Design, Synthesis, invitro MAO-B Inhibitory Evaluation, and Computational Studies of Some 6-Nitrobenzothiazole-Derived Semicarbazones

Article abstract of DOI:10.1002/cmdc.201200484

Monoamine oxidaseB (MAO-B) is an important drug target for the treatment of neurological disorders. A series of 6-nitrobenzothiazole-derived semicarbazones were designed, synthesized, and evaluated as inhibitors of the rat brain MAO-B isoenzyme. Most of the compounds were found to be potent inhibitors of MAO-B, with IC₅₀ values in the nanomolar to micromolar range. Molecular docking studies were performed with AutoDock4.2 to deduce the affinity and binding mode of these inhibitors toward the MAO-B active site. The free energies of binding (ΔG) and inhibition constants (K_i) of the docked compounds were calculated by the Lamarckian genetic algorithm (LGA) of AutoDock4.2. Good correlations between the calculated and experimental results were obtained. 1-[(4-Chlorophenyl)(phenyl)methylene]-4-(6-nitrobenzothiazol-2-yl)semicarbazide emerged as the lead MAO-B inhibitor, with top ranking in both the experimental MAO-B assay (IC₅₀: 0.004±0.001μM) and in computational docking studies (K_i: 1.08μM). Binding mode analysis of potent inhibitors suggests that these compounds are well accommodated by the MAO-B active site through stable hydrophobic and hydrogen bonding interactions. Interestingly, the 6-nitrobenzothiazole moiety is stabilized in the substrate cavity with the aryl or diaryl residues extending up into the entrance cavity of the active site. According to our results, docking experiments could be an interesting approach for predicting the activity and binding interactions of this class of semicarbazones against MAO-B. Thus, a binding site model consisting of three essential pharmacophoric features is proposed, and this can be used for the design of future MAO-B inhibitors.

Full text of DOI:10.1002/cmdc.201200484

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DOI: 10.1002/cmdc.201200484
Design, Synthesis, in vitro MAO-B Inhibitory Evaluation,
and Computational Studies of Some 6-Nitrobenzothiazole-
Derived Semicarbazones
Rati K. P. Tripathi, Omprakash Goshain, and Senthil Raja Ayyannan*[a]
Monoamine oxidase B (MAO-B) is an important drug target for
the treatment of neurological disorders. A series of 6-nitroben-
zothiazole-derived semicarbazones were designed, synthesized,
and evaluated as inhibitors of the rat brain MAO-B isoenzyme.
Most of the compounds were found to be potent inhibitors of
MAO-B, with IC50 values in the nanomolar to micromolar range.
Molecular docking studies were performed with AutoDock 4.2
to deduce the affinity and binding mode of these inhibitors
toward the MAO-B active site. The free energies of binding
(DG) and inhibition constants (Ki) of the docked compounds
were calculated by the Lamarckian genetic algorithm (LGA) of
AutoDock 4.2. Good correlations between the calculated and
experimental results were obtained. 1-[(4-Chlorophenyl)-
(phenyl)methylene]-4-(6-nitrobenzothiazol-2-yl)semicarbazide
emerged as the lead MAO-B inhibitor, with top ranking in both
the experimental MAO-B assay (IC50: 0.004Æ0.001 mm) and in
computational docking studies (Ki: 1.08 mm). Binding mode
analysis of potent inhibitors suggests that these compounds
are well accommodated by the MAO-B active site through
stable hydrophobic and hydrogen bonding interactions. Inter-
estingly, the 6-nitrobenzothiazole moiety is stabilized in the
substrate cavity with the aryl or diaryl residues extending up
into the entrance cavity of the active site. According to our re-
sults, docking experiments could be an interesting approach
for predicting the activity and binding interactions of this class
of semicarbazones against MAO-B. Thus, a binding site model
consisting of three essential pharmacophoric features is pro-
posed, and this can be used for the design of future MAO-B in-
hibitors.
Introduction
Monoamine oxidases (MAOs; amine–oxygen oxidoreductase;
EC 1.4.3.4) contain the flavin adenine dinucleotide (FAD) cofac-
tor covalently bonded to a cysteine residue,[1] and are involved
in catalyzing the oxidative deamination of monoamine neuro-
transmitters, resulting in the modulation of their concentra-
tions in brain and peripheral tissues.[2,3] In mammals, MAOs are
present in two isoenzyme forms, namely MAO-A and MAO-B,
which are located predominantly in the outer mitochondrial
membrane of neuronal, glial, and other cells.[4] Physiologically,
MAOs cause oxidation of biogenic neurotransmitters such as
dopamine, norepinephrine, 5-hydroxytryptamine (5-HT, seroto-
nin), and b-phenethylamine, as well as xenobiotic amines such
as tyramine and benzylamine.[5,6] MAO-A has greater affinity for
serotonin and norepinephrine and is more sensitive to inhibi-
tors like clorgyline and moclobemide. On the other hand,
MAO-B is selectively inhibited by low concentrations of selegi-
line (l-deprenyl) and rasagiline.[7,8]
ble for mitochondrial damage and neurodegenerative distur-
bances. Therefore, the development of MAO inhibitors (MAOIs)
represents a very important and useful approach for the treat-
ment of various neurological and neuropsychiatric disor-
ders.[9,10] Nonselective or irreversible inhibitors have shown var-
ious drawbacks such as cumulative effects and the so-called
“cheese effect”, whereby the catabolism of ingested amines is
blocked; these have rendered the therapeutic use of nonselec-
tive MAOIs obsolete. MAO-A-selective inhibitors are used for
the treatment of mental disorders, and have found use as anti-
depressants and anxiolytics,[11–16] whereas MAO-B-selective in-
hibitors are used as adjuncts for the treatment of Parkinson’s,
Alzheimer’s, and Huntington’s diseases.[17–23]
The determination of the crystal structure of MAO-B by
Binda and colleagues has resolved the catalytic mechanism in-
volved in the selective interactions between the enzymes and
their ligands or substrates, and has provided a better under-
standing of the pharmacophoric requirements for the rational
design of potent and selective inhibitors with therapeutic po-
tential.[24] The RCSB Protein Data Bank (PDB) availability of ex-
perimentally determined co-crystals of MAO-B with various in-
hibitors has allowed researchers to also perform computational
studies with the aim of proposing preferred binding modes,
thereby aiding in the rational design of new MAO-B inhibitors.
There have been reports on the influence of hydrophobicity
at the active site on the affinity of various types of MAO inhibi-
tors or substrates.[25,26] A wide variety of structurally unrelated
MAO-mediated oxidations lead to the formation of aldehyde
as the main product along with several neurotoxic chemical
species such as hydrogen peroxide and ammonia as byprod-
ucts. Given the formation of these neurotoxic byproducts, pro-
longed or excessive activity of these enzymes may be responsi-
[a] R. K. P. Tripathi, O. Goshain, S. R. Ayyannan
21, Pharmaceutical Chemistry Research Laboratory
Department of Pharmaceutics, Indian Institute of Technology
Banaras Hindu University, Varanasi 221005, Uttar Pradesh (India)
E-mail: asraja.phe@itbhu.ac.in
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selective inhibitors of MAO containing a basic nitro-
gen atom held in a relatively rigid environment, e.g.,
as part of a heterocycle, are well known.[27–29] A di-
verse class of MAO-B inhibitors including aryl hydra-
zines[30–32] and their heterocyclic analogues[33,34] bear-
ing thiazole and pyrazole rings have been studied,
and their specific mechanism of action has been fully
investigated. Several aryl hydrazones and semicarba-
zones possessing lipophilic substituents such as
a benzyloxy group were found to possess activity
against MAO-B with IC50 values in the range of 10À8
to >10À5 m.[35] The structures of some heterocyclic
MAO-B inhibitors and aryl hydrazones and semicarba-
zones are shown in Figure 1 (compounds 1–6).[36–39]
The extraction of pharmacophore features of
Figure 2. Design of heterocyclic semicarbazones bearing 6-nitrobenzothiazole and isatin
moieties.
depicted in Figure 2. As a stable electron-rich region, the versa-
known potent MAO-B inhibitors has revealed the importance
tile semicarbazone moiety can serve as a hydrogen bonding
site, whereas the substituted aryl
systems at the amido and carbi-
mino termini can offer hydro-
phobic interactions to the active
site of MAO-B. The work present-
ed herein includes the synthesis,
in vitro MAO-B inhibition, and
computational studies of 6-nitro-
benzothiazole-derived semicar-
bazones 10–34 along with a com-
parison of their MAO-B binding
and inhibitory potential against
those of the reference drugs.
Computational analysis of
MAO-B inhibitors by the docking
Figure 1. Structures of some heterocycles, hydrazones, and semicarbazones possessing potent MAO-B inhibitory
simulation
software
Auto-
properties.
Dock 4.2 was performed to gain
structural insight into the bind-
ing modes and possible interac-
of two elements: namely hydrophobic and hydrogen bonding
(acceptor and donor) sites in their structures. Further in silico
modeling studies of known MAO-B inhibitors against MAO-B
have suggested that these compounds occupy both the sub-
strate and entrance cavities, and a majority of them are stabi-
lized through hydrophobic and hydrogen bonding interactions
with the complementary residues in the active site.[40]
tions of the inhibitor compounds with the active site of human
recombinant MAO-B and to determine the binding orientation,
free energy of binding (DG), and inhibition constants (Ki) of the
experimentally tested MAO-B inhibitors.
Results and Discussion
Semicarbazones[41,42] possessing a R-NH-(C=O)-NH-N=C-R1R2
moiety have been well documented as novel scaffolds for the
development of new therapeutic agents for the treatment of
CNS disorders. Both isatin-[41a] and benzothiazole-derived[42]
semicarbazones have been reported to possess potential anti-
convulsant and antidepressant activities with no or minimal
neurotoxicity. On the other hand, isatin (indol-2,3-dione), an
endogenous compound, was found to possess both MAO-A
and MAO-B inhibitory properties with no neurotoxicity.[43] With
a view to identify novel heterocyclic compounds as potent
MAO-B inhibitors that could serve as potential lead molecules
for drug discovery, we designed a series of heteroaryl semicar-
bazones by incorporating 6-nitrobenzothiazole and arylidene,
diarylmethylene, and isatin residues at amido and carbimino
termini in the semicarbazone scaffold. The design approach is
Chemistry
(6-Nitro-1,3-benzothiazol-2-yl)semicarbazones 10–30 were syn-
thesized by the method outlined in Scheme 1 from 1-(6-nitro-
1,3-benzothiazol-2-yl)semicarbazide (9).[42] The intermediate 1-
(6-nitro-1,3-benzothiazol-2-yl)urea (8) was prepared by treating
2-amino-6-nitrobenzothiazole (7) with aqueous sodium cyanate
in the presence of glacial acetic acid; compound 9 was ob-
tained by treating 1-(6-nitro-1,3-benzothiazol-2-yl)urea (18)
with hydrazine hydrate at reflux. The final products 10–34
were obtained in good yields by condensation of compound 9
with the appropriate aldehydes or ketones.
Isatin-substituted semicarbazones 31–34 were synthesized
by the conventional condensation reaction between com-
pound 9 and 5-(un)substituted isatin in ethanol (Scheme 2).[42]
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lowed by 25 (1-[1-(4-fluorophenyl)ethylidene]-4-(6-nitrobenzo-
thiazol-2-yl)semicarbazide, IC50 =0.017Æ0.005 mm) and 27 (1-
(diphenylmethylene)-4-(6-nitrobenzothiazol-2-yl)semicarbazide,
IC50 =0.018Æ0.002 mm). All three inhibitors were found to be
more potent than selegiline.
In general, improvement in MAO-B inhibitory activity was
observed by substituting the H atom with a methyl group at
R2 (compare 23–25, 27, 28, and 10–22). Particularly compound
28 (IC50 =0.004Æ0.001 mm) was found to be nearly fivefold
more potent than selegiline (IC50 =0.020Æ0.008 mm). An in-
crease in the methoxy substitutions on the phenyl ring (at the
carbimino terminus) caused a decrease in activity toward
MAO-B (compare 15, 20, 21, and 22 with 23–25, 27, and 28).
In contrast, introduction of a fluoro or chloro group at the
para position of the phenyl ring at R1 along with the methyl
group at R2 (25 and 28) increased potency. Thus, substitution
of fluoro, bromo, and chloro groups on the distal phenyl ring,
preferably at the para position, enhanced MAO-B inhibitory ac-
tivity, whereas the presence of bulkier substituents such as me-
thoxy or nitro groups caused decreased activity.
Scheme 1. Synthesis of compounds 10–30. Reagents and conditions: a) Gla-
cial AcOH/NaOCN, RT, 3 h; b) EtOH, NH2NH2·H2O, reflux, 6–7 h; c) EtOH,
reflux, glacial AcOH, 30–52 h.
Among the isatin-3-substituted semicarbazones 31–34, 5-ni-
troisatin derivative 34 showed the most potent MAO-B inhibi-
tion followed by bromo- and chloro-substituted derivatives 32
and 33, respectively. The unsubstituted derivative 30 was
found to be least active. A more detailed discussion on the
SAR of these inhibitors is included along with their computa-
tional binding and experimental activity data in the next sec-
tion. The synthesized compounds were also assayed for their in
vitro MAO-A inhibitory activity, and the data (unpublished re-
sults) revealed that some of the compounds (namely, 18, 28,
27, and 33) showed good selectivity (56- to 102-fold) toward
the MAO-B isoenzyme.
Scheme 2. Synthesis of compounds 31–34. Reagents and conditions: a) EtOH,
glacial AcOH, reflux, 10–30 h.
Computational studies
Analytical and spectral data for all synthesized compounds are
in good agreement with their composition. Physicochemical
characteristic data for all final compounds 10–34, along with
their MAO-B IC50 values, are listed in Table 1.
In silico docking studies were carried out to evaluate the affini-
ty and binding interactions of experimentally tested 6-nitro-
benzothiazole-derived semicarbazones 10–34 against human
recombinant MAO-B. The structural and binding mode analy-
ses of compounds 10–34 with MAO-B were carried out under
an automated molecular docking simulation by AutoDock 4.2.
The best-scoring molecules from the largest cluster were con-
sidered for further structural and interaction studies. The theo-
retical Ki values and free energies of binding are listed in
Table 2.
In vitro MAO-B inhibition studies
Table 1 lists the in vitro rat brain MAO-B inhibitory activity of
final compounds 10–34 as well as the reference compounds
selegiline, safinamide, and isatin. The inhibition data are ex-
pressed as IC50 values. All the synthesized compounds 10–34
showed MAO-B inhibition in the micromolar to nanomolar
range; IC50 values toward MAO-B range from 0.004Æ0.001 mm
for compound 28 to 2.154Æ0.095 mm for compound 31.
Indeed, structural modifications attempted in all compounds
of the series generally resulted in improved activity. In particu-
lar, enhancement of lipophilicity by incorporating monoaryl or
diaryl rings or an isatin residue at the carbimino terminus of
semicarbazone resulted in increased inhibitory activity against
MAO-B.
To validate the docking protocol, the AutoDock-generated
docked pose of the reference inhibitor safinamide (2-[({4-[(3-
fluorophenyl)methoxy]phenyl}methyl)amino]propanamide) into
the MAO-B active site was compared with the orientation of
co-crystallized safinamide (gold standard pose, GSP) MAO-B
complex (PDB ID: 2V5Z) (Figure 3a). The docked safinamide
shared a similar orientation and binding mode as those of the
co-crystallized safinamide. The binding orientation of safina-
mide in MAO-B traverses both entrance and substrate cavities.
The nonpolar 3-fluorobenzyloxy moiety is located in the en-
trance cavity space surrounded by residues Pro102, Leu164,
Phe168, Ile198, Ile199, Trp199, Ile316, and Tyr326, whereas
Compound 28 (1-[(4-chlorophenyl)(phenyl)methylene]-4-(6-
nitrobenzothiazol-2-yl)semicarbazide, IC50 =0.004Æ0.001 mm)
emerged as the most active MAO-B inhibitor in the study, fol-
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found with inhibitors 13 and 34,
possessing polar hydroxy and
nitro groups respectively, for
which the binding orientation is
completely the reverse; eventu-
ally these inhibitors showed
lower binding affinity than the
others.
Table 1. Physicochemical characteristics and MAO-B inhibitory data for compounds 10–34.
Poor binding affinity and
higher Ki values are observed
with inhibitors 13, 21, 16, 20,
14, 10, and 22, which bear one
or more alkoxy, nitro, or hydroxy
substituents on the distal aryl
rings present at the carbimino
terminus of semicarbazones. In
contrast, the compounds bear-
ing either a chloro or bromo
substituent on the carbimino
terminal aryl ring (12, 17, 18, 28,
and 30) displayed better binding
affinity toward MAO-B. This
clearly demonstrates the role of
Compd
R1
R2
R3
Yield [%]
Rf[a]
IC50 [mm][b]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CH3
C6H5
CH3
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
75.20
56.67
58.67
49.02
49.85
54.87
78.89
64.94
68.36
56.19
69.90
63.56
68.78
62.97
57.25
65.43
58.78
53.45
62.56
76.45
73.34
72.59
75.92
76.22
74.34
–
0.75
0.74
0.71
0.68
0.66
0.72
0.71
0.69
0.72
0.73
0.73
0.67
0.70
0.71
0.69
0.63
0.76
0.71
0.66
0.77
0.74
0.61
0.72
0.70
0.65
–
0.132Æ0.027
0.069Æ0.006
0.095Æ0.067
0.133Æ0.016
0.299Æ0.051
0.286Æ0.081
0.064Æ0.001
0.406Æ0.023
0.063Æ0.018
0.126Æ0.009
0.091Æ0.004
0.757Æ0.052
0.836Æ0.192
0.022Æ0.004
0.054Æ0.003
0.017Æ0.005
1.070Æ0.647
0.018Æ0.002
0.004Æ0.001
0.114Æ0.035
0.512Æ0.002
2.154Æ0.095
0.055Æ0.010
0.188Æ0.024
0.029Æ0.007
0.020Æ0.008
0.100
4-Br C6H4
4-OH C6H4
4-NO2 C6H4
4-OCH3 C6H4
4-N(CH3)2 C6H4
2,3-Cl2 C6H3
2,6-Cl2 C6H3
2,4-Cl2 C6H3
3,4-(OCH3)2 C6H3
2,5-(OCH3)2 C6H3
3,4,5-(OCH3)3 C6H2
C6H5
4-Br C6H4
4-F C6H4
4-OH C6H4
C6H5
4-Cl C6H4
4-OH C6H4
4-Cl C6H4
–
electronegative
chloro
and
bromo substituents, which en-
hance the stabilization of hydro-
phobic aryl rings into the com-
plementary regions of the en-
trance cavity. In addition, the
substitution of the H atom at R2
(compound 13) with a methyl
group (compounds 28, 27, and
25) caused improved binding
and inhibitory potency. Com-
pared with the monoaryl-substi-
tuted derivatives (at the carbimi-
no terminus), the diaryl deriva-
tives showed better binding and
Ki values, which may be due to
the additional hydrophobic in-
teractions with the complemen-
CH3
C6H5
C6H5
C6H5
–
–
–
–
4-Cl C6H4
–
–
–
–
–
–
–
–
H
Br
Cl
NO2
–
–
–
–
–
–
–
34
selegiline
safinamide[48a]
isatin[43]
–
–
–
–
–
–
3.00
[a] Solvent system: CH3Cl/CH3OH/toluene 5:4:1. [b] Values represent the assay drug concentration that give
50% inhibition of MAO-B activity, and are the mean ÆSEM; statistical significance: p<0.05 versus the corre-
sponding IC50 values obtained against MAO-B, as determined by ANOVA/Dunnett’s test.
the polar propanamidyl moiety occupies the substrate cavity
embedded around Tyr60, Cys172, Tyr188, Gln206, Phe343,
Tyr398, and Tyr435.
tary hydrophobic residues of the entrance cavity.
Semicarbazones 31–34 possessing both benzothiazole and
isatin residues at amido and carbimino termini, respectively,
generally showed a similar binding orientation, but higher af-
finity toward MAO-B than the mono- or diaryl-substituted com-
pounds. The ranking of these inhibitors according to computa-
tional data is 32 > 33 > 31 > 34, whereas the ranking as per
the experimental inhibitory data is 34 > 32 > 33 > 31. Inter-
estingly, among compounds 31–33, an excellent correlation
between virtual and experimental data is observed. Except for
the binding mode of 34, in which the isatin group is stabilized
into substrate cavity while the benzothiazole is caged in the
entrance cavity, all others (compounds 31–33) shared similar
binding modes as those of 11–30 except compound 13. This
rather unusual reversed binding mode for 34 may be due to
the presence of a 5-nitro substituent, which enhances the po-
Visual inspection of computationally docked binding poses
of all compounds within the active site of MAO-B resulted in
the following observations: All test compounds are well stabi-
lized in both the substrate and entrance cavities of the MAO-B
active site. The binding mode of best-scored inhibitors further
suggests that the rigid hydrophobic 6-nitrobenzothiazole
moiety is caged into the substrate cavity, while the hydropho-
bic arylidene or diarylmethylene or isatin residue present in
the carbimino terminus is stabilized in the entrance cavity. The
selective and preferential stabilization of the 6-nitrobenzothia-
zole moiety may be due to the presence of a polar nitro
group, which may be predicted to interact with the water mol-
ecules present in the substrate cavity. A few exceptions are
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hibitors are stabilized by p–p stacking and hydrogen bonding
interactions.
Table 2. Free energies of binding and calculated inhibition constants for
compounds 10–34, listed in ranking order.
All compounds showed p–p interactions; a p–p interaction
with the Tyr398 residue are observed for compounds 10, 12–
14, 16–21, 24–28, 31–33, and 34, and with Tyr435 for com-
pounds 11, 13, 15, 17, 18, 20, 22, 23, 25–30, 33, and 34. More-
over, 15 and 22 were found to undergo p–p interactions with
Tyr326. Moreover, compounds 12 and 18 are stabilized by p–
s and p–cation interactions, respectively. A p–s interaction
with FAD was observed for compounds 19–24, 26, 29, and 32.
Thus, in most of the potent compounds, preferably p–p hydro-
phobic and hydrogen bonding interactions were observed to
be responsible for mediating biological activity. A graphical in-
spection was made to identify the possible binding orientation
and interactions of 10–34 within the MAO-B active site. We fo-
cused our attention only on the binding modes of the most
active compounds 28, 27, 18, and 32.
Rank
Compd
DG [kcalmolÀ1
]
Ki [mm]
1
2
3
4
5
6
7
8
28
27
18
29
30
32
33
23
24
26
31
19
34
12
15
17
25
11
13
21
16
20
14
10
À8.14
À7.99
À7.92
À7.73
À7.73
À7.60
À7.58
À7.11
À7.08
À7.01
À6.93
À6.92
À6.79
À6.78
À6.75
À6.67
À6.64
À6.62
À6.39
À6.19
À6.13
À6.04
À5.67
À5.46
À5.43
À5.93
À4.74
1.08
1.39
1.55
2.15
2.15
2.69
2.77
6.16
6.43
7.32
8.39
8.47
10.51
10.8
11.24
12.94
13.6
13.94
20.79
28.89
32.07
37.69
70.36
100.27
104.38
45.23
333.71
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
–
Binding mode of 28 and 27
Examination of one of the best-ranked docking solutions of 28
(Figure 3c) and 27 (Figure 3d) revealed that the 6-nitrobenzo-
thiazole nucleus is located in the substrate cavity, while the
carbimino side chain is extended toward the entrance cavity of
MAO-B. Furthermore, the diarylidene-bearing carbimino side
chain of 28 occupies the entrance hydrophobic cleft formed
by Pro102, Pro104, Trp119, Leu164, Ile198, Ile199, Thr201,
Ile316, and Tyr326, whereas the benzothiazole moiety in the
substrate cavity is embedded in a large hydrophobic cage
formed by residues Ser59, Tyr60, Cys172, Gln206, Tyr398, and
Tyr435. The benzene and thiazole rings of the benzothiazole
nucleus are involved in a p–p interaction, with Tyr398 at inter-
plane distances of ~3.81 and ~4.99 ꢁ, respectively. In addition,
a p–p interaction was also observed between Tyr435 and the
benzene ring of benzothiazole, with a distance of ~5.24 ꢁ. Im-
portantly, the chlorine atom of 28 is stabilized by hydrogen
bond interaction with the hydroxy group of Thr201 (d=
2.49 ꢁ) located in the entrance cavity. Because the addition of
chlorine would enhance the lipophilicity of a phenyl ring,
these interactions may explain the observation that chlorine
substitution of the phenyl side chain of 28 enhances MAO-B
inhibitory potency. Besides this, electrostatic interactions with
Leu164 and Tyr326 were also evident (Figure 3c).
22
safinamide[a]
isatin
–
[a] IC50 =100 nm.[48a]
larity of the hydrophobic isatin residue (even more polar than
the 6-nitrobenzothiazole ring), thereby guiding it to the sub-
strate cavity. Furthermore, the binding mode of 34 is found to
match that of isatin (PDB ID: 1OJA), and this may be the
reason for its greater potency in the experimental MAO-B
assay (IC50 =0.029Æ0.007 mm) than others (compounds 31–
33). The superimposed binding modes of 31–34 along with
the reference GSP of isatin are shown in Figure 3b.
Important is the observation that the 6-nitro group present
on the benzothiazole moiety may be involved in interactions—
possibly hydrogen bonding—with the fluxional water mole-
cules present in the large substrate cavity of the MAO-B active
site. This interaction may be responsible for the preferential
and selective orientation of 6-nitrobenzothiazole (for 10–33)
and 5-nitroisatin (for 34) moieties into the substrate cavity.
Further investigation of the interactions of all inhibitors 10–
34 with the anchoring amino acid residues present in the
active site of MAO-B led to the following observations: The po-
tential binding sites of these inhibitors were found to be occu-
pied in the wide active site cavity surrounded by residues
Ser59, Tyr60, Phe168, Leu171, Cys172, Tyr188, Ile199, Gln206,
Lys 296, Tyr326, Tyr398, and Tyr435; this is very similar to the
binding sites of the standard inhibitor safinamide. This proves
that the effective binding sites are present in the 6-nitrobenzo-
thiazole-derived semicarbazones, and that these compounds
possess the potential to inhibit MAO-B. Furthermore, these in-
Conversely, the benzene moiety of 27 (Figure 3d) is possibly
stabilized by p–p interactions with Tyr398 at an interplane dis-
tance of ~4.82 ꢁ. Varying degrees of electrostatic and van der
Waals interactions with residues Trp119, Leu164, Ile198,
Cys172, Ile199, Gln206, and Ile316 may further contribute to
the binding and stabilization of 37 in the cavity space. In addi-
tion, a hydrogen bond interaction with the oxygen atom of
Ile198 at an interplane distance of 2.49 ꢁ is observed.
Binding mode of 18
Visual inspection of the pose of 18 in the MAO-B binding site
revealed that the binding orientation is similar to that of the
above compounds. That is, the benzothiazole nucleus binds
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Figure 3. a) AutoDock-generated pose of safinamide (pink) with the GSP of safinamide (brown) (PDB ID: 2V5Z) in the MAO-B active site. b) Superimposed
binding modes of isatin-substituted MAO-B inhibitors 31 (violet), 32 (pink), 33 (yellow), and 34 (maroon) along with the GSP of isatin (green). c) Binding inter-
actions of 28 (pink) in the MAO-B active site. d) Binding interactions of 27 (pink) in the MAO-B active site. Selected residues are labeled and displayed in wire-
frame, and the FAD cofactor is shown as blue sticks in each case.
within the substrate cavity space and is surrounded by resi-
dues Ser59, Tyr60, Cys172, Gln206, Tyr398, and Tyr435,
whereas the arylidene-bearing carbimino side chain occupies
the entrance cavity and is lined by residues Pro104, Trp119,
Leu164, Phe168, Ile198, Ile199, Ile316, and Tyr326. The benzo-
thiazole system is sandwiched between the aromatic residue
Tyr435, forming p–p stacking interactions. The amino nitrogen
atom of the semicarbazone linker is stabilized by hydrogen
bond interactions with the oxygen atom of Ile198 (Figure 4a).
In addition, a p–cation interaction was observed between
Tyr326 and the carbimino nitrogen atom, with an interplane
distance of ~2.47 ꢁ.
Figure 4. a) Binding interactions of 18 (pink) in the MAO-B active site. b) Binding interactions of 32 (pink) in the MAO-B active site. c) Superimposed binding
modes of 28 (pink) and 27 (yellow) with the GSP of safinamide (orange) (PDB ID: 2V5Z) in the MAO-B active site. d) Superimposed binding modes of 18
(pink) and 32 (yellow) with the GSP of safinamide (orange) in the MAO-B active site. e) Superimposed binding modes of the most active MAO-B inhibitors 28
(pink), 27 (maroon), 18 (violet), and 32 (yellow) along with the GSP of safinamide (orange) in the MAO-B active site. Selected residues are labeled and dis-
played in wireframe, and the FAD cofactor is shown as blue sticks in each case.
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Binding mode of 32
range. Computational docking studies with AutoDock 4.2 were
carried out for the first time on semicarbazone-based MAO-B
inhibitors against MAO-B. The comparative binding mode anal-
ysis of potent MAO-B inhibitors suggests that the optimal
binding to the large hydrophobic pocket of the substrate
cavity requires a large, rigid heterocyclic ring substituted with
a polar nitro group. This polar nitro group may guide and sta-
bilize the benzothiazole ring to the substrate cavity, possibly
through interaction with fluxional water molecules present in
this cavity. On the other hand, the entrance cavity requires
chloro- or bromo-substituted mono or diaryl rings for effective
binding and stabilization. Both chloro and bromo substituents
on the aryl ring at the carbimino terminus of the semicarba-
zones enhanced MAO-B inhibitory potencies. Thus, the design
approach used on the semicarbazone scaffold produced
potent MAO-B inhibitors. Compound 28 (1-[(4-chlorophenyl)-
(phenyl)methylene]-4-(6-nitrobenzothiazol-2-yl)semicarbazide)
emerged as the lead MAO-B inhibitor, which ranked at the top
in both experimental MAO-B assays (IC50: 0.004Æ0.001 mm)
and computational docking studies (Ki: 1.08 mm). The potential
binding of this lead inhibitor may be attributed to the pres-
ence of a large hydrophobic aryl binding site in the form of 6-
nitrobenzothiazole and a diaryl binding sites at the amido and
carbimino termini, respectively.
Analysis of the most active benzothiazole- and isatin-derived
inhibitor 32 in complex with MAO-B revealed that the benzo-
thiazole ring is oriented in the substrate cavity, whereas the
carbimino side chain bearing the isatin residue is directed
toward the entrance cavity (Figure 4b). This orientation contra-
dicts that of the most active inhibitor 34 of same class. The
isatin moiety stabilized in the entrance cavity is surrounded by
residues Trp119, Pro104, Leu164, Phe168, Ile198, Ile199,
Ile316, and Tyr326. The benzothiazole ring is inserted into the
substrate cavity, forming an aromatic cage framed by Ser59,
Tyr60, Gln206, Tyr398, and Tyr435, and the binding is further
stabilized by p–p interactions between Tyr435 and the ben-
zene ring at an interplane distance of ~4.85 ꢁ. Moreover, two
hydrogen bonds are also observed for the carbonyl oxygen
atom of the isatin moiety and the carbonyl oxygen of Ile199,
and between the carbonyl oxygen of the carbimino side chain
and the hydroxy group of Tyr326. In addition, a p–cation inter-
action between the amino nitrogen atom of the side chain
and Tyr326 is also observed, with an interplane distance of
5.36 ꢁ. It is observed that the bromine atom of 32 undergoes
significant van der Waals interactions with Leu164, Phe168,
and Ile316. However, the lower affinity of 32 relative to 28, 27,
and 18 may be due to the steric bulk imparted by the large
hydrophobic isatin moiety. (Figure 4b).
By the analysis of experimental and computational data, the
following significant pharmacophoric elements influencing the
binding affinity and potency of these MAO-B inhibitors are
identified: 1) A large hydrophobic heterocyclic ring preferably
substituted with a polar nitro group at the amido terminus of
semicarbazone is essential for binding with the substrate
cavity of the MAO-B active site. 2) Another hydrophobic aryl
ring with electronegative substituents such as chloro and
bromo groups is crucial for effective binding and stabilization
in the entrance cavity space of MAO-B. 3) A flexible linker pos-
sessing hydrogen bonding regions is also essential for guiding
the optimal orientation of both hydrophobic aryl residues in
their respective binding pockets within the active site of MAO-
B. Thus, a binding site model consisting of three essential
pharmacophoric features is proposed (Figure 5), and this
model can be used in the design of future potent MAO-B in-
hibitors.
Thus, in this study we have tried to establish how these
compounds interact differently in the MAO-B binding pocket.
Docking of 27 showed that its stability is due to p–p interac-
tions, van der Waals, and electrostatic forces, whereas the sta-
bility of 28, 18, and 22 also includes hydrogen bonding inter-
actions along with the above interactions. Primarily to check
the congruence of the observed results with the predicted re-
sults in terms of binding modes to the MAO-B receptor, super-
imposition of 28 and 27 (Figure 4c) and 18 and 32 (Figure 4d)
was carried out separately and altogether (Figure 4e) with the
GSP of safinamide within the MAO-B active site. Good agree-
ment was observed in both cases, as evidenced from the struc-
tural screenshots.
The difference in the experimental and theoretical results is
due to the difference in the sources of MAO-B enzymes.
Hence, we did not expect to obtain exact experimental values
by computational calculations. This difference may also arise in
the approximations and simplifications made during the com-
putation process; for example, no explicit water molecules
were considered during docking simulation. Furthermore, Au-
toDock 4.2 uses empirical scoring functions for free energy cal-
culations. Considering all these factors, a very reasonable pre-
diction of IC50 values, inhibition constants (Ki values), and bind-
ing energy was obtained.
Conclusions
In this study, a series of 6-nitrobenzothiazole-based semicarba-
zones 10–34 were synthesized and evaluated as inhibitors of
MAO-B. Most synthesized semicarbazones were found to ex-
hibit inhibitory potencies in the micromolar to nanomolar
Figure 5. Proposed binding site model for MAO-B inhibitors showing the es-
sential pharmacophoric features.
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Final products
Experimental Section
General procedure for the conventional synthesis of com-
pounds 10–30
Chemistry
All commercial reagents and solvents used were of laboratory
grade, obtained from Sigma–Aldrich or Merck. Melting points were
determined in open capillary tubes in a Sonar melting point appa-
ratus and are uncorrected. IR and H and 13C NMR spectra were re-
The final compounds 10--30 (substituted monoaryl/diaryl semicar-
bazones) were synthesized by the reaction of 1-(6-nitro-1,3-benzo-
thiazol-2-yl)semicarbazide 9 (0.003 mol) with appropriate aldehydes
or ketones (0.003 mol). The reaction mixture was adjusted to pH 5–
6 by adding glacial acetic acid and held at reflux for 30–52 h.[43]
The solvent was evaporated, and the crude product was purified
and recrystallized from 95% EtOH.
1
corded in a Shimadzu FT-IR 8400S IR spectrophotometer (KBr) and
a JEOL AL300 FT-NMR spectrometer (300 MHz, [D6]DMSO), respec-
tively, at ambient temperature. Chemical shifts (d) are reported in
parts per million (ppm) using tetramethylsilane (TMS) as an internal
standard. Signals are designated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet. All exchangeable protons were confirmed by
the addition of D2O. FAB MS data were recorded on a JEOL SX
102/Da-600 mass spectrometer/data system using Ar/Xe (6 kV,
10 mA) as the FAB gas. The accelerating voltage was 10 kV, and
spectra were recorded at room temperature. Elemental analyses (C,
H, and N) were undertaken with a model CE-440 CHN analyzer
(Exeter Analytical Inc.). All products had satisfactory C, H and N
analyses (within Æ0.4%).
4-(6-Nitrobenzothiazol-2-yl)-1-(propan-2-ylidene)semicarbazide
(10): Yellow solid; mp: 2308C; IR (KBr) n˜ =3460.41 (NÀH str),
3061.13 (aromatic CÀH str), 2947.33 (alkane CÀH str), 1654.98 (C=O
str), 1570.11 (C=N str), 1535.39, 1329.00 (ArÀNO2 str), 1301.99 cmÀ1
1
(CÀN str); H NMR ([D6]DMSO, D2O exchange, 300 MHz): d=0.90 (s,
6H, CH3), 6.0 (s, 1H, CONH), 6.99 (s, 1H,=NNH, D2O exchangeable),
8.48–8.49 (d, 2H, benzothiazole CÀH), 9.05 ppm (s, 1H, benzothia-
zole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=16.4 (C’’1, CH3), 22.3
(C’1, CH3), 117.94 (C7), 120.17 (C5), 122.12 (C4), 126.96 (C7a), 145.81
(C6), 149.68 (C=N), 155.43 (C3a), 157.62 (C=O), 173.91 ppm (C2);
FAB: m/z 293.24 [M+1]+; Anal. for C11H11N5O3S: calcd: C 45.04, H
3.76, N 23.88, found: C 45.01, H 3.78, N 23.85.
Starting materials
1-Benzylidene-4-(6-nitrobenzothiazol-2-yl)semicarbazide
(11):
Yellow solid; mp: 2108C; IR (KBr): n˜ =3460.41 (NÀH str), 3074.66
2-Amino-6-nitrobenzothiazole 7 (0.03 mol) was dissolved in glacial
acetic acid (25 mL) and diluted with distilled H2O (46–50 mL). To
this, a warm solution of NaOCN (0.03 mol, 2.2 equiv) in H2O
(70 mL) was added with vigorous stirring. The mixture was stirred
for 3–4 h before being left to stand overnight. The resultant solid
was collected by filtration, washed with cold H2O to remove excess
glacial acetic acid, and dried.[43] The yellow-colored crude product
1-(6-nitro-1,3-benzothiazol-2-yl)urea 8 obtained was then recrystal-
lized from 95% EtOH. Thereafter, to a solution of 1-(6-nitrobenzo-
(aromatic CÀH), 1654.12 (C=O), 1570.11 (C=N str), 1531.53, 1329.00
1
(ArÀNO2 str), 1294.28 cmÀ1 (CÀN str); H NMR ([D6]DMSO, D2O ex-
change, 300 MHz): d=6.02 (s, 1H, CONH), 7.18 (s, 1H,=NNH, D2O
exchangeable), 7.41–7.73 (m, 8H, Ar CÀH), 8.19 (s, 1H, CÀH), 8.45–
8.49 (d, 2H, benzothiazole CÀH), 9.15 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.80 (C7), 120.13 (C5),
122.64 (C4), 125.43 (C7a), 128.89 (C’3, C’5), 129.25 (C’2, C’6), 131.08
(C’4), 133.92 (C’1), 144.31 (C=N), 145.74 (C6), 155.36 (C3a), 157.21
(C=O), 174.28 ppm (C2); FAB: m/z 341.33 [M+1]+; Anal. for
C15H11N5O3S: calcd: C 52.78, H 3.25, N 20.52, found: C 52.75, H 3.27,
N 20.50.
thiazol-2-yl)urea
8 (0.03 mol) in EtOH, NH2NH2·H2O (0.03 mol,
2.0 equiv) was added. The content was heated at reflux for 6–7 h
and cooled. The crystallized solid yellow-colored product 1-(6-
nitro-1,3-benzothiazol-2-yl)semicarbazide 9 formed was filtered,
dried, and recrystallized from 95% EtOH. The presence of single
spot in TLC denoted completion of reaction.
1-(4-Bromobenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicarba-
zide (12): Yellow solid; mp: 2378C; IR (KBr): n˜ =3510.56 (NÀH str),
3091.99 (aromatic CÀH str), 1647.26 (C=O str), 1594.88 (C=C str),
1570.11 (C=N str), 1527.67, 1327.07 (ArÀNO2 str), 1296.21 (CÀN str),
553.59 cmÀ1 (CÀBr str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=6.15 (s, 1H, CONH), 6.58 (s, 1H,=NNH, D2O ex-
changeable), 7.45–7.55 (d, 4H, Ar CÀH), 8.23 (s, 1H, CÀH), 8.25–
8.28 (d, 2H, benzothiazole CÀH), 9.19 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.76 (C7), 120.11 (C5),
122.34 (C4), 125.36 (C’4), 125.46 (C7a), 131.34 (C’2, C’6), 131.89 (C’3,
C’5), 133.06 (C’1), 144.01 (C=N), 145.83 (C6), 155.10 (C3a), 157.42
(C=O), 174.17 ppm (C2); FAB: m/z 420.04 [M+1]+; Anal. for
C15H10BrN5O3S: calcd: C 42.87, H 2.40, N 16.67, found: C 42.89, H
2.43, N 16.64.
1-(6-Nitrobenzothiazol-2-yl)urea 8: Yellow solid; mp: 224–2268C;
IR (KBr) n˜ =3514.42 (18 NÀH str), 3327.32 (28 NÀH str), 3093.92 (aro-
matic CÀH str), 2906.33 (alkane CÀH str), 1647.26 (C=O str), 1570.11
(C=N str), 1529.60, 1329.00 (ArÀNO2 str), 1298.14 cmÀ1 (CÀN str);
1H NMR ([D6]DMSO, 300 MHz): d=5.88 (s, 1H, NÀH), 6.01 (s, 2H,
NH2), 8.48–8.49 (d, 2H, benzothiazole CÀH), 9.03 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.84 (C7),
120.27 (C5), 122.16 (C4), 126.01 (C7a), 145.64 (C6), 155.81 (C3a),
156.22 (C=O), 174.31 ppm (C2); FAB: m/z 238.99 [M+1]+; Anal. for
C8H6N4O3S: calcd: C 40.33, H 2.54, N 23.52, found: C 40.35, H 2.53,
N 23.51.
1-(6-Nitrobenzothiazol-2-yl)semicarbazide 9: Yellow solid; mp:
258–2608C; IR (KBr) n˜ =3458.48 (18 NÀH str), 3394.53 (28 NÀH str),
3091.13 (aromatic CÀH str), 2941.54 (alkane CÀH str), 1651.12 (C=O
str), 1597.11 (C=N str), 1533.46, 1325.14 (ArÀNO2 str), 1294.28 cmÀ1
1-(4-Hydroxybenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicarba-
zide (13): Yellow solid; mp: 2398C; IR (KBr): n˜ =3743.96 (OÀH str),
3514.42 (NÀH str), 3097.78 (aromatic CÀH str), 1647.26 (C=O str),
1600.97 (C=C str), 1594.88 (C=N str), 1529.60, 1327.07 (ArÀNO2 str),
1294.28 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=5.12 (s, 1H, OÀH), 6.10 (s, 1H, CONH), 7.34 (s, 1H,=
NNH, D2O exchangeable), 6.83–7.55 (d, 4H, Ar CÀH), 8.24 (s, 1H, CÀ
H), 8.36–8.39 (d, 2H, benzothiazole CÀH), 9.12 ppm (s, 1H, benzo-
thiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=116.95 (C’3, C’5),
117.74 (C7), 119.34 (C5), 122.02 (C4), 126.13 (C7a), 126.74 (C’1),
130.56 (C’2, C’6), 142.68 (C=N), 146.81 (C6), 155.41 (C3a), 158.62
1
(CÀN str); H NMR ([D6]DMSO, D2O exchange, 300 MHz): d=2.23 (d,
2H, NH2), 6.08 (s, 1H, CONH), 8.48–8.49 (d, 2H, benzothiazole CÀH),
9.07 ppm (s, 1H, benzothiazole CÀH); 13C NMR ([D6]DMSO,
300 MHz): d=117.50 (C7), 120.37 (C5), 122.83 (C4), 125.56 (C7a),
145.91 (C6), 155.18 (C3a), 154.14 (C=O), 174.62 ppm (C2); FAB: m/z
254.24 [M+1]+; Anal. for C8H7N5O3S: calcd: C 37.94, H 2.79, N
27.66, found: C 37.92, H 2.83, N 27.65.
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(C=O), 160.81 (C’4), 173.81 ppm (C2); FAB: m/z 357.16 [M+1]+;
Anal. for C15H11N5O4S: calcd: C 50.42, H 3.10, N 19.60, found: C
50.45, H 3.08, N 19.57.
str), 1570.11 (C=N str), 1535.74, 1325.14 (ArÀNO2 str), 1290.42 (CÀN
str), 752.26 cmÀ1 (CÀCl str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=6.10 (s, 1H, CONH), 6.19 (s, 1H,=NNH, D2O ex-
changeable), 7.26–7.29 (m, 3H, Ar CÀH), 8.15 (s, 1H, CÀH), 8.59–
8.61 (d, 2H, benzothiazole CÀH), 9.18 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.54 (C7), 120.02 (C5),
122.41 (C4), 125.83 (C7a), 127.19 (C’3, C’5), 132.01 (C’1), 134.08
(C’4), 135.62 (C’2, C’6), 143.55 (C=N), 145.98 (C6), 155.12 (C3a),
157.07 (C=O), 174.59 ppm (C2); FAB: m/z 409.39 [M+1]+; Anal. for
C15H9Cl2N5O3S: calcd: C 43.92, H 2.21, N 17.07, found: C 43.89, H
2.23, N 17.05.
1-(4-Nitrobenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicarbazide
(14): Yellow solid; mp: 2318C; IR (KBr): n˜ =3527.92 (NÀH str),
3095.85 (aromatic CÀH str), 1647.26 (C=O str), 1596.88 (C=C str),
1570.11 (C=N str), 1525.74, 1327.07 (ArÀNO2 str), 1296.21 cmÀ1 (CÀ
N str); 1H NMR ([D6]DMSO, D2O exchange, 300 MHz): d=6.09 (s,
1H, CONH), 6.46 (s, 1H,=NNH, D2O exchangeable), 7.95–8.68 (d,
4H, Ar CÀH), 8.11 (s, 1H, CÀH), 8.41–8.48 (d, 2H, benzothiazole CÀ
H), 9.16 ppm (s, 1H, benzothiazole CÀH); 13C NMR ([D6]DMSO,
300 MHz): d=117.65 (C7), 120.91 (C5), 121.1 (C’3, C’5), 123.04 (C4),
125.43 (C7a), 130.31 (C’2, C’6), 140.05 (C’1), 144.17 (C=N), 145.86
(C6), 150.74 (C’4), 155.06 (C3a), 157.03 (C=O), 174.12 ppm (C2);
FAB: m/z 386.18 [M+1]+; Anal. for C15H10N6O5S: calcd: C 46.63, H
2.61, N 21.75, found: C 46.61, H 2.64, N 21.73.
1-(2,4-Dichlorobenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicar-
bazide (19): Yellow solid; mp: 2428C; IR (KBr): n˜ =3462.34 (NÀH
str), 3082.35 (aromatic CÀH str), 1653.05 (C=O str), 1592.95 (C=C
str), 1570.11 (C=N str), 1535.39, 1329.00 (ArÀNO2 str), 1301.99 (CÀN
str), 750.33 cmÀ1 (CÀCl str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=6.07 (s, 1H, CONH), 6.49 (s, 1H,=NNH, D2O ex-
changeable), 7.23–7.69 (m, 3H, Ar CÀH), 8.13 (s, 1H, CÀH), 8.57–
8.64 (d, 2H, benzothiazole CÀH), 9.08 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.58 (C7), 120.28 (C5),
122.46 (C4), 125.80 (C7a), 126.99 (C’5), 130.48 (C’3), 131.43 (C’1),
132.56 (C’6), 135.65 (C’2), 137.98 (C’4), 142.95 (C=N), 145.85 (C6),
155.09 (C3a), 156.87 (C=O), 174.33 ppm (C2); FAB: m/z 409.45 [M+
1]+; Anal. for C15H9Cl2N5O3S: calcd: C 43.92, H 2.21, N 17.07, found:
C 43.91, H 2.24, N 17.04.
1-(4-Methoxybenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicar-
bazide (15): Yellow solid; mp: 2578C; IR (KBr): n˜ =3460.41 (NÀH
str), 3068.85 (aromatic CÀH str), 2733.22 (alkane CÀH), 1654.98 (C=
O str), 1593.95 (C=C str), 1570.11 (C=N str), 1533.46, 1332.86 (ArÀ
NO2 str), 1294.28 (CÀN str), 1122.61 cmÀ1 (CÀO str); 1H NMR
([D6]DMSO, D2O exchange, 300 MHz): d=3.76 (s, 3H, OCH3), 6.23 (s,
1H, CONH), 6.28 (s, 1H,=NNH, D2O exchangeable), 6.87–7.63 (d,
4H, Ar CÀH), 8.20 (s, 1H, CÀH), 8.41–8.46 (d, 2H, benzothiazole CÀ
H), 9.06 ppm (s, 1H, benzothiazole CÀH); 13C NMR ([D6]DMSO,
300 MHz): d=55.93 (C’’1, OCH3), 114.35 (C’3, C’5), 117.81 (C7),
120.21 (C5), 122.46 (C4), 125.43 (C7a), 125.95 (C’1), 130.54 (C’2, C’6),
143.09 (C=N), 145.48 (C6), 155.32 (C3a), 157.33 (C=O), 163.25 (C’4),
174.56 ppm (C2); FAB: m/z 371.28 [M+1]+; Anal. for C16H13N5O4S:
calcd: C 51.75, H 3.53, N 18.86, found: C 51.72, H 3.55, N 18.89.
1-(3,4-Dimethoxybenzylidene)-4-(6-nitrobenzothiazol-2-yl)semi-
carbazide (20): Yellow solid; mp: 2408C; IR (KBr): 3460.41 (NÀH
str), 3057.27 (aromatic CÀH), 2947.33 (alkane CÀH), 1654.98 (C=O
str), 1568.18 (C=N str), 1533.46, 1329.00 (ArÀNO2 str), 1301.99 (CÀN
str), 1292.35 cmÀ1 (CÀO str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=3.79 (s, 6H, OCH3), 6.25 (s, 1H, CONH), 7.38 (s, 1H,=
NNH, D2O exchangeable), 6.69–7.24 (dd, 3H, Ar CÀH), 8.15 (s, 1H,
CÀH), 8.41–8.44 (d, 2H, benzothiazole CÀH), 9.01 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=56.28 (C’’1,
C’’’1, OCH3), 114.38 (C’2), 115.33 (C’5), 117.92 (C7), 119.91 (C5),
122.13 (C4), 122.53 (C’6), 125.41 (C7a), 127.39 (C’1), 143.18 (C=N),
145.83 (C6), 149.83 (C’3), 152.18 (C’4), 155.21 (C3a), 157.16 (C=O),
173.93 ppm (C2); FAB: m/z 401.15 [M+1]+; Anal. for C17H15N5O5S:
calcd: C 50.87, H 3.77, N 17.45, found: C 50.89, H 3.74, N 17.48.
1-(4-(Dimethylamino)benzylidene)-4-(6-nitrobenzothiazol-2-yl)-
semicarbazide (16): Yellow solid; mp: 2078C; IR (KBr): n˜ =3458.48
(NÀH str), 3061.13 (aromatic CÀH), 2943.47 (alkane CÀH), 1570.11
(C=N str), 1654.98 (C=O str), 1533.46, 1329.00 (ArÀNO2 str),
1300.07 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=2.98 (s, 6H, CH3), 6.09 (s, 1H, CONH), 6.53–7.34 (d,
4H, Ar CÀH), 7.17 (s, 1H,=NNH, D2O exchangeable), 8.16 (s, 1H,
CÀH), 8.33–8.42 (d, 2H, benzothiazole CÀH), 8.97 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=40.35 (C’’1,
C’’’1, CH3), 113.98 (C’3, C’5), 117.46 (C7), 119.75 (C5), 121.99 (C4),
123.43 (C’1), 125.49 (C7a), 129.92 (C’2, C’6), 143.94 (C=N), 145.79
(C6), 152.08 (C’4), 155.17 (C3a), 156.89 (C=O), 174.56 ppm (C2);
FAB: m/z 384.07 [M+1]+; Anal. for C17H16N6O3S: calcd: C 53.12, H
4.20, N 21.86, found: C 53.09, H 4.22, N 21.89.
1-(2,5-Dimethoxybenzylidene)-4-(6-nitrobenzothiazol-2-yl)semi-
carbazide (21): Yellow solid; mp: 2388C; IR (KBr): n˜ =3510.56 ((NÀ
H str), 3099.71 (aromatic CÀH str), 1653.05 (C=O str), 1591.02 (C=C
str), 1570.11 (C=N str), 1527.67, 1327.07 (ArÀNO2 str), 1298.14 (CÀN
str), 1124.54 cmÀ1 (CÀO str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=3.78 (s, 6H, OCH3), 5.99 (s, 1H, CONH), 7.03 (s, 1H,=
NNH, D2O exchangeable), 6.75–7.13 (m, 3H, Ar CÀH), 8.14 (s, 1H,
CÀH), 8.35–8.69 (d, 2H, benzothiazole CÀH), 9.13 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=55.96 (C’’1,
C’’’1, OCH3), 114.38 (C’6), 115.32 (C’3), 117.48 (C’4), 117.88 (C7),
118.21 (C’1), 120.16 (C5), 122.19 (C4), 125.42 (C7a), 143.62 (C=N),
145.92 (C6), 152.93 (C’2), 153.81 (C’5), 155.18 (C3a), 157.14 (C=O),
174.37 ppm (C2); FAB: m/z 401.38 [M+1]+; Anal. for C17H15N5O5S:
calcd: C 50.87, H 3.77, N 17.45, found: C 50.84, H 3.78, N 17.48.
1-(2,3-Dichlorobenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicar-
bazide (17): Yellow solid; mp: 2548C; IR (KBr): n˜ =3460.41 (NÀH
str), 3066.92 (aromatic CÀH str), 1654.98 (C=O str), 1591.02 (C=C
str), 1570.11 (C=N str), 1531.53, 1325.14 (ArÀNO2 str), 1288.49 (CÀN
str), 752.26 cmÀ1 (CÀCl str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=6.03 (s, 1H, CONH), 6.66 (s, 1H,=NNH, D2O ex-
changeable), 7.14–7.49 (m, 3H, Ar CÀH), 8.17 (s, 1H, CÀH), 8.51–
8.56 (d, 2H, benzothiazole CÀH), 8.99 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.36 (C7), 120.32 (C5),
122.94 (C4), 125.58 (C7a), 127.93 (C’5), 128.67 (C’6), 130.86 (C’2),
132.86 (C’4), 133.59 (C’3), 134.42 (C’1), 143.25 (C=N), 145.82 (C6),
155.24 (C3a), 157.34 (C=O), 174.53 ppm (C2); FAB: m/z 409.36 [M+
1]+; Anal. for C15H9Cl2N5O3S: calcd: C 43.92, H 2.21, N 17.07, found:
C 43.95, H 2.20, N 17.04.
1-(3,4,5-Trimethoxybenzylidene)-4-(6-nitrobenzothiazol-2-yl)se-
micarbazide (22): Yellow solid; mp: 2478C; IR (KBr): n˜ =3527.92
(NÀH str), 3093.92 (aromatic CÀH str), 1647.26 (C=O str), 1594.88
(C=C str), 1570.11 (C=N str), 1525.74, 1327.07 (ArÀNO2 str), 1296.21
(CÀN str), 1124.54 cmÀ1 (CÀO str); 1H NMR ([D6]DMSO, D2O ex-
change, 300 MHz): d=3.78 (s, 9H, OCH3), 6.14 (s, 1H, CONH), 6.24
(s, 1H,=NNH, D2O exchangeable), 6.65 (s, 2H, Ar CÀH), 8.16 (s, 1H,
CÀH), 8.47–8.50 (d, 2H, benzothiazole CÀH), 8.98 ppm (s, 1H, ben-
1-(2,6-Dichlorobenzylidene)-4-(6-nitrobenzothiazol-2-yl)semicar-
bazide (18): Yellow solid; mp: 2368C; IR (KBr): n˜ =3460.41 (NÀH
str), 3066.92 (aromatic CÀH str), 1653.05 (C=O str), 1591.05 (C=C
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zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=55.91 (C’’3,
C’’5, OCH3), 56.29 (C’’4, OCH3), 108.19 (C’2, C’6), 117.23 (C7), 120.13
(C5), 122.39 (C4), 125.47 (C7a), 126.21 (C’1), 140.97 (C’4), 143.01 (C=
N), 145.64 (C6), 150.95 (C’3, C’5), 155.03 (C3a), 157.23 (C=O),
174.75 ppm (C2); FAB: m/z 431.40 [M+1]+; Anal. for C18H17N5O6S:
calcd: C 50.11, H 3.97, N 16.23, found: C 50.08, H 3.94, N 16.26.
1-(Diphenylmethylene)-4-(6-nitrobenzothiazol-2-yl)semicarba-
zide (27): Yellow solid; mp: 1908C; IR (KBr): n˜ =3460.41 (NÀH str),
3057.27 (aromatic CÀH str), 1653.05 (C=O str), 1570.11 (C=N str),
1533.46, 1327.07 (ArÀNO2 str), 1284.63 cmÀ1 (CÀN str); 1H NMR
([D6]DMSO, D2O exchange, 300 MHz): d=6.34 (s, 1H, CONH), 7.41
(s, 1H,=NNH, D2O exchangeable), 7.59–8.01 (m, 10H, Ar CÀH),
8.49–8.53 (d, 2H, benzothiazole CÀH), 9.17 ppm (s, 1H, benzothia-
zole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.89 (C7), 120.13
(C5), 122.72 (C4), 126.03 (C7a), 128.73(C’3, C’5, C’’3, C’’5), 129.32
(C’2, C’6, C’’2, C’’6), 131.27 (C’4, C’’4), 133.11 (C’1, C’’1), 146.07 (C6),
155.32 (C3a), 155.76 (C=N), 157.53 (C=O), 173.99 ppm (C2); FAB: m/
z 417.39 [M+1]+; Anal. for C21H15N5O3S: calcd: C 60.42, H 3.62, N
16.78, found: C 60.40, H 3.65, N 16.75.
4-(6-Nitrobenzothiazol-2-yl)-1-(1-phenylethylidene)semicarba-
zide (23): Yellow solid; mp: 2028C; IR (KBr): n˜ =3460.41 (NÀH str),
3064.99 (aromatic CÀH str), 1654.98 (C=O str), 1568.18 (C=N str),
1531.53, 1329.00 (ArÀNO2 str), 1292.35 cmÀ1 (CÀN str); 1H NMR
([D6]DMSO, D2O exchange, 300 MHz): d=0.81 (s, 6H, CH3), 6.21 (s,
1H, CONH), 7.13 (s, 1H,=NNH, D2O exchangeable), 7.45–7.73 (m,
5H, Ar CÀH), 8.68–8.71 (d, 2H, benzothiazole CÀH), 9.67 ppm (s,
1H, benzothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=20.01
(C’’1, CH3), 117.34 (C7), 120.21 (C5), 122.02 (C4), 124.96 (C7a),
128.32 (C’3, C’5), 129.1 (C’2, C’6), 129.9 (C’4), 133.91 (C’1), 145.75
(C6), 154.93 (C3a), 156.79 (C=O), 167.98 (C=N), 174.80 ppm (C2);
FAB: m/z 355.29 [M+1]+; Anal. for C16H13N5O3S: calcd: C 54.08, H
3.69, N 19.71, found: C 54.10, H 3.68, N 19.73.
1-((4-Chlorophenyl)(phenyl)methylene)-4-(6-nitrobenzothiazol-2-
yl)semicarbazide (28): Yellow solid; mp: 2458C; IR (KBr): n˜ =
3514.42 (NÀH str), 3091.99 (aromatic CÀH str), 1649.19 (C=O str),
1589.10 (C=C str), 1587.47 (C=N str), 1529.60, 1398.44 (ArÀNO2 str),
1
1280.78 (CÀN str), 692.47 cmÀ1 (CÀCl str); H NMR ([D6]DMSO, D2O
exchange, 300 MHz): d=6.37 (s, 1H, CONH), 7.28 (s, 1H,=NNH,
D2O exchangeable), 7.43–7.95 (m, 9H, Ar CÀH), 8.53–8.57 (d, 2H,
benzothiazole CÀH), 9.32 ppm (s, 1H, benzothiazole CÀH); 13C NMR
([D6]DMSO, 300 MHz): d=117.73 (C7), 120.45 (C5), 122.69 (C4),
125.64 (C7a), 128.97 (C’’3, C’’5), 128.99 (C’3, C’5), 129.32 (C’’2, C’’6),
130.59 (C’2, C’6), 130.97 (C’1), 131.21 (C’’4), 132.94 (C’’1), 136.58
(C’4), 145.68 (C6), 155.13 (C3a), 155.64 (C=N), 157.37 (C=O),
174.85 ppm (C2); FAB: m/z 451.76 [M+1]+; Anal. for C21H14ClN5O3S:
calcd: C 55.82, H 3.12, N 15.50, found: C 55.80, H 3.15, N 15.48.
1-(1-(4-Bromophenyl)ethylidene)-4-(6-nitrobenzothiazol-2-yl)se-
micarbazide (24): Yellow solid; mp: 2358C; IR (KBr): n˜ =3406.40
(NÀH str), 3095.85 (aromatic CÀH str), 1654.98 (C=O str), 1587.17
(C=C str), 1572.04 (C=N str), 1529.60, 1334.78 (ArÀNO2 str), 1288.49
(CÀN str), 549.73 cmÀ1 (CÀBr str); 1H NMR ([D6]DMSO, D2O ex-
change, 300 MHz): d=0.89 (s, 3H, CH3), 6.17 (s, 1H, CONH), 7.23 (s,
1H,=NNH, D2O exchangeable), 7.59 (dd, 4H, Ar CÀH), 8.24–8.29 (d,
2H, benzothiazole CÀH), 8.90 ppm (s, 1H, benzothiazole CÀH);
13C NMR ([D6]DMSO, 300 MHz): d=19.11 (C’’1, CH3), 117.79 (C7),
120.18 (C5), 122.31 (C4), 125.42 (C’4), 125.56 (C7a), 131.39 (C’2, C’6),
131.85 (C’3, C’5), 133.24 (C’1), 145.90 (C6), 155.74 (C3a), 157.26 (C=
O), 168.83 (C=N), 173.98 ppm (C2); FAB: m/z 433.95 [M+1]+; Anal.
for C16H12BrN5O3S: calcd: C 44.25, H 2.79, N 16.13, found: C 44.28,
H 2.77, N 16.11.
1-((4-Hydroxyphenyl)(phenyl)methylene)-4-(6-nitrobenzothiazol-
2-yl)semicarbazide (29): Yellow solid; mp: 2088C; IR (KBr): n˜ =
3512.49 (OÀH str), 3336.98 (NÀH str), 3090.07 (aromatic CÀH str),
1645.33 (C=O str), 1566.25 (C=N str), 1529.60, 1329.00 (ArÀNO2 str),
1294.28 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=4.99 (s, 1H, Ar OÀH), 6.09 (s, 1H, CONH), 7.23 (s,
1H,=NNH, D2O exchangeable), 6.99–7.86 (m, 9H, Ar CÀH), 8.28–
8.39 (d, 2H, benzothiazole CÀH), 8.98 ppm (s, 1H, benzothiazole
CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=116.11 (C’3, C’5), 117.81
(C7), 120.72 (C5), 122.63 (C4), 125.43 (C’1), 126.61 (C7a), 128.33
(C’’3, C’’5), 129.12 (C’’2, C’’6), 130.58 (C’2, C’6), 131.21 (C’’4), 132.11
(C’’1), 146.23 (C6), 155.28 (C=N), 155.38 (C3a), 157.25 (C=O), 160.74
(C’4), 160.77 (C’4), 174.59 ppm (C2); FAB: m/z 433.41 [M+1]+; Anal.
for C21H15N5O4S: calcd: C 58.19, H 3.49, N 16.16, found: C 58.17, H
3.51, N 16.19.
1-(1-(4-Fluorophenyl)ethylidene)-4-(6-nitrobenzothiazol-2-yl)se-
micarbazide (25): Yellow solid; mp: 2418C; IR (KBr): n˜ =3458.48
(NÀH str), 3095.85 (aromatic CÀH str), 1654.98 (C=O str), 1592.95
(C=C str), 1570.11 (C=N str), 1531.53, 1327.07 (ArÀNO2 str),
1290.42 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=0.94 (s, 3H, CH3), 6.21 (s, 1H, CONH), 7.29 (s, 1H,=
NNH, D2O exchangeable), 7.04–7.93 (dd, 4H, Ar CÀH), 8.35–8.69 (d,
2H, benzothiazole CÀH), 9.13 ppm (s, 1H, benzothiazole CÀH);
13C NMR ([D6]DMSO, 300 MHz): d=19.23 (C’’1, CH3), 114.52 (C’3,
C’5), 117.81 (C7), 120.28 (C5), 122.13 (C4), 125.31 (C7a), 129.63 (C’1),
130.49 (C’2, C’6), 145.73 (C6), 155.67 (C3a), 156.87 (C=O), 165.19
(C’4), 168.74 (C=N), 173.85 ppm (C2); FAB: m/z 373.17 [M+1]+;
Anal. for C16H12FN5O3S: calcd: C 51.47, H 3.24, N 18.76, found: C
51.50, H 3.22, N 18.73.
1-(Bis(4-chlorophenyl)methylene)-4-(6-nitrobenzothiazol-2-yl)se-
micarbazide (30): Yellow solid; mp: 2148C; IR (KBr): n˜ =3514.42
(NÀH str), 3093.92 (aromatic CÀH), 1647.26 (C=O), 1566.25(C=N str),
1529.60, 1329.00 (ArÀNO2 str), 1294.28 (CÀN str), 750.33 cmÀ1 (CÀCl
1
str); H NMR ([D6]DMSO, D2O exchange, 300 MHz): d=6.32 (s, 1H,
CONH), 7.26 (s, 1H,=NNH, D2O exchangeable), 7.29–7.67 (m, 8H,
Ar CÀH), 8.36–8.42 (d, 2H, benzothiazole CÀH), 9.22 ppm (s, 1H,
benzothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.85 (C7),
120.16 (C5), 122.77 (C4), 126.01 (C7a), 128.94 (C’3, C’5, C’’3, C’’5),
130.54 (C’2, C’6, C’’2, C’’6), 131.18 (C’1, C’’1), 136.76 (C’4, C’’4),
146.14 (C6), 155.17 (C3a), 155.66 (C=N), 157.05 (C=O), 174.49 ppm
(C2); FAB: m/z 485.93 [M+1]+; Anal. for C21H13Cl2N5O3S: calcd: C
51.86, H 2.69, N 14.40, found: C 51.89, H 2.68, N 14.43.
1-(1-(4-Hydroxyphenyl)ethylidene)-4-(6-nitrobenzothiazol-2-yl)-
semicarbazide (26): Yellow solid; mp: 1768C; IR (KBr): n˜ =3510.56
(OÀH str), 3392.60 (NÀH str), 3093.92 (aromatic CÀH str)1647.26
(C=O str), 1575.89 (C=N str), 1531.53, 1329.00 (ArÀNO2 str),
1294.28 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=0.86 (s, 6H, CH3), 5.25 (s, 1H, Ar OÀH), 6.14 (s, 1H,
CONH), 7.22 (s, 1H,=NNH, D2O exchangeable), 7.40–7.63 (m, 5H,
Ar CÀH), 8.54–8.59 (d, 2H, benzothiazole CÀH), 9.35 ppm (s, 1H,
benzothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=19.43 (C’’1,
CH3), 116.5 (C’3, C’5), 117.46 (C7), 120.32 (C5), 122.41 (C4), 126.33
(C’1), 126.38 (C7a), 130.57 (C’2, C’6), 145.57 (C6), 155.11 (C3a),
157.24 (C=O), 161.02 (C’4), 168.76 (C=N), 173.96 ppm (C2); FAB: m/
z 371.35 [M+1]+; Anal. for C16H13N5O4S: calcd: C 51.75, H 3.53, N
18.86, found: C 51.78, H 3.55, N 18.89.
General procedure for the conventional synthesis of com-
pounds 31–34
To
a
solution of 1-(6-nitrobenzothiazol-2-yl)semicarbazide
9
(0.003 mol) in EtOH, was added an equimolar quantity (0.003 mol)
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of the appropriate 5-substituted isatin. The reaction mixture was
adjusted to pH 5–6 by adding glacial acetic acid and held at reflux
for 10–30 h. The contents of the flask were then poured into cold
H2O (30–60 mL), and the amorphous precipitate formed was fil-
tered, dried, and recrystallized from 95% EtOH to produce the final
products 31–34.
174.57 ppm (C2); FAB: m/z 427.26 [M+1]+; Anal. for C16H9N7O6S:
calcd: C 44.97, H 2.12, N 22.94, found: C 44.95, H 2.16, N 22.93.
In vitro MAO-B inhibition studies
Permission for animal studies was obtained from the Central
Animal Ethical Committee of the University, Institute of Medical Sci-
ences, Banaras Hindu University. Albino Wistar rats weighing be-
tween 200–220 g were obtained from Central Animal House, Insti-
tute of Medical Sciences, Banaras Hindu University (Registration
No. 542/AB/CPCSEA).
4-(6-Nitrobenzothiazol-2-yl)-1-(2-oxoindolin-3-ylidene)semicar-
bazide (31): Yellow solid; mp: 2098C; IR (KBr): n˜ =3460.41 (NÀH
str), 3059.20 (aromatic CÀH), 1747.57, 1653.05 (C=O str), 1570.11
(C=N str), 1533.46, 1329.00 (ArÀNO2 str), 1290.42 cmÀ1 (CÀN str);
1H NMR ([D6]DMSO, D2O exchange, 300 MHz): d=6.18 (s, 1H,
CONH), 7.27 (s, 1H,=NNH, D2O exchangeable), 7.05–7.79 (m, 4H,
oxindole CÀH), 8.04 (s, 1H, NÀH), 8.51–8.56 (d, 2H, benzothiazole
CÀH), 9.19 ppm (s, 1H, benzothiazole CÀH); 13C NMR ([D6]DMSO,
300 MHz): d=117.76 (C’3a), 117.83 (C7), 120.16 (C5), 121.59 (C’7),
122.32 (C4), 124.31 (C’5), 125.43 (C7a), 129.23 (C’4), 130.99 (C’6),
132.86 (C’3), 133.67 (C=N), 145.75 (C6), 146.87 (C’7a), 155.30 (C3a),
157.05 (C=O), 167.48 (C’2), 174.53 ppm (C2); FAB: m/z 382.14 [M+
1]+; Anal. for C16H10N6O4S: calcd: C 50.26, H 2.64, N 21.98, found: C
50.23, H 2.67, N 21.96.
Rat brain mitochondria were used as a source of the MAO-B iso-
form. Determination of IC50 values for the inhibition of MAO-B as
well as the preparation of rat brain mitochondrial MAO-B were car-
ried out under all suitable laboratory conditions. The final com-
pounds 10–34 were evaluated for their in vitro MAO-B inhibitory
activity according to the procedure reported by Tabor et al.[44] with
necessary modifications using benzylamine as a substrate. Opti-
mum conditions for the measurement of enzyme activity and IC50
values were sought in the experiment. The rat mitochondrial pro-
tein content was determined according to Lowry et al.[45] with
bovine albumin as the standard. The test compounds 10–34 were
dissolved in DMSO and added to the buffered incubation mixture
such that the final DMSO concentration was 4%, which caused no
MAO inhibition. Selegiline, safinamide, and isatin were taken as ref-
erence compounds for the determination of MAO-B activity. IC50
values were calculated at 95% confidence limits by using Prism
GraphPad Software (version 5.0) from plots of inhibition percentag-
es (calculated in relation to a sample of the enzyme treated under
the same conditions without inhibitors) versus the logarithm of
the inhibitor concentration.
1-(5-Bromo-2-oxoindolin-3-ylidene)-4-(6-nitrobenzothiazol-2-yl)-
semicarbazide (32): Red solid; mp: 1788C; IR (KBr): n˜ =3408.33
(NÀH str), 3095.85 (aromatic CÀH str), 1747.57, 1710.92 (C=O str),
1612.54 (C=C str), 1570.11 (C=N str), 1527.67, 1329.35 (ArÀNO2 str),
1
1292.35 (CÀN str), 462.93 cmÀ1 (CÀBr str); H NMR ([D6]DMSO, D2O
exchange, 300 MHz): d=6.21 (s, 1H, CONH), 7.09 (s, 1H,=NNH,
D2O exchangeable), 7.45–7.88 (m, 3H, oxindole CÀH), 8.12 (s, 1H,
NÀH), 8.49–8.53 (d, 2H, benzothiazole CÀH), 9.06 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.92 (C7),
118.44 (C’5), 120.07 (C’3a), 120.15 (C5), 122.53 (C4), 123.94 (C’7),
125.46 (C7a), 132.83 (C’3), 132.85 (C=N), 132.87 (C’4), 134.19 (C’6),
145.69 (C’7a), 145.79 (C6), 155.14 (C3a), 157.28 (C=O), 167.58 (C’2),
174.32 ppm (C2); FAB: m/z 460.19 [M+1]+; Anal. for C16H9BrN6O4S:
calcd: C 41.66, H 1.97, N 18.22, found: C 41.68, H 1.94, N 18.19.
Isolation of rat brain mitochondria[46]
All operations were carried out at 48C. Male and female adult
Wistar rats weighing 200–220 g were decapitated. All brains were
rapidly removed and homogenized with a Potter–Elvehjem ho-
mogenizer in cold 0.32m sucrose and 50 mm Tris·HCl, pH 8.2 (10:1,
v/w). The homogenate was centrifuged twice at 1000 g for 5 min
at 48C. The resulting supernatant was centrifuged at 20000 g for
20 min. The mitochondrial pellet obtained was suspended in
100 mm sodium phosphate buffer, pH 7.4 (4:1, v/w), fractionated in
plastic vials to 500 mL samples, and stored at À808C. Before use,
mitochondria were diluted with 100 mm sodium phosphate buffer
1-(5-Chloro-2-oxoindolin-3-ylidene)-4-(6-nitrobenzothiazol-2-yl)-
semicarbazide (33): Brown solid; mp: 2488C; IR (KBr): n˜ =3516.35
(NÀH str), 3093.92 (aromatic CÀH), 1741.78, 1647.26 (C=O str),
1570.11(C=N str), 1529.60, 1329.00 (ArÀNO2 str), 1298.14 (CÀN str),
1
1494.88 (C=C str), 750.33 cmÀ1 (CÀCl str); H NMR ([D6]DMSO, D2O
exchange, 300 MHz): d=6.13 (s, 1H, CONH), 7.41 (s, 1H,=NNH,
D2O exchangeable), 7.43–7.68 (m, 3H, oxindole CÀH), 8.08 (s, 1H,
NÀH), 8.46–8.59 (d, 2H, benzothiazole CÀH), 9.11 ppm (s, 1H, ben-
zothiazole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.89 (C7),
119.21 (C’3a), 120.18 (C5), 122.34 (C4), 122.91 (C’7), 125.39 (C7a),
129.51 (C’4), 129.98 (C’5), 131.31 (C’6), 132.75 (C’3), 132.83 (C=N),
144.93 (C’7a), 145.97 (C6), 155.24 (C3a), 157.03 (C=O), 167.52 (C’2),
174.48 ppm (C2); FAB: m/z 416.75 [M+1]+; Anal. for C16H9ClN6O4S:
calcd: C 46.11, H 2.18, N 20.16, found: C 46.13, H 2.20, N 20.15.
to give a working solution of 0.84 (mg protein)mLÀ1
.
MAO-B inhibitory activity assay[47]
An aliquot was made to contain a mixture of 55 mL mitochondrial
suspension [0.84 (mg protein)mLÀ1], 90 mL 50 mm Tris·HCl buffer
pH 8.2, and 30 mL solubilizing solution (control or inhibitor solution
at five different concentrations). The reaction was started by
adding 25 mL benzylamine (0.1m, substrate for MAO-B) for the de-
termination of MAO-B activity. The mixture was incubated at 378C
for 30 min, and the reaction was stopped by the addition of 1 mL
of 3% ice-cold ZnSO4 solution. It was subsequently mixed by vor-
texing for 10 s and then centrifuged at 3000 rpm for 15 min. The
supernatant was taken up, and the absorbance was taken at
l 250 nm (formation of benzaldehyde) for MAO-B estimation. All
the assays were performed in duplicate, and were repeated twice.
Control experiments were carried out without inhibitor, and blanks
were run without mitochondrial suspension.
1-(5-Nitro-2-oxoindolin-3-ylidene)-4-(6-nitrobenzothiazol-2-yl)se-
micarbazide (34): Brown solid; mp: 2128C; IR (KBr): n˜ =3527.92
(NÀH str), 3097.78 (aromatic CÀH str), 1651.12, 1622.19 (C=O str),
1614.88 (C=C str), 1572.04 (C=N str), 1531.53, 1330.93 (ArÀNO2 str),
1300.07 cmÀ1 (CÀN str); 1H NMR ([D6]DMSO, D2O exchange,
300 MHz): d=6.05 (s, 1H, CONH), 7.11 (s, 1H,=NNH, D2O ex-
changeable), 7.98–8.64 (m, 3H, oxindole CÀH), 8.23 (s, 1H, NÀH),
8.48–8.52 (d, 2H, benzothiazole CÀH), 9.26 ppm (s, 1H, benzothia-
zole CÀH); 13C NMR ([D6]DMSO, 300 MHz): d=117.84 (C7), 118.76
(C’3a), 120.04 (C5), 122.37 (C4), 122.56 (C’7), 123.48 (C’6), 124.83
(C’4), 125.45 (C7a), 132.73 (C=N), 132.77 (C’3), 144.32 (C’5), 145.56
(C6), 153.12 (C’7a), 155.13 (C3a), 157.26 (C=O), 167.49 (C’2),
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The refined protein molecule (PDB ID: 2V5Z) was initially subjected
to addition of all hydrogen atoms to the macromolecule, which is
a step necessary for correct calculation of partial atomic charges
followed by the calculation of Gasteiger charges for each atom of
the macromolecule in AutoDock 4.2. The grid maps were calculat-
ed using AutoGrid (version 4.2), one of the utility programs of Au-
toDock. In all docking, a grid of size 50ꢂ50ꢂ50 points in x, y, and
z directions was built, then the maps were centered on the N5
atom of the flavin (FAD) in the catalytic site of the protein. A grid
spacing of 0.375 ꢁ (approximately one fourth of the length of
a carbon–carbon covalent bond) and a distance-dependent func-
tion of the dielectric constant were used for the calculation of the
energetic map. Additionally, an electrostatic map and a desolvation
map were also calculated.[51] Rapid energy evaluation was achieved
by pre-calculating atomic affinity potentials for each atom in the
ligand molecule. In the AutoGrid procedure, the MAO-B receptor
was embedded on a three-dimensional grid point,[52] and the
energy of interaction for each atom in the ligand was encountered.
The grid maps were calculated by AutoGrid, one for each atom
type present in the ligand being docked. Thus, the grid parameter
file (receptor.gpf) was generated and saved. This is followed by the
generation of docking parameter file (ligand.dpf). This helps to
make the docking calculations fast.
Computational studies
In an attempt to obtain insight into the affinity and possible bind-
ing modes of 6-nitrobenzothiazole-derived semicarbazones 10–34
within the active site of MAO-B, molecular docking experiments
were performed. All computational studies were carried out on PC-
based machines running Windows 7 (x86) as operating system. The
MarvinSketch 5.6 module of Chemaxon tools was obtained http://
www.chemaxon.com. Python 2.7: language was obtained from
http://www.python.com, Cygwin C:\program and Python 2.5 were
simultaneously obtained from http://www.cygwin.com. Molecular
Graphics Laboratory (MGL) tools 1.5.4 and AutoDock 4.2 were ob-
tained from http://www.scripps.edu. Discovery Studio Visualizer 3.1
and PyMOL 1.3 were obtained from http://www.accelrys.com and
http://www.pymol.org, respectively.
Protein preparation
The X-ray crystallographic structure of human recombinant MAO-B
was obtained from the PDB (http://www.rcsb.org). The structure of
MAO-B co-crystallized (in complex) with safinamide (PDB ID: 2V5Z,
resolution: 1.6 ꢁ)[48] was selected as receptor model. This model
was selected due to the relatively high resolution of the crystallo-
graphic structure and also on the basis of observation that in the
complex between MAO-B and safinamide, the side chain of Ile199
is rotated out of the normal conformation, thus allowing fusion of
the entrance and substrate cavities. This is the preferred conforma-
tion when the relatively large inhibitors, which span both the en-
trance and substrate cavities, bind to the active site of MAO-B.
The various important docking parameters selected for LGA in-
cludes population size of 150 individuals, 2.5 million energy evalua-
tions, maximum of 27000 generations, maximum number of top
individuals to automatically survive to next generation of 1, gene
mutation rate of 0.02, crossover rate of 0.8, 10 docking runs and
random initial positions and conformations. The probability of per-
forming local search on an individual in the population was set to
0.06.
Coordinate file preparation
AutoDock was run several times to get various docked conforma-
tions, and used to analyze the predicted docking energy. The bind-
ing sites for these molecules were selected based on the ligand
binding pocket of the templates. AutoDock tools provide various
methods to analyze the results of docking simulations, such as
conformational similarity, visualizing the binding site and its
energy, and other parameters like intermolecular energy and inhib-
ition constant. For each ligand, ten best poses were generated and
scored using AutoDock 4.2 scoring functions.[53] At the end of
docking, ligands with the most favorable free energy of binding
were selected as the resultant complex structure. The same above
procedure was repeated separately for all the ligands.
Computational studies were carried out on only one subunit of the
MAO-B enzyme. The PDB file of MAO-B was edited, and the a-
chain was removed together with the complexed inhibitor (safina-
mide). All the water molecules and all non-interacting ions were
also removed. This refinement in the crystal structure of MAO-B
was carried out with the help of Discovery Studio Visualizer. An ex-
tended PDB format, termed as a PDBQT file, was used for coordi-
nate files which includes atomic partial charges. AutoDock Tools
was used for creating PDBQT files from traditional PDB files.[49]
Docking methodology
To determine the accuracy of this docking protocol, the reference
inhibitor safinamide was docked into the active site of MAO-B, and
the resulting binding pose was compared with that of co-crystal-
lized safinamide. This procedure was repeated three times, and the
best ranked solutions of safinamide exhibited an RMSD value of
1.80 ꢁ from the position of the co-crystallized safinamide (Fig-
ure 3a). Because RMSD values <2.0 ꢁ generally indicate that the
docking protocol is capable of accurately predicting the binding
orientation of the ligand,[54] this protocol was deemed suitable for
the docking of inhibitors into the MAO-B active site. The docking
solutions were ranked according to their respective dock score
values.
Lamarckian genetic algorithm (LGA)[50] for ligand conformational
searching, which is a hybrid of a genetic algorithm and a local
search algorithm, was employed in this study. This algorithm first
builds a population of individuals (genes), each being a different
random conformation of the docked molecule. Each individual is
then mutated to acquire a slightly different translation and rota-
tion, and the local search algorithm then performs energy minimi-
zations on a user-specified proportion of the population of individ-
uals. The individuals with low resulting energy are transferred to
the next generation, and the process is then repeated. The algo-
rithm is called Lamarckian because every new generation of indi-
viduals is allowed to inherit the local search adaptations of their
parents.
Acknowledgements
The test ligands 10–34 and standard ligand (safinamide) were built
using MarvinSketch module of Chemaxon tools and optimized
using “Prepare Ligands” in AutoDock 4.2 for docking studies. The
optimized ligand molecules in PDBQT format were docked into the
refined MAO-B model using AutoDock 4.2.[50]
The authors thank Dr. Sairam K., Department of Pharmaceutics,
Indian Institute of Technology (BHU), Varanasi for his assistance
in MAO-B screening. R.K.P.T. is thankful to the University Grants
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 462 – 474 473

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
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