Synthesis and evaluation of human monoamine oxidase inhibitory activities of some 3,5-diaryl-N-substituted-4,5-dihydro-1H-pyrazole-1-carbothioamide derivatives

Article abstract of DOI:10.1002/ardp.201100448

Sixteen 3-aryl-5-(4-fluorophenyl)-N-substituted-4,5-dihydro-1H-pyrazole-1- carbothioamide derivatives were synthesized and their structure were identified by UV, IR, ¹H NMR, mass spectra, and microanalyses. The compounds were evaluated in vitro for their human monoamine oxidase (hMAO) inhibitory activities and their MAO-A and -B selectivity. All the compounds were found to potently inhibit MAO-A isoforms. 5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-N- methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (1.0 × 10^-3 μM) was found to inhibit hMAO-A most selectively and potently. The binding mode of 5-(4-fluorophenyl)-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-1H-pyrazole- 1-carbothioamide to hMAO-A was also predicted using docking studies.

Full text of DOI:10.1002/ardp.201100448

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Synthesis and Evaluation of Human Monoamine Oxidase
Inhibitory Activities of Some 3,5-Diaryl-N-substituted-4,5-
dihydro-1H-pyrazole-1-carbothioamide Derivatives
1
Kerem S¸entu¨rk , Oya Unsal Tan , Samiye Yabanoglu C¸ iftc¸i , Gu¨lberk Uc¸ar , and Erhan Palaska
1
2
2
1
˘
1 Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hacettepe University, Ankara, Turkey
2 Faculty of Pharmacy, Department of Biochemistry, Hacettepe University, Ankara, Turkey
Sixteen 3-aryl-5-(4-fluorophenyl)-N-substituted-4,5-dihydro-1H-pyrazole-1-carbothioamide derivatives
were synthesized and their structure were identified by UV, IR, 1H NMR, mass spectra, and
microanalyses. The compounds were evaluated in vitro for their human monoamine oxidase
(hMAO) inhibitory activities and their MAO-A and -B selectivity. All the compounds were found to
potently inhibit MAO-A isoforms. 5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (1.0 ꢀ 10ꢁ3 mM) was found to inhibit hMAO-A most selectively and
potently. The binding mode of 5-(4-fluorophenyl)-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide to hMAO-A was also predicted using docking studies.
Keywords: 2-Pyrazoline / 4,5-Dihydropyrazole / Docking / Monoamine oxidase
Received: December 8, 2011; Revised: April 18, 2012; Accepted: April 25, 2012
DOI 10.1002/ardp.201100448
Introduction
have numerous, significant interactions with dietary sympa-
thomimetic amines such as tyramine and cause hypertensive
crises, so-called ‘‘cheese effect’’, blurred vision, and dizziness.
They also potentiate the effects of alcohol and antihist-
amines, tricyclic antidepressants, tranquilizers, some pain
relievers, and muscle relaxants. In the last decades, efforts
have been directed to the design, synthesis, and study of new
MAO inhibitors which display low toxicity and relative selec-
tivity for the treatment of various psychiatric and neuro-
degenerative disorders. These inhibitors belong to various
chemical classes and show different modes of enzyme
inhibition resulting from the covalent mechanism-based
interaction of the compounds (clorgyline, selegiline, and
rasagiline) to non-covalent interaction-based ones (brofaro-
mine, toloxatone, and moclobemide) with MAO isoforms.
Despite considerable progress in understanding the inter-
actions of the two enzyme forms with their preferred sub-
strates and inhibitors, no general rules are yet available for
the rational design of potent, reversible, and selective inhibi-
tors of MAO, possibly due the fact that the mechanism of
interaction of the new drugs with MAO isoforms have not
been fully characterized.
The monoamine oxidase (MAO) catalyzes the oxidative deam-
ination of biogenic amines to aldehydes with radicalic and
polar nucleophilic mechanisms. MAO exists in two isoforms,
MAO-A and MAO-B. These isoforms are encoded by two
different genes and distinguished by different substrate spe-
cificities and sensivities to the selective inhibitors. MAO-A
deaminates seratonin and norephinephrine [1–5]. Reversible
inhibition of MAO-A in the brain causes increased brain levels
of transmitters such as dopamine and serotonin. Selective
MAO-A inhibitors, such as clorgyline and moclobemide, are
useful in the treatment of depression. MAO-B selectively
catalyses the oxidation of 2-phenylethylamine and benzyl-
amine. The abnormal MAO-B activity is implicated in neuro-
degenerative disorders and the selective MAO-B inhibitor
l-deprenyl (selegiline) is widely used in the treatment of
Alzheimer’s and Parkinson’s disease [4–9].
Adverse side effects of non-selective and irreversible MAO
inhibitors such as iproniazid, isocarboxazid, and phenelzine
During recent years there has been an increasing amount
of work done on 3,5-diaryl-4,5-dihydro-1H-pyrazole deriva-
tives, many of which were found to possess selective MAO
inhibitory properties. 1,3,5-Triphenyl-, 1-acyl-, and 3,5-diaryl-
Correspondence: Dr. Erhan Palaska, Faculty of Pharmacy, Department
of Pharmaceutical Chemistry, University of Hacettepe, 06100 Ankara,
Turkey.
E-mail: epalaska@hacettepe.edu.tr
Fax: þ90-3123051872
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
K. S¸entu¨rk et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
4,5-dihydro-1H-pyrazole-1-carbothioamide derivatives
showed remarkable MAO inhibitory [10–14] and antidepress-
ant [11, 15, 16] activities. The substitution of N1 in the 4,5-
dihydro-1H-pyrazole nucleus with the carbothioamide group
favored the inhibitory activity toward the MAOs [11, 13, 14,
17]. Also, in our previous study in which we evaluated MAO
inhibitory activities of 3,5-diaryl-4,5-dihydro-1H-pyrazole
derivatives having a carbothioamide substituent at the N1
position, we observed that the compounds mostly had selec-
tive inhibitory activity against the MAO-A enzyme [18].
Pursuing our studies on MAO inhibitor compounds, in the
present work, we synthesized 3-aryl-5-(4-fluorophenyl)-N-sub-
on the production of hydrogen peroxide from p-tyramine,
using the Amplex-Red MAO assay kit and hMAO isoforms in
microsomes prepared from insect cells (BTI-TN-5B1-4) infected
with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B. The inhibition of hMAO activity was
evaluated using a fluorimetric method. The synthesized
derivatives and reference inhibitors (selegiline and moclobe-
mide) were unable to react directly with Amplex-Red reagent.
The compounds did not react with resorufin used in the
method and did not quench the fluorescence generated by
this product.
The hMAO-A and hMAO-B inhibition data are reported in
Table 1 together with the selectivity index (SI). According to
the enzymatic assays, all the synthesized compounds were
found to be potent (Ki: 1 ꢀ 10ꢁ3–1.16 mM) and selective
(SI: 4.65 ꢀ 10ꢁ4–0.89) MAO-A inhibitors. The inhibition pro-
file was also found to be competitive and reversible for all
compounds.
stituted-4,5-dihydro-1H-pyrazole-1-carbothioamide
deriva-
tives 1–16 and assayed their MAO-A and MAO-B inhibitory
activities by in vitro tests. Docking studies for the most potent
and selective derivative were performed in order to gain
more insight toward its binding mode.
Results
Among them, 5-(4-fluorophenyl)-3-(4-methoxyphenyl)-N-
methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide 4 is the
most effective (Ki: 1.0 ꢀ 10ꢁ3 mM) compound in this series.
The synthetic route used to synthesize 1–16 is outlined in
Scheme 1. The starting compounds, 1-aryl-3-(4-fluorophenyl)-
2-propen-1-ones III, were synthesized by condensing 4-fluo-
robenzaldehyde I with acetophenones II according to the
previously reported Claisen–Schmidt condensation reaction
procedures [19, 20]. The N-substituted carbothioamide deriva-
tives 1–16 were synthesized by the reaction of appropriate
1-aryl-3-(4-fluorophenyl)-2-propen-1-ones III and hydrazine
hydrate followed by the addition of isothiocyanates.
Although the syntheses of 5 and 9 in the series have been
reported previously [21], they were also synthesized in the
present study to investigate their human monoamine
oxidase (hMAO) inhibitory activities.
The SI of 4 toward hMAO-A was calculated as 4.65 ꢀ 10ꢁ4
,
which is smaller than that of moclobemide.
As observed from the data, the introduction of a 4-methoxy
group on the 3-phenyl ring increased the MAO-A inhibitory
activity (Ki: 1.0 ꢀ 10ꢁ3, 0.21, 0.24, and 0.12 mM for 4, 8, 12,
and 16, respectively) and selectivity (SI: 4.65 ꢀ 10ꢁ4, 0.11,
0.15, and 0.13 for 4, 8, 12, and 16, respectively) of the deriva-
tives. In addition, the substitution by a chlorine atom on
the phenyl ring (except 2) produced comparable hMAO-A
inhibitory activity to the substitution by
a methoxy
group. However, the inhibitory potencies of 3-phenyl (1, 5,
9, and 13) and 3-(4-methylphenyl) derivatives (3, 7, 11,
and 15) were found to be lower than those of the other
derivatives.
The potential inhibitory actions of novel compounds on
hMAO activity were investigated by measuring their effects
O
i
R1
C
CH CH
F
R1
COCH3
F
CHO
III
I
II
R1
R1
iii
HA
HB
ii
HX
F
F
N
N
S
N
N
H
C
NH R2
1:R1=H, R2= CH3; 2:R1=Cl, R2= CH3; 3:R1=CH3, R2= CH3; 4:R1=CH3O, R2= CH3; 5:
R1=H, R2= C2H5; 6:R1=Cl, R2= C2H5; 7:R1=CH3, R2= C2H5; 8:R1=CH3O, R2= C2H5;
9:R1=H, R2= C6H5; 10:R1=Cl, R2= C6H5; 11:R1=CH3, R2= C6H5; 12:R1=CH3O, R2= C6H5;
13:R1=H, R2= C3H5; 14:R1=Cl, R2= C3H5; 15:R1=CH3, R2= C3H5; 16:R1=CH3O, R2= C3H5;
Scheme 1. Synthesis of the compounds. (i)
KOH, 108C, 1 h, (ii) H2NNH2, reflux, 2 h, and
(iii) R2-NCS, rt, 3 h.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
New 3,5-Diaryl-4,5-dihydropyrazoles as MAO-A Inhibitors
3
Table 1. Experimental Ki values corresponding to the inhibition of hMAO isoforms by 1-N0-substituted thiocarbamoyl-3-aryl-5-(4-
fluorophenyl)-4,5-dihydro-(1H)-pyrazole derivatives
Comp
R1
R2
Ki value for
MAO-A (mM)
Ki value for
MAO-B (mM)
SIa)
MAO selectivity, inhibition type
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H–
Cl–
CH3–
CH3O–
H–
CH3–
CH3–
CH3–
0.75 ꢂ 1.0 ꢀ 10ꢁ3
0.55 ꢂ 0.02
1.20 ꢂ 0.11
2.20 ꢂ 0.13
1.30 ꢂ 0.08
2.15 ꢂ 0.11
1.30 ꢂ 0.14
0.20 ꢂ 0.01
0.85 ꢂ 0.06
1.90 ꢂ 0.05
1.10 ꢂ 0.09
0.70 ꢂ 0.05
0.80 ꢂ 0.03
1.58 ꢂ 0.11
1.50 ꢂ 0.12
0.23 ꢂ 0.01
1.02 ꢂ 0.08
0.95 ꢂ 0.03
0.09 ꢂ 4.0 ꢀ 10ꢁ3
1.22 ꢂ 0.08
0.63
0.25
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive and reversible
MAO-A, competitive And reversible
MAO-A, competitive and reversible
MAO-B, competitive and irreversible
MAO-A, competitive and reversible
0.60 ꢂ 0.03
0.46
4.65 ꢀ 10ꢁ4
CH3–
1.0 ꢀ 10ꢁ3 ꢂ 1.0 ꢀ 10ꢁ4
C2H5–
C2H5–
C2H5–
C2H5–
C6H5–
C6H5–
C6H5–
C6H5–
C3H5–
C3H5–
C3H5–
C3H5–
1.16 ꢂ 0.08
0.89
0.50
Cl–
0.10 ꢂ 0.07
CH3–
CH3O–
H–
0.33 ꢂ 0.01
0.39
0.11
0.71
0.30
0.44
0.15
0.66
0.52
0.78
0.13
99.92
4 ꢀ 10ꢁ3
0.21 ꢂ 0.01
0.78 ꢂ 0.05
Cl–
0.21 ꢂ 0.01
CH3–
CH3O–
H–
Cl–
CH3–
CH3O–
0.35 ꢂ 0.01
0.24 ꢂ 0.02
0.61 ꢂ 0.04
0.12 ꢂ 0.01
0.80 ꢂ 0.04
0.12 ꢂ 0.01
Selegiline
Moclobemide
9.06 ꢂ 0.44
5 ꢀ 10ꢁ3 ꢂ 1.3 ꢀ 10ꢁ4
Ki values were determined from the kinetic experiments in which p-tyramine (substrate) was used at 500 mM to measure MAO-A and at
2.5 mM to measure MAO-B. Pargyline or clorgyline were added at 0.50 mM to determine the isoforms A and B. Newly synthesized
compounds and the known inhibitors were preincubated with the homoganates for 60 min at 378C. Each value represents the
mean ꢂ SEM of three independent experiments. Ki values corresponding to the inhibition of MAO isoforms by moclobemide as
selective MAO-A inhibitor, and selegiline as selective MAO-B inhibitor, were also determined to assess the inhibitory potencies of the
novel compounds.
a)
Selectivity index. It was calculated as Ki (MAO-A)/Ki (MAO-B).
It was also observed that the presence of groups smaller
than methoxy (H, Cl, and CH3) on the phenyl ring caused a
noticeable decrease in selectivity toward MAO-A.
For the R enantiomer, it was observed that the 4-fluorophenyl
ring at position 5 was oriented close to the hydrophobic
cavity obtained by Tyr407 and Tyr444 instead of the
4-methoxyphenyl ring at position 3 in the S enantiomer.
The R enantiomer did not make any favorable interaction
with the enzyme. Considering their orientations and S scores
(data not shown) we infered that the S enantiomer of 4
was more suitable in structure for the MAO-A enzyme than
the R enantiomer. In order to visualize the selectivity of 4,
the structure of MAO-A docked with the S enantiomer of 4
was superimposed with MAO-B. In Fig. 2, it is seen that
Tyr326, known as an amino acid responsible for selectivity
[22], clashed with the ligand.
On the other hand, when the substitution of carbothio-
amide group is concerned, it can be said that smaller groups
are preferable for the activity of MAO-A. The introduction of a
bulky group on the carbothioamide moiety did not make any
contribution to the activity.
The docking study was performed by using MOE software to
understand the orientations of the most active ligand in the
MAO-A enzyme. 2Z5X, a high resolution crystallographic struc-
ture, was used as receptor model after correcting the FAD
double bonds. Since the compounds were racemic mixtures
we constructed the docking studies with two enantiomers of
the ligand.
Discussion
Because 4 was the most active compound on MAO-A, we
mainly considered about its orientations. For the S enan-
tiomer, it was observed that the 4-methoxyphenyl ring at
position 3 was oriented close to the hydrophobic cavity
obtained by Tyr407 and 444 while the 4-fluorophenyl ring
at position 5 was face to face with Phe208. The oxygen of the
methoxy group interacted with the OH group of Tyr444
In this study, a series of new 3-aryl-5-(4-fluorophenyl)-N-sub-
stituted-4,5-dihydro-1H-pyrazole-1-carbothioamide
deriva-
tives was synthesized in order to evaluate their inhibitory
activities on hMAO-A and hMAO-B enzymes. The binding
mode of the most potent compound inside the MAO-A active
site was predicted using a docking technique.
˚
(<3 A). In addition to these, the N–H proton of the thiocar-
Considering the inhibitory activities and docking studies
of the compounds together, it can be presumed that: (i) the 3-
˚
bamoyl moiety approached the C O of Thr336 (<4 A; Fig. 1).
–
–
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4
K. S¸entu¨rk et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Figure 1. The orientation of S (ball and stick)
and R (stick) enantiomers of 4 in MAO-A active
pocket.
Figure 2. Superimposition of MAO-A (thin)
docked with the S (ball and stick) enantiomer
of 4 and MAO-B (stick).
aryl-5-(4-fluorophenyl)-N-substituted-4,5-dihydro-1H-pyrazole-
1-carbothioamide structure is a suitable scaffold for the MAO-A
enzyme; (ii) the presence of a hydrogen bond acceptor group
such as methoxy or chloro on the 3-phenyl ring increases the
potency and selectivity toward MAO-A; (iii) substituents bulk-
ier than methyl on the carbothioamide moiety do not make
any contribution to the selectivity; and (iv) the synthesized
pyrazole derivatives may be promising candidates as selective
and potent antidepressant agents since 4 in this series
appeared as a more selective novel MAO-A inhibitor than
moclobemide, the known potent MAO-A inhibitor.
Experimental
Chemistry
All chemicals were purchased from Fluka, E. Merck, and
Aldrich Chemical Company. Melting points were determined
with
a
Thomas-Hoover capillary melting point apparatus
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
New 3,5-Diaryl-4,5-dihydropyrazoles as MAO-A Inhibitors
5
(Philadelphia, PA, USA) and are uncorrected. IR spectra were
recorded on a Perkin Elmer FTIR system, Spectrum BX IR spectro-
photometer. The 1H NMR and 13C NMR spectra were recorded on
a Varian Mercury 400 FT NMR spectrophotometer using TMS
400 MHz): d 3.08 (3H; d; NH–CH3), 3.17 (1H; dd; HA-pyrazole-
H4), 3.85 (3H; s; OCH3), 3.92 (1H; dd; HB-pyrazole-H4), 6.01 (1H;
dd; HX-pyrazole-H5) (JAB: 18, JAX: 4.0, JBX: 11.2 Hz), 6.98–7.82
(8H; m; aromatic prot.), 8.16 (1H; q; CS–NH–CH3). ESI-MS (m/z);
as an internal reference (chemical shift represented in d ppm). 366 [MþNa]þ (100%), 344 [MþH]þ. Anal. Calcd. for C18H18FN3OS:
The ESI-MS spectra were measured on a Micromass ZQ MS
C, 62.95; H, 5.28; N, 12.24; S, 9.34. Found: C, 62.84; H, 5.18; N,
spectrometer (MassLynx 4.1 software). Elemental analyses (C, 12.16; S, 9.23.
H, N) were performed on a Leco CHNS 932 analyzer.
N-Ethyl-5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (5)
Preparation of 3-aryl-5-(4-fluorophenyl)-N-substituted-4,5-
Yield: 41.0%, mp 83–848C. IR: (cmꢁ1): 3345 (N–H), 1601 (C N),
–
–
dihydro-1H-pyrazole-1-carbothioamide derivatives (1–16)
A solution of appropriate 1-aryl-3-(4-fluorophenyl)-2-propen-1-one
(10 mmol) and hydrazine hydrate (20 mmol) was refluxed in
ethanol for 2 h. The reaction mixture was cooled to ꢁ188C,
and the solid mass separated out was filtered off and dissolved
in dry diethyl ether. Substituted isothiocyanate (10 mmol) and
three drops of triethylamine were added and stirred for 3 h at
room temperature. The mixture was evaporated to dryness and
the residue was crystallized from suitable solvents.
4
5
1
1377 (C –H), 1316 (C S), 1036 (C –N ). 1H NMR (acetone-d6,
–
–
400 MHz): d 1.20 (3H; t; CH2–CH3) 3.20 (1H; dd; HA-pyrazole-
H4), 3.65 (2H; m; NH–CH2–CH3), 3.97 (1H; dd; HB-pyrazole-H4),
6.04 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 4.0, JBX: 11.6 Hz),
7.03–7.87 (9H; m; aromatic prot.), 8.29 (1H; s; CS–NH–C2H5).
ESI-MS (m/z); 350 [MþNa]þ (100%), 328 [MþH]þ. Anal. Calcd.
for C18H18FN3S: C, 66.03; H, 5.54; N, 12.83; S, 9.79. Found: C,
66.29; H, 5.36; N, 12.79; S, 9.39.
3-(4-Chlorophenyl)-N-ethyl-5-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (6)
5-(4-Fluorophenyl)-N-methyl-3-phenyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (1)
Yield: 27.7%, mp 123–1248C. IR: (cmꢁ1): 3363 (N–H), 1605 (C N),
Yield: 28.4%, mp 151–1538C. IR: (cmꢁ1): 3363 (N–H), 1605 (C N),
–
–
–
–
4
5
1
1397 (C –H), 1347 (C S), 1105 (C –N ). 1H NMR (DMSO-d6,
4
5
1
–
–
–
–
1374 (C –H), 1333 (C S), 1087 (C –N ). 1H NMR (DMSO-d6,
400 MHz): d 1.11 (3H; t; CH2–CH3) 3.09 (1H; dd; HA-pyrazole-
H4), 3.53 (2H; m; NH–CH2–CH3), 3.83 (1H; dd; HB-pyrazole-H4),
5.92 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 4, JBX: 11.6 Hz),
7.07–7.89 (8H; m; aromatic prot.), 8.29 (1H; s; CS–NH–C2H5).
13C NMR (DMSO-d6): d 175.79, 161.77 (d, J ¼ 240.8 Hz), 153.87,
139.99 (d, J ¼ 3.1 Hz), 135.76, 130.58, 129.48, 129.43, 128.14
(d, J ¼ 8.4 Hz), 115.87 (d, J ¼ 21.3 Hz), 63.43, 42.36, 40.19,
15.19. ESI-MS (m/z); 386 [MþNaþ2]þ, 384 [MþNa]þ (100%),
362 [MþH]þ. Anal. Calcd. for C18H17ClFN3S: C, 59.74; H,
4.74; N, 11.61; S, 8.86. Found: C, 59.42; H, 4.67; N, 11.67; S,
400 MHz): d 2.95 (3H; d; NH–CH3), 3.11 (1H; dd; HA-pyrazole-
H4), 3.86 (1H; dd; HB-pyrazole-H4), 5.91 (1H; dd; HX-pyrazole-H5)
(JAB: 18.4, JAX: 3.6, JBX: 11.2 Hz), 7.07–7.87 (9H; m; aromatic prot.),
8.54 (1H; q; CS–NH–CH3). ESI-MS (m/z); 336 [MþNa]þ (100%), 314
[MþH]þ. Anal. Calcd. for C17H16FN3S: C, 65.15; H, 5.15; N, 13.41;
S, 10.23. Found: C, 64.28; H, 5.16; N, 13.20; S, 10.44.
3-(4-Chlorophenyl)-5-(4-fluorophenyl)-N-methyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (2)
Yield: 37.8%, mp 138–1398C. IR: (cmꢁ1): 3271 (N–H), 1601 (C N), 9.01.
–
–
1388 (C –H), 1356 (C S), 1089 (C –N ). H NMR (CDCl , 400 MHz):
4
5
1 1
–
–
d 2.96 (3H; d; NH–CH3), 3.14 (1H; dd; HA-pyrazole-H4), 3.87 (1H;
3
N-Ethyl-5-(4-fluorophenyl)-3-(4-methylphenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (7)
dd; HB-pyrazole-H4), 5.92 (1H; dd; HX-pyrazole-H5) (JAB: 18.4, JAX
:
3.6, JBX: 11.6 Hz), 7.10–7.92 (8H; m; aromatic prot.), 8.62 (1H; q;
CS–NH–CH3). ESI-MS (m/z); 372 [MþNaþ2]þ, 370 [MþNa]þ (100%),
348 [MþH]þ. Anal. Calcd. for C17H15ClFN3S: C, 58.70; H, 4.35;
N, 12.08; S, 9.22. Found: C, 58.16; H, 4.42; N, 11.97; S, 9.26.
Yield: 33.9%, mp 121–1228C. IR: (cmꢁ1): 3372 (N–H), 1603 (C N),
–
–
4
5
1
1374 (C –H), 1366 (C S), 1094 (C –N ). 1H NMR (DMSO-d6,
–
–
400 MHz): d 1.10 (3H; t; CH2–CH3), 2.32 (3H; s; Ar–CH3), 3.07
(1H; dd; HA-pyrazole-H4), 3.50 (2H; m; NH–CH2–CH3), 3.82 (1H;
dd; HB-pyrazole-H4), 5.89 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 3.6,
JBX: 11.6 Hz), 7.07–7.77 (8H; m; aromatic prot.), 8.49 (1H; s;
CS–NH–C2H5). ESI-MS (m/z); 364 [MþNa]þ (100%), 342 [MþH]þ.
Anal. Calcd. for C19H20FN3S: C, 66.83; H, 5.90; N, 12.31; S, 9.39.
Found: C, 66.92; H, 5.73; N, 12.29; S, 9.45.
5-(4-Fluorophenyl)-N-methyl-3-(4-methylphenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (3)
Yield: 13.3%, mp 125–1278C. IR: (cmꢁ1): 3276 (N–H), 1603 (C N),
–
–
4
5
1
1392 (C –H), 1317 (C S), 1108 (C –N ). 1H NMR (DMSO-d6,
–
–
400 MHz): d 2.35 (3H; s; Ar–CH3), 2.97 (3H; d; NH–CH3), 3.11
(1H; dd; HA-pyrazole-H4), 3.85 (1H; dd; HB-pyrazole-H4), 5.91
(1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 3.6, JBX: 11.6 Hz), 7.10–
7.78 (8H; m; aromatic prot.), 8.51 (1H; q; CS–NH–CH3). ESI-MS
(m/z); 350 [MþNa]þ (100%), 328 [MþH]þ. Anal. Calcd.
for C18H18FN3S: C, 66.03; H, 5.54; N, 12.83; S, 9.79. Found: C,
66.01; H, 5.59; N, 12.92; S, 9.84.
N-Ethyl-5-(4-fluorophenyl)-3-(4-methoxylphenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (8)
Yield: 36.7%, mp 1398C. IR: (cmꢁ1): 3344 (N–H), 1602 (C N), 1369
–
–
(C –H), 1334 (C S), 1111 (C –N ). H NMR (DMSO-d , 400 MHz): d
4
5
1
1
–
–
6
1.13 (3H; t; CH2–CH3), 3.10 (1H; dd; HA-pyrazole-H4), 3.54 (2H; m;
NH–CH2–CH3), 3.81 (3H; s; OCH3), 3.84 (1H; dd; HB-pyrazole-H4),
5.91 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 3.6, JBX: 11.6 Hz),
6.99–7.85 (8H; m; aromatic prot.), 8.46 (1H; s; CS–NH–C2H5).
ESI-MS (m/z); 380 [MþNa]þ (100%), 358 [MþH]þ. Anal. Calcd.
5-(4-Fluorophenyl)-3-(4-methoxylphenyl)-N-methyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (4)
Yield: 42.4%, mp 128–1298C. IR: (cmꢁ1): 3362 (N–H), 1605 (C N), for C19H20FN3OS: C, 63.84; H, 5.64; N, 11.76; S, 8.97. Found: C,
–
–
4
5
1
1398 (C –H), 1344 (C S), 1034 (C –N ). 1H NMR (acetone-d6, 64.03; H, 5.29; N, 11.81; S, 8.96.
–
–
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
K. S¸entu¨rk et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
5-(4-Fluorophenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-
1-carbothioamide (9)
N-Allyl-3-(4-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (14)
Yield: 22.3%, mp 178–1798C. IR: (cmꢁ1): 3343 (N–H), 1603 (C N),
Yield: 27.5%, mp 187–1888C. IR: (cmꢁ1): 3377 (N–H), 1600 (C N),
–
–
–
–
1396 (C -H), 1334 (C S), 1096 (C -N ). 1H NMR (DMSO-d6,
400 MHz): d 3.23 (1H; dd; HA-pyrazole-H4), 3.97 (1H; dd; HB-pyr-
1372 (C –H), 1339 (C S), 1090 (C –N ). 1H NMR (acetone-d6,
400 MHz): d 3.23 (1H; dd; HA-pyrazole-H4), 4.00 (1H; dd; HB-pyr-
4
5
1
4
5
1
–
–
–
–
–
azole-H ), 4.28 (2H; m; CH ), 5.08 (1H; dd; CH , H ), 5.21 (1H; dd;
–
4 2 2 a
azole-H4), 6.05 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 3.6, JBX
:
–
–
11.2 Hz), 7.14–8.01 (14H; m; aromatic prot.), 10.19 (1H; s;
CS–NH–C6H5). ESI-MS (m/z); 398 [MþNa]þ (100%), 376 [MþH]þ.
Anal. Calcd. for C22H18FN3S: C, 70.38; H, 4.83; N, 11.19; S, 8.54.
Found: C, 70.04; H, 4.84; N, 11.02; S, 8.57.
CH , H ) 5.96 (1H; m; –CH , H ) (Jab: 1.6, Jax: 10.0, Jbx: 17.2 Hz),
–
–
2 b x
6.07 (1H; dd; HX-pyrazole-H5) (JAB: 17.6, JAX: 3.6, JBX: 11.6 Hz),
7.04–7.91 (8H; m; aromatic prot.), 8.39 (1H; s; CS–NH–CH2–).
ESI-MS (m/z); 398 [MþNaþ2]þ, 396 [MþNa]þ (100%), 374
[MþH]þ. Anal. Calcd. for C19H17ClFN3S: C, 61.04; H, 4.58; N,
11.24; S, 8.58. Found: C, 61.00; H, 4.61; N,11.25; S, 8.60.
3-(4-Chlorophenyl)-5-(4-fluorophenyl)-N-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (10)
Yield: 20.7%, mp 134–1358C. IR: (cmꢁ1): 3314 (N–H), 1594 (C N),
N-Allyl-5-(4-fluorophenyl)-3-(4-methylphenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (15)
–
–
4
5
1
1372 (C –H), 1319 (C S), 1091 (C –N ). 1H NMR (acetone-d6,
400 MHz): d 3.31 (1H; dd; HA-pyrazole-H4), 4.08 (1H; dd; HB-
–
–
Yield: 19.0%, mp 134–1358C. IR: (cmꢁ1): 3354 (N–H), 1603 (C N),
–
–
4
5
1
–
pyrazole-H4), 6.18 (1H; dd; HX-pyrazole-H5) (JAB: 18, JAX: 3.6, JBX
:
1366 (C –H), 1328 (C S), 1096 (C –N ). 1H NMR (DMSO-d6,
400 MHz): d 2.35 (3H; s; Ar–CH3), 3.12 (1H; dd; HA-pyrazole-H4),
–
11.6 Hz), 7.06–8.01 (13H; m; aromatic prot.), 9.97 (1H; s;
CS–NH–C6H5). ESI-MS (m/z); 434 [MþNaþ2]þ, 432 [MþNa]þ
(100%), 410 [MþH]þ. Anal. Calcd. for C22H17ClFN3S: C, 64.46;
H, 4.18; N, 10.25; S, 7.82. Found: C, 63.93; H, 4.12; N, 10.41; S,
7.82.
3.87 (1H; dd; HB-pyrazole-H4), 4.16 (2H; m; CH2), 5.08 (1H; dd;
–
–
–
CH , H ), 5.15 (1H; dd; CH , H ), 5.88 (1H; m; –CH , H ) (Jab: 1.6,
a
–
–
–
2
2
b
x
Jax: 10.0, Jbx: 17.2 Hz), 5.92 (1H; dd; HX-pyrazole-H5) (JAB: 18,
JAX: 3.6, JBX: 11.6 Hz), 7.10–7.80 (8H; m; aromatic prot.), 8.67
(1H; s; CS–NH–CH2–). 13C NMR (DMSO-d6); d 175.96, 161.76
(d, J ¼ 241.1 Hz), 155.29, 141.22, 139.99, 135.88, 129.94, 128.82,
128.07 (d, J ¼ 7.7 Hz), 127.803, 116.14, 115.92 (d, J ¼ 21.2 Hz),
63.27, 46.90, 42.523, 21.75. ESI-MS (m/z); 376 [MþNa]þ (100%),
354 [MþH]þ. Anal. Calcd. for C20H20FN3S: C, 67.96; H, 5.70;
N, 11.89; S, 9.07. Found: C, 67.72; H, 5.40; N,11.88; S, 9.26.
5-(4-Fluorophenyl)-3-(4-methylphenyl)-N-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (11)
Yield: 39.4%, mp 161–1628C. IR: (cmꢁ1): 3338 (N–H), 1603 (C N),
–
–
1388 (C –H), 1334 (C S), 1096 (C –N ). 1H NMR (DMSO-d6,
400 MHz): d 2.36 (3H; s; Ar–CH3), 3.20 (1H; dd; HA-pyrazole-H4),
4
5
1
–
–
3.94 (1H; dd; HB-pyrazole-H4), 6.03 (1H; dd; HX-pyrazole-H5) (JAB
:
N-Allyl-5-(4-fluorophenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (16)
18, JAX: 3.6, JBX: 11.2 Hz), 7.13–7.90 (13H; m; aromatic prot.), 10.15
(1H; s; CS–NH–C6H5). ESI-MS (m/z); 412 [MþNa]þ (100%), 390
[MþH]þ. Anal. Calcd. for C23H20FN3S: C, 70.93; H, 5.18; N,
10.79; S, 8.23. Found: C, 70.56; H, 5.07; N, 10.90; S, 8.59.
Yield: 20.1%, mp 87–888C. IR: (cmꢁ1): 3374 (N–H), 1606 (C N), 1371
–
–
(C –H), 1334 (C S), 1139 (C –N ). H NMR (DMSO-d , 400 MHz): d
4
5
1
1
–
–
3.08 (1H; dd; HA-pyrazole-H4), 3.78 (3H; s; OCH3), 3.83 (1H; dd; 05
6
–
–
–
(1H; dd; CH , H ), 5.12 (1H; dd; CH , H ), 5.84 (1H; m; –CH , H )
x
–
–
–
2
a
2
b
5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-N-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (12)
(Jab: 1.6, Jax: 10.4, Jbx: 17.2 Hz), 5.89 (1H; dd; HX-pyrazole-H4) (JAB: 18,
JAX: 3.6, JBX: 11.2 Hz), 6.98–7.84 (8H; m; aromatic prot.), 8.59 (1H; s;
CS–NH–CH2–). ESI-MS (m/z); 392 [MþNa]þ (100%), 370 [MþH]þ.
Anal. Calcd. for C20H20FN3OS: C, 65.02; H, 5.46; N, 11.37;
S, 8.68. Found: C, 64.91; H, 5.31; N,11.63; S, 9.11.
Yield: 34.7%, mp 176–1778C. IR: (cmꢁ1): 3312 (N–H), 1606 (C N),
–
–
4
5
1
1391 (C –H), 1315 (C S), 1114 (C –N ). 1H NMR (acetone-d6,
–
–
400 MHz): d 3.27 (1H; dd; HA-pyrazole-H4), 3.87 (3H; s; OCH3),
4.04 (1H; dd; HB-pyrazole-H4), 6.14 (1H; dd; HX-pyrazole-H5)
(JAB: 18, JAX: 4, JBX: 11.2 Hz), 7.02–7.92 (13H; m; aromatic prot.),
9.86 (1H; s; CS–NH–C6H5). ESI-MS (m/z); 428 [MþNa]þ (100%),
406 [MþH]þ. Anal. Calcd. for C23H20FN3OS: C, 68.13; H,
4.97; N, 10.36; S, 7.91. Found: C, 67.52; H, 4.87; N, 9.82; S,
7.51.
Biochemistry
Chemicals
hMAO-A (recombinant, expressed in baculovirus infected BTI
insect cells), hMAO-B (recombinant, expressed in baculovirus
infected BTI insect cells), R-(–)-deprenyl hydrochloride, resorufin,
dimethyl sulfoxide, and some other chemicals were purchased
from Sigma–AldrichTM (Germany). Moclobemide was a gift
(Roche Pharmaceuticals, Germany). The Amplex1-Red MAO
Assay Kit (Molecular Probes, USA) contained benzylamine,
p-tyramine, Clorgyline (MAO-A inhibitor), pargyline (MAO-B
inhibitor), and horseradish peroxidase.
N-Allyl-5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-
pyrazole-1-carbothioamide (13)
Yield: 36.2%, mp 115–1168C. IR: (cmꢁ1): 3275 (N–H), 1602 (C N),
–
–
4
5
1
1370 (C –H), 1323 (C S), 1123 (C –N ). 1H NMR (DMSO-d6,
400 MHz): d 3.11 (1H; dd; HA-pyrazole-H4), 3.87 (1H; dd; HB-pyr-
–
–
–
azole-H ), 4.16 (2H; m; CH ), 5.06 (1H; dd; CH , H ), 5.13 (1H; dd;
–
–
CH , H ) 5.85 (1H; m; –CH , H ) (Jab: 1.6, Jax: 10.0, Jbx: 17.2 Hz),
4
2
2
a
–
–
–
2
b
x
Determination of inhibitory activities of the compounds on
human MAO-A and -B
The activity of hMAO-A and hMAO-B (using p-tyramine as
common substrate for both isoforms) was found as
185.60 ꢂ 9.50 pmol/mg/min (n ¼ 3).
5.93 (1H; dd; HX-pyrazole-H5) (JAB: 18.4, JAX: 4, JBX: 11.6 Hz),
7.08–7.89 (9H; m; aromatic prot.), 8.69 (1H; t; CS–NH–CH2–).
ESI-MS (m/z); 362 [MþNa]þ (100%), 340 [MþH]þ. Anal. Calcd.
for C19H18FN3S: C, 67.23; H, 5.35; N, 12.38; S, 9.45. Found: C,
67.07; H, 5.35; N,12.54; S, 9.63.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
New 3,5-Diaryl-4,5-dihydropyrazoles as MAO-A Inhibitors
7
The interactions of the synthesized compounds with hMAO
isoforms were determined by a fluorimetric method described
and modified previously [23–25]. The production of H2O2
catalyzed by MAO isoforms was detected using 10-acetyl-3,7-dihy-
droxyphenoxazine (Amplex1-Red reagent), a non-fluorescent,
highly sensitive, and stable probe that reacts with H2O2 in the
presence of horseradish peroxidase to produce the fluorescent
product resorufin.
two enantiomers of the ligand and the final refined poses were
ranked by the MM/GBVI binding free energy estimation, written
in the S field. The best scores of ligand–enzyme complexes were
selected and minimized by using the LigX utility which holds the
receptor atoms far from the ligand fixed while allowing receptor
atoms near the ligand to move.
The authors have declared no conflict of interest.
Kinetic experiments
References
Newly synthesized compounds were dissolved in dimethyl sulf-
oxide, maximum concentration 1%, and used in the concen-
tration range of 1–100 mM. Kinetic data for interaction of the
enzyme with the compounds were determined using the
Microsoft Excel package program. Lineweaver–Burk plots were
used to estimate the inhibition constant (Ki) of the inhibitors. SI
(Ki (MAO-A)/Ki (MAO-B)) was also calculated. The protein was
determined according to the Bradford method [26], in which
bovine serum albumin was used as a standard.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
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ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com