Discovery of 3-Hydroxy-3-phenacyloxindole Analogues of Isatin as Potential Monoamine Oxidase Inhibitors

Article abstract of DOI:10.1002/cmdc.201500443

A series of 3-hydroxy-3-phenacyloxindole analogues of isatin were designed, synthesized, and evaluated in vitro for their inhibitory activity toward monoamine oxidase (MAO) A and B. Most of the synthesized compounds proved to be potent and selective inhibitors of MAO-A rather than MAO-B. 1-Benzyl-3-hydroxy-3-(4′-hydroxyphenacyl)oxindole (compound 18) showed the highest MAO-A inhibitory activity (IC₅₀: 0.009±0.001 μm, K_i: 3.69±0.003 nm) and good selectivity (selectivity index: 60.44). Kinetic studies revealed that compounds 18 and 16 (1-benzyl-3-hydroxy-3-(4′-bromophenacyl)oxindole) exhibit competitive inhibition against MAO-A and MAO-B, respectively. Structure-activity relationship studies suggested that the 3-hydroxy group is an essential feature for these analogues to exhibit potent MAO-A inhibitory activity. Computational studies revealed the possible molecular interactions between the inhibitors and MAO isozymes. The computational data obtained are congruent with experimental results. Further studies on the lead inhibitors, including co-crystallization of inhibitor-MAO complexes and in vivo evaluations, are essential for their development as potential therapeutic agents for the treatment of MAO-associated neurological disorders.

Full text of DOI:10.1002/cmdc.201500443

DOI: 10.1002/cmdc.201500443
Full Papers
Discovery of 3-Hydroxy-3-phenacyloxindole Analogues of
Isatin as Potential Monoamine Oxidase Inhibitors
Rati K. P. Tripathi,[a] Sairam Krishnamurthy,[b] and Senthil R. Ayyannan*[a]
A series of 3-hydroxy-3-phenacyloxindole analogues of isatin
were designed, synthesized, and evaluated in vitro for their in-
hibitory activity toward monoamine oxidase (MAO) A and B.
Most of the synthesized compounds proved to be potent and
selective inhibitors of MAO-A rather than MAO-B. 1-Benzyl-3-
Structure–activity relationship studies suggested that the 3-hy-
droxy group is an essential feature for these analogues to ex-
hibit potent MAO-A inhibitory activity. Computational studies
revealed the possible molecular interactions between the in-
hibitors and MAO isozymes. The computational data obtained
are congruent with experimental results. Further studies on
the lead inhibitors, including co-crystallization of inhibitor–
MAO complexes and in vivo evaluations, are essential for their
development as potential therapeutic agents for the treatment
of MAO-associated neurological disorders.
hydroxy-3-(4’-hydroxyphenacyl)oxindole
(compound
18)
showed the highest MAO-A inhibitory activity (IC50: 0.009Æ
0.001 mm, Ki: 3.69Æ0.003 nm) and good selectivity (selectivity
index: 60.44). Kinetic studies revealed that compounds 18 and
16 (1-benzyl-3-hydroxy-3-(4’-bromophenacyl)oxindole) exhibit
competitive inhibition against MAO-A and MAO-B, respectively.
Introduction
Monoamine oxidases (MAOs, amine-oxygen oxidoreductase, EC
1.4.3.4) are flavin adenine dinucleotide (FAD)-containing en-
zymes bound to the outer mitochondrial membrane of neuro-
nal, glial, and other cells.[1,2] These enzymes are responsible for
the oxidative deamination of neurotransmitters and other diet-
ary amines.[3,4] Two isoforms of MAO have been identified,
MAO-A and MAO-B, based on their primary sequences, 3D
structures, substrate sensitivity, and inhibitor selectivity.[5–8]
MAO-A has a higher affinity for serotonin and norepinephrine,
whereas MAO-B preferentially catalyzes the deamination of b-
phenylethylamine.[9] These properties determine the clinical in-
terest of MAO inhibitors (MAOIs). Selective MAO-A inhibitors,
such as clorgyline and moclobemide are used for the treat-
ment of depression and anxiety,[10,11] whereas selective MAO-B
inhibitors, such as selegiline and rasagiline, are useful as adju-
vants for the treatment of Parkinson’s[12,13] and Alzheimer’s dis-
eases.[14,15] Because of the poor efficacy of the currently avail-
able drugs, an intensive search for new and innovative MAOIs
is still needed. Efforts toward the discovery of ideal drug candi-
dates for the treatment of neurological disorders have in-
creased considerably in recent years.[16–20]
Isatin (2,3-dioxindole) has been documented as a well-
known pharmacological agent that possesses a broad range of
actions in biological systems. Isatin has also been identified as
a major component of tribulin, a family of naturally occurring
low-molecular-weight non-peptide inhibitors of MAO.[21] This
endogenous compound has been reported as a moderately
potent inhibitor of human MAO-B, with an enzyme–inhibitor
dissociation constant (Ki value) of 3 mm. It also inhibits human
MAO-A with a Ki value of 15 mm.[22] Isatin and its natural and
synthetic derivatives display diverse pharmacological activities.
The isatin scaffold also appears as a component of many syn-
thetic compounds that exhibit a wide range of effects[23,24] in-
cluding anticonvulsant, anxiolytic, antitubercular, and antitu-
mor activities.
Compounds with the isatin moiety have been well docu-
mented as promising scaffolds for the development of new
therapeutic agents for the treatment of central nervous system
disorders. 3-Hydroxy-3-substituted oxindoles derived from
isatin were reported to possess anticonvulsant activity with no
or minimal toxicity. Within the context of enzyme inhibitors,
isatins have seen applications in the inhibition of monoamine
oxidases. Several isatin derivatives were found to possess both
MAO-A and MAO-B inhibitory properties with very low or no
neurotoxicity.[25–27] Figure 1 illustrates a few examples of isatin
derivatives (1 and 2) that possess good activity against MAO,
and few reference MAO inhibitors (compounds 3–6).[28–32]
Isatin is therefore a biologically validated starting point for
the design of chemical libraries directed at targeting the MAO
isozymes.[22,23] Due to the privileged nature of isatin, it can be
predicted that chemical libraries of this scaffold should yield
medicinally active compounds with high hit rates.
[a] R. K. P. Tripathi, Dr. S. R. Ayyannan
Pharmaceutical Chemistry Research Laboratory
Department of Pharmaceutics, Indian Institute of Technology
Banaras Hindu University, Varanasi 221005, Uttar Pradesh (India)
E-mail: asraja.phe@iitbhu.ac.in
[b] Dr. S. Krishnamurthy
Neurotherapeutics Laboratory
Department of Pharmaceutics, Indian Institute of Technology
Banaras Hindu University, Varanasi 221005, Uttar Pradesh (India)
ORCID(s) from the author(s) for this article is/are available on the WWW
under http://dx.doi.org/10.1002/cmdc.201500443.
ChemMedChem 2016, 11, 119 – 132
119
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 1. MAO inhibitory activity of a few isatin derivatives and reference inhibitors.
The solution of crystal structures of MAO enzymes alone and
in complex with their respective inhibitors has significantly en-
hanced the rate of identification of new potent inhibitors. The
high-resolution three-dimensional (3D) structure of a complex
between isatin and human recombinant MAO-B shows that
isatin binds within the substrate cavity in close proximity to
the FAD co-factor with the dioxoindolyl NH group and the C2
carbonyl oxygen atom hydrogen bonded to conserved water
molecules within the active site. This binding mode leaves the
entrance cavity of MAO-B unoccupied (Figure 2).[8]
libus et al.[6] at a resolution of 3.0 Š. In 2008, Son et al. reported
the X-ray crystal structure of human MAO-A co-crystallized
with a potent MAO-A-specific inhibitor, harmine, at 2.2 Š reso-
lution.[7] Harmine, a b-carboline alkaloid found in plants,[33] se-
lectively binds to MAO-A, but does not inhibit the variant
MAO-B.[34] The high-resolution structure of harmine in complex
with MAO-A has provided greater insight into the enzymatic
properties and substrate/inhibitor binding specificities of MAO-
A. Harmine is found to be located in the active site cavity of
the enzyme and interacts with several residues: Tyr69, Asn181,
Phe208, Val210, Gln215, Cys323, Ile325, Ile335, Phe352, Tyr407,
Tyr444, and FAD.
With a view to identify novel heterocyclic compounds as
potent MAO inhibitors that could serve as potential chemical
templates for drug discovery, we designed a series of 3-hy-
droxy-3-phenacyloxindoles by incorporating a benzyl moiety at
the 1-position and/or a bromo group at position 5, and (un)-
substituted phenacyl group at position 3. The design approach
is depicted in Figure 3. It is presumed that the versatile isatin
Figure 3. Structural modifications to the isatin scaffold.
Figure 2. AutoDock-generated pose of isatin with gold-standard pose (GSP)
of isatin (PDB ID: 1OJA) in the MAO-B active site. Selected residues are de-
picted in black. Isatin, GSP of isatin, and FAD are shown in pink, yellow, and
cyan, respectively.
moiety, being a rigid stable electron-rich region, can serve as
a hydrophobic and/or hydrogen bonding site while the 3-phe-
nacyl-substituted systems can offer hydrophobic interactions
with the active site of MAO.
A high-resolution 3D structure of isatin bound to MAO-A has
not yet been determined. However, a lower-resolution struc-
ture of MAO-A was determined in order to understand the cat-
alytic and inhibitory mechanisms. Ma et al.[5] first determined
the X-ray crystal structure of rat MAO-A at 3.2 Š resolution,
and later the structure of human MAO-A was solved by De Co-
The present work includes the synthesis, in vitro and in silico
MAO-A and MAO-B inhibition studies of 3-hydroxy-3-phenacy-
loxindoles (compounds 9–27) derived from isatin and compari-
son of their MAO-A and MAO-B binding and inhibitory poten-
tials with those of the reference inhibitors. Kinetic studies were
carried out to examine the modes of inhibition of the most
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
120
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
active inhibitors (compounds 18 and 16) against MAO-A and
MAO-B, respectively. Furthermore, computational evaluations
of MAO-A and MAO-B inhibitors by docking simulations (Auto-
Dock 4.2) were performed to gain structural insight into the
binding modes and possible interactions of the inhibitor com-
pounds within the active sites of MAO-A and MAO-B and also
to determine the free energy of binding (DG) and inhibition
constants (Ki) of the experimentally tested MAO-A and MAO-B
inhibitors.
mechanism of this condensation reaction is depicted in
Scheme 2. Details of the physicochemical and spectral charac-
terization of the synthesized compounds are given in Experi-
mental Section below, and are in good agreement with the
composition of the synthesized compounds.
Enzyme inhibition assays
MAO-A and MAO-B inhibitory activities of test compounds 9–
27 expressed in terms of IC50 values are listed in Table 1. The
inhibitory activity of test compounds was measured in vitro by
a spectrophotometric method, by using crude rat brain mito-
chondrial suspensions, as reported. For comparison, the inhibi-
tory potencies of reference inhibitors isatin (1), clorgyline (3),
harmine (4), selegiline (5), and rasagiline (6) are also presented
in Table 1.
Results and Discussion
Chemistry
The intermediates and final compounds were synthesized ac-
cording to well-established procedures,[25–27] but with modifica-
tions wherever needed. The synthetic route followed for com-
pounds 7–27 is outlined in Scheme 1. N-Benzylisatin 7 and 5-
The test compounds exhibited significant MAO inhibitory ac-
tivity, with IC50 values in the micromolar to sub-micromolar
range. The IC50 values are in the range of 0.009Æ
0.001 mm (compound 18) to 1.057Æ0.019 mm (com-
pound 9) for MAO-A, and 0.111 Æ0.001 mm (com-
pound 16) to 5.654Æ0.043 mm (compound 9) for
MAO-B. To gain more insight into the structure–activ-
ity relationships (SARs), focused structural modifica-
tions were attempted on the isatin scaffold to vary
the lipophilic, electronic, and steric properties. In
general, all structural variations attempted in the
scaffold resulted in improved inhibitory activity. In
particular, the enhancement of lipophilicity (com-
pounds 14–20) by incorporating a benzyl moiety at
position N1 and bromoaryl or hydroxyaryl substitu-
ents at position 3 of the isatin scaffold resulted in im-
proved inhibitory activity against the MAO isozymes.
1-Benzyl-3-hydroxy-3-(4’-hydroxyphenacyl)oxindole
(18, IC50: 0.009Æ0.001 mm) emerged as the most
active inhibitor examined toward MAO-A, followed
by 1-benzyl-3-hydroxy-3-(4’-bromophenacyl)oxindole
(16, IC50: 0.013Æ0.001 mm). The most active MAO-B
inhibitor, compound 16, was found to exhibit an IC50
Scheme 1. Synthesis of 3-hydroxy-3-phenacyloxindole analogues 9–27. Reagents and
conditions: a) C6H5CH2Cl, DMF, K2CO3, reflux, 4 h; b) Br2, 08C, glacial acetic acid; c) acetone
or substituted acetophenones (R-COCH3), HN(C2H5)2, C2H5OH, reflux, 30 min; d) R’-COCH3,
HN(C2H5)2, C2H5OH; e) R’’-COCH3, HN(C2H5)2, C2H5OH, reflux, 30 min.
value of 0.111 Æ0.001 mm followed by 1-benzyl-3-hy-
droxy-3-phenacyloxindole (15, IC50: 0.184Æ0.005 mm).
Notably, all compounds displayed mild-to-moder-
ate selectivity for MAO-A over MAO-B (Table 1). Alter-
bromoisatin 8 were obtained by reaction of isatin 1 with
benzyl chloride and bromine, respectively.[35,36] 3-Hydroxy-3-
phenacyloxindole analogues of unsubstituted isatin (9–13), N-
benzylisatin (14–20), and 5-bromoisatin (21–27) were obtained
through Knoevenagel condensation of the appropriate isatin
with acetone or substituted acetophenones in the presence of
diethylamine as basic catalyst. When an ethanolic solution of
isatin or substituted isatin and acetone or substituted aceto-
phenone containing a small quantity of diethylamine was held
at reflux for 30 min and then allowed to stand for few days, co-
pious yields of product compounds 9–27 were obtained. The
products were evidently the result of interaction of reactive b-
carbonyl group of isatin or substituted isatin with the methyl
group of acetone or substituted acetophenone. The proposed
ations in the electronic and steric features afforded by the
presence of various mono- or disubstituted phenyl rings at C3
significantly influenced the MAO inhibitory profile of these
compounds. Interestingly, the 4-hydroxy-substituted analogue
(compound 18) was found to be the most potent MAO-A in-
hibitor among the other analogues with the highest selectivity
index of 60.44. This potential activity and selectivity can be at-
tributed to the presence of a 4-hydroxy substituent on the
phenacyl side chain. However, bromo (16 and 20, 1.5- and 2-
fold less potent than 18) and fluoro (17, nearly 11-fold less
potent than 18) substitutions on the phenacyl side chain re-
sulted in decreased inhibitory potency against MAO-A, which
may be due to variations in the electronic properties imparted
by these groups. Among the 5-bromoisatin analogues (21–27),
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
121
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Scheme 2. Proposed condensation reaction mechanism between isatin and substituted acetophenone.
the chloro-substituted analogue (compound 24) was found to
be the most active, with an IC50 value of 0.071 mm; this com-
pound was found to be almost 8-fold less potent than the
lead MAO-A inhibitor, compound 18.
moderately inhibit MAO-B, with an IC50 value of 0.111 mm, ~6-
fold less potent than the potency of selegiline.
Thus, substitution with functional groups such as hydroxy,
bromo, chloro, and fluoro at the C3 phenacyl ring increased
potency toward MAO-A. Among the non-functionalized phe-
nacyl analogues 9, 15, and 22, compound 15, bearing an N-
benzyl moiety, was more active (IC50: 0.059 mm), which further
supports the above observation claiming the crucial role of the
N-benzyl group for potent MAO-A inhibitory activity. Moreover,
the presence of a bulkier substituent such as a nitro group re-
sulted in less potent analogues (compounds 19 and 27). These
results guide us toward the influence of the steric groups and
electronic substituents on the MAO inhibitory profile.
The inhibitory data indicate that N-benzylisatin analogues
14–20 are more active than N-unsubstituted isatins 9–13 and
5-bromoisatin analogues 21–27. Thus, the ranking order of
these inhibitors for potency against MAO-A as per experimen-
tal inhibitory data is: 18 > 16 > 20 > 15 > 24 > 13 > 17 >
19 > 14 > 22 > 21 > 27 > 10 > 23 > 26 > 25 > 11 > 12 >
9, whereas the ranking according to computational data is: 18
> 20 > 16 > 15 > 17 > 19 > 22 > 14 > 24 > 13 > 21 > 27
> 10 > 23 > 26 > 25 > 11 > 12 > 9. Therefor, a good corre-
lation of the in vitro and in silico data is observed for MAO-A
inhibition.
Kinetic studies
In contrast to its effect on MAO-A inhibitory potency, the
presence of a 4-hydroxy group on the phenacyl side chain
(compound 18) did not exhibit a pronounced enhancement in
MAO-B inhibitory potency. Compound 16 was found to only
To further examine the modes of MAO-A and MAO-B inhibition,
sets of Lineweaver–Burk plots were constructed for the inhibi-
tion of MAO-A by compound 18 and MAO-B by compound 16,
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
122
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Table 1. In vitro and computational MAO inhibition data for compounds 9–27 along with reference inhibitors.
Compd
MAO-A
MAO-B
SI[a]
In vitro
IC50 [mm][b]
In silico
In vitro
IC50 [mm][b]
In silico
DG [kcalmolÀ1
]
Ki [mm]
DG [kcalmolÀ1
]
Ki [mm]
9
10
11
1.057Æ0.019
0.265Æ0.003
0.738Æ0.008
0.911Æ0.006
0.092Æ0.002
0.128Æ0.007
0.059Æ0.003
0.013Æ0.001
0.102Æ0.004
0.009Æ0.001
0.118Æ0.001
0.018Æ0.006
0.142Æ0.009
0.138Æ0.005
0.301Æ0.012
0.071Æ0.003
0.681Æ0.007
0.591Æ0.002
0.148Æ0.004
31.8Æ4.50
À4.95
À6.31
À5.63
À5.57
À6.43
À6.5
À7.67
À8.34
À7.39
À9.19
À6.94
À9.08
À6.38
À6.56
À6.14
À6.49
À5.86
À6.06
À6.31
–
235.65
23.51
74.05
82.75
19.41
17.08
2.4
0.77
3.83
0.18
8.15
0.22
21.05
15.59
31.57
17.36
50.62
35.85
23.66
–
5.654Æ0.043
0.391Æ0.005
0.787Æ0.013
3.615Æ0.052
0.192Æ0.006
0.931Æ0.016
0.184Æ0.005
0.111 Æ0.001
0.199Æ0.008
0.544Æ0.004
0.314Æ0.012
0.677Æ0.002
2.799Æ0.034
0.592Æ0.024
0.342Æ0.008
0.235Æ0.003
0.925Æ0.017
1.326Æ0.029
0.513Æ0.016
12.4Æ0.693
0.26
À6.21
À7.08
À6.93
À6.6
28.28
6.47
8.26
14.49
2.38
12.57
1.22
0.56
3.4
5.35
1.48
1.07
3.97
2.09
7.27
3.12
8.54
1.95
60.44
2.66
37.61
19.71
4.29
1.14
3.31
1.36
2.24
3.47
–
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1[28]
3[29]
4[30]
5[31]
6[32]
À7.67
À6.69
À8.02
À8.53
À7.46
À9.1
0.21
4.9
À7.24
À8.67
À5.95
À6.71
À7.27
À7.33
À6.89
À6.81
À7.04
À4.74
–
0.44
43.2
11.99
4.69
4.27
8.86
10.18
6.92
333.71
–
0.00042
–
–
–
–
–
–
3.00
À5.3
130.82
–
7000
–
–
–
–
16.8
67.25Æ1.02
0.412Æ0.123
–
0.019Æ0.0008
0.004Æ0.0009
–
–
À6.51
[a] Selectivity index for MAO-A: IC50(MAO-B)/IC50(MAO-A). [b] Test compound concentration required for 50% inhibition of enzyme activity; values are the
meanÆSEM; p<0.05 versus the corresponding IC50 values obtained against MAO-A and MAO-B, as determined by ANOVA/Dunnett’s.
the respective selected representative MAO-A and MAO-B in-
hibitors (Figure 4). Analysis of the Lineweaver–Burk plots sug-
gests that compounds 18 and 16 inhibit MAO-A and MAO-B
competitively, as the plots are linear and intersect at the
y axis.[28]
15, 30, and 60 min). From this result it may be concluded that
the inhibition of MAO-A is reversible, at least for the maximum
time period assayed (60 min). Interestingly, a marked increase
in the MAO-A catalytic rate with increased pre-incubation time
with compound 18 was observed.
Determination of Ki
In silico studies
For each type of mode of inhibition, Ki values can be calculat-
ed, reflecting the strength of the interactions between the
enzyme and the inhibitor. Inhibition constants for competitive
inhibitors were calculated (GraphPad Prism software), resulting
in Ki values of 3.69Æ0.003 nm for compound 18 and 5.6Æ
0.12 nm for compound 16. In comparing the IC50 values with
the Ki values, a 2.5-fold difference was observed for compound
18, and a 20-fold difference was observed for compound 16,
which reflects tight binding of the inhibitor to the enzyme.
Computational studies were carried out to shed some light on
the peculiar binding modes and interactions of MAO inhibitors
with the MAO isozymes in addition to affinity and selectivity.
All experimentally tested inhibitors (compounds 9–27) were
docked successfully into the catalytic sites of MAO-A and
MAO-B using the automated docking program AutoDock 4.2
as per the protocol described.[38] The best-scoring conformers
from the largest cluster were considered for further structural
and interaction studies. The results of the docking studies for
both MAO-A and MAO-B, expressed in terms of theoretical in-
hibition constants (Ki values) and estimated binding energies
(DG) for each virtual enzyme–inhibitor complex, are listed in
Table 1.
Reversibility and irreversibility experiments
Compound 18, the most active MAO-A inhibitor, was further
subjected to time-dependent inhibition studies to investigate
whether the observed enzyme inhibition is reversible or irre-
versible. The reversibility test was performed by using a slightly
modified method described by Legoabe et al.[37] As shown in
Figure 5, there is no time-dependent decrease in the rates of
MAO-A-catalyzed oxidation of serotonin if compound 18 was
pre-incubated with the enzyme for various periods of time (0,
Pose analysis of MAO-A inhibitors
Visual inspection of binding modes of all inhibitors within the
active site of MAO-A and MAO-B was done to gain more in-
sight into the binding orientation and potential inhibitor–
enzyme interactions. The inhibitor binding site of MAO-A is
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
123
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
3
~500 Š and is quite hydrophobic.[6] All inhibitors are located
in the active site cavity of MAO-A. Compounds 15, 17, and 20
(Figure 6A) and 9–14, 16, 18, 19, and 21–27 (Figure 6B) share
a common binding mode within the MAO-A active site.
Further investigation of the interactions of all inhibitors 9–
27 with the anchoring residues present in the MAO-A active
site led to the following observations: The potential binding
site of these inhibitors was found to be surrounded by the res-
idues Tyr69, Ile180, Asn181, Phe208, Val210, Gln215, Cys323,
Ile325, Ile335, Met350, Phe352, Tyr407, Tyr444, and FAD
(Figure 6) which is very similar to the binding site environment
of the reference MAO-A inhibitor, harmine. In nearly all com-
pounds, the oxindole moiety occupies the center of the cavity
with the substituted phenacyl group occupying the cavity ex-
tending toward FAD, while the N-benzyl group is situated
toward the opening of the cavity. At the bottom of the cavity,
Tyr444, FAD, and Tyr407 form an aromatic cage, which deter-
mines the final orientation of the inhibitors and hence their
stabilization and activity. This proves that the effective compli-
mentary binding groups for MAO-A are present in the 3-hy-
droxy-3-phenacyloxindole analogues. Furthermore, these inhib-
itors are stabilized by p–p stacking and hydrogen bonding (H-
bond) interactions.
All compounds showed p–p interactions except compounds
9, 10, 12, and 16. A p–p interaction with Tyr407 was observed
with compounds 11, 13–15, 20, and 22–27, and with Tyr444
for compounds 15, 17, 20, 23, 25, and 26. The binding of com-
pound 18 is also stabilized by a p–s interaction with Tyr407;
the same is true for 24 with Phe208, and for 21 and 25 with
FAD. All compounds showed one or more H-bond interaction
except compound 23. Thus, in most of the potent compounds,
preferably p–p, p–s hydrophobic, and H-bond interactions
were found to be responsible for mediating the inhibitory ac-
tivity against MAO-A.
Figure 4. Kinetics of rat brain MAO isozyme inhibition by compounds 18
and 16: A) Lineweaver–Burk plot of MAO-A-catalyzed oxidation of serotonin
in the absence (control) and presence of various concentrations of com-
pound 18 as indicated. B) Lineweaver–Burk plot of MAO-B-catalyzed oxida-
tion of benzylamine in the absence (control) and presence of various con-
centrations of compound 16 as indicated. Rates (V) are expressed as (nmol
Examination of one of the best-ranked docking solutions of
lead MAO-A inhibitor 18 (Figure 6C) revealed that the oxindole
nucleus is located at the center of the cavity, while the phenac-
yl side chain extends toward the FAD cofactor, and the benzyl
moiety is situated toward the opening of the MAO-A active
site. From the molecular modeling studies, it was shown that
18 can access deep inside the cavity of MAO-A. The entire mol-
ecule appears to be entrapped by residues Tyr197, Asn181,
Phe208, Ile325, Ile335, Phe352, Tyr407, Tyr444, and FAD. The
C2 atom of the phenyl ring of the phenacyl side chain shows
a p–s interaction with FAD at an inter-plane distance of 3.89 Š.
The molecule is also stabilized by several H-bond interactions.
The carbonyl oxygen atom of the phenacyl side chain forms
an H-bond with H5 and O4 of FAD. The oxygen atom of the 4-
OH group on the phenyl ring of the phenacyl side chain is in-
volved in H-bond interactions with the OH groups of Tyr444 at
inter-plane distances of 2.41 and 2.38 Š, and with OH groups
of Tyr197 at inter-plane distances of 3.19 and 2.24 Š, respec-
tively. Moreover, the hydrogen atom of the 4-OH group on the
phenyl ring of the phenacyl side chain also stabilizes the mole-
cule by forming H-bond interactions with the oxygen atom of
Asn181. As a whole, these interactions resulted in the rigidity
of the ligand in the active site of MAO-A and hence demon-
product formed)minÀ1 (mg protein)À1
.
Figure 5. Time-dependent inhibition of MAO-A-catalyzed oxidation of sero-
tonin by compound 18.
a single cavity that extends from the flavin ring to the cavity-
shaping loop. The volume of this cavity is estimated to be
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
124
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 6. Superimposed MAO-A inhibitors docked into the active site of MAO-A. FAD is shown in cyan, and selected active site residues are highlighted.
A) Compounds 15, 17, and 20 are displayed in violet, orange, and green, respectively. B) Compounds 9–14, 16, 18, 19, and 21–27 are shown in fluorescent
green, violet, blue, crimson yellow, red, dark blue, brown, maroon, light blue, light pink, dark green, black, grey, yellow, orange, and light beige, respectively.
C) Binding orientation of 18 (maroon) within the MAO-A active site, showing p–s (orange lines) and H-bond (green dashes) interactions. D) Superimposed
binding mode of 18 (maroon) in MAO-A originally docked with harmine (yellow).
strates the greater stability of the complex. The docked pose
of 18 in the MAO-A active site superimposed with the pose of
harmine (yellow) shows that both have a similar binding orien-
tation (Figure 6D), which further supports our findings.
ponents indicated a complete preference of compound 18
with respect to the A isoform in terms of both van der Waals
and electrostatic contributions.
We also decided to consider both global minimum energy
structures for the next docking simulations. These calculations
were performed with hMAO-A and hMAO-B models, and the
recognition of compound 18 was evaluated with the interac-
tion energy parameter (Table 2).[39] Compound 18 was found to
undergo a more favorable interaction with hMAO-A than
hMAO-B, which is in agreement with the experimental data, as
reported in Table 2. An analysis of the interaction energy com-
Pose analysis of MAO-B inhibitors
In contrast to MAO-A, the active site of MAO-B consists of two
3
cavities: a 420 Š hydrophobic substrate cavity connected to
an entrance cavity of 290 Š3.[40] Compounds 14, 15, and 17
(Figure 7A) and compounds 9–13, 16, and 18–27 (Figure 7B)
were observed to have a common binding mode within the
active site cavity of MAO-B. Almost all the inhibitors are situat-
ed in the active site of MAO-B. Compounds 9–27 were found
to be embedded in the space surrounded by residues Leu171,
Cys172, and Tyr398 on one side, and by Ile198, Ile199, and
Tyr435 on the other side, in addition to the aromatic residues
Tyr60, Tyr326, and Phe343. Visual inspection revealed that the
binding sites of inhibitors 9–27 are roughly similar to the bind-
ing sites of the standard MAO-B inhibitor, rasagiline. Further-
more, p–p stacking and H-bond interactions impart stability to
these inhibitors.
Table 2. Interaction energies of compound 18 within hMAO-A and
hMAO-B binding pockets.
[a]
[b]
[c]
DEint [kcalmolÀ1
]
vdW [kcalmolÀ1
]
El [kcalmolÀ1
]
hMAO-A
hMAO-B
À9.79
À9.69
À9.63
À9.59
À0.16
À0.1
[a] Total interaction energy. [b] van der Waals contribution. [c] Electrostatic
contribution.
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
125
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 7. Superimposed MAO-B inhibitors docked into the active site of MAO-B. FAD is shown in cyan, and selected active site residues are highlighted.
A) Compounds 14, 15, and 17 are displayed in dark blue, violet, and orange, respectively. B) Compounds 9–13, 16, and 18–27 are shown in fluorescent
green, dark pink, blue, crimson yellow, red, dark green, maroon, light blue, violet, light pink, dark blue, black, grey, yellow, orange, and light beige, respective-
ly. C) Binding orientation of 16 (dark green) within the MAO-B active site, showing p–p interactions (orange lines). D) Superimposed binding mode of 16
(dark green) in MAO-B originally docked with rasagiline (pink).
All compounds showed p–p interactions except compounds
9, 22, 26, and 27; p–p interactions with residue Tyr326 are ob-
served with compounds 16 and 19, and with Tyr398 for com-
pounds 10–13, 20, and 23–25. Moreover, compounds 15 and
17 were found to have p–p interactions with Phe343; likewise
compounds 10–13, 24, and 25 with Tyr435. Compounds 18
and 24 are stabilized by a p–s interaction with Tyr326; likewise
compounds 11 and 23 with FAD, and compound 23 with
Ile199. In addition, H-bond interactions were observed with
Tyr435 for compounds 14, 18, 21, 22 and 24; with FAD for 14,
18, 21, 26, and 27; with Cys172 for 10, 11, 13, 18, 22, and 26;
with Tyr188 for 12 and 21; with Tyr326 and Pro102 for 9; with
Gln206 for 10, 11, 13, 23, and 26; with Phe168 for 27; with
Ile198 for 10, 11, 13, 25, 26, and 27; with Tyr398 for 12 and
25; and with Leu171 for 12. However, compounds 15–17, 19,
and 20 lack H-bond interactions. Thus, in most of the potent
compounds, preferably p–p and H-bond interactions were
found to be responsible for mediating the inhibitory activity
against MAO-B. Good van der Waals and electrostatic interac-
tions with Pro102, Ile316, and Phe343 were also observed for
all the docked molecules.
molecule is stabilized in both the cavities surrounded by resi-
dues Leu171, Cys172, Ile198, Ile199, and Tyr326 toward the en-
trance cavity space, and residues Tyr60, Tyr398, and Tyr435
frame the aromatic cage in the substrate cavity. The binding is
further stabilized by various p–p interactions, one of which
was observed between Tyr326 and the N-benzyl ring with an
inter-plane distance of ~4.64 Š. Of importance is the observa-
tion that no H-bond interactions were found to be involved in
displaying inhibitors activity toward MAO-B.
An overall analysis of the interactions of all inhibitors 9–27
with the anchoring residues present in the active site of MAO-
B lead to the following observations: 1) The most potent com-
pounds lack H-bond interactions (compounds 15–17, 19, and
20). 2) p–p interactions were found to play an important role
in mediating activity against MAO-B (all compounds except 9,
22, 26, and 27). 3) In addition to the oxindole skeleton, anoth-
er hydrophobic aryl ring with electronegative substituents
such as bromo, chloro, and fluoro groups, is crucial for their ef-
fective binding and stabilization in the active site cavity of
MAO-B.
The dissimilarity between the experimental and computa-
tional results is due to variation in the sources of MAO-A and
MAO-B isozymes. Therefore, exact experimental values were
not expected from the computational work. This difference
may also arise from the approximations and simplifications
made during the computation process; for example, no explicit
water molecules were considered during docking simulations.
Inspection of the virtual complex between the lead MAO-B
inhibitor 16 and MAO-B reveals that, in the substrate cavity,
the oxindole ring is oriented with the N-benzyl moiety directed
toward the entrance cavity and the 3-phenacyl side chain
pointing toward the FAD cofactor (Figure 7C). This suggests
that the whole molecule traverses both cavities. The entire
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
126
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Furthermore, AutoDock 4.2 uses an empirical scoring function
for free energy calculations. Considering all these factors,
a very reasonable prediction of IC50 values, inhibition constants
(Ki values), and binding energies was obtained.
Experimental Section
Chemistry
Unless otherwise specified, all starting materials and solvents were
of commercial quality of laboratory grade purchased from Sigma–
Aldrich and Merck and were used without purification. Melting
points (mp) were determined in open capillary tubes on a Sonar
melting point apparatus and are uncorrected. All intermediates
and final compounds were characterized by IR, H NMR, 13C NMR,
1
and FAB-MS analyses. IR spectra were recorded on a Shimadzu FT-
IR 8400S infrared spectrophotometer using KBr pellets. 1H and
13C NMR spectra were recorded on a Jeol AL300 FT-NMR spectrom-
eter at frequencies of 300 and 75 MHz, respectively. All NMR meas-
urements were conducted in [D6]DMSO. Chemical shifts (d) are re-
ported in parts per million (ppm) downfield from the signal of tet-
ramethylsilane added to the deuterated solvent. Spin multiplicities
are given as s (singlet), d (doublet), t (triplet), or m (multiplet).
Mass spectra were recorded on a Thermo LCQ Advantage Max ion
trap mass spectrometer. Elemental analyses (C, H, and N) were un-
dertaken with an Exeter Analytical Inc. Model CE-440 CHN analyzer,
and the observed values were within Æ0.4% of calculated values.
Conclusions
In this study, we designed and synthesized a new series of 3-
hydroxy-3-phenacyloxindole analogues 9–27. Interestingly,
evaluation of the MAO-A and MAO-B inhibitory activities of
these compounds revealed some highly potent and selective
MAO-A inhibitors. The most active compound, 1-benzyl-3-hy-
droxy-3-(4’-hydroxyphenacyl)oxindole (18), possessing a hy-
droxy group at the para position of the 3-phenacyl ring,
showed strong inhibitory activity against MAO-A (IC50:
0.009 mm) and has a selectivity index (SI) of 60.44 followed by
16 (IC50: 0.013 mm, SI: 8.54) and 20 (IC50: 0.018 mm, SI: 37.61),
both of which have a bromo substituent in place of a hydroxy
group. Furthermore, kinetic studies suggest that these com-
pounds exhibit a competitive mode of enzyme inhibition. The
N-benzylisatin-based 3-hydroxy-3-phenacyloxindole analogues
were found to be more potent than N-unsubstituted isatin and
5-bromoisatin-based analogues. SAR studies revealed several
structural factors important for the potency and selectivity of
the designed analogues. First are multiple hydroxylations, one
of which should be the hydroxylation at C3 of isatin in addition
to hydroxylation at the para position of the 3-phenacyl ring.
Second, bromination at the para position of the 3-phenacyl
side chain is also important for MAO-A potency and selectivity,
apart from the hydroxy group. Computational studies guided
by an established docking protocol yielded a good correlation
between the theoretical and experimental inhibitory data.
Docking data and binding pose analysis of docked conforma-
tions revealed the importance of p–p and H-bond interactions
for the effective stabilization of virtual inhibitor–MAO-A com-
plexes. Furthermore, the binding orientation adopted by the
oxindole moiety appears to be unimportant for modulating
MAO-A inhibitory activity, because it assumes an opposite ori-
entation between the two most active MAO-A inhibitors, com-
pounds 18 and 20. Besides these, the high MAO-A selectivity
of 3-hydroxy-3-phenacyloxindole analogues could be ascribed
to their molecular shape, enabling a better accommodation
into the MAO-A binding site, which is wider and less flat than
that of MAO-B.[6,40]
Intermediates
General procedure for the synthesis of N-benzylisatin 7: To a so-
lution of isatin (0.0029 mol) in DMF (10 mL) containing potassium
carbonate (0.007 mol), benzyl chloride (0.0029 mol) was added
slowly with simultaneous stirring. The mixture was then heated on
a water bath for ~4 h. The contents of the flask were then poured
into cold water (50 mL), and the amorphous precipitate formed
was filtered, dried, and recrystallized from acetonitrile to produce
crystalline N-benzylisatin.[35]
N-benzylisatin 7: Yield: 79.88%; mp: 112–1168C; IR (KBr): n˜ =
3454.62 (38N str), 3030.27 (CÀH arom str), 1734.06 (2 C=O str),
1612.54 (3 C=O str), 1467.88 (CH2 bend), 1309.71 cmÀ1 (CÀN str);
1H NMR (300 MHz, [D6]DMSO): d=4.48 (s, 2H, Ar-CH2), 7.19 (dd, J=
7.1, 2.2 Hz, 1H, ArCÀH), 7.23 (dd, J=7.3, 2.4 Hz 2H, ArCÀH), 7.27 (d,
J=6.2 Hz, 2H, ArCÀH), 7.32 (dd, J=7.4, 2.3 Hz, 1H, oxindole CÀH),
7.84 (dd, J=7.6, 3.2 Hz, 1H, oxindole CÀH), 7.85 (d, J=7.6 Hz, 1H,
oxindole CÀH), 8.13 ppm (d, J=2.6 Hz, 1H, oxindole CÀH); 13C NMR
([D6]DMSO): d=43.29 (Ar-CH2), 115.35 (oxindole C-3a), 123.72 (oxin-
dole C-7), 124.68 (oxindole C-5), 125.81 (C’-4), 126.58 (C’-2, C’-6),
128.98 (C’-3, C’-5), 130.42 (oxindole C-4), 136.23 (oxindole C-6),
143.37 (C’-1), 145.67 (oxindole C-7a), 165.21 (oxindole C-2),
178.54 ppm (oxindole C-3).
General procedure for the synthesis of 5-bromoisatin 8: To a so-
lution of isatin (0.1 mol) in glacial acetic acid at 08C in a conical
flask, bromine solution was added from the burette, until the loss
of all color. The reaction mixture was then allowed to stand for 10–
15 min and then poured into ice-cold water. The orange-colored
precipitate was separated, filtered, and recrystallized from glacial
acetic acid.[36]
5-bromoisatin 8: Yield: 89.43%; mp: 224–2288C; IR (KBr): n˜ =
3205.80 (NÀH str), 2996.55 (arom CÀH str), 1755.28 (2 C=O str),
1710.92 (3 C=O str), 1282.71 (CÀN str), 886.52 (CÀBr str),
464.86 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=7.43 (d,
J=6.8 Hz, 1H, oxindole CÀH), 7.58 (d, J=7.1 Hz, 1H, oxindole CÀ
H), 8.01 (s, 1H, oxindole CÀH), 7.93 ppm (s, 1H, oxindole NÀH);
13C NMR ([D6]DMSO): d=121.52 (oxindole C-3a), 123.27 (oxindole
C-5), 125.78 (oxindole C-7), 132.15 (oxindole C-4), 136.32 (oxindole
C-6), 145.23 (oxindole C-7a), 160.37 (oxindole C-2), 175.28 ppm (ox-
indole C-3).
These results provide a better understanding of the molecu-
lar fragments that are essential to maintain and/or improve
MAO inhibitory activity and selectivity. These findings encour-
age us to continue our efforts toward optimization of the ac-
tivity profile of this important scaffold in designing selective
MAO inhibitors for the potential treatment of behavior- and
age-related disorders associated with MAO activity.
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
127
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
993.37 cmÀ1 (CÀF str); 1H NMR (300 MHz, [D6]DMSO): d=3.51 (s,
1H, CH2), 3.57 (s, 1H, CH2), 4.05 (s, 1H, OÀH), 6.86 (dd, J=7.5,
2.4 Hz, 1H, oxindole CÀH), 7.17 (d, J=6.5 Hz, 1H, oxindole CÀH),
7.20 (dd, J=7.7, 2.3 Hz, 1H, oxindole CÀH), 7.46 (d, J=7.2 Hz, 1H,
oxindole CÀH), 7.12 (s, 1H, oxindole NÀH), 7.32 (d, J=7.0 Hz, 2H,
Ar’CÀH), 7.95 ppm (d, J=7.7 Hz, 2H, Ar’CÀH); 13C NMR ([D6]DMSO):
d=45.66 (-CH2), 72.97 (oxindole C-3), 115.78 (C’-3, C’-5), 121.06 (ox-
indole C-7), 123.58 (oxindole C-5), 128.87 (oxindole C-3a, oxindole
C-6), 130.86 (oxindole C-4), 131.62 (C’-2, C’-6), 132.96 (C’-1), 142.84
(oxindole C-7a), 166.73 (C’-4), 178.19 (oxindole C=O), 195.04 ppm
(C=O); Anal. calcd for C16H12FNO3: C 67.36, H 4.24, N 4.91, found: C
67.42, H 4.26, N 4.95.
Final products
General procedure for the synthesis of 3-hydroxy-3-phenacylox-
indole analogues of isatin 9–13: Isatin (0.005 mol) and an equi-
molecular amount of unsubstituted or 4-substituted acetophenone
(0.005 mol) were dissolved in absolute ethanol (10–12 mL) and di-
ethylamine (10–15 drops) was added. The mixture was heated at
reflux on a steam bath for 30 min. After standing for 5–7 days at
room temperature, the products were collected by filtration, dried,
and recrystallized from 95% ethanol.[25–27]
3-hydroxy-3-phenacyloxindole 9: Yield: 42.67%; mp: 166–1688C;
IR (KBr): n˜ =3306.10 (OÀH str), 3254.02 (NÀH str), 3097.78 (arom CÀ
H str), 2914.54 (CH2 str), 1712.85 (2 C=O str), 1681.98 (C=O str),
1597.11 (C=C str), 1477.52 cmÀ1 (CH2 bend); 1H NMR (300 MHz,
[D6]DMSO): d=1.17 (s, 1H, CH2), 1.19 (s, 1H, CH2), 4.23 (s, 1H, OÀ
H), 6.03 (dd, J=7.2, 2.1 Hz, 1H, oxindole CÀH), 6.77 (d, J=6.5 Hz,
1H, oxindole CÀH), 6.77 (dd, J=7.3, 2.4 Hz, 1H, oxindole CÀH),
6.80 (d, J=6.5 Hz, 1H, oxindole CÀH), 7.34 (dd, J=7.8, 2.9 Hz, 2H,
Ar’CÀH), 7.47 (dd, J=8.1, 3.3 Hz, 1H, Ar’CÀH), 7.75 (d, J=7.5 Hz,
2H, Ar’CÀH), 7.85 ppm (s, 1H, oxindole NÀH); 13C NMR ([D6]DMSO):
d=64.29 (-CH2), 79.60 (oxindole C-3), 102.57 (oxindole C-7), 116.58
(oxindole C-5), 121.01 (oxindole C-6, oxindole C-3a), 122.38 (C’-3,
C’-5), 126.04 (C’-2, C’-6), 131.89 (oxindole C-4), 153.38 (C’-4), 154.32
(C’-1), 154.85 (oxindole C-7a), 155.89 (oxindole C=O), 190.29 ppm
(C=O); Anal. calcd for C16H13NO3: C 71.90, H 4.90, N 5.24, found: C
71.95, H 4.93, N 5.20.
3-hydroxy-3-(2’,4’-dibromophenacyl)oxindole 13: Yield: 32.35%;
mp: 163–1658C; IR (KBr): n˜ =3368.54 (OÀH str), 3210.14 (NÀH str),
3095.85 (arom CÀH str), 2970.48 (CH2 str), 1732.13 (2 C=O str),
1685.84 (C=O str), 1581.68 (C=C str), 1465.95 (CH2 bend),
648.10 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=3.34 (s,
1H, CH2), 3.90 (s, 1H, CH2), 5.12 (s, 1H, OÀH), 6.93 (dd, J=7.6,
2.3 Hz, 1H, oxindole CÀH), 7.25 (d, J=6.7 Hz, 1H, oxindole CÀH),
7.25 (dd, J=7.8, 2.1 Hz, 1H, oxindole CÀH), 7.71 (d, J=7.3 Hz, 1H,
oxindole CÀH), 7.27 (d, J=6.8 Hz, 2H, Ar’CÀH), 7.74 (d, J=7.5 Hz„
2H, Ar’CÀH), 7.78 (s, 1H, Ar’CÀH), 7.82 ppm (s, 1H, oxindole NÀH);
13C NMR ([D6]DMSO): d=60.43 (-CH2), 63.50 (oxindole C-3), 111.01
(oxindole C-7), 119.16 (C’-2), 122.04 (oxindole C-5), 123.09 (oxindole
C-3a), 123.09 (oxindole C-6), 128.67 (C’-4), 129.92 (oxindole C-4),
131.12 (C’-5), 132.22 (C’-6), 133.63 (C’-3), 143.99 (C’-1), 148.08 (oxin-
dole C-7a), 170.64 (oxindole C=O), 190.36 ppm (C=O); Anal. calcd
for C16H11Br2NO3: C 45.21, H 2.61, N 3.30, found: C 45.18, H 2.63, N
3.34.
3-hydroxy-3-(4’-bromophenacyl)oxindole 10: Yield: 23.81%; mp:
176–1798C; IR (KBr): n˜ =3371.68 (OÀH str), 3200.01 (NÀH str),
3061.13 (arom CÀH str), 2983.98 (CH2 str), 1701.27 (2 C=O str),
1687.77 (C=O str), 1583.61 (C=C str), 1469.81 (CH2 bend),
657.75 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=4.20 (s,
1H, OÀH), 3.06 (s, 1H, CH2), 3.25 (s, 1H, CH2), 6.59 (dd, J=7.3,
2.2 Hz, 1H, oxindole CÀH), 7.17 (d, J=6.5 Hz, 1H, oxindole CÀH),
7.21 (dd, J=7.7, 2.5 Hz, 1H, oxindole CÀH), 7.39 (d, J=6.9 Hz, 1H,
oxindole CÀH), 7.49 (d, J=7.1 Hz, 2H, Ar’CÀH), 7.16 (d, J=6.6 Hz,
2H, Ar’CÀH), 7.69 ppm (s, 1H, oxindole NÀH); 13C NMR ([D6]DMSO):
d=45.69 (-CH2), 72.69 (oxindole C-3), 116.86 (C’-3, C’-5), 122.59 (ox-
indole C-7), 125.56 (oxindole C-5), 127.88 (oxindole C-6), 127.88
(oxindole C-3a), 128.72 (C’-4), 130.81 (oxindole C-4), 131.96 (C’-2,
C’-6), 132.92 (C’-1), 143.28 (oxindole C-7a), 178.60 (oxindole C=O),
195.98 ppm (C=O); Anal. calcd for C16H12BrNO3: C 55.51, H 3.49, N
4.05, found: C 55.58, H 3.46, N 4.09.
General procedure for the synthesis of 3-hydroxy-3-phenacylox-
indole analogues of N-benzylisatin 14–20: N-benzylisatin
(0.005 mol) and an equimolecular amount of acetone or 4-substi-
tuted acetophenone (0.005 mol) were dissolved in absolute etha-
nol (10–12 mL), and diethylamine (10–15 drops) was added. After
standing for 5–7 days at room temperature, the products were col-
lected by filtration, dried and recrystallized from 95% ethanol.[25–27]
1-benzyl-3-hydroxy-3-(2-oxopropyl)indolin-2-one
14:
Yield:
53.0%; mp: 128–1328C; IR (KBr): n˜ =3313.82 (OÀH str), 3091.99
(arom CÀH str), 3063.06 (alkane CÀH str), 1720.56 (2 C=O str),
1618.33 (C=O str), 1494.88 (C=C str), 1465.95 cmÀ1 (CH2 bend);
1H NMR (300 MHz, [D6]DMSO): d=2.17 (s, 3H, CH3), 3.28 (s, 1H,
CH2), 3.76 (s, 1H, CH2), 4.15 (s, 1H, OÀH), 4.91 (s, 2H, Ar-CH2), 7.11
(d, J=6.3 Hz, 1H, oxindole CÀH), 7.23 (dd, J=6.9, 2.1 Hz, 1H, oxin-
dole CÀH), 7.33 (dd, J=7.1, 1.9 Hz, 1H, oxindole CÀH), 7.38 (d, J=
6.8 Hz„ 1H, oxindole CÀH), 7.49 (d, J=8.2, 2.8 Hz, 2H, ArCÀH), 7.59
(dd, J=7.9, 2.2 Hz, 1H, ArCÀH), 7.91 ppm (dd, J=8.2, 2.8 Hz, 2H,
ArCÀH); 13C NMR ([D6]DMSO): d=33.71 (-CH3), 42.87 (Ar-CH2), 45.58
(-CH2), 72.30 (oxindole C-3), 118.68 (oxindole C-5), 120.07 (oxindole
C-7), 123.81 (C’-4), 127.38 (C’-2, C’-6), 128.43 (oxindole C-3a), 129.94
(oxindole C-6), 130.94 (oxindole C-4), 131.78 (C’-3, C’-5), 141.59 (C’-
1), 144.29 (oxindole C-7a), 176.79 (oxindole C=O), 195.34 ppm (C=
O); Anal. calcd for C18H17NO3: C 73.20, H 5.80, N 4.74, found: C
73.16, H 5.82, N 4.73.
3-hydroxy-3-(4’-chlorophenacyl)oxindole 11: Yield: 59.86%; mp:
171–1748C; IR (KBr): n˜ =3375.54 (OÀH str), 3196.15 (NÀH str),
3059.20 (arom CÀH str), 2902.96 (CH2 str), 1699.34 (2 C=O str),
1683.91 (C=O str), 1587.47 (C=C str), 1471.74 (CH2 bend),
750.33 cmÀ1 (CÀCl str); 1H NMR (300 MHz, [D6]DMSO): d=3.50 (s,
1H, CH2), 3.57 (s, 1H, CH2), 4.05 (s, 1H, OÀH), 6.86 (dd, J=7.5,
2.4 Hz, 1H, oxindole CÀH), 7.17 (d, J=6.5 Hz, 1H, oxindole CÀH),
7.24 (dd, J=7.1, 1.9 Hz, 1H, oxindole CÀH), 7.55 (d, J=7.4 Hz, 1H,
oxindole CÀH), 7.26 (d, J=6.8 Hz, 2H, Ar’CÀH), 7.86 (d, J=7.6 Hz,
2H, Ar’CÀH), 7.89 ppm (s, 1H, oxindole NÀH); 13C NMR ([D6]DMSO):
d=45.69 (-CH2), 72.96 (oxindole C-3), 109.37 (oxindole C-7), 121.08
(oxindole C-5), 123.62 (oxindole C-3a), 128.74 (oxindole C-6), 128.91
(C’-3, C’-5), 129.82 (oxindole C-4), 131.53 (C’-2, C’-6), 134.85 (C’-1),
138.28 (C’-4), 142.79 (oxindole C-7a), 178.14 (oxindole C=O),
195.49 ppm (C=O); MS: m/z=302.78 [M+1]+; Anal. calcd for
C16H12ClNO3: C 63.69, H 4.01, N 4.64, found: C 63.74, H 4.06, N 5.67.
1-benzyl-3-hydroxy-3-phenacyloxindole 15: Yield: 84.03%; mp:
170–1748C; IR (KBr): n˜ =3327.32 (OÀH str), 3080.27 (arom CÀH str),
1695.49 (2 C=O str), 1616.40 (C=O str), 1496.81 (C=C str),
1
1465.95 cmÀ1 (CH2 bend); H NMR (300 MHz, [D6]DMSO): d=3.69 (s,
1H, CH2), 3.75 (s, 1H, CH2), 4.22 (s, 1H, OÀH), 4.91 (s, 2H, Ar-CH2),
6.94 (d, J=6.7 Hz, 2H, ArCÀH), 7.13 (dd, J=6.9, 2.0 Hz, 2H, ArCÀH),
7.16 (dd, J=7.0, 1.8 Hz, 1H, ArCÀH), 6.77 (d, J=6.5 Hz, 1H, oxin-
dole CÀH), 7.29 (dd, J=7.3, 1.9 Hz, oxindole CÀH), 7.35 (d, J=
3-hydroxy-3-(4’-fluorophenacyl)oxindole 12: Yield: 89.76%; mp:
204–2068C; IR (KBr): n˜ =3385.18 (OÀH str), 3198.08 (NÀH str),
3064.99 (arom CÀH str), 2901.04 (CH2 str), 1701.27 (2 C=O str),
1681.98 (C=O str), 1599.04 (C=C str), 1471.74 (CH2 bend),
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
128
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
6.8 Hz, 1H, oxindole CÀH), 7.35 (dd, J=7.5, 2.3 Hz, 1H, oxindole CÀ
H, 1H, oxindole CÀH), 7.48 (dd, J=7.9, 2.9 Hz, 2H, Ar’CÀH), 7.61
(dd, J=8.3, 3.2 Hz, 1H, Ar’CÀH), 7.91 ppm (d, J=7.6 Hz, 2H, Ar’CÀ
H); 13C NMR ([D6]DMSO): d=42.71 (Ar-CH2), 45.87 (-CH2), 72.73 (ox-
indole C-3), 108.86 (oxindole C-7), 121.89 (oxindole C-5), 123.31 (C’-
4), 127.19 (C’-2, C’-6), 127.90 (oxindole C-3a), 127.90 (oxindole C-6),
128.39 (C’-3, C’-5), 128.67 (C’’-3, C’’-5), 128.89 (C’’-2, C’’-6), 131.05
(oxindole C-4), 133.45 (C’’-4), 135.98 (C’’-1), 136.39 (C’-1), 143.41
(oxindole C-7a), 176.77 (oxindole C=O), 196.44 ppm (C=O); Anal.
calcd for C23H19NO3: C 77.29, H 5.36, N 3.92, found: C 77.32, H 5.34,
N 3.93.
142.36 (oxindole C-7a), 162.24 (C’’-4), 177.69 (oxindole C=O),
194.73 ppm (C=O); Anal. calcd for C23H19NO4: C 73.98, H 5.13, N
3.75, found: C 74.01, H 5.11, N 3.74.
1-benzyl-3-hydroxy-3-(4’-nitrophenacyl)oxindole
19:
Yield:
66.96%; mp: 136–1408C; IR (KBr): n˜ =3460.41 (OÀH str), 3097.78
(arom CÀH str), 1703.20 (2 C=O str), 1656.76 (C=O str), 1606.76 (C=
C str), 1523.82, 1346.36 (Ar-NO2 str), 1465.95 cmÀ1 (CH2 bend);
1H NMR (300 MHz, [D6]DMSO): d=3.25 (s, 1H, CH2), 3.56 (s, 1H,
CH2), 4.23 (s, 1H, OÀH), 4.86 (s, 2H, Ar-CH2), 6.85 (d, J=6.3 Hz, 2H,
ArCÀH), 7.11 (dd, J=6.8, 1.7 Hz, 1H, ArCÀH), 7.25 (dd, J=7.2,
1.9 Hz, 2H, ArCÀH), 7.36 (d, J=7.1 Hz, 2H, Ar’CÀH), 7.01 (d, J=
6.0 Hz, 1H, oxindole CÀH), 7.31 (d, J=6.8 Hz, 1H, oxindole CÀH),
7.57 (dd, J=7.8, 2.6 Hz, 1H, oxindole CÀH), 7.69 (dd, J=8.1, 3.1 Hz,
1H, oxindole CÀH), 7.76 ppm (d, J=7.5 Hz, 2H, Ar’CÀH); 13C NMR
([D6]DMSO): d=43.27 (Ar-CH2), 44.11 (-CH2), 73.68 (oxindole C-3),
122.82 (oxindole C-7), 123.07 (C’’-3, C’’-5), 125.35 (oxindole C-5),
126.13 (C’-4), 127.10 (C’-3, C’-5), 128.29 (oxindole C-3a), 128.44 (ox-
indole C-6), 128.91 (C’-2, C’-6), 131.58 (oxindole C-4), 132.30 (C’’-2,
C’’-6), 135.32 (C’-1), 143.86 (C’’-1), 144.74 (oxindole C-7a), 155.15
(C’’-4), 169.54 (oxindole C=O), 190.41 ppm (C=O); Anal. calcd for
C23H18N2O5: C 68.65, H 4.51, N 6.96, found: C 68. 63, H 4.50, N 6.98.
1-benzyl-3-hydroxy-3-(4’-bromophenacyl)oxindole 16: Yield:
62.30%; mp: 228–2308C; IR (KBr): n˜ =3462.34 (OÀH str), 3086.21
(arom CÀH str), 1710.92 (2 C=O str), 1610.61 (C=O str), 1585.54 (C=
1
C str), 1465.95 (CH2 str), 698.25 cmÀ1 (CÀBr str); H NMR (300 MHz,
[D6]DMSO): d=3.48 (s, 1H, CH2), 3.65 (s, 1H, CH2), 4.02 (s, 1H, OÀ
H), 4.69 (s, 2H, Ar-CH2), 6.81 (dd, J=7.1, 1.8 Hz„ 1H, ArCÀH), 6.91
(d, J=6.7 Hz„ 2H, ArCÀH), 6.71 (d, J=6.5 Hz, 1H, oxindole CÀH),
7.25 (d, J=6.9 Hz, 1H, oxindole CÀH), 7.35 (dd, J=7.5, 2.3 Hz, 1H,
oxindole CÀH), 7.47 (dd, J=7.7, 2.1 Hz„ 1H, oxindole CÀH), 7.49
(dd, J=7.8, 2.4 Hz, 2H, ArCÀH), 7.68 (d, J=7.3 Hz, 2H, Ar’CÀH),
7.90 ppm (d, J=7.6 Hz, 2H, Ar’CÀH); 13C NMR ([D6]DMSO): d=
43.89 (Ar-CH2), 45.71 (-CH2), 72.54 (oxindole C-3), 121.62 (oxindole
C-7), 123.13 (C’-4), 124.28 (oxindole C-5), 125.46 (C’’-4), 127.80 (ox-
indole C-3a), 127.86 (C’’-3, C’’-5), 127.94 (C’-2, C’-6), 128.13 (C’-3, C’-
5), 128.16 (oxindole C-6), 128.97 (C’’-2, C’’-6), 130.14 (oxindole C-4),
135.55 (C’’-1), 136.91 (C’-1), 143.00 (oxindole C-7a), 176.85 (oxindole
C=O), 196.78 ppm (C=O); Anal. calcd for C23H18BrNO3: C 63.32, H
4.16, N 3.21, found: C 63.30, H 4.17, N 3.22.
1-benzyl-3-hydroxy-3-(2’,4’-dibromophenacyl)oxindole 20: Yield:
91.36%; mp: 190–1928C; IR (KBr): n˜ =3448.84 (OÀH str), 3092.87
(arom CÀH str), 1726.35 (2 C=O str), 1681.98 (C=O str), 1612.54 (C=
C str), 1469.81 (CH2 str), 499.58, 565.16 cmÀ1 (CÀBr str); 1H NMR
(300 MHz, [D6]DMSO): d=3.32 (s, 1H, CH2), 3.86 (s, 1H, CH2), 5.02
(s, 2H, Ar-CH2), 5.29 (s, 1H, OÀH), 6.96 (d, J=6.4 Hz„ 2H, ArCÀH),
7.06 (dd, J=6.4, 2.0 Hz„ 2H, ArCÀH), 7.27 (dd, J=7.1, 1.8 Hz„ 1H,
ArCÀH), 6.88 (d, J=6.5 Hz, 1H, oxindole CÀH), 7.32 (dd, J=7.3,
2.0 Hz, 1H, oxindole CÀH), 7.36 (d, J=6.9 Hz, 1H, oxindole CÀH),
7.36 (dd, J=7.5, 2.1 Hz, 1H, oxindole CÀH, 1H, oxindole CÀH), 7.37
(d, J=7.2 Hz, 1H, Ar’CÀH), 7.76 (d, J=7.5 Hz, 1H, Ar’CÀH),
7.83 ppm (s, 1H, Ar’CÀH); 13C NMR ([D6]DMSO): d=43.41 (Ar-CH2),
60.21 (-CH2), 63.79 (oxindole C-3), 108.69 (C’’-2), 109.71 (oxindole C-
7), 110.48 (oxindole C-5), 118.80 (C’-4), 122.78 (C’-2, C’-6), 122.95
(oxindole C-3a), 122.95 (oxindole C-6), 127.23 (C’-3, C’-5), 127.60
(C’’-4), 128.76 (oxindole C-4), 129.98 (C’’-5), 131.11 (C’’-6), 132.19
(C’’-3), 133.59 (C’’-1), 135.77 (C’-1), 144.35 (oxindole C-7a), 169.40
(oxindole C=O), 190.14 ppm (C=O); Anal. calcd for C23H17Br2NO3: C
53.62, H 3.33, N 2.72, found: C 53.65, H 3.32, N 2.73.
1-benzyl-3-hydroxy-3-(4’-fluorophenacyl)oxindole
17:
Yield:
63.35%; mp: 164–1688C; IR (KBr): n˜ =3352.39 (OÀH str), 3093.92
(arom CÀH str), 1705.13 (2 C=O str), 1681.98 (C=O str), 1599.04 (C=
C
str), 1467.88 (CH2 bend), 1003.02 cmÀ1 (CÀF str); 1H NMR
(300 MHz, [D6]DMSO): d=3.67 (s, 1H, CH2), 3.73 (s, 1H, CH2), 4.19
(s, 1H, OÀH), 4.89 (s, 2H, Ar-CH2), 6.76 (d, J=7.8 Hz, 2H, Ar’CÀH),
6.93 (d, J=7.2 Hz, 2H, ArCÀH), 7.12 (dd, J=6.9, 1.9 Hz, 2H, ArCÀH),
7.26 (dd, J=7.2, 1.8 Hz, 1H, ArCÀH), 6.27 (d, J=6.1 Hz, 1H, oxin-
dole CÀH), 7.31 (dd, J=7.1, 1.8 Hz, 1H, oxindole CÀH), 7.44 (d, J=
6.9 Hz, 1H, oxindole CÀH), 7.47 (dd, J=7.7, 2.1 Hz, 1H, oxindole CÀ
H), 7.95 ppm (d, J=7.7 Hz, 1H, oxindole CÀH, 2H, Ar’CÀH);
13C NMR ([D6]DMSO): d=42.72 (Ar-CH2), 45.83 (-CH2), 72.75 (oxin-
dole C-3), 108.89 (C’’-3, C’’-5), 115.83 (oxindole C-7), 121.91 (oxin-
dole C-5), 123.36 (C’-4), 127.19 (C’-2, C’-6), 128.39 (oxindole C-3a,
oxindole C-6), 128.94 (C’-3, C’-5), 130.99 (oxindole C-4), 131.07 (C’’-
2, C’’-6), 132.80 (C’’-1), 136.37 (C’-1), 143.36 (oxindole C-7a), 165.53
(C’’-4), 176.72 (oxindole C=O), 195.07 ppm (C=O); Anal. calcd for
C23H18FNO3: C 73.59, H 4.83, N 3.73, found: C 73.63, H 4.82, N 3.71.
General procedure for the synthesis of 3-hydroxy-3-phenacylox-
indole analogues of 5-bromoisatin 21–27: 5-Bromoisatin
(0.003 mol) and an equimolecular amount of acetone or 4-substi-
tuted acetophenone (0.003 mol) were dissolved in absolute etha-
nol (10–12 mL), and diethylamine (10–15 drops) was added. The re-
action mixture was heated at reflux on a steam bath for 30 min
and allowed to stand for 7–10 days at room temperature, whereby
the products formed were collected by filtration, dried and recrys-
tallized from 95% ethanol.[25–27]
1-benzyl-3-hydroxy-3-(4’-hydroxyphenacyl)oxindole 18: Yield:
22.43%; mp: 120–1228C; IR (KBr): n˜ =3325.39 (OÀH str), 3090.07
(arom CÀH str), 1701.27 (2 C=O str), 1618.33 (C=O str), 1492.95 (C=
5-bromo-3-hydroxy-3-(2-oxopropyl)indolin-2-one
21:
Yield:
1
C str), 1467.88 cmÀ1 (CH2 str); H NMR ([D6]DMSO): d=3.24 (s, 1H,
58.37%; mp: 184–1888C; IR (KBr): n˜ =3581.93 (OÀH str), 3228.95
(NÀH str), 3078.49 (arom CÀH str), 2893.32 (alkane CÀH str),
1701.27 (2 C=O str), 1618.33 (C=O str), 1479.45 (C=C str), 1448.59
CH2), 3.64 (s, 1H, CH2), 4.40 (s, 1H, OÀH), 4.86 (s, 2H, Ar-CH2), 5.09
(s, 1H, Ar’OÀH), 6.73 (d, J=7.4 Hz, 2H, Ar’CÀH), 6.99 (d, J=6.9 Hz,
2H, ArCÀH), 6.87 (d, J=6.3 Hz, 1H, oxindole CÀH), 7.00 (dd, J=6.9,
1.9 Hz, 1H, oxindole CÀH), 7.38 (d, J=6.8 Hz, 1H, oxindole CÀH),
7.69 (dd, J=8.1, 2.6 Hz, 1H, oxindole CÀH), 7.26 (dd, J=7.2, 1.8 Hz,
2H, ArCÀH), 7.73 (dd, J=8.3, 2.9 Hz, 1H, ArCÀH), 7.92 ppm (d, J=
7.6 Hz, 2H, Ar’CÀH); 13C NMR ([D6]DMSO): d=45.81 (-CH2), 48.96
(Ar-CH2), 73.02 (oxindole C-3), 115.42 (C’’-3, C’’-5), 121.42 (oxindole
C-7), 124.19 (oxindole C-5), 127.37 (oxindole C-3a), 127.47 (C’-4),
127.83 (oxindole C-6), 129.94 (C’-2, C’-6), 130.26 (C’-3, C’-5), 131.75
(C’’-1), 133.26 (C’’-2, C’’-6), 134.65 (oxindole C-4), 141.11 (C’-1),
1
(CH2 bend), 840.99 cmÀ1 (CÀBr str); H NMR (300 MHz, [D6]DMSO):
d=2.18 (s, 3H, CH3), 2.91 (s, 1H, CH2), 2.97 (s, 1H, CH2), 4.02 (s, 1H,
OÀH), 7.75 (d, J=7.5 Hz, 1H, oxindole CÀH), 7.26 (s, 1H, oxindole
CÀH), 7.85 (d, J=7.7 Hz, 1H, oxindole CÀH), 7.98 ppm (s, 1H, oxin-
dole NÀH); 13C NMR ([D6]DMSO): d=33.59 (-CH3), 44.99 (-CH2),
79.64 (oxindole C-3), 121.51 (oxindole C-5), 125.00 (oxindole C-7),
128.84 (oxindole C-6), 131.00 (oxindole C-3a), 135.08 (oxindole C-4),
140.58 (oxindole C-7a), 175.78 (oxindole C=O), 190.97 ppm (C=O);
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
129
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Anal. calcd for C11H10BrNO3: C 46.50, H 3.55, N 4.93, found: C 46.53,
H 3.57, N 4.91.
(oxindole C-6), 134.46 (oxindole C-4), 142.63 (oxindole C-7a), 162.54
(C’’-4), 177.37 (oxindole C=O), 194.74 ppm (C=O); Anal. calcd for
C16H11BrFNO3: C 52.77, H 3.04, N 3.85, found: C 52.81, H 3.07, N
3.90.
5-bromo-3-hydroxy-3-phenacyloxindole 22: Yield: 83.26%; mp:
192–1968C; IR (KBr): n˜ =3320.67 (OÀH str), 3223.16 (NÀH str),
3096.78 (arom CÀH str), 2893.32 (alkane CÀH str), 1708.99 (2 C=O
str), 1683.91 (C=O str), 1618.33 (C=C str), 1448.59 (CH2 bend),
842.92 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=3.59 (s,
1H, CH2), 3.65 (s, 1H, CH2), 4.18 (s, 1H, OÀH), 6.79 (dd, J=6.6,
2.1 Hz„ 2H, Ar’CÀH), 7.35 (d, J=6.8 Hz, 1H, oxindole CÀH), 7.35 (s,
1H, oxindole CÀH), 7.61 (d, J=7.1 Hz, 1H, oxindole CÀH), 7.52 (dd,
J=7.1, 1.9 Hz„ 1H, Ar’CÀH), 7.63 (d, J=7.2 Hz, 2H, Ar’CÀH),
7.89 ppm (s, 1H, oxindole NÀH); 13C NMR ([D6]DMSO): d=45.70
(-CH2), 73.00 (oxindole C-3), 111.81 (oxindole C-5), 112.86 (oxindole
C-7), 126.67 (C’’-3, C’’-5), 127.93 (C’’-2, C’’-6), 128.74 (oxindole C-3a),
131.49 (oxindole C-6), 133.54 (C’’-4), 134.36 (oxindole C-4), 135.91
(C’’-1), 142.32 (oxindole C-7a), 177.92 (oxindole C=O), 196.58 ppm
(C=O); Anal. calcd for C16H12BrNO3: C 55.51, H 3.49, N 4.05, found:
C 55.47, H 3.51, N 3.99.
5-bromo-3-hydroxy-3-(4’-hydroxyphenacyl)oxindole 26: Yield:
42.81%; mp: 168–1708C; IR (KBr): n˜ =3368.54 (OÀH str), 3210.14
(NÀH str), 3095.85 (arom CÀH str), 2970.48 (CH2 str), 1732.13 (2 C=
O str), 1685.84 (C=O str), 1581.68 (C=C str), 1465.95 (CH2 bend),
648.10 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=2.96 (s,
1H, Ar’-OH), 3.46 (s, 1H, CH2), 3.52 (s, 1H, CH2), 4.03 (s, 1H, OÀH),
6.79 (d, J=6.4 Hz„ 2H, Ar’CÀH), 7.30 (d, J=6.9 Hz, 1H, oxindole CÀ
H), 7.33 (d, J=7.1 Hz, 1H, oxindole CÀH), 7.45 (s, 1H, oxindole CÀ
H), 7.74 (d, J=7.5 Hz, 2H, Ar’CÀH), 8.33 ppm (s, 1H, oxindole NÀH);
13C NMR ([D6]DMSO): d=45.18 (-CH2), 73.05 (oxindole OH), 111.26
(C’’-3, C’’-5), 112.72 (oxindole C-5), 115.27 (oxindole C-7), 126.49
(C’’-1), 127.34 (oxindole C-3a), 130.45 (C’’-2, C’’-6), 131.30 (oxindole
C-6), 134.57 (oxindole C-4), 142.31 (oxindole C-7a), 162.75 (C’’-4),
177.97 (oxindole C=O), 194.38 ppm (C=O); Anal. calcd for
C16H12BrNO4: C 53.06, H 3.34, N 3.87, found: C 53.02, H 3.36, N 3.90.
5-bromo-3-hydroxy-3-(4’-bromophenacyl)oxindole 23: Yield:
12.36%; mp: 159–1618C; IR (KBr): n˜ =3471.98 (OÀH str), 3365.90
(NÀH str), 3082.35 (arom CÀH str), 2983.98 (CH2 str), 1701.56 (2 C=
O str), 1685.84 (C=O str), 1587.47 (C=C str), 1483.31 (CH2 bend),
628.81, 588.31 cmÀ1 (CÀBr str); 1H NMR (300 MHz, [D6]DMSO): d=
3.04 (s, 1H, CH2), 3.42 (s, 1H, CH2), 4.04 (s, 1H, OÀH), 6.69 (d, J=
6.3 Hz, 1H, oxindole CÀH), 7.47 (s, 1H, oxindole CÀH), 7.56 (d, J=
7.1 Hz, 1H, oxindole CÀH), 7.32 (d, J=6.7 Hz„ 2H, Ar’CÀH), 7.97 (s,
1H, oxindole NÀH), 7.98 ppm (d, J=7.8 Hz, 2H, Ar’CÀH); 13C NMR
([D6]DMSO): d=48.50 (-CH2), 70.32 (oxindole OH), 112.61 (oxindole
C-5), 118.68 (oxindole C-7), 128.93 (C’’-4), 130.12 (oxindole C-3a),
132.69 (C’’-2, C’’-6), 133.53 (C’’-3, C’’-5), 134.11 (oxindole C-6),
138.38 (C’’-1), 140.46 (oxindole C-4), 142.02 (oxindole C-7a), 175.67
(oxindole C=O), 196.47 ppm (C=O); Anal. calcd for C16H11Br2NO3: C
45.21, H 2.61, N 3.30, found: C 45.26, H 2.58, N 3.26.
5-bromo-3-hydroxy-3-(4’-nitrophenacyl)oxindole
27:
Yield:
13.52%; mp: charred at 188–1908C; IR (KBr): n˜ =3367.98 (OÀH str),
3173.01 (NÀH str), 3103.57 (arom CÀH str), 2985.91 (CH2 str),
1720.56 (2 C=O str), 1697.41 (C=O str), 1602.90 (C=C str), 1514.17
(CH2 bend), 1464.02, 1346.36 (NO2 str), 663.53 cmÀ1 (CÀBr str);
1H NMR (300 MHz, [D6]DMSO): d=3.26 (s, 1H, CH2), 3.55 (s, 1H,
CH2), 4.07 (s, 1H, OÀH), 7.28 (d, J=7.0 Hz„ 2H, Ar’CÀH), 7.63 (d, J=
7.5 Hz, 1H, oxindole CÀH), 7.56 (s, 1H, oxindole CÀH), 7.21 (d, J=
6.6 Hz, 1H, oxindole CÀH), 7.86 (d, J=7.7 Hz, 2H, Ar’CÀH),
7.98 ppm (s, 1H, oxindole NÀH); 13C NMR ([D6]DMSO): d=45.35
(-CH2), 72.91 (oxindole OH), 120.86 (C’’-3, C’’-5), 121.08 (oxindole C-
5), 123.07 (oxindole C-7), 128.88 (oxindole C-3a, oxindole C-6),
130.51 (oxindole C-4), 131.67 (C’’-2, C’’-6), 142.61 (C’’-1), 143.58 (ox-
indole C-7a), 150.76 (C’’-4), 178.17 (oxindole C=O), 195.80 ppm (C=
O); Anal. calcd for C16H11BrN2O5: C 42.13, H 2.83, N 7.16, found: C
42.17, H 2.88, N 7.21.
5-bromo-3-hydroxy-3-(4’-chlorophenacyl)oxindole 24: Yield:
19.71%; mp: 116–1188C; IR (KBr): n˜ =3363.97 (OÀH str), 3275.24
(NÀH str), 3080.42 (arom CÀH str), 2982.05 (CH2 str), 1703.62 (2 C=
O str), 1687.77 (C=O str), 1587.47 (C=C str), 1477.52 (CH2 bend),
817.85 (CÀCl str), 692.47 cmÀ1 (CÀBr str); 1H NMR (300 MHz,
[D6]DMSO): d=2.01 (s, 1H, CH2), 2.04 (s, 1H, CH2), 4.23 (s, 1H, OÀ
H), 7.37 (d, J=6.8 Hz„ 2H, Ar’CÀH), 6.76 (d, J=6.5 Hz, 1H, oxindole
CÀH), 7.74 (s,1H, oxindole CÀH), 7.57 (d, J=7.0 Hz„ 1H, oxindole
CÀH), 7.94 (d, J=7.6 Hz, 2H, Ar’CÀH), 7.96 ppm (s, 1H, oxindole NÀ
H); 13C NMR ([D6]DMSO): d=48.45 (-CH2), 60.20 (oxindole OH),
102.14 (oxindole C-5), 118.78 (oxindole C-7), 124.12 (oxindole C-3a),
128.69 (C’’-3, C’’-5), 131.35 (C’’-2, C’’-6), 132.87 (C’’-1), 138.39 (C’’-4),
144.61 (oxindole C-6), 145.06 (oxindole C-4), 152.02 (oxindole C-7a),
165.71 (oxindole C=O), 194.61 ppm (C=O); MS: m/z=381.88 [M+
1]+; Anal. calcd for C16H11BrClNO3: C 50.49, H 2.91, N 3.68, found: C
50.46, H 2.94, N 3.70.
Enzyme inhibition assays
Materials: 5-HT (serotonin) for MAO-A assay, benzylamine for MAO-
B assay, and the reference inhibitors clorgyline (MAO-A) and selegi-
line (MAO-B) were purchased from Sigma–Aldrich. Tris·HCl, sodium
phosphate and zinc sulfate were obtained from Merck and SD
Fine.
Animal ethical approval: The Ethics Committee of Laboratory Ani-
mals at Banaras Hindu University, Varanasi (India), approved the
animal experimentation reported herein. Albino Wistar rats weigh-
ing between 200 and 220 g were obtained from Central Animal
House, Institute of Medical Sciences, Banaras Hindu University
(Registration No. Dean/12-13/CAEC/23).
5-bromo-3-hydroxy-3-(4’-fluorophenacyl)oxindole
25:
Yield:
25.34%; mp: 156–1598C; IR (KBr): n˜ =3483.56 (OÀH str), 3373.61
(NÀH str), 3080.42 (arom CÀH str), 2982.05 (CH2 str), 1718.63 (2 C=
O str), 1685.84 (C=O str), 1597.11 (C=C str), 1467.88 (CH2 bend),
1031.95 (CÀF str), 590.24 cmÀ1 (CÀBr str); 1H NMR (300 MHz,
[D6]DMSO): d=3.28 (s, 1H, CH2), 3.31 (s, 1H, CH2), 4.25 (s, 1H, OÀ
H), 6.77 (s, 1H, oxindole CÀH), 6.79 (d, J=6.5 Hz, 1H, oxindole CÀ
H), 7.35 (d, J=7.1 Hz„ 1H, oxindole CÀH), 6.73 (d, J=6.3 Hz„ 2H,
Ar’CÀH), 7.74 (d, J=7.5 Hz, 2H, Ar’CÀH), 7.96 ppm (s, 1H, oxindole
NÀH); 13C NMR ([D6]DMSO): d=45.78 (-CH2), 73.95 (oxindole OH),
111.60 (C’’-3, C’’-5), 112.19 (oxindole C-5), 115.742 (oxindole C-7),
126.54 (C’’-2, C’’-6), 127.43 (oxindole C-3a), 130.99 (C’’-1), 131.43
Isolation of rat brain mitochondria:[41] Rat brain mitochondria were
used as a source of the two MAO isoforms. All operations were car-
ried out at 48C. Male and female adult Wistar rats weighing 200–
220 g were decapitated. All brains were rapidly removed and ho-
mogenized with a Potter-Elvehjem homogenizer in cold 0.32m su-
crose 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 super-
natant 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 sam-
ples and stored at À808C. Before use, mitochondria were diluted
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
130
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
with 100 mm sodium phosphate buffer to give a working solution
of 0.84 mg protein per mL.
the test inhibitors within the active sites of MAO-A and MAO-B.
The molecular docking studies were carried out with PC-based ma-
chines running Windows 7 (”86) as operating system.
In vitro MAO inhibition assays: Experiments were carried out under
all suitable laboratory conditions. The final compounds 9–27 were
evaluated for their in vitro MAO-A inhibitory activity according to
a method reported by Sjoerdsma et al.[42] for the metabolism of se-
rotonin, and by the UV spectrophotometric method described by
Udenfriend et al.,[43] which was applied for the determination of se-
rotonin. Inhibitory activity of MAO-B was measured using benzyla-
mine as a substrate according to the procedure reported by Tabor
et al.[44] with necessary modifications. The rat mitochondrial protein
content was determined according to Lowry et al.,[45] with bovine
serum albumin as the standard. The test compounds were dis-
solved in DMSO and added to the buffered incubation mixture
such that the final DMSO concentration was 4%, which caused no
MAO inhibition. Clorgyline, selegiline, and isatin were taken as ref-
erence inhibitors for the determination of MAO-A and MAO-B ac-
tivity, respectively. An aliquot was made to contain a mixture of
55 mL mitochondrial suspension (0.84 mg protein per mL), 90 mL
50 mm Tris·HCl buffer, pH 8.2 and 30 mL solubilizing solution (con-
trol or inhibitor solution at five different concentrations). The reac-
tion was initiated by adding 25 mL of serotonin (4 mm, substrate
for MAO-A) for evaluation of MAO-A activity, and 25 mL benzyla-
mine (0.1m, substrate for MAO-B) for the determination of MAO-B
activity. The mixture was incubated at 378C for 30 min, and the re-
action was stopped by the addition of 1 mL 3% ice-cold zinc sul-
fate solution. It was subsequently mixed by vortexing for 10 s and
then centrifuged using a fixed-angle rotor at 3000 rpm for 15 min.
The supernatant was taken up, and the absorbance was read at
l 280 nm (due to formation of 5-hydroxyindolacetic acid) for MAO-
A estimation and at 250 nm (due to the formation of benzalde-
hyde) for MAO-B estimation. All assays were performed in dupli-
cate and were repeated twice. Control experiments were carried
out without inhibitor, and blanks were run without mitochondrial
suspension.[46]
Software: The molecular modelling software included MGL tools
1.5.4 based AutoDock 4.2 (www.scripps.edu) which uses Python 2.7
language—Cygwin C:\ program (www.cygwin.com) and Python 2.5
(www.python.com). Discovery Studio Visualizer 3.1 (www.accelry-
s.com) was used for visualizing the docked molecules.
Protein preparation: The X-ray crystallographic structures of
human MAO-A co-crystallized with harmine (PDB ID: 2Z5X, resolu-
tion=2.20 Š)[7] and human MAO-B co-crystallized with rasagiline
(PDB ID: 1S2Q, resolution=2.07 Š)[47] were retrieved from the RCSB
Protein Data Bank (www.rcsb.org/pdb). These models were select-
ed based on the relatively high resolution of the crystallographic
structures. Furthermore, in the complex between MAO-B and rasa-
giline, the side chain of Ile199 is rotated out of the normal confor-
mation. This allows fusion of the entrance and substrate cavities
which is a necessity for the binding of relatively bulky ligands that
span both the entrance and substrate cavities.[47] In contrast, the
active site of MAO-A consists of a single cavity.
Coordinate file preparation: Computational studies were carried
out on only one subunit of the MAO isozymes. The PDB files ob-
tained from Protein Data Bank were edited, and the b-chain was
removed together with the complexed inhibitor except FAD from
the active site. All water molecules and all non-interacting ions
were also removed. This refinement in the crystal structure of both
isozymes was carried out with the help of Discovery Studio Visual-
izer. An extended PDB format, termed as a PDBQT file, was used
for coordinate files which includes atomic partial charges. Auto-
Dock Tools was used for creating PDBQT files from traditional PDB
files.[48]
Ligand preparation: The structures of the test ligand and standard
ligand molecules were built with the aid of the MarvinSketch 5.6
module of ChemAxon tools (www.chemaxon.com) and optimized
using “Prepare Ligands” in AutoDock 4.2 and saved in PDB format.
The partial charges of the PDB files were further modified using
the ADT package (version 1.4.6) so that the charges of the nonpo-
lar hydrogen atoms were assigned to the atom to which the hy-
drogen is attached. The resulting files were saved as PDBQT files.[38]
Data analysis: IC50 values were calculated with 95% confidence
limits by using Prism GraphPad Software (version 5.0), from plots
of inhibition percentages (calculated in relation to a sample of the
enzyme treated under the same conditions without inhibitor)
versus the logarithm of the inhibitor concentration.
Reversibility and irreversibility experiments: To determine whether
the observed enzyme inhibition is reversible or irreversible, a time-
dependent inhibition study was carried out with a representative
MAO-A inhibitor, compound 18. Reversibility tests were performed
by using a slightly modified method described by Legoabe et al.[37]
Compound 18 was allowed to pre-incubate with the mitochondrial
working solution (0.84 mg protein per mL) for various periods of
time (0, 15, 30, 60 min) at 378C in Tris·HCl buffer (50 mm, pH 8.2).
For this purpose, the concentration of compound 18 was equal to
twofold the measured IC50 value for the inhibition of MAO-A
(18 nm). The reactions were subsequently diluted twofold by the
addition of 4 mm serotonin to yield a final enzyme concentration
of 0.41 mgmLÀ1 and concentrations of compound 18 that are
equal to the IC50 values. The reactions were incubated at 378C for
a further 15 min, and the residual enzyme activities were mea-
sured; from these, bar graphs were constructed. All measurements
were carried out in triplicate and are expressed as the meanÆSEM.
Docking methodology: The refined protein molecules (2Z5X for
MAO-A and 1S2Q for MAO-B) were used, and the docking method-
ology was performed according to a previously reported proto-
col.[49]
Acknowledgements
R.K.P.T. is thankful to the Indian Council of Medical Research,
New Delhi for the award of a Senior Research Fellowship.
Keywords: 3-hydroxy-3-phenacyloxindoles · inhibitors · isatin ·
molecular docking · monoamine oxidase
[1] C. Binda, J. Wang, L. Pisani, C. Caccia, A. Carotti, P. Salvati, D. E. Edmond-
son, A. Mattevi, J. Med. Chem. 2007, 50, 5848–5852.
[2] L. Novaroli, A. Daina, E. Favre, J. Bravo, A. Carotti, F. Leonetti, M. Catto,
P. Carrupt, M. Reist, J. Med. Chem. 2006, 49, 6264–6272.
In silico studies
[3] P. Dostert, M. Strolin Benedetti, M. Jafre in Monoamine Oxidase: Basic
and Clinical Frontiers (Eds.: K. Kamijo, E. Usdin, T. Nagausu), Excerpta
Medica, Amsterdam, 1982, pp. 197–208.
Platform: A computational approach through docking calculations
was used to better understand the possible binding orientations of
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
131
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[4] T. P. Singer in Chemistry and Biochemistry of Flavoenzymes II (Ed.: F.
Muller), CRC Press, London, 1991, pp. 437–470.
[5] J. Ma, M. Yoshimura, E. Yamashita, A. Nakagawa, A. Ito, T. Tsukihara, J.
Mol. Biol. 2004, 338, 103–114.
[6] L. De Colibus, M. Li, C. Binda, A. Lustig, D. E. Edmondson, A. Mattevi,
Proc. Natl. Acad. Sci. USA 2005, 102, 12684–12689.
[7] S. Y. Son, J. Ma, Y. Kondou, M. Yoshimura, E. Yamashita, T. Tsukihara,
Proc. Natl. Acad. Sci. USA 2008, 105, 5739–5744.
[8] C. Binda, M. Li, F. Hubalek, N. Restelli, D. E. Edmondson, A. Mattevi, Proc.
Natl. Acad. Sci. USA 2003, 100, 9750–9755.
[9] M. Yamada, H. Yasuhara, Neurotoxicology 2004, 25, 215–221.
[10] M. V. Rudorfer, W. Z. Potter, Drugs 1989, 37, 713–738.
[11] S. Palhagen, E. Heinonen, J. Hagglund, T. Kaugesaar, O. Maki-Ikola, R.
Palm, Neurology 2006, 66, 1200–1206.
[12] D. R. Guay, Am. J. Geriatr. Pharmacother. 2006, 4, 330–346.
[13] P. Riederer, W. Danielczyk, E. Grunblatt, Neurotoxicology 2004, 25, 271–
277.
[29] L. H. Mu, B. Wang, H. Y. Ren, P. Liu, D. H. Guo, F. M. Wang, L. Bai, Y. S.
Guo, Bioorg. Med. Chem. Lett. 2012, 22, 3343–3348.
[30] J. Wouters, Curr. Med. Chem. 1998, 5, 137–162.
[31] M. J. Matos, D. Vina, C. Picciau, F. Orallo, L. Santana, E. Uriarte, Bioorg.
Med. Chem. Lett. 2009, 19, 5053–5055.
[32] M. B. H. Youdim, A. Gross, J. P. M. Finberg, British J. of Pharmacol. 2001,
132, 500–506.
[33] D. J. McKenna, G. H. Towers, F. Abbott, J. Ethnopharmacol. 1984, 10,
195–223.
[34] J. Gerardy, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 1994, 18, 793–
802.
[35] J. F. M. Da Silva, S. J. Garden, C. Da, A. Pinto, J. Braz. Chem. Soc. 2001,
12, 273–324.
[36] S. N. Pandeya, A. S. Raja, J. P. Stables, J. Pharm. Pharm. Sci. 2002, 5, 266–
271.
[37] L. J. Legoabe, A. Petzer, J. P. Petzer, Eur. J. Med. Chem. 2012, 49, 343–
353.
[14] M. B. H. Youdim, M. Fridkin, H. Zheng, J. Neural Transm. 2004, 111,
1455–1471.
[15] A. M. Cesura, A. Pletscher, Prog. Drug Res. 1992, 38, 171–297.
[16] L. Santana, H. Gonzalez-Diaz, E. Quezada, E. Uriarte, M. Yanez, D. Vina, F.
Orallo, J. Med. Chem. 2008, 51, 6740–6751.
[17] S. Carradori, J. P. Petzer, Expert Opin. Ther. Pat. 2015, 25, 91–110.
[18] A. Bolasco, R. Fioravanti, S. Carradori, Expert Opin. Ther. Pat. 2005, 15,
1763–1782.
[38] D. S. Goodsell, G. M. Morris, A. J. Olson, J. Mol. Recognit. 1996, 9, 1–5.
[39] F. Chimenti, A. Bolasco, D. Secci, P. Chimenti, A. Granese, S. Carradori, M.
YµÇez, F. Orallo, F. Ortuso, S. Alcaro, Bioorg. Med. Chem. 2010, 18, 5715–
5723.
[40] C. Binda, P. Newton-Vinson, F. Hubalek, D. E. Edmondson, A. Mattevi,
Nat. Struct. Biol. 2002, 9, 22–26.
[41] P. R. Reinhart, W. M. Taylor, F. L. Bygrave, Biochem. J. 1982, 204, 731–
735.
[19] A. Bolasco, S. Carradori, R. Fioravanti, Expert Opin. Ther. Pat. 2010, 20,
909–939.
[42] A. Sjoerdsma, T. E. Smith, T. D. Stevenson, S. Udenfriend, Proc. Soc. Exp.
Biol. Med. 1955, 89, 36–38.
[20] S. Carradori, D. Secci, A. Bolasco, P. Chimenti, M. D’Ascenzo, Expert Opin.
Ther. Pat. 2012, 22, 759–801.
[43] S. Udenfriend, H. Weissbach, C. T. Clark, J. Biol. Chem. 1955, 215, 337–
344.
[21] F. Hubalek, C. Binda, A. Khalil, M. Li, A. Mattevi, N. Castagnoli, Jr., D. E.
Edmondson, J. Biol. Chem. 2005, 280, 15761–15766.
[22] V. Glover, J. Halket, P. J. Watkins, A. Clow, B. L. Goodwin, M. Sandler, J.
Neurochem. 1988, 51, 656–659.
[23] S. N. Pandeya, S. Smitha, M. Jyoti, S. K. Sridhar, Acta Pharm. 2005, 55,
27–46 and references therein.
[44] C. Tabor, W. H. Tabor, S. M. Rosenthal, J. Biol. Chem. 1954, 208, 645–661.
[45] O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. T. Randall, J. Biol. Chem.
1951, 193, 265–275.
[46] H. Park, M.-U. Choi, Korean J. Biochem. 1983, 16, 189–198.
[47] C. Binda, F. Hubalek, M. Li, Y. Herzig, J. Sterling, D. E. Edmondson, J.
Med. Chem. 2004, 47, 1767–1774.
[24] V. Glover, S. K. Bhattacharya, M. Sandler, Indian J. Exp. Biol. 1991, 29, 1–
5.
[25] a) F. D. Popp, R. Parson, B. E. Donigan, J. Pharm. Sci. 1980, 69, 1235–
1237; b) F. D. Popp, R. Parson, B. E. Donigan, J. Heterocycl. Chem. 1980,
17, 1329–1330.
[48] A. Madeswaran, M. Umamaheswari, K. Asokkumar, T. Sivashanmugam, V.
Subhadradevi, P. Jagannath, J. Comput. J. Comput. Methods Mol. Des.
2011, 1, 65–72.
[49] R. K. P. Tripathi, O. Goshain, S. R. Ayyannan, ChemMedChem 2013, 8,
462–474.
[26] a) F. D. Popp, H. Pajouhesh, J. Pharm. Sci. 1982, 71, 1052–1054; b) F. D.
Popp, J. Heterocycl. Chem. 1982, 19, 589–592.
Received: September 27, 2015
Revised: November 1, 2015
[27] H. Pajouhesh, R. Parson, F. D. Popp, J. Pharm. Sci. 1983, 72, 318–321.
[28] B. Strydom, S. F. Malan, N. Castagnoli, Jr., J. J. Bergh, J. P. Petzer, Bioorg.
Med. Chem. 2010, 18, 1018–1028.
Published online on November 23, 2015
ChemMedChem 2016, 11, 119 – 132
www.chemmedchem.org
132
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim