Exploring 4-substituted-2-thiazolylhydrazones from 2-, 3-, and 4-acetylpyridine as selective and reversible hMAO-B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.05.032

A series of 4-substituted-2-thiazolylhydrazone derivatives have been synthesized and tested in vitro for their human monoamine oxidase (hMAO) A and B inhibitory activity. Our findings confirmed that the substitution at C4 of the thiazole ring was important to obtain highly potent and selective hMAO-B inhibitors with IC50 values in the nanomolar range. Moreover, these derivatives were endowed with a reversible mechanism of enzyme inhibition. Molecular modelling studies were performed to rationalize the recognition of all inhibitors with respect to hMAO-A and -B isoforms.

Full text of DOI:10.1016/j.ejmech.2013.05.032

Accepted Manuscript
Exploring 4-substituted-2-thiazolylhydrazones from 2-, 3-, and 4-acetylpyridine as
selective and reversible hMAO-B inhibitors
Paola Chimenti, Ané l Petzer, Simone Carradori, Melissa D’Ascenzio, Romano
Silvestri, Stefano Alcaro, Francesco Ortuso, Jacobus P. Petzer, Daniela Secci
PII:
S0223-5234(13)00341-3
10.1016/j.ejmech.2013.05.032
EJMECH 6210
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 January 2013
Revised Date: 20 May 2013
Accepted Date: 22 May 2013
Please cite this article as: P. Chimenti, A. Petzer, S. Carradori, M. D’Ascenzio, R. Silvestri, S. Alcaro,
F. Ortuso, J.P. Petzer, D. Secci, Exploring 4-substituted-2-thiazolylhydrazones from 2-, 3-, and 4-
acetylpyridine as selective and reversible hMAO-B inhibitors, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.05.032.
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ACCEPTED MANUSCRIPT
3
CH3
N
R
N
N
S
reversible
hMAO-B inhibitors
NH

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Highlights
•
•
•
•
Synthesis and in vitro MAO inhibitory activity of new 2-thiazolylhydrazones
hMAO-B selectivity was also corroborated by molecular modelling studies
Thiazole substitution is important for the activity of this scaffold
They were endowed with a reversible mechanism of enzyme inhibition

ACCEPTED MANUSCRIPT
Exploring 4-substituted-2-thiazolylhydrazones from 2-, 3-, and 4-
acetylpyridine as selective and reversible hMAO-B inhibitors
Paola Chimenti,a* Anél Petzer,b Simone Carradori,a Melissa D’Ascenzio,a Romano Silvestri,a Stefano
Alcaro,c Francesco Ortuso,c Jacobus P. Petzer,b Daniela Seccia
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5,
b
00185 Rome, Italy. Pharmaceutical Chemistry and Unit for Drug Research and Development, School
of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa.
cDipartimento di Scienze della Salute, “Magna Graecia” University of Catanzaro, Campus Universitario
“S. Venuta”, Viale Europa Loc. Germaneto, 88100 Catanzaro, Italy.
*Corresponding author: Phone: +39
paola.chimenti@uniroma1.it
6
49913149; Fax: +39
6
49913923; e-mail:
Keywords: Monoamine oxidase; Acetylpyridine; Reversibility; Molecular modelling; Thiazole.
Abstract
A series of 4-substituted-2-thiazolylhydrazone derivatives have been synthesized and tested in vitro for
their human monoamine oxidase (hMAO) A and B inhibitory activity. Our findings confirmed that the
substitution at C4 of the thiazole ring was important to obtain highly potent and selective hMAO-B
inhibitors with IC50 values in the nanomolar range. Moreover, these derivatives were endowed with a
reversible mechanism of enzyme inhibition. Molecular modelling studies were performed to rationalize
the recognition of all inhibitors with respect to hMAO-A and -B isoforms.
1.
Introduction
Monoamine oxidase metabolizes monoamine neurotransmitters and dietary amines by oxidative
deamination, and thus plays an important role in regulating emotional and other brain functions.
Substrate oxidation is accompanied by the production of potentially harmful species such as H2O2,
which may contribute to cellular degeneration. There are two isoforms, MAO-A and MAO-B,
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distinguished by their different substrate and inhibitor specificities and tissue distribution [1].
Immunohistochemical studies revealed that hMAO-A is the main isoform in the gastrointestinal tract,
where it regulates the metabolism of tyramine, and in catecholaminergic neurons in the brain. Since
hMAO-A inhibition is associated with the risk for hypertensive crisis, selective hMAO-B inhibitors are
more frequently employed to modulate central monoamine levels [2]. In fact, hMAO-B is responsible
for >80% of the MAO activity in the brain, where it metabolizes dopamine and other amines in
serotonergic and histaminergic neurons and astrocytes [1]. This enzymatic activity was shown to
increase with age, a process associated with gliosis. Additionally, hMAO-B activity is significantly
higher in the brains of patients with Alzheimer's and Parkinson’s disease (PD) [3]. Human MAO-B
inhibitors may also have neuroprotective and neurorestorative properties. Although these effects may be
the result of the inhibition of the formation of neurotoxic by-products derived from the MAO catalytic
cycle, evidence suggests that mechanisms not related to MAO inhibition may be involved [4]. Several
large-scale, randomized placebo-controlled clinical trials (DATATOP, TEMPO, ADAGIO) have
demonstrated that treatment with selective hMAO-B inhibitors leads to a symptomatic amelioration of
early PD with disease-modifying capabilities in the clinical settings [5,6].
In the course of our research [7], we have reported on the synthesis and MAO inhibitory activities of a
large number of 2-thiazolylhydrazone derivatives. A 3D-QSAR study [8] indicated that the 2-
thiazolylhydrazone moiety, in itself, contributes significantly towards the inhibitory activity against
hMAO-B. We were particularly interested in also examining the effects that different substituents at C2,
C4, and C5 of the thiazole nucleus have on MAO inhibitory activity and selectivity. On the basis of the
information extrapolated from the interaction pattern among these isoforms and their inhibitors [9], we
placed various moieties on these positions (aliphatic, cycloaliphatic, and aromatic groups) and examined
the MAO inhibitory properties of the resulting compounds. The best moieties attached to the hydrazonic
nitrogen were found to be derived from 2-, 3-, and 4-acetylpyridine interacting with the substrate-
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orienting Tyr326 in hMAO-B active site. Substituents on the phenyl ring at C4 of the thiazole ring
influenced the activity as demonstrated by the introduction of several groups (NO2, CN, CH3, OCH3) or
halogens (F, Cl) in the ortho, meta, and para positions [10-16], while the introduction of more sterically
hindered moieties (coumarin and naphthalene) led to a decreased hMAO inhibitory activity [12,17]. The
C5 of the thiazole ring must be unsubstituted [17]. Encouraged by these results, in this paper, we
designed a series of 2-thiazolylhydrazone derivatives, in which the ethylpyridine moiety on the
hydrazonic nitrogen was retained for all homologues. The aim of this study was to explore how the
steric hindrance and the electronic properties of the substituent at C4 position (methyl, ethyl ester, and
phenyl) affect hMAO-A and hMAO-B inhibitory activity and selectivity. For this purpose IC50 values
for the inhibition of the MAO enzymes were measured. Moreover, the time-dependence of inhibition of
the series of 2-thiazolylhydrazone derivatives was also investigated and the Ki values determined for
selected compounds.
2.
Chemistry
4-Substituted-2-thiazolylhydrazone derivatives (1-9) were synthesized in high yields (81-99%) as
reported in our previous communications (Table 1) [18]. 2-, 3-, and 4-Acetylpyridine reacted directly
with thiosemicarbazide in ethanol in the presence of catalytic amounts of acetic acid at room
temperature, and the obtained thiosemicarbazones were subsequently converted into 4-substituted-2-
thiazolylhydrazones by reaction with the appropriate α-haloketone or α-haloketoester under the same
conditions (Hantzsch reaction). All the synthesized products were washed with petroleum ether and
diethyl ether and purified by chromatography (ethyl acetate/petroleum ether) before characterization by
spectroscopic methods and elemental analysis.
3.
Results and discussion
The hMAO inhibitory properties of the pyridine derivatives (1-9) were investigated using commercially
available (Sigma-Aldrich) microsomes from insect cells containing recombinant hMAO-A and hMAO-
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B with kynuramine as the substrate [19]. All inhibition data (IC50 values determined with kynuramine at
~1 x Km) are presented in Table 1. Six out of nine derivatives were found to be relatively weak hMAO-
A inhibitors. With the exception of compound 4, all compounds displayed selective inhibition of the
hMAO-B isoform with the most potent MAO-B inhibitors identified in the present study, 5, 8, and 9,
exhibiting binding affinities that are much higher for human MAO-B than for MAO-A. Once again, the
best results have been obtained with compounds deriving from 3- and 4-acetylpyridine and bearing a
phenyl ring or an ethyl ester group at C4 of the thiazole nucleus. To determine if the inhibition of
hMAO-B by the test compounds is time-dependent, 5, 8, and 9 at concentrations approximately equal to
twofold their respective IC50 values, were preincubated with hMAO-B for 0, 15, 30, and 60 min and the
residual enzyme activities were subsequently determined. As shown in Fig. 1, the enzyme activities are
not reduced with increased preincubation time which indicates that these inhibitors are not time-
dependent inhibitors of hMAO-B over the period evaluated. Since irreversible inhibitors display a time-
dependent reduction of enzyme activity, it can be concluded that these derivatives act as reversible
human MAO-B inhibitors (the only possible exception is compound 5 which may possess a slow-tight
binding component towards hMAO-A). To provide further evidence that compounds 5, 8 and 9 are
reversible MAO-B inhibitors, a set of Lineweaver-Burk plots were constructed for the inhibition of
MAO-B by each inhibitor. As shown in Fig. 2, the plots obtained for 8 and 9 are linear and intersect on
the y-axis. This is indicative of competitive inhibition and is further support for a reversible interaction
of these inhibitors with MAO-B. The plots obtained for compound 5 intersect to the right of the y-axis,
which suggests a partial mixed inhibition mechanism. The deviation from strict competitive pattern can
also arise from the failure of the MAOs to fulfill the assumptions of the Michaelis-Menten equation in
the presence of inhibitor 5. Intersection to the right of the y-axis may also be suggestive of either non-
linear plots or differential binding of the inhibitor to different forms of the enzyme [20]. Further
investigation is necessary to clarify this point.
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In order to derive structure-activity relationship information, the recognition of the 2-thiazolylhydrazone
derivatives by hMAO-A and -B was investigated by means of docking experiments. With the aim to
evaluate the stability of the enzyme-inhibitor complexes and possible induced fit phenomena, the best
scored configuration of each ligand within both isozymes was further examined via molecular dynamics
simulations. To facilitate discussion of the results, our inhibitors were classified, on the basis of the R
substituent, in three groups: (I) R= methyl, (II) R= ethyl ester, and (III) R= phenyl ring. The results
showed that the isoform selectivity can be attributed to hydrogen bonds between the 2-
thiazolylhydrazone moiety and the hMAO-B Cys172 (Asn181 in hMAO-A). Additional productive
interactions with two other hMAO-B exclusive residues (Ile199 and Tyr326) also contributed to
complex stabilization. In hMAO-A the corresponding residues are Phe208 and Ile335 (Fig. 3). Residues
Ile199 and Tyr326 determine, to a large degree, the shape and size of the hMAO-B binding site, and
allow the placement of the ligands close to the hMAO-B Cys172 side chain. In hMAO-A, the Phe208
residue, which is sterically larger than Ile199 in hMAO-B, does not permit binding of the inhibitors in
the proximity of Asn181. Instead the 2-thiazolyl moiety of this scaffold occupies space in a lipophilic
cage opened by Ile335. This area is not accessible in the hMAO-B active site due to the presence of the
Tyr326 at this position [21]. All inhibitors exhibited similar orientations in both hMAOs active sites and
their docking complexes were not significantly modified by molecular dynamics simulations. The
pyridine rings of the inhibitors were close to the FAD cofactor, while the R moiety was located towards
the entrance of the active site. The only exception was the group I inhibitors. In hMAO-A these
inhibitors exhibited an opposite orientation with the R group extending towards the FAD and,
consequently, the pyridine ring extending towards the access of the cleft. Also, the group I inhibitors do
not undergo hydrogen bonding to Cys172 in hMAO-B. Since this interaction makes a remarkable
thermodynamic contribution to complex stabilization, group I inhibitors are comparatively weaker
hMAO-B inhibitors.
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We also investigated the role of the R moiety in the binding of the inhibitors to the MAO enzymes.
Visual inspection of the most stable enzyme-inhibitor complexes revealed that, in both MAO-A and -B,
small groups, such as the methyl, do not interact with the enzyme as strongly as ethyl ester or phenyl.
As already described, in hMAO-A, group I inhibitors exhibit a reversed binding orientation, which
places the methyl moiety towards the FAD where it can establish relatively few Van der Waals contacts
to the cofactor, Tyr407, and Tyr444. In hMAO-B, the methyl group of group I inhibitors is located at
the entrance cavity and undergoes Van der Waals contacts to Leu171, Tyr326, and Ile199. The balance
of these two effects makes compound 4, the only active derivative of group I, and at variance with the
other compounds, more selective with respect to isoform A. The ethyl ester moiety (group II) exhibits
more interactions with both enzyme isoforms. The steric hindrance and the electronic properties of the
ethyl ester prevent the reversed orientation observed with the group I in hMAO-A. Although these
compounds display similar binding orientations to both MAO-A and -B active sites, the R moiety forms
a larger interaction network in hMAO-B. In hMAO-A, the ethyl ester moiety of group II inhibitors is
located at the entrance gorge interacting to Leu97, Ala111, Val210, Gln215, Leu325, and Leu337, while
in hMAO-B productive contacts are formed with Phe103, Pro104, Trp119, Leu164, Leu167, Phe168,
Ile199, and Ile316. The binding modes of the group III molecules (R= phenyl) were similar to those of
the group II inhibitors: the phenyl ring occupied the same areas as those occupied by the ethyl ester
substituent, but its conformational rigidity reduced the number of interacting residues in both enzymes.
The phenyl moiety, however, overcame such a disadvantage in terms of complex stability by forming
stronger T-shape π-π interactions to hMAO-A Phe208 and hMAO-B Tyr326. It may thus be concluded
that both the, ethyl ester and phenyl moieties contribute equally to complex stabilization, and this degree
of stabilization is greater than that obtained with the methyl group.
With the aim to rationalize the different MAO inhibitory potencies of the compounds containing the 2-,
3-, and 4-pyridine moieties, the molecular interactions of these moieties with MAO-A and -B were
6

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investigated. The active site affinities were found to be dependent on the position of the nitrogen atom
in the pyridine ring: in general, the highest affinity was observed for the 3- or 4-pyridine analogues
while the lowest affinity was observed for the 2-pyridine analogues. The only exception was observed
for the group III inhibitors for which the 2-pyridine derivative interacted slightly better with hMAO-A
than the 3-pyridine derivative. Among the group III inhibitors, the 4-pyridine (9) was still the best
inhibitor. This behavior may be attributed to the strong contribution of the R moiety (phenyl) that
reduced the influence of the pyridine. Visual inspection of all complexes revealed that pyridine rings
can establish one hydrogen bond to the side chains of hMAO-A Tyr197 and hMAO-B Tyr188 (Fig. 3).
Such a productive contribution was geometrically prevented for 2-pyridine derivatives. The importance
of the pyridine ring confirmed our previous results which suggested that this part of the molecule
interacts with the “aromatic cage” (towards the flavin coenzyme), thereby stabilizing the inhibitor
within the MAO enzyme active site. In conclusion, this study confirms the role of the 2-
thiazolylhydrazone scaffold to selectively recognize hMAO-B. Insertion of phenyl or ethyl ester
substituent at position R allowed for additional productive interactions and led to an enhancement in
hMAO-B inhibition potency. MAO-B selectivity was further enhanced by the introduction of 3-/4-
ethylpyridine moieties.
4.
Experimental protocol
Chemistry
4.1
Starting materials and reagents used in the synthetic procedures were obtained from commercial
suppliers and were used without purification. Melting points (mp) were determined by the capillary
1
13
method on an FP62 apparatus (Mettler-Toledo) and are uncorrected. H and C NMR spectra were
recorded on 400 MHz Bruker spectrometer using DMSO-d6 as the solvent. Chemical shifts are reported
in parts per million (δ) downfield from the signal of tetramethylsilane (TMS) which was added to the
deuterated solvent. Coupling constants J are expressed in hertz (Hz). IR spectra were registered on a
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PerkinElmer FTIR Spectrometer Spectrum 1000 in anhydrous potassium bromide (KBr). Elemental
analyses for C, H, and N were determined with a Perkin-Elmer 240 B microanalyzer and the analytical
results were ≥95% purity for all compounds. All reactions were monitored by TLC performed on 0.2
mm thick silica gel plates (60 F254 Merck). Preparative flash column chromatography was carried out on
silica gel (230-400 mesh, G60 Merck). Organic solutions were dried over anhydrous sodium sulfate.
Concentration and evaporation of the solvent after reaction or extraction was carried out on a rotary
evaporator (Büchi Rotavapor). Fluorescence spectrophotometry was conducted with a Varian Cary
Eclipse fluorescence spectrophotometer. Microsomes from insect cells containing recombinant human
MAO-A and -B (5 mg/mL) and kynuramine 2HBr were obtained from Sigma-Aldrich.
4.2
General procedure for the synthesis of derivatives 1-9.
The appropriate 2-, 3-, or 4-acetylpyridine (50 mmol) was dissolved in 50 mL of ethanol and
magnetically stirred with an equimolar quantity of thiosemicarbazide for 24 h at room temperature with
catalytic amounts of acetic acid. The desired thiosemicarbazone was a coloured powder which
precipitated from reaction mixture. It was filtered, washed with adequate solvents (n-hexane, petroleum
ether, and diethyl ether), and dried under vacuum. The prepared thiosemicarbazone (50 mmol) reacted
with equimolar amounts of the corresponding α-haloketone or α-haloketoester (50 mmol) dissolved in
ethanol (50 mL) under magnetic stirring at room temperature. The reaction was monitored until
completion by TLC. The resulting 2-thiazolylhydrazone derivative was filtered, washed with petroleum
ether and diethyl ether, and, if necessary, purified by chromatography using ethyl acetate/n-hexane (3/1)
as mobile phase, to give all compounds in high yields (81-99%). Some compounds (2, 5, 8) have been
already synthesized and characterized by us in a previous study [18].
Characterization data for new compounds:
4.2.1. 4-Methyl-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (1).
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Dark yellow solid, yield 99%; mp 231-233 °C; IR cm-1 (KBr): 3107 (NH), 3055 (Csp2-H), 2912 (Csp3-
1
H), 1607 (C=C), 1555 (C=N); H NMR (DMSO-d6): 2.28 (s, 3H, CH3), 2.43 (s, 3H, CH3), 6.66 (s, 1H,
C5H-thiazole), 7.72-7.73 (t, 1H, Ar), 8.27-8.28 (m, 1H, Ar), 8.31-8.33 (m, 1H, Ar), 8.72-8.73 (d, 1H,
Ar), 10.78 (bs, 1H, NH, D2O exch.); 13C NMR (DMSO-d6): 12.14, 17.86, 107.25, 124.32, 128.87,
130.65, 131.99, 135.54, 144.45, 146.46, 158.78, 170.41. Anal. Calcd for C11H12N4S: C, 56.87; H, 5.21;
N, 24.12. Found: C, 56.99; H, 5.09; N, 24.01.
4.2.2. 4-Phenyl-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (3).
Yellow solid, yield 97%; mp 252-254 °C; IR cm-1 (KBr): 3135 (NH), 3093 (Csp2-H), 2924 (Csp3-H),
1
1617 (C=C), 1534 (C=N); H NMR (DMSO-d6): 2.43 (s, 3H, CH3), 7.33 (s, 1H, C5H-thiazole), 7.40-
7.44 (m, 3H, Ar), 7.62-7.63 (m, 1H, Ar), 7.87-7.89 (m, 2H, Ar), 8.17-8.18 (m, 2H, Ar), 8.67-8.68 (m,
1H, Ar), 11.85 (bs, 1H, NH, D2O exch.); 13C NMR (DMSO-d6): 14.35, 105.34, 126.74, 128.11, 129.01,
134.25, 136.85, 140.77, 142.74, 160.54, 169.15. Anal. Calcd for C16H14N4S: C, 65.28; H, 4.79; N,
19.03. Found: C, 65.13; H, 5.00; N, 19.19.
4.2.3. 4-Methyl-2-(2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)thiazole (4).
Dark red solid, yield 99%; mp 114-116 °C; IR cm-1 (KBr): 3117 (NH), 3026 (Csp2-H), 2924 (Csp3-H),
1619 (C=C), 1548 (C=N); 1H NMR (DMSO-d6): 2.24 (s, 3H, CH3), 2.45 (s, 3H, CH3), 6.55 (s, 1H, C5H-
thiazole), 7.95-7.98 (t, 1H, Ar), 8.80-8.82 (m, 2H, Ar), 9.26 (s, 1H, Ar), 10.70 (bs, 1H, NH, D2O exch.);
13C NMR (DMSO-d6): 12.02, 17.45, 107.44, 124.23, 128.74, 130.11, 131.78, 135.99, 144.56, 146.74,
158.36, 170.44. Anal. Calcd for C11H12N4S: C, 56.87; H, 5.21; N, 24.12. Found: C, 56.99; H, 5.07; N,
24.01.
4.2.4. 4-Phenyl-2-(2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)thiazole (6).
Yellow solid, yield 90%; mp 256-257 °C; IR cm-1 (KBr): 3105 (NH), 3035 (Csp2-H), 2926 (Csp3-H),
1
1610 (C=C), 1533 (C=N); H NMR (DMSO-d6): 2.40 (s, 3H, CH3), 7.32 (s, 1H, C5H-thiazole), 7.39-
7.44 (m, 3H, Ar), 7.87-7.91 (m, 3H, Ar), 8.60-8.62 (m, 1H, Ar), 8.77-8.78 (m, 1H, Ar), 9.07 (s, 1H, Ar),
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13
11.71 (bs, 1H, NH, D2O exch.); C NMR (DMSO-d6): 14.22, 105.18, 126.03, 126.91, 128.14, 129.12,
134.86, 136.96, 140.76, 142.74, 169.64. Anal. Calcd for C16H14N4S: C, 65.28; H, 4.79; N, 19.03. Found:
C, 65.04; H, 4.56; N, 18.87.
4.2.5. 4-Methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (7).
Red solid, yield 98%; mp 247-249 °C; IR cm-1 (KBr): 3163 (NH), 3057 (Csp2-H), 2914 (Csp3-H), 1620
1
(C=C), 1552 (C=N); H NMR (DMSO-d6): 2.18 (s, 3H, CH3), 2.35 (s, 3H, CH3), 6.43 (s, 1H, C5H-
13
thiazole), 8.17-8.18 (m, 2H, Ar), 8.78-8.80 (m, 2H, Ar), 10.72 (bs, 1H, NH, D2O exch.); C NMR
(DMSO-d6): 12.74, 17.23, 107.45, 124.55, 128.47, 130.36, 131.25, 135.89, 144.77, 146.74, 158.62,
170.37. Anal. Calcd for C11H12N4S: C, 56.87; H, 5.21; N, 24.12. Found: C, 56.75; H, 5.01; N, 24.28.
4.2.6. 4-Phenyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (9).
Yellow solid, yield 95%; mp 246-248 °C; IR cm-1 (KBr): 3114 (NH), 3025 (Csp2-H), 2923 (Csp3-H),
1
1636 (C=C), 1525 (C=N); H NMR (DMSO-d6): 2.36 (s, 3H, CH3), 7.34 (s, 1H, C5H-thiazole), 7.89-
7.91 (m, 1H, Ar), 8.14-8.16 (m, 1H, Ar), 8.37 (bs, 2H, Ar), 8.61 (s, 1H, Ar), 8.80 (bs, 2H, Ar), 10.67
(bs, 1H, NH, D2O exch.); 13C NMR (DMSO-d6): 14.35, 105.44, 126.02, 128.10, 129.78, 134.56, 136.64,
140.63, 142.88, 160.76, 169.03. Anal. Calcd for C16H14N4S: C, 65.28; H, 4.79; N, 19.03. Found: C,
65.47; H, 4.93; N, 19.25.
4.3. hMAO-A and -B inhibition studies
As enzyme sources, commercially available (Sigma-Aldrich) microsomes from insect cells, containing
recombinant human MAO-A and -B, were employed. The enzyme activity measurements were based on
the MAO-A or MAO-B (0.0075 mg protein/mL) catalyzed oxidation of kynuramine to 4-
hydroxyquinoline. The incubations were conducted in 500 ꢀL potassium phosphate buffer (100 mM, pH
7.4, made isotonic with KCl 20.2 mM) in the presence of various concentrations of the test inhibitors (0-
100 ꢀM) and 4% DMSO as the cosolvent. For MAO-A activity measurements the reactions contained
45 ꢀM kynuramine while for the for MAO-B activity measurements the kynuramine concentration was
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30 ꢀM. Following a 20 min incubation at 37 ºC, the reactions were terminated by the addition of 400 ꢀL
NaOH (2N) and 1000 ꢀL water. The reactions were centrifuged for 10 minutes at 16,000 g and
concentration measurements of 4-hydroxyquinoline in the supernatants of the samples were carried out
spectrofluorometrically at excitation and emission wavelengths of 310 nm and 400 nm, respectively.
The quantitative estimations were made with the aid of a linear calibration curve constructed with
known amounts (0.047-1.56 ꢀM) of the authentic metabolite dissolved in 500 ꢀL potassium phosphate
buffer (100 mM, pH 7.4, made isotonic with KCl 20.2 mM). To each calibration standard, volumes of
400 ꢀL NaOH (2N) and 1000 ꢀL water were added. The necessary control samples were included to
confirm that the test inhibitors do not fluoresce or quench the fluorescence of 4-hydroxyquinoline under
these assay conditions. IC50 values were calculated by plotting the initial rate of kynuramine oxidation
versus the logarithm of the inhibitor concentration to obtain a sigmoidal dose-response curve. For each
sigmoidal curve, six different inhibitor concentrations spanning at least 3 orders of a magnitude were
used. These values were selected to bracket the IC50 values of the test inhibitors and to properly define
the upper and lower plateaus of the sigmoidal curve. The inhibitor concentrations were also selected so
that they are equally spaced on a logarithmic scale. These kinetic data were fitted to the one site
competition model incorporated into the Prism software package (GraphPad). Each IC50 value was
measured using six single inhibitor concentrations as described above. Three such experiments were
performed for each compound yielding three IC50 values. The mean and standard deviation (SD) of
these three IC50 values are reported in the results section.
4.3.1. Time-dependent studies
This study shows that the test pyridine derivatives act as moderate to potent hMAO-B inhibitors while
possessing little or weak hMAO-A inhibition potential. To determine whether the inhibitors interact
reversibly or irreversibly with hMAO-B, time-dependent inhibition studies were carried out with three
selected inhibitors (5, 8, and 9). They were preincubated with recombinant human MAO-B (0.03 mg
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protein/mL) for periods of 0, 15, 30, 60 min at 37 ºC in potassium phosphate buffer (100 mM, pH 7.4,
made isotonic with KCl 20.2 mM) at concentrations which are approximately 2 fold the measured IC50
values for the inhibition of human MAO-B (Table 1). The reactions were diluted twofold with the
addition of kynuramine (30 ꢀM), and then incubated for a further 20 min at 37 °C. After dilution, the
final volumes of these incubations were 500 ꢀL, the concentrations of the test inhibitors were
approximately equal to the IC50 values for the inhibition of hMAO-B by the respective inhibitors, and
the enzyme concentration was 0.015 mg protein/mL. The reactions were terminated with 200 ꢀL NaOH
(2N) and a volume of 1200 ꢀL distilled water and the rates of formation of 4-hydroxyquinoline were
measured and quantified as described above. All measurements were carried out in triplicate and are
expressed as mean ± SD.
4.3.2. Lineweaver-Burk plots
A set consisting of four Lineweaver-Burk plots were constructed for the inhibition of human MAO-B by
compounds 5, 8 and 9. For this purpose, the rates of the hMAO-B-catalyzed oxidation of kynuramine
were measured in the absence and presence of three different concentrations of the test compounds. The
hMAO-B incubations and rate measurements were carried out as described above. The reactions
contained kynuramine (15-90 µM), the test inhibitors, and hMAO-B (0.015 mg protein/mL). DMSO
(4%) was added as cosolvent to all incubations. Linear regression analysis was performed using
GraphPad Prism 5. Ki values were estimated from the x-axis intercepts (–Ki) of replots of the slopes of
the Lineweaver–Burk plots versus inhibitor concentration.
4.4. Molecular modelling
The 3D models of our compounds were built in E configuration by means of the Maestro GUI [22] and
were energy optimized using the OPLS-AA force field [23] coupled to the GB/SA [24] water implicit
solvation model as implemented in MacroModel software [25]. High resolution Protein Data Bank [26]
(PDB) crystallographic structures, deposited with codes 2Z5X [9a] and 2V60 [27], were adopted as
receptor models of hMAO-A and hMAO-B, respectively. Taking into account the limits of the PDB file
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ACCEPTED MANUSCRIPT
format, both structures were manipulated to be used in our docking: missing atoms and FAD bond order
were fixed, hydrogen atoms were added and co-crystallized water solvent molecules and ligands,
harmine and 7-(3-chlorobenzyloxy)-4-carboxaldehyde-coumarin, for 2Z5X and 2V60 respectively, were
removed. The recognition with respect to both targets was investigated by means of docking
experiments carried out using the flexible algorithm of Glide software [28]. The binding site, of both
targets, was defined by a regular box of about 110,000 Å3 centred onto the FAD N5 atom. The docking
complexes scoring was performed using the default Glide XP function. The best docking scored
complex of each inhibitor into both targets was submitted to 1.2 ns of molecular dynamics (MD)
simulations, at 300 K, carried out with Desmond package [29]. The final MD structures were considered
in our binding mode analysis.
Acknowledgements
This work was supported by MIUR (Italy).
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Table 1.
Synthetic route and hMAO inhibition data of compounds 1-9. IC50 values were determined with kynuramine at ~1 x Km.
Compound
Pyridine isomer
2-acetylpyridine
2-acetylpyridine
2-acetylpyridine
3-acetylpyridine
3-acetylpyridine
3-acetylpyridine
4-acetylpyridine
4-acetylpyridine
4-acetylpyridine
R
CH3
COOEt
Ph
CH3
COOEt
Ph
CH3
COOEt
Ph
IC50 hMAO-A (µM)
No inhibition
No inhibition
16.6 ± 2.01
IC50 hMAO-B (µM)
No inhibition
1
2
3
4
5
6
7
8
9
No inhibition
3.84 ± 0.133
6.910 ± 0.227
6.571 ± 0.296
21.3 ± 0.88
No inhibition
6.630 ± 0.667
2.67 ± 0.082
13.633 ± 0.870
0.0722 ± 0.0057
0.944 ± 0.075
No inhibition
0.1274 ± 0.0028
0.013 ± 0.0012
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6
5
4
3
2
1
0
25
20
15
10
5
5
0
No Inhibitor
0
15
30
60
No Inhibitor
0
15
30
60
Incubation time (min)
Incubation time (min)
25
20
15
10
5
4
3
2
1
0
8
9
0
No Inhibitor
0
15
30
60
No Inhibitor
0
15
30
60
Incubation time (min)
Incubation time (min)
6
5
4
3
2
1
0
25
20
15
10
5
0
No Inhibitor
0
15
30
60
No Inhibitor
0
15
30
60
Incubation time (min)
Incubation time (min)
Fig. 1. The time-dependency of hMAO-A (left) and hMAO-B (right) inhibition by compounds 5 (top, 13.14 ꢀM for hMAO-
A and 0.144 ꢀM for hMAO-B), 8 (middle, 13.26 ꢀM for hMAO-A and 0.255 ꢀM for hMAO-B), and 9 (bottom, 5.33 ꢀM for
hMAO-A and 0.026 ꢀM for hMAO-B). The test inhibitors were preincubated with the hMAO isoforms for various periods of
times (0-60 min) and the residual enzyme catalytic rates (nmol 4-hydroxyquinoline formed/min/mg protein) were measured.
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100
75
50
25
0
-0.02
0.00
0.02
0.04
0.06
1/[S]
100
75
50
25
0
-0.02
0.00
0.02
0.04
0.06
1/[S]
100
75
50
25
0
-0.02
0.00
0.02
0.04
0.06
1/[S]
Fig. 2. Lineweaver-Burk plots of the oxidation of kynuramine by recombinant human MAO-B. The plots were constructed in
the absence (asterisk) and presence of various concentrations of compounds 5 (top), 8 (middle) and 9 (bottom). The inhibitor
concentrations employed were 0.25 x IC50 (open squares), 0.5 x IC50 (filled circles) and 1 x IC50 (open circles). Ki values
were estimated from the x-axis intercepts (–Ki) of replots of the slopes of the Lineweaver–Burk plots versus inhibitor
concentration (r2 > 0.993). These values are 0.048 µM, 0.048 µM and 0.011 µM for compounds 5, 8 and 9, respectively.
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Fig. 3. Docking best poses of 9 in a) hMAO-A and b) hMAO-B and 4 in c) hMAO-A and d) hMAO-B. Interacting residues
and ligands are reported in CPK and green carbon coloured sticks respectively. FAD cofactor is displayed in spacefill. Yellow
dotted lines indicate hydrogen bonds.
20