Synthesis, in vitro lipoxygenase inhibition, docking study and thermal stability analyses of novel indole derivatives: Non-isothermal kinetic study of potent LOX inhibitor N'-(diphenylmethylene)-2-(1H-indole-3-yl) acetohydrazide

Article abstract of DOI:10.1007/s13738-013-0308-3

A series of indole derivatives has been synthesized and biologically evaluated to identify potent new lipoxygenase (LOX) inhibitors. All selected indole derivatives were screened for their LOX inhibition studies. Most of compounds showed good in vitro LOX inhibition properties exhibiting IC ₅₀ values in the range of 53.61 ± 0.14 to 198.61 ± 0.11 μM (mean ± SEM), as compared to the standard inhibitor baicalein with IC₅₀ value 22.4 ± 1.3 μM. Structure activity relationship has been discussed and docking stimulation of most active compound 4f has also performed. Thermal stability and melting point of indole derivatives have been performed by thermal gravimetric analysis and differential scanning calorimetry analysis under nitrogen atmosphere at heating rate of 20 C min^-1. Compound 4f bearing bis-phenyl moiety has been found to be the most potent (IC₅₀ 53.61 ± 0.14 μM) and thermally most stable among the tested compounds. Imine (C=N) was found to be the key moiety for increasing the thermal stabilities of indole derivatives. FT-IR, NMR and elemental analysis techniques were performed for structural characterization.

Full text of DOI:10.1007/s13738-013-0308-3

J IRAN CHEM SOC
DOI 10.1007/s13738-013-0308-3
ORIGINAL PAPER
Synthesis, in vitro lipoxygenase inhibition, docking study
and thermal stability analyses of novel indole derivatives
Non-isothermal kinetic study of potent LOX inhibitor N’-(diphenylmethylene)
-2-(1H-indole-3-yl) acetohydrazide
•
•
•
Muhammad Yar Lala Rukh Sidra Eleni Pontiki Nafeesa Mushtaq
•
•
Muhammad Ashraf Rumana Nasar Islam Ullah Khan Nasir Mahmood
•
•
•
•
Syed Ali Raza Naqvi Zulfiqar Ali Khan Sohail Anjum Shahzad
•
Received: 26 March 2013 / Accepted: 25 June 2013
Ó Iranian Chemical Society 2013
Abstract A series of indole derivatives has been syn-
thesized and biologically evaluated to identify potent
new lipoxygenase (LOX) inhibitors. All selected indole
derivatives were screened for their LOX inhibition studies.
Most of compounds showed good in vitro LOX inhibi-
tion properties exhibiting IC50 values in the range of
53.61 0.14 to 198.61 0.11 lM (mean SEM), as
compared to the standard inhibitor baicalein with IC50
value 22.4 1.3 lM. Structure activity relationship has
been discussed and docking stimulation of most active
compound 4f has also performed. Thermal stability and
melting point of indole derivatives have been performed by
thermal gravimetric analysis and differential scanning
calorimetry analysis under nitrogen atmosphere at heating
rate of 20 °C min-1. Compound 4f bearing bis-phenyl
moiety has been found to be the most potent (IC50
53.61 0.14 lM) and thermally most stable among the
tested compounds. Imine (C=N) was found to be the key
moiety for increasing the thermal stabilities of indole
derivatives. FT-IR, NMR and elemental analysis tech-
niques were performed for structural characterization.
Keywords Differential scanning calorimetry ꢀ Thermal
gravimetric analysis ꢀ Lipoxygenase inhibitor ꢀ Imine ꢀ
Indole-3-acetic acid
Introduction
Lipoxygenase (LOX) is a lipid peroxidizing enzyme, which
shows an essential role in leukotriene (LTs) biosynthesis
and mediates pathophysiology of allergic disorders,
inflammatory reactions and neoangiogenesis [1–3]. LOX
specifically is involved in the auto-oxidation of polyun-
saturated fatty acids that cause food spoilage by creating
Electronic supplementary material The online version of this
article (doi:10.1007/s13738-013-0308-3) contains supplementary
material, which is available to authorized users.
N. Mahmood
Department of Allied Sciences and Chemical Pathology,
University of Health Sciences, Lahore 54600, Pakistan
M. Yar (&) ꢀ L. R. Sidra ꢀ N. Mushtaq
Interdisciplinary Research Center in Biomedical Materials,
COMSATS Institute of Information Technology,
Lahore 54000, Pakistan
S. A. R. Naqvi ꢀ Z. A. Khan
e-mail: drmyar@ciitlahore.edu.pk
Department of Chemistry, Government College University,
Faisalabad 38000, Pakistan
E. Pontiki
Department of Pharmaceutical Chemistry, School of Pharmacy,
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
S. A. Shahzad
Department of Chemistry, COMSATS Institute of Information
Technology, Abbottabad 22060, Pakistan
M. Ashraf ꢀ R. Nasar
Department of Biochemistry and Biotechnology,
The Islamia University of Bahawalpur,
Bahawalpur 63100, Pakistan
I. U. Khan
Department of Chemistry, Government College University,
Lahore 54000, Pakistan
123

J IRAN CHEM SOC
Thermal study of pharmaceutical compounds has
become a reliable method for purity control and charac-
terization of novel compounds with potential bioactivity.
Thermogravimetric analysis/Differential scanning micros-
copy (TGA/DSC) is widely used for the study of the sta-
bility and decomposition of solid biological active
compounds used in medicine [13]. It is also a reliable
method for the study of interaction of active pharmaceutical
ingredients with the excipients by correlation of dissolution
and thermal rate constants of active ingredients and their
tablets formulation [14, 15]. Although thermal studies are
considered to be important and play a vital role in the
development of medicinal compounds. However, there are
only few reports published on the thermal analysis of newly
synthesized potentially biologically active compounds. The
promising biological potentials of indole alkaloids and the
interest for observing their thermal behavior have prompted
us to biologically evaluate a series of new indole derivatives
(Table 1) as potential LOX inhibitors and also to test their
thermal stabilities. In this paper, we report the synthesis of a
series of new indole derivatives (Scheme 1), LOX inhibi-
tory activities, docking study, thermal studies and kinetic
study of these indole derivatives.
Table 1 The quantitative LOX inhibition of indole derivatives
Entry Sample
code
Conc.
(mM)
Inhibition
(%)
IC50
(lM SEMa)
1
2
3
4
5
6
7
8
9
10
a
2
0.5
66.09 0.61
123.41 0.31
3
0.5
55.78 0.18 \400
56.01 0.44 \400
42.15 0.52 [400
4a
0.5
4b
0.5
4c
0.5
67.71 0.61
198.61 0.11
4d
0.5
34.59 0.14 [400
53.26 0.15 \400
4e
0.5
4f
0.5
92.55 0.18
53.61 0.14
4g
Baicaleinb
0.5
25.66 0.62 [500
0.5 mM
93.79 1.27
22.41 1.3
All reactions were performed in triplicates and averaged, and SEM
is standard mean error of the three experiments
b
Standard used for LOX inhibition
off-odor and off-flavors [4]. Moreover, the study of role of
LTs in allergic disorders reveals that 5-LOX inhibitors
have a therapeutic potential in asthma, rheumatoid arthritis
and ulcerative colitis [5].
There is a great interest for the search of new and potent
LOX inhibitors. Indole derivatives exhibit a broad range of
biological activities such as antiviral, antibacterial, anti-
oxidant, anti-inflammatory, anticancer, analgesic, agonis-
tic, hypotensive, hallucinogenic and antimetastatic
activities [6–10]. Indole moiety has been incorporated as a
core skeleton to achieve active sites involved in enzyme
inhibitions [11]. In addition, some indole derivatives such
as thiopyranol-indoles and their analogs are also identified
as good inhibitors of 5-LOX [12].
Results and discussion
Chemistry
Indole-3-carboxylic acid was heated under reflux in the
presence of methanol and sulfuric acid which resulted in
indole-3-methyl acetate 2 in good yield (95 %) [16].
Scheme 1 Synthetic strategy of
indole derivatives
NH-NH2
O
OCH3
80% N2H4
OH
O
CH3OH
O
H2SO4/reflux
4-5 h
EtOH, reflux,
4-5 h
N
H
(1)
N
H
N
H
[60%]
[95%]
2
( )
3
( )
CH3OH,
Aldehyde
GAA
R2
4a R1 = H, R2 = H
4b R1 = H, R2 = 2-OH
N
R1 = H, R2 = 4-OCH3
4c
4d
NH
R1
R1 = H, R2 = 4- CH3
R1 = CH3, R2 = H
4e
O
4f R1 = Ph, R2 = H
4g R1 = H, R2 = 4-Cl
N
H
56-95 %
4a-g
123

J IRAN CHEM SOC
Table 2 TG and DSC data of compounds 1–4g
Compounds
DSC
TG
Tonset (°C)
Stages
Tpeak (°C)
Peak nature
Phenomenon
To (°C)
Residue (%)
2.9
1
122.3
175.7
126.1
153.2
I
144.2
194.3
135.1
166.3
312.4
180.9
318.8
201.9
83.3
Endo
Endo
Endo
Endo
Exo
Crystallization
Melting
141.8
Ii
I
3
Melting
165.6
254.6
16.2
12.5
4a
I
Melting
ii
i
Boiling
4b
4c
4d
4e
4f
174.1
196.5
169.8
147.7
145.1
202.5
Endo
Exo
Melting
270.5
17.2
ii
i
Boiling
Endo
Endo
Endo
Exo
Melting
277.4
255.2
17.2
10.8
i
Glass Transition
Melting
ii
iii
i
183.2
319.5
158.0
335.4
152.8
335.4
209.9
Boiling
Endo
Endo
Endo
Endo
Endo
Melting
258.2
287.2
281.4
5.4
11.3
4.5
ii
i
Decomposition
Melting
ii
i
Decomposition
Melting
4g
The resulting ester 2 was further reacted with hydrazine
in the presence of methanol (Scheme 1) which afforded
1H-indole-3-carbohydrazide 3 [17]. In order to synthesize a
series of indole Schiff base derivatives, 1H-indole-3-car-
bohydrazide 3 was selected as a starting hydrazide and was
treated with a range of selected aldehydes and ketones
(Scheme 1). The reaction was carried out in the presence of
methanol and few drops of glacial acetic acid; this resulted
in good to excellent yield of the corresponding Schiff bases
4a–4h (56–95 %).
Schiff base 4c (Table 1, Entry 5) showed almost four-
fold less activity (4 times more IC50 value) as compared to
Schiff base 4f, this decrease in activity might be due to
mono substitution at imine carbon (C=N), and less rigid
structure as compared to 4f which decreases binding with
enzyme. Comparison of IC50 value due to presence of
different groups is presented in Scheme 2. Indole hydra-
zide 3 and Schiff bases 4a and 4e (Table 1, entries 2, 3 and
7) showed IC50 value of less than 400 lM. Poor inhibition
of indole hydrazide 3 might be due to the absence of ter-
minal N-substituent. The order of LOX inhibition is
LOX inhibition
4f [ 4c [ 2 [ 4a [ 3 [ 4e [ 4b [ 4d [ 4g
Schiff bases 4b, 4d and 4g were found to be inactive
against LOX inhibition. This poor enzyme inhibition was
probably due to the presence of different degrees of sub-
stituents which cause steric hindrance and binding with
enzyme becomes difficult.
The whole series of compounds 1–4g shown in Scheme 1
was screened for their LOX activities, and compared to
baicalein, a potent lipoxygenase inhibitor used as a standard
inhibitor [18]. Obtained IC50 values (concentration to
inhibit 50 % enzyme activity) of derivatives are listed in
Table 2. Indole ester 2 (Table 1, Entry 1) showed % inhi-
bition of 66.09 0.61 and an IC50 value of 123.41
0.31 lM. Among tested indole derivatives, Schiff base 4f
(Table 1, Entry 8) showed the highest LOX inhibition with
% inhibition of 92.55 0.18 and IC50 value of 53.61
0.14 lM. This higher inhibition may be attributed due to its
rigid structure and presence of greater delocalized electron
density because of bis-phenyl moiety attached at imine
carbon, which may enhance bonding with active site (iron
atom) of the enzyme. The catalytic mechanism of LOX is
well known and it explains the oxidation of iron of LOX
that later on accelerates enzyme activity, while bonding
with iron inhibits enzyme activity [19].
In addition to LOX inhibition of theses selected indole
derivatives, tyrosinase inhibition has also been carried out
and all the compounds were found to be inactive (See SI
for details).
Molecular docking studies on LOX
Soybean enzyme was used because of its easily availability
and highly characterized structure [20]. It is believed that
all LOXs follow the same catalytic mechanism [21];
however, it is probably the vicinity of the iron site that
determines the regio and stereospecificity of the particular
enzyme. In this contradictorily state of facts soybean lip-
oxygenases stand as a control point in terms of structure,
123

J IRAN CHEM SOC
R
O
N
H
4f
4c
2
OCH3
OCH3
HN
N
HN
N
IC50=123.41 0.31
IC50=198.61 0.11
IC50=53.61 0.14
Scheme 2 Effects of various groups on LOX inhibition
activity and thus inhibition [22]. For the docking studies we
selected 4f which presents a good combination of in vitro
anti-LOX result. The possible mechanism of action, as well
as the differences in activity toward soybean LOX com-
pared to the other derivatives could be explained by
docking calculations.
Fig. 1 Docked poses of conjugate 4f (Magenta) in the LOX binding
site. Side-chains of relevant binding site residues are rendered as ball
and stick models. The iron ion is rendered as an orange sphere
Thermogravimetric analysis
For the docking studies of LOX we have used the 1RRH
(soybean lipoxygenase) available from the Protein Data
All indole derivatives 1–4g were thermally studied by TGA
and DSC at heating rate of 20 °C min-1 under nitrogen
atmosphere. For illustration, TG curves of 1–4g are shown
in Fig. 3. Indole acid 1 and indole hydrazide 3 exhibited
comparatively poor thermal stability than synthesized
indole Schiff bases. Indole acid 1 started weight loss at
180 °C and until 200 °C almost 95 % weight was lost and
only 5 % weight left. This sharp weight loss appears due to
loss of CO2. It looks feasible that the removal of CO2 is
responsible for poor stability of indole acid 1. Hydrazide 3
formed from indole acid exhibited much higher thermal
stability. Interestingly, in the case of Schiff bases, thermal
stability was further increased and degradation was started
above 250 °C. Here, imine (C=N) was identified to be
important functional group for increasing the thermal sta-
bilities of indole derivatives.
˚
Bank (PDB) with a resolution of 2 A [23]. We used the
1RRH from PDB with Fe?3 running blind docking. Two
soybean LOX models were derived from 1RRH. One with
a ligand taken from 1IK3 (ligand ID: 9OH) for the docking
studies into the catalytic cavity and another one without
any ligand in the catalytic cavity for the blind docking
simulation.
The metal center in both models has been considered as
a cation, with charge q = ?3 and no bond restraint was
applied between the iron and the ligands. Lennard-Jones’
parameters for Fe(III) force field can be resumed as:
rvdw = 2.138157e-01 nm, evdw = 2.092e-1 kJ/mol.
The validation of our docking study was made with the
ligands crystallized in the protein of complex 1RRH, where
our docking results were in good agreement with the
structure of the molecules in the active site of the protein.
The docking orientations of 4f are given in Fig. 1. It is
important to mention that conjugate 4f presents significant
high binding energy to the protein (-8.8 kcal/mol). A
number of H-bonds were developed. These H-bonds were
observed between: (a) the nitrogen of the indole group with
LEU258, (b) the nitrogen of the hydrazide group with
VAL256 and (c) the nitrogen of the hydrazide group
adjacent to the carbonyl group with GLY265. Weak
hydrophobic interactions were also observed, supporting an
even stronger binding of the compound 4f to the LOX
cavity. As for baicalein (Fig. 2), a natural flavonoid and
well-known lipoxygenase inhibitor, used as a reference,
presents binding energy to the protein (-9.1 kcal/mol)
developing a number of H-bonds. These H-bonds were
observed between the 5-OH of the 5,6,7-trihydroxyflavone
with ASN146, ASN788 and CYS145.
TG study revealed that 4f having bis-phenyl group
among all Schiff bases exhibited the highest thermal sta-
bility. Among the tested compounds, following order of
thermal stability was observed; imines 4a–g [ hydrazide
3 [ carboxylic acid 1. Onset degradation temperature (To)
and residue yield calculated at 450 °C from TG curves are
summarized in Table 2. The order of calculated residue
is 4f [ 4g [ 4c [ 4b [ 4e [ 4d [ 4a [ 3 [ 1. Residue
data show that more than 80 % mass loss has taken place
up to 450 °C. It is well known that higher the value of
residue higher will be thermal stability, also clear from
comparative TG curves of derivatives (Fig. 3).
Differential scanning calorimetry
DSC thermograms of indole derivatives 1–4g are shown in
Fig. 4. It was generally observed that most of derivatives
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J IRAN CHEM SOC
1.4
1.3
1.2
1.1
1
α value
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.9
0.8
0.7
0.6
1.58
1.63
1.68
1.73
1.78
1.83
103 T-1(K-1)
Fig. 2 Docked poses of Baicalein (yellow) in the LOX binding site.
Side-chains of relevant binding site residues are rendered as ball and
stick models. The iron ion is rendered as an orange sphere
Fig. 5 Iso-conversional plot of O–F–W method for compound 4f
4e [ 4f [ 3. This can be attributed to various inter or intra
molecular interactions and the degree of various substitu-
ents in the compounds. It was seen that bulkier R1 substi-
tution greatly reduced meting point, for example in case of
4e and 4f.
1
3
4a
4b
4c
4d
4e
4f
4g
100
80
60
40
Kinetic study of compound 4f
20
0
On the basis of good LOX inhibition, the compound 4f was
selected to study the kinetics of thermal degradation. TG
curves of 4f were recorded at three multiple heating rates,
i.e., 5, 15 and 20 °C min-1 to meet the requirement of
kinetic methods (Figs. 5, 6, 7) [15].
0
100
200
300
400
500
Temprature (°C)
Fig. 3 TG curves of indole derivatives
The TG curves shift towards higher temperature with
increase in heating rates as shown in Fig. 8. This might be
due to different heat transfer and kinetic rates which delay
the decomposition of sample.
25
15
5
1
3
Iso-conversional plots of O–F–W and modified Coats–
Redfern for 4f are shown in Figs. 5 and 6, respectively. The
Ea and R2 values calculated by iso-conversional model-free
methods at a constant a value with mean and standard
deviation are given in Table 3.
The Ea values lie in the range of 7.8–8.9 kJ mol-1
calculated by iso-conversional methods. The mean value of
Ea calculated by O–F–W and modified Coats–Redfern
methods are 69.5 2.4 and 64.8 1.8 kJ mol-1, respec-
tively, corresponding to decomposition of 4f. Ea values
calculated by modified Coats–Redfern method are in good
agreement with those calculated by O–F–W [24]. The plot
of Ea as a function of a based on iso-conversional methods
is shown in Fig. 9.
-5
4a
4b
4c
4d
4e
4f
-15
-25
-35
-45
-55
4g
0
100
200
300
400
500
Temprature (°C)
Fig. 4 DSC curves
showed first endothermic peaks for melting and second
peak for decomposition.
Whereas compounds 4a, 4b and 4d in addition to two
endothermic peaks showed one extra exothermic peak of
boiling. Tonset of melting and Tpeak values corresponding to
the first, second and third DSC peaks are summarized in
Table 2. The Tonset of melting for compounds 1–4g are
122.3, 175.7, 126.1, 153.2, 174.1, 196.5, 169.8, 147.7,
145.1 and 202.5 °C. Unlike thermal stability; melting point
order of compounds is 4g [ 4c [ 1 [ 4b [ 4d [ 4a [
There is a small increment in Ea with progress of con-
version. This might be due to weaker bonds ruptures at
lower conversion values (a) and requires low Ea, but with
increase in conversion, residue left with stronger bonds will
ultimately require high Ea.
Also, the small standard deviation value and insignifi-
cant change in Ea value (Table 3) clearly demonstrate
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J IRAN CHEM SOC
-9.6
-9.8
Table 3 Activation energy and correlation coefficient calculated by
model-free iso-conversional methods for 4f under nitrogen
α value
0.1
0.2
0.3
0.4
0.5
0.6
0.7
-10
a Value O–F–W method
Ea (KJ/mol) R2
Modified Coats–Redfern
method
-10.2
-10.4
-10.6
-10.8
-11
Ea (KJ/mol) R2
0.1
8.9
0.994
0.996
0.999
0.997
1
8.6
8.8
8.2
8.4
8.5
8.6
7.8
0.987
0.2
8.8
0.999
-11.2
-11.4
0.3
8.3
0.997
1.58
1.63
1.68
1.73
1.78
1.83
0.4
8.5
0.993
103T-1(K-1)
0.5
8.6
0.998
0.6
8.7
1
0.999
Fig. 6 Iso-conversional plot of modified Coats–Redfern method of 4f
0.7
8.2
0.998
0.996
Mean
8.6 0.2
0.99 0.002 8.5 0.3
0.99 0.004
-9
-9.2
10
-9.4
9
8
7
6
5
4
3
2
1
0
-9.6
-9.8
-10
-10.2
-10.4
-10.6
-10.8
O-F-W method
Modified Coats–
Redfern method
1.58
1.6
1.62
1.64
1.66
1.68
1.7
0
0.2
0.4
0.6
0.8
103T-1(K-1)
α value
Fig. 7 A Kissinger plot for compound 4f
Fig. 9 A comparison and variation of activation energy calculated by
O–F–W and modified Coats–Redfern methods with degree of
conversion 4f
5 °C/min
15 °C/min
20 °C/min
100
80
60
40
20
0
obtained in kinetic investigation using different methods
are similar, it can be used in quality control of the drug,
with a view to attain the improvement of final product and
for the determination of drug quality via technological
parameters.
Conclusion
50
150
250
350
450
Temp (°C)
In summary, a series of new indole derivatives were syn-
thesized, tested in vitro against LOX inhibition and also
investigated their thermal behavior over a wide temp range.
Schiff base 4f exhibited comparatively best LOX inhibition
whereas indole ester 3 and Schiff bases 4c showed sig-
nificant to good LOX inhibition. Size and nature of sub-
stituent at 3-carbon of indole effectively control the LOX
inhibition. Docking simulation studies were carried out on
the 4f to find the preferable binding sites. The order of
LOX inhibition was 4f [ 4c [ 2 [ 4a [ 3 [ 4e [ 4b [
4d [ 4g while order of thermal stability was 4f [ 4g [
4c [ 4b [ 4e [ 4d [ 4a [ 3 [ 1. Among this series, the
Fig. 8 TG curves of 4f at heating rates of 5, 15 and 20 °C/min under
nitrogen atmosphere
about the possibility of single reaction mechanism
throughout the whole degradation process.
A plot of Kissinger method is shown in Fig. 7. The Ea
value calculated by Kissinger is 8.4 kJ mol-1 and corre-
sponding R2 is 0.997. The Ea Value calculated by Kissinger
method is very close to those Ea values obtained by O–F–
W and modified Coats–Redfern methods. Since results
123

J IRAN CHEM SOC
most potent compound, 4f with IC50 53.61 0.14 lM,
may act as potential lead molecules for the future research
in the field of LOX inhibitors. Work is ongoing to make
more derivatives of 4f to further increase the enzyme
inhibition properties.
washed with water (200 mL) and n-hexane (30 mL) to
purify the product.
N0-benzylidene-2-(1H-Indole-3-yl) acetohydrazide (4a)
The compound 4a was prepared by general method using
1H-indole-3-carbohydrazide
3 (500 mg, 2.6 mmol, 1
equiv.) and benzaldehyde (0.29 mL, 2.9 mmol) after re-
fluxing for 8 h to give the required product as white
amorphous solid; Yield: 62 %; m.p. 179–180 °C; Rf (n-
hexane: EtOAc 1:1) 0.475; IR (mmax, KBr, cm-1): 1,660
Experimental
All chemicals and solvents used are of analytical grade and
were purchased from Sigma, Aldrich and Merck Chemical
Co. and were used without further purification. Melting
points were taken in open capillary tubes and are uncor-
rected. TLC was run on the silica-coated aluminum sheets
(silica gel 60 F254, E Merck, Germany) and visualized in
low UV light. IR spectra in KBr pellets were recorded on
the FT-IR Perkin Elmer spectrum BX spectrophotometer.
1H NMR and 13C NMR spectra were recorded at 500 MHz
on JEOL-Lambda NMR instrument. Chemical shifts are
quoted as d ppm and the coupling constants J in Hz. Sig-
nals are described as s (singlet), d (doublet), m (multiplet)
and br (broad). Melting points were measured on a Bu¨chi
434 melting point apparatus and uncorrected.
1
(C=O), 1,551 (C=N), 1,199 (C–N), 3,412 (NH). H NMR
(500 MHz, DMSO-d6) d = 3.15 (2H, s, CH2–CO), 6.0–6.8
(10H, m, Ar–H), 7.30 (1H, broad s, NH), 7.09 (1H, s,
CH=N), 10.0 (1H, broad s, NH indole ring) ppm; 13C NMR
(DMSO-d6) d = 29.0, 108.1, 111.2, 118.3, 123.1, 126.1,
127.0 (2 9 C), 128.1 (2 9 C), 129.4, 130.1, 134.1, 136.2,
142.1, 146.2, 167.1 ppm. Anal. calc. for C17H15N3O
(277.32): C, 73.63; H, 5.45; N, 15.15; O, 5.77; found: C,
73.64; H, 5.44; N, 15.17; O, 5.76.
N0-(2-hydroxybenzylidene)-2-(1H-Indole-3-yl) acetohyd-
razide (4b) The compound 4b was prepared by general
method A using 1H-indole-3-carbohydrazide 3 (500 mg,
2.6 mmol) and salicylaldehyde (0.27 mL, 2.9 mmol) after
refluxing for 5 h to give the product as white amorphous
solid. Yield: 95 %; m.p: 188–189 °C; Rf (n-hexane:
EtOAc, 1:1) 0.42; IR (mmax, KBr, cm-1): 1,663 (C=O),
1,607 (C=N), 1,254 (C–N), 3,497(NH); 1H NMR
(500 MHz, DMSO-d6) d = 2.89 (1H, s, OH), 3.13 (2H,
s,CH2–CO), 6.0–7.09 (9H, m, Ar–H), 7.2 (1H, s, CH = N),
10.0 (1H, broad s, NH indole ring), 10.2 (1H, broad s, NH-
CO) ppm; 13C NMR (125 MHz, DMSO-d6): d = 29.2,
55.1, 108.3, 114.2, 118.1, 123.2, 124.1, 126.0, 127.2,
128.1, 128.3, 129.1, 130.2, 142.1, 146.2, 160.1, 166.2 ppm.
Anal. calc. for C17H15N3O2 (293.32): C, 69.61; H, 5.15; N,
14.33; O, 10.91; found: C, 69.62; H, 5.16; N, 14.35; O,
10.90.
Indole-3-methylacetate (2) Indole 3-methyl acetate 2 was
prepared using the reported method. 1 Yield: 95 %; m.p:
48–48.3 °C (Lit. m.p: 48–48.5 °C) [16]; Rf (EtOAc) 0.9; IR
(mmax, KBr pellets, cm-1): 3,472, 3,304, 2,986, 1,737,
1,641, 1,483; 1H NMR (500 MHz, CDCl3) dH: 3.58 (2H, s,
CH2–CO), 3.7 (3H, s, CH3–O), 6.9–7.4 (5H, m, Ar–H),
10.9 (1H, NH in indole ring).
2-(1H-indol-3-yl) acetohydrazide (3) 2-(1H-indol-3-yl)
acetohydrazide 3 was prepared using the reported method.
2 Yield: 60 %; m.p:142–143 °C (Lit. m.p: 142–145 °C)
[17]; Rf (n-hexane: EtOAc, 1:1) 0.18; IR (mmax, KBr pellets,
cm-1): 3,472 (NH), 3,304 (NH2), 2,986 (CH), 1,737
(C=O), 1,641 (C–N), 1,483 (C=C); 1H NMR (500 MHz,
CDCl3) dH: 3.7 (2H, s, CH2–CO), 4.1 (2H, s, NH2NH),
6.9–7.4 (5H, m, Ar–H), 9.1 (1H, s, NHNH2), 10.8 (1H,
broad s, NH in indole ring).
2-(1H-Indole-3-yl)-N0-(4-methoxybenzlidene) acetohydraz-
ide (4c) The compound 4c was prepared by general
method using 1H-indole-3-carbohydrazide 3 (500 mg,
2.6 mmol) and p-anisaldehyde (0.34 mL, 2.9 mmol) after
refluxing for 5 h to give the product as white amorphous
solid. Yield: 95 %, m.p: 190–191 °C; Rf (n-hexane: EtOAc,
1:1) 0.4; IR (mmax, KBr, cm-1): 1,660 (C=O), 1,607 (C=N),
1,254 (C–N), 3,355 (NH); 1H NMR (500 MHz, DMSO-d6)
d = 2.89 (3H, s, CH3–O), 3.13 (2H, s, CH2 –CO), 6.0–7.0
(9H, m, Ar–H), 7.20 (1H, s, CH=N), 10.0 (1H, broad s, NH
indole ring), 10.5 (1H, s, NH–CO) ppm; 13C NMR
(125 MHz, DMSO-d6) d: 29.1, 55.3, 108.2, 111.1, 114.2,
118 (2 9 C), 120.0, 126.5, 127.2, 128.1 (2 9 C), 130.2,
136.1, 142.2, 146.1, 167.1, 172.2; Anal. calc. for
C18H17N3O2 (307.35): C, 70.34; H, 5.58; N, 13.67; O,
10.41; found: C, 70.33; H, 5.57; N, 13.65; O, 10.43.
General procedure A for the synthesis of compounds
(4a–4g)
The compounds 4a–4g were prepared by following the
method reported by Joshi et al. with some modifications
starting from compound 3.3. 1H-Indole-3-carbohydrazide 3
(1 equiv) was dissolved into absolute methanol (50 mL) in
the presence of glacial acetic acid (2–3 mL). The reaction
mixture was stirred at r.t and then treated with suitable
aldehyde or ketone (1.1 equiv). After being refluxed for
5–8 h, the reaction mixture was then cooled overnight in
refrigerator at 0 °C. The resulting solid was filtered,
123

J IRAN CHEM SOC
2-(1H-Indole-3-yl)-N0-(4-methylbenzlidene) acetohydraz-
ide (4d) The compound 4d was prepared by general
method A using 1H-indole-3-carbohydrazide 3 (500 mg,
2.6 mmol) and p-tolualdehyde (0.34 mL, 2.9 mmol) after
refluxing for 5 h to give the product as white amorphous
solid. Yield: 63 %; Rf (n-hexane: EtOAc, 1:1) 0.65; m.p:
199–200 °C; IR (mmax, KBr, cm-1): 1,650 (C=O), 1,607
(C=N), 1,229 (C–N), 3,400 (NH); 1H NMR (500 MHz,
DMSO-d6): d = 1.40 (3H, s, CH3-Ph), 3.70 (2H, s, CH2–
CO), 6.0–7.0 (9H, m, Ar–H), 7.10 (1H, s, CH=N), 9.98
(1H, broad s, NH indole ring), 10.4 (1H, s, NH-CO) ppm;
13C NMR (125 MHz, DMSO-d6): d = 21.1, 29.2, 108.1,
111.2, 118.1, 121.2, 123.1, 127.2 (2 9 C), 129.1 (2 9 C),
130.1, 131.1, 136.1, 139.6, 142.1, 146.1, 172.3 ppm; Anal.
calc. for C18H17N3O (291.35): C, 74.20; H, 5.88; N, 14.42;
O, 5.49; found: C, 74.18; H, 5.90; N, 14.43; O, 5.51.
after refluxing for 8 h to give the product as white amor-
phous solid. Yield: 56 %; m.p: 212–214 °C; Rf (n-hexane:
EtOAc, 1:1) 0.55; IR (mmax, KBr, cm-1):1,660 (C=O), 742
1
(C–Cl), 1,557 (C=N), 1,200 (C–N), 3,400 (NH); H NMR
(500 MHz, DMSO-d6) dH: 3.60 (2H, s, CH2–CO),
6.0–7.10 (9H, m, Ar–H), 8.1 (1H, s, CH=N), 10.8 (1H,
broad s, NH indole ring), 11.2 (1H, s, NH–CO); 13C NMR
(125 MHz, DMSO-d6): 29, 108, 111, 118, 123, 128
(2 9 C), 126, 129, 130 (2 9 C), 132, 136, 139, 142, 146,
172; Anal. calc. for C17H14N3OCl (311.77): C, 65.49; H,
4.53; N, 13.48; O, 5.13; found: C, 65.47;H, 4.55; N, 13.46;
O, 5.16.
Thermal study
Differential scanning microscopy and thermogravimetric
analysis of all derivatives were carried out from ambient
temperature to 500 °C under nitrogen atmosphere at a
heating rate of 20 °C min-1 using SDT Q-600 instrument.
Platinum sample holder (90 lL) was used for taking curves
and dried platinum pan as a reference material. To achieve
the good reproducibility, small amounts of indole deriva-
tives were taken (2–9 mg).
2-(1H-Indole-3-yl)-N0-(1-phenylethyldene) acetohydrazide
(4e) The compound 4e was prepared by general
method A using 1H-indole-3-carbohydrazide 3 (500 mg,
2.6 mmol) and acetophenone (0.34 mL, 2.9 mmol) after
refluxing for 8 h to give the product as white amorphous
solid. Yield: 64 %; m.p: 54–55 °C; Rf (n-hexane: EtOAc,
1:1) 0.475; IR (mmax, KBr, cm-1): 1,664 (C=O), 1,545
1
(C=N), 1,199 (C–N), 3,448 (NH); H NMR (DMSO-d6)
Non-isothermal model-free kinetic methods
d = 1.30 (1H, s, CH3-C), 3.19 (2H, s, CH2–CO), 6.0–7.1
(10H, m, Ar–H,), 9.90 (1H, broad s, NH indole ring), 9.50
(1H, broad s, NH–CO) ppm; 13C NMR (125 MHz, DMSO-
d6): d = 14.1, 29.0, 108.2, 111.1, 118.1, 120.2, 123.1,
126.1 (2 9 C), 128.2 (2 9 C), 129.1, 131.1, 136.1, 138.1,
142.2, 146.1, 167 ppm; Anal. calc. for C18H17N3O
(291.35): C, 74.20; H, 5.78; N, 14.42; O, 5.49; found: C,
74.18; H, 5.76; N, 14.42; O, 5.50.
TG curves were analyzed using Qseries600 software from
TG analyzer, and data were used in MS Excel software to
appraise activation energy (Ea) and correlation coefficient
(R2). The Ea value of sample was calculated by ‘‘model
free’’ methods, namely, Ozawa–Flynn–Wall (O–F–W),
Coats–Redfern (modified), and Kissinger using Eqs. 1–3,
respectively [25–27]. In these equations, b is the heating
rate, R is the universal gas constant, Tm is temperature
corresponding to DSC peak maxima, A is the pre expo-
nential factor, a is the degree of conversion, and g(a) is a
function of a. In iso-conversional O–F–W method, log b
was plotted against 1/T which leads to slope –0.4567 Ea/R
of a line at a particular conversion value.
N0-(diphenylmethylene)-2-(1H-indole-3-yl) acetohydrazide
(4f) The compound 4f was prepared by general method A
using 1H-indole-3-carbohydrazide 3 (500 mg, 2.6 mmol)
and benzophenone (422 mg, 2.926 mmol) after refluxing
for 8 h to give the product as white amorphous solid. Yield:
88 %; m.p: 161–163 °C; Rf (n-hexane: EtOAc, 1:1) 0.625;
IR (mmax, KBr pellets, cm-1): 1,684 (C=O), 1,505 (C=N),
ꢀ
ꢁ
ꢀ
ꢁ
AEa
R
Ea
1
ðlog gðaÞÞ ¼ log
ꢁ logðbÞ ꢁ 2:315 ꢁ 0:4567
1,223 (C–N), 3,474 (NH); H NMR (500 MHz, DMSO-
RT
d6): d = 3.2 (2H, s, CH2–CO), 6.0–7.1 (15H, m, Ar–H),
8.30 (1H, broad s, NH indole ring), 9.90 (1H, broad s, NH–
CO) ppm; 13C NMR (125 MHz, DMSO-d6) d: 29, 106,
107, 111, 118, 121, 124, 127 (4 9 C), 128 (2 9 C), 129
(4 9 C), 131 (2 9 C), 137, 154, 167,172 ppm; Anal. calc.
for C23H19N3O (353.42): C, 78.16; H, 5.42; N, 11.89; O,
4.53; found: C, 78.18; H, 5.40, N, 11.88; O, 4.55.
ð1Þ
2
3
ꢄ
ꢅ
b
AR
Ea
4
d ln
5
ꢂ
ꢃ
ln
¼ ln ꢁ
ꢁ
ð2Þ
2RT
Ea
Ealnð1 ꢁ aÞ
RT
T2 1 ꢁ
ꢂ ꢂ ꢃꢃ
b
Tm2
Ea
R
ꢂ ꢃ
¼ ꢁ
ð3Þ
N0-(4-chlorobenzylidene)-2-(1H-Indole-3-yl) acetohydraz-
ide (4g) The compound 4g was synthesized by general
method A using 1H-indole-3-carbohydrazide 3 (500 mg,
2.6 mmol) and p-chlorobenzaldehyde (422 mg, 2.9 mmol)
1
Tm
d
The modified Coats–Redfern method is a multiple
heating rate application of the Coats–Redfern equation.
123

J IRAN CHEM SOC
In this method, ln (b/T2) is plotted against 1/T which gives
slope –Ea/R of line at any degree of conversion. In
Kissinger method, a linear plot of ln (b/T2m) against 1/Tm
gives a slope -Ea/R. Using the value of slope of each line,
Ea was calculated by each method.
solutions ranked according to the corresponding scoring
function values, each defined by the 3D coordinates of its
atoms and expressed as a PDB file.
Acknowledgments We acknowledge Aamir Saghir for his guidance
and Higher Education Commission and Ministry of Science and
Technology Pakistan for financial support.
Lipoxygenase assay
LOX inhibition was assayed according to the reported
method [28] but with slight modification. A total volume of
200 lL was prepared by mixing 140 lL of sodium phos-
phate buffer (100 mM, pH 8.0), 20 lL test compound and
15 lL (600 U) purified lipoxygenase enzyme (Sigma,
USA). The mixture was pre-read at 234 nm and pre-incu-
bated for 10 min at 25 °C. Then, reaction was initiated by
the addition of 25-lL substrate solution (linoleic acid in
Tween 20). The change in absorbance was observed after
6 min at 234 nm. Synergy HT (BioTek, USA) 96-well
plate reader was used in all experiments. All reactions were
performed in triplicates. The positive and negative controls
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123