Novel monoamine oxidase inhibitors based on the privileged 2-imidazoline molecular framework

Article abstract of DOI:10.1016/j.bmcl.2018.11.018

Series of structurally diverse 2-imidazoline derivatives have been synthesized by condensation of substituted aldehydes with ethylenediamine, Pd-catalyzed N-arylation of 2-imidazolines and by the formation of 1,2,4-oxadiazoles and benzoxazepines from 2-imidazoline-containing precursors. The 2-imidazoline derivatives were evaluated as potential inhibitors of human monoamine oxidase (MAO) A and B. Among the 2-imidazolines, good potency inhibitors were discovered with compound 9p (IC₅₀ = 0.012 μM) being the most potent MAO-B inhibitor, while compound 9d (IC₅₀ = 0.751 μM) was the most potent MAO-A inhibitor of the series. These potencies are in the same range as those of reference MAO inhibitors used in the clinic. Among 33 compounds evaluated, 13 exhibited IC₅₀ values in the submicromolar range for the inhibition of an MAO isoform. It is postulated that the imidazoline moieties of some of these inhibitors may be recognized by the imidazoline I₂-binding site of MAO. Good potency MAO inhibitors may be useful for the treatment of neuropsychiatric and neurodegenerative disorders such as depression and Parkinson's disease, and future application for the treatment of prostate cancer, congestive heart failure and Alzheimer's disease. In addition, high potency 2-imidazoline-derived MAO inhibitors may be used as potential probes for the imidazoline binding sites of the MAOs, as well as to determine alternative binding regions of imidazoline within the MAO active site.

Full text of DOI:10.1016/j.bmcl.2018.11.018

Accepted Manuscript
Novel monoamine oxidase inhibitors based on the privileged 2-imidazoline
molecular framework
Anton Shetnev, Angelina Osipyan, Sergey Baykov, Alexander Sapegin, Zhanna
Chirkova, Michail Korsakov, Anél Petzer, Idalet Engelbrecht, Jacobus P. Petzer
PII:
S0960-894X(18)30883-7
https://doi.org/10.1016/j.bmcl.2018.11.018
BMCL 26129
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 September 2018
6 November 2018
8 November 2018
Please cite this article as: Shetnev, A., Osipyan, A., Baykov, S., Sapegin, A., Chirkova, Z., Korsakov, M., Petzer,
A., Engelbrecht, I., Petzer, J.P., Novel monoamine oxidase inhibitors based on the privileged 2-imidazoline
molecular framework, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.11.018
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Novel monoamine oxidase inhibitors based on the privileged 2-
imidazoline molecular framework
Anton Shetneva, Angelina Osipyanb, Sergey Baykovb, Alexander Sapeginb, Zhanna
Chirkovac, Michail Korsakova, Anél Petzerd, Idalet Engelbrechtd, Jacobus P. Petzerd,*
a
Pharmaceutical Technology Transfer Center, Ushinsky Yaroslavl State Pedagogical University, 108
Respublikanskaya St., Yaroslavl, 150000, Russian Federation
b
Institute of Chemistry, Saint Petersburg State University, 26 Universitetskii Prospect, Peterhof, Saint
Petersburg, 198504, Russian Federation
c
Yaroslavl State Technical University, 150023, Yaroslavl, Russian Federation
d
Pharmaceutical Chemistry and Centre of Excellence for Pharmaceutical Sciences, North-West University,
Potchefstroom 2520, South Africa
*Corresponding author.
E-mail address: jacques.petzer@nwu.ac.za
E-Mail Addresses
Anton Shetnev
a.shetnev@yspu.org
Angelina Osipyan
Sergey Baykov
Alexander Sapegin
Zhanna Chirkova
Michail Korsakov
Anél Petzer
angelina.osipyan@gmail.com
sergei.v.baikov@yandex.ru
sapegin_yar@mail.ru
chirkovazhv@ystu.ru
mkkors@mail.ru
12264954@nwu.ac.za
idalet.engelbrecht@gmail.com
jacques.petzer@nwu.ac.za
Idalet Engelbrecht
Jacobus P. Petzer
1

Abstract:
Series of structurally diverse 2-imidazoline derivatives have been synthesized by condensation of
substituted aldehydes with ethylenediamine, Pd-catalyzed N-arylation of 2-imidazolines and by the
formation of 1,2,4-oxadiazoles and benzoxazepines from 2-imidazoline-containing precursors. The 2-
imidazoline derivatives were evaluated as potential inhibitors of human monoamine oxidase (MAO)
A and B. Among the 2-imidazolines, good potency inhibitors were discovered with compound 9p
(IC50 = 0.012 µM) being the most potent MAO-B inhibitor, while compound 9d (IC50 = 0.751 µM)
was the most potent MAO-A inhibitor of the series. These potencies are in the same range as those of
reference MAO inhibitors used in the clinic. Among 33 compounds evaluated, 13 exhibited IC50
values in the submicromolar range for the inhibition of an MAO isoform. It is postulated that the
imidazoline moieties of some of these inhibitors may be recognized by the imidazoline I2-binding site
of MAO. Good potency MAO inhibitors may be useful for the treatment of neuropsychiatric and
neurodegenerative disorders such as depression and Parkinson’s disease, and future application for the
treatment of prostate cancer, congestive heart failure and Alzheimer’s disease. In addition, high
potency 2-imidazoline-derived MAO inhibitors may be used as potential probes for the imidazoline
binding sites of the MAOs, as well as to determine alternative binding regions of imidazoline within
the MAO active site.
Keywords: monoamine oxidase; MAO; inhibition; selective; 2-imidazoline
2

2-Imidazolines belong to a class of so-called privileged structures, since they can act as modulators of
a wide range of biological targets.1–3 It has been postulated that imidazoline compounds exert their
pharmacological actions by binding to at least three types of imidazoline binding sites, termed the I1-,
I2-, and I3-binding sites.4,5 The mitochondrial bound monoamine oxidase (MAO) enzymes have been
identified as possessing I2-binding sites to which imidazole ligands bind either competitively or non-
competitively. Structural evidence shows that for MAO-B, the I2-site corresponds to the “entrance”
cavity of the bipartite active site of the enzyme.5 In this respect, X-ray crystal structures show that 2-
(2-benzofuranyl)-2-imidazoline (2-BFI, 1) is bound with the imidazoline moiety hydrogen bonded to
the carbonyl oxygen of Pro102 and the phenolic hydrogen of Tyr326, while the benzofuran ring
potentially interacts via van der Waals interactions with Ile199 and Ile 316 (Fig. 1). 2-BFI is a
moderately potent competitive inhibitor of the MAOs with Ki values of 17 and 8.3 µM for human
MAO-A and MAO-B, respectively.5 Interestingly, covalent modification of the FAD cofactor in
MAO-B by the mechanism-based inhibitor, tranylcypromine, enhances the affinity of the enzyme for
2-BFI, possibly by inducing the closed conformation of the Ile199 side chain and altering the
conformation of the side chain of Gln206. Molecular dynamic simulation suggests that after
tranylcypromine modification, the MAO-B entrance cavity is smaller and less flexible, which may
contribute the greater affinity for imidazoline ligands.6 Human MAO-B inhibited by tranylcypromine
thus binds 2-BFI with a Kd of 9 nM.5 These findings suggest that highly potent MAO-B specific
inhibitors may be designed that specifically bind to the entrance cavity.
Fig. 1. The structure of 2-BFI (1).
The MAOs are of interest to medicinal chemists since they are key metabolic enzymes for amine
neurotransmitters and dietary amines. MAO-A catalyzes the oxidation of serotonin, while
benzylamine and β-phenylethylamine are MAO-B specific substrates. The catecholamines, dopamine,
noradrenaline and adrenaline, as well as the dietary amines, tryptamine and tyramine, are substrates
3

for both MAO isoforms.7 Inhibitors of MAO-A, by enhancing central serotonin levels, are thus of
value for the treatment of major depression,8,9 while MAO-B inhibitors are used in the treatment of
Parkinson’s disease where they block the metabolism of dopamine.7 MAO-B inhibitors may also
afford neuroprotection by reducing the central formation of hydrogen peroxide and aldehyde species,
by-products of MAO catalysis. MAO inhibitors have also been investigated as potential therapy for
Alzheimer’s disease,10 while MAO-A inhibitors in particular may provide cardioprotection in
cardiovascular disease by reducing hydrogen peroxide formation in the heart.11 MAO-A is also
considered to be a target for the treatment of cancer since MAO-A levels are reported to be elevated
in certain types of cancer tissue such as prostate cancer, and is associated with enhanced growth,
invasiveness, and metastasis of prostate cancer cells.12
Based on academic interest in the imidazoline class of inhibitors and the possibility of designing
MAO inhibitors that specifically target the imidazoline binding site, a number of studies have
explored the MAO inhibition properties of synthetic 2-imidazoline compounds. For example, a series
of 2-imidazoline derivatives have recently been shown to inhibit rat MAO-A and MAO-B with the
most potent inhibitor being compound 2 with Ki values of 12.8 and 1.38 µM, respectively (Fig. 2).13
Other 2-imidazoline inhibitors of the MAOs include cirazoline, naphazoline and clonidine.14,15 The
present study synthesizes four series of 2-imidazoline derivatives, which included 2-imidazolines
annulated with the benzoxazepine moiety (3a–c), benzonitrile-2-imidazoline derivatives substituted
with heteroaryl groups on N1 (4a–b), 2-imidazolines substituted with side chains containing two or
more linked aromatic moieties (5a–c), and 2-imidazoline derivatives that incorporates the 1,2,4-
oxadiazole moiety (6–10). MAO inhibitors incorporating the 1,3,4-oxadiazole moiety has been
described.16 Although these series are structurally highly diverse, the objective of this study was to
discover new 2-imidazoline-containing MAO inhibitors, compounds that may be used as future
probes for the imidazoline binding sites of the MAOs, and as potential leads for the development of
new therapeutic agents. As will be shown, numerous 2-imidazolines considered in this study are good
potency MAO inhibitors with submicromolar IC50 values. Although speculative, the good inhibition
4

potencies of such inhibitors may, at least in part, be attributed to recognition of the imidazoline
moiety by the I2-binding site.
Fig. 2. The structures of selected 2-imidazoline derivatives.
The first series of 2-imidazolines that was considered for this study, was benzoxazepine derivatives
3a–c (Fig. 3). These imidazolines, annulated with the benzoxazepine moiety, were obtained by the
metal free dehalocyclization reaction according to literature.17 The synthesis was carried out by
reacting 2-(2-hydroxyphenyl)imidazoline 11 with bis-electrophilic aromatic substrates 12a–c in DMF,
in the presence of K2CO3. In the first step of this reaction, nucleophilic aromatic substitution of the
halogen atom for the phenoxy group took place, which was followed by the Smiles rearrangement
leading to the regioselective formation of the 1,4-benzoxazepine ring. The second series, the N-
arylated 2-imidazolines 4a and 4b, was synthesized according to the previously developed procedure,
which consisted of the Pd-catalyzed coupling between imidazoline 13 and heteroaryl halides 14a,b
(Fig. 4).18 The third series, compounds 5a–c, was synthesized by condensation of substituted
aldehydes (15a–c) with ethylenediamine in the presence of the NBS as dehydrogenating agent (Fig.
5).19 Finally, a fourth series of structurally diverse 2-imidazoline derivatives that incorporates the
1,2,4-oxadiazole moiety (6–10) were synthesized by the condensation of amidoximes and carboxylic
acid esters in superbase medium, NaOH/DMSO (Figs. 6–8). The use of a superbasic medium for the
synthesis of the 1,2,4-oxadiazoles offers the possibility of obtaining these compounds from
amidoxime precursors in high yields and in the presence of other nucleophilic centers under mild
reaction conditions (rt, 2–8 h reaction time).20 Carrying out the synthesis at room temperature reduces
5

the probability of oxidation reactions which could lead to the conversion of the 2-imidazoline
precursors to the corresponding imidazoles. This new approach thus allowed for the synthesis of the
oxadiazole-containing imidazolines in good yields, employing simple procedures for isolating the
target products. Interestingly, oxadiazole-containing imidazolines have been reported to act as
potential factor Xa inhibitors and as modulators of sphingosine phosphate receptors,21,22 however,
their actions on MAO have not yet been examined. The synthesized compounds were characterized by
NMR, MS and elemental analysis, while a crystal structure (CCDC 1847812) was obtained for
imidazoline 9p (Fig. 9).
Fig. 3. The synthesis of benzoxazepine derivatives 3a–c. Key: (i) K2CO3, DMF, 50–90 °C.
Fig. 4. The synthesis of N-arylated 2-imidazolines 4a–b. Key: (i) Pd(CH3COO)2 (2%), BINAP (4%),
Cs2CO3, toluene, 100 °C, 20 h.
6

Fig. 5. The synthesis of 2-imidazolines 5a–c. Key: (i) NBS, CH3CN, 0–20 °C, 12 h.
Fig. 6. The synthesis of 2-imidazolines 6a–c, 7 and 8a–c. Key: (i) NH2OH∙HCl, NaHCO3, C2H5OH;
(ii) NaOH, DMSO.
7

Fig. 7. The synthesis of 2-imidazolines 9a–p. Key: (i) NaOH, DMSO.
Fig. 8. The synthesis of 2-imidazoline 10. Key: (i) NaOH, DMSO.
8

Fig. 9. ORTEP representation of the X-ray crystal structure of compound 9p displaying thermal
ellipsoids at 50% probably level.
The MAO inhibition properties of the 2-imidazolines were determined according the literature
protocol.23 Recombinant human MAO-A and MAO-B served as enzyme sources and the MAO-A/B
nonselective substrate, kynuramine, was used as substrate for both MAO isoforms.24 The MAO-
catalyzed oxidation of kynuramine ultimately yields 4-hydroxyquinoline, which may be measured by
fluorescence spectrophotometry after alkalization of the enzyme reaction mixtures. MAO activity was
thus measured in the absence and presence of different concentrations of the test inhibitors (0.0003–
100 µM), and sigmoidal plots of enzyme catalytic rate versus logarithm of inhibitor concentration
were constructed. From these plots, the IC50 values were estimated and are given the mean ± standard
deviation of triplicate experiments. An example of sigmoidal dose–response curves for the
determination of IC50 values is given in fig. 10.
9

100
75
50
25
0
-3
-2
-1
0
1
2
Log[I]
Fig. 10. Sigmoidal dose-response curves for the inhibition of MAO-A by 9d (open circles), and
MAO-B by compounds 6b (filled circles) and 9p (triangles), respectively.
The results of the MAO inhibition studies are given in table 1, and show that 2-imidazolines 3–5 are
weak inhibitors of MAO-A and MAO-B with IC50 values >17.2 µM. Most compounds did not inhibit
MAO-A up to a maximal tested concentration of 100 µM. Among derivatives 6–10, however, good
potency MAO inhibition was observed with 9p being the most potent inhibitor with an IC50 value of
0.012 µM for MAO-B. This inhibition potency is similar to those of the reference MAO-B inhibitors,
lazabemide (IC50 = 0.091 µM) and safinamide (IC50 = 0.048 µM), which were evaluated under
identical experimental conditions.25 Four additional derivatives also displayed IC50 values <0.1 µM
for the inhibition of MAO-B (e.g. 6b, 8a, 9g, 9h), while only one derivative inhibited MAO-A with a
submicromolar IC50 value (9d; IC50 = 0.751 µM). With regards to isoform specific inhibition, 9a and
9j are noteworthy as compounds with high MAO-B specificity and good inhibition potency. These
compounds inhibited MAO-B with IC50 values of 0.608 and 0.756 µM, while displaying no inhibition
of MAO-A at a maximal tested concentration of 100 µM. Compound 9h is also noteworthy as a
particularly potent and specific MAO-B inhibitor (IC50 = 0.030 µM) with an SI of 570. The most
potent inhibitor, 9p, also displays good specificity for MAO-B inhibition (SI = 92). Derivative 9p,
however, also may be viewed as a relatively potent MAO-A inhibitor since its inhibition potency is
10

similar to the reference MAO-A inhibitor, toloxatone (IC50 = 3.92 µM), which was evaluated under
identical experimental conditions.25
Table 1. The human MAO inhibition potencies of 2-imidazoline derivatives 3–10, 19 and reference
compounds.
IC50 (µM)a
MAO-A
MAO-B
SIb
–
> 100
> 100
3a
3b
3c
4a
4b
5a
5b
5c
6a
6b
6c
7
> 100
> 100
–
32.0 ± 4.31
> 100
> 100
<0.32
>5.8
>2.4
–
17.2 ± 19.5
41.8 ± 2.30
> 100
> 100
> 100
> 100
> 100
–
> 100
18.4 ± 0.106
4.28 ± 0.269
0.071 ± 0.005
39.7 ± 0.276
0.160 ± 0.0096
0.030 ± 0.0025
1.06 ± 0.111
23.9 ± 2.97
0.608 ± 0.007
0.230 ± 0.022
9.98 ± 1.78
0.411 ± 0.011
1.37 ± 0.033
0.890 ± 0.121
0.064 ± 0.0054
0.030 ± 0.0067
1.26 ± 0.122
0.756 ± 0.044
0.216 ± 0.019
9.02 ± 0.107
3.64 ± 0.428
47.6 ± 15.8
2.28 ± 0.486
0.012 ± 0.00061
0.300 ± 0.054
1.87 ± 0.222
–
>5.4
1.7
32
7.37 ± 0.330
2.31 ± 0.123
41.1 ± 2.82
14.7 ± 1.85
1.39 ± 0.067
88.3 ± 5.78
62.2 ± 15.1
> 100
1.0
91
46
8a
8b
8c
9a
9b
9c
9d
9e
9f
83
2.6
>164
97
22.5 ± 1.33
> 100
>10
1.8
2.0
1.7
85
0.751 ± 0.023
2.77 ± 0.454
1.57 ± 0.148
5.47 ± 0.518
17.1 ± 0.325
> 100
9g
9h
9i
570
>79
>132
55
> 100
9j
12.0 ± 0.205
24.6 ± 3.38
55.5 ± 3.17
> 100
9k
9l
2.7
15
9m
9n
9o
9p
10
19
>2.1
5.4
92
12.4 ± 0.099
1.11 ± 0.007
24.7 ± 3.53
147 ± 35.7
82
78
Toloxatone 3.92c
Lazabemide
–
–
0.091c
0.048c
Safinamide
a
All values are expressed as the mean ± standard deviation (SD) of triplicate determinations.
11

b
c
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of
IC50(MAO-A)/IC50(MAO-B).
Value obtained from reference.25
Although the series investigated here are structurally diverse, some structure-activity relationships
(SARs) may be derived. For example, the active 2-imidazolines are specific for the MAO-B isoform,
possibly due to the relatively large size of the compounds studied here. In general, the MAO-B active
site is better able to accommodate larger inhibitors than MAO-A.26 Compounds 3–5 are weak MAO
inhibitors, which may be due to the non-linear arrangement of the rings that constitute the structures
of these compounds. The MAO-A and MAO-B active sites are relatively flat elongated cavities and
MAO inhibitors are for the most part either small molecules or structures that can adopt a linear
arrangement of the ring systems and functional groups.27,28 The 1,2,4-oxadiazole compounds (6–10)
are in general good potency MAO-B inhibitors since all of the compounds are relatively linear. The
arrangement of the functional groups on the 1,2,4-oxadiazole does not seem to affect inhibition
activity to a high degree, and placement of the imidazolylphenyl substituent on either the 3 (e.g. 6b,
8a) or 5 (e.g. 9g, 9h, 9p) positions yields good potency inhibitors. The finding that 8b is a good
potency MAO-B inhibitor shows that substitution with the 2-imidazoline ring on the phenyl may
occur either meta or para to the 1,2,4-oxadiazole moiety. With methylation of the 2-imidazoline (e.g.
7 and 10), good potency MAO-B inhibition is retained. Considering the structures of the five most
potent MAO-B inhibitors of this study it is observed that, with the exception of 6b, all compounds are
substituted with a chlorophenyl on the 1,2,4-oxadiazole moiety (8a, 9g, 9h), with the most potent
inhibitor 9p, being substituted with dichlorophenyl. Halogen-containing phenyl rings are thus suitable
substituents where good potency MAO-B inhibition is desired. Other aromatic (e.g. 6b, 9a, 9d, 9j, 9k)
as well as heteroaromatic (e.g. 9f, 10) and aliphatic substitution (e.g. 9b) on the 1,2,4-oxadiazole
moiety, however also yield good potency MAO-B inhibition.
This study selected one of the most potent MAO-B inhibitors, 6b, to investigate the reversibility of
MAO-B inhibition. A mixture of compound 6b (at a concentration of 4 × IC50) and MAO-B was pre-
incubated for 15 min and subsequently dialyzed for 24 h. After dialysis, the samples were diluted
12

twofold to yield an inhibitor concentration of 2 × IC50, and the residual MAO-B activities were
measured. For this purpose, kynuramine was added to the dialysis samples and the reactions were
incubated for 20 min. 4-Hydroxyquinoline generated from the MAO-B-catalyzed oxidation of
kynuramine was subsequently quantified by fluorescence spectrophotometry. The residual MAO-B
activities in non-dialyzed mixtures of 6b and MAO-B were also recorded for comparison while, as
negative and positive controls, dialysis experiments were carried out in the absence of inhibitor and
with the irreversible MAO-B inhibitor, (R)-deprenyl, respectively. The results of the dialysis
experiments are given in Fig. 11 and show that 6b is a reversible inhibitor of MAO-B since enzyme
activity is partially recovered by dialysis, with the activity at 66% compared to the negative control
value (100%). In contrast, a greater degree of inhibition of MAO-B (by 6b) is observed in undialyzed
samples with the activity at 36%. As expected, dialysis does not restore catalytic activity when MAO-
B was incubated in the presence of (R)-deprenyl (residual activity of 4.7%).
100
75
50
25
0
NI
6b depr
dialyzed
6b
undialyzed
Fig. 11. Reversibility of inhibition of MAO-B by 6b. MAO-B and 6b (at a concentration of 4 × IC50)
were incubated for 15 min, dialyzed for 24 h and the residual enzyme activity was measured (6b
dialyzed). Similar incubation and dialysis of the enzyme in the absence (NI dialyzed) and presence of
the irreversible inhibitor, (R)-deprenyl (depr dialyzed), were also carried out. The residual activity of
undialyzed mixtures of the enzyme and 6b was also recorded (6b undialyzed).
13

To investigate the mode of MAO-B inhibition by 6b, sets of Lineweaver-Burk plots were constructed.
For this purpose, the catalytic activities of MAO-B were recorded at 8 different kynuramine
concentrations (15–250 µM). These measurements were carried out in the absence as well as presence
of five different concentrations of the inhibitor (¼ × IC50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50).
The Lineweaver-Burk plots are given in fig. 12 and suggest that 6b is a competitive inhibitor of
MAO-B since the plots are linear and intersect at the y-axis. The competitive mode of inhibition
further supports the finding that 6b acts as a reversible inhibitor of MAO-B. From the Lineweaver-
Burk plots a Ki value of 0.038 µM is estimated for the inhibition of MAO-B by 6b.









[I], M






1/[S]


Fig. 12. Lineweaver-Burk plots of MAO-B activity in the absence (filled squares) and presence of
various concentrations of 6b (¼ × IC50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50.). The inset is a
graph of the slopes of the Lineweaver-Burk plots versus inhibitor concentration.
In conclusion, the present study discovers, among structurally highly diverse series of 2-imidazolines,
compounds that are good potency MAO inhibitors with submicromolar IC50 values. These are
exemplified by compounds 6b, 8a, 9g, 9h and 9p (IC50 < 0.1 µM) which represent good potency
inhibitors of MAO-B. It is tempting to speculate that the 2-imidazoline moiety of these inhibitors are
recognized by the I2-binding site, leading to greater binding affinity. Being relatively large molecules,
for these inhibitors, such a binding mode would place the terminal phenyl ring in proximity to the
14

FAD in the substrate cavity of MAO-B. These inhibitors thus represents, in addition to several known
MAO-B inhibitors, cavity-spanning inhibitors. Structural evidence suggests that such cavity-spanning
inhibitors display, in general, higher potency inhibition compared to inhibitors that bind to only the
substrate cavity.26 From this it may be postulated that the MAO-B inhibition potencies of imidazoline
inhibitors which bind to the I2-binding site, may be enhanced by substitution with appropriate side
chains that are recognized by the substrate binding site. It may thus be concluded that 2-imidazoline
derivatives such as those discovered here would be suitable leads for the design of cavity-spanning
MAO-B inhibitors that are possibly recognized by the I2-binding site as well as residues in the
substrate cavity. For this design approach, however, further structural information is required to guide
the selection of suitable substituents and substitution patterns. It should be kept in mind that the 2-
imidazoline moiety may also be recognized by the aromatic sandwich residues of MAO-B, Tyr435
and Tyr-398, particularly since this is the most polar region of the active site. For example, for 9p the
imidazoline moiety is more polar compared to the dichlorophenyl, which suggests that it should be
placed in proximity to the FAD where hydrogen bonding with Tyr435 and Tyr398 as well as active
site waters may occur. The dichlorophenyl moiety, on the other hand, may better interact with the
entrance cavity which is hydrophobic in nature. To illustrate this, 9p was docked into the active site of
MAO-B (PDB: 2Z5X)29 with the CDOCKER module of Discovery Studio 3.1 (Accelrys) according to
the reported protocol.23 The result is given in Fig. 13, and shows that the 2-imidazoline moiety of 9p
is indeed placed in the substrate cavity with hydrogen bonding occurring with Tyr435, Tyr398 and a
water molecule. The dichlorophenyl partially overlaps with the benzofuran of 2-BFI.
15

Fig. 13. Overlay of the binding orientation of 2-BFI (cyan; PDB: 2XFN) and the predicted binding
orientation of 9p (green) in the MAO-B active site.
To further investigate the proposal that some of the 2-imidazoline derivatives may be suitable leads
for drug design, the logP value of 9p was calculated (ChemSketch 14, Advanced Chemistry
Development). In the absence of pharmacokinetic and in vitro property data, much may be concluded
from the lipophilicity of a compound. With a logP of 3.93, 9p is expected to exhibit high permeability
through biological membranes, a property required for absorption from the gastrointestinal tract and
access to the brain. Future design strategies should, however, focus on reducing the logP value in
order to promote aqueous solubility, a key determinant of absorption. Among the most potent
inhibitors of this study, 9g (logP = 2.67) has the most favorable lipophilicity (6b, logP = 2.95; 8a,
logP = 3.29; 9h, logP = 3.29).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was financially supported by the Russian Foundation for Basic Research (RFBR) grant No.
18-33-01108. Further support was obtained from the National Research Foundation of South Africa
[Grant specific unique reference numbers (UID) 85642 and 96180]. The Grantholders acknowledge
16

that opinions, findings and conclusions 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. Physicochemical studies were performed at the Magnetic Resonance
Research Centre, Center for X-ray Diffraction Studies and Chemical Analysis and Materials Research
Centre (Saint Petersburg State University).
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Highlights


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
2-Imidazolines were examined as potential MAO inhibitors
2-Imidazolines (e.g. 2-BFI) are known to bind to I2-binding sites
This study finds 9p to be the most MAO-B inhibitor (IC50 = 0.012 µM)
Alternative binding regions of imidazoline within the MAO active site are proposed
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Graphical Abstract
100
75
50
25
0
-3
-2
-1
0
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2
Log[I]
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