Synthesis and monoamine oxidase inhibitory activities of 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole derivatives

Article abstract of DOI:10.1002/ardp.200700159

Ten new 1-thiocarbamoyl-3-(phenyl and/or 4-substituted phenyl)-5-(3,4- dimethoxyphenyl and/or 2-chloro-3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole derivatives were synthesized by reacting 1,3-diphenylpropen-1-ones and thiosemicarbazide. The chemical stru

Full text of DOI:10.1002/ardp.200700159

Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
E. Palaska et al.
209
Full Paper
Synthesis and Monoamine Oxidase Inhibitory Activities of
1-Thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole
Derivatives
Erhan Palaska1, Firat Aydin1, Gꢀlberk Uꢁar2, and Dilek Erol3
1 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
2 Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, Ankara, Turkey
3 Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey
Ten new 1-thiocarbamoyl-3-(phenyl and/or 4-substituted phenyl)-5-(3,4-dimethoxyphenyl and/or
2-chloro-3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole derivatives were synthesized by reacting
1,3-diphenylpropen-1-ones and thiosemicarbazide. The chemical structures of the compounds
were verified by means of their IR, 1H-NMR, ESI-MS spectroscopic data and elementary analyses.
All the compounds were investigated for their ability to selectively inhibit monoamine oxidase
(MAO) by in-vitro tests. Monoamine oxidase was isolated and purified from the mitochondrial
extracts of rat-liver homogenates and human platelets. Monoamine oxidase inhibitory activities
of the compounds were compared with pargyline and clorgyline. Most of the compounds inhib-
ited the total activity of rat liver homogenates. The monoamine oxidase-A inhibitory effects of 1-
thiocarbamoyl-3-(4-methoxyphenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole and 1-thio-
carbamoyl-3-(4-methoxyphenyl)-5-(2-chloro-3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole were
detected as potent as clorgyline. Selective and irreversible inhibition of rat liver monoamine oxi-
dase-A by synthesized compounds have promising features for designing the new selective
monoamine oxidase A inhibitors as potent and reliable anti-depressants in the future.
Keywords: 4,5-Dihydro-1H-pyrazole / MAO (Monoamine oxidase) / 2-Pyrazoline / Synthesis /
Received: July 29, 2007; accepted: November 19, 2007
DOI 10.1002/ardp.200700159
dized by both forms of the enzyme in most species. Rever-
sible inhibition of MAO-A in the central nervous system
leads to increased brain levels of dopamine, noradrena-
line, and serotonin (5-hydroxytryptamine, 5-HT), which is
useful in the treatment of depression in elderly patients,
anxiety, and atypical and bipolar depressions [1–6].
MAO-B mainly deaminates trace amines (benzylamine, 2-
phenylethylamine, tyramine). Numerous studies have
established the potential utility of reversible and selec-
tive MAO-B inhibitors, in particular in the treatment of
neurodegenerative disorders, including Parkinson's dis-
ease (PD) and Alzheimer's disease (AD) [5–10].
Introduction
Monoamine oxidases (MAO; EC 1.4.3.4) are widely distrib-
uted enzymes among mammals, plants and prokaryotic
and eukaryotic microrganisms that catalyse the oxida-
tive deamination of a variety of monoamines to alde-
hydes with radicalic (iminium cation as intermediate)
and polar nucleophilic mechanisms. MAO enzymes can
be sub-classified into A and B isoforms. Dopamine, nora-
drenaline, adrenaline, tryptamine, and tyramine are oxi-
MAO inhibitory antidepressant drugs carry an amine,
hydrazine, and hydrazide moiety on a structural basis.
Non-selective and irreversible MAO inhibitors such as
iproniazid, isocarboxazid, and phenelzine have numer-
ous, significant interactions with dietary sympathomi-
Correspondence: Prof. Dr. Erhan Palaska, Hacettepe University Faculty
of Pharmacy Department of Pharmaceutical Chemistry, 06100 Ankara,
Turkey.
E-mail: epalaska@hacettepe.edu.tr
Fax: +90 312 305-1872
Abbreviations: monoamine oxidase (MAO); platelet-rich plasma (PRP)
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E. Palaska et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
Figure 1. Structural formulas of selec-
tive MAO-A and MAO-B inhibitors.
metic amines, such as tyramine, and cause considerably flavin-adenine dinucleotide (FAD) [23, 25, 30]. Recently,
increase in blood pressure, dizziness, blurred vision, and Chimenti et al. elucidated the role of this asymmetric
weakness. They also potentiate the effects of alcohol and centre (C5 position of the pyrazole moiety) in MAO inhib-
other drugs that slow the central nervous system, such as ition for 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyra-
antihistamines, cold medicine, sleep aids, medicine for zole derivatives. Based on computational studies and
seizures, tricyclic antidepressants, tranquilizers, some docking experiments, the contribution to MAO selectiv-
pain relievers, and muscle relaxants. In the last decades, ity appears greatly affected by the S(-)-enantiomer [26].
efforts have been directed to the design, synthesis, and Keeping the above facts in view, we carried out studies to
study of new MAO inhibitors which displayed low toxic- synthesize ten new 1-thiocarbamoyl-3,5-diphenyl-4,5-
ity and relative selectivity for the treatment of various dihydro-1H-pyrazole derivatives and investigate them
psychiatric and neurodegenerative disorders. They rearding their MAO inhibitory activities.
belong to various chemical classes with different modes
of enzyme inhibition. These range from covalent mecha-
nism-based interaction (clorgyline, selegiline, rasagiline)
to non-covalent interaction (brofaromine, toloxatone,
Results and discussion
and moclobemide) (Fig. 1).
Ten new 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-
Despite considerable progress in understanding the pyrazole derivatives 4a–j were synthesized by the reac-
interactions of the two enzyme forms with their pre- tion of 1,3-diphenyl-2-propenone (chalcones) 3 with thio-
ferred substrates and inhibitors, no general rules are yet semicarbazide, and evaluated for their MAO-inhibitory
available for the rational design of potent, reversible, activities by in-vitro tests. The 1,3-diphenyl-2-propenone
and selective inhibitors of MAO, possibly due the fact derivatives were prepared by the reaction of 2-chloro-3,4-
that the mechanism of interaction of the new drugs with dimethoxy- and 3,4-dimethoxybenzaldehyde with appro-
MAO isoforms have not been fully characterized.
4,5-Dihydro-1H-pyrazole derivatives exert various bio- described in the literature [31, 32] (Scheme 1).
logical activities such as antipyretic [11], anti-inflamma- The IR spectra of the compounds showed C=N stretch-
priate acetophenones according to the methods
tory [12–14], hypotensive [15, 16], antimicrobial-antipro- ing bands at 1607-1591 cm– 1 because of ring closure. In
tozoal [14, 17–21], anticonvulsant [22] activities. It has the 1H-NMR spectrum, C4 (HA, HB) and C5 (HX) protons of
been reported that 1,3,5-triphenyl-, 1-acetyl-, and 1-thio- 4,5-dihydro-1H-pyrazole ring resonated as a pair of dou-
carbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives blets of doublets at d 2.98–3.15, 3.65–3.95 and 5.75–
have potent MAO inhibitory [23–27] and antidepressant 6.23 ppm, respectively (JAB: 16.90–18.20, JAX: 3.25–4.15,
[24, 28, 29] activities. The substitution of the aromatic JBX: 11.70–12.05 Hz). These splittings are due to vicinal
ring on N1 of the 4,5-dihydro-1H-pyrazole nucleus with coupling with the two magnetically non-equivalent C4
an acetyl and thiocarbamoyl groups favored inhibitory methylene protons of the ring. The protons belonging to
activity toward the MAOs. This substitution reduces the the aromatic ring and the other aliphatic groups were
steric hindrance of the molecules and raises the positive observed with the expected chemical shift and integral
charge of N1 of the heterocycle, which strengthens the values. In mass spectra, the ions produced under ESI
charge-transfer bond with the isoalloxazine nucleus of showed both characteristic [M + H]+, [M + Na]+ ion peaks
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Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
1-Thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
211
Scheme 1. Synthesis of the presented com-
pounds.
and contain [M–SH]+ fragments that confirm the 1-thio-
carbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole.
Table 1. Kinetic parameters of rat liver MAO-A and B activities
towards p-tyramine.a)
MAO was purified from mitochondrial extracts of rat
liver homogenates and from human platelets according
to the method of Holt et al. [33] with some modifications.
Membranes were incubated with 1% CHAPS for 60
minutes at 378C for the extraction of the membrane-
bound enzyme. The specific MAO activity was found to be
43 nmol/mg protein for rat liver homogenates when p-
tyramine was used as substrate. MAO activity of human
platelets were calculated as 24 nmol/108 platelets/h when
benzylamine was used as substrate.
Inhibitor
Form
remaining
Km (lM)
Vmax (nmol/h/
mg protein)
None
Pargyline
Clorgyline
A and B
A
B
35.00 l 2.10
21.50 l 1.40
45.80 l 2.20
55.22 l 2.80
11.01 l 2.30
40.00 l 3.20
a)
Values represent the mean l SE of six measurements. Rat
liver homogenates were incubated for 60 min at 378C with
either water or 50 lM inhibitor before activities were deter-
mined. p-Tyramine concentrations were chosen as 10-100 lM
for MAO-A and 50–500 lM for MAO-B.
The initial rates of the p-tyramine metabolism by both
MAO-A and B were found to increase linearly with
increasing enzyme concentration. Kinetic parameters of
rat liver MAO-A and MAO-B towards p-tyramine are
shown in Table 1.
phenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole
4b inhibited the MAO-A isoform selectively (IC50:
29.75 l 2.56 lM, Ki: 4.81 l 0.36 lM). An additional chloro
substituent at 5-phenyl ring 4g, showed a stronger inhib-
itory activity towards MAO-A (IC50: 18.45 l 2.46 lM, Ki:
2.60 l 0.18 lM) when compared with that of 4b. Com-
pounds 4c and 4h, which were the 4-bromide substitu-
ents at 3-phenyl ring, also appeared as MAO-A inhibitors
(Ki: 19.51 l 1.50 lM and 21.70 l 2.08 lM). However, their
inhibitory potencies were found to be significantly lower
than those of the chloro-substituents. 1-Thiocarbamoyl-3-
(4-methylphenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-
1H-pyrazole and 5-(2-chloro-3,4-dimethoxyphenyl) deriv-
atives 4d, 4i were found to be non-competitive and irre-
versible inhibitors of rat liver MAO-B. IC50 values for the
inhibition of platelet MAO-B by these compounds were
calculated as IC50: 52.26 l 9.21 and 63.80 l 8.06 lM (Ki:
17.85 l 1.52 lM and 3.80 l .30 lM), respectively. The 4-
methoxy group on the 3-phenyl ring seemed to increase
the MAO-A inhibitory activity of the derivatives. Chloro
substitution also enhanced the MAO-A activity, whereas
the replacement of the chlorides by bromo groups caused
a decrease in their MAO-A inhibitory activities (Table 2).
MAO-A and MAO-B which are encoded by two different
genes and distinguished by different substrate specific-
ities and sensivities to the selective inhibitors [35, 36].
Since platelets have been reported previously to con-
tain only the B-form of MAO [34], benzylamine metabo-
lism of platelet MAO was also determined. The Km and
Vmax values were calculated as 0.19 mM and
48.18 l 4.26 nmol/108 platelets/h. Compounds which
appeared as MAO-B inhibitors were tested by using ben-
zylamine as substrate with the MAO-B isoform obtained
from human platelets. IC50 values of the newly synthe-
sized compounds are shown in Table 2. All the com-
pounds studied, except 4a and 4f, inhibited the total
MAO activity of rat liver homogenates. The compounds
with MAO inhibitory activity were found as time-depend-
ent and irreversible inhibitors since their MAO inhibi-
tory activities markedly increased parallel to the
increased incubation time.
Compounds having a 4-methoxyphenyl ring at posi-
tion 3 of 4,5-dihydro-1H-pyrazole ring (4e and 4j, IC50: 4.
80 l 0.73 lM and 3.14 l 0.83 lM; Ki: 2.45 l 0.16 lM and
2.08 l 0.10 lM, respectively) inhibited MAO-A selectively,
which is close to that caused by clorgyline (IC50:
2.72 l 0.98 lM and Ki: 2.75 l 0.17 lM). Inhibition of liver
MAO-A by these compounds were found to be non-com-
petitive and irreversible. 1-Thiocarbamoyl-3-(4-chloro-
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E. Palaska et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
Table 2. Ki and IC50 values for inhibition and MAO-A and MAO-B selectivity of rat liver MAO isoforms by the newly synthesized com-
pounds.a)
Comp.
Ki valuesb) (lM)
IC50 for MAO-Ac) (lM)
Preincubation Preincubation
IC50 for MAO-Bc) (lM)
Preincubation Preincubation
MAO Inhibitory selectivity
B/A
A/B
0 min.
60 min
0 min.
60 min
4a
4b
4c
4d
4e
4f
4g
4h
–
511.60 l 15.80
38.20 l 3.99
285.27 l 12.34
501.23 l 12.70
6.70 l 0.85
510.80 l 14.90
34.20 l 2.86
305.50 l 12.20
512.90 l 15.20
4.96 l 1.10
509.45 l 12.09
29.75 l 2.56
209.10 l 13.80
488.70 l 12.56
4.80 l 0. 73
511.60 l 12. 56
18.45 l 2. 46
220.09 l 13. 80
490.35 l 21. 94
3.14 l 0. 83
510.78 l 13.89
500.90 l 18.24
511.80 l 12. 0
65.88 l 6.02
530.80 l 14.25
500.90 l 16.86
510.90 l 15.07
490.0 l 28.0
508.34 l 13.80
485.70 l 17.19
499.00 l 13.05
52.26 l 9.21
500.22 l 15.89
499.80 l 12.70
500.50 l 16.30
402.0 l 29.0
No inhibition
4.81 l 0.36
19.51 l 1.50
17.85 l 1.52
2.45 l 0.16
–
2.60 l 0.18
21.70 l 2.08
3.80 l 0.30
2.08 l 0.10
3.87 l 0.31
2.75 l 0.17
16.33
2.39
0.11
0.06
0.42
9.35
0.01
104.21
No inhibition
27.13
1.83
0.04
0.55
4i
4j
68.66 l 6.81
485.06 l 15.40
4.05 l 0.95
63.80 l 8.06
470.60 l 18.22
2.85 l 0.83
0.13
7.69
149.87
0.01
147.39
0.01
136.91
0.01
Pargyline
Clorgyline
432.26 l 18.60
4.16 l 0.93
390.20 l 17. 23
2.72 l 0.98
410.15 l 16.78
400.90 l 15.54
a)
The IC50 values were determined from the kinetic experiments, in which inhibitors were initially pre-incubated with the homo-
genates before adding substrate p-tyramine, used at 500 lM to measure MAO-A and 2.5 mM to measure MAO-B. Pargyline or clor-
gyline were added at 0. 50 lM to determine the isoenzymes A and B.
b)
c)
Ki values were determined from the kinetic experiments, in which p-tyramine was used at 500 lM to measure MAO-A and 2.5 mM
to measure MAO-B. Pargyline or clorgyline were added at 0.50 lM to determine the isoenzymes A and B. Compounds 4a–j and
pargyline or clorgyline were pre-incubated with the homogenates for 60 min. at 378C.
Each value represents the mean l SEM of three independent experiments.
MAO-A was suggested to be inhibited by e. g. clorgyline inhibition possibly cannot enter the small active-site cav-
and prefers serotonin and nor-epinephrine as substrates, ity of the enzyme and may interact tightly with another
whereas MAO-B is inhibited by e. g. pargyline and prefers binding site or with some other reactive groups present,
dopamine and benzylamine as substrates [37]. Although possibly close to the active site in the molecule. Our bio-
both forms of the enzyme are present in the liver and chemical studies carried out with MAOs partially puri-
brain of mammals, it was suggested that only the B-form fied from rat liver showed that the novel compounds
is present in human platelets and only the A-form is strongly inhibited MAO isoforms selectively, and our pre-
present in placenta. The active site of MAO-B is suggested liminary kinetic studies indicated that these derivatives
to consist of two cavities; an entrance cavity of 290 ꢀ3 in appeared as non-competitive and irreversible inhibitors.
volume and a substrate cavity of 420 ꢀ3 in volume. The Further detailed kinetic experiments are needed to clar-
substrate cavity is hydrophobic and an aromatic cage ify their kinetic behavior. The selective and irreversible
formed by the amino acid residues of Tyr398, Tyr435, inhibition of rat liver MAO-A by synthesized pyrazoline
and flavin is important for recognition and direction of derivatives have promising features for designing new
the amine group of the substrate [38, 39]. MAO-A has only selective MAO-A inhibitors in the future and screen them
one active-site cavity of 550 ꢀ3. It has been shown that the for their MAO inhibitory actions toward highly purified
sizes of MAO-A and MAO-B active sites differ in structure MAO isoforms.
to show different interaction profiles with various inhib-
itors [40, 41].
The authors have declared no conflict of interest.
It was recently reported that a novel series of 1-thiocar-
bamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazole
derivatives
also appeared as potent non-competitive and irreversible
inhibitors [42]. The authors suggested that these com-
pounds were time-dependent inhibitors of MAO since
their inhibitory activities were significantly increased
parallel to the increased incubation time. The authors
also indicated that non-competitive and irreversible
inhibition of rat liver MAO by these derivatives suggested
that especially the compounds which showed MAO-B
Experimental
Chemistry
All chemicals were purchased from Fluka (Buchs SG, Switzer-
land), E. Merck (Darmstadt, Germany) and Aldrich (Steinheim,
Germany) Chemical Company. Melting points are uncorrected
and were recorded on a Thomas Hoover Capillary Melting Point
Apparatus (Philadelphia, PA, USA). IR spectra on KBr disks were
recorded on a Bruker Vector 22 IR Spectrophotometer (Ettlin-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
1-Thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
213
gen, Germany). 1H-NMR spectra were recorded on a Bruker
AC200, 200 MHz Spectrometer (Karlsruhe, Germany) in CDCl3
using tetramethylsilane as an internal standard. Splitting pat-
terns are designated as follows; s, singlet; dd, doublet of doublet;
m, multiplet. Coupling constants (J) are given in Hertz. Electro-
spray ionization mass spectra (ESI-MS) of the compounds were
recorded in ESI positive ion mode on a Micromass ZQ Mass Spec-
trometer (Manchester, UK) in methanol. Elemental analyses for
C, H, and N were performed on a LECO CHNS-932 microanalyzer
(Leco, St. Joseph, MI, USA) and the analytical results were within
l 0.4% of the theoretical values. The starting compounds (1,3-di-
aryl-2-propen-1-ones, chalcones) 3 were synthesized by condens-
ing acetophenone 1 with aryl aldehydes 2 according to the
Claisen–Schmidt condensation [31, 32].
1-Thiocarbamoyl-3-(4-methylphenyl)-5-(3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4d
Yield: 88%, mp. 1388C. IR (cm– 1): 3511, 3430 (N-H), 1596, 1565
1
(C=N, C=C), 1354 (C=S), 1265 (C-O), 1039 (C5-N1). H-NMR: d 2.40
(3H, s, -CH3), 3.15 (1H, dd, HA), 3.78 (1H, dd, HB), 3.80 (3H, s,
-OCH3), 3.85 (3H, s, -OCH3), 5.95 (1H, dd, HX) (JAB: 16.90, JAX: 4.10,
JBX: 12.05 Hz), 6.80–7.80 (7H, m, aromatic prot.). ESI-MS (m/z):
324 [M – SH], 356 [M + H]+, 378 [M + Na]+. Anal. Calcd. for
C19H21N3O2S: C, 64.20; H, 5.95; N, 11.82; Found: C, 63.82; H, 5.84;
N, 12.20.
1-Thiocarbamoyl-3-(4-methoxylphenyl)-5-(3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4e
Yield: 65%, mp. 1628C. IR (cm– 1): 3503, 3377 (N-H), 1607, 1561
(C=N, C=C), 1361 (C=S), 1253, 1140 (C-O), 1038 (C5-N1). 1H-NMR: d
3.10 (1H, dd, HA), 3.70 (3H, s, -OCH3), 3.72 (3H, s, -OCH3), 3.85 (3H,
s, -OCH3), 3.97 (1H, dd, HB), 5.75 (1H, dd, HX) (JAB: 17.30, JAX: 3.90,
JBX: 11.90 Hz), 6.70–7.75 (7H, m, aromatic prot.). ESI-MS (m/z):
340 [M – SH], 372 [M + H]+, 394 [M + Na]+. Anal. Calcd. for
C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31; Found: C, 61.48; H, 5.71;
N, 11.34.
Preparation of 1-Thiocarbamoyl-3,5-diphenyl-4,5-
dihydro-1H-pyrazole derivatives 4a–j
A mixture of the appropriate 1-(phenyl and/or 4-substituted phe-
nyl)-3-(3,4-dimethoxy and/or 2-chloro-3,4-dimethoxyphenyl)-2-
propen-1-one 3 (10 mmol) and 1.09 g of thiosemicarbazide
(12 mmol) was refluxed in ethanol (50 mL). After dissolution of
the reactans, a solution of KOH (25 mmol) in 5 mL of water was
added dropwise. The solution was refluxed for a further 4 h. The
reaction mixture allowed to cool, poured into crushed ice, and
the solid mass seperated out was filtered, washed with cold etha-
nol, dried, and crystallized from ethanol / water (70 : 30).
1-Thiocarbamoyl-3-phenyl-5-(2-chloro-3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4f
Yield: 77%, mp. 2328C. IR (cm– 1): 3404, 3266 (N-H), 1600, 1540
(C=N, C=C), 1367 (C=S), 1272, 1217 (C-O), 1033 (C5-N1). 1H-NMR: d
3.04 (1H, dd, HA), 3.74 (1H, dd, HB), 3.80 (3H, s, -OCH3), 3.85 (3H, s, -
OCH3), 6.22 (1H, dd, HX) (JAB: 16.95, JAX: 4.15, JBX: 11.95 Hz), 7.20–
8.45 (7H, m, aromatic prot.), 11.40 (2H, s, -NH2). ESI-MS (m/z): 376
[M + H]+, 398 [M + Na]+. Anal. Calcd. for C18H18ClN3O2S: C, 57.52; H,
4.83; N, 11.18; Found: C, 57.56; H, 4.53; N, 11.07.
1-Thiocarbamoyl-3-phenyl-5-(3,4-dimethoxyphenyl)-4,5-
dihydro-1H-pyrazole 4a
Yield: 84%, mp. 1638C. IR (cm– 1): 3426, 3266 (N-H), 1591, 1516
(C=N, C=C), 1342 (C=S), 1237, 1140 (C-O), 1024 (C5-N1). 1H-NMR: d
3.08 (1H, dd, HA), 3.68 (1H, dd, HB), 3.75 (3H, s, -OCH3), 3.87 (3H, s, -
OCH3), 6.04 (1H, dd, HX) (JAB: 17.10, JAX: 3.45, JBX: 11.95 Hz), 7.00-
7.80 (8H, m, aromatic prot.). ESI-MS (m/z): 310 [M – SH], 342 [M +
H]+, 364 [M + Na]+. Anal. Calcd. for C18H19N3O2S: C, 63.32; H, 5.61;
N, 12.31; Found: C, 63.27; H, 5.64; N, 12.39.
1-Thiocarbamoyl-3-(4-chlorophenyl)-5-(2-chloro-3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4g
Yield: 71%, mp. 1538C. IR (cm– 1): 3511, 3430, 3384 (N-H), 1596,
1565 (C=N, C=C), 1355 (C=S), 1256 (C-O), 1038 (C5-N1). H-NMR: d
1
3.03 (1H, dd, HA), 3.80 (3H, s, -OCH3), 3.85 (3H, s, -OCH3), 3.95 (1H,
dd, HB), 6.23 (1H, dd, HX) (JAB: 16.90, JAX: 3.95, JBX: 11.90 Hz), 6.70–
7.80 (6H, m, aromatic prot.). ESI-MS (m/z): 378 [M – SH], 410 [M +
H]+, 432 [M + Na]+. Anal. Calcd. for C18H17Cl2N3O2S: C, 52.69; H,
4.18; N, 10.24; Found: C, 52.49; H, 4.19; N, 10.31.
1-Thiocarbamoyl-3-(4-chlorophenyl)-5-(3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4b
Yield: 69%, mp. 1588C. IR (cm– 1): 3494, 3372 (N-H), 1595, 1515
(C=N, C=C), 1356 (C=S), 1236, 1141 (C-O), 1026 (C5-N1). 1H-NMR: d
3.15 (1H, dd, HA), 3.70 (1H, dd, HB), 3.75 (3H, s, -OCH3), 3.85 (3H, s, -
OCH3), 5.93 (1H, dd, HX) (JAB: 17.05, JAX: 3.55, JBX: 11.95 Hz), 6.80-
7.85 (7H, m, aromatic prot.). ESI-MS (m/z): 344 [M – SH], 376 [M +
H]+, 398 [M + Na]+. Anal. Calcd. for C18H18ClN3O2S: C, 57.52; H,
4.83; N, 11.18; Found: C, 57.35; H, 4.83; N, 11.40.
1-Thiocarbamoyl-3-(4-bromophenyl)-5-(2-chloro-3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4h
Yield: 86%, mp. 2348C. IR (cm– 1): 3411, 3269 (N-H), 1600, 1540
(C=N, C=C), 1366 (C=S), 1272, 1218 (C-O), 1041 (C5-N1). 1H-NMR: d
3.00 (1H, dd, HA), 3.75 (3H, s, -OCH3), 3.79 (1H, dd, HB), 3.85 (3H, s, -
OCH3), 6.20 (1H, dd, HX) (JAB: 17.60, JAX: 4.05, JBX: 11.80 Hz), 7.20–
8.45 (6H, m, aromatic prot.), 11.42 (2H, s, -NH2). ESI-MS (m/z): 454
[M + H]+, 476 [M + Na]+. Anal. Calcd. for C18H17BrClN3O2S: C, 47.54;
H, 3.77; N, 9.24; Found: C, 47.11; H, 4.16; N, 9.51.
1-Thiocarbamoyl-3-(4-bromophenyl)-5-(3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4c
Yield: 74%, mp. 1678C. IR (cm– 1): 3491, 3371 (N-H), 1597, 1567
(C=N, C=C), 1356 (C=S), 1236, 1140 (C-O), 1027 (C5-N1). 1H-NMR: d
3.05 (1H, dd, HA), 3.65 (1H, dd, HB), 3.75 (3H, s, -OCH3), 3.85 (3H, s, -
OCH3), 5.95 (1H, dd, HX) (JAB: 17.50, JAX: 3.25, JBX: 11.90 Hz), 6.60-
7.90 (7H, m, aromatic prot.). ESI-MS (m/z): 388 [M – SH], 420 [M +
H]+, 442 [M + Na]+. Anal. Calcd. for C18H18BrN3O2S: C, 51.43; H,
4.32; N, 10.00; Found: C, 51.37; H, 4.30; N, 10.19.
1-Thiocarbamoyl-3-(4-methylphenyl)-5-(2-chloro-3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4i
Yield: 94%, mp. 2298C. IR (cm– 1): 3413, 3272 (N-H), 1601, 1541
(C=N, C=C), 1367 (C=S), 1272, 1219 (C-O), 1042 (C5-N1). 1H-NMR: d
3.05 (1H, dd, HA), 3.15 (3H, s, -CH3), 3.70 (3H, s, -OCH3), 3.80 (1H,
dd, HB), 3.85 (3H, s, -OCH3), 6.22 (1H, dd, HX) (JAB: 18.20, JAX: 3.85,
JBX: 11.70 Hz), 7.12–8.40 (6H, m, aromatic prot.), 11.42 (2H, s, -
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214
E. Palaska et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
NH2). ESI-MS (m/z): 390 [M + H]+, 412 [M + Na]+. Anal. Calcd. for
C19H20ClN3O2S: C, 58.53; H, 5.17; N, 10.78; Found: C, 58.69; H,
5.10; N, 10.57.
Measurement of MAO activity
Total MAO activity was measured spectrophotometrically
according to the method of Holt et al. [39]. The chromogenic sol-
ution which is prepared for inclusion in the assay mixture con-
sisted of 1 mM vanillic acid, 500 lM 4-aminoantipyrine, 4 U/
mL– 1 peroxidase in 0.2 M potassium phosphate buffer, pH 7.6.
Chromogenic solution was prepared daily and kept at 48C until
used. A standard assay mixture contained 167 lL chromogenic
solution, 667 lL substrate (500 lM p-tyramine or 450 lM benzyl-
amine, pH 7.6) and 133 lL 0.2 M potassium phosphate buffer,
pH 7.6. The mixture was pre-incubated at 378C for 10 min before
the addition of enzyme. The reaction was initiated by addition
of the homogenate (100 lL) and the absorbance increase was fol-
lowed at 498 nm at 378C for 60 min. The molar absorption coef-
ficient of 4654 M– 1cm– 1 was used to calculate the initial velocity
of the reaction.
1-Thiocarbamoyl-3-(4-methoxyphenyl)-5-(2-chloro-3,4-
dimethoxyphenyl)-4,5-dihydro-1H-pyrazole 4j
Yield: 82%, mp. 1518C. IR (cm– 1): 3445, 3269 (N-H), 1607, 1561
(C=N, C=C), 1353 (C=S), 1256, 1179 (C-O), 1038 (C5-N1). 1H-NMR: d
2.98 (1H, dd, HA), 3.67 (1H, dd, HB), 3.70 (3H, s, -OCH3), 3.75 (3H, s, -
OCH3), 3.85 (3H, s, -OCH3), 6.18 (1H, dd, HX) (JAB: 17.35, JAX: 3.95, JBX:
12.05 Hz), 6.80–7.80 (6H, m, aromatic prot.). ESI-MS (m/z): 374
[M
–
SH], 406 [M + H]+, 428 [M + Na]+. Anal. Calcd. for
C19H20ClN3O3S: C, 56.22; H, 4.97; N, 10.35; Found: C, 55.90; H,
4.97; N, 10.72.
Biochemistry
Clorgyline, pargyline, vanillic acid, 4-aminoantipyrine, peroxi-
dase (type II, horseradish), p-tyramine hydrochloride, benzyl-
amine hydrochloride, TRIS, EDTA, CHAPS, and Triton X-100
were purchased from Sigma-Aldrich,Co (Steinheim, Germany).
All other reagents were of analytical grade.
Selective measurement of MAO-A and MAO-B activities
Rat liver homogenates were incubated with the mixed substrate
p-tyramine at 500 lM to measure MAO-A and at 2.5 mM to meas-
ure MAO-B; 450 lM for benzylamine following inhibition of
MAO with inhibitors (50 lM) or at 500 lM to measure total MAO
when no inhibitor had been included. Substrate concentrations
were chosen with respect to our previous studies on MAO activ-
ity of rat liver homogenates [44].
Aqueous solutions of clorgyline or pargyline (50 lM) were
added to homogenates at the ratio of 1 : 100 (v/v) so the final
inhibitor concentrations were 500 nM. Homogenates were incu-
bated with inhibitors at 378C for 60 min prior to activity meas-
urement.
In order to test the dependence of reaction upon enzyme con-
centration, a range of volumes of inhibitor-treated homogenates
(25-100 lL) was made up to total volumes of 100 lL with potas-
sium phosphate buffer and then incubated with p-tyramine, at
500 lM or 2.5 mM, to assay MAO-A and B activities, respectively.
The kinetic behavior of the tested compounds was determined
in the same mixture above by pre-incubating the homogenates
with newly synthesized compounds dissolved in methanol in
the concentration range of 0–1000 lM for 0, 10, 20, 30, 45, and
60 min at 378C. The IC50 (concentration of the test compound
that reduces the enzyme activity by 50%) values were deter-
mined from plots of residual activity percentage, calculated in
relation to a sample of the enzyme treated under the same con-
ditions without inhibitors, versus-log [I].
Purification of MAO from the liver homogenates
MAO was purified from the rat liver (100–150 g) (Hacettepe Uni-
versity Ethics Committee: 2001/25-4) Homogenates of the liver
were prepared with 1 : 25 (w/v) in ice-cold potassium phosphate
buffer, pH 7.4 with a polytron mechanical homogenizer. Homo-
genates were centrifuged at 1000 g, at 48C for 15 min. and the
supernatant was used as the source of MAO and kept at –708C.
Since MAO-A activity decreased rapidly following homogeniza-
tion, freshly prepared homogenates were used in the studies.
These crude homogenates were used as the MAO source for pre-
liminary kinetic assays.
Mitochondrial MAO was purified by isolation of mitochondria
from liver homogenates. Liver tissue (5–8 g) was homogenized
1 : 40 (w/v) in 0.3 M sucrose. Following centrifugation at 1.000 g
for 10 min., the supernatant was centrifuged at 10000 g for
30 min. to obtain crude mitochondrial pellet. The pellet was
incubated with CHAPS of 1% at 378C for 60 min. and centrifuged
at 1000g for 15 min. The pellet was resuspended in 0.3 M
sucrose and layered onto 1.2 M sucrose, centrifuged at 53000 g
for 2 h and resuspended in potassium phosphate buffer. It was
kept at –708C until used.
Reversibility of the inhibiton of rat liver MAO isoformes by the
newly synthesized inhibitors was assessed by dilution. Enzyme
samples (10 times the final concentration) were incubated for
60 min. at 378C with different concentrations of the inhibitors
related to their IC50 values. The samples were then diluted 10
times into the assay mixture. A parallel experiment was carried
out where the enzyme (10 times the final concentration) was
incubated for 60 min. at 378C with an equivalent amount of
water. The samples were then diluted 10 times into the assay
mixture containing the same final concentrations of the inhibi-
tors. Both sets were assayed for the MAO-A and B activities.
Kinetic data for interaction of the enzyme with the com-
pounds were determined using the Lineweaver–Burk double
reciprocal plot, by plotting 1/v vs 1/s, analyzed over a range of
substrate concentrations and also evaluated using Microsoft
Excel package program.
Purification of MAO-B from human platelets
Venous blood samples were pooled in stoppered laboratory vacu-
tainer tubes containing sodium citrate as anticoagulant. Plate-
let-rich plasma (PRP) was prepared by sequential centrifugation.
Blood was centrifuged at 200 g for 15 min. at room temperature.
Supernatant was aspirated, recentrifuged at 1200 g for 10 min.
and kept. The pellet was resuspended in 0.5 mL of TRIS buffer
(15 mM TRIS-HCl, 14 mM NaCl, 10 mM EDTA), pH 7.4 and centri-
fuged at 10000 g for 10 min. Supernatants were pooled. Platelet
counts were determined on aliquots of pooled PRP diluted in Iso-
ton II and counted twice on a thrombocounter (Coulter STKS
Counter, Coulter Electronics, Hialeah, FL, USA) [43]. The kinetic
behavior of MAO-B in the pooled PRP was determined as
described below by using benzylamine (0–10 mM) as substrate
and the results were expressed as nmol/108 platelets/h.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 209–215
1-Thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
215
[21] M. Abid, A. Azam, Eur. J. Med. Chem. 2005, 40, 935–942.
Protein determination
Protein contents were determined according to the method of
Bradford with bovine serum albumin used as standard [45].
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