1H, H-5), 7.67–7.60 (m, 1H, H-7),

7.48–7.38 (m, 4H, H-2′/6′, H-3′/5′)*, 7.32–7.23 (m, 2H, H-6,

H-8), 4.80 (s, 2H, H-10). Minor isomer: 10.66 (br s, 1H,

NH), 8.71 (br s, 1H, H-9), 7.95–7.86 (m, 1H, H-5), 7.67–

7.60 (m, 1H, H-7), 7.48–7.38 (m, 4H, H-2′/6′, H-3′/5′)*,

7.32–7.23 (m, 2H, H-6, H-8), 4.80 (s, 2H, H-10). ¹³C NMR

(151 MHz, DMSO-d₆) δ Major isomer: 179.5 (C-4), 162.8

(3E/Z)‑3‑{[(Pyridin‑2‑yl)amino]methylidene}‑3,4‑dihy-

dro‑2H‑1‑benzopyran‑2,4‑dione (14i) The title compound

was prepared from chromone-3-carboxylic acid and 2-ami-

nopyridine in a yield of 65%: mp. 212.2–214.9 °C (meth-

anol), (lit. 237–240 °C (DMF), [47]), yellow solid. Ratio

of major isomer: minor isomer 1:0.4. ¹H NMR (600 MHz,

CDCl₃) δ Major isomer: 13.57 (d, J= 12.5 Hz, 1H, NH),

9.61 (d, J=12.8 Hz, 1H, H-9), 8.49–8.43 (m, 1H, H-6′), 8.05

(dd, J=7.8, 1.7 Hz, 1H, H-5), 7.77 (ddd, J=8.1, 7.4, 1.9 Hz,

1H, H-4′), 7.63–7.56 (m, 1H, H-7), 7.32–7.23 (m, 2H, H-6,

H-8), 7.20 (ddd, J=7.4, 4.8, 0.9 Hz, 1H, H-5′), 7.10 (br d,

J=8.1 Hz, 1H, H-3′). Minor isomer: 11.91 (d, J=13.7 Hz,

1H, NH), 9.71 (d, J=13.7 Hz, 1H, H-9), 8.49–8.43 (m, 1H,

H-6′), 8.13 (dd, J=7.8, 1.7 Hz, 1H, H-5), 7.77 (ddd, J=8.1,

1 3

Molecular Diversity

7.4, 1.9 Hz, 1H, H-4′), 7.63–7.56 (m, 1H, H-7), 7.32–7.23

(m, 2H, H-6, H-8), 7.20 (ddd, J=7.4, 4.8, 0.9 Hz, 1H, H-5′),

7.12 (br d, J = 8.1 Hz, 1H, H-3′). ¹³C NMR (151 MHz,

CDCl₃) δ Major isomer: 182.4 (C-4), 163.3 (C-2), 155.1

(C-9), 154.4 (C-8a), 149.4 (C-6ʹ), 149.1 (C-2′), 139.0 (C-4′),

135.1 (C-7), 126.0 (C-5), 124.2 (C-6), 122.1 (C-5′), 120.3

(C-4a), 117.5 (C-8), 113.4 (C-3′), 99.7 (C-3). Minor isomer:

178.8 (C-4), 165.3 (C-2), 154.7 (C-9), 154.5 (C-8a), 149.3

(C-6ʹ), 149.25 (C-2ʹ), 139.0 (C-4′), 134.9 (C-7), 126.7 (C-5),

124.5 (C-6), 122.0 (C-5′), 120.7 (C-4a), 117.4 (C-8), 113.2

(C-3′), 100.0 (C-3). IR ν_max(cm⁻¹) 3218, 1685, 1640, 1459,

760. APCI-HRMS m/z calcd. for C₁₅H₁₁N₂O₃, 267.0764,

[M+H]⁺found 267.0748. Purity (HPLC): 99%.

of the test inhibitors were prepared in DMSO and added

to the reactions to yield a fnal concentration of 4%. Reac-

tions serving as negative controls were carried out in the

absence of inhibitor. The enzyme reactions were initiated

with the addition of the MAO enzymes and were subse-

quently incubated for 20 min at 37 °C in a convection oven.

At endpoint, the reactions were terminated with the addition

of 80 µL sodium hydroxide (2 N) and the concentration of

4-hydroxyquinoline, the product of kynuramine oxidation

by MAO, was measured by fuorescence spectrophotom-

etry (λ_ex=310 nm; λ_em=400 nm). For this purpose, a linear

calibration curve was constructed with authentic 4-hydrox-

yquinoline (0.047–1.56 μM). The rates of MAO-catalysed

4-hydroxyquinoline formation thus measured were ftted to

the one-site competition model of the Prism 5 software pack-

age (GraphPad). This gave sigmoidal plots of rate versus

logarithm of inhibitor concentration from which the IC₅₀

values were estimated. IC₅₀values were measured in tripli-

cate and are reported as the mean standard deviation (SD).

(3E/Z)‑3({[2‑(Pyridin‑2‑yl)ethyl]amino}methylidene)‑3,4‑di-

hydro‑2H‑1‑benzopyran‑2,4‑dione (14j) The title com-

pound was prepared from chromone-3-carboxylic acid

and 2-(2-pyridyl)ethylamine in a yield of 53%: mp. 144.7–

145.8 °C (methanol), colourless crystals. Ratio of major iso-

mer: minor isomer 1:0.4. ¹H NMR (600 MHz, DMSO-d₆) δ

Major isomer: 11.81 (s, 1H, NH), 8.56–8.49 (m, 1H, H-6′),

8.40 (br s, 1H, H-9), 7.93–7.86 (m, 1H, H-5), 7.76–7.69

(m, 1H, H-4′), 7.66–7.58 (m, 1H, H-7), 7.33–7.21 (m, 4H,

H-3′, H-5′, H-6, H-8), 4.01 (t, J=6.7 Hz, 2H, H-10), 3.16

(t, J=6.6 Hz, 2H, H-11). Minor isomer: 10.61 (s, 1H, NH),

8.56–8.49 (m, 2H, H-9, H-6′), 7.93–7.86 (m, 1H, H-5), 7.76–

7.69 (m, 1H, H-4′), 7.66–7.58 (m, 1H, H-7), 7.33–7.21 (m,

4H, H-3′, H-5′, H-6, H-8), 4.01 (t, J= 6.7 Hz, 2H, H-10),

3.16 (t, J=6.6 Hz, 2H, H-11). ¹³C NMR (151 MHz, DMSO-

d₆) δ Major isomer: 179.2 (C-4), 162.7 (C-2), 162.2 (C-9),

157.8 (C-2′), 154.1 (C-8a), 149.1 (C-6′), 136.7 (C-4′), 134.2

(C-7), 125.3 (C-5), 123.9 (C-6), 123.6, 121.9 (C-3′, C-5′)*,

120.3 (C-4a), 116.9 (C-8), 95.6 (C-3), 49.3 (C-10), 36.97

(C-11). Minor isomer: 176.9 (C-4), 163.2 (C-2), 160.6 (C-9),

157.9 (C-2′), 154.2 (C-8a), 149.0 (C-6′), 136.7 (C-4′), 134.3

(C-7), 125.7 (C-5), 124.0 (C-6), 123.6, 121.9, (C-3′, C-5′)*,

120.4 (C-4a), 117.0 (C-8), 95.4 (C-3), 49.5 (C-10), 37.1

(C-11). IR ν_max(cm⁻¹): 3197, 1708, 1605, 1463, 762. APCI-

HRMS m/z calcd. for C₁₇H₁₅N₂O₃, 295.1077, [M + H]⁺

found 295.1071. Purity (HPLC): 100%. *In no particular

order.

Dialysis studies

Dialysis was carried out with Slide-A-Lyzer dialysis cas-

settes (Thermo Scientifc) with a molecular weight cut-of

of 10 000 and a sample volume capacity of 0.5–3 mL [49].

The test inhibitor (at a concentration equal to 4×IC₅₀) and

MAO-B (0.03 mg protein/mL) were prepared to a fnal vol-

ume of 0.8 mL in the dialysis bufer (potassium phosphate

bufer, 100 mM, pH 7.4, 5% sucrose). Stock solutions of the

inhibitor were prepared in DMSO and added to the bufer

to yield 4% DMSO. These samples were preincubated for

15 min at 37 °C and were subsequently dialysed at 4 °C in

80 mL of dialysis bufer. The dialysis bufer was replaced

with fresh bufer at 3 h and 7 h after the start of dialysis.

As positive control, MAO-B was similarly preincubated

and dialysed in the presence of the irreversible inhibitor,

(R)-deprenyl (IC₅₀= 0.079 µM) [57]. As negative control

dialysis of the enzyme was carried out in the absence of

the inhibitor. After 24 h of dialysis, 250 µL of the dialysed

samples was diluted twofold with the addition of 250 µL

kynuramine (dissolved in potassium phosphate buffer,

100 mM, pH 7.4, made isotonic with KCl) to yield reactions

with a fnal volume of 500 µL and containing kynuramine

(50 µM), MAO-B (0.015 mg protein/mL) and test inhibitor

(2×IC₅₀). The reactions (contained in 1.5 mL microcentri-

fuge tubes) were incubated at 37 °C in a waterbath and after

20 min were terminated with the addition of 400 µL sodium

hydroxide (2 N). After addition of 1000 µL water, the con-

centrations of 4-hydroxyquinoline were measured by fuo-

rescence spectrophotometry (λ_ex= 310 nm; λ_em= 400 nm)

employing a 3.5-mL quartz cuvette (pathlength 10×10 mm).

To quantify 4-hydroxyquinoline, a linear calibration

curve was constructed with authentic 4-hydroxyquinoline

Measurement of IC₅₀values for the inhibition

of MAO

Recombinant human MAO-A and MAO-B enzymes served

as enzyme sources [49]. The enzyme reactions were car-

ried out in white 96-well microtiter plates (Eppendorf) in

potassium phosphate bufer (pH 7.4, 100 mM, made iso-

tonic with KCl). The fnal volume of the reactions was

200 µL and contained kynuramine (50 μM), the test inhibi-

tors (0.003–100 µM) and MAO-A (0.0075 mg protein/

mL) or MAO-B (0.015 mg protein/mL). Stock solutions

1 3

Molecular Diversity

5. Schwarzschild MA, Agnati L, Fuxe K, Chen JF, Morelli M (2006)

Targeting adenosine A2A receptors in Parkinson’s disease. Trends

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(0.047–1.56 μM). For comparison, undialysed mixtures of

MAO-B and the test inhibitor were maintained at 4 °C for

24 h and subsequently diluted twofold and assayed as above.

All reactions were carried out in triplicate, and the residual

enzyme catalytic rates were expressed as mean SD.

Lineweaver–Burk plots and K_ivalue calculations

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A set consisting of four Lineweaver–Burk plots were con-

structed. The frst plot was constructed in the absence of

inhibitor, while the remaining three plots were constructed

in the presence of the test inhibitor at concentrations of

½ × IC₅₀, 1 × IC₅₀and 1¼ × IC₅₀. The enzyme–substrate,

kynuramine, was used at concentrations ranging from 15 to

250 µM, while the fnal enzyme concentration was 0.015 mg

protein/mL. All incubations were carried out in 1.5 mL

microcentrifuge tubes to a volume of 500 µL. The MAO-B

catalytic activity was measured by fuorescence spectropho-

tometry as described for the dialysis study. K_ivalues were

estimated from plots of the slopes of the Lineweaver–Burk

plots versus inhibitor concentration, where the x-axis inter-

cept equals −K_i.

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T (2008) Structure of human monoamine oxidase A at 2.2-A reso-

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pnas.0710626105

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Acknowledgements The NMR and MS spectra were recorded by

André Joubert and Johan Jordaan of the Laboratory for Analytical

Services (LAS) in the focus area Chemical Resource Benefciation,

while HPLC analysis was carried out by Jan du Preez of the Analytical

Technology Laboratory (ATL) at the North-West University (NWU).

X-ray crystallography was carried out by Johan Jordaan.

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and inhibitors in Parkinson’s disease and depressive illness. Br

J Pharmacol 147(Suppl 1):S287–S296. https://doi.org/10.1038/

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MAO-B inhibitors. J Med Chem 58:6717–6732. https://doi.

org/10.1021/jm501690r

Funding The fnancial assistance of the National Research Foundation

(NRF) of South Africa [Grant specifc unique reference numbers (UID)

85642, 96180, 76308] towards this research is hereby acknowledged.

The Grantholders acknowledge that opinions, fndings and conclu-

sions or recommendations expressed in any publication generated by

the NRF supported research are that of the authors, and that the NRF

accepts no liability whatsoever in this regard.

16. Carradori S, Petzer JP (2015) Novel monoamine oxidase inhibi-

tors: a patent review (2012–2014). Expert Opin Ther Pat 25:91–

110. https://doi.org/10.1517/13543776.2014.982535

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oxidase B in living healthy human subjects. Neurobiol Aging

18:431–435

Compliance with ethical standards

Conflict of interest The authors declare that they have no confict of

19. deSouza RM, Schapira A (2017) Safnamide for the treatment of

Parkinson’s disease. Expert Opin Pharmacother 18:937–943. https

://doi.org/10.1080/14656566.2017.1329819

interest.

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tyramine, food, beverages and the reversible MAO inhibitor

moclobemide. J Neural Transm Suppl 26:31–56

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Synthesis and evaluation of chromone derivatives as inhibitors of monoamine oxidase

DOI: 10.1007/s11030-019-09917-8

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