Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.02.038

A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the

Full text of DOI:10.1016/j.ejmech.2016.02.038

Accepted Manuscript
Design and synthesis of novel chalcones as potent selective monoamine oxidase-B
inhibitors
Arwa Hammuda, Raed Shalaby, Stefano Rovida, Dale E. Edmondson, Claudia Binda,
Dr. Ashraf Khalil
PII:
S0223-5234(16)30118-0
10.1016/j.ejmech.2016.02.038
EJMECH 8389
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2015
Revised Date: 13 February 2016
Accepted Date: 15 February 2016
Please cite this article as: A. Hammuda, R. Shalaby, S. Rovida, D.E. Edmondson, C. Binda, A. Khalil,
Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.038.
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O
O
O
F3C
No
Ki = 0.78 µM
effect
MAO-A

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Design and synthesis of novel chalcones as potent
selective monoamine oxidase-B inhibitors
Arwa Hammudaa, Raed Shalabya, Stefano Rovidab,
Dale E. Edmondsonc, Claudia Bindab, and Ashraf Khalila,*
aCollege of Pharmacy, Qatar University, Doha, Qatar
bDepartment of Biology and Biotechnology, University of Pavia, Via Ferrata
1, 27100 Pavia, Italy
cDepartment of Biochemistry, Emory University School of Medicine,
Atlanta, Georgia 30322, USA
* Corresponding author: Dr. Ashraf Khalil
Qatar University, College of Pharmacy
P.O. Box 2713
Doha, Qatar
E-mail: akhalil@qu.edu.qa
Tel: +974-44035579
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Abstract
A novel series of substituted chalcones were designed and synthesized to be evaluated
as selective human MAO-B inhibitors. A combination of either methylsulfonyl or
trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on
the other end of the chalcone scaffold was investigated. The compounds were tested for
their inhibitory activities on both human MAO-A and B. All compounds appeared to be
selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range.
Molecular modeling studies have been performed to get insight into the binding mode of
the synthesized compounds to human MAO-B active site.
Keywords
Monoamine oxidase, neuroprotection, drug design, synthesis, enzyme, chalcones.
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1. Introduction
Monoamine oxidases (MAO1, EC 1.4.3.4) are mitochondrial membrane-bound,
flavin-dependent enzymes responsible for the metabolism of arylalkylamine
neurotransmitters (epinephrine, norepinephrine, β-phenylethylamine, serotonin and
dopamine)[1] and a variety of xenobiotic amines[2] in the CNS and peripheral tissues by
catalyzing their oxidative deamination to the corresponding aldehydes (Fig. 1). Two
MAO isoforms (MAO-A and MAO-B) have been identified based on substrate specificity,
inhibitor selectivity[3], amino acid sequence and tissue distribution.[4] MAO-A
preferentially catalyzes the deamination of serotonin, epinephrine and norepinephrine
and is specifically inhibited by clorgyline, while MAO-B preferentially oxidizes β-
phenylethylamine and benzylamine and is inhibited by L-deprenyl. Both isoforms are
active towards dopamine and tyramine.[5] The well-known role of MAOs in the
metabolism of biogenic amines in neuronal tissues makes these enzymes important
drug targets for diseases such as depression and Parkinson’s disease.
Currently, the Protein Data Bank contains more than 40 crystal structures of MAO
(most of them MAO-B) in complex with different reversible and irreversible inhibitors
with resolutions ranging from 1.6 to 3 Å.[6] MAO-A and MAO-B share 70% sequence
identity and a similar overall fold topology with the main differences consisting in details
of the respective active sites, which accounts for their divergence in substrate and
inhibitor specificity. The availability of such structural data paved the way for rationalized
drug design of isoform-selective MAO inhibitors. Targeting MAO-A
exerts an
1
Abbreviations: MAO, monoamine oxidase; MMTP, 1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-
tetrahydropyridine; MMDP+, 1-methyl-4-(1-methylpyrrol-2-yl)-dihydropyridinium cation
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antidepressant effect, while selective MAO-B inhibitors are being used to treat
Parkinson’s disease.[7] In addition, the MAO-catalyzed deamination reactions produce
hydrogen peroxide as a byproduct (Fig. 1), which contributes to the oxidative stress
condition typical of many pathologies. In this regard, MAO inhibitors are considered to
act as neuroprotective agents in degenerative processes. [5, 8, 9]
Figure 1- MAO-catalyzed oxidative deamination of dopamine to 3,4-
dihydroxyphenylacetaldehyde with the production of hydrogen peroxide as a byproduct
Based on the finding that MAO-B expression in neuronal tissue increases about
4-fold with age especially in glial cells[10], interest has increased in developing selective
MAO-B inhibitors for neurodegenerative diseases. The majority of known MAO-B
inhibitors act in an irreversible manner by forming a N(5)-flavocyanine adduct with the
enzyme leading to potential immunogenicity effects in addition to long-lasting enzyme
inhibition[11]. For this reason, over the last years, drug design studies have been
focused on the development of reversible inhibitors that are devoid of these
disadvantages, such as the recently approved new anti-Parkinson drug safinamide[12].
Along this line, new scaffolds have been explored including dihydropyrazoles[13],
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chromones[14], coumarins[15], xanthines[16], thiazolidindiones[17] and chalcones[18].
The last ones, which chemically correspond to trans-1,3-diphenyl-2-propen-1-ones,
represent an interesting group of natural compounds that are abundant in fruits and
vegetables and possess a wide range of biological activities such as, anti-
inflammatory[19], antimalarial[20], antifungal[21], anticancer[22] and antidepressant
activities.[23]. They are open chain flavonoids and act as precursors in the biosynthetic
pathways of this plant secondary metabolites[24]. With their structure consisting of two
aromatic rings linked through a three-carbon α,β-unsaturated carbonyl system (C6-C3-
C6), they possess the required structural features for selective binding to human MAO-
B[18, 25]. In this study, we report the synthesis of a series of novel chalcones that
proved promising in terms of binding affinity and A/B isoform selectivity.
2. Results and Discussion
2.1.Chemistry
Ten novel chalcones were designed and prepared by Claisen-Schmidt condensation
reaction of equimolar amounts of the appropriate substituted benzaldehydes and
acetophenones in 1 mmol, 50% ethanolic NaOH solution as shown in Scheme-1.
Scheme 1- Synthetic route to the selected chalcones
The filtered crude products were crystallized from ethanol to give crystalline
compounds. The structures of the obtained compounds were confirmed by pertinent
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spectroscopic techniques e.g. FT-IR, GC-MS and NMR. To assign the chemical shifts of
all protons and carbons, compound-1 was subjected to 2D-NMR (HMQC and HMBC;
Fig. 2). The olefinic protons appeared as doublet. The proton signal at δ 7.55 (J= 16
Hz) was assigned to the α-H and the carbon signal at δ 122.9 ppm to the α-C. While
the proton signal at δ 8.12 (J= 16 Hz) and the carbon signal at δ 141 were assigned to
the β-H and β-C respectively. The large coupling constant of the olefinic protons
confirms the trans configuration.
Figure 2 - Key HMBC correlations of compound 1
2.2.Biochemistry
The prepared compounds were screened for their inhibitory activities against human
MAO-A and MAO-B at 10 µM final concentration by using an assay based on 1-methyl-
4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP; Scheme 2) as substrate for
both MAO isoforms (measured Km values for human MAO-A and MAO-B are 88 µM and
101 µM, respectively, in agreement with previous reports [26-28]). The MAO-catalyzed
oxidation of MMTP substrate is monitored at a wavelength (420 nm) that does not
overlap with the inhibitor spectrum, therefore providing a direct and convenient assay
for inhibition studies.
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Scheme 2 - MAO-catalyzed oxidation of MMTP (a) to MMDP+ (b)
Two series of chalcone derivatives were tested at a final concentration of 10 µM
(Table 1). Derivatives 1-5, which feature different substituents on the aromatic ketone
moiety (A) and a dimethoxyphenyl ring (B) on the other end, showed poor MAO-B
inhibition except for compound 3 and, in part, for 1. Instead, the series 6-10 resulted to
be more active, which suggests that the chalcone scaffold bearing a benzodioxol ring
binds to human MAO-B with higher affinity than the dimethoxyphenyl moiety. In
particular, the same substituent on ring A produces different inhibition effects when
combined with either a methoxyphenyl or a benzodioxol units on ring B, as evinced by
comparing 1 with 6, 4 with 9 and 5 with 10. The only exceptions are represented by a
trifluoromethyl in ortho position that prevents binding in both cases (2 and 7) and a
methylsulfonyl group in para that improves inhibition for either derivative (3 and 8).
None of these molecules inhibits human MAO-A, except for a limited effect exerted by
6.
Experiments to determine the IC50 values were carried out on those compounds that
showed a MAO-B inhibitory potency higher than 60% (1, 3, 6, 8, 9, 10; Table 1). For all
of them the IC50 values are in the micromolar range with inhibitor 6 being the most
active (IC50 = 0.55 µM) and 1 the weakest (~ 8-fold less potent than 6). A time-
dependent analysis was performed by incubating MAO-B with compound 6 at a
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concentration of 0.55 µM final concentration (IC50) for varying periods of time (up to 1
hour) and measuring the residual enzymatic activity to investigate the binding mode of
these class of molecules. The MAO-B residual enzymatic activity was unchanged at the
various time points indicating that the inhibitory properties of 6 (and presumably those of
the other compounds under investigation) are not time-dependent and do not involve
any covalent adduct with either the flavin cofactor or protein residues (Fig. 3).
Figure 3 - Time-dependent inhibition of human MAO-B catalyzed oxidation of MMTP by
compound 6. The enzyme was preincubated for various periods of time (5 - 60 min) with
6 at 0.55 ꢀM final concentration. The rates are expressed as apparent kcat values (min-
1). Error bars are shown and correspond to SEM from two replicates.
To investigate the inhibition of the most potent compounds in more detail and to
provide a comparative analysis with other known MAO-B inhibitors, Ki values were
determined (Fig. 4) for compounds 3, 6, 8, 9, 10 (Table 1). The inhibition constants are
approximately in line with the IC50 values. Compound 9 represents the most potent
inhibitor with a Ki value of 0.55 µM, although 6 and 8 essentially inhibit MAO-B at a
similar extent and even the inhibition constant for derivative 9 falls in the micromolar
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range. This suggests that any substituent on the ring A either at meta- or para- positions
(but not in ortho- as in 7) exerts a similar effect on the binding affinity to MAO-B.
Instead, within series 1-5 a methylsulfonyl substituent (as in 3) improves significantly the
inhibitory potency with respect to the other substituents. Altogether, these observations
suggest that a finely tuned combination of different chemical modifications on the
ketone aromatic ring with a benzodioxol ring on the other end of the chalcone molecule
leads to inhibitors with good human MAO-B binding affinity.
Figure 4 - Original curves for human MAO-B competitive inhibition by compound 9. The
inhibition constant (Ki) was determined by fitting the apparent kcat values measured at
varying MMTP substrate concentrations to the equation describing competitive
inhibition: app. kcat = kcat [S] / ([S] + Km + Km/Ki), where S is substrate concentration
whereas kcat and Km are the steady-state parameters for each of the curves plotted at
different inhibitor concentrations (inset in the plot) [29].
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Table 1
Structures and human MAO inhibitory properties of the synthesized compounds.
MAO-B
MAO-A
Inhibition
at 10 µM
(%)
IC50
Ki
Inhibition
at 10 µM
(%)
Compound
R1
R2
R3
(µM)a
(µM)
1
2
H
CF3
H
CF3
H
63
27
90
36
41
96
7
4.49 ± 0.03
n.d.b
n.d.b
n.d.b
0
0
H
H
3
H
H
CH3-SO2-
1.97 ± 0.04 1.15 ± 0.12
0
4
H
CH3-S-
n.d.b
n.d.b
n.d.b
n.d.b
0
5
H
F3C-O-
CF3
H
H
0
6
H
H
H
0.55 ± 0.07 0.78 ± 0.11
n.d.b n.d.b
16
n.d.b
0
7
CF3
H
8
H
CH3-SO2-
CH3-S-
H
93
86
88
1.00 ± 0.07 0.72 ± 0.07
0.95 ± 0.07 0.55 ± 0.05
1.51 ± 0.05 2.36 ± 0.34
9
H
H
0
10
H
F3C-O-
0
a All IC50 values shown in the table are the mean ± SEM from two experiments.
b n.d., not determined.
2.3.Molecular docking simulation
A docking simulation of the most active (3, 6, 8, 9, 10) among the synthesized
compounds was performed to investigate their binding mode inside human MAO-B
active site. In this enzyme either substrates or competitive inhibitors bind to a highly
hydrophobic cavity that may exist as two entities: an “entrance cavity” separated from
the outer surface by the cavity gating loop (residues 99–110), and a “substrate cavity”
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that extends from FAD to the entrance cavity. Residues Tyr398 and Tyr435, together
with FAD, form an “aromatic cage” where the catalyzed reaction occurs[30], while Ile199
plays an important role as a gating residue between the two cavities by adopting either
a closed or an open conformation.[27] The docking simulation with compound 6 showed
that the aromatic amino acids present in the binding site aid in the accommodation of
the benzodioxol ring within the aromatic sandwich formed by Tyr398 and Tyr435,
whereas ring A is directed towards the entrance cavity (Figs. 5 and 6). The same result
was obtained with inhibitors 9 and 10 (Fig. 7), whereas compounds 3 and compound 8
are exceptions as they may adopt two major poses where the methylsulfonyl group is
predicted to be either directed towards the substrate- or the entrance-cavity. This may
be due to the ability of this group to form hydrogen bonds with Tyr398 and with the
water molecules present in the aromatic cage (Fig. 8). This result predicts that the
orientation of both rings mainly depends on the substituents and their ability to form
binding contacts with the aromatic cage. In all poses the carbonyl oxygen anchors the
inhibitors to the bottom of the binding site by forming hydrogen bond with Cys172. The
position of the substituent on ring A was showed to be critical for inhibition, as it is
favorable to be on meta- or para- position to be perfectly surrounded by the hydrophobic
amino acids forming the entrance cavity (residues: Leu164, Leu167, Phe168 and
Trp119). In contrast, substitution on ortho- position of ring A would cause a steric clash
with carbonyl group that distorts the co-planar ring system and does not satisfy the
appropriate distance between the carbonyl group as a hydrogen bond acceptor and the
hydrophobic group. This may explain the lower activity of compound 2 and 7 compared
to compound 1 and 6.
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Figure 5 - Ribbon stereoview of human MAO-B structure with the docked pose of
compound 6 (stick representation), FAD is represented as yellow spheres. The inset
shows a zoomed view of the active site highlighting the docked pose of compound 6 in
MAO-B binding site with Ile199 adopting the open conformation. A hydrogen bond
between Cys172 and carbonyl oxygen is shown as a green line.
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Figure 6 - 2D representation of the interactions of compound 6 with residues of human
MAO-B binding site according to the docking results. The red arrow represents H-bond
and yellow rings represent hydrophobic interactions. Key interacting amino acids are
shown.
Figure 7 - Superimposition of docked poses of compounds 6 (green), 8 (purple), 9
(cyan) and 10 (pink) inside MAO-B binding site showing the same binding mode where
ring A occupies the entrance cavity and ring B is directed towards FAD (yellow). Key
interacting amino acids are shown in stick representation.
Figure 8 – a) The atypical binding mode of compound 3 inside MAO-B binding site as
predicted by the docking calculations: ring A is directed towards FAD forming hydrogen
bonds (green dashed lines) with Tyr398 and a water molecule. The methoxylated ring
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occupies the entrance cavity. Key interacting amino acids are shown in stick
representation. b) 2D representation of the atypical binding mode of compound 3 inside
MAO-B binding site. The red arrows represent H-bonds and yellow rings represent
hydrophobic interactions.
Conclusion
Chalcones are aromatic compounds which were shown to exert inhibitory properties
on the MAO drug targets[18]. In this study, ten new chalcone analogs were evaluated
for their activity on recombinant human MAO-A and B. The obtained data confirmed that
all the prepared chalcone analogs are selective MAO-B inhibitors with negligible or no
inhibitory activity on MAO-A, which is in agreement with previous studies[18, 25, 31]. In
the present study a higher level of novelty was explored by introducing a benzodioxol
ring in the molecule combined with different substituent on the other aromatic ring
(Table 1). This is relevant considering that this chemical entity is present also in 3,4-
methylenedioxymethamphetamine (the well-known illicit drug “ecstasy”) which was
showed to inhibit MAOs[32]. Our analysis revealed that the same substituent on the
ketone aromatic ring exerts a different effect when combined with either a
methoxyphenyl or a benzodioxol moiety, with the latter being more inhibitory.
The most active compounds were selected for further analyses, including both
enzymatic assays to measure their inhibition potency and docking simulation to predict
the binding mode. The inhibition of the chalcone derivatives is not time-dependent,
indicating that their binding is reversible. The Ki values range from 0.55 to 2.36 µM with
compound 9 being the most potent and having the same inhibition potency of the new
anti-Parkinson drug safinamide[33]. These values are also comparable to those recently
reported[34] for other chalcone derivatives, but a higher level of MAO-B/MAO-A
selectivity was herewith achieved. In this regard, it is important to notice that the
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hydrogen bond predicted to form between the inhibitor carbonyl oxygen and Cys172 is
common to all the studied inhibitors. This protein residue is not conserved in human
MAO A (corresponding to Asn181), which might represent an important element in
determining isozyme selectivity[35]. The molecular docking simulation provided an
explanation also for the crucial correlation between the inhibitory activity and the
position of the substituent on the aromatic ketone ring, because it showed as either
meta- or para- substitution would allow an inhibitor orientation perfectly surrounded by
the hydrophobic active site residues. Moreover, an interesting result was obtained with
compounds 3 and 8 that are predicted to bind in two opposite conformations including
that with the methylsulphonyl-substituted ring within the MAO-B aromatic sandwich in
front of the flavin ring. This observation will prompt further structural studies in order to
investigate this aspect.
3. Experimental protocols
3.1.Materials and methods
All fine chemicals and solvents were obtained from Sigma–Aldrich (unless specified
otherwise) and were used without further purification. For screening and IC50
determination, insect cell microsomes containing recombinant human MAO-A and
MAO-B (5 mg/mL) from Sigma–Aldrich were used. For Ki measurements, recombinant
human MAO-A and MAO-B were over-expressed in Pichia pastoris and purified as
detergent-solubilized homogeneous samples by using published protocols .[27]
Analytical thin layer chromatography was performed on precoated silica gel 60 F254
aluminum plates purchased from Fisher Scientific. Melting points were determined on a
Stuart SMP40 automatic melting point apparatus and are uncorrected. All compounds
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were analyzed for the presence of functional groups using Perkin Elmer Spotlight 400
Fourier transform infrared (FTIR) Imaging System. GC-MS analysis was performed on
an Agilent 5973 GC/MS equipment. Proton (1H) and carbon (13C) NMR spectra were
recorded on a Bruker Avance III spectrometer at 400 MHz using DMSO-d6 as a solvent
or on a Jeol 500 in CDCl3. Coupling constants are reported in hertz (Hz). The chemical
shifts are reported as parts per million (δ) relative to the solvent peak. Purity of
compounds were confirmed with C, H and S analysis performed on Thermo Scientific
FLASH 2000 CHNS/O analyzer.
3.2.Chemistry
3.2.1. General Procedure for the synthesis of chalcones (1-10)
Chalcones were prepared utilizing Claisen-Schmidt condensation. To a stirred solution
of the substituted acetophenone (1 mmol) and the substituted benzaldehyde (1 mmol) in
ethanol at 0 °C, sodium hydroxide solution (0.5 ml, 50%) was added dropwise. The
solution was then stirred at room temperature overnight. The formed precipitate was
filtered and washed with cold water and recrystallized from ethanol, to afford chalcones
1-10.
3.2.1.1.
(1)
(E
)-3-(2,3-dimethoxyphenyl)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one
1
Yellowish white solid, yield = 57%, m.p. = 84 – 88 °C, H NMR (400 MHz, CDCl3)
δ
8.42
-Olefinic), 7.83 (d, J =
7.8 Hz, 1H, H4), 7.64 (dd, J = 8.2 Hz, 7.8 Hz, 1H, H5), 7.55 (d, J = 16 Hz, 1H,
(s, 1H, H2), 8.18 (d, J = 7.8 Hz, 1H, H6), 8.12 (d, J = 16 Hz, 1H,
β
α-
Olefinic), 7.28 (dd, J= 7.1 Hz, 1.4 Hz, 1H, H6`), 7.11 (dd, J= 7.8 Hz, 7.8 Hz, 1H, H5`),
6.99 (dd, J= 7.6 Hz, 1.4 Hz, 1H, H4`), 3.89 (s, 6H, -OCH3). 13C NMR (101 MHz, CDCl3)
189.7, 152, 149, 141.1, 138, 131.8, 129.4, 129.2, 128, 125.5, 124, 123, 119.7, 114.6,
61.5, 56. Anal. calcd. for C18H15F3O3: C, 64.29; H, 4.50. Found: C, 64.79; H, 4.54. EI-
MS (m/z): calculated 336.1, observed 336.1 (M).
δ
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3.2.1.2.
(E
)-3-(2,3-dimethoxyphenyl)-1-(2-(trifluoromethyl)phenyl)prop-2-en-1-
one (2)
1
Buff solid, yield = 80%, m.p. = 63 - 66 °C, H NMR (400 MHz, DMSO-d6)
7.7 Hz, 1H, H6), [7.83 (dd, J = 7.3 Hz, 1H), 7.77 (dd, J = 7.6 Hz, 1H), H4 and H5], 7.68
(d, J = 7.4 Hz, 1H, H3), 7.52 (d, J = 16.4 Hz, 1H, -Olefinic), 7.45 (dd, J = 7.4, 1.6 Hz,
1H, H6`), 7.24 (d, J = 16.4 Hz, 1H, -Olefinic), 7.20 – 7.09 (m, 2H, H4` and H5`), 3.82 (s,
3H, -OCH3), 3.65 (s, 3H, -OCH3). 13C NMR (101 MHz, DMSO-d6)
188.23, 152.76,
δ 7.91 (d, J =
β
α
δ
148.44, 139.05, 138.29, 132.48, 130.14, 129.80, 129.48, 127.99, 125.27, 124.88,
124.29, 122.41, 119.30, 115.35, 61.06, 55.83. Anal. calcd. for C18H15F3O3: C, 64.29; H,
4.50. Found: C, 65.11; H, 4.46. EI-MS (m/z): calculated 336.1, observed 336.1 (M).
3.2.1.3. (
E
)-3-(2,3-dimethoxyphenyl)-1-(4-(methylsulfonyl)phenyl)prop-2-en-1-one
(3)
1
Pale yellow solid, yield = 67%, m.p. = 171 - 175 °C , H NMR (400 MHz, DMSO-d6)
δ
8.35 (d, J = 8.3 Hz, 2H, H3 and H5), 8.11 (d, J = 8.3 Hz, 2H, H2 and H6), 8.04 (d, J =
15.8 Hz, 1H, -Olefinic), 7.91 (d, J = 15.8 Hz, 1H, -Olefinic), 7.64 (dd, J = 6.4, 2.8 Hz,
1H, H6`), 7.25 – 7.10 (m, 2H, H4` and H5`), 3.85 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3),
3.31 (s, 3H, -SO2CH3). 13C NMR (101 MHz, DMSO-d6)
188.76, 152.78, 148.48,
β
α
δ
144.19, 141.39, 139.25, 129.36, 127.92, 127.42, 124.36, 122.69, 119.30, 115.45, 61.07,
55.86, 43.21. Anal. calcd. for C18H18O5S: C, 62.41; H, 5.24; S, 9.26. Found: C, 62.31; H,
5.27; S, 10.03. EI-MS (m/z): calculated 346.1, observed 346.1 (M).
3.2.1.4. (
E
)-3-(2,3-dimethoxyphenyl)-1-(4-(methylthio)phenyl)prop-2-en-1-one (4)
1
Pale yellow solid, yield = 83%, m.p. = 82 - 86 °C, H NMR (400 MHz, DMSO-d6)
δ
8.08
-Olefinic), 7.88 (d, J = 15.8 Hz,
-Olefinic), 7.61 (dd, J = 9.0, 4.3 Hz, 1H, H5`), 7.40 (d, J = 8.5 Hz, 2H, H3 and H5),
7.15 (d, J = 4.7 Hz, 2H, H4` and H6`), 3.84 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 2.55 (s,
3H, -S-CH3). 13C NMR (101 MHz, DMSO-d6)
188.01, 152.77, 148.25, 145.61, 137.65,
(d, J = 8.5 Hz, 2H, H2 and H6), 7.98 (d, J = 15.8 Hz, 1H,
1H,
β
α
δ
133.73, 129.03, 128.24, 124.96, 124.30, 122.73, 119.18, 114.96, 60.98, 55.81, 13.92.
Anal. calcd. for C18H18O3S: C, 68.77; H, 5.77; S, 10.20. Found: C, 68.97; H, 5.74; S,
11.25. EI-MS (m/z): calculated 314.1, observed 314.1 (M).
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3.2.1.5. (
E
)-3-(2,3-dimethoxyphenyl)-1-(3-(trifluoromethoxy)phenyl)prop-2-en-1-
one (5)
1
White solid, yield = 31%, m.p. = 81 - 84 °C, H NMR (400 MHz, DMSO-d6)
1H), 8.09 – 7.99 (m, 2H), 7.9 (d, 1H), 7.77 – 7.60 (m, 3H), 7.21 – 7.10 (m, 2H), 3.83 (s,
3H, -OCH3), 3.79 (s, 3H, -OCH3). 13C NMR (101 MHz, DMSO-d6)
187.94, 152.76,
δ 8.19 (d,
δ
148.72, 148.43, 139.61, 138.95, 131.05, 127.99, 127.65, 125.55, 124.29, 122.39,
120.77, 119.28, 115.32, 61.03, 55.82. Anal. calcd. for C18H15F3O4: C, 61.39; H, 4.29.
Found: C, 61.97; H, 4.32. EI-MS (m/z): calculated 352.1, observed 352.1 (M).
3.2.1.6. (E)-3-(benzo[d][1,3]dioxol-4-yl)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one(6)
Deep yellow solid, yield = 45%, m.p. = 102 – 105 °C , 1H NMR (400 MHz, DMSO-d6)
8.36 (d, J = 7.8 Hz, 1H, H4), 8.31 (s, 1H, H2), 8.03 (d, J = 7.8 Hz, 1H, H6), 7.90 (d, J =
15.7 Hz, 1H, -Olefinic), 7.82 (dd, J = 7.8 Hz, 1H, H5), 7.74 (d, J = 15.7 Hz, 1H,
Olefinic), 7.40 (d, J = 8.0 Hz, 1H, H6`), 7.01 (d, J = 7.5 Hz, 1H, H4`), 6.92 (dd, J = 7.9
Hz, 1H, H5`), 6.18 (s, 2H, -O-CH2-O-). 13C NMR (101 MHz, DMSO-d6) 188.07,
δ
β
α-
δ
147.69, 146.96, 138.46, 138.14, 132.34, 130.22, 129.52, 124.65, 122.93, 121.95,
121.86, 116.86, 110.39, 101.78. Anal. calcd. for C17H11F3O3: C, 63.76; H, 3.46. Found:
C, 63.74; H, 3.41. EI-MS (m/z): calculated 320.1, observed 320.1 (M).
3.2.1.7.
(E)-3-(benzo[d][1,3]dioxol-4-yl)-1-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (7)
1
Yellow solid, yield = 62%, m.p. = 70 - 74 °C, H NMR (400 MHz, DMSO-d6)
δ 7.91 (d, J
= 7.7 Hz, 1H, H6), [7.84 (d, J = 7.5 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H), H4 and H5] 7.67 (d,
J = 7.4 Hz, 1H, H3), 7.26 (s, 2H, Olefinic), 7.20 (d, J = 7.8 Hz, 1H, H6`), 7.03 (d, J = 7.6
Hz, 1H, H4`), 6.90 (dd, J = 7.9 Hz, 1H, H5`), 6.15 (s, 2H, -O-CH2-O-). 13C NMR (101
MHz, DMSO-d6)
δ 194.43, 147.74, 146.67, 141.33, 138.16, 132.60, 130.56, 128.30,
126.77, 122.35, 122.08, 116.30, 110.69, 101.84.Anal. calcd. for C17H11F3O3: C, 63.76;
H, 3.46. Found: C, 64.39; H, 3.53. EI-MS (m/z): calculated 320.1, observed 320.1 (M).
3.2.1.8.
(E)-3-(benzo[d][1,3]dioxol-4-yl)-1-(4-(methylsulfonyl)phenyl)prop-2-en-1-one (8)
8.27 (d, J
Yellow solid, yield = 45%, m.p. = 126 - 130 °C, 1H NMR (400 MHz, DMSO-d6)
δ
= 7.4 Hz, 2H, H3 and H5), 8.13 (d, J = 7.3 Hz, 2H, H2 and H6), 7.85 (d, J = 15.6 Hz, 1H,
Olefinic), 7.73 (d, J = 15.6 Hz, 1H, -Olefinic), 7.37 (d, J = 7.4 Hz, 1H, H6`), 7.03 (d, J = 7.0 Hz,
1H, H4`), 6.94 (d, J = 7.4 Hz, 1H, H5`), 6.20 (s, 2H, -O-CH2-O-), 3.36 (s, 3H, -SO2-CH3). 13C
NMR (101 MHz, DMSO-d6) 188.85, 147.72, 146.93, 144.17, 141.32, 138.94, 129.22, 127.52,
β-
α
δ
123.49, 122.18, 122.03, 116.85, 110.50, 101.82, 59.76, 43.24, 20.75, 14.08.Anal. calcd. for
18

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C17H14O5S: C, 61.81; H, 4.27; S, 9.70. Found: C, 61.67; H, 4.25; S, 8.87. EI-MS (m/z):
calculated 330.1, observed 330.1 (M).
3.2.1.9.
(E)-3-(benzo[d][1,3]dioxol-4-yl)-1-(4-(methylthio)phenyl)prop-2-en-1-one (9)
1
Greenish yellow solid, yield = 23%, m.p. = 83 – 87 °C, H NMR (400 MHz, DMSO-d6)
δ
7.99 (d, J = 8.3 Hz, 2H, H2 and H6), 7.85 (d, J = 15.7 Hz, 1H,
15.7 Hz,1H, -Olefinic), 7.39 (d, J = 8.3 Hz, 2H, H3 and H5), 7.32 (d, J = 7.7 Hz, 1H,
H6`), 6.99 (d, J = 7.5 Hz, 1H, H4`), 6.90 (dd, J = 7.8 Hz, 1H, H5`), 6.17 (s, 2H, -O-CH2-O-
), 2.54 (s, 3H, -S-CH3). 13C NMR (101 MHz, DMSO-d6)
187.87, 147.67, 146.66,
β-Olefinic), 7.66 (d, J =
α
δ
145.73, 137.34, 133.57, 128.87, 125.04, 123.45, 122.11, 121.97, 117.17, 110.10,
101.71, 13.93. Anal. calcd. for C17H14O3S: C, 68.44; H, 4.73; S, 10.75. Found: C, 69.05;
H, 4.87; S, 11.01. EI-MS (m/z): calculated 298.1, observed 298.1 (M).
3.2.1.10. (E)-3-(benzo[d][1,3]dioxol-4-yl)-1-(3-(trifluoromethoxy)phenyl)prop-2-en-1-one
(10)
1
Deep yellow solid, yield = 30%, m.p. = 80 - 84 °C, H NMR (400 MHz, DMSO-d6)
δ
8.10
-Olefinic), 7.71 (d, J
-Olefinic), 7.68 (m, 2H, H4 and H5), 7.37 (d, J = 8.0 Hz, 1H, H6`), 7.00
(d, J = 7.6 Hz, 1H, H4`), 6.90 (dd, J = 7.9 Hz, 1H, H5`), 6.16 (s, 2H, -O-CH2-O-). 13C NMR
(101 MHz, DMSO-d6) 187.84, 148.74, 147.69, 146.94, 139.48, 138.41, 131.15,
(d, J = 7.4 Hz, 1H, H6), 7.94 (s, 1H, H2), 7.85 (d, J = 15.7 Hz, 1H,
= 15.7 Hz, 1H,
β
α
δ
127.49, 125.56, 122.98, 121.95, 121.88, 121.35, 120.46, 116.87, 110.37, 101.78. Anal.
calcd. for C17H11F3O4: C, 60.72; H, 3.30. Found: C, 60.42; H, 3.18. EI-MS (m/z):
calculated 336.1, observed 336.1 (M).
3.2.1.11.
3.3.
Biochemistry
3.3.1. MAO inhibition screening
All synthesized compounds were evaluated for their ability to inhibit the A and B
isoforms of human MAO by a spectrophotometric assay in which MMTP was used as a
non-selective substrate (see text above in paragraph 2.2) and whose MAO-catalyzed
oxidation product (dihydropyridinium species) was monitored by measuring absorbance
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ACCEPTED MANUSCRIPT
at 420 nm with a microplate and cuvette reader (SpectraMax M2, Molecular Devices).
Due to their limited aqueous solubility, compounds 1-10 were dissolved in DMSO whose
final concentration of 3.3% (v/v) which does not affect MAO activity. Control
experiments were carried out in absence of the tested compound and adding an equal
volume of DMSO to assure constant conditions in all measurements. Each assay was
performed in a quartz cuvette containing 0.1 M potassium phosphate buffer (150 µL
total volume) in which 25 µg of human MAO-A or MAO-B was incubated with 10 µM
inhibitor at 37 °C for 5 minutes. The reaction was started by adding MMTP at a
concentration of 0.15 mM. The increase in absorbance was recorded at 420 nm at 37
°C and the enzyme initial velocity (v 0) was calculated by using the extinction coefficient
of 25000 M-1 cm-1. The ratio between the v0 value determined for each chalcone
derivative and that in the control experiment (100% activity) was used to comparatively
evaluate the inhibitory power of the tested compounds (Table 1). For those molecules
that produced more than 60% of enzyme inhibition further experiments at different
concentrations were carried out to determine IC50 by fitting the remaining activities (%)
at different inhibitor concentrations [I] with respect to the reference assay (100%) as a
function of log10[I] in a dose-response curve with the Graphpad Prism 5.0 software.
3.3.2. Determination of the mode of inhibition of MAO-B
To investigate the mode of inhibition of the synthesized compounds, compound 6
(the most potent inhibitor in the presented study) was incubated with human MAO-B at
a concentration of 0.55 µM (IC50) for different times (5, 15, 30 and 60 minutes) and the
enzymatic activity was measured as indicated above.
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3.3.3 Determination of Ki for the most potent compounds
Further analysis to determine Ki was carried out by selecting the chalcone
derivatives whose IC50 values were below 2 µM (Table 1). For these experiments
purified samples of recombinant human MAO B were used with the reaction buffer
containing also 0.25% v/v of reduced Triton X-100. The same MMTP assay as that
described above was followed and the time-courses of the reaction were measured in
quartz cuvettes under aerobic conditions by using
a
Cary 100 UV/Vis
spectrophotometer (Agilent) equipped with thermostated cell holder (T=25 °C). Varied
concentrations (0.07-2.00 mM) of MMTP and of the inhibitor under analysis (range 3-40
µM) were tested. Apparent kcat values (calculated for each measurement as v0/[E],
where v0 is the initial velocity and [E] corresponds to enzyme concentration) were
plotted as a function of substrate concentration and fit to equations describing
competitive, uncompetitive, and noncompetitive inhibition patterns using Graphpad
Prism 5.0 software. For all inhibitors the best fit was obtained with the equation of
competitive inhibition and Ki values are reported in Table 1.
3.4.Molecular docking simulation
A molecular docking study was performed on the synthesized compounds to gain
more insight of the binding mode with human MAO-B active site. The total procedure
was conducted using Autodock 4.2 software[36]. The crystal structure of human MAO-B
in complex with a coumarin analog was obtained from the PDB (2v61)[33] and was
used throughout the molecular modeling study. Of the MAO-B dimeric structure chain A
was selected for this study and prepared by adding hydrogen atoms and partial charges
keeping FAD in an oxidized state. By investigation of various x-ray structures of MAO-B
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ACCEPTED MANUSCRIPT
co-crystalized with different inhibitors, it was observed that there are some conserved
water molecules in proximity of the FAD, that appear to be important for mediating
interactions with the inhibitors.[30] They are involved in a network of hydrogen bonds
which stabilize the ligand into the binding site. Nine conserved water molecules were
therefore included in the docking process.[37] 3D structures of the synthesized
compounds were built using Openbabel[38] then imported into Autodock software. All
compounds were prepared by adding hydrogen atoms and Gasteiger partial charges
followed by merging non-polar hydrogens. All single bonds of the compounds were
considered rotatable during the docking simulation where residues of the binding site
were considered rigid. To define the binding site, a grid box of 60, 60, and 60 points in
x, y, and z directions was centered on N5 atom of FAD with grid spacing of 0.375 Å. A
total of hundred Lamarckian genetic algorithm runs were conducted for each compound
with the following parameters: population size: 150, number of evaluations: 2,500,000
and number of generations: 27,000, returning the pose with the best binding energy in
the largest cluster. Key interactions between docked poses and binding site residues
were identified using UCSF Chimera.[39]
Acknowledgments
This work was supported by internal grants (# QUST-CPH-FALL-14\15-9 and QUST-
CPH-SPR-14/15-9) from the Office of Academic Research, Qatar University, Doha,
Qatar. C.B. is member of the collaborative network within the COST Action CM1103,
Structure-based drug design for diagnosis and treatment of neurological diseases. We
are grateful to Professor Neal Castagnoli Jr. Professor Emeritus, Virginia Tech for
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ACCEPTED MANUSCRIPT
providing the MMTP substrate. The 2D-NMR spectra were recorded by Professor Khalid
Elsayed, University of Louisiana at Monroe, USA.
Supporting Information Available
Analytical and spectral data for the 10 compounds.
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Highlights:
•
Novel chalcone analogs were designed and synthesized as human MAO-B reversible
inhibitors.
•
•
•
The inhibitors bind with micromolar to submicromolar potency.
The molecular scaffold determines high MAO-A/MAO-B selectivity.
A double conformation in MAO-B active site is predicted by molecular docking.