Synthesis and molecular modeling of some novel hexahydroindazole derivatives as potent monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.07.033

A novel series of 2-thiocarbamoyl-2,3,4,5,6,7-hexahydro-1H-indazole and 2-substituted thiocarbamoyl-3,3a,4,5,6,7-hexahydro-2H-indazoles derivatives were synthesized and investigated for the ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). The target molecules were identified on the basis of satisfactory analytical and spectra data (IR, ¹H NMR, ¹³C NMR, ²D NMR, DEPT, EI-MASS techniques and elemental analysis). Synthesized compounds showed high activity against both the MAO-A (compounds 1d, 1e, 2c, 2d, 2e) and the MAO-B (compounds 1a, 1b, 1c, 2a, 2b) isoforms. In the discussion of the results, the influence of the structure on the biological activity of the prepared compounds was delineated. It was suggested that non-substituted and N-methyl/ethyl bearing compounds (except 2c) increased the inhibitory effect and selectivity toward MAO-B. The rest of the compounds, carrying N-allyl and N-phenyl, appeared to select the MAO-A isoform. The inhibition profile was found to be competitive and reversible for all compounds. A series of experimentally tested (1a-2e) compounds was docked computationally to the active site of the MAO-A and MAO-B isoenzyme. The autodock 4.01 program was employed to perform automated molecular docking. In order to see the detailed interactions of the docked poses of the model inhibitors compounds 1a, 1d, 1e and 2e were chosen because of their ability to reversibly inhibit the MAO-B and MAO-A and the availability of experimental inhibition data. 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. 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. Excellent to good correlations between the calculated and experimental K_i values were obtained. In the docking of the MAO-A complex, the trans configuration of compound 1e made various very close interactions with the residues lining the active site cavity these interactions were much better than those of the other compounds tested in this study. This tight binding observation may be responsible for the nanomolar inhibition of form of MAOA. However, it binds slightly weaker (experimental K_i = 1.23 μM) to MAO-B than to MAO-A (experimental K_i = 4.22 nM).

Full text of DOI:10.1016/j.bmc.2009.07.033

Bioorganic & Medicinal Chemistry 17 (2009) 6761–6772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and molecular modeling of some novel hexahydroindazole
derivatives as potent monoamine oxidase inhibitors
a
b
c
d
Nesrin Gökhan-Kelekçi a, , Ö. Özgün Sßimßsek , Aysße Ercan , Kemal Yelekçi , Z. Sibel Sßahin ,
*
Sßamil Isßık d, Gülberk Uçar b, A. Altan Bilgin a
a Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey
b Faculty of Pharmacy, Department of Biochemistry, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey
c
_
Kadir Has University, The Faculty of Arts and Sciences, 34080 Fatih-Istanbul, Turkey
d Ondokuz MayısUniversity, Faculty of Arts and Sciences,, Department of Physics, 55139 Kurupelit, Samsun, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 February 2009
Revised 8 July 2009
Accepted 16 July 2009
Available online 23 July 2009
A novel series of 2-thiocarbamoyl-2,3,4,5,6,7-hexahydro-1H-indazole and 2-substituted thiocarbamoyl-
3,3a,4,5,6,7-hexahydro-2H-indazoles derivatives were synthesized and investigated for the ability to
inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). The target molecules were
identified on the basis of satisfactory analytical and spectra data (IR, 1H NMR, 13C NMR, 2D NMR, DEPT,
EI-MASS techniques and elemental analysis). Synthesized compounds showed high activity against both
the MAO-A (compounds 1d, 1e, 2c, 2d, 2e) and the MAO-B (compounds 1a, 1b, 1c, 2a, 2b) isoforms. In
the discussion of the results, the influence of the structure on the biological activity of the prepared com-
pounds was delineated. It was suggested that non-substituted and N-methyl/ethyl bearing compounds
(except 2c) increased the inhibitory effect and selectivity toward MAO-B. The rest of the compounds, car-
rying N-allyl and N-phenyl, appeared to select the MAO-A isoform. The inhibition profile was found to be
competitive and reversible for all compounds. A series of experimentally tested (1a–2e) compounds was
docked computationally to the active site of the MAO-A and MAO-B isoenzyme. The AUTODOCK 4.01 pro-
gram was employed to perform automated molecular docking. In order to see the detailed interactions
of the docked poses of the model inhibitors compounds 1a, 1d, 1e and 2e were chosen because of their
ability to reversibly inhibit the MAO-B and MAO-A and the availability of experimental inhibition data.
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. 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. Excellent to good correlations between the calculated and experimental Ki values were
obtained. In the docking of the MAO-A complex, the trans configuration of compound 1e made various
very close interactions with the residues lining the active site cavity these interactions were much better
than those of the other compounds tested in this study. This tight binding observation may be responsible
for the nanomolar inhibition of form of MAOA. However, it binds slightly weaker (experimental
Keywords:
Hexahydroindazole
MAO-A/MAO-B inhibition
Docking
X-ray crystallographic model
Ki = 1.23
l
M) to MAO-B than to MAO-A (experimental Ki = 4.22 nM).
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
(ssAOs) that contain copper II-2,4,5-trihydroxyphenylalanine qui-
none as a cofactor (TPQ-Cu AOs). Both classes have been isolated
and characterized from micro-organisms, plants and mammals.
Monoamine oxidase (MAO, E.C. 1.4.3.4) is a flavin adenine dinucle-
otide (FAD)-containing enzyme present in the outer mitochondrial
membranes of neuronal, glial and other cells.1–3 It catalyzes the
oxidative deamination of biogenic amines in the brain and the
peripheral tissues, regulating their level. MAO exists in two forms,
namely, MAO-A and MAO-B.4 MAO-A catalyzes the oxidative
deamination of serotonin (5-HT), adrenaline (A), and noradrenaline
(NA) and is selectively inhibited by irreversible inhibitor clorgyline
and reversible inhibitor moclobemide. MAO-B catalyzes the
Amine oxidases (amine: oxygen oxidoreductases, AOs) are a
heterogeneous superfamily of enzymes that catalyze the oxidative
deamination of mono-, di-, and polyamines. AOs differ because of
their molecular architecture, catalytic mechanisms and subcellular
localizations. On the basis of the chemical nature of the cofactor,
AOs fall into two classes: AOs that contain flavin adenine dinucle-
otide as a cofactor (FAD-AOs), and semicarbazide sensitive AOs
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.033

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
oxidative deamination of b-phenylethylamine and benzylamine
and is selectively inhibited by irreversible inhibitor selegiline.5
MAO-A and MAO-B have essential roles in vital physiological
processes and are involved in the pathogenesis of various human
diseases. Due to their key role, MAO inhibitors represent a useful
tool for the treatment of several psychiatric and neurological dis-
eases. In particular, reversible and selective MAO-A inhibitors are
used as antidepressant and antianxiety drugs6–8 while MAO-B
inhibitors have been found to be useful as coadjuvants in the
treatment of Parkinson’s disease (PD) and Alzheimer’s disease
(AD).9–11
A recent description of the crystal structure of the two isoforms
of human MAO by Binda et al. provides a better understanding of
the pharmacophoric requirements needed for the rational design
of potent and selective enzyme inhibitors.12–17
It was reported that numerous compounds among the great
variety of substituted hydrazines behave as MAO inhibitors18–20
and a common structural feature of substrates and inhibitors is
an amino or imino group playing in interaction at the active site
of the enzyme.21 2-Pyrazolines can also be considered as a cyclic
hydrazine moiety. For this reason, researchers have investigated
MAO and other amine oxidase inhibition activities of 2-pyrazolines
and found high activity (Fig. 1).22–31
The discovery of this class of drugs has led to a considerable in-
crease in modern drug development and also pointed out the
unpredictability of biological activity arising from structural mod-
ifications of a prototype drug molecule.
In light of the aforementioned findings, in order to increase our
knowledge of the MAO inhibitory activity and selectivity of hydra-
zine-containing compounds, and to continue our study of pyrazo-
line derivatives as inhibitors of MAO-A and MAO-B isoforms, we
reported here the synthesis and the evaluation of the MAO-A and
MAO-B inhibitor activity of the hexahydroindazol derivatives
(1a–2e). Furthermore, molecular modeling work was performed
utilizing docking techniques to explain the selective inhibitory
activity toward the MAO-A and MAO-B enzymes.
2.1. Chemistry
The arylidenecyclohexanones (1,2) were synthesized by meth-
ods found in the literature.32,33 The reaction of arylidenecyclohexa-
none with hydrazine and subsequent isothiocyanate derivatives
yielded 2-substituted thiocarbamoyl-3,3a,4,5,6,7-hexahydro-2H-
indazoles (compounds 1b–1e, 2b–2e) while the condensation of
arylidenecyclohexanone with thiosemicarbazide was ascribed to
2-thiocarbamoyl-2,3,4,5,6,7-hexahydro-1H-indazole
derivatives
(compounds 1a, 2a). These reactions were probably involved in
the intermediate formation of hydrazones and subsequent addition
of N–H on the olefinic bond of the ethylenic moiety (Scheme 1).
The formation of this bicycle in the course of the addition reac-
tion of arylidenecyclohexanones with hydrazine derivatives re-
sulted in the formation of two stereoisomers: namely, the cis
isomer (compounds 1b–1d, 2b–2d), and the trans isomer (com-
pounds 1e) which is an isomer that had been separated by crystal-
lization from methanol. If the addition reaction is preceded by
trans addition to the double band, the ring closure of the phen-
ylhydrazone gives rise to the cis isomer (1b–1d, 2b–2d). If addition
occurs at the opposite side, the trans isomer is obtained (1e).
However, we were unable to separate the isomers of compound
2e owing to its close Rf values although compound 2e after the
work up was seen as two spots which were not equal in intensity.
We performed crystallization, preparative chromatography and
column chromatography many times in order to separate it, but
we were not successful. On the other hand, we have concluded that
trans isomer might be dominant in the diastereomeric mixture
from integral values obtained in 1H NMR where the ratio of the
heights of the cis/trans isomers was 0.13/1. Hence, because of the
failure to separate the mixture, analytical and spectroscopical mea-
surements in this case have been performed on the mixture as it is.
The structure of the compounds was elucidated by IR, 1H NMR,
13C NMR, DEPT, 2D NMR and EI-MASS techniques.
UV and IR spectra do not serve to distinguish between cis and
trans isomers of 2-thiocarbamoyl-3,3a,4,5,6,7-hexahydro-2H-inda-
zoles derivatives. We have turned to NMR to provide additional
support for structure assignments based on chemical evidence.
Chemical shift and proton coupling constant differences have been
observed for cis/trans isomer pairs in olefins and cyclic compounds
and have been used in structure assignments. The 1H NMR spectra
of compounds 1b–1d, 2b–2d display the signals from vicinal pro-
tons at C-3 and C-3a (d 5.8–6.1 and 3–3.40 ppm, respectively) with
J = 10.8–11.2 Hz, which are indicative of their cis configuration. In
2. Results and discussion
Our initial goal in this study was to prepare the N-substituted
hexahydroindazoles derivatives (Scheme 1), evaluate their mono-
amine oxidase (MAO) A and B inhibitory activity and carry out
docking studies.
Figure 1. Pyrazole derivatives having MAO, SSAO and BSAO inhibition.

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
6763
Scheme 1. Synthetic pathway of compounds la–2e.
compound 1e, H-3 and H-3a protons resonated 5.41–5.65 and
2.2. X-ray crystal analysis of compound 1e
3.05–3.12 ppm with J = 4.8–5.2 Hz predicating its trans configura-
tion. In the 1H NMR spectrum, the proton at C-3 and the proton
on the ring nitrogen and hydrogen atoms attached to the nitrogen
atom in the thiocarbamoyl moiety resonated at 8.69–9.97 and
6.45–8.21 ppm, respectively. Chemical shifts of the cyclohexane
ring protons also showed characteristic differences in cis and trans
isomers, such that the H-4ax proton shifted to a considerably high-
er field (0.53–0.85 ppm) in cis isomers due to the shielding effect of
the 3-aryl ring which is oriented close to this proton, while in the
trans isomer it overlapped with H-5ax and H-6ax protons in the
range 1.4–1.6 ppm. The protons belonging to the aromatic ring
and the other aliphatic groups were observed with the expected
chemical shift and integral values. All compounds gave satisfactory
elemental analyses.
The molecular structure of compound 1e was determined by X-
ray crystallographic analysis. The compound crystallizes in the
tetragonal system, space group P41 with a = 10.600(2) Å,
b = 10.600(2) Å, c = 14.863(4) Å,
a = 90.00°, b = 90.00°, c = 90.00°.
Ortep depiction with atom numbering of compound 1e is shown
in Figure 2.
The S1–C8 and N1–C7A bond lengths are both indicative of a
significant double bond character (Table 1). All the C–C bond
distances in the benzene ring have typical Csp2–Csp2 values. The
average C–C bond distance within benzene ring is 1.370(2) Å.
The C10–N9–C8–N2 torsion angle is 177.7(8)°.
The furan and benzene rings are planar, the maximum devia-
0
tions from the least-squares planes being 0.0063(3) ÅA for atom
0
In the 13C NMR of the cis isomer, the thiophen ring attached to
C-3 and H-4ax appeared to have steric interactions, as was evident
from the lower values of the chemical shifts for C-4 for the cis- than
for the trans isomer of the compound 1e with the furan ring at-
tached to C-3. In the DEPT spectra of compound 1a, four methylene
and four methine carbons were seen in expected values. For the
complete assignment of all protons, two dimensional NMR tech-
niques (1H–1H-COSY and 13C–1H COSY) were utilized.
C50 and 0.0072(2) ÅA for atom C15. The dihedral angles are as fol-
lows: 84.74(2)° between furan and N2–N1–C7A–C3A–C3 ring and
43.07(3)° between benzene and furan rings.
The hexahydroindazol ring exhibits a puckered conformation,
0
0
with puckering parameters33 q2 = 0.069 (10) ÅA, q3 = 0.537 (10) ÅA,
0
QT = 0.541 (10) AÅ, u = 224 (8)° and h = 7.0 (11)°, which indicates
that the hexahydroindazol ring has a chair conformation. The larg-
0
est deviat0ions from the best plane are ꢀ0.253 (2) ÅA for C4 and
0.254 (2) ÅA for C5. The hexahydroindazol ring makes a dihedral an-
gle of 37.22 (3)° with the N2–N1–C7A–C3A–C3 ring. It has also
been found that the H3A and H3 atoms deviate from the latter
Inspection of the 1H–13C NMR spectrum of compound 1a re-
vealed a correlation from signal at 2.43 and 2.77 ppm to signal at
27 and 29 ppm of C-4/C-7 and vice versa. But which signal was
H-7 or H-4 was unknown. Because of this, we utilized 1H–1H COSY
spectrum for understanding which of the signal at 2.43 or
2.77 ppm belong to H-4 or H-7 protons. The correlation between
the signal of H-3 (6.45–6.48 ppm)/H-30,40,50 (6.45–6.48/6.45–
6.48/7.47 ppm) and the signal at 2.43 ppm confirmed that that sig-
nal (at 2.43 ppm) corresponded to axial and equatorial protons of
H-4. Therefore, the other signal at 2.77 ppm could be assigned
the axial and equatorial protons of H-7.
In the mass spectra, molecular ion signals were prominent for
all compounds for which six of them were also base signal (com-
pounds 1a–1e, 2c). Two sets of fragments were detected belonging
to the fragmentation of the thiocarbamoyl and bicyclic dihydropy-
razole structure. Fragments resulting from the loss of an SH ion
from the thiocarbamoyl group were observed for most compounds
plane in opposite directions. The magnitudes of the deviations of
0
H3A and H3 atoms in opposite directions are equal to 0.39 ÅA and
0
0.4 ÅA, respectively, constituting a trans configuration.
2.3. Biochemistry
MAO-A and MAO-B inhibitory activities of newly synthesized
hexahydroindazole derivatives were determined using MAO-A
and -B isoforms of rat liver mitochondrial pellets. Liver tissue
was used to screen the MAO-inhibitory actions of these novel com-
pounds since liver was reported to be a good source for both iso-
forms of the enzyme. According to the IC50 values corresponding
to the inhibition of rat liver MAO by the newly synthesized hexa-
hydroindazole derivatives, all of the compounds were found to in-
hibit rat liver MAO. All novel compounds were reversible inhibitors
of rat liver MAO since the enzyme activity (approx. 98–100%) was
restored after 24 h dialysis (Table 2).
in different intensity (compounds 1a, 2a–e). In addition to,
a-
cleavage of the C@S group in both sides causing ejection of the
NHR (compounds 1d, 2d) or CSNHR (compounds 1a, 1b, 2a, 2e)
type of ions were also observed.
The X-ray diffraction data of the crystal obtained for compound
1e showed that compound 1e is in the trans configuration (Fig. 2).
The thiosemicarbazide moiety in the parent structure was sug-
gested to be responsible for the MAO inhibitory activity of the
newly synthesized compounds. According to our experimental

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
Figure 2. The molecular structure of le, showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 40% probability.
Table 1
data, compounds 1d (cis), 1e (trans), 2c (cis), 2d (cis) and 2e
Selected geometric parameters (Å) and crystallographic parameters for the compound
(mixture) inhibited MAO-A while 1a (cis), 1b (cis), 1c (cis), 2a and
2b (cis) inhibited MAO-B selectively. The mode of inhibition was
found to be competitive and reversible for all compounds tested.
Since IC50 value for a compound is generally calculated from the
experiments which a wide range of inhibitor concentration used
in, and this value cannot give an idea about the kinetic behavior
of the inhibitor, we preferred to discuss the data upon Ki values.
In respect to the Ki values experimentally found (Table 2), com-
pounds 1e [3-(2-furyl)-2-(N-phenylthiocar-bomoyl)-3,3a,4,5,6,7-
hexahydro-2H-indazole] and 2e [3-(2-thienyl)-2-(N-phenylthioc-
1e
Selected bond lengths (Å)
Crystallographic parameters for the compound 1e
C1–N1
N1–C7
N2–N3
N2–C14
S1–C7
1.430(10)
1.355(10)
1.400(8)
1.500(9)
1.666(9)
1.35(1)
Chemical formula sum
Chemical formula weight
Symmetry cell setting
Space group
C18H19N3OS
325.42
Tetragonal
P41
a (Å)
b (Å)
c (Å)
10.600(2)
10.600(2)
14.863(4)
90.00
90.00
90.00
C7–N2
N3–C8
1.277(9)
a
(°)
b (°)
(°)
arba-moyl)-3,3a,4,5,6,7-hexahydro-2H-indazole], which carry
a
c
phenyl substituent on the nitrogen atom were found to be highly
Table 2
Experimental IC50 and Ki values corresponding to the inhibition of rat liver MAO isoforms by the newly synthesized hexahydroindazole derivativesa
Compounds
IC50 for MAO-Ab
M)
Ki value for
IC50 for MAO-Bb
Ki value for
Inhibition
type
Reversibility
SIc MAO-A/
MAO-B
MAO inhibitory
selectivity
(
l
MAO-Ab
(lM)
MAO-Bb
1a
1b
1c
1d
1e
2a
2b
2c
80.22 6.91
3.90 0.28
8.82 0.61
4.26 0.30
0.65 0.03
l
l
l
l
M
M
M
M
1.37 0.10
2.99 0.15
2.77 0.96
0.96 0.01 nM
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
4062.50
4.20
2.19
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
108.34 7.09
110.45 8.56
1.78 0.17
0.78 0.08
95.20 6.89
62.34 5.10
3.01 0.29
2.05 0.17
0.70 0.02
90.55 7.05
5.70 0.37
2.10 0.01
1.95 0.05
4.05 0.28
1.23 0.01
lM
lM
lM
lM
113.88 9.70
80.45 7.45
1.26 0.01
2.61 0.18
90.12 8.72
78.62 5.03
75.12 5.87
1.60 0.10
89.55 6.30
0.16
4.22 0.33 nM
0.0034
3322.22
2.96
0.57
0.30
0.004
78.26
0.005
2.99 0.17
5.01 0.38
1.72 0.01
0.88 0.01
39.15 2.70 nM
105.66 9.21
5.53 0.27 nM
lM
lM
lM
lM
0.90 0.01 nM
1.69 0.09
3.01 0.23
2.90 0.17
92.26 7.13
1.35 0.12
1.08 3.00
lM
lM
lM
lM
lM
lM
2d
2e
Selegiline
Moclobemide
lM
a
Each value represents the mean SEM of three independent experiments.
IC50 and Ki values were determined from the kinetic experiments in which p-tyramine (substrate) was used at 500
b
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. Newly synthesized compounds and the known inhibitors were preincubated with the
homoganates for 60 min at 37 °C.
c
Selectivity index. It was calculated as Ki (MAO-A)/Ki (MAO-B).

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
6765
potent MAO-A inhibitors. These two molecules differ from each
other with the existence of furyl or thienyl groups at the 3-position
of the hexahydroindazole ring. MAO-A/MAO-B selectivities of com-
pound 1e (bearing a furyl group) and 2e (bearing a thienyl group)
were found be 0.0034 and 0.004, respectively, while MAO-A/MAO-
B selectivity of moclobemide, the known selective MAO-A inhibi-
tor, was calculated as 0.005. Since the trans and cis forms of com-
pound 2e could not be separated following the synthesis,
experimental inhibition studies were conducted only with the
mixture obtained.
was calculated as 2.46. Compound 2a was found to be the most po-
tent MAO-B inhibitor with an experimental Ki value of
0.90 0.01 nM. This new compound inhibited rat liver MAO-B
more potently than selegiline (Ki value was determined as
1.35 0.12 lM), the well known MAO-B inhibitor (Fig. 3).
Among all novel derivatives studied, compound 1e was found to
be the most potent MAO-A inhibitor with an experimental Ki value
of 4.22 0.33 nM. This new compound was more potent than moc-
lobemide, the well known MAO-A inhibitor (Ki value was deter-
mined as 5.53 .0.27).
According to the experimental findings, compound 1e was
found to inhibit rat liver MAO-A potently and selectively with a
Ki value of 4.22 0.33 nM. Compound 2e also inhibited rat liver
MAO-A potently. The Ki values corresponding to the inhibition of
rat liver MAO-A with compound 2e were calculated as 20.95 nM
The results presented here show that newly synthesized hexa-
hydroindazole derivatives may be promising candidates as potent
anti-depressant/anti-parkinson agents. At the same time, this
study indicates a significant correlation between the docking re-
sults and experimental ones. However, further experiments are
necessary to fully elucidate the binding characteristics of the novel
compounds to MAO isoforms purified from different sources since
it was recently shown that the similarities and differences between
human MAO-A (hMAO-A) and rat MAO-A (rMAO-A) might be
important in drug development.34 Although these two enzymes
exhibit ꢁ90% sequence identity, they reveal significant differences
in their quaternary structures. The volume of the active site cavity
of rMAO-A (ꢁ450 Å) is smaller than that of hMAO-A (ꢁ550 Å). It
should be kept in mind that the results obtained with non-human
forms of MAO (e.g., the evaluation of the inhibitory properties of a
compound) may not be extrapolated to the same situation in
humans.35
and 31.17 lM for its trans and cis configurations (Table 3), respec-
tively, from the docking studies. Thus, this cross tabulation and the
integration values obtained in 1H NMR support the hypothesis that
the compound 2e might be mainly in trans configuration. Since
compounds 1e and 2e, which bear a phenyl group on thiosemicar-
bazide moiety appeared as potent MAO-A inhibitors (experimental
and calculated Ki values are in nM range), it was suggested that
phenyl substitution increases the MAO-A inhibitory potency of
the derivatives studied.
Compounds 1d and 2d which contain an allyl derivative on the
nitrogen atom also inhibited rat liver MAO-A selectively and
reversibly in a competitive manner. These two molecules also dif-
fer from each other with respect to furyl and thienyl groups at the
hexahydroindazole ring. Although the MAO-A/MAO-B selectivities
of compounds 1d and 2d were calculated from the experimental
data as 0.16 and 0.30, and as 0.15 and 0.13, from the docking stud-
ies, respectively. These two compounds therefore are also potent
MAO-A inhibitors among the novel compounds studied in respect
to both calculated and experimental data (Table 3).
2.4. Molecular docking studies
Compound 1a is docked in the active site of the MAO-A enzyme
as shown in Figure 4 (Ki = 1.36 lM). When examined closely, it is
observable that the furane ring is oriented horizontally between
the phenolic side chains of Tyr444 and Tyr407 residues and it is
approaching from the re face of FAD. The hydrogen atom on the
amine group on the side chain makes a hydrogen bond with the
backbone carbonyl group of Ile180 (2.16 Å). Ile207, Asn181 and
Tyr197 are the other active site residues interacting with the inhib-
itor. The same compound shows different binding patterns with
Compound 1c carrying an ethyl group on the nitrogen atom and
a furyl group on the hexahydroindazole ring inhibited rat liver
MAO-B while compound 2c carrying a thienyl group instead of fur-
yl inhibited rat liver MAO-A selectively and reversibly in a compet-
itive manner.
Compounds 1a and 1b bearing non-substituted or N-meth-
ylthiocarbamoyl and a furyl group on the hexahydroindazole ring
inhibited rat liver MAO-B potently as compounds 2a and 2b, the
thienyl bearing ones. Compound 1a appeared as the most potent
compound in this group. Its experimental SI (MAO-B/MAO-A)
the MAO-B enzyme as shown in Figure 5 (Ki = 1.27 lM). The inhib-
itor is placed far distant from the hydrophobic cage surrounded by
Tyr398, Tyr435 and FAD. In this case the inhibitor is located close
to the entrance cavity. One of the thiocarbamoyl hydrogens of the
inhibitor makes a hydrogen bond with the entrance site residue
Table 3
Calculated and experimental Ki values corresponding to the inhibition of MAO isoforms by the newly synthesized hexahydroindazole derivativesa
Compound
Calculated Ki values
Experimental Ki
Calculated Ki values
Experimental Ki
Calculated SIc
MAO-A/MAO-B
Experimental SIc
MAO-A/MAO-B
Selectivity
for MAO-Aa
values for MAO-Ab
for MAO-Ba
values for MAO-Bb
1a
1b
1c
1d
1e
2a
2b
2c
1.36
3.18
2.80
0.525
8.77 nM
2.07
2.62
0.422
0.268
20.95 nM
31.17
—
l
l
l
M
M
M
3.90 0.28
8.82 0.61
4.26 0.30
0.65 0.03
l
l
l
l
M
M
M
M
1.27
2.90
2.73
3.58
l
l
l
l
M
M
M
M
0.96 0.01 nM
1.07
1.09
1.02
0.14
0.12
1.86
1.54
0.17
0.13
0.3
4062.50
4.20
2.19
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
2.10 0.01
1.95 0.05
4.05 0.28
1.23 0.01
lM
lM
lM
lM
l
M
0.16
4.22 0.33 nM
70.72 nM
0.0034
3322.22
2.96
0.57
0.30
0.004
—
78.26
0.005
l
l
M
M
2.99 0.17
5.01 0.38
1.72 0.01
0.88 0.01
lM
lM
lM
lM
1.11
1.70
2.47
1.99
69.46 nM
93.08 nM
lM
lM
lM
lM
0.90 0.01 nM
1.69 0.09
3.01 0.23
2.90 0.17
92.26 7.13
—
lM
lM
lM
lM
l
l
M
M
2d
2e trans
2e cis
Selegiline
Moclobemide
39.15 2.70 nM
l
M
—
340.5
—
—
105.66 9.21
5.53 0.27 nM
l
M
—
—
1.35 0.12
1.08 3.00
l
l
M
M
—
a
Ki values were determined from the kinetic experiments in which p-tyramine (substrate) 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. Newly synthesized compounds and the known inhibitors were preincubated with the
homoganates for 60 min at 37 °C.
b
Each value represents the mean SEM of three independent experiments.
Selectivity index. It was calculated as Ki (MAO-A)/Ki (MAO-B).
c

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
Figure 3. Lineweaver–Burk plot for the inhibition of rat liver MAO-B by the compound 2a (0–0.5 nM) with 60 min of preincubation at 37 °C. p-Tyramine was used as
substrate (0.01–0. l mM). Values are the mean of three independent experiments. Second graph represents the plot of the slope of reciprocal plot, v = velocity (nmol/saat/mg).
Figure 4. Binding mode of compound la in MAO-A active site.
Ile199 (2.10 Å). The second hydrogen bond forms between Pro102
backbone carbonyl group and amine hydrogen of the indazole ring.
Leu88, Gln206, Tyr326, Ile316, Leu164 and Leu171 are the other
Figure 5. Binding mode of compound la in MAO-B active site.
residues stabilizing the inhibitor tightly at this volume.
Figure 6 shows the binding pose of 1d in the active site of MAO-
A. The N-allylthiocarbamoyl moiety of compound 1d is sandwiched
between Tyr407 and Tyr444 and it approaches to FAD as closely as
possible (Ki = 0.525 M). In addition, the N-H group of the thiocar-
MAO-A in Figure 7 (Ki = 3.58
is sandwiched between Tyr435 and Tyr398 and it is not as close to
FAD as in MAO-A, causing a low potency (Ki = 3.58 M) when com-
pared with MAO-A (Ki = 0.525 M). The major contribution of
binding energy comes from the hydrogen bond resulting from
the Tyr326 side chain OH and thiocarbamoyl hydrogen (2.05 Å).
The other contributing van der Waals interactions are between
lM). None of the groups of the ligand
l
bamoyl moiety and OH group of the Tyr407 makes a strong hydro-
gen bond (1.96 Å). Asn181, Phe208, Ile207 and Glu216, Ile180 and
Ser209 side chains are the other residues interacting with the
inhibitor in the active site of the MAO-A. On the other hand 1d is
oriented differently in the active site of MAO-B than that of
l
l

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
6767
Figure 8. Binding pose of compound le in the active site ofMAO-A.
Figure 6. Binding mode of compound Id in MAO-A active site.
Figure 9. Binding pose of compound le in the active site of MAO-B.
Figure 7. Binding mode of compound Id in MAO-B active site.
the residues of Leu88, Ile316, Pro104, Pro102, Ile199, Leu164 and
Gln206.
Figure 8 shows the compound 1e in the active site of MAO-A
(Ki = 8.77 nM). The hexahydo-1H-indazole ring of compound 1e is
approached to FAD as close as possible and perfectly sandwiched
between Tyr407 and Tyr444. In addition to that, hydrogen atom
of the N–H group is making an excellent hydrogen bond with the
O–H group of Tyr407 (1.98 Å) in the hydrophobic cage. Ile180,
Asn181, Leu337, Phe208, Ile207 and Gln215 are the other residues
having very close contact with the inhibitor. This compound has
the lowest inhibition constant value among the compounds calcu-
lated computationally. In the MAO-B complex of the same com-
pound, inhibitor occupies
a
volume far from FAD ring
Figure 10. Binding mode of compound 2e-trans in MAO-A active site.
(Ki = 70.72 nM). The phenyl ring is oriented toward Tyr435 and
surrounded by Gln206, Cys172, Ile198 and Gln206. Sulfur atom
of the thioamide moiety group is making two close interaction;
one with the backbone carbonyl group of Ile199 and the other with
the hydroxyl group of Tyr326. The hexahydo-1H-indazole ring is,
on the other hand, surrounded by Leu88, Ile316, Pro102, Leu167,
Leu164 and Phe168 (Fig. 9).
thiophene ring is snugly sandwiched between Tyr407, Tyr444 in
the hydrophobic package vertically to the re face of FAD. The phe-
nyl ring is placed between Glu 216 and Phe 208 at the entrance
cavity. There is one close interaction between the Tyr407 side
chain and benzylic N-H moiety (1.81 Å). Tyr69, Gln215, Phe208,
Ile207 and Asn181 are the other residues having close contact with
the inhibitor. In the MAO-B complex of the same compound, the
In Figure 10 the compound 2e-trans is shown (Ki = 20.95 nM). In
the complex of MAO-A with the trans isomer of compound 2e, the

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N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
phenyl ring is surrounded by Ile198, Gln206 and Cys172. The
cyclohexane ring is, on the other hand, surrounded by Leu167,
Leu164 and Ile316. The thiophene ring has neighboring Ile199,
Pro102 and Leu88 residues (Fig. 11).
4.1.1. Synthesis of 2-furfurylidene/2-(2-thienylmethylene)cyclo-
hexanone
2-Furfurylidene/2-(2-thienylmethylene)cyclohexanone
was
synthesized as a result of the reaction of cyclohexanone and furfu-
ral/thiophene-2-aldehyde in basic media with 1 N NaOH at room
temperature according to the method reported earlier.36
3. Conclusion
4.1.2. General procedure for the preparation of 3-(2-furyl/
thienyl)-2-thiocarbamoyl-1,3,4,5,6,7-hexahydro-1H-indazoles
(1a, 2a)
One gram (0.025 mol) NaOH in 5 ml water was added to
0.01 mol of 2-furfurylidene/2-(2-thienylmethylene)cyclohexanone
and 1.092 g (0.012 mol) thiosemicarbazide. The reaction mixture
was refluxed for 8 h than poured into 200 ml of cold water. The
precipitate was filtered and recrystallized from methanol.
The biological behavior of the hexahydroindazol derivatives
was investigated against both MAO-A and -B isoforms. Most of
them showed potent inhibition activities in the micromolar range
with selectivity against the MAO-A and MAO-B isoform. It was
determined that the length of the lateral chain of the aryl diazo
derivatives should not be longer than two atoms as was observed
for the substrates of MAO-B (benzylamine, phenylethylamine).
The presence of the longer and volumed groups on thiocarbamoyl
nitrogen could be indicated as a requisite for the selective MAO-A
activity. The docking studies carried out on the most active and
selective compounds 1a, 1d, 1e and 2e-trans provided us new
and complementary insights into the inhibition mechanism and
patterns. These results encouraged us to pursue our molecular
modeling studies in designing more potent/selective MAO inhibi-
tors based on the hexahydroindazole scaffold.
4.1.3. General procedure for the preparation of 3-(2-furyl/
thienyl)-2-(N-substitued thiocarbamoyl)-3,3a,4,5,6,7-
hexahydro-2H-indazoles (1b–e, 2b–e)
One gram (0.02 mol) hydrazine hydrate (100%) was added to
0.01 mol of 2-furfurylidene/2-(2-thienylmethylene)cyclohexanone
in 20 ml ethanol and refluxed for 2 h. Reaction mixture was cooled
and solvent evaporated under vacuum. The intermediate 3-(2-fur-
yl/thienyl)-3,3a,4,5,6,7-hexahydro-2H-indazole was solved in
20 ml dry ether. 0.01 mol appropriate isothiocyanate and four
drops of triethylamine was added into this solution and the reac-
tion mixture was mixed for 4 h at room temperature. The precipi-
tate was filtered and recrystallized from appropriate solvents.
4. Experimental
4.1. Chemistry
All chemicals were obtained from Merck Co. except 2-furalde-
hyde. 2-Furaldehyde was obtained from Sigma–Aldrich Co. Melting
points were determined through a Thomas Hoover capillary melt-
ing point apparatus and are uncorrected. Ultraviolet (UV) spectra
were recorded with an Agilent 8453 UV–vis spectra spectrometer
in methanol approximately 4 ꢂ 10ꢀ5 M concentration. Infrared
(IR) spectra were obtained with a Perkin Elmer Spectrum BX FT-
IR spectrometer using potassium bromide plates and the results
were expressed in wave number (cmꢀ1). Nuclear magnetic reso-
nance (1H NMR and 13C NMR) spectra were scanned on a Varian
400 and 100 MHz High Performance spectrometer, respectively,
using dimethylsulfoxide (DMSO-d6) as a solvent. Chemical shifts
are expressed in d (parts per million) relative to tetramethylsilane.
Splitting patterns are as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; dq, doublet of quartet; dt, doublet of triplet; sb, singlet
broad. The mass spectra were obtained with electron impact tech-
nique using a Direct Insertion Probe and Agilent 5973-Network
Mass Selective Dedector at 70 eV. Elemental analyses (C, H, N, S)
were performed on a Leco CHNS 932 analyzer.
4.1.3.1. 3-(2-Furyl)-2-thiocarbamoyl-2,3,4,5,6,7-hexahydro-1H-
indazol (1a). A dark yellow solid substance with a yield of 54%
and recrystallized from methanol. Mp 184–5 °C. UV (CH3OH) nm:
202 (log e: 4.24) and 3 nm. (log e
: 4.32); IR (KBr) cmꢀ1, 34, 3211,
3130, 3035, 29, 2864, 1595, 1497, 1261, 1151, 1080; 1H NMR
(DMSO-d6, 400 MHz) d (ppm): 1.59–1.64 (4H; m; H5ax, H5eq, H6ax,
H6eq), 2.50 (2H; m; H4ax, H4eq), 2.68 (2H; t; H7ax, H7eq), 6.58 (2H;
m; furan H3, furan H4), 7.21 (1H; m; H3), 7.74 (1H; d; furan H5),
7.85 and 8.21 (1H; s; NH2), 9.97 (1H; s; H1); 1H NMR (CDCl3,
400 MHz) d (ppm): 1.69–1.80 (4H; m; H5ax, H5eq, H6ax, H6eq), 2.43
(2H; t; H4ax, H4eq), 2.77 (2H; dt; H7ax, H7eq), 6.45–6.48 (3H; m; fur-
an H3, furan H4, NH2), 7.05 (1H; m; H3), 7.26 (1H; s; NH2), 7.46 (1H;
d; furan H5), 8.69 (1H; s; H1); 13C NMR (400 mHz, DMSO-d6) d
(ppm); 22 (C5), 23 (C6), 27 (C4), 29 (C7), 112 (furan-C3, furan-C4),
116 (C3), 133 (C3a), 144 (furan-C5), 151 (C7a), 153 (furan-C2), 179
(C@S); 13C NMR (400 mHz, CDCl3) d (ppm); 22 (C5), 23 (C6), 27
(C4), 29 (C7), 112 (furan-C3, furan-C4), 117 (C3), 132 (C3a), 143 (fur-
an-C5), 152 (C7a), 153 (furan-C2), 179 (C@S); HSQC (1H–13
NMR)(CDCl3); 1.69–1.180/22 (H5ax, H5eq/C5), 1.69–1.180/23 (H6ax
C
,
H6eq/C6), 2.43/27 (H4ax, H4eq,/C4), 2.68/29 (H7ax, H7eq/C7), 7.05/117
(H3/C3), 6.45–6.48/112 (furan H3/furan C3), 6.45–6.48/112 (furan
H4/furan C4), 7,46/143 (furan H5/furan C5); MS (70 eV, EI): m/e
(%) 249: (M+, 100%), 216 (MꢀSH, %18.), 189 (MꢀCSNH2, %67.82),
181 (MꢀC4H4O;%15.78), 161 (MꢀCH2N3S, %31.57). Anal. Calcd for
C12H15N3OS: C, 57.81; H, 6.06; N, 16.85; S, 12.86. Found: C,
57.71; H, 6,181; N, 16.; S, 12.64.
4.1.3.2. 3-(2-Furyl)-2-(N-methylthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (1b). A dirty white solid substance with a
yield of 31% and recrystallized from methanol–water. Mp 167–
8 °C. UV (CH3OH) nm: 201 (log e: 4.36) and 273 nm. (log e: 4.31);
IR (KBr) cmꢀ1; 3345, 3112, 2987, 2953, 2864, 1649, 1535, 1322,
1216, 1144,1106; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J in
Hz): 0.55 (1H; dq; H4ax), 1.10–1.40 (2H; m; H5ax, H6ax), 1.66 (2H;
m; H4eq, H5eq), 1.92 (1H; m; H6eq), 2.26 (1H; dt; H7ax), 2.58 (1H;
d; H7eq), 2.86 (3H; d; –CH3), 3.40 (1H; m; H3a), 5.80 (1H; d; H3; J:
10.8), 6.08 (1H; d; furan H4), 6.35 (1H; dd; furan H3), 7.51 (1H;
Figure 11. Binding mode of compound 2e-trans in MAO-B active site.

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
6769
m; furan H5), 8.12 (1H; q; NH); MS (70 eV, EI): m/e (%): 263 (M+,
100%), 195 (MꢀC4H4O, %23.68), 189 (MꢀC2H4NS, %17.54), 181
(MꢀC6H10, %27.19), 167 (MꢀC6H10N, %67.82), 107 (MꢀC7H12N2S,
%14.), 81 (MꢀC8H12N3S, %30.70). Anal. Calcd for C13H17N3OS: C,
59.29; H, 6.51; N, 15.96; S, 12.18. Found: C, 59.09; H, 5.68; N,
15.62; S, 11.38.
169 (MꢀC6H10N, %25.74), 123 (MꢀC6H10N2S, %33.61), 97
(MꢀC7H10N3S, %30). Anal. Calcd for C12H15N3S2: C, 54.31; H, 5.70;
N, 15.83; S, 24.16. Found: C, 54.35; H, 5.756; N, 15.71; S, 24.02.
4.1.3.7. 3-(2-Thienyl)-2-(N-methylthiocarbamoyl)-3,3a,4,5,6,7-
hexahydro-2H-indazol (2b). A dirty white solid substance with
a yield of 50% and recrystallized from methanol. Mp 177–8 °C.
4.1.3.3. 3-(2-Furyl)-2-(N-ethylthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (1c). A white solid substance with a yield of
51% and recrystallized from ethanol. Mp 186 °C. UV (CH3OH) nm:
UV (CH3OH) nm: 201 (log
e: 4.29), 245 (log e: 4.20) and 274 nm.
(log
e
:4.29); IR (KBr) cmꢀ1; 3361, 3066, 2952, 2940, 2865, 16, 15,
1345, 1232, 1133, 1106; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J
in Hz): 0.60 (1H; dq; H4ax), 1.11 (1H; m; H5ax), 1.32 (1H; m;
H6ax) 1.65 (2H; m; H4eq, H5eq), 1.89 (1H; m; H6eq), 2.28 (1H; dt;
H7ax), 2.60 (1H; d; H7eq), 2.87 (3H; d; –CH3), 3.40 (1H; m; H3a),
6.09 (1H; d; H3; J: 10.8), 6.76 (1H; d; thiophene H4), 6.94 (1H;
dd; thiophene H3), 7.33 (1H; m; thiophene H5),, 8.18 (1H; q;
NH); MS (70 eV, EI): m/e (%): 279 (M+, %68), 246 (MꢀSH, %49),
197 (MꢀC6H10, %28), 183 (MꢀC6H10N, 100%), 123 (MꢀC7H12N2S,
%18), 97 (MꢀC8H12N3S, %19). Anal. Calcd for C13H17N3S2: C,
55.88; H, 6.13; N, 15.04; S, 22.95. Found: C, 55.05; H, 6.304; N,
14.99; S, 22.97.
202 (log e: 4.34) and 274 nm. (log e
: 4.33); IR (KBr) cmꢀ1; 3339,
3110, 2975, 2927, 2862, 1637, 1533, 1323, 1214, 1145, 1112; 1H
NMR (DMSO-d6, 400 MHz) d (ppm) (J in Hz): 0.53 (1H; dq; H4ax),
1.06 (3H; t; –CH3), 1.10–1.40 (2H; m; H5ax, H6ax), 1.65 (2H; m;
H4eq, H5eq), 1.92 (1H; m; H6eq), 2.26 (1H; dt; H7ax), 2.60 (1H; d;
H7eq), 3.40 (1H; m; H3a), 3.50 (2H; m; –CH2–), 5.81 (1H; d; H3; J:
10.8), 6.08 (1H; d; furan H4), 6.35 (1H; dd; furan H3), 7.51 (1H;
m; furan H5),, 8.10 (1H; t; NH); MS (70 eV, EI): m/e (%): 277 (M+,
100%), 209 (MꢀC4H4O, %18), 195 (MꢀC6H10, %22), 181 (MꢀC6H10N,
%53.47), 107 (MꢀC8H14N2S, %15), 81 (MꢀC9H14N3S, %31.57), 44
(MꢀC12H13N2OS, %29). Anal. Calcd for C14H19N3OS: C, 60.62; H,
6.90; N, 15.15; S, 11.56. Found: C, 60; H, 6.97; N, 15.05; S, 10.88.
4.1.3.8. 3-(2-Thienyl)-2-(N-ethylthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (2c). A white solid substance with a yield of 51%
and recrystallized from methanol. Mp 194 °C. UV (CH3OH) nm: 201
4.1.3.4. 3-(2-Furyl)-2-(N-allylthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (1d). A white solid substance with a yield of
45% and recrystallized from methanol–water. Mp 161 °C. UV
(log e: 4.33), 246 (log e: 4.24) and 275 nm. (log e:4.24); IR (KBr)
cmꢀ1; 3356, 3075, 2974, 2940, 2863, 1643, 1528, 1320, 1228, 1149,
1110; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J in Hz): 0.60 (1H;
dq; H4ax), 1.06 (3H; t; –CH3), 1.10–1.40 (2H; m; H5ax, H6ax), 1.60–
1.70 (2H; m; H4eq, H5eq), 1.90 (1H; m; H6eq), 2.28 (1H; dt; H7ax),
2.60 (1H; dd; H7eq), 3.40 (1H; m; H3a), 3.50 (2H; m; –CH2–), 6.10
(1H; d; H3; J: 11.2), 6.76 (1H; d; thiophene H4), 6.95 (1H; dd; thio-
phene H3), 7.33 (1H; m; thiophene H5), 8.18 (1H; t; NH); 1H NMR
(CDCl3, 400 MHz) d (ppm) (J in Hz): 0.85 (1H; dq; H4ax), 1.24 (3H;
t; –CH3), 1.27–1.41 (2H; m; H5ax, H6ax), 1.72–1.79 (2H; m; H4eq,
H5eq), 1.97 (1H; m; H6eq), 2.20 (1H; dt; H7ax), 2.72 (1H; dd; H7eq),
3.24 (1H; m; H3a), 3.64 (2H; m; –CH2–), 6.20 (1H; d; H3; J: 11.2),
6.80 (1H; d; thiophene H4), 6.95 (1H; dd; thiophene H3), 7.17 (1H;
m; thiophene H5), 7.27 (1H; t; NH); 13C NMR (400 mHz, CDCl3) d
(ppm); 15 (CH3), 24 (C5), 25 (C6), 27.2 (C4), 27.8 (C7), 39 (CH2), 50
(C3a), 62 (C3), 124.1 (thiophene-C5), 124.7 (thiophene-C4), 127 (thio-
(CH3OH) nm: 202 (log e: 4.32) and 274 nm. (log e: 4.27); IR (KBr)
cmꢀ1; 3345, 3112, 2981, 2939, 2863, 16, 1528, 1322, 1217,
1147, 1119; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J in Hz):
0.56 (1H; dq; H4ax), 1.17 (1H; m; H5ax), 1.32 (1H; m; H6ax), 1.67
(2H; m; H4eq, H5eq), 1.92 (1H; m; H6eq), 2.28 (1H; dt; H7ax), 2.60
(1H; d; H7eq), 3. (1H; m; H3a), 4.10 (2H; m; –CH2–), 5.06 (2H;
dq; @CH2), 5.80–5.87 (2H; m; –CH@, H3; J: 10.8), 6.09 (1H; d; fur-
an H4), 6.36 (1H; dd; furan H3), 7.52 (1H; m; furan H5), 8.19 (1H; t;
NH); MS (70 eV, EI): m/e (%): 289 (M+, 100%), 274 (MꢀCH3,
%37.72), 233 (MꢀC3H6N, %36), 193 (MꢀC6H10N, %43), 107
(MꢀC9H14N2S, %48.74), 81 (MꢀC10H14N3S, %63.47). Anal. Calcd
for C15H19N3OS: C, 62.25; H, 6.62; N, 14.52; S, 11.08. Found: C,
62.37; H, 6.491; N, 14.48; S, 11.19.
4.1.3.5. 3-(2-Furyl)-2-(N-phenyllthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (1e). A white needles with a yield of 73% and
recrystallized from ethanol. Mp 157–8 °C. UV (CH3OH) nm: 203
phene-C3), 1 (C7a), 161 (thiophene-C2), 175 (C@S); HSQC (1H–13
NMR)(CDCl3): 1.24/15 (CH3/CH3) 0.85, 1.72–1.79/27.2 (H4ax, H4eq
C
–
C4), 1.27–1.41, 1.72–1.79 /24 (H5ax, H5eq/C5), 1.27–1.41, 1.97/25
(H6ax, H6eq/C6), 2.20, 2.72/27.8 (H7ax, H7eq/C7), 3.24/50 (H3a-C3a),
3.64/39 (–CH2–/–CH2–), 6.20/62 (H3/C3), 6.95/127 (thiophene H3/thi-
ophene C3), 6.80/124.7 (thiophene H4/thiophene C4), 7,17/124.1 (thi-
ophene H5/thiophene C5); MS (70 eV, EI): m/e (%): 293 (M+, 100%),
260 (MꢀSH, %59.13), 197 (MꢀC6H10N, %90.32), 123 (MꢀC8H14N2S,
%32.57), 97 (MꢀC9H14N3S, %30.43), 44 (MꢀC12H13N2S2, %55.91). Anal.
Calcd for C14H19N3S2: C, 57.30; H, 6.53; N, 14.32; S, 21.85. Found: C,
57.44; H, 6.307; N, 14.26; S, 21.82.
(log e: 4.44) and 278 nm. (log e
: 4.36); IR (KBr) cmꢀ1; 3490, 3189,
2937, 2856, 1636, 1509, 1329, 1214, 1147, 1089; 1H NMR (DMSO-
d6, 400 MHz) d (ppm) (J in Hz): 1.40–1.60 (3H; m; H4ax, H5ax, H6ax),
1.75 (1H; m; H4eq), 2.03 (1H; m; H5eq), 2.15 (1H; m; H6eq), 2.45
(1H; m; H7ax), 2.60 (1H; d; H7eq), 3.12 (1H; m; H3a), 5.41 (1H; d;
H3; J: 5.2), 6.28 (1H; d; furan H4), 6.39 (1H; dd; furan H3), 7.10
(1H; m; furan H5), 7,26 (2H; m; phenyl), 7.54 (3H; m; phenyl), 9.88
(1H; s; NH); MS (70 eV, EI): m/e (%): 325 (M+, 100%), 257 (MꢀC4H4O,
%28), 243 (MꢀC6H10
,
%37), 229 (MꢀC6H10N%60.34), 81
(MꢀC13H14N3S, %34.21), 77 (MꢀC12H14N3OS%51.72). Anal. Calcd for
C18H19N3OS: C, 66.43; H, 5.88; N, 12.91; S, 9.85. Found: C, 66.26; H,
5.504; N, 13.02; S, 9.54.
4.1.3.9. 3-(2-Thienyl)-2-(N-allylthiocarbamoyl)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol (2d). A white solid substance with a yield of
42% and recrystallized from methanol. Mp 181–2 °C. UV (CH3OH)
nm: 201 (log e: 4.26), 246 (log e: 4.15) and 275 nm. (log e: 4.23);
4.1.3.6. 3-(2-Thienyl)-2-thiocarbamoyl-2,3,4,5,6,7-hexahydro-1H-
indazol (2a). A yellow solid substance with a yield of 55% and
recrystallized from methanol. Mp 191–2 °C. UV (CH3OH) nm: 202
IR (KBr) cmꢀ1; 3360, 3074, 2975, 2938, 2860, 16, 1526, 1320,
1257, 1116, 1149; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J in Hz):
0.60 (1H; dq; H4ax), 1.11 (1H; tq; H5ax), 1.32 (1H; m; H6ax) 1.65
(2H; m; H4eq, H5eq), 1.89 (1H; m; H6eq), 2.29 (1H; dt; H7ax), 2.61
(1H; d; H7eq), 3. (1H; m; H3a), 4.09 (2H; m; –CH2–), 5.06 (2H; dq;
@CH2), 5.84 (1H; m; –CH@), 6.10 (1H; d; H3; J: 10.8), 6.77 (1H; d;
thiophene H4), 6.94 (1H; dd; thiophene H3), 7.33 (1H; d; thiophene
H5), 8.27 (1H; t; NH); MS (70 eV, EI): m/e (%): 305 (M+, %55.07), 290
(MꢀCH3, %45.63), 272 (MꢀSH, %84.05), 249 (MꢀC3H6N, %88.40), 123
(MꢀC9H14N2S, 100%), 97 (MꢀC10H14N3S%66.66). Anal. Calcd for
(log e: 4.31) and 344 nm. (log e
: 4.37); IR (KBr) cmꢀ1; 3401, 3236,
31, 2926,2861, 1585, 1500, 1278, 1205, 1071; 1H NMR (DMSO-d6,
400 MHz) d (ppm): 1.63 (4H; m; H5ax, H5eq, H6ax, H6eq), 2.50 (6H;
m; H4ax, H4eq), 2.63 (2H; m; H7ax, H7eq), 7.14 (H; dd; furan H3),
7.30 (1H; d; furan H4), 7.65 (2H; m; H3-furan H5), 7.90 and 8.27
(1H; sb; NH2), 9.97 (1H; s; H1); MS (70 eV, EI): m/e (%):265 (M+,
%64.03), 232 (MꢀSH, %39.60), 205 (MꢀCSNH2, %98.52), 189(,100%),

6770
N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
C15H19N3S2: C, 58.98; H, 6.27; N, 13.76; S, 20.99. Found: C, 59.17; H,
6.086; N, 13.73; S, 21.05.
mixture was preincubated at 37 °C for 10 min before the addition
of enzyme. The reaction was initiated by adding the homogenate
(100 ll), and an increase in absorbance was monitored at 498 nm
4.1.3.10. 3-(2-Thienyl)-2-(N-phenylthiocarbamoyl)-3,3a,4,5,6,7-
hexahydro-2H-indazol (2e). A yellowish white solid substance
with a yield of 70% and recrystallized from methanol. Mp: 140–
at 37 °C for 60 min. A molar absorption coefficient of 4654 Mꢀ1
cmꢀ1 was used to calculate the initial velocity of the reaction.
Results were expressed as nmol hꢀ1 mgꢀ1
.
1 °C. UV (CH3OH) nm: 202 (log e: 4.50) and 278 nm (log e: 4.32);
IR (KBr) cmꢀ1; 3313, 3059, 2941, 2855, 1640, 1516, 1330, 1229,
1192, 1152; 1H NMR (DMSO-d6, 400 MHz) d (ppm) (J in Hz): 1.50
(3H; m; H4ax, H5ax, H6ax), 1.75 (1H; m; H4eq), 2.03 (1H; m; H5eq),
2.22 (1H; m; H6eq), 2.45 (1H; m; H7ax), 2.60 (1H; m; H7eq), 3.05
(1H; m; H3a), 5.65 (1H; d; H3trans; J: 4.8), 6.22 (1H; d; H3cis; J:
11.2), 6.98 (1H; m; thiophene H4), 7.10 (1H; m; thiophene H3),
7,28 (2H; m; phenyl), 7.37 (1H; m; thiophene H5), 7.54 (3H; m;
phenyl), 9.96 (1H; s; NH); MS (70 eV, EI): m/e (%): 341 (M+,
4.3.3. Selective measurement of MAO-A and MAO-B activities
Homogenates were incubated with the substrate p-tyramine
(500
lowing the inhibition of one of the MAO isoforms with selective
inhibitors. Aqueous solution of clorgyline or pargyline (50 M),
lM to measure MAO-A and 2.5 mM to measure MAO-B) fol-
l
as selective MAO-A and -B inhibitors were added to homogenates
at the ratio of 1:100 (v/v), yielding the final inhibitor concentra-
tions of 0.50 lM. Homogenates were incubated with these inhibi-
%59.09), 308 (MꢀSH, 100%), 259 (MꢀC6H10
,
%21.81), 245
tors at 37 °C for 60 min prior to activity measurement. After
incubation of homogenates with selective inhibitors, total MAO
activity was determined by the method described above.
(MꢀC6H10N, %79.09), 205 (MꢀC7H6NS, %26.36). Anal. Calcd for
C15H19N3S2: C, 63.31; H, 5.61; N, 12.30; S, 18.78. Found: C, 63.48;
H, 5.457; N, 12.29; S, 18.86.
4.3.4. Analysis of the kinetic data
Newly synthesized compounds were dissolved in dimethyl sulf-
oxide (DMSO), to a maximum concentration of 1% and used in the
4.2. Single crystal X-ray crystallographic data of 1e
The data collection was performed on a STOE IPDS-II diffrac-
concentration range of 1–1000 lM. Inhibitors were incubated with
tometer with graphite-monochromated MoK
a
radiation
the purified MAO at 37 °C for 0–60 min prior to adding them to the
assay mixture. The reversibility of the inhibition of the enzyme by
novel compounds was assessed by dialysis performed over 24 h at
37 °C relative to a potassium phosphate buffer, pH 7.6 capable of
restoring 98–100% of the enzyme activity.
(k = 0.71073 Å) at 296 K. Crystallographic and refinement parame-
ters are summarized in Table 1.
The structure was solved by direct methods using SHELXS-9737
and refined by full-matrix least-squares procedures on F2, using
the program SHELXL-97.37 All non-hydrogen atoms were refined
anisotropically. All hydrogen atom positions were refined using a
riding model. An empirical w scan absorption correction was ap-
plied. Molecular diagram (Fig. 2) was created using ORTEP-III.38
Geometric calculations were performed with PLATON.39
Crystallographic data for compound 1e reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication number CCDC 717517. 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).
Kinetic data for interaction of the enzyme with the compounds
were determined using the Microsoft Excel package program. The
inhibitory activities of the novel compounds for MAO-A and -B
were determined at 37 °C after incubation of the homogenates
(previously treated with clorgyline for MAO-A or –B measurement)
with the compounds for 60 min. Lineweaver–Burk plots were used
to estimate the inhibition constant (Ki) of the inhibitors. IC50 values
were determined from plots of residual activity percentage, calcu-
lated in relation to a sample of the enzyme treated under the same
conditions without inhibitor, versus inhibitor concentration [I].
IC50 values were determined using non-linear regression analysis
according to the equation for a sigmoid plot. Logarithmic transfor-
mation was also used for the determination of IC50 values for some
compounds which showed their inhibition in a large concentration
range.
4.3. Biochemistry
All chemicals used were purchased from Sigma–Aldrich Co.
(Germany).
4.3.5. Protein determination
4.3.1. Isolation of MAO from rat liver homogenates
The protein was determined according to the Bradford meth-
The ethics Committee of Laboratory Animals at Hacettepe Uni-
versity, Turkey (2001/25-4), approved the animal experimentation.
MAO was purified from the rat liver according to the Holt method
with some modifications.40 Liver tissue was homogenized 1:40 (w/
v) in 0.3 M sucrose. Following centrifugation 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. Pel-
let 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 potas-
sium phosphate buffer, pH 7.4, kept at ꢀ70 °C until used.
od,41 in which bovine serum albumin was used as a standard.
4.4. Molecular docking
4.4.1. Protein setup
MAO-A (pdb code: 2z5x, resolution: 2.2 Å, cocrystalized with
harmine) and MAO-B (pdb code: 1s3e, resolution: 1.6 Å, cocrystal-
ized with the inhibitor 6-hydroxy-N-propargyl-1(R)-aminoindan)
were obtained from the Protein Data Bank (http://www.rcsb.
org).12,14,15,42,43 Studies were carried out on only one subunit of
the enzymes. The pdb files were edited and the b-chains were re-
moved together with their irreversible inhibitors. All the water
and all non-interacting ions were also removed.
4.3.2. Measurement of MAO activity
Total MAO activity was measured spectrophotometrically
according to the Holt method.40 The assay mixture contained a
4.4.2. Simulations of enzymes
chromogenic solution consisting 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
Cleaned MAO-A and MAO-B and their cofactors FAD are equili-
brated via energy minimization and Molecular Dynamics (MD)
using NAMD v2.6 simulation package.44 MD simulations were per-
formed at constant NPT at 310 K using Langevin dynamics for all
167
ll chromogenic solution, 667
ll substrate solution (500 lM
p-tyramine) and 133
l
l potassium phosphate buffer, pH 7.6. The

N. Gökhan-Kelekçi et al. / Bioorg. Med. Chem. 17 (2009) 6761–6772
6771
non-hydrogen atoms, with a Langevin damping coefficient of
5 psꢀ1. The system was kept at a constant pressure of 1 atm by
using a Nose–Hoover Langevin piston45 with a period of 100 fs
and damping timescale of 50 ps. To simulate the cytoplasmic envi-
ronment, the system was first solvated in a water box with dimen-
sions of 107.6 Å ꢂ 96.5 Å ꢂ 84.2 Å and ions are added to make the
overall system neutral using the plug-ins of VMD molecular visual-
ization program (http://www.ks.uiuc.edu/Research/vmd).46 The
system is comprised of the protein and its cofactor with 8253
atoms, 74458 water molecules and 4 ions.
The resultant structure files were analyzed by using DISCOVERY
STUDIO VISUALIZER 2.1 (http://accelrys.com) visualization program.
Acknowledgment
This study was supported by the Hacettepe University Research
Fund (08D09301002 (4686)).
References and notes
CHARMM22 forcefield47,48 was used to describe the interaction
potential of the protein, and waters are treated explicitly using
TIP3P model.49 Long-range electrostatic interactions were treated
by the particle mesh Ewald (PME) method with a grid point density
of over 1/Å. A cutoff of 12 Å was used for van der Waals and short-
range electrostatics interactions; a switching function was started
at 10 Å for van der Waals interactions to ensure a smooth cutoff.
Simulation was performed under periodic boundary conditions to
prevent surface effects. The hydrogen–oxygen and hydrogen–
hydrogen distances in waters are constrained to the nominal
length or angle specified in the parameter file, making the water
molecules completely rigid. Also, the bond between each hydrogen
and the (one) atom to which it is bonded is similarly constrained.
Time step was 2 fs and the data was taken every 1 ps. The num-
ber of time steps between each full electrostatics evaluation is set
to 2. Short-range non-bonded interactions are calculated every
time step.
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4.4.3. Ligand setups
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in dockings. In all docking a grid box size of 80 ꢂ 80 ꢂ 80 points in
x, y, and z directions was built, and because the location of the inhib-
itor in the complex was known, the maps were centered on the N5
atom of FAD in the catalytic site of the protein. A grid spacing of
0.375 Å (approximately one forth of the length of a carbon–carbon
covalent bond) and a distances-dependent function 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 initial population of 50 randomly
placed individuals, a maximum number of 2.5 ꢂ 107 energy evalua-
tions, and a maximum number of 2.7 ꢂ 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 positional 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.
From the ‘Estimated Free Energy of Binding’ (kcal/mol) program
calculates the inhibition constants taking into consideration of the
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