New pyrazoline bearing 4(3H)-quinazolinone inhibitors of monoamine oxidase: Synthesis, biological evaluation, and structural determinants of MAO-A and MAO-B selectivity

Article abstract of DOI:10.1016/j.bmc.2008.11.068

A new series of pyrazoline derivatives were prepared starting from a quinazolinone ring and evaluated for antidepressant, anxiogenic and MAO-A and -B inhibitory activities by in vivo and in vitro tests, respectively. Most of the synthesized compounds show

Full text of DOI:10.1016/j.bmc.2008.11.068

Bioorganic & Medicinal Chemistry 17 (2009) 675–689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New pyrazoline bearing 4(3H)-quinazolinone inhibitors of monoamine oxidase:
Synthesis, biological evaluation, and structural determinants of MAO-A
and MAO-B selectivity
b
c
d
e
Nesrin Gökhan-Kelekçi a, , Semra Koyunog˘lu , Samiye Yabanog˘lu , Kemal Yelekçi , Özen Özgen ,
*
Gülberk Uçar c, Kevser Erol f, Engin Kendi e, Akgül Yeßsilada b
a Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhıye, Ankara, Turkey
b Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Sıhhıye, Ankara, Turkey
c Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Sıhhıye, Ankara, Turkey
d
_
Kadir Has University, The Faculty of Arts and Sciences, 34080 Fatih, Istanbul, Turkey
e Hacettepe University, Faculty of Engineering, Department of Physics Engineering, 06800 Beytepe, Ankara, Turkey
f Osmangazi University, Faculty of Medicine, Eskißsehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2008
Revised 17 November 2008
Accepted 21 November 2008
Available online 3 December 2008
A new series of pyrazoline derivatives were prepared starting from a quinazolinone ring and evaluated
for antidepressant, anxiogenic and MAO-A and -B inhibitory activities by in vivo and in vitro tests, respec-
tively. Most of the synthesized compounds showed high activity against both the MAO-A (compounds
4a–4h, 4j–4n, and 5g–5l) and the MAO-B (compounds 4i and 5a–5f) isoforms. However, none of the
novel compounds showed antidepressant activity except for 4b. The reason for such biological properties
was investigated by computational methods using recently published crystallographic models of MAO-A
and MAO-B. The differences in the intermolecular hydrophobic and H-bonding of ligands to the active site
of each MAO isoform were correlated to their biological data. Compounds 4i, 4k, 5e, 5i, and 5l were cho-
sen for their ability to reversibly inhibit MAO-B and MAO-A and the availability of experimental inhibi-
tion data. Observation of the docked positions of these ligands revealed interactions with many residues
previously reported to have an effect on the inhibition of the enzyme. Among the pyrazoline derivatives,
it appears that the binding interactions for this class of compounds are mostly hydrophobic. All have
Keywords:
2-Pyrazoline
MAO-A/MAO-B inhibition
Docking
Antidepressant-anxiogenic activities
Crystallographic model
potential edge-to-face hydrophobic interactions with F343, as well as p–p stacking with Y398 and other
hydrophobic interactions with L171. Strong hydrophobic and H-bonding interactions in the MAO recog-
nition of 4i could be the reason why this compound shows selectivity toward the MAO-B isoform. The
very high MAO-B selectivity for 4i can be also explained in terms of the distance between the FAD and
the compound, which was greater in the complex of MAO-A-4i as compared to the corresponding
MAO-B complex.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
knowledge of the enzyme, in particular the identification of two iso-
forms (MAO-A and MAO-B) that can be selectively inhibited, and the
Inhibitors of monoamine oxidase have shown therapeutic value
in a variety of neurodegenerative diseases.1 The discovery in the
1950s of the antidepressant properties of MAO inhibitors (MAOIs)
was the major finding that led to the monoamine theory of depres-
sion. Earlier MAO inhibitors introduced into clinical practice for
the treatment of depression were abandoned due to adverse side-ef-
fects, such as hepatotoxicity, orthostatic hypotension and the so-
called ‘cheese effect’ characterized by hypertensive crises.2 This
handicap was thought to be related to nonselective and irreversible
monoamine oxidase inhibition. However, more recently, a better
development of new generations of inhibitors have led to a renewed
interest in the therapeutic potential of these compounds. These two
forms of MAO are characterized by their different sensitivities to
inhibitors and their different specificities to substrates.3 MAO-A
preferably metabolizes serotonin, adrenalin, and noradrenalin,4
whereas b-phenylethylamine and benzylamine are predominantly
metabolized by MAO-B.5 Tyramine, dopamine, and some other
important amines are common substrates for both isoenzymes.6
Nowadays, the therapeutic interest of MAOIs falls into two
major categories. MAO-A inhibitors have been used mostly in the
treatment of mental disorders, in particular depression and anxi-
ety,7–9 while MAO-B inhibitors could be used in the treatment of
Parkinson’s disease and perhaps, Alzheimer’s disease.10,11 Despite
* Corresponding author. Tel.: +90 312 305 30 17; fax: +90 312 311 47 77.
E-mail address: onesrin@hacettepe.edu.tr (N. Gökhan-Kelekçi).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.068

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
the considerable progress in understanding the interactions of the
two enzyme forms with their preferred substrates and inhibitors,
few general rules are yet available for the rational design of potent
and selective inhibitors of MAO-A and MAO-B. Despite these diffi-
culties, efforts have been oriented toward the discovery of revers-
ible and selective inhibitors of MAO-A/MAO-B leading to a new
generation of compounds.
anxiogenic activities of 26 2-(10-substituted phenyl-30-heteroaryl-
20-propenylidene)hydrazine-3-methyl-4(3H)-quinazolinone and
2-(3-substituted phenyl-50-heteroaryl-20-pyrazoline-1-yl)-3-meth-
yl-4(3H)-quinazolinone derivatives, 12 new ones have been syn-
thesized. In particular, the influence on biological behavior (such
as MAO inhibition, antidepressant, and anxiogenic activities) of
the introduction of different aromatic rings in the 3 and 5 positions
The recent determination of the crystal structure of the two iso-
forms of human MAO by Binda et al. elucidates the mechanism
underlying the selective interactions between these proteins and
their ligands, probes the catalytic mechanism, and provides a bet-
ter understanding of the pharmacophoric requirements needed for
a rational design of potent and selective enzyme inhibitors with a
therapeutic potential.12–15
of
the
4,5-dihydro-(1H)-pyrazole
nucleus
containing
3-methyl-4(3H)-quinazolinone has been investigated.
2.1. Chemistry
We described here a convenient approach to the preparation of
2-(10-substituted phenyl-30-heteroaryl-20-propenylidene)hydra-
The classical period of the MAO inhibitors started with hydra-
zine derivatives. Originally proposed as a tuberculostatic agent,
their prototype, iproniazid, was the first modern antidepressant
and was introduced into the market under the trade name Marsi-
lidÒ.16 Subsequently, research has been directed at the preparation
of heterocyclic hydrazines and hydrazides and their potential as
therapeutic agents for the treatment of CNS depression.17–19
2-Pyrazolines can be considered as a cyclic hydrazine moiety.
For this reason, some authors investigated MAO-inhibitory proper-
ties of 1,3,5-triphenyl-2-pyrazolines and found high activity.20–27
The discovery of this class of drugs has led to a considerable
increase in modern drug development in this area and has also
pointed out the unpredictability of biological activity from struc-
tural modification for a prototype drug molecule.
All these findings have pushed us to synthesize various pyra-
zoles derivatives and examine their different amine oxidase inhibi-
tion activities including bovine serum amine oxidase (BSAO), MAO,
and semicarbazide sensitive amine oxidase (SSAO). A considerable
number of the prepared compounds were found to have BSAO,
SSAO, and MAO-inhibitory activity comparable to or higher than
the reference compounds.28–30 At the same time, some of the pyr-
azoles, which were presented as selective MAO-B inhibitors,24
were found to inhibit the AChE activities of human erythrocyte
and plasma, approved for the treatment of cognitive dysfunction
in AD, selectively, and noncompetetively31 (Fig. 1).
zine-3-methyl-4(3H)-quinazolinone and 2-(3-substituted phenyl-
50-heteroaryl-20-pyrazoline-1-yl)-3-methyl-4(3H)-quinazolinone
4a–4f/5a–5f. For the synthesis of the desired compounds, Scheme
1 was followed.
The key reactions involved are the intermediate formation of
hydrazones and subsequent addition of N–H on the olefinic bond
of the propenone moiety that form the ring-closed final products
5. In the 1H NMR spectra of the compounds 4, olefinic protons
(20-CH and 30-CH) appeared as doublets at about 6.8 and
7.8 ppm, respectively (Jtrans = 14–16 Hz). After the ring closure, ring
protons (HA and HB) of the compounds 5 showed at around 3.4 and
3.7 ppm and also HX gave triplet/multiplet at about 6.3 ppm due to
vicinal coupling with the two magnetically nonequivalent protons
of the methylene group. The quinazolinone and phenyl protons
were observed at the expected chemical shifts and integral values.
In the IR spectra of the compounds 5a–5f the disappearance of
C@C (olefinic) and N–H stretching bands at 1584–1608 and
3257–3366 cmꢀ1 is due to the ring closure.
The characteristic peaks were observed in the mass spectra of
the compounds. Molecular ion peaks (M+) provided the molecular
formula of all synthesized compounds 4a–4f/5a–5f. Characteristic
M+2 isotope peaks were observed in the mass spectra of the com-
pounds having a halogen or a sulfur. As for the compounds 5a–5f,
the 2-pyrazoline moiety have shown a fragmentation pattern giv-
ing rise to desired peaks at m/e 250, 252, 268, and 282 (Scheme 2).
The fragmentation of compounds 4a–4f occurs via cleavage of
quinazolinone moiety from the other group giving desired peaks
at m/z 159. This is followed by the loss of a C2H2N radical to pro-
duce an ion at m/z 119 (Scheme 3).
Moreover, a number of quinazolinones and their analogues have
shown clinical importance through the exhibition of monoamine
oxidaseinhibitory activity. Itwasobservedthat the2ndand3rdposi-
tions of quinazolinones have been the target for the substitution of
different heterocyclic moieties.32–36 However, a 5-membered pyraz-
oline ring has not appeared to be linked with quinazolinones so far.
These observations motivated us to link different heterocyclic moie-
ties to synthesize a new series of pyrazole derivatives by combining
the quinazolinone moiety at the 1st position in order to evaluate the
effect of this substitution on monoamine oxidase inhibitory effects.
2.2. X-ray crystal analysis of 5a
A colorless suitable crystal with dimensions 0.42 ꢁ 0.48 ꢁ
0.48 mmwas chosen for thestructuredetermination. The diffraction
intensity data were collected at room temperature on an Enraf-Non-
ius CAD-4 diffractometer using graphite-monochromated radiation
(CuKa, k = 1.54184 Å). w/2h scan mode was employed for data col-
lection. The cell parameters were refined from accurately deter-
2. Results and discussion
mined 25 reflections in the range of 3.7° 6 h 6 74.2°. A total of
4320 reflections (4128 unique, 3088 observed [I > 2r(I)]) were col-
To verify the effects of structural modifications on both inhibi-
tion and selectivity toward MAO-A/MAO-B, and antidepressant and
Figure 1. Pyrazole derivatives having MAO, SSAO, and BSAO inhibition.

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
677
Scheme 1. Synthetic pathway of compounds 4a–4n/5a–5i.
Scheme 2. Fragmentation pattern of the compounds (5a–5f).
lected for ꢀ11 6 h 6 11, ꢀ12 6 k 6 11, 0 6 l 6 16 with Rint = 0.0148.
Extinction correction was not applied.
All hydrogen atoms were positioned geometrically and refined
using a riding model with (C–H (aromatic) = 0.93, 97, 98, and
0.96 Å for methyl H atoms). After a few cycle refinements, an
The crystal structure was solved by direct methods using the
SHELXS-97 program37 which revealed the positions off all the non-
hydrogen atoms and these were refined isotropically and then
anisotropically by full matrix least-squares calculations (based on
|F2|) using SHELXL-97.38 The experimental crystallographic data
and refinement parameters are given in Table 1.
empirical
W
scan absorption correction was applied.39 The view
of the molecule performed using ORTEP40 and some selected bond
lengths, angles, and torsion angles are given in Figure 2.
The 2-pyrazoline ring deviates markedly from planarity with
the torsion angles N1–C12–C11–C10 of 15.6(3)°. The pyrazoline

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
Scheme 3. Fragmentation pattern of the compounds (4a–4f).
Table 1
Crystallographic and refinement parameters of the molecule
Molecular formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Cell dimensions
a (Å)
C23H20N4OS
400.50
293 (2)
1.54184
Triclinic
ꢀ
P1
8.889 (2)
b (Å)
9.838 (3)
c (Å)
12.871 (3)
110.86 (2)
92.35 (2)
a
(°)
b (°)
c
(°)
100.15 (2)
1028.9 (5)
2
1.36
1.564
Volume (Å3)
Z
Density (calculated) (Mg mꢀ3
)
)
Absorption coefficient (mmꢀ1
F000
420
Crystal size (mm)
Crystal color
0.42 ꢁ 0.48 ꢁ 0.48
Colorless
h range for data collection (°)
Index ranges
3.7–74.2
ꢀ11 6 h 6 11
ꢀ12 6 k 6 11
0 6 l 6 16
4320
4128 [Rint = 0.0148]
Full-matrix least-squares on F2
4128/0/262
1.055
Reflections collected
Independent reflections
Refinement method
Figure 2. QRTEP drawing of the molecule indicating atom numbering scheme with
30% probability. Selected intramolecular bond lengths (A), bond angles (°) and
torsion angles (°) of 5a SI–C16 = 1.678(4). S1–C13 = 1.691(3), C13–C14 = 1.457(4),
C15–C16 = 1.311(7), N1–N2 = 1.416(3) N2–C10 = 1.288(4), C10–C11 = 1.503(4),
C12–C11 = 1.534(4). N1–C12 = 1.488(3), C12–C13 = 1.501(4), N4–C3 = 1.470(4).
N3–C3 = 1.390(4), N4–C2 = 1.387(4), N3–C1 = 1.283(3), C1–C2 = 1.230(3), C13–S1–
C16 = 92.6(2), S1–C16–C15 = 112.7(3), C16–C15–C14 = 116.4(4), C15–C14–C13 =
106.5(3), C14–C13–S1 = 111.7(2), N1–N2–C10 = 108.4(2), N2–C10–C11 = 113.5(2).
C10–C11–012 = 102.9(2), C11–C12–N1 = 101.1(2). C12–N1–N2 = 110.9(2), C11–
C10–C17 = 125.1(2), C13–C12–N12 = 109.8(2), C13–C12–C11 = 116.1(2), C1–N1–
C12 = 118.8(2), C1–N1–N2 = 113.9(2), O1–C2–N4 = 120 0(3), N1–C1–N3 =
119.2(2), N4–C1–N1 = 115.7(2), S1–C13–C14–C15 = 17(3). N1–N2–C10–011 =
ꢀ1.1(3). C10–C11–C12–N1 = 15.6(3). C1–N4–C2–O1 = ꢀ179.3(3), S1–C13–C12–
N1 = 53.3(3), C10–C11–C12–C13 = 134.4(3), N2–N1–C12–C13 = ꢀ141.0(2), N2–
C10–C17–C22 = 173.3(3), N2–N1–C1–N4 = 617(3).
Data/restraints/parameters
Goodness-of-fit on F2
R indices [I > 2
r
(I)]
R1 = 0.065, wR2 = 0.194
0
0.574
ꢀ0.531
(D/r)
D
D
qmax (eÅꢀ3
qmin (eÅꢀ3
)
)
ring is in a distorted envelope conformation. Maximum deviation
of the C12 atom from the N1–N2–C10–C11 plane is 0.283(3) Å.
The bond lengths and angles of the pyrazoline ring are compared

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
679
with the corresponding values of similar compounds.41,27 In the
present study, all the geometric parameters of the pyrazoline ring
are in agreement with the reported values in 2-[3-phenyl-5-(m-
chlorophenyl)-2-pyrazolin-1-yl]-3-methyl-4(3H)-quinazolinone41
and a new therapeutic approach in the treatment of Alzheimer dis-
ease: some novel pyrazole derivatives as a dual MAO-B inhibitors
and antiinflammatory analgesics.27
In the thienyl ring, though the C15–C16 [1.311(7) Å] bond
shows a double bond character and is shorter than the values of
1.339(3) and 1.365(3) Å reported for 3-phenyl-5(2-thienyl)-2-pyr-
azoline-1-thioamide,43 the C13–C14 [1.457(4) Å] bond shows a
single bond character. The S1–C13 [1.691(3)Å] and S1–C16
[1.678(4) Å] bonds are in agreement with the values reported for
3-phenyl-5(2-thienyl)-2-pyrazoline-1-thioamide [S1–C10 = 1.703
(2) and S1–C13 = 1.716(2) Å].43 The thienyl ring is almost perpen-
dicular to the pyrazoline ring. The dihedral angle between two
rings is 84.8(1)°. The dihedral angle between the pyrazoline ring
and the phenyl ring is 6.7(1)° and these rings are nearly coplanar.
The determination of the structure indicates the absolute R config-
uration of the chiral atom C12.
The 4(3H)-quinazolinone group attached to the N1 atom. The
N1–N2 bond shows a single bond character with the value of
1.416(3) Å. The N1–N2 bond length is almost similar to the values
of 1.411(7) and 1.413(7) Å reported in 2-[3-phenyl-5-(m-chloro-
phenyl)-2-pyrazolin-1-yl]-3-methyl-4(3H)-quinazolinone41
and
the value of 1.400(2) Å reported in a new therapeutic approach
in Alzheimer disease: some novel pyrazole derivatives as a dual
MAO-B inhibitors and antiinflammatory analgesics.27
The N2–C10 [1.288(4) Å] and N1–C12 [1.488(3) Å] bonds are
well in agreement with the values of 1.285(2) and 1.483(2)
Å reported in 10,20,30,40-tetrahydro-1,3-diphenyl-4-chlorospiro[2-
pyrazoline-5,2-napthalen]10one.42
Furthermore van der Waals forces, the crystal packing is
stabilized by C–Hꢂ ꢂ ꢂO and C–Hꢂ ꢂ ꢂN intramolecular interactions
[for C9–H9Aꢂ ꢂ ꢂO1; D–H = 0.96, H. . .A = 2.23, D. . .A = 2.688(5),
D–H. . .A = 108 [for C9–H9Aꢂ ꢂ ꢂN2; D–H = 0.96, H. . .A = 2.34,
D. . .A = 2.909(5), D–H. . .A = 117].
Whereas the 3-methyl-4(3H)-pyrimidine ring deviates slightly
from the planarity, the phenyl ring of the quinazolinone moiety
adopts nearly planar conformation. The C3 atom deviates from
the plane of the pyrimidine ring with the distance of ꢀ0.0314(2) Å.
The ketone group located C2 is in the same plane with the pyrim-
idine structure with a C1–N4–C2–O1 torsion angle of ꢀ179.3(3)°.
The C1–N3 [1.283(3) Å] and C2–N4 [1.387(4) Å] bond lengths are
in agreement with the values of 1.277(8) and 1.277(8) Å and
1.401(9) and 1.408(9) Å reported for 2-[3-phenyl-5-(m-chloro-
phenyl)-2-pyrazolin-1-yl]-3-methyl-4(3H)-quinazolinone.41
2.3. Biochemistry
MAO-A and MAO-B inhibitory activities of newly synthesized
4(3H)-quinazolinone derivatives were determined using MAO iso-
forms of rat liver (Table 2). Liver tissue was used to screen the
MAO-inhibitory actions of these novel compounds since liver was
reported to be a good source for both isoforms of the enzyme.
According to the IC50 values corresponding to the inhibition of
rat liver MAO-A and -B by the newly synthesized quinazolinone
derivatives, compounds 2, 4a–4h, 4j–4n, and 5g–5l inhibited rat
Table 2
Kinetic data corresponding to the inhibition of rat liver MAO isoforms by the newly synthesized quinazoline derivativesa
Compound
Ki valuesa
for MAO-A
(
lM)
IC50 for MAO-A (
preincubation 60 min
l
M)b
Ki valuesa
for MAO-B
(lM)
IC50 for MAO-B (
preincubation 60 min
l
M)b
MAO-inhibitory selectivity
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
2
3
190.12 10.26
28.16 1.50
185.13 12.16
180.80 11.10
80.13 5.16
70.26 6.48
34.20 2.60
1.05 0.95
33.16 2.50
28.05 3.56
27.18 1.76
90.80 8.63
31.60 2.80
34.20 2.76
35.10 3.90
3.56 0.28
188.22 9.80
69.12 5.01
184.40 12.00
179.11 10.05
125.13 10.55
90.55 7.89
70.55 5.80
7.80 0.67
419.12 33.25
415.70 17.19
299.30 21.25
320.34 21.20
400.05 23.35
419.30 26.76
415.20 40.85
377.75 16.98
1.15 0.12
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
5.10 0.23
30.16 0.17
1.15 0.10
468.00 20.66
30.50 1.90
9.05 0.18
0.16 0.08
64.56 5.32
10.58 1.19
69.57 5.54
30.56 2.75
65.12 5.18
90.68 7.47
25.56 1.96
16.27 1.20
88.56 7.16
9.25 0.71
470 27.16
399.00 19.00
487.05 47.83
429.00 28.50
470.50 30.00
70.83 7.16
27.56 1.36
15.23 1.60
77.13 6.98
7.22 0.70
40.27 3.05
80.11 6.13
90.67 8.40
130.45 10.77
65.33 5.13
25.28 2.01
140.23 10.44
28.74 2.05
6.80 0.52
9.90 0.80
2.70 0.20
0.90 0.07
2.56 0.31
30.50 1.90
29.00 2.50
68.01 5.88
414.75 34.00
420.23 27.21
439.93 30.18
410.11 17.59
450.24 30.55
418.00 20.20
9.46 0.72
3.70 0.27
1.02 0.09
4.51 0.33
4.51 0.33
1.68 0.18
3.42 0.47
1.48 0.66
2.03 0.27
25.20 1.99
18.43 1.60
5.80 0.75
7.90 0.52
9.90 0.81
10.55 0.89
78.34 5.09
18.58 1.25
3.36 0.27
5.60 0.44
1.35 0.12
10.21 0.83
409.76 10.70
415.70 17.19
399.90 12.30
483.77 36.50
400.22 20.89
485.26 26.71
120.35 11.60
21.16 6.83
1.03 0.10
1.17 0.19
30.18 1.97
25.23 2.46
8.50 0.89
1.20 0.09
15.70 1.04
5.53 0.27
37.41 2.05
390.20 17.23
2.05 0.19
499.01 18.20
3.90 0.19
Pargyline
Clorgyline
Selegiline
Moclobemide
3.85 0.23
400.90 15.54
2.01 0.15
480.12 21.05
a
Ki values were determined from the kinetic experiments in which p-tyramine was used at 500
l
M 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. Newly synthesized compounds and the known inhibitors were preincubated with the homogenates
for 60 min at 37 °C.
b
IC50 values were determined from plots of residual activity percentage, calculated in relation to a sample of the enzyme treated under the same conditions without
inhibitor, versus inhibitor concentration [I]. Newly synthesized compounds and the known inhibitors were preincubated with the homogenates for 60 min at 37 °C.

680
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
liver MAO-A selectively while compounds 3, 4i, and 5a–5f inhib-
ited rat liver MAO-B selectively (Table 2).
90.68 7.47; 25.56 1.96; 16.27 1.20; 88.56 7.16; 9.25 0.71;
and 1.48 0.66 M, respectively. The most potent compound in
l
These compounds were found to be time-dependent inhibitors
of MAO-A since their inhibitory activities were significantly
increased parallel to the increased incubation time. Homogenates
were preincubated with the compounds for 0, 10, 20, 40, and
60 min (Table 2). After 60 min of preincubation, activity was found
to be unchanged and the most potent inhibitory activities of these
compounds were found by incubation at 37 °C for 60 min.
The starting compound entitled quinazolinone ring (compound
2) inhibited MAO-A competitively and reversibly whereas quinaz-
olinone hydrazine (compound 3) inhibited MAO-B competitively
and irreversibly with the Ki values of 30.18 1.97 and
this group was compound 5f, which bears a methoxy group on
the phenyl at 3 position of the pyrazoline and furyl at its 5 position.
Compounds 5g, 5h, 5i, 5j, 5k, and 5l, which have a phenyl and
substituted phenyl at 3 and 5 positions, respectively, inhibited
MAO-A competitively and reversibly (Table 3). The most potent
inhibitor in this group was compound 5i, which bears a chloride
group at 3 position on the phenyl ring (Table 2 and Fig. 3).
As a result, it is worth noting that the main difference in the
MAO-inhibitory activities of pyrazoline type compounds 5a–5l is
found when the 5-membered heterocycle at the fifth position of
the pyrazoline ring is replaced by a 6-membered heterocycle. This
replacement appeared to be responsible for the remarkable differ-
ences in the inhibition activity mainly against MAO-A/MAO-B
isoforms, respectively.
18.58 1.25 lM, respectively.
Hydrazone derivatives bearing quinazolinone ring, compounds
4a–4n except that 4i, were found as selective MAO-A inhibitors
irrespective of the substituents with the Ki values of
190.12 10.26; 28.16 1.50; 185.13 12.16; 180.80 11.10;
80.13 5.16; 70.26 6.48; 34.20 2.60; 1.05 0.95; 0.16 0.08;
30.16 0.17; 1.15 0.10; 40.27 3.05; 30.56 2.75; and
65.12 5.18 lM, respectively. Compounds 4a–4g inhibited MAO
irreversibly in a noncompetitive manner whereas compounds
4h–4l inhibited MAO irreversibly in a competitive manner.
These results suggested that substitutions on quinazolinone
hydrazone prevent the interaction of the molecule with the com-
pact active site of MAO-B, but, contrarily, may enable the interac-
tion of the compounds with the relatively larger catalytic cavity of
MAO-A. Quite the opposite of these, compound 4i acted as a selec-
tive MAO-B inhibitor with a Ki value of 0.16 0.08 lM in a compet-
itive and reversible manner.
It seemed that the compounds bearing phenyl moiety at the 3
position and substituted phenyl at the 5 position (compounds
4g–4n) were more effective than those which carried substituted
phenyl at the 3 position and furyl/thienyl at the 5 position. The
main difference in the MAO-inhibitory activities of these com-
pounds seemed to originate from the replacement of the methyl,
bromo, ethoxy, and 3,4-dimethoxy on the aromatic moiety at the
5 position by a chloride atom or a methoxy group. The position
of chloride on the phenyl ring of the molecule seems to be respon-
sible for the MAO-inhibitory activity since compound 4i, which has
a chloride at m-position of the phenyl ring inhibits MAO-B potently
while p-chloride derivatives inhibit MAO-A (Tables 2 and 3).
o-Chloride substitution may block the compounds’ fit in the
narrow active cavity of the MAO-B molecule.
Figure 3. Lineweaver-Burk plot for the inhibition of rat liver MAQ-A by the
compound 5i (2–20
used as substrate in the concentration range of 50–500
independent experiments. *Equations corresponding to the lines at the first graph
2 = 0.3990 for 10
are y = 0.0044x + 0.012 inhibitor; y = 0.0026x + 0.0114
2 = 0.9968 for 10 M inhibitor; 0.0015x + 0.0103 R2 = 0.9968 for 12
l lM inhibitor;
lM) with 60 min of preincubation at 37 °C. p-Tyramine was
lM. Value are means of the
R
lM
Compounds 5a, 5b, and 5c carrying thienyl moiety and 5d, 5e,
and 5f carrying furyl moiety at fifth position of the molecule inhib-
ited MAO-B competitively and reversibly with the Ki values of
R
and 0.007 ꢁ 0.0106 R2 = 0.9950 for control. **Second graph represents the plot of the
slope of reciprocal plot.
Table 3
Calculated and experimental Ki values for the inhibition of rat liver MAO isoforms of the selected quinazoline derivatives
Compound Experimental Ki values*
(l
M) Calculated Ki values for MAO-A (lM) Calculated Ki values for MAO-B (lM) Inhibition type
4h
4i
4k
5e
5f
5g
5h
5i
5j
5k
5l
2
1.05 0.95 (for MAO-A)
0.16 0.08 (for MAO-B)
1.15 0.10 (for MAO-A)
9.25 0.7 (for MAO-B)
1.48 0.22 (for MAO-B)
9.90 0.80 (for MAO-A)
2.70 0.20 (for MAO-A)
0.90 0.07 (for MAO-A)
2.56 0.31 (for MAO-A)
1.03 0.10 (for MAO-A)
1.17 0.19 (for MAO-A)
0.088
0.020
0.045
6.41
0.94
37.31
2.12
0.79
2.5
0.25
0.57
2.45
0.36 (nM)
2.57
3.74
0.98
79.96
58.53
0.96
12.6
10.33
583
Selective for MAO-A competitive reversible
Selective for MAO-B competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-B competitive reversible
Selective for MAO-B competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-A competitive reversible
Selective for MAO-B competitive irreversible
30.18 1.97 (for MAO-A)
18.58 1.25 (for MAO-B)
48.63
23.78
39.89
19.08
3
*
Ki values were determined from the kinetic experiments in which p-tyramine was used at 500
M to determine the isoenzymes A and B. Newly synthesized compounds and the known inhibitors were preincubated with the homogenates
for 60 min at 37 °C. Each value represents means SEM of three independent experiments.
lM to measure MAO-A and 2.5 mM to measure MAO-B. Pargyline or
clorgyline were added at 0.50
l

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
681
2.4. Molecular docking studies
hydrogen of Tyr435 (1.94 Å). Cys172 (a distance of 2.90 Å), Leu171
(a distance of 2.59 Å), Tyr326 (a distance of 2.51 Å), and Ile199 (a
distance of 3.54 Å) are the other close contact residues in the active
site. Varying degrees of electrostatic and van der Waals interac-
tions may contribute to the binding and stabilization of 4k with
MAO-A at almost two order of magnitude better than MAO-B.
The binding mode of inhibitor 5e within the MAO-A cavity is
shown in Figure 6a. The chlorophenyl group of 5e was sandwiched
between Tyr444 (4.35 Å), Tyr407 (5.50 Å), and FAD (4.04 Å). Ile335,
Gln215, Val210, and Cys323 are the other residues in the active
site, however, they are not contributing much to the binding and
stabilization of 5e.
Figure 6b shows the optimal binding mode of 5e in the active
site of the MAO-B. The chlorophenyl group of 5e was inserted
much further into the hydrophobic cage, lined with Tyr398
(4.20 Å) and Tyr435 (4.16 Å), than that of MAO-A. Gln206
(4.10 Å) and Tyr326 (3.56 Å) are interacting with N2 nitrogen of
the pyrazole ring. The other important interactions between 5e
and the side chains of Ile199 (2.28 Å) and Thy326 (3.56 Å) have
considerable effect on the inhibitions of this compound. The effec-
tive binding mode of 5e with MAO-B makes this compound a
better MAO-B inhibitor than MAO-A.
Figure 7a shows the binding modes of compound 5i (a) into the
MAO-A binding cavity and (b) into the MAO-B binding cavity. A
close look at the docking results of compound 5i in complex with
MAO-A shows that its chlorophenyl group at the 3 position of
the pyrazole ring complexed with MAO-A shows that its chloro-
phenyl group at 3 position of the pyrazole ring inserted between
Tyr407, Tyr444, and the FAD hydrophobic region. It was seen that
the Cl atom of the inhibitor is making important van der Waals
interactions with the hydroxyl group of the side chain of Tyr407
(3.72 Å), Tyr444 (6.62 Å), and FAD (3.55 Å). Hydroxyl group of
Glu216 is interacting very closely with the N2 nitrogen of the pyr-
azole ring (2.31 Å). The phenyl moiety of the Phe352 is in close
proximity (2.95 Å) to chlorophenyl group at 3 position of the pyr-
In order to gain more insight into the binding mode and obtain
additional validations for a few unexpected results, molecular
docking studies were also performed for the compounds ( 4i, 4k,
5e, 5i, 5l, 13, and 14) experimentally tested. The calculated inhibi-
tion constants (Ki) as well as their experimental values for each
enzyme–inhibitor complex are shown in Table 3. In general, the
results obtained computationally are in good agreement with the
experimental values. All compounds experimentally tested were
found to be MAO-A selective except compounds 4i, 5e, 5f, and 14
which were found to be MAO-B selective. These results were sup-
ported by the computational modeling studies. The exception to
this was compound 2 which showed slightly higher MAO-B activ-
ity. The compound 5f showed the same potency toward both MAO-
A and MAO-B. In order to see the binding pose of these types of
compounds in detail five representative ligands 4i, 4k, 5e, 5i, and
5l were chosen.
Figure 4a–c shows the binding mode of compound 4i in the
MAO-B binding cavity. To date 4i shows the highest MAO-B
potency (Ki = 363 pmol) among the compounds tested in this
study. Analysis of the docking results of compound 4i in complex
with MAO-B revealed that the 4(3H)-quinazolinone ring system
of the ligand was inserted into the hydrophobic aromatic cage sur-
rounded by FAD, Tyr435, and Tyr398. The inhibitor snugly fits the
active site making various close contacts with the active site resi-
dues. One important interaction between 4i and MAO-B indicates
a strong stabilizing hydrogen bond between the azo group hydro-
gen next to quinazolinone ring and the backbone carbonyl of Ile
199 [1.76 Å Fig. 4a and c].
The lack of this hydrogen bond between 4i and MAO-A makes
this inhibitor 56-fold more selective toward MAO-B as a result of
this computational work. The 4(3H)-quinazolinone ring of the
inhibitor makes some important van der Waals interactions with
FAD (2.43 Å), Tyr435 (2.61 Å), Tyr398 (3.97 Å), Cys172 (2.61 and
3.23 Å). Gln206 plays an important role in this inhibition, contrib-
uting two important interactions (2.64 Å between carbonyl of the
Gln206 and N1 nitrogen of the 4(3H)-quinazolinone ring and
2.87 Å between the azo group of the ligand and side chain of
Gln206). Phe343 (4.15 Å), Tyr326 (3.28 Å), Leu171 (3.48 Å),
Phe168 (3.28 Å), and Ile199 (3.03 Å) are the other residues making
close interactions resulting high inhibition potency (Fig. 4a and b).
On the other hand 4i is oriented differently in the active site of
MAO-A than that of MAO-B in Figure 4d and e. The 4(3H)-quinaz-
olinone ring system of the ligand is not sandwiched between
Tyr407 (3.67 Å) and Tyr444 (3.11 Å) and it is not as close to FAD
azole ring making
p–p interaction. The 4(3H)-quinazolinone ring
system of the ligand was positioned in the entrance cavity making
some van der Waals interactions with Phe209 (4.05 Å), Cys323
(2.90 Å), and Ile355 (3.63 Å).
Contrary to this pose, the same ligand was oriented in MAO-B in
an upside down position in Figure 7b. Its 4(3H)-quinazolinone ring
inserted into the ‘aromatic cage’ lined with Tyr398 (3.43 Å), Tyr435
(5.12 Å), and FAD. Phe343 (3.38 Å), Gln206 (3.80 Å), Ile199
(2.26 Å), Try326 (3.80 Å), and Ile316 (4.24 Å) are the surrounding
residues of pyrazole ring. The better binding mode of 5i with
MAO-A explains the observation of the slightly higher MAO-A
inhibitory potency of this compound.
(2.79 Å) like in MAO-B causing low potency (Ki = 0.020
lM). This
value is much lower potency than that of 4i in MAO-B
(Ki = 0.36 nM). The other contributing van der Waals interactions
are between the residues of Phe352 (3.04 Å), Asn181 (4.72 Å),
Cys323 (3.37 Å), and Val210 (3.06 Å) and the various atoms of
the inhibitor shown in Figure 4d and e.
Figure 8a shows the binding modes of compound 5l into the
MAO-A binding cavity. Analysis of the docking results of com-
pound 5l complexed with MAO-A reveals that its 4(3H)-quinaz-
olinone ring was oriented among Tyr407 (2.49 Å), Phe352
(3.05 Å), side chains, and FAD (2.72 Å). The hydroxyl moiety of
Glu216 and N1 nitrogen of the pyrazole ring is also in close
proximity (2.88 Å). Ser209 (2.92 Å), Cys323 (3.15 Å), and Ile335
(3.41 Å) are the other favorable interactions stabilizing 5l in
the MAO-A cavity.
The binding mode of inhibitor 5l within the MAO-B cavity is
shown in Figure 8b. Contrary to most of the docking modes, the
4(3H)-quinazolinone moiety was not stacked between Tyr 398
and Tyr435. The aromatic ring of 4(3H)-quinazolinone is vertically
oriented away from these two side chains and is much closer to the
Tyr398 side chain (3.10 Å) than Tyr435 (5.13 Å). It was seen that
the Gln206 (3.98 Å), Ile199 (2.17 Å) are the other important inter-
actions with 5l. The better binding mode of 5l with MAO-A
explains the observation of the much higher MAO-A inhibitory
potency of this compound than that of MAO-B.
The binding mode of the compound 4k with MAO-A observed at
the end of docking simulation was shown in Figure 5a.
It is interesting to see that the phenyl moiety in 4k was sand-
wiched between Tyr407 (a distance of 3.43 Å) and Tyr444 (a dis-
tance of 3.58 Å). The methoxy phenyl group is in close proximity
with Glu216 (a distance of 2.79 Å). The other important van der
Waals interactions between the 4k and the various residues are
Phe352 (a distance of 3.10 Å), Ile355 (a distance of 3.54 Å),
Phe208 (a distance of 3.794.85 Å) and Cys323 (a distance of
3.12 Å). In Figure 5b, the docked mode of compound 4k with
MAO-B was shown.
The 4(3H)-quinazolinone ring was oriented across Tyr398 and
Tyr435 making one important hydrogen bond between the car-
bonyl group at the 4 position of 4(3H)-quinazolinone and hydroxyl

682
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
Figure 4. (a) Compound 4i in the active site- of MAO-B. The shaded volume shows the van der Waals volume of the inhibitor 4i. The hydrogen bond distance was indicated as
1.76 Å. Figure was generated by Using AutoDock Tools 1.5.1 (Ref. 68). (b) Binding model of 4i in MAO-B active site. (c) Inhibitor 4i in the active site of MAO-B. Here, only the
hydrogen bonding residues were shown for clearness. (d) Compound 4i in the active site of MAO-A. The shaded volume shows the van der Waals volume of the inhibitor 41.
Figure was generated by using AutoDock Tools 1.5.1 (Ref. 68). (e) Binding model of 4i in MAO-1A active site.
2.5. Pharmacology
Although the use of monoamine oxidase inhibitors (MAOIs) for
patients with depressive disorders is well established,46,47 most of
the newly synthesized compounds (4a, 4c–4f, and 5a–5f) which
carry furyl, thienyl group on C-5 did not show a marked antide-
pressant activity in Porsolt’s forced swimming test in contrast to
previously synthesized compounds carrying phenyl groups at C-5
of the 2-pyrazoline ring where the activity observed may be mainly
Due to the lipophilic character of the compounds, pharmacolog-
ical activity tests were directed to assess the various effects of these
compounds on the central nervous system. The effects of the
compounds on anxiety and depression were evaluated by Porsolt’s
forced swimming test44 and plus-maze,45 respectively (Table 4).

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
683
Figure 5. (a) Binding model of 4k in MAO-A active site. (b) Binding model of 4k in MAO-B active site.
Figure 6. (a) Binding model of 5e in MAO-A active site. (b) Binding model of 5e in MAO-B active site.
based on the substituent effect (4b, 4g, 4i, 4j, 4k, 4l, and 5g–5l).
These active compounds decreased the duration of immobility.
The activities of 5g and 5i are more than that of moclobemide;
the others are as active as moclobemide.
In this study the use of furyl and thienyl rings instead of phenyl
moiety gives us the opportunity to discuss the ring effect which
brings about sterical and electronic differences in the molecules
and it seems that these rings do not contribute to antidepressant
activity. But this result may also be related to the test method used.
The application of the modified behavioral despair test method48 is
our future aim in order to confirm these results.
plus-maze test to get additional evidence for the elevation level
of cathechol amines by the synthesized compounds. The experi-
mental data have shown that most of the new and previously syn-
thesized compounds possessed no anxiolytic activity except 4b,
4g, 4n, 5a, 5d, 5h, and 5j. Because these compounds (4b, 4g,
4n, 5a, 5d, 5h, and 5j) decreased the time spent in the closed
arm. But 5b seems an anxiogenic agent because of the decrease
of the number of the entries and insignificant increase of the time
spent in the closed arm.
Despite the clinical efficacy of MAOIs in some anxiety disor-
ders,51 it was reported that that these compounds do not usually
show anxiolytic-like effects in experimental models of anxi-
ety.52,53 However, it was also reported that the MAOIs such as
befloxatone and moclobemide exhibited anxiolytic-like effects
in the elevated plus-maze in rats and the light/dark choice test
Although there is evidence of a direct relationship between the
plasma concentration of MAO and the antidepressant response of
MAOIs49,50 a precise mechanism underlying the anxiolytic effects
of MAOIs has not been well defined. Nevertheless we applied the

684
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
Figure 7. (a) Binding model of 5i in MAO-A active site. (b) Binding model of 5i in MAO-B active site.
Figure 8. (a) Binding model of 5l in MAO-A active site. (b) Binding model of 5l in MAO-B active site.
in mice.54,55 The reasons for this inconsistency on drug effects
are not yet clear since it has been suggested that classical animal
models of anxiety are insensitive to the action of these
compounds.55
The comparison of in vitro and in vivo test results shows that
previously synthesized compounds ( 4g, 4i–4l, and 5g–5l) seem
to be active in the applied pharmacological activity tests in parallel
to MAO-A inhibition. The lack of in vivo activity of compounds
4a–4f/5a–5f might be related to their MAO-B inhibition. As a result
these new synthetic compounds proved to be reversible, potent,
and selective inhibitors of monoamine oxidase-B rather than of
monoamine oxidase-A, and are promising candidates to further
advance drug discovery efforts.
The results obtained in the monoamine oxidase inhibitory study
show that compound 4i is able to differentially inhibit MAO-B, in
preference to MAO-A, a behavior that can be rationalized on the
basis of the structural differences of MAO-B and MAO-A active
sites. The binding pose resulting from the docking studies of these
compounds revealed the detailed structural information between
the active site residues and the inhibitors. Nevertheless, many
experimental studies have shown that MAOs isolated from differ-
ent sources/species (rat, human or mouse liver, etc.) exhibit differ-
ent binding modes to the MAO inhibitors possibly resulting from
the differences in the volume and shape of the active sites of the
3. Conclusion
It is worth noting that the main difference in the MAO-inhibi-
tory activities of pyrazole type compounds 5a–5l was found when
the 5-membered heterocycle on the pyrazole ring is replaced by a
6-membered heterocycle. This replacement appeared to be respon-
sible for the remarkable differences in the inhibition activity
mainly against MAO-A/MAO-B isoforms, respectively. Compounds
5a–5f bearing a 5-membered ring at 5 the position showed
MAO-B inhibition while compounds 5g–5l carrying a 6-membered
ring at the same position were inhibiting MAO-A. From a careful
examination of the reported data, it can be emphasized that some
substituents, in particular of the 3- and 5-phenyl groups, have a
strong influence on A-selectivity.

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
685
Table 4
Antidepressant and anxiolytic activities of newly synthesized compound
Compounda
n
Anxiolytic activity
Antidepressant activity
Time spent in the closed arm SDa
Number of the entriesb
X
SD
Duration of immobility (s) mean SDc
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
7
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
7
204.71 55.09
162.17 36.83*
206.29 23.51
238.67 20.85
219.50 15.73
237.25 17.45
188.14 31.61*
244.50 62.75
248.50 12.64
208.57 20.60*
204.00 39.80
226.00 29.39
228.63 11.55
191.00 49.39*
284.20 38.08
246.87 31.81
216.12 47.69
225.38 51.50
218.71 45.97
218.88 35.83
209.63 45.98*
222.27 38.58
154.75 76.5*
218.25 93.51
178.25 82.77*
162.43 35.72
230.00 46.83
254.15 31.77
250.72 17.20
5.57 2.94
8.50 2.26
8.00 2.31
5.14 1.95
6.75 1.83
4.63 1.60
5.14 2.61
2.29 1.11*
5.13 1.64
9.00 0.82*
4.86 2.27
5.57 2.76
6.25 2.05
5.37 2.56*
2.51 1.52
3.15 1.55
6.37 2.93*
4.50 3.67
3.87 1.73
5.75 2.18*
5.63 1.19*
4.37 1.84
4.12 2.69
3.75 2.37
3.87 1.72
5.37 2.58*
3.25 1.90
3.00 1.41
6.15 2.15
65.57 15.90
50.39 12.90*
49.29 11.71
82.43 29.44
67.88 12.12
80.38 20.69
74.14 32.62
61.83 22.82
61.13 16.56
64.00 28.31
74.00 27.57
69.00 15.98
67.75 9.75
Control (DMSO/water 1:4)
4g
4h
4i
4j
4k
4l
4m
4n
5g
5h
5i
5j
5k
5l
55.43 21.55*
125.86 38.65
54.57 22.58*
79.25 30.18*
86.75 25.77*
65.57 12.51*
90.23 26.58
130.57 56.63
27.00 22.12**
42.37 17.58*
26.25 19.12**
43.25 26.54*
58.00 25.10*
65.12 30.78*
122.00 37.84
54.30 9.73*
Control (DMSO/water 1:4)
Moclobemide (5 mg/kg)
a
The amount of time spent in closed arms is decreased in anxiolytic agents and decreased in anxiogenic agents.
The number of entries is increased in anxiolytic agents and is decreased in anxiogenic agents.
The reduction time of immobility time in the FST is an established way to evaluate effectiveness of potential antidepressant drugs.
p < 0.05.
p < 0.01.
b
c
*
**
MAOs isolated.14 Thus, further docking studies with the different
models are needed. We think that the experimental data herein
should be the most reliable unless these models were to be fully
described. However, compound 4i may be considered a promising
lead in the treatment of Parkinson’s disease and worth testing in
future.
4.1.1. Synthesis of 3-methyl-2(1H)-thioxo-4(3H)-quinazolinone
(2)
3-Methyl-2(1H)-thioxo-4(3H)-quinazolinone was synthesized
as a result of the reaction of anthranilic acid with methyl isothio-
cyanate according to the method reported earlier (mp 261 °C).56
Overall, the results of this work will be useful in the rational
design of novel selective and potent MAO inhibitors.
4.1.2. 2-Hydrazino-3-methyl-4(3H)-quinazolinone (3)
2-Hydrazino-3-methyl-4(3H)-quinazolinone was synthesized
by refluxing of 3-methyl-2(1H)-thioxo-4(3H)-quinazolinone and
hydrazine hydrate in 2-propanol for 4 h as published before (mp
256 °C).57
4. Experimental
4.1. Chemistry
4.1.3. Preparation of 1,3-diphenyl-2-propen-1-ones (chalcones)
(general procedure)
Chalcone derivatives were synthesized by condensing aceto-
phenone or 4-substituted acetophenones with thiophen aldehyde
or furfural previously according to the methods given.58
All chemicals were obtained from Aldrich Chemical Co.
(Steinheim, Germany). Melting points were determined through a
Thomas Hoover capillary melting point apparatus and are uncor-
rected. Infrared (IR) spectra were obtained with a Bruker Vector 22
IR (Opus Spectroscopic Software Version 2.0) spectrometer using
potassium bromide plates and the results were expressed in wave
number (cmꢀ1). 1H Nuclear magnetic resonance spectra were
scanned on a Bruker 400 MHz UltraShield spectrometer or Bruker
AC 80 MHz NMR instrument using chloroform (CDCl3) as solvent.
Chemical shifts are expressed in d (parts per million) relative to tetra-
methylsilane. Splitting patterns are as follows: s, singlet; d, doublet;
m, multiplet; b, broad; dd (doublet in doublet). The mass spectra
were obtained with electron impact technique using a Direct Inser-
tion Probe and Agilent 5973-Network Mass Selective Dedector at
70 eV. Elemental analyses (C, H, N) were performed on Leco CHNS
932 analyzer. The elemental analysis results for C, H, N were in the
range of 0.4% of the theoretical value for compounds 5a–5f.
4.1.4. 2-(10-Substituted phenyl-30-heteroaryl-20-propenylidene)
hydrazine-3-methyl-4(3H)-quinazolinone derivatives (4)
A mixture of chalcones (0.01 mol) and 2-hydrazino-3-methyl-
4(3H)-quinazolinone (0.01) and 2 mL of acetic acid in n-propanol
(100 mL) was stirred under reflux for 4–7 h. After the reaction
was cooled to room temperature, the resulting residue was filtered,
washed with n-propanol and dried. Recrystallization of the crude
product from ethanol gave 4 as a yellow solid.
4.1.4.1. 2-[10-(4-Methylphenyl)-30-thienyl-20-propenylidene]
hydrazine-3-methyl-4(3H)-quinazolinone (4a). 51%; mp 223
(chloroform–methanol); IR (KBr): 3366 (NH), 1678 (C@O),

686
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
1603 (C@C), 1495 cmꢀ1 (C@N); 1H NMR d (ppm): 2.2 (3H, s,
Ph–CH3), 3.6 (3H, s, N–CH3), 6.6 (1H, d, J = 13 Hz, 20-CH), 7.85
(1H, d, J = 13 Hz, 30-CH), 6.9–8.2 (13H, m, Arom-H); MS (70 eV,
EI): 400 (M+), 291 (100%), 284, 283, 119. Calculated for
C23H20N4OS (400.50): C, 68.98; H, 5.03; N, 13.99. Found: C,
68.72; H, 4.85; N, 13.77.
4.1.4.11. 2-[10-Phenyl-30-(4-methoxyphenyl)-20-propenylidene]
hydrazine-3-methyl-4(3H)-quinazolinone (4k). 65%; mp 160,
C25H22N4O; Ref. 59.
4.1.4.12. 2-[10-Phenyl-30-(4-bromophenyl)-20-propenylidene]
hydrazine-3-methyl)-4(3H)-quinazolinone (4l). 61%; mp
217, C25H22N4O2; Ref. 59.
4.1.4.2. 2-[10-(4-Chlorophenyl)-30-thienyl-20-propenylidene]hy-
drazine-3-methyl)-4(3H)-quinazolinone (4b). 79%; mp 210
(chloroform–methanol); IR (KBr): 3366 (NH), 1679 (C@O),1604
(C@C), 1492 cmꢀ1 (C@N); 1H NMR d (80 MHz) (ppm): 3.6 (3H, s,
N–CH3), 6.9–8.2 (13H, m, Arom-H); MS (70 eV, EI): 422 (M++2),
420 (M+), 283, 197 (100%), 159, 119. Calculated for C22H17ClN4OS
(420.92): C,62.78; H,4.07; N,13.31. Found: C, 62.92; H, 4.32; N,
13.11 .
4.1.4.13. 2-[10-Phenyl-30-(4-ethoxyphenyl)-20-propenylidene]
hydrazine-3-methyl-4(3H)-quinazolinone (4m). 50%; mp
180, C26H24N4O3; Ref. 59.
4.1.4.14. 2-[10-Phenyl-30-(3,4-dimethoxyphenyl)-20-propenylid-
ene]hydrazine-3-methyl-4(3H)-quinazolinone (4n). 47%; mp
170, C26H24N4O3; Ref. 59.
4.1.4.3. 2-[10-(4-Methoxyphenyl)-30-thienyl-20-propenylidene]
hydrazine-3-methyl-4(3H)-quinazolinone (4c). 55%; mp 188
(chloroform–methanol); IR (KBr): 3342 (NH), 1684 (C@O), 1586
(C@C), 1503 cmꢀ1 (C@N); 1H NMR d (ppm): 3.84 (3H, s, N–CH3),
3.89 (3H, s, Ph–OCH3), 7 (1H, d, J = 16 Hz, 20-CH), 7.6 (1H, d,
J = 16 Hz, 30-CH), 6.9–8.2 (11H, m, Arom-H); MS (70 eV, EI): 416
(M+,100%), 283, 159, 119. Calculated for C23H20N4O2S (416.50):
C, 66.33; H, 4.84; N, 13.45. Found: C, 66.46; H, 5.24; N, 13.22.
4.1.5. 2-(3-Substituted phenyl-50-heteroaryl-20-pyrazoline-1-yl)
-3-methyl-4(3H)-quinazolinone derivatives (5)
A
solution of 2-(10-substituted phenyl-30-heteroaryl-20-pro-
penylidene)hydrazine-3-methyl-4(3H)-quinazolinones
4
(0.01 mol) in glacial acetic acid was stirred under reflux for 72 h.
The resulting solution was poured into ice-water, neutralized with
concentrated NaOH solution, and extracted with ethyl acetate. The
organic phase was dried over magnesium sulfate, filtered, and
evaporated to dryness to yield the oily residue. The crude product
was further purified by chromatography on a silica gel column
(elution with chloroform) followed by crystallization from diethyl
ether to give 5 as a white solid.
4.1.4.4. 2-[10-(4-Methylphenyl)-30-furyl-20-propenylidene]hydr-
azine-3-methyl-4(3H)-quinazolinone (4d). 56%; mp 199 (chlo-
roform–methanol); IR (KBr): 3257 (NH), 1678 (C@O),1584 (C@C),
1486 cmꢀ1 (C@N); 1H NMR d (ppm): 2.4 (3H, s, Ph–CH3), 3,6 (3H,
s, N–CH3), 6.7 (1H, d, J = 16 Hz, 20-CH), 7.9 (1H, d, J = 16 Hz, 30-
CH), 6.4–8.2 (11H, m, Arom-H); MS (70 eV, EI): 384 (M+), 291,
192 (100%), 159, 119. Calculated for C23H20N4O2 (384.44): C,
71.86; H, 5.24; N, 14.57. Found: C, 72.05; H, 5.44; N, 14.34.
4.1.5.1. 2-[(3-(4-Methylphenyl)-50-thienyl-20-pyrazoline-1-yl]-
3-methyl-4(3H)-quinazolinone (5a). 94%; mp 201 (chloro-
form–methanol); IR (KBr): 1671 (C@O), 1563 (C@C), 1474 (C@N);
1H NMR d (ppm): 2.7 (3H, s, Ph–CH3), 3.6 (1H, t, HA), 4.05 (4H, m, HB
and N–CH3), 6.5 (1H, t, HX), 7.2–8.5 (11H, m, Arom-H); MS (70 eV,
EI): 400, 282, 268 (100%), 250. Calculated for C23H20N4OS (400.50):
C, 68.98; H, 5.03; N, 13.99. Found: C, 68.86; H, 4.86; N, 13.98.
4.1.4.5. 2-[10-(4-Chlorophenyl)-30-furyl-20-propenylidene]hydr-
azine-3-methyl)-4(3H)-quinazolinone (4e). 64%; mp 190
(chloroform–methanol); IR (KBr): 3325 (NH), 1677 (C@O), 1608
(C@C), 1489 cmꢀ1 (C@N); 1H NMR d (ppm): 3.8 (3H, s, N–CH3),
6.8 (1H, d, J = 14 Hz, 20-CH), 7.2–8.5 (10H, m, 30-CH, Arom-H).;
MS (70 eV, EI): 406 (M+2), 404, 267, 252 (100%), 159, 119. Calcu-
lated for C22H17ClN4O2 (404.86): C, 65.27; H, 4.23; N, 13.84.
Found: C, 65.43; H, 4.32; N, 14.10.
4.1.5.2. 2-[(3-(4-Chlorophenyl)-50-thienyl-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5b). 93%; mp 191 (chloroform–
methanol); IR (KBr): 1671 (C@O), 1563 (C@C), 1473 (C@N); 1H
NMR d (80 MHz) (ppm): 3.4 (1H, s, HA), 3.6 (1H, s, HB), 3.8 (1H, s,
N–CH3), 6.5 (1H, m, HX), 7.1–8.4 (11H, m, Arom-H); MS (70 eV,
EI): 422 (M++2), 420 (M+), 282, 268 (100%), 250. Calculated for
C22H17ClN4OS (420.92): C, 62.78; H, 4.07; N, 13.31. Found: C,
62.50; H, 3.73; N, 13.26 .
4.1.4.6. 2-[10-(4-Methoxyphenyl)-30-furyl-20-propenylidene]hyd-
razine-3-methyl-4(3H)-quinazolinone (4f). 72%; mp 178 (chloro-
form–methanol); IR (KBr): 3337 (NH), 1670 (C@O), 1586 (C@C), 1505
(C@N); 1H NMR d (ppm): 3.6 (3H, s, N–CH3), 3.87 (3H,s, Ph–OCH3), 6.7
(1H, d, J = 14 Hz, 20-CH), 7.9 (1H, d, J = 14 Hz, 30-CH), 6.4–8.1 (11H, m,
Arom-H), 9.4 (1H, br s, NH); MS (70 eV, EI): 400 (M+), 225 (100%), 169,
119. Calculated for C23H20N4O3 (400.44): C, 68.99; H, 5.03; N, 13.99.
Found: C, 68.86; H, 4.86; N, 13.98.
4.1.5.3. 2-[(3-(4-Methoxyphenyl)-50-thienyl-20-pyrazoline-1-yl]-
3-methyl-4(3H)-quinazolinone (5c). 86%; mp 100 (chloro-
form–methanol); IR (KBr): 1669 (C@O), 1593 (C@C), 1471 (C@N);
1H NMR d (ppm): 3.4 (1H, t, HA), 3.8 (1H, m, HB and N–CH3), 3.9
(3H, s, Ph–OCH3), 6.2 (1H, t, HX), 6.94–8.22 (11H, m, Arom-H);
MS (70 eV, EI): 416 (M+), 418:(M+2), 282, 268 (100%), 250. Calcu-
lated for C23H20N4O2S (416.50): C, 66.33; H, 4.84; N, 13.45. Found:
C, 66.33; H, 5.73; N, 13.10.
4.1.4.7. 2-(1,30-Diphenyl-20-propenylidene)hydrazine-3-methyl
-4(3H)-quinazolinone (4g). 40%; mp 173, C24H20N4O; Ref. 59.
4.1.4.8. 2-[10-Phenyl-30-(4-chlorophenyl)-20-propenylidene]hyd-
razine-3-methyl-4(3H)-quinazolinone (4h). 60%; mp 153,
C24H19N4OCl; Ref. 59.
4.1.5.4. 2-[(3-(4-Methylphenyl)-50-furyl-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5d). 83%; mp 151 (chloro-
form–methanol); IR (KBr): 1672 (C@O), 1561 (C@C), 1474
(C@N); 1H NMR d (80 MHz) (ppm): 2.3 (3H, s, Ph–CH3), 3.3 (1H,
s, HA), 3.4 (1H, s, HB), 3.60 (3H, s, N–CH3), 6 (1H, m, HX), 6.8–
8.1 (11H, m, Arom-H); MS (70 eV, EI): 384 (M+), 252 (100%),
119. Calculated for C23H20N4O2 (384.44): C, 71.86; H, 5.24; N,
14.57. Found: C, 72.20; H, 5.72; N, 14.46.
4.1.4.9. 2-[10-Phenyl-30-(3-chlorophenyl)-20-propenylidene]hyd-
razine-3-methyl-4(3H)-quinazolinone (4i). 46%; mp 175,
C24H19N4OCl; Ref. 59.
4.1.4.10. 2-[10-Phenyl-30-(4-methylphenyl)-20-propenylidene]
hydrazine-3-methyl)-4(3H)-quinazolinone (4j). 91%; mp
181, C24H19N4OBr; Ref. 59.
4.1.5.5. 2-[(3-(4-Chlorophenyl)-50-furyl-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5e). 98%; mp 195 (chloro-

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
687
form–methanol); IR (KBr): 1671 (C@O), 1563 (C@C), 1472 (C@N);
1H NMR d (ppm): 3.4 (1H, t, HA), 3.8 (4H, m, HB and N–CH3), 6.4
(1H, t, HX), 7.0–8.1 (11H, m, Arom-H) 406 (M+2), 404 (M+), 268,
252 (100%), 215, 119; MS (70 eV, EI): 406 (M+2), 404 (M+), 268,
252 (100%), 215, 119. Calculated for C22H17ClN4O2 (404.86): C,
65.27; H, 4.23; N, 13.84. Found: C, 64.89; H, 4.19; N, 13.47.
experimentation. MAO was purified from the rat liver according
to the method of Holt with some modifications.60 Liver tissue
was homogenized 1:40 (w/v) in 0.3 M sucrose. Following centrifu-
gation at 1000g for 10 min, the supernatant was centrifuged at
10,000g for 30 min to obtain crude mitochondrial pellet.
The pellet was incubated with CHAPS of 1% at 37 °C for 60 min.
and centrifuged at 1000g for 15 min. Pellet was resuspended in
0.3 M sucrose and was layered onto 1.2 M sucrose, centrifuged at
53,000g for 2 h and resuspended in potassium phosphate buffer,
pH 7.4, kept at ꢀ70 °C until used.
4.1.5.6. 2-[(3-(4-Methoxyphenyl)-50-furyl-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5f). 85%; mp 168 (chloroform–
methanol); IR (KBr): 1686 (C@O), 1596 (C@C), 1471 (C@N); 1H
NMR d (ppm): 3.4 (1H, s, HA), 3.7 (1H, s, HB), 3.76 (3H, s, N–CH3),
3.84 (3H, s, Ph–OCH3), 6.1 (1H, t, HX), 6.3–8.2 (11H, m, Arom-H);
MS (70 eV, EI): 400 (M+), 267, 252 (100%). Calculated for
C23H20N4O3 (400.44): C, 68.99; H, 5.03; N, 13.99. Found: C, 68.86;
H, 4.86; N, 13.98.
4.3.2. Measurement of MAO activity
Total MAO activity was measured spectrophotometrically
according to the method of Holt.60 The assay mixture contained a
chromogenic solution consisted of 1 mM vanillic acid, 500 lM 4-
aminoantipyrine, and 4 U mLꢀ1 peroxidase type II in 0.2 M potas-
sium phosphate buffer, pH 7.6. The assay mixture contained
4.1.5.7. 2-(3,50-Diphenyl-20-pyrazoline-1-yl)-3-methyl-4(3H)-
quinazolinone (5g). 59%; mp 165, C24H20N4O; Ref. 59.
167
ll chromogenic solution, 667
ll substrate solution (500 lM
p-tyramine), and 133
mixture was preincubated at 37 °C for 10 min before the addition
of enzyme. Reaction was initiated by adding the homogenate
(100 ll), and increase in absorbance was monitored at 498 nm at
l
l potassium phosphate buffer, pH 7.6. The
4.1.5.8. 2-[(3-Phenyl-50-(4-chlorophenyl)-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5h). 79%; mp 125, C24H19N4OCl;
Ref. 59.
37 °C for 60 min. The molar absorption coefficient of
4.1.5.9. 2-[(3-Phenyl-50-(3-chlorophenyl)-20-pyrazoline-1-yl]-3-
methyl-4(3H)-quinazolinone (5i). 99%; mp 149, C24H19N4OCl;
Ref. 59.
4654 Mꢀ1 cmꢀ1 was used to calculate the initial velocity of the
reaction. The results were expressed as nmol hꢀ1 mgꢀ1
.
4.3.3. Selective measurement of MAO-A and MAO-B activities
Homogenates were incubated with the substrate p-tyramine
(500 lM to measure MAO-A and 2.5 mM to measure MAO-B)
4.1.5.10. 2-[(3-Phenyl-50-(4-methylphenyl)-20-pyrazoline-1-yl]-
3-methyl)-4(3H)-quinazolinone (5j). 98%; mp 170, C26H24N4OBr;
Ref. 59.
following the inhibition of one of the MAO isoforms with selec-
tive inhibitors. Aqueous solutions of clorgyline or pargyline
4.1.5.11. 2-[(3-Phenyl-50-(4-methoxyphenyl)-20-pyrazoline-1-yl]-
3-methyl-4(3H)-quinazolinone (5k). 81%; mp 120, C25H22N4O;
Ref. 59.
(50
homogenates at the ratio of 1:100 (v/v), yielding the final inhib-
itor concentrations of 0.50 M. Homogenates were incubated
lM), as selective MAO-A and -B inhibitor, were added to
l
with these inhibitors at 37 °C for 60 min prior to activity mea-
surement. After incubation of homogenates with selective inhib-
itors, total MAO activity was determined by the method
described above.
4.1.5.12. 2-[(3-Phenyl-50-(4-bromophenyl)-20-pyrazoline-1-yl]-
3-methyl-4(3H)-quinazolinone (5l). 66%; mp 140, C25H22N4O2;
Ref. 59.
4.2. Single crystal X-ray crystallographic data of 5a
4.3.4. Analysis of the kinetic data
Newly synthesized compounds were dissolved in dimethyl sulf-
oxide (DMSO), maximum concentration 1% and used in the con-
The data collection was performed on a CAD-4 diffractometer
employing graphite-monochromated CuK
a
radiation (k = 1.54184
centration range of 1–1000 lM. Inhibitors were incubated with
Å). Crystallographic and refinement parameters are summarized in
Table 1. Three standard reflections were measured every 2 h. The
structure was solved by direct methods. The refinement was made
with anisotropic temperature factors for all nonhydrogen atoms.
the purified MAO at 37 °C for 0, 10, 20, 40, and 60 min prior to add-
ing to the assay mixture. Reversibility of the inhibition of the
enzyme by novel compounds was assessed by dialysis performed
over 24 h at 4 °C relative to a potassium phosphate buffer, pH
7.6, capable of restoring 98–100% of the enzyme activity.
The hydrogen atoms were generated geometrically. An empirical
W
scan absorption correction was applied. Some selected bond lengths,
angles and torsion angles are listed in Figure 2.
Kinetic data for interaction of the enzyme with the compounds
were determined using Microsoft Excel package program. The ini-
tial rates were obtained at five different substrate concentrations
Crystallographic data (excluding structure factors) for com-
pound 5a reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publi-
cation number CCDC-690526. Copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: +44 (0) 1223 336033 or e-mail: deposit@
ccdc.cam.ac.uk).
(50–500 lM) in the absence and presence of four different inhibi-
tor concentrations. The slopes of the Lineweaver–Burk plots were
plotted versus the inhibitor concentration and the Ki value was
determined from the x-axis intercept as ꢀKi. Each Ki value is the
representative of single determination where the correlation coef-
ficient (R2) of the replot of the slopes versus the inhibitor concen-
trations was at least 0.98.
4.3. Biochemistry
IC50 values were determined from plots of residual activity per-
centage, calculated in relation to a sample of the enzyme treated
under the same conditions without inhibitor, versus inhibitor con-
centration [I].
All chemicals used were purchased from Sigma–Aldrich Co.
(Germany).
4.3.1. Isolation of MAO from rat liver homogenates
4.3.5. Protein determination
The Ethics Committee of Laboratory Animals at Hacettepe
Protein was determined according to the method of Bradford,61
in which bovine serum albumin was used as standard.
University,
Turkey
(2001/25-4),
approved
the
animal

688
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 675–689
4.4. Molecular docking
and injected to the animals intraperitoneally at 100 mg/kg doses
in approximately 0.1 mL volume 1 h before the test. 0.1 mL
DMSO/water (1:4) was given to the control animals. A plus-maze
test was applied for anxiolitic activity of the compounds.76
Antidepressant activity was tested by Porsolt forced swimming
test (behavioral despair test).77 Each animal was used for the plus-
maze at first and then for the forced swimming test. All experi-
ments for animal testing were approved by Osman Gazi University
School of Medicine Animal Use and Care Committee.
4.4.1. Protein setup
The crystal structures human monoamine oxidase-A (PDB entry
code: 2BXS, co-crystalized with the inhibitor clorgyline) and
human monoamine oxidase-B (PDB entry code: 1S3E co-crystal-
ized with the inhibitor 6-hydroxy-N-propargyl-1(R)-aminoindan)
were obtained from the Protein Data Bank (http://
www.rcsb.org).62–65 Studies were carried out on only one subunit
of the enzymes. The PDB files were edited and the b-chains were
removed together with their irreversible inhibitors.
4.5.1. Elevated plus-maze test
In order to relieve the crystal structure tension and to make the
protein available to use in the AutoDock docking simulation pro-
gram, all polar hydrogens were added with the GROMACS model-
ing package.66,67 The obtained structure was optimized in 400
steps of conjugate gradient minimization, employing the GRO-
MOS-87 force field. During minimization the heavy atoms were
kept fixed at their initial crystal coordinates, but added hydrogens
were made free to move. Minimization was performed under a
vacuum medium.
The elevated plus-maze is used to determine the mouse’s uncon-
ditioned response to a potentially dangerous environment and anx-
iety-related behavior is measured by the degree to which the mouse
avoids the unenclosed arms of the maze. The test is particularly use-
ful in testing the effects of anxiolytic and anxiogenic drugs.
It is a standard test of fear and anxiety in which the animal is
placed in the center of an elevated 4-arm maze in which 2 arms
are open 50 ꢁ 10 cm and 2 are enclosed 50 ꢁ 10 ꢁ 40 cm with an
open roof. The two open arms were opposite to each other. The
maze was elevated to a height of 50 cm. The measures indicated
in the procedure section were taken by two observers, sitting in
the same room with the maze.
Mice were placed in the center of the maze and the following
measures scored by two observers for 5 min. The number of times
the animal enters each of the arms and the time spent in each arm
is noted.76 Two indices of anxiety are obtained: the amount of time
spent in closed arms and the number of entries into the arms. The
test is rapid and to be sensitive to the effects of both anxiolytic and
anxiogenic agents, anxiolytic agents decreasing, and anxiogenic
agents increasing the amount of time spent in closed arms; anxio-
lytic agents increasing and anxiogenic agents decreasing the num-
ber of entries.78,79
The AutoDockTools (ADT),68 graphical user interface, program
was employed to setup the enzymes: all hydrogens were added,
Gasteiger69 charges were calculated and nonpolar hydrogens were
merged to carbon atoms. For macromolecules, generated pdbqt
files were saved.
4.4.2. Ligands
The 3D structures of ligand molecules were built, optimized
(PM3) level, and saved in mol2 format with the aid of the molecu-
lar modeling program Spartan (Wavefunction Inc.).70 These partial
charges of Mol2 files were further modified by using the ADT pack-
age (version 1.4.6) so that the charges of the nonpolar hydrogens
atoms assigned to the atom to which the hydrogen is attached.
The resulting files were saved as pdbqt files.
4.5.2. Forced swimming test
4.4.3. Molecular docking
Individual mice were forced to swim vertical plexiglass cylin-
ders (height: 25 cm, diameter 10 cm) containing 6 cm of water at
21–23 °C 1 h after a single ip injection. Mice were dropped into
the cylinder and left there for 6 min. An observer counted the total
amount of time that the mice spent immobile. Immobility was
defined as ‘floating motionless or making only those movements
necessary to keep its head above the water’.80 Because little immo-
bility is observed during the first 2 min the total duration of immo-
bility was measured during the last 4 min. The moused was judged
to be immobile whenever it remained floating passively in the
water in a slightly hunched but upright position, while its head
was just above the surface.77
AutoDock 4.01,71,72 was employed for all docking calculations.
The AutoDockTools program was used to generate the docking
input files. In all docking a grid box size of 80 ꢁ 80 ꢁ 80 points in
x, y, and z directions was built, and because the location of the
inhibitor in the complex was known,73,74 the maps were centered
on N5 atom of the flavin (FAD) in the catalytic site of the protein.
A grid spacing of 0.375 Å (approximately one forth of the length
of carbon–carbon covalent bond) and a distances-dependent func-
tion of the dielectric constant were used for the calculation of the
energetic map. Ten runs were generated by using Lamarckian
genetic algorithm searches. Default settings were used with an ini-
tial population of 50 randomly placed individuals, a maximum
number of 2.5 ꢁ 106 energy evaluations, and a maximum number
of 2.7 ꢁ 104 generations. A mutation rate of 0.02 and a crossover
rate of 0.8 were chosen. Results differing by less than 0.5 Å in posi-
tional root-mean-square deviation (RMSD) were clustered together
and the results of the most favorable free energy of binding were
selected as the resultant complex structures. All calculations were
carried out on an Mac OS X machine of intel core duo processor at
2 GHz with 1 GB of RAM. The resultant structure files were ana-
lyzed using VMD75 (Visual Molecular Dynamics) visualization
programs.
Statistical analysis results were expressed as means SD and
evaluated by Student’s t test.
Acknowledgment
This study was supported by the Hacettepe University Research
Fund (Project No. 07 D03 301 003).
References and notes
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Oxidase Inhibitors. In Developments of Drugs and Modern Medicines; Gorrod, J.
W., Gibson, G. G., Mitchard, M., Eds.; Part I. Drug Design; Ellis Horwood Ltd:
Chichester, 1986; p 40.
4.5. Pharmacology
Local bred albino mice of either sex weighing 20–25 g were
used in all experiments. They were housed eight per cage and kept
in a room with controlled temperature (20 2 °C) and 12 h light/
dark cycle. All animals were allowed ad libitum access to food
and water. All compounds were dissolved in DMSO/water (1:4)
3. Kalgutkar, A. S.; Castagnoli, N., Jr. Med. Res. Rev. 1995, 15, 325.
4. Johston, J. P. Biochem. Pharmacol. 1968, 17, 1285.
5. Knoll, J.; Magyar, K. Adv. Biochem. Psychophys. 1972, 5, 393.
6. O’Carroll, A.; Fowler, C. J.; Phillips, J. P.; Tobbia, I.; Tipton, K. F. N.-S. Arch.
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