Design, synthesis and QSAR study of arylidene indoles as anti-platelet aggregation inhibitors

Article abstract of DOI:10.1007/s00044-015-1440-7

A series of novel substituted indole carbohydrazide was synthesized and evaluated for anti-platelet aggregation activity. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis and were evaluated for their ability to inhibit platelet aggregation induced by adenosine diphosphate, arachidonic acid (AA) and collagen. Compounds 3e and 3b exhibited the highest activities against the platelet aggregation induced by collagen with IC₅₀ values of 12.7 and 13.3 μM, respectively, and 2h with IC₅₀ value of 51.88 μM and 2i with IC₅₀ of 44.38 μM efficiently inhibited platelet aggregation induced by AA. The QSAR investigation indicated the importance of the topological, constitutional and geometrical parameters (PW3, PW4, LP1 and GATS6v) in describing the anti-platelet aggregation activity of the synthesized hydrazides. Evaluation of cytotoxic activity of the compounds against L929 cell line and three cancer cell lines revealed that none of the compounds have significant cytotoxicity. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-015-1440-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1440-7
ORIGINAL RESEARCH
Design, synthesis and QSAR study of arylidene indoles
as anti-platelet aggregation inhibitors
Seyedeh Sara Mirfazli1 Mehdi Khoshneviszadeh2
•
•
Mohammad Jeiroudi1 Alireza Foroumadi1
Farzad Kobarfard3,4 Abbas Shafiee1
•
•
•
Received: 29 September 2014 / Accepted: 18 August 2015
Ó Springer Science+Business Media New York 2015
Abstract A series of novel substituted indole carbohy-
drazide was synthesized and evaluated for anti-platelet
aggregation activity. The structures of the synthesized
compounds were confirmed by spectral data and elemental
analysis and were evaluated for their ability to inhibit
platelet aggregation induced by adenosine diphosphate,
arachidonic acid (AA) and collagen. Compounds 3e and 3b
exhibited the highest activities against the platelet aggre-
gation induced by collagen with IC50 values of 12.7 and
13.3 lM, respectively, and 2h with IC50 value of 51.88 lM
and 2i with IC50 of 44.38 lM efficiently inhibited platelet
aggregation induced by AA. The QSAR investigation
indicated the importance of the topological, constitutional
and geometrical parameters (PW3, PW4, LP1 and
GATS6v) in describing the anti-platelet aggregation
activity of the synthesized hydrazides. Evaluation of
cytotoxic activity of the compounds against L929 cell line
and three cancer cell lines revealed that none of the com-
pounds have significant cytotoxicity.
Graphical Abstract
O
OH
HN
H
N
O
O
N
O
O
HO
Aspirin
IC50= 9.7 µM
IC50= 12.7 µM
Keywords Anti-platelet aggregation Á Cytotoxicity Á
Indole Á N-acylhydrazone Á QSAR
Introduction
A primary factor in the development of thrombotic disor-
ders, such as unstable angina, myocardial infarction, stroke
and peripheral vascular diseases, is platelet aggregation
which plays a central role in thrombosis (clot formation)
(Reddy et al., 2011).
& Farzad Kobarfard
kobarfard@sbmu.ac.ir
Blood is not aggregated in the blood vessels, but when
bleeding occurs, blood aggregation is occurred as a phys-
iological defense reaction. Activation of the platelets by a
complex of biochemical pathways and multitude of medi-
ators is the primary step in the progress of these diseases
(Mashayekhi et al., 2013a, b). However, platelet aggrega-
tion is also caused by physiological substances, such as
thrombin and prostaglandin endoperoxide, and it leads to
an arterial thrombosis. Platelets can be activated by a
number of endogenous agonists such as arachidonic acid
(AA), adenosine 50-diphosphate (ADP), thromboxane A2
(TxA2), thrombin, platelet activating factor (PAF),
& Abbas Shafiee
ashafiee@ams.ac.ir
1
Department of Medicinal Chemistry, Faculty of Pharmacy
and Pharmaceutical Sciences Research Center, Tehran
University of Medical Sciences, Tehran, Iran
2
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
3
Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
4
Phytochemistry Research Center, Shahid Beheshti University
of Medical Sciences, Vali Asr Ave. Niayesh Junction,
P.O. Box 14155-6153, Tehran, Iran
123

Med Chem Res
epinephrine (EPN) and collagen (DeCandia et al., 2009;
Reddy et al., 2011).
such as arachidonic acid. Furthermore, these fragments
have an important role as pharmacophore cores with anti-
inflammatory, anti-nociceptive and anti-platelet aggrega-
tion activity (Silva et al., 2004).
Acetyl salicylic acid (a non-selective irreversible COX
inhibitor) and clopidogrel (an irreversible antagonist of
P2Y12) are the most commonly prescribed oral anti-pla-
telet drugs to date (Mashayekhi et al., 2013a, b). However,
definite disadvantageous effects of these compounds, for
instance, a number of drug-class-specific adverse reactions
such as gastrointestinal ulcers, bleeding, thrombocytopenic
purpura and skin necrosis are repeatedly reported. Also,
inefficient therapies and their related problems cause anti-
platelet therapy more complicated.
Nazare et al., (2004) introduced indolecarboxamides as
a rigid a-amino acid mimetic and very flexible moiety,
enabling to represent different structures at its nitrogen or
also carboxy group (Nazare et al., 2004). Moreover, in a
successful study, indole ring is demonstrated as a structural
moiety which has anti-platelet aggregation effect. Indole-3-
carbinol, a natural compound found in cruciferous veg-
etables, is known to have anti-platelet and anti-thrombotic
activities in vitro and in vivo. Indole-3-carbinol has been
shown to inhibit collagen-induced platelet aggregation in
human platelet-rich plasma (PRP) in dose-dependent
manner (Mashayekhi et al., 2013a, b; Park et al., 2008).
In our ongoing research addressed toward new anti-platelet
aggregation agents which showed inhibitory effects on AA-,
collagen- and ADP-induced aggregation, a series of indole
hydrazonecompoundshavebeendesignedandsynthesizedby
molecular hybridization between indole and hydrazones
moieties, as potential anti-platelet aggregation derivatives
(Haj Mohammad Ebrahim Tehrani et al., 2013; Mashayekhi
et al., 2013a, b). In order to investigate the potential correla-
tion between activity and positional isomerism in the deriva-
tives, two groups of compounds were synthesized with
various B-ring substituents on position 2 and position 3 of
indole ring, and the compounds were screened for their inhi-
bitory effects on platelet aggregation, induced by arachidonic
acid, ADP and collagen. Thus, in this research, we report the
synthesis, platelet aggregation inhibitory effects and SAR
study of new indole acylhydrazone derivatives (Fig. 2).
Therefore, many research groups have made significant
attempts to modify the potency and platelet anti-aggregatory
activity of common drugs (Siwek et al., 2011).
N-acylheteroarylhydrazones (NAH) have been recently
reported as platelet aggregation inhibitors. Bezerra-Neto
et al., (2006) have previously described the N-acylhydrazone
subunit as a pharmacophore group for analgesic, anti-in-
flammatory and anti-platelet properties, and it is also consid-
ered a privileged structure in the design of new bioactive
compounds (Duarte et al., 2007; Brito et al., 2010). Coquelet
et al. have reported some acylhydrazone derivatives as inhi-
bitors of platelet cyclooxygenase (COX) and lipooxigenase
(LOX) blocking the transformation of arachidonic acid to its
proaggregatory metabolites. In light of the finding of this
study,Barreiroetal.,ina seriesofstudies, managedtodevelop
some new hydrazones which effectively inhibited platelet
aggregation with selective inhibitory activity toward platelet
aggregation induced by arachidonic acid. They found active
group of hydrazones with arylsulfonate acylhydrazone, phe-
nothiazine-1-acylhydrazone, N-substituted-phenyl-1,2,3-tri-
azole-4-acylhydrazone and pyrazolylhydrazone structures.
Although a diverse group of derivatives have been found to
exhibit anti-platelet activity in these studies, they share a
preserved structural backbone that is two (hetero)aromatic
ring systems linked by a hydrazone bond (Haj Mohammad
Ebrahim Tehrani et al., 2013) (Fig. 1).
Materials and methods
General
Coquelet et al. reported the most active derivatives with
R = CH3 and NH2, while Barreiro et al. have mainly
focused on the derivatives with R = H. Cunha et al.,
(2003) also synthesized N-phenylpyrazolyl moiety which
has the NAH chain that exhibited inhibitory effect on AA-,
collagen- and ADP-induced platelet aggregation so, rep-
resented a novel family of anti-platelet agents. Silva et al.
suggest that the hydrazone and acylhydrazone moieties
demonstrate a subunit which stabilize free radicals that
mimicking bis-allyl fragment of unsaturated fatty acids
All commercial solvents, chemicals and reagents were
purchased from either Merck or Sigma-Aldrich with the
highest purity and used without further purification. Proton
nuclear magnetic resonance (1H NMR) spectra were
H
N
H
H
O
Ar
O
Ar
N
4
7
H
N
3a
7a
N
5
6
N
H
7a
2
7
3
N
H
6
Fig. 1 Schematic
3a
Ar1
Ar2
4
representation of the general
hydrazone structural backbone
with anti-platelet activity
N
3
2
5
HN
R
Fig. 2 Main scaffolds of target compounds
123

Med Chem Res
Table 1 In vitro anti-platelet aggregation activity of the synthesized compounds (2a-m and 3a-r) induced by AA, ADP and collagen
Comp.
Ar
IC50(lM)a
Inhibition (%)b
Collagen
AA
ADP
2a
Phenyl
134 4.7
796 19.9
705 21.1
[1000
94 2.5
50.6 2.8
47.6 0.8
21.5 1.5
66.7 3.0
0
2b
2,4-Dichlorophenyl
2,6-Dichlorophenyl
4-Methoxyphenyl
2-Nitro-5-thiophene
2-Hydroxy-5-ethoxyphenyl
2,4-Dihydroxyphenyl
2,5-Dihydroxyphenyl
2,3-Dihydroxyphenyl
5-Chloro-2-nitrophenyl
2-Nitro-5-furane
880 8.9
837 4.6
142 1.8
731 4.7
[1000
2c
2d
2e
328 2.1
[1000
2f
0
2g
416.5 12.4
173.2 10.4
239.4 6.3
[1000
414 5.2
51.88 0.8
44.38 0.9
[1000
53.6 2.0
57.2 1.8
58.6 0.7
58.7 2.3
37.7 0.6
61.2 3.3
20.6 1.0
80.3 1.0
71.24 0.8
Not dissolved
83 1.3
2h
2i
2j
2k
[1000
618 4.6
[1000
2l
5-Bromo-2-hydroxyphenyl
3-Methoxyphenyl
Phenyl
520.1 17.7
446.7 15.1
20.9 1.4
13.3 1.0
Not dissolved
19.5 1.2
12.7 0.9
23.3 1.1
106.8 4.9
70 1.4
2m
137 2.1
182.6 6.1
251.2 5.8
Not dissolved
497 3.9
219.8 1.9
292 2.7
326 8.0
190 1.5
292.5 3.4
169.8 2.2
102.5 6.7
229 1.1
237 0.8
530 1.9
307.3 5.0
Not dissolved
Not dissolved
346 1.7
3a
3b
2,6-Dichlorophenyl
2,4-Dichlorophenyl
4-Methoxyphenyl
2-Hydroxy-5-methoxyphenyl
4-Nitrophenyl
3c
3d
3e
55 0.5
3f
50 1.1
3g
3-Indole
77 0.9
3h
2-Thiophene
80 1.4
3i
5-Chloro-2-nitrophenyl
2,3-Dihydroxyphenyl
2,5-Dihydroxyphenyl
2,4-Dihydroxyphenyl
2,3-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
3-Nitrophenyl
20.3 1.4
66.8 2.6
31.5 0.8
64.7 2.8
124.8 3.5
227.9 9.1
95.6 3.0
Not dissolved
Not dissolved
295 5.9
1.2 0.1
70 0.7
3j
64.7 2.3
98 2.6
3k
3l
75.2 3.1
18 1.4
3m
3n
10 0.4
3o
77.8 2.6
Not dissolved
Not dissolved
35.7 1.9
42 1.1
3p
2-Nitro-5-thiophene
5-Nitrofuran
3q
3r
Indomethacinc
3-Methoxyphenyl
3
0.2
Aspirinc
9.7 0.6
30 2.6
21 0.6
a
Values are presented as mean SD of three separate determination
b
Inhibition (%) values for ADP as a platelet aggregation inducer represent at 1 mM concentration for (2a–m) and at 250 lM concentration for
(3a–r)
c
Aspirin and indomethacin were used as a positive control
recorded on a Bruker 500 MHz spectrometers (Bruker,
Rheinstetten, Germany), pick positions are illustrated in
parts per million (d) in DMSO-d6 solution and tetram-
ethylsilane (0.05 % v/v) as internal standard, and coupling
constant values (J) are given in Hertz. Signal multiplicities
are reported by: s (singlet), d (doublet), t (triplet), q
(quadruplet), m (multiplet) and brs (broad signal). For
NMR spectral data assignments, the atom numbering of
compounds is depicted in Table 1. Analytical thin-layer
chromatography (TLC) was performed with Merck silica
gel plates and visualized with UV irradiation (254 nm) or
iodine. Electrospray ionization mass spectra (ESI–MS)
were obtained using Agilent 6410 Triple Quad. LC/MS.
Melting points were obtained by an Electrothermal 9100
apparatus and are uncorrected. The IR spectra were taken
by a PerkinElmer 843 spectrometer with KBr as diluent.
123

Med Chem Res
Scheme 1 The synthesis pathway for indole N-acylhydrazones
General procedure for the preparation of N-acylhydrazone
derivatives (2a–m) and (3a–r) Titled compounds (N-
acylhydrazone derivatives) were obtained according to the
literature method (Mirfazli et al., 2014).
The elemental analysis for C, H and N was performed by a
Costech model 4010, and the percentage values agreed
with the proposed structures within 0.4 % of the theo-
retical values. All described products showed 1H NMR
spectra according to the assigned structures.
N0-benzylidene-1H-indole-3-carbohydrazide
(3a) The
derivative 3a was obtained as a white solid by condensa-
tion of B3 with benzaldehyde in 82.5 % yield, mp
232–234 °C. IR (KBr) cm-1: 3282, 3206, 3115, 3059,
3032, 1629, 1601, 1576, 1547; 1H NMR (400 MHz,
DMSO-d6): d 11.80 (brs, 1H, Indole-NH), 11.44 (s, 1H,
CONH), 8.31 (bs, 1H, –N=CH–), 8.21–8.42 (m, 2H, Indole
H2, H4), 7.72 (d, 2H, J = 6.8 Hz, –N=CH–C6H5 H2, H6),
7.41–7.49 (m, 4H, Indole H7, –N=CH–C6H5 H3, H4, H5),
7.18 (m, 2H, Indole H5, H6); 13C NMR (125 MHz, DMSO-
d6) d, 161.25 (C=O), 144.72 (C=N), 142.55 (C3a), 135.95
(C10 benzyl), 134.76 (C40 benzyl), 134.52 (C2), 132.22
(C7a), 129.47 (C20 and C60 benzyl), 129.19 (C30 and C50
benzyl), 128.78 (C4), 122.21 (C6), 121.16 (C5), 111.94
(C3), 108.50 (C7). ESI-Mass m/z: 264 [M ? H]?, 286
[M ? Na]?, 302 [M ? K]?; Anal. Calcd. for C16H13N3O:
C, 72.99; H, 4.98; N, 15.96. Found: C, 72.62; H, 5.08; N,
15.72 (Alemany et al., 1966).
Chemistry
The synthetic procedure planned to obtain the desired
indole N-acylhydrazone derivatives is shown in Scheme 1.
The key intermediates were obtained by hydrazinolysis of
methyl 1H-indole-3-carboxylate and ethyl 1H-indole-2-
carboxylate in 96 and 91 % yield, respectively, using
hydrazine monohydrate 99 % in ethanol. The final indole
N-acylhydrazone derivatives were obtained by condensing
the hydrazide intermediates with the proper aromatic
aldehydes (W-ArCHO) in water and glacial acetic acid as
the solvent, in good yields.
The derivatives were obtained by the reaction of 1H-
indole-3-carbohydrazide (B3) and 1H-indole-2-carbohy-
drazide (B2) with the proper aldehydes. Synthesis of Schiff
bases was performed in ethanol with a few drops of glacial
acetic acid. This reaction in the majority of the cases was
straightforward, but the products were soluble in ethanol
and their separation was difficult. Thus, water was used as
solvent and a few drops of glacial acetic acid were added to
the reaction mixture and heated to complete the reaction.
N0-(2,6-dichlorobenzylidene)-1H-indole-3-carbohydrazide (3b)
The derivative 3b was obtained as a white solid by con-
densation of B3 with 2,6-dichlorobenzaldehyde in 86 %
yield, mp 281–283 °C. IR (KBr) cm-1: 3412, 3234, 3180,
3086, 1651, 1614, 1592, 1556; 1H NMR (500 MHz,
DMSO-d6): d 11.79 (bs, 1H, Indole-NH), 11.40 (s, 1H,
CONH), 8.48 (bs, 1H, –N=CH–), 8.35 (bs, 1H, Indole H2),
8.24 (d, 1H, J = 8.0 Hz, Indole H4), 7.58 (d, 2H,
J = 8 Hz, –N=CH–C6H5, H3, H5), 7.48 (d, 1H,
J = 8.0 Hz, Indole H7), 7.43 (t, 1H, J = 8 Hz, –N=CH–
C6H5 H4), 7.22–7.15 (m, 2H, Indole H5, H6). 13C NMR
(125 MHz, DMSO-d6): d 153.25 (C=O), 139.57 (C=N),
137.81 (C3a), 135.77 (C20 and C60 2,6-dichlorophenyl),
133.76 (C2), 130.78 (C10 2,6-dichlorophenyl), 129.13 (C40
General procedure for the preparation of carbohydrazides
(B2 and B3)
1H-indole-3-carbohydrazide (B3) and 1H-indole-2-carbo-
hydrazide (B2) were prepared according to the literature
method (Mirfazli et al., 2014, Bao et al., 2013). Com-
pounds 2b, 2j and 2k in which E/Z ratio was higher than 10
percent were distinguished through their H and 13C NMR
1
spectra.
123

Med Chem Res
2,6-dichlorophenyl), 126.84 (C7a),123.09 (C30 and C50 2,6-
dichlorophenyl), 122.25 (C4), 121.16 (C6), 120.85 (C5),
111.91 (C3), 107.48 (C7). ESI-Mass m/z: 354 [M ? Na]?;
Anal. Calcd. for C16H11Cl2N3O: C, 57.85; H, 3.34; N,
12.65. Found: C, 57.63; H, 3.48; N, 13.02.
d 11.79 (bs, 1H, Indole-NH), 11.69 (s, 1H, CONH), 10.88
(bs, 1H, OH), 8.50 (s, 1H, –N=CH–), 8.22 (bs, 1H, Indole
H2), 8.19 (d, 1H, J = 7.80 Hz, Indole H4), 7.48 (d, 1H,
J = 7.80 Hz, Indole H7), 7.22–7.15 (m, 2H, Indole H5,
H6), 7.12 (s, 1H, –N=CH–C6H4, H6), 6.91–6.85 (m, 2H,
–N=CH–C6H4, H3, H4), 3.74 (s, 3H, –OCH3). 13C NMR
(125 MHz, DMSO-d6) d: 160.77 (C=O), 159.10 (C20
2-hydroxy-5-methoxyphenyl), 151.17 (C50 2-hydroxy-5-
methoxyphenyl), 144.97 (C=N), 136.08 (C3a), 128.77
(C2), 126.43 (C7a), 122.32 (C4), 121.00 (C6), 120.84 (C5),
119.26 (C10 2-hydroxy-5-methoxyphenyl), 117.38 (C40
2-hydroxy-5-methoxyphenyl), 117.12 (C30 2-hydroxy-5-
methoxyphenyl), 112.46 (C60 2-hydroxy-5-methox-
yphenyl), 112.02 (C3), 108.18 (C7), 55.43 (O-CH3). ESI-
Mass m/z: 310 [M ? H]?, 332 [M ? Na]?; Anal. Calcd.
for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58. Found: C,
66.36; H, 5.03; N, 13.70.
N0-(2,4-dichlorobenzylidene)-1H-indole-3-carbohydrazide (3c)
The derivative 3c was obtained as a white solid by con-
densation of B3 with 2,4-dichlorobenzaldehyde in 78 %
yield, mp 288–291 °C. IR (KBr) cm-1: 3472, 3316, 3282,
3126, 3103, 1659, 1612, 1594,1569.1540; 1H NMR
(500 MHz, DMSO-d6): d 11.79 (s, 1H, Indole-NH), 11.64
(s, 1H, CONH), 8.66 (bs, 1H, –N=CH–), 8.25 (bs, 1H,
Indole H2), 8.19 (d, 1H, J = 7.6 Hz, Indole H4), 8.02 (d,
1H, J = 8.5 Hz, –N=CH–C6H5, H6), 7.71 (d, 1H,
J = 2.25 Hz, –N=CH–C6H5, H3), 7.53 (dd, 1H, J = 8.55,
2.07 Hz, –N=CH–C6H5, H5), 7.48 (d, 1H, J = 7.6 Hz,
Indole H7), 7.19 (m, 2H, Indole H5, H6). 13C NMR
(125 MHz, DMSO-d6): d 157.15 (C=O), 139.57 (C=N),
137.20 (C3a), 135.77 (C20 2,4-dichlorophenyl), 133.76
(C2), 129.13 (C10 2,4-dichlorophenyl), 128.35 (C30 2,4-
dichlorophenyl), 127.23 (C60 2,4-dichlorophenyl), 126.84
(C7a), 125.12 (C40 2,4-dichlorophenyl), 122.25 (C50 2,4-
dichlorophenyl), 121.16 (C4), 120.85 (C6), 111.91 (C5),
108.01 (C3), 107.48 (C7). ESI-Mass m/z: 354 [M ? Na]?;
Anal. Calcd. for C16H11Cl2N3O: C, 57.85; H, 3.34; N,
12.65. Found: C, 57.71; H, 3.20; N, 12.92.
N0-(4-nitrobenzylidene)-1H-indole-3-carbohydrazide (3f)
The derivative 3f was obtained as a yellow solid by con-
densation of B3 with 4-nitrobenzaldehyde in 89 % yield,
mp 289–291 °C. IR (KBr) cm-1: 3387, 3250, 3126, 3044,
1638, 1584, 1552, 1520 and 1343 (NO2); 1H NMR
(500 MHz, DMSO-d6): d 11.79 (bs, 2H, CONH, Indole-
NH), 8.41 (bs, 1H, Indole H2), 8.30–8.27 (m, 3H, –N=CH–
C6H5, H3, H5, –N=CH–), 8.23 (d, 1H, J = 7.7 Hz, Indole
H4), 7.96 (d, 2H, J = 8.6 Hz, –N=CH–C6H5, H2, H6), 7.50
(d, 1H, J = 7.7 Hz, Indole H7), 7.23–7.16 (m, 2H, Indole
H5, H6). 13C NMR (125 MHz, DMSO-d6) d: 161.76
(C=O), 147.38 (C40 4-nitrophenyl), 141.57 (C=N), 141.18
(C10 4-nitrophenyl), 135.98 (C3a), 129.33 (C2), 127.50
(C7a), 126.67 (C20 and C60 4-nitrophenyl), 124.00 (C30 and
C50 4-nitrophenyl), 122.35 (C4), 121.09 (C6), 120.90 (C5),
112.02 (C3), 108.03 (C7). ESI-Mass m/z: 309 [M ? H]?;
Anal. Calcd. for C16H12N4O3: C, 62.33; H, 3.92; N, 18.17.
Found: C, 62.54; H, 3.63; N, 18.52 (Alemany et al., 1966).
N0-(4-methoxybenzylidene)-1H-indole-3-carbohydrazide (3d)
The derivative 3d was obtained as a white solid by con-
densation of B3 with 4-methoxybenzaldehyde in 61 %
yield, mp 239–241 °C. IR (KBr) cm-1: 3386, 3300, 3163,
3137, 1667, 1629, 1594, 1558; 1H NMR (500 MHz,
DMSO-d6): d 11.71 (s, 1H, Indole-NH), 11.27 (s, 1H,
CONH), 8.30–8.20 (m, 3H, Indole H2, H4, –N=CH–), 7.66
(d, 2H, J = 8.70 Hz, –N=CH–C6H5, H2, H6), 7.48 (d, 1H,
J = 7.85 Hz, Indole H7), 7.21–7.14 (m, 2H, Indole H5,
H6), 7.03 (d, 2H, J = 8.70 Hz, –N=CH–C6H5, H3, H5),
3.82 (s, 3H, –OCH3). 13C NMR (125 MHz, DMSO-d6) d:
161.11 (C=O), 160.39 (C40 4-methoxyphenyl), 144.75
(C=N), 144.31 (C3a), 135.93 (C2), 128.24 (C20 and C60
4-methoxyphenyl), 127.33 (C7a), 126.73 (C10 4-methox-
yphenyl), 122.15 (C4), 121.13 (C6), 120.65 (C5), 114.29
(C30 and C50 4-methoxyphenyl), 111.89 (C3), 108.59 (C7),
55.25 (O-CH3). ESI-Mass m/z: 294 [M ? H]?; Anal.
Calcd. for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33.
Found: C, 69.36; H, 5.43; N, 14.70 (Alemany et al., 1966).
N0-((1H-indol-3-yl)
methylene)-1H-indole-3-carbohy-
drazide (3g) The derivative 3g was obtained as a white
solid by condensation of B3 with 1H-indole-3-carbalde-
hyde in 63.5 % yield, mp 279–281 °C. IR (KBr) cm-1
:
3387, 3361, 3210, 3110, 3068, 1632, 1607, 1577, 1541; 1H
NMR (500 MHz, DMSO-d6): d 12.03 (s, 1H, Indole-NH),
11.81 (s, 1H, –Indole-NH), 10.31 (s, 1H, CONH), 8.99 (s,
1H, –N=CH–), 8.60 (s, 1H, –Indole H2), 7.68 (d, 1H,
J = 7.9 Hz, –Indole H4), 7.48 (d, 1H, J = 8.1 Hz, Indole
H4), 7.33 (s, 1H, Indole H2), 7.24 (t, 2H, J = 7.4 Hz,
–Indole H7, Indole H7), 7.04–7.09 (m, 2H –Indole H5,
Indole H5), 6.79–6.73 (m, 2H, –Indole H6, Indole H6). 13C
NMR (125 MHz, DMSO-d6) d: 157.34 (C=O), 150.05
(C=N), 149.89 (C3a), 146.57 (C30a 1H-indole-3-yl), 136.85
(C2), 129.74 (C20 1H-indole-3-yl), 126.96 (C7a), 123.89
(C70a 1H-indole-3-yl), 121.78 (C4), 119.98 (C40 1H-
N0-(2-hydroxy-5-methoxybenzylidene)-1H-indole-3-carbohy-
drazide (3e) The derivative 3e was obtained as a pale
yellow solid by condensation of B3 with 2-hydroxy-5-
methoxybenzaldehyde in 97 % yield, mp 263–266 °C. IR
(KBr) cm-1: 3576 (m OH), 3380–3260 (m NH), 3200, 3058,
1
1663, 1640, 1614, 1577. H NMR (500 MHz, DMSO-d6):
123

Med Chem Res
indole-3-yl), 119.23 (C6), 118.82 (C60 1H-indole-3-yl),
118.23 (C5), 118.00 (C50 1H-indole-3-yl), 117.04 (C3),
113.44 (C30 1H-indole-3-yl), 112.37 (C7), 103.67 (C70 1H-
indole-3-yl). ESI-Mass m/z: 303 [M ? H]?; Anal. Calcd.
for C18H14N4O: C, 71.51; H, 4.67; N, 18.53. Found: C,
71.16; H, 4.39; N, 18.89 (Alemany et al., 1966).
71 % yield, mp 229–231 °C. IR (KBr) cm-1: 3737 and
3682 (m OH), 3420–3225 (m NH), 3146, 3077, 1675, 1623,
1
1589, 1573. H NMR (500 MHz, DMSO-d6): d 11.79 (s,
1H, Indole-NH), 11.70 (s, 1H, CONH), 11.41 (s, 1H, OH),
9.16 (s, 1H, OH), 8.47 (s, 1H, –N=CH–), 8.21–8.18 (m, 2H,
Indole H4, H2), 7.49 (d, 1H, J = 7.9 Hz, Indole H7), 7.21
(t, 1H, J = 7.9 Hz, Indole H5), 7.17 (t, 1H, J = 7.9 Hz,
Indole H6), 6.95 (m, 1H, –N=CH–C6H4, H6), 6.84 (d, 1H,
J = 7.8 Hz, –N=CH–C6H4, H4), 6.74 (t, 1H, J = 7.8 Hz,
–N=CH–C6H4, H5).13C NMR (125 MHz, DMSO-d6) d:
160.64 (C=O), 146.29 (C20 2,3-dihydroxyphenyl), 145.74
(C30 2,3-dihydroxyphenyl), 145.51 (C=N), 136.09 (C3a),
128.72 (C2), 126.33 (C7a), 122.33 (C4), 120.96 (C6),
120.85 (C5), 119.94 (C60 2,3-dihydroxyphenyl), 119.04
(C50 2,3-dihydroxyphenyl), 117.95 (C10 2,3-dihydrox-
yphenyl), 116.86 (C40 2,3-dihydroxyphenyl), 112.03 (C3),
108.13 (C7). ESI-Mass m/z: 296 [M ? H]?, 318
[M ? Na]?; Anal. Calcd. for C16H13N3O3: C, 65.08; H,
4.44; N, 14.23. Found: C, 65.32; H, 4.09; N, 14.56.
N0-(thiophen-2-ylmethylene)-1H-indole-3-carbohydrazide (3h)
The derivative 3h was obtained as a pale yellow solid by
condensation of B3 with thiophene-2-carbaldehyde in
93 % yield, mp 269–271 °C. IR (KBr) cm-1: 3382–3200
1
(m NH), 3156, 3123, 1644, 1612, 1600, 1575. H NMR
(500 MHz, DMSO-d6): d 11.79 (s, 1H, Indole-NH), 11.39
(s, 1H, CONH), 8.23–8.61 (m, 2H, Indole H4, –N=CH–),
7.62 (d, 1H, J = 4.0 Hz, Thiophene H5), 7.49 (d, 1H,
J = 7.8 Hz, Indole H7), 7.42 (s, 1H, J = 4.0 Hz, Thio-
phene H3), 7.21 (t, 1H, J = 7.6 Hz, Indole H5) 7.16 (t, 1H,
J = 7.6 Hz, Indole H6), 7.13 (t, 1H, J = 4.0 Hz, Thio-
phene H4).13C NMR (125 MHz, DMSO-d6) d: 161.03
(C=O), 140.56 (C20 thiophen-2-yl), 139.70 (C3a), 135.91
(C2), 129.75 (C30 thiophen-2-yl), 127.98 (C50 thiophen-2-
yl), 127.80 (C=N), 127.07 (C40 thiophen-2-yl), 126.67
(C7a), 122.23 (C4), 121.13 (C6), 120.76 (C5), 111.92 (C3),
108.51 (C7). ESI-Mass m/z: 270 [M ? H]?, 292
[M ? Na]?; Anal. Calcd. for C14H11N3OS: C, 62.44; H,
4.12; N, 15.60. Found: C, 62.06; H, 4.39; N, 15.92
(Alemany et al., 1966).
N0-(2,5-dihydroxybenzylidene)-1H-indole-3-carbohydrazide
(3k) The derivative 3k was obtained as a yellow-white
solid by condensation of B3 with 2,5-dihydroxybenzalde-
hyde in 81 % yield, mp 265–266 °C. IR (KBr) cm-1: 3678
and 3576 (m OH), 3390–3257 (m NH), 3200, 3067, 1663,
1640, 1614, 1577. 1H NMR (500 MHz, DMSO-d6): d
11.78 (s, 1H, Indole-NH), 11.60 (s, 1H, CONH), 10.68 (s,
1H, OH2), 8.97 (s, 1H, OH5), 8.45 (s, 1H, –N=CH–),
8.21–8.19 (m, 2H, Indole H4, H2), 7.49 (d, 1H, J = 7.8 Hz,
–N=CH–C6H4, H4), 7.22–7.15 (m, 2H, Indole H5, H6), 6.95
(s, 1H, –N=CH–C6H4, H6), 6.76 (d, 1H, J = 8.7 Hz, Indole
H7), 6.72 (dd, 1H, J = 8.7, 2.7 Hz, –N=CH–C6H4, H3).
13C NMR (125 MHz, DMSO-d6) d: 160.60 (C=O), 150.09
(C20 2,5-dihydroxyphenyl), 149.79 (C50 2,5-dihydrox-
yphenyl), 145.22 (C=N), 136.06 (C3a), 128.61 (C2),
126.39 (C7a), 122.29 (C4), 120.99 (C6), 119.24 (C5),
118.97 (C30 2,5-dihydroxyphenyl), 118.19 (C10 2,5-dihy-
droxyphenyl), 116.92 (C40 2,5-dihydroxyphenyl), 114.00
(C60 2,5-dihydroxyphenyl), 112.00 (C3), 108.24 (C7). ESI-
Mass m/z: 296 [M ? H]?, 318 [M ? Na]?; Anal. Calcd.
for C16H13N3O3: C, 65.08; H, 4.44; N, 14.23. Found: C,
64.82; H, 4.73; N, 14.52.
N0-(5-chloro-2-nitrobenzylidene)-1H-indole-3-carbohy-
drazide (3i) The derivative 3i was obtained as a yellow
solid by condensation of B3 with 5-chloro-2-nitroben-
zaldehyde in 90 % yield, mp 267–269 °C. IR (KBr) cm-1
:
3450–3335 (m NH), 3189, 3086, 1646, 1607, 1581, 1563,
1530 and 1339 (m NO2). 1H NMR (500 MHz, DMSO-d6): d
11.90 (s, 1H, Indole-NH), 11.86 (s, 1H, CONH), 8.74 (s,
1H, –N=CH–), 8.28 (d, 1H, J = 2.35 Hz, –N=CH–C6H4
H6), 8.21 (d, 1H, J = 7.6 Hz, –N=CH–C6H4 H3),
8.10–8.12 (m, 2H, Indole H4, H2), 7.69 (dd, 1H, J = 8.7,
2.95 Hz, –N=CH–C6H4 H4), 7.50 (d, 1H, J = 8.0 Hz,
Indole H7), 7.23–7.16 (m, 2H, Indole H5, H6). 13C NMR
(125 MHz, DMSO-d6) d: 161.25 (C=O), 146.26 (C=N),
138.28 (C20 5-chloro-2-nitrophenyl), 137.85 (C50 5-chloro-
2-nitrophenyl), 136.00 (C3a), 131.35 (C2), 130.82 (C40
5-chloro-2-nitrophenyl), 129.55 (C10 5-chloro-2-nitro-
phenyl), 126.93 (C60 5-chloro-2-nitrophenyl), 126.78
(C7a), 126.60 (C30 5-chloro-2-nitrophenyl), 122.38 (C4),
121.03 (C6), 120.95 (C5), 112.03 (C3), 107.91 (C7). ESI-
Mass m/z: 365 [M ? Na]?; Anal. Calcd. for C16H11ClN4
O3: C, 56.07; H, 3.24; N, 16.35. Found: C, 55.82; H, 3.56;
N, 16.80.
N0-(2,4-dihydroxybenzylidene)-1H-indole-3-carbohydrazide
(3l) The derivative 3l was obtained as a pale yellow-
white solid by condensation of B3 with 2,4-dihydroxy-
benzaldehyde in 88 % yield, mp 293–295 °C. IR (KBr)
cm-1: 3658 and 3552 (m OH), 3407, 3327, 3239, 3159,
1
1619, 1595, 1576, 1543. H NMR (500 MHz, DMSO-d6):
d 11.77 (s, 1H, Indole-NH), 11.71(s, 1H, CONH), 11.56 (s,
1H, OH2), 9.93 (s, 1H, OH4), 8.44 (s, 1H, –N=CH–),
8.23–8.21 (m, 2H, Indole H4, H2), 7.50 (d, 1H, J = 7.8 Hz,
–N=CH–C6H4, H6), 7.30 (d, 1H, J = 3.0 Hz, –N=CH–
C6H4, H3), 7.22–7.16 (m, 2H, Indole H5,H6), 6.42–6.39 (m,
N0-(2,3-dihydroxybenzylidene)-1H-indole-3-carbohydrazide
(3j) The derivative 3j was obtained as a white-gray solid
by condensation of B3 with 2,3-dihydroxybenzaldehyde in
123

Med Chem Res
2H, –N=CH–C6H4, H5, Indole H7). 13C NMR (125 MHz,
DMSO-d6) d: 160.57 (C=O), 160.21 (C20 2,4-dihydrox-
yphenyl), 159.23 (C40 2,4-dihydroxyphenyl), 146.72
(C=N), 136.11 (C3a), 131.08 (C60 2,4-dihydroxyphenyl),
128.47 (C2), 126.34 (C7a), 122.28 (C4), 120.98 (C6),
120.79 (C5), 112.01 (C3), 110.85 (C10 2,4-dihydrox-
yphenyl), 108.33 (C7), 107.52 (C50 2,4-dihydroxyphenyl),
102.69 (C30 2,4-dihydroxyphenyl). ESI-Mass m/z: 296
[M ? H]?, 318 [M ? Na]?; Anal. Calcd. for C16H13N3O3:
C, 65.08; H, 4.44; N, 14.23. Found: C, 65.41; H, 4.02; N,
14.46.
C, 64.58; H, 5.42; N, 11.89. Found: C, 64.12; H, 5.73 N,
12.05.
N0-(3-nitrobenzylidene)-1H-indole-3-carbohydrazide (3o)
The derivative 3o was obtained as a yellow solid by con-
densation of B3 with 3-nitrobenzaldehyde in 98 % yield,
mp 278–281 °C. IR (KBr) cm-1: 3531, 3461, 3285, 3117,
1629, 1591, 1577, 1541 and 1364 (NO2). 1H NMR
(500 MHz, DMSO-d6): d 11.84 (s, 1H, Indole-NH), 11.69
(s, 1H, CONH), 8.51 (s, 1H, –N=CH–C6H5, H2), 8.43 (bs,
1H, –N=CH–),8.31 (bs, 1H, Indole H2), 8.25 (d, 1H,
J = 7.6 Hz, Indole H4), 8.19 (d, 1H, J = 7.7 Hz, –N=CH–
C6H5 H6) 8.10 (d, 1H, J = 7.5 Hz, –N=CH–C6H5 H4), 7.68
(t, 1H, J = 7.7 Hz, –N=CH–C6H5, H5), 7.49 (d, 1H,
J = 7.6 Hz, Indole H7), 7.22–7.16 (m, 2H, Indole H5, H6).
13C NMR (125 MHz, DMSO-d6) d: 161.43 (C=O), 148.17
(C30 3-nitrophenyl), 141.93 (C=N), 136.65 (C3a), 136.00
(C10 3-nitrophenyl), 132.89 (C60 3-nitrophenyl), 130.25
(C2), 129.16 (C7a), 126.66 (C50 3-nitrophenyl), 123.56
(C40 3-nitrophenyl), 122.20 (C4), 121.10 (C6), 120.86 (C20
3-nitrophenyl), 120.50 (C5), 111.99 (C3), 108.21 (C7).
ESI-Mass m/z: 309 [M ? H]?, 331 [M ? Na]?; Anal.
Calcd. for C16H12N4O3: C, 62.33; H, 3.92; N, 18.17.
Found: C, 62.64; H, 3.83; N, 17.92.
N0-(2,3-dimethoxybenzylidene)-1H-indole-3-carbohydrazide
(3m) The derivative 3m was obtained as an off-white
solid by condensation of B3 with 2,3-dimethoxybenzalde-
hyde in 85 % yield, mp 224–226 °C. IR (KBr) cm-1
:
3417–3272, 3197, 3094, 1652, 1619, 1615, 1544. 1H NMR
(500 MHz, DMSO-d6): d 11.84 (s, 1H, Indole-NH), 11.56
(s, 1H, CONH), 8.62 (bs, 1H, –N=CH–), 8.26 (bs, 2H,
Indole H4, H2), 7.51–7.49 (m, 2H, –N=CH–C6H4, H6,
Indole H7), 7.22–7.16 (m, 2H, –N=CH–C6H4, H5, Indole
H5), 7.12 (t, 1H, J = 8.0 Hz, Indole H6), 7.07 (d, 1H,
J = 7.9 Hz, –N=CH–C6H4, H4) 3.83 (s, 3H, OCH3), 3.75
(s, 3H, OCH3). 13C NMR (125 MHz, DMSO-d6): d 161.04
(C=O), 152.63 (C30 2,3-dimethoxyphenyl), 147.67 (C20
2,3-dimethoxyphenyl), 140.09 (C=N), 135.96 (C3a),
131.51 (C2), 128.20 (C7a), 126.60 (C60 2,3-dimethox-
yphenyl), 124.28 (C10 2,3-dimethoxyphenyl), 122.21 (C4),
121.16 (C6), 120.74 (C5), 116.89 (C40 2,3-dimethox-
yphenyl), 113.63 (C50 2,3-dimethoxyphenyl), 110.92 (C3),
108.48 (C7), 61.09(O-CH3), 55.68 (O-CH3). ESI-Mass m/
z: 324 [M ? H]?, 346 [M ? Na]?; Anal. Calcd. for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C, 66.52;
H, 5.58; N, 12.79.
N0-((5-nitrothiophen-2-yl)methylene)-1H-indole-3-carbohy-
drazide (3p) The derivative 3p was obtained as a yellow-
orange solid by condensation of B3 with 5-nitrothiophene-
2-carbaldehyde in 93 % yield, mp 285–286 °C. IR (KBr)
cm-1: 3425, 3232, 3119, 3067, 1653, 1592, 1573, 1536 and
1
1334 (NO2). H NMR (400 MHz, DMSO-d6): d 11.88 (s,
1H, Indole-NH), 11.81 (s, 1H, CONH), 8.56 (bs, 1H,–N=
CH–), 8.26 (s, 1H, Indole H2), 8.20 (d, 1H, J = 7.7 Hz,
Indole H4), 8.14 (d, 1H, J = 4.4 Hz, Thiophene H4), 7.54
(d, 1H, J = 4.4, Thiophene H3), 7.49 (d, 1H, J = 7.7 Hz,
Indole H7), 7.23–7.16 (m, 2H, Indole H5, H6). 13C NMR
(125 MHz, DMSO-d6) d: 166.95 (C=O), 155.32 (C50
5-nitrothiophen-2-yl), 152.88 (C20 5-nitrothiophen-2-yl),
135.83 (C3a), 133.74 (C30 5-nitrothiophen-2-yl), 132.98
(C40 5-nitrothiophen-2-yl), 131.21 (C2), 129.85 (C7a),
128.72 (C=N), 122.67 (C4), 121.29 (C6), 120.61 (C5),
112.28 (C3), 108.07 (C7). ESI-Mass m/z: 315 [M ? H]?,
337 [M ? Na]?; Anal. Calcd. for C14H11N3OS: C, 53.50;
H, 3.21; N, 17.83. Found: C, 53.16; H, 2.99; N, 17.98.
N0-(3,4,5-trimethoxybenzylidene)-1H-indole-3-carbohydrazide
(3n) The derivative 3n was obtained as a white solid by
condensation of B3 with 3, 4, 5-trimethoxybenzaldehyde in
96 % yield, mp 245–246 °C. IR (KBr) cm-1: 3200–3111,
1
3048, 3005, 1686, 1639, 1617, 1571. H NMR (500 MHz,
DMSO-d6): d 11.78 (s, 1H, CONH), 11.42 (s, 1H, Indole-
NH), 8.29–8.21 (m,3H, Indole H2, –N=CH–, Indole H4),
7.49 (d, 1H, J = 8.0 Hz, Indole H7), 7.22–7.15 (m, 2H,
Indole H5, H6), 7.02 (s, 2H, –N=CH–C6H4, H2, H6), 3.89
(s, 6H, OCH3), 3.76 (s, 3H, OCH3). 13C NMR (125 MHz,
DMSO-d6): d 161.04 (C=O), 153.17 (C30 and C50 3,4,5-
trimethoxyphenyl), 144.78 (C=N), 143.85 (C40 3,4,5-tri-
methoxyphenyl), 135.91 (C3a), 130.33 (C2), 128.49 (C7a),
126.62 (C10 3,4,5-trimethoxyphenyl), 122.18 (C4), 121.13
(C6), 120.71 (C5), 111.92 (C3), 108.42 (C7), 103.89 (C20
and C60 3,4,5-trimethoxyphenyl), 60.08 (C40 O-CH3),
55.85 (C30 and C50 O-CH3). ESI-Mass m/z: 354
[M ? H]?, 393 [M ? K]?; Anal. Calcd. for C19H19N3O4:
N0-((5-nitrofuran-2-yl)methylene)-1H-indole-3-carbohydrazide
(3q) The derivative 3q was obtained as a yellow solid by
condensation of B3 with 5-nitrofuran-2-carbaldehyde in
98 % yield, mp 285–286 °C. IR (KBr) cm-1: 3420–3372,
1
3188, 3120, 1633, 1586, 1566, 1523 and 1355 (NO2). H
NMR (400 MHz, DMSO-d6): d 11.88 (s, 1H, Indole-NH),
11.81 (s, 1H, CONH), 8.26 (s, 1H,–N=CH–), 8.21 (d, 1H,
J = 8.0 Hz, Indole H4), 7.81 (s, 1H, Indole H2), 7.49 (d,
1H, J = 6.8 Hz, Furan H4), 7.19–7.23 (m, 3H, Indole H5,
123

Med Chem Res
H6, H7), 7.16 (d, 1H, J = 7.2 Hz, Furan H3). 13C NMR
(125 MHz, DMSO-d6) d: 161.65 (C=O), 152.46 (C20
5-nitrofuran-2-yl), 151.63 (C50 5-nitrofuran-2-yl), 135.94
(C3a), 131.51 (C=N), 130.17 (C2), 126.66 (C7a), 122.43
(C4), 121.02 (C6), 117.18 (C5), 114.88 (C30 5-nitrofuran-
2-yl), 114.13 (C40 5-nitrofuran-2-yl), 112.04 (C3), 107.84
(C7). ESI-Mass m/z: 299 [M ? H]?, 321 [M ? Na]?;
Anal. Calcd. for C14H11N3OS: C, 56.38; H, 3.38; N, 18.79.
Found: C, 56.02; H, 3.79; N, 18.52.
N0-(2,4-dichlorobenzylidene)-1H-indole-2-carbohydrazide (2b)
The derivative 2b was obtained as a pale yellow-white
solid by condensation of B2 with 2,4-dichlorobenzalde-
hyde in 98 % yield, as a 80:20 mixture of E and Z isomers,
mp 228–230 °C. IR (KBr) cm-1: 3472, 3316, 3126, 3103,
1659, 1612, 1594,1569.1540; 1H NMR (500 MHz, DMSO-
d6): d 12.18 (s, 1H, CONH), 11.86 (s, 1H, Indole-NH), 8.80
(s, 1H, –N=CH–), 8.05 (d, 1H, J = 8.5 Hz, –N=CH–C6H5,
H6 (E)), 7.86 (d, 1H, J = 8.5 Hz, –N=CH–C6H5, H6 (Z)),
7.81 (d, 1H, J = 2.1 Hz, –N=CH–C6H5, H3 (Z)), 7.71 (d,
1H, J = 2.1 Hz, –N=CH–C6H5, H3 (E)), 7.69 (d, 1H,
J = 8.1 Hz, Indole H7), 7.61 (dd, 1H, J = 7.4, 2.1 Hz,
–N=CH–C6H5, H5 (Z)), 7.53 (d, 1H, J = 7.4 Hz, –N=CH–
C6H5, H5 (E)), 7.48 (d, 1H, J = 7.8 Hz, Indole H4), 7.36 (s,
1H, Indole H3), 7.24 (t, 1H, J = 7.8 Hz, Indole H6), 7.08
(t, 1H, J = 7.8 Hz, Indole H5). 13C NMR (125 MHz,
DMSO-d6) d: 157.68 (C=O), 141.74 (C7a), 139.70 (C=N),
136.92 (C2), 134.94 (C10 2,4-dichlorophenyl), 133.74
(C3a), 131.02 (C20 2,4-dichlorophenyl (Z)), 130.70 (C20
2,4-dichlorophenyl (E)), 130.23 (C30 2,4-dichlorophenyl
(Z)), 129.68 (C60 2,4-dichlorophenyl (Z)), 129.34 (C30 2,4-
dichlorophenyl (E)), 128.23 (C60 2,4-dichlorophenyl (E)),
128.03 (C40 2,4-dichlorophenyl), 126.93 (C50 2,4-dichlor-
ophenyl), 124.03 (C6), 121.85 (C4), 120.00 (C5), 112.40
(C3), 103.96 (C7). ESI-Mass m/z: 332 [M ? H]?, 354
[M ? Na]?; Anal. Calcd. for C16H11Cl2N3O: C, 57.85; H,
3.34; N, 12.65. Found: C, 58.03; H, 3.18; N, 12.92.
N0-(3-methoxybenzylidene)-1H-indole-3-carbohydrazide (3r)
The derivative 3r was obtained as a pale pink-white solid
by condensation of B3 with 3-methoxybenzaldehyde in
72 % yield, mp 239–241 °C. IR (KBr) cm-1: 3252, 3156,
1
3063, 3009, 1625, 1618, 1603, 1573. H NMR (500 MHz,
DMSO-d6): d 11.75 (s, 1H, Indole-NH), 11.36 (s, 1H,
CONH), 8.28–8.19 (m, 3H, Indole H2, H4, –N=CH–), 7.54
(s, 1H, –N=CH–C6H5, H2), 7.51–7.48 (m, 2H, –N=CH–
C6H5, H6, Indole H7), 7.35 (t, 1H, J = 7.6 Hz, –N=CH–
C6H5, H5), 7.23 (t, 1H, J = 7.7 Hz, Indole H5), 7.19 (t, 1H,
J = 7.7 Hz, Indole H6), 7.15 (d, 1H, J = 7.6 Hz, –N=CH–
C6H5, H4), 2.37 (s, 3H, –CH3). 13C NMR (125 MHz,
DMSO-d6): d 162.14 (C=O), 161.09 (C30 3-methox-
yphenyl), 148.65 (C=N), 140.13 (C3a), 138.33 (C10
3-methoxyphenyl), 130.24 (C2), 129.75 (C50 3-methox-
yphenyl), 127.43 (C7a), 122.55 (C4), 122.14 (C60
3-methoxyphenyl), 121.89 (C6), 119.57 (C5), 117.02 (C40
3-methoxyphenyl), 112.39 (C20 3-methoxyphenyl), 110.09
(C3), 108.99 (C7), 54.29 (O-CH3). ESI-Mass m/z: 294
[M ? H]?, 316 [M ? Na]?; Anal. Calcd. for C17H15N3O2:
C, 69.61; H, 5.15; N, 14.33. Found: C, 69.49; H, 4.87; N,
13.98.
N0-(2,6-dichlorobenzylidene)-1H-indole-2-carbohydrazide
(2c) The derivative 2c was obtained as a white solid by
condensation of B2 with 2,6-dichlorobenzaldehyde in
98 % yield, mp 224–227 °C. IR (KBr) cm-1: 3426, 3283,
3103, 3023, 1667, 1624, 1610, 1574,1547; 1H NMR
(500 MHz, DMSO-d6): d 12.19 (s, 1H, CONH), 11.84 (s,
1H, Indole-NH), 8.66 (s, 1H, –N=CH–), 7.69 (d, 1H,
J = 8.1 Hz, Indole H7), 7.58 (d, 2H, J = 8.5 Hz, –N=CH–
C6H5, H3, H5), 7.49 (d, 1H, J = 8.1 Hz, Indole H4),7.45 (t,
1H, J = 8.5 Hz, –N=CH–C6H5, H4), 7.38 (bs, 1H, Indole
H3), 7.24 (t, 1H, J = 8.1 Hz, Indole H6), 7.08 (d, 1H,
J = 8.1, Indole H5). 13C NMR (125 MHz, DMSO-d6) d:
157.76 (C=O), 142.35 (C7a), 136.92 (C=N), 134.53 (C2),
133.90 (C20 and C60 2,6-dichlorophenyl), 131.11 (C3a),
130.65 (C10 2,6-dichlorophenyl), 129.92 (C40 2,6-dichlor-
ophenyl), 129.05 (C30 and C50 2,6-dichlorophenyl), 124.04
(C6), 121.89 (C4), 119.98 (C5), 112.40 (C3), 104.03 (C7).
ESI-Mass m/z: 332 [M ? H]?, 354 [M ? Na]?; Anal.
Calcd. for C16H11Cl2N3O: C, 57.85; H, 3.34; N, 12.65.
Found: C, 57.63; H, 3.48; N, 13.02.
N0-benzylidene-1H-indole-2-carbohydrazide
(2a) The
derivative 2a was obtained as an off-white solid by con-
densation of B2 with benzaldehyde in 95 % yield, mp
197–199 °C. IR (KBr) cm-1: 3404, 3264, 3125, 3064,
1656, 1623, 1610, 1593, 1575; 1H NMR (500 MHz,
DMSO-d6): d 11.90 (s, 1H, CONH), 11.80 (s, 1H, Indole-
NH), 8.47 (s, 1H, –N=CH–), 7.77 (d, 2H, J = 7.1 Hz, –N=
CH–C6H5, H2, H6), 7.69 (d, 1H, J = 7.2 Hz, Indole H7),
7.49–7.44 (m, 4H, Indole H4, –N=CH–C6H5, H3,H4,H5),
7.33 (s, 1H, Indole H3), 7.23 (t, 1H, J = 7.2 Hz, Indole
H5), 7.08 (t, 1H, J = 7.2 Hz, Indole H6). 13C NMR
(125 MHz, DMSO-d6) d: 161.11 (C=O), 157.63 (C=N),
147.08 (C7a), 144.63 (C2), 136.81 (C10 benzyl), 134.30
(C3a), 129.98 (C40 benzyl), 128.83 (C20 and C60 benzyl),
127.03 (C30 and C50 benzyl), 123.84 (C6), 121.75 (C4),
119.94 (C5), 112.36 (C3), 103.58 (C7). ESI-Mass m/z: 264
[M ? H]?, 286 [M ? Na]?; Anal. Calcd. for C16H13N3O:
C, 72.99; H, 4.98; N, 15.96. Found: C, 72.52; H, 5.08; N,
15.72 (Alemany et al., 1966).
N0-(4-methoxybenzylidene)-1H-indole-2-carbohydrazide (2d)
The derivative 2d was obtained as a white solid by con-
densation of B2 with 4-methoxybenzaldehyde in 94 %
yield, mp 213–215 °C. IR (KBr) cm-1: 3442–3209, 3109,
123

Med Chem Res
1
3053, 1669, 1635, 1596, 1577, 1541; H NMR (500 MHz,
DMSO-d6): d 11.77 (s, 1H, CONH), 11.62 (s, 1H, Indole-
NH), 8.40 (s, 1H, –N=CH–), 7.71 (d, 2H, J = 8.4 Hz, –N=
CH–C6H5, H2, H6), 7.68 (d, 1H, J = 7.9 Hz, Indole H7),
7.47 (d, 1H, J = 7.9 Hz, Indole H4), 7.30 (s, 1H, Indole
H3), 7.22 (t, 1H, J = 7.9 Hz, Indole H6), 7.09–7.04 (m, 3H,
151.33 (C50 2-hydroxy-5-methoxyphenyl), 146.60 (C=N),
136.88 (C7a), 129.65 (C2), 126.95 (C3a), 123.95 (C6),
121.80 (C4), 120.01 (C5), 119.14 (C10 2-hydroxy-5-meth-
oxyphenyl), 118.11 (C40 2-hydroxy-5-methoxyphenyl),
117.23 (C30 2-hydroxy-5-methoxyphenyl), 112, 39 (C3),
111.94 (C60 2-hydroxy-5-methoxyphenyl), 103.84 (C7),
55.47 (O-CH3). ESI-Mass m/z: 310 [M ? H]?, 332
[M ? Na]?; Anal. Calcd. for C17H15N3O3: C, 66.01; H,
4.89; N, 13.58. Found: C, 66.36; H, 5.03; N, 13.70.
–N=CH–C6H5, H3, H5, Indole H5), 3.83 (s, 3H, –CH3). 13
C
NMR (125 MHz, DMSO-d6) d: 160.77 (C40 4-methox-
yphenyl), 157.50 (C=O), 147.04 (C=N), 136.77 (C7a),
130.18 (C2), 128.65 (C3a), 126.98 (C20 and C60 4-meth-
oxyphenyl), 126.86 (C10 4-methoxyphenyl), 123.73 (C6),
121.68 (C4), 119.91 (C5), 114.32 (C30 and C50 4-methox-
yphenyl), 112.35 (C3), 103.35 (C7), 55.26 (O-CH3). ESI-
Mass m/z: 294 [M ? H]?, 316 [M ? Na]?; Anal. Calcd.
for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C,
69.36; H, 5.43; N, 14.70 (Alemany et al., 1966).
N0-(2,4-dihydroxybenzylidene)-1H-indole-2-carbohydrazide
(2g) The derivative 2g was obtained as a pale yellow
solid by condensation of B2 with 2,4-dihydroxybenzalde-
hyde in 95 % yield, mp 292–293 °C. IR (KBr) cm-1: 3571,
3499 (m OH), 3410–3158 (m NH), 3133, 3061, 1658, 1625,
1601, 1576, 1543. 1H NMR (500 MHz, DMSO-d6): d
11.96 (s, 1H, CONH), 11.79 (s, 1H, Indole-NH), 11.38 (s,
1H, OH2), 9.96 (s, 1H, OH4), 8.52 (s, 1H, –N=CH–), 7.68
(d, 1H, J = 8.0 Hz, Indole H7), 7.47 (d, 1H, J = 8.0 Hz,
Indole H4), 7.35 (d, 1H, J = 8.4 Hz, –N=CH–C6H4, H6),
7.29 (s, 1H, Indole H3), 7.23 (t, 1H, J = 8.0 Hz, Indole
H6), 7.07 (t, 1H, J = 8.0 Hz, Indole H5), 6.38 (dd, 1H,
J = 8.5, 2.2 Hz, –N=CH–C6H4, H5), 6.32 (s, 1H, –N=CH–
C6H4, H3).
13C NMR (125 MHz, DMSO-d6) d: 160.63 (C20 2,4-
dihydroxyphenyl), 159.29 (C40 2,4-dihydroxyphenyl),
157.09 (C=O), 148.21 (C=N), 136.79 (C7a), 131.02 (C20
2,4-dihydroxyphenyl), 129.79(C2), 126.96 (C3a), 123.78
(C6), 121.72 (C4), 119.95 (C5), 112.35 (C3), 110.65 (C10
2,4-dihydroxyphenyl), 107.69 (C50 2,4-dihydroxyphenyl),
103.42 (C7), 102.69 (C30 2,4-dihydroxyphenyl). ESI-Mass
m/z: 296 [M ? H]?, 318 [M ? Na]?; Anal. Calcd. for
C16H13N3O3: C, 65.08; H, 4.44; N, 14.23. Found: C, 64.82;
H, 4.75; N, 14.83.
N0-((5-nitrothiophen-2-yl)methylene)-1H-indole-2-carbohy-
drazide (2e) The derivative 2e was obtained as a primrose
yellow solid by condensation of B2 with 5-nitrothiophene-
2-carbaldehyde in 88 % yield, mp 275–276 °C. IR (KBr)
cm-1: 3420, 3304, 3237, 3214, 1628, 1584, 1559, 1548,
1
1524 and 1353 (NO2); H NMR (500 MHz, DMSO-d6): d
12.28 (s, 1H, CONH), 11.86 (s, 1H, Indole-NH), 8.69 (s,
1H, –N=CH–), 8.16 (d, 1H, J = 4.3 Hz, Thiophene H4),
7.70 (d, 1H, J = 7.8 Hz, Indole H7), 7.61 (d, 1H,
J = 4.3 Hz, Thiophene H3), 7.48 (d, 1H, J = 7.8 Hz,
Indole H4), 7.36 (s, 1H, Indole H3), 7.25 (t, 1H,
J = 7.8 Hz, Indole H5), 7.09 (t, 1H, J = 7.8 Hz, Indole
H6). 13C NMR (125 MHz, DMSO-d6) d: 157.74 (C=O),
150.67 (C50 5-nitrothiophen-2-yl), 146.73 (C20 5-nitroth-
iophen-2-yl), 140.35 (C7a), 137.00 (C2), 130.49 (C3a),
129.44 (C40 5-nitrothiophen-2-yl), 126.89 (C30 5-nitroth-
iophen-2-yl), 124.23 (C=N), 122.19 (C6), 121.94 (C4),
120.09 (C5), 112.42 (C3), 104.37 (C7). ESI-Mass m/z: 315
[M ? H]?, 337 [M ? Na]?; Anal. Calcd. for C14H10N4-
O3S: C, 53.50; H, 3.21; N, 17.83. Found: C, 53.87; H, 3.60;
N, 17.72.
N0-(2,5-dihydroxybenzylidene)-1H-indole-2-carbohydrazide
(2h) The derivative 2h was obtained as a green-gray solid
by condensation of B2 with 2,5-dihydroxybenzaldehyde in
95 % yield, mp 304–305 °C. IR (KBr) cm-1: 3572 (m OH),
3413, 3337, 3127, 3100, 1699, 1651, 1614, 1575; 1H NMR
(500 MHz, DMSO-d6): d 12.01 (s, 1H, CONH), 11.81 (s,
1H, Indole-NH), 10.27 (s, 1H, OH), 8.98 (s, 1H, OH), 8.58
(s, 1H, –N=CH–), 7.67 (d, 1H, J = 8.0 Hz, Indole H7),
7.46 (d, 1H, J = 8.0 Hz, Indole H4), 7.31 (s, 1H, Indole
H3), 7.22 (t, 1H, J = 8.0 Hz, Indole H6), 7.07 (t, 1H,
J = 8.0 Hz, Indole H5), 7.03 (d, 1H, J = 2.6 Hz, –N=CH–
C6H4, H6), 6.76 (d, 1H, J = 8.7 Hz, –N=CH–C6H4, H3),
6.71 (dd, 1H, J = 8.7, 2.6 Hz, –N=CH–C6H4, H4). 13C
NMR (125 MHz, DMSO-d6) d: 161.77 (C=O), 149.91 (C20
2,5-dihydroxyphenyl), 146.93 (C50 2,5-dihydroxyphenyl),
144.28 (C=N), 137.91 (C7a), 130.51 (C2), 127.13 (C3a),
122.54 (C6), 120.82 (C30 2,5-dihydroxyphenyl), 120.41
(C4), 119.09 (C5), 118.65 (C10 2,5-dihydroxyphenyl),
117.93 (C40 2,5-dihydroxyphenyl), 117.05 (C60 2,5-
N0-(2-hydroxy-5-methoxybenzylidene)-1H-indole-2-carbo-
hydrazide (2f) The derivative 2f was obtained as a pale
yellow solid by condensation of B2 with 2-hydroxy-5-
methoxybenzaldehyde in 93 % yield, mp 276–278 °C. IR
(KBr) cm-1: 3594 (m OH), 3431–3300 (m NH), 3097, 3041,
1641, 1598, 1571, 1540,1512; 1H NMR (500 MHz,
DMSO-d6): d 12.10 (s, 1H, CONH), 11.80 (s, 1H, Indole-
NH), 10.59 (s, 1H, OH), 8.63 (s, 1H, –N=CH–), 7.68 (d,
1H, J = 8.0 Indole H7), 7.47 (d, 1H, J = 8.0 Hz, Indole
H4), 7.33 (s, 1H, Indole H3), 7.22 (t, 1H, J = 8.0 Hz,
Indole H6), 7.16 (s, 1H, –N=CH–C6H4, H6), 7.07 (t, 1H,
J = 8.0 Hz, Indole H5), 6.91 (dd, 1H, J = 8.8, 3.0, –N=
CH–C6H4, H4), 6.86 (d, 1H, J = 8.8, –N=CH–C6H4, H3),
3.74 (s, 3H, –CH3). 13C NMR (125 MHz, DMSO-d6): d
157.43 (C=O), 152.15 (C20 2-hydroxy-5-methoxyphenyl),
123

Med Chem Res
dihydroxyphenyl), 113.39 (C3), 104.51 (C7). ESI-Mass m/
z: 296 [M ? H]?, 318 [M ? Na]?, 334 [M ? K]?; Anal.
Calcd. for C16H13N3O3: C, 65.08; H, 4.44; N, 14.23.
Found: C, 65.53; H, 4.12; N, 14.62.
5-chloro-2-nitrophenyl (E)), 124.17 (C6), 121.92 (C4),
120.06 (C5), 112.42 (C3), 104.36 (C7). ESI-Mass m/z: 343
[M ? H]?, 365 [M ? Na]?; Anal. Calcd. for C16H11
ClN4O3: C, 56.07; H, 3.24; N, 16.35. Found: C, 56.33; H,
3.02; N, 16.79.
N0-(2,3-dihydroxybenzylidene)-1H-indole-2-carbohydrazide
(2i) The derivative 2i was obtained as a gray solid by
condensation of B2 with 2,3-dihydroxybenzaldehyde in
89 % yield, mp 238–240 °C. IR (KBr) cm-1: 3799 and
3671 (m OH), 3406, 3267, 3044, 1644, 1605, 1577, 1542.
1H NMR (500 MHz, DMSO-d6): d 11.05 (s, 1H, OH2),
10.51 (s, 1H, Indole-NH), 10.21 (s, 1H, CONH), 9.29 (s,
1H, OH3), 8.60 (s, 1H, –N=CH–), 7.71 (d, 1H, J = 7.4 Hz,
Indole H7), 7.51 (d, 1H, J = 7.4 Hz, Indole H4), 7.33 (s,
1H, Indole H3), 7.22 (d, 1H, J = 7.4 Hz, Indole H6), 7.04
(d, 1H, J = 7.4 Hz, Indole H5), 6.91–6.65 (m, 3H, –N=
CH–C6H4, H4, H5, H6). 13C NMR (125 MHz, DMSO-d6) d:
157.37 (C=O), 148.01(C20 2,3-dihydroxyphenyl), 145.93
(C30 2,3-dihydroxyphenyl), 145.58 (C=N), 136.90 (C7a),
129.57 (C2), 126.93 (C3a), 123.94 (C6), 121.82 (C60 2,3-
dihydroxyphenyl), 120.01 (C4), 119.79 (C50 2,3-dihy-
droxyphenyl), 119.15 (C5), 118.93 (C10 2,3-dihydrox-
yphenyl), 117.25 (C40 2,3-dihydroxyphenyl), 112.39 (C3),
103.80 (C7). ESI-Mass m/z: 296 [M ? H]?, 318
[M ? Na]?; Anal. Calcd. for C16H13N3O3: C, 65.08; H,
4.44; N, 14.23. Found: C, 65.42; H, 4.24; N, 13.92.
N0-((5-nitrofuran-2-yl)methylene)-1H-indole-2-carbohydrazide
(2k) The derivative 2k was obtained as a yellow solid by
condensation of B2 with 5-nitrofuran-2-carbaldehyde in
87 % yield, as a 86:14 mixture of E and Z isomers, mp
273–274 °C. IR (KBr) cm-1: 3482, 3363, 3280, 3096,
1688, 1632, 11,616, 1595, 1564 and 1386 (NO2); 1H NMR
(500 MHz, DMSO-d6): d 12.29 (s, 1H, Indole-NH (E)),
11.98 (s, 1H, Indole-H (Z)), 11.91 (s, 1H, CONH (E)),
11.53 (s, 1H, CONH (Z)), 8.40 (s, 1H, –N=CH–), 7.88 (d,
1H, J = 3.1 Hz, Furan H4 (Z)), 7.81 (d, 1H, J = 3.1 Hz,
Furan H4 (E)), 7.75 (s, 1H, Indole H3 (Z)), 7.69 (d, 1H,
J = 7.5 Hz, Indole H7), 7.46 (d, 1H, J = 7.5 Hz, Indole
H4), 7.39 (s, 1H, Indole H3 (E)), 7.35 (d, 1H, J = 3.1 Hz,
Furan H3 (Z)), 7.29 (d, 1H, J = 3.1 Hz, Furan H3 (E)), 7.25
(t, 1H, J = 7.5 Hz, Indole H6), 7.09 (t, 1H, J = 7.5 Hz,
Indole H5). 13C NMR (125 MHz, DMSO-d6): d 157.81
(C=O), 151.80 (C20 5-nitrofuran-2-yl (E)), 151.28 (C20
5-nitrofuran-2-yl (Z)), 148.11 (C50 5-nitrofuran-2-yl (E)),
137.21 (C50 5-nitrofuran-2-yl (Z)), 137.01 (C7a), 134.65
(C=N), 126.88 (C2), 124.41 (C3a (Z)), 124.26 (C3a (E)),
121.94 (C6), 121.78, 120.29 (C4 (Z)), 120.11 (C4 (E)),
118.01 (C5), 115.23 (C40 5-nitrofuran-2-yl (Z)), 114.68
(C40 5-nitrofuran-2-yl (E)), 113.79 (C30 5-nitrofuran-2-yl),
112.42 (C3), 104.97 (C7 (Z)), 104.42 (C7 (E)). ESI-Mass
m/z: 299 [M ? H]?, 321 [M ? Na]?; Anal. Calcd. for
C14H10N4O4: C, 56.38; H, 3.38; N, 18.79. Found: C, 56.87;
H, 3.60; N, 18.40.
N0-(5-chloro-2-nitrobenzylidene)-1H-indole-2-carbohydrazide
(2j) The derivative 2j was obtained as a yellow solid by
condensation of B2 with 5-chloro-2-nitrobenzaldehyde in
92 % yield, as a 84:16 mixture of E and Z isomers, mp
278–280 °C. IR (KBr) cm-1: 3424, 3265, 3078, 1655,
1
1613, 1544, 1540 and 1378 (NO2); H NMR (500 MHz,
DMSO-d6): d 12.36 (s, 1H, Indole-NH), 11.87 (s, 1H,
CONH), 10.21 (s, 1H, –N=CH– (Z)), 8.87 (s, 1H, –N=CH–
(E)), 8.18 (d, 1H, J = 8.7 Hz, –N=CH–C6H4 H3 (Z)), 8.15
(d, 1H, J = 8.7 Hz, –N=CH–C6H4 H3 (E)), 8.11 (d, 1H,
J = 1.9 Hz, –N=CH–C6H4 H6 (E)), 8.05 (d, 1H,
J = 1.9 Hz, –N=CH–C6H4 H6 (Z)), 7.95 (dd, 1H, J = 8.7,
1.9 Hz, –N=CH–C6H4 H4 (Z)), 7.76 (dd, 1H, J = 8.7,
1.9 Hz, –N=CH–C6H4 H4 (E)), 7.69 (d, 1H, J = 7.5 Hz,
Indole H7), 7.48 (d, 1H, J = 7.5 Hz, Indole H4), 7.39 (s,
1H, Indole H3), 7.25 (t, 1H, J = 7.5 Hz, Indole H6), 7.08
(t, 1H, J = 7.5 Hz, Indole H5).13C NMR (125 MHz,
DMSO-d6): d 158.67 (C=O), 146.46 (C20 5-chloro-2-ni-
trophenyl (E)), 140.93 (C=N), 138.98 (C20 5-chloro-2-ni-
trophenyl (Z)), 138.40 (C7a), 136.98 (C2), 133.53 (C50
5-chloro-2-nitrophenyl (E)), 132.42(C50 5-chloro-2-nitro-
phenyl (Z)), 132.27 (C40 5-chloro-2-nitrophenyl (Z)),
131.39(C60 5-chloro-2-nitrophenyl (Z)), 130.97 (C40
5-chloro-2-nitrophenyl (E)), 130.06 (C60 5-chloro-2-nitro-
phenyl (E)), 129.42(C10 5-chloro-2-nitrophenyl (Z)),
129.18 (C10 5-chloro-2-nitrophenyl (E)), 126.99 (C3a),
126.90 (C30 5-chloro-2-nitrophenyl (Z)), 126.45 (C30
N0-(5-bromo-2-hydroxybenzylidene)-1H-indole-2-carbohy-
drazide (2l) The derivative 2l was obtained as an off-
white solid by condensation of B2 with 5-chloro-2-ni-
trobenzaldehyde in 76 % yield, mp 278–280 °C. IR (KBr)
cm-1: 3564 (m OH), 3355, 3216, 3122, 3058, 1623, 1602,
1565, 1538, 1520. 1H NMR (500 MHz, DMSO-d6): d
12.21 (s, 1H, Indole-NH), 11.85 (s, 1H, CONH), 11.21 (s,
1H, OH), 8.63 (s, 1H, –N=CH–), 7.83 (d, 1H, J = 2.6 Hz,
–N=CH–C6H4, H6), 7.69 (d, 1H, J = 8.0 Hz, Indole H7),
7.48 (d, 1H, J = 8.0 Hz, Indole H4), 7.44 (dd, 1H, J = 8.7,
2.5 Hz, –N=CH–C6H4 H4), 7.35 (s, 1H, Indole H3), 7.24 (t,
1H, J = 8.0 Hz, Indole H6), 7.08 (t, 1H, J = 8.0 Hz,
Indole H5), 6.92 (d, 1H, J = 8.7 Hz, –N=CH–C6H4,
H3).13C NMR (125 MHz, DMSO-d6): d 157.50 (C=O),
156.25 (C20 5-bromo-2-hydroxyphenyl), 144.55 (C=N),
136.92 (C7a), 133.43 (C40 5-bromo-2-hydroxyphenyl),
130.06 (C2), 129.53 (C60 5-bromo-2-hydroxyphenyl),
126.92 (C3a), 124.00 (C6), 121.84 (C4), 121.53 (C20
5-bromo-2-hydroxyphenyl), 120.01 (C5), 118.61 (C30
5-bromo-2-hydroxyphenyl), 112.40 (C3), 110.48 (C50
123

Med Chem Res
5-bromo-2-hydroxyphenyl), 103.97 (C7). ESI-Mass m/z:
358 [M ? H]?, 365 [M ? Na]?; Anal. Calcd. for C16-
H12BrN43O2: C, 53.65; H, 3.38; N, 11.73. Found: C, 53.89;
H, 3.02; N, 12.02.
plates and allowed to incubate for 24 h. Then, medium was
aspirated and various concentrations of test compounds in
complete medium were added to each well. After further
incubation at 37 °C for 24 h/48 h, the medium was discarded
and MTT solution (2 mg/mL, 100 lL) was added to the wells
and the mixture was incubated for 3 h at 37 °C. The produced
formazan crystals were dissolved in 100 lL of DMSO. Plates
were incubated for 20 min at 37 °C, and the optical densities
were read at 570 nm with a reference wavelength of 630 nm
as background using a spectrophotometer plate reader (In-
finiteÒ M200; TECAN, Morrisville, NC, USA). DMSO was
used as the solvent of the test compounds, and its final con-
centration was less than 0.2 %. IC50 values were calculated by
GraphPad Prism 5.04. All the tests were performed in tripli-
cate (Mashayekhi et al., 2013a, b).
N0-(3-methoxybenzylidene)-1H-indole-2-carbohydrazide (2m)
The derivative 2m was obtained as an off-white solid by
condensation of B2 with 3-methoxybenzaldehyde in 93 %
yield, mp 129–130 °C. IR (KBr) cm-1: 3407–3271, 3061,
2929, 1628), 1610, 1599, 1574, 1529; 1H NMR (500 MHz,
DMSO-d6): d 11.89 (s, 1H, Indole-NH), 11.82 (s, 1H,
CONH), 8.43 (s, 1H, –N=CH–), 7.69 (d, 1H, J = 7.5 Hz,
Indole H7), 7.59 (s, 1H, –N=CH–C6H5, H2), 7.54 (d, 1H,
J = 7.5 Hz, Indole H4), 7.48 (d, 1H, J = 8.0 Hz, –N=CH–
C6H5, H6), 7.37 (t, 1H, J = 8.0 Hz, –N=CH–C6H5, H5),
7.33 (s, 1H, Indole H3), 7.27 (d, 1H, J = 7.45 Hz, –N=
CH–C6H5, H4), 7.23 (t, 1H, J = 7.5 Hz, Indole H6), 7.08
(t, 1H, J = 7.5 Hz, Indole H5), 2.39 (s, 3H, –CH3). 13C
NMR (125 MHz, DMSO-d6) d: 157.89 (C=O), 153.50 (C30
3-methoxyphenyl), 148.74 (C=N), 146.32 (C7a), 138.07
(C10 3-methoxyphenyl), 133.41 (C2), 131.72 (C50
3-methoxyphenyl), 127.57 (C3a), 126.18 (C60 3-methox-
yphenyl), 124.96 (C6), 122.63 (C4), 121.58 (C5), 118.19
(C40 3-methoxyphenyl), 114.57 (C20 3-methoxyphenyl),
111.35 (C3), 104.05 (C7), 55.76 (O-CH3). ESI-Mass m/z:
294 [M ? H]?, 315 [M ? Na?]; Anal. Calcd. for
C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C, 69.84;
H, 4.93; N, 14.53.
Results and discussion
Chemistry
The N-acylhydrazones were synthesized through a classic
imine formation reaction between indolehydrazides and
1
different aldehydes. The H NMR and ESI-mass data of
compounds approved the exact structures. In the 1H NMR
spectra of these compounds, the existence of two singlets at
11–12 ppm was assigned to hydrazide NH and indole-NH
also. Singlet signal at 8.2–8.8 ppm was assigned to H–
C=N. Molecular mass of all the derivatives was detected by
electron spray ionization mass spectrometry (ESI–MS) as
M ? 1 and/or M ? 23 and/or M ? 39 relating to hydro-
gen and sodium and potassium adducts of the intact
molecules, respectively.
Biological activity evaluation
In vitro platelet aggregation
Platelet aggregation was evaluated by turbidimetric method
reported by Born (Born 1962) using APACT 4004 aggre-
gometer. All the synthesized compounds were evaluated
for their ability to inhibit platelet aggregation of human
platelet-rich plasma (PRP) induced by AA, ADP and col-
lagen as potent aggregation inducers, and using indo-
methacin and aspirin as standards.
The acylhydrazone structures gave two sets of signals
in NMR spectra which their intensities depend on solvent
and the pair of signals coalesces on warming the NMR
tube. The existence of the carbonyl oxygen atom and the
imine nitrogen atom in N-acylhydrazones indicates geo-
metrical isomers (E/Z). Syakaev et al. (2006) also
noticed that derivatives of acylhydrazones from aromatic
aldehydes showed two rotamers (cis and trans) due to
the N–C(O) bond (Syakaev et al. 2006; Unsal-Tan et al.,
2010).
In vitro cytotoxicity assay
To determine the potential cytotoxicity of synthesized
derivatives against fibroblast L929, HepG2 (human liver
carcinoma), MCF7(humanbreastadenocarcinoma)andHeLa
(human cervix adenocarcinoma) cell lines, MTT [3-(4,5-
dimethylthiazol-2-yl-2,5-tetrazolium bromide)] method was
used. The cells were maintained in RPMI1640 medium con-
taining 10 % (v/v) heat-inactivated fetal bovine serum (FBS)
supplemented with penicillin (100 U/mL) and streptomycin
(100 g/mL) at 37 °C under 5 % CO2. Cells dispersed at a
concentration of 6000 cells/mL were seeded into 96-well
In our study, the 1H NMR spectra at room temperature
for some compounds of b and c series represented E/Z
isomerization which was related to C=N double bonds.
Right after dissolution of the synthesized compounds in
DMSO-d6, the double signals of EC=N/ZC=N conformers are
registered by NMR. The two conformers show two sets of
signals at different chemical shifts, and integration of the
two sets of signals indicates that the EC=N conformer is the
predominant conformer. Based on the earlier report that
N-acylhydrazones derived from aromatic aldehydes in
123

Med Chem Res
QSAR study
solution remained mostly in the E form because of the
hindered rotation on the imine bond (Palla et al., 1986), we
considered E geometry in our cases.
In order to explore the important physicochemical and
structural parameters responsible for the modulation of
the anti-platelet aggregation activities of the synthesized
compounds, quantitative structure–activity relationship
(QSAR) analysis with different types of molecular
descriptors was carried out. Various physicochemical
descriptors as independent variables (e.g., topological
indices, hydrophobic and steric parameters) were used in
QSAR modeling studies to find the impacts of different
structural properties on the biological activity of the
synthesized (except 3c, 3p and 3q which did not dis-
solved). The IC50 values were transformed into pIC50
values (i.e., -log IC50) and determined as a dependent
variable in the QSAR study. In the present study, a data
set of the twenty-eight structures of titled compounds was
subjected to stepwise linear regression analysis for model
generation. During this analysis, it was observed that the
response value of one compound only in first model (AA
data set) was outside the limits of response values of
other synthesized compounds. Thus, the compound was
considered as outliers and was not involved in the data
set for QSAR model generation. Since the calculated
values of some electronic descriptors depend on the
three-dimensional molecular geometry, the optimum 3-D
geometry of the molecules was obtained by Hyperchem
software (version 7, Hypercube Inc, USA), using AM1
semi-empirical method. Dragon software (Todeschini
http://www.disat.unimib.it/vhm/) was employed to obtain
constitutional, functional, geometrical and topological
descriptors of the synthesized compounds 2 and 3.
Meanwhile, some electronic descriptors such as frontier
molecular orbital (HOMO, LUMO), dipole moment
(MDP) and partial charges were calculated by the
Hyperchem software. Activity data were converted to
logarithmic scale (i.e., pIC50 for platelet aggregation
induced by AA, collagen and ADP). For each set of
descriptors, the best multi-linear regression equation was
obtained by multiple linear regression (MLR) methods
subroutine of SPSS software (SPSS Inc., version 18). The
correlation coefficient (R2), standard error of regression
(SE), root-mean-square error (RMS) and significant level
(p value) were employed to judge the validity of
regression equation. Correlation analysis was done to
indicate the co-linear descriptors, since colinearity
declines the efficiency of the MLR-based QSAR equa-
tion. Therefore, the descriptors that correlated with each
other and with activity data were determined and the
highest correlated descriptor among the co-linear
descriptors was hold and the others were removed
(Aggarwal et al., 2011; Firoozpour et al., 2012; Nakhjiri
et al., 2012; Singh et al., 2010). The calculated
Anti-platelet aggregation activity
Anti-platelet aggregation activity of the synthesized com-
pounds is shown in Table 1. All derivatives were initially
tested at 250 lM concentration, and IC50 values were
calculated for those with more than 50 % platelet aggre-
gation inhibitory effect.
The inhibition of platelet aggregation induced by col-
lagen, AA and ADP was evaluated for 2 series of acylhy-
drazone compounds as indole-3-carbaldehyde and indole-
2-carbaldehyde derivatives. In general, indole-3-carbalde-
hyde derivatives demonstrated higher activity against pla-
telet aggregation induced by collagen than the other group.
Compounds 3e and 3b exhibited the highest activities
against the platelet aggregation induced by collagen with
IC50 values of 12.7 lM and 13.3 lM, respectively, which
are comparable to that of Aspirin (9.7 lM), a well-known
non-selective cyclooxygenase inhibitor. Nevertheless,
compounds in c series showed higher activities against the
platelet aggregation induced by arachidonic acid. In this
group, 2h with IC50 value of 51.88 lM and 2i with IC50 of
44.38 lM were the most potent compounds among indole-
2-carbaldehyde derivatives which are comparable with
aspirin (30 2.6). Compound 3k exhibited promising
inhibitory effect against platelet aggregation agonist of
ADP which shows considerable activity in comparison
with indomethacin (42 %) and aspirin (21 %) as standards.
Mirfazli et al. have reported a group of N-acylhydra-
zones of indole ring, among which the most active com-
pound has been compound 2a with the IC50 value of
94 1.9 lM against the aggregation induced by arachi-
donic acid (AA). In the present study, a larger group of N-
acylhydrazone derivatives of indole has been synthesized
with more diverse structures. Compound 2i with a 2,3-
dihydroxyphenyl moiety has shown a twofold activity
against AA-induced aggregation when compared with
compound 2a. Furthermore, in the new series, some of the
compounds such as 3e, 3b and 3d have shown great
activities against collagen-induced platelet aggregation
(Table 1).
Cytotoxicity assay
The synthesized derivatives were evaluated for their cyto-
toxicity against three cancer cell lines and L929 as a non-
cancer one. As evident from the activity data listed in
Table 2, none of the compounds showed a significant
cytotoxic effect either against the normal cell L929 or
cancerous cell lines.
123

Med Chem Res
Table 2 Cytotoxic activity of the synthesized compounds (2a–m) and (3a–r)
Compounds
Anti-cancer activity, IC50 (lM)
Non-cancer cell line
L929
Cancer cell lines
HeLa
HepG2
MCF7
3a
3b
3c
3d
3e
3f
301.23
265.34
C500
249.53
278.89
291.32
276.90
148.89
180.96
159.11
182.57
280.90
C500
228.57
253.78
230.34
234.78
129.90
367.76
239.91
C500
263.14
248.78
201.05
288.90
133.33
259.55
199.23
166.85
C500
C500
165.45
277.98
222.77
197.74
C500
3g
3h
3i
197.44
159.90
190.98
361.78
C500
3j
C500
172.57
C500
3k
3l
277.90
384,25
C500
250.67
359.90
C500
247.90
C500
3m
3n
3o
3p
3q
3r
2a
2b
2c
2d
2e
2f
169.45
C500
160.13
C500
167.89
C500
194.12
C500
200.35
189.21
C500
C500
378.90
291.35
398.32
133.89
222.67
188.98
C500
356.01
301.40
320.46
C500
323.23
401.21
160.90
261.88
272.25
291.44
C500
C500
358.11
C500
193.89
248.67
C500
373.50
C500
C500
298.89
167.23
262.10
C500
144.79
291.29
C500
209.71
198.99
320.55
167.23
C500
188.91
323.67
290.45
203.45
233.80
299.80
201.45
391.45
2g
2h
2i
C500
391.89
C500
2j
390.43
250.04
130.45
C500
2k
2l
302.44
221.67
201.34
C500
214.01
268.28
2m
descriptors in separated equation related to different
inducers (AA, collagen and ADP) were shown in data
matrices for retained descriptors in Table 3 for Eqs. (1),
(2) and (3), respectively. Finally, we selected similar
molecules reported in pervious study as external
prediction set (Mirfazli et al., 2014). The experimental
and predicted activity was shown in Table 4. According
to predicted activity, Eqs. (1) and (2) represented good
predictive ability; however, Eq. (3) did not show appro-
priate prediction for selected compounds.
123

Med Chem Res
In this study, we used the pIC50 of indole acylhydrazone
derivatives against AA-induced platelet aggregation as the
dependent variable, and Eq. 1 was obtained from the pool
of calculated descriptors.
Molecular topology as numerical quantifiers is sensitive
to bonding pattern, content of heteroatom (complexity of
atomic neighborhoods) and symmetry (Lather and Madan,
2005) (Kumar et al., 2014; Rathod et al., 2012). In this
case, topological index, PW3, was the most dominant
parameter in describing the anti-platelet activity of the
synthesized compounds against the aggregation induced by
AA and a positive correlation was observed. The molecular
path/walk indices are specified as the ratio between the
atomic path count and the atomic walk count of the same
length. Whereas the number of paths in a molecule is
bounded and determined by the molecule’s diameter, the
number of walks is unbounded. However, being interested
only in quotients, the walk count is terminated when it
exceeds the maximum allowed length of the corresponding
path. The average sum of atomic path/walk indices of equal
length and its count ratio is independent of molecular size.
Therefore, PW3 can be considered as shape descriptor and
is related to transport phenomena and interaction capabil-
ities between ligands and receptors (Vahdani and Bayat,
2011; Todeschini and Consonni, 2000). As is apparent in
AA model equation, the PW3-positive sign means that
increasing the PW3 of molecules leads to decreasing in
IC50 values.
pIC50 ¼ 2:93 ðÆ1:842ÞÀ0:491 ðÆ0:064Þ X2sol
þ 0:199 ðÆ0:042Þ RDF135u
þ 0:002 ðÆ0:001ÞTIEÀ0:088 ðÆ0:027Þ RDF120u
þ 15:128 ðÆ5:863Þ PW3
n ¼ 27 R2 ¼ 0:791 Se ¼ 0:191
F ¼ 15:93 R2cv ¼ 0:67 RMScv ¼ 0:218
PE ¼ 0:018
ð1Þ
Here and thereafter, ‘n’ is the number of data points, R—
correlation coefficient, Se—standard error of the estimate,
F—Fischer statistic, PE—probable error of the coefficient
of correlation.
As seen in Eq. (1), this model shows good statistical
quality (R2 = 0.791, Se = 0.191, F = 15.93) that con-
tains: solvation connectivity index of order 2 (X2sol),
Radial Distribution Function-135 unweighted (RDF135u),
E-state topological parameter (TIE), Radial Distribution
Function-120 unweighted (RDF120u) and path/walk
3-Randic shape index (PW3).
Table 3 Correlation coefficient (R2) matrix for some of descriptors used in this study
X2sol
RDF135u
TIE
RDF120u
PW3
Table Eq. (1): Anti-platelet aggregation activity induced by AA data set
X2sol
1
0.204
1
0.183
0.071
1
0.146
0.582
0.055
1
0.361
0.320
0.001
0.521
1
RDF135u
TIE
RDF120u
PW3
LP1
PW4
RDF145u
PJI2
RDF100u
Table Eq. (2) Anti-platelet aggregation activity induced by collagen data set
LP1
1
0.691
1
0.251
-0.018
-0.068
-0.070
1
-0.102
0.284
-0.236
-0.120
1
PW4
-0.080
RDF145u
PJI2
1
RDF100u
RDF100v
RDF065u
nR06
RDF115u
GATS6v
Table Eq. (3) Anti-platelet aggregation activity induced by ADP data set
RDF100v
RDF065u
nR06
1
0.701
1
0.209
0.274
1
0.111
0.233
0.343
1
0.256
0.165
0.063
-0.518
1
RDF115u
GATS6v
123

Med Chem Res
Table 4 Experimental and predicted activity of external prediction set
Compounds
AA
Collagen
Experimental
Predicted
3.83
Experimental
Predicted
4.29
3.50
3.53
3.74
3.73
3.91
3.72
3.91
4.67
3.73
3.55
3.66
4.46
4.53
4.50
3.54
3.69
3.88
3.48
3.14
3.72
3.48
3.63
Equation (2) describes the most reliable model for
inhibitory effect of the synthesized compound in platelet
aggregation induced by collagen as below:
increasing the branching of molecules leads to a decrease
in their IC50 (Du et al., 2011). Also in this model, PW4
is another descriptor which can show significant effect in
IC50 value, but, with negative sign, it has reverse effect
in IC50 values in collagen-induced platelet aggregation
equation.
pIC50 ¼ À207:85 ðÆ18:126Þ þ 90:871 ðÆ8:02Þ LP1
À52:043 ðÆ8:85Þ PW4
þ 0:625 ðÆ0:161Þ RDF145m þ 1:697 ðÆ0:631Þ PJI2
þ 0:050 ðÆ0:019Þ RDF100u
For Eq. (3), the percent of inhibition for platelet
aggregation induced by ADP of the compounds as depen-
dent variable was used and Eq. (3) as ADP model was
determined.
n ¼ 28 R2 ¼ 0:883 Se ¼ 0:238 F ¼ 33:316
R2cv ¼ 0:807 RMScv ¼ 0:277 PE ¼ 0:014
ð2Þ
Log ðp=100 À pÞ ¼ À0:591 ðÆ0:783Þ À 0:863 ðÆ0:105Þ
RDF100v þ 0:331 ðÆ0:048Þ RDF065u
þ 1:894 ðÆ0:367Þ nR06 À 0:299 ðÆ0:065Þ RDF115u
À 1:240 ðÆ0:310Þ GATS6v
This equation has high statistical quality (R2 = 0.883,
Se = 0.238, F = 33.316) which has Lovasz–Pelikan index
(leading eigenvalue) (LP1), path/walk 4-Randic shape
index (PW4), Radial Distribution Function-145 unweighted
(RDF145u), 2D Petitjean shape index (PJI2) and Radial
Distribution Function-100 unweighted (RDF100u) as
important parameters in the Equation.
n ¼ 28 R2 ¼ 0:838 Se ¼ 0:534 F ¼ 22:781
R2cv ¼ 0:686 RMScv ¼ 0:672 PE ¼ 0:020
ð3Þ
LP1 is the Lovasz–Pelikan index of leading eigen-
value and is a topological descriptor. It has been sug-
gested as an index of molecular branching, the smallest
values corresponding to chain graphs and the highest to
the most branched graphs. In Eq. (2), the LP1 contri-
bution has a positive sign, which indicates that the IC50
is inversely related to this descriptor; therefore,
Third equation, Eq. (3), with Radial Distribution Function-
100 weighted by van der Waals volume (RDF100v), Radial
Distribution Function-065 unweighted (RDF065u), number
of 6-membered rings (nR06), Radial Distribution Function-
115 unweighted (RDF115u) and Geary autocorrelation of
lag 6 weighted by van der Waals volume (GATS6v) as
ADP model descriptors defines best statistical quality with
123

Med Chem Res
R2 = 0.838, Se = 0.534, F = 22.781. Ring descriptor
(nR06) as a constitutional descriptor has detrimental effects
on percent inhibition activity of the compounds and
GATS6v with negative sign determines that decreasing the
GATS6v (van der Waals volume) leads to increase in IC50
values.
The correlation between the experimental and predicted
activities for all of 28 compounds is represented graphi-
cally in Fig. 3a, b, c for AA, collagen and ADP model,
respectively.
Equations (1) and (2) illustrate that spatial and topo-
logical descriptors have important role in anti-platelet
aggregation activity of the synthesized compounds. The
similarity of descriptors in Eqs. (1) and (2) represents that
structures inhibit platelet aggregation through approxi-
mately same mechanism. In Eq. (3), the autocorrelation
vectors represent the degree of similarity between mole-
cules. Therefore, anti-platelet aggregation activity of these
compounds is mainly dependent on molecular shape of the
structure. The other important descriptors that presented in
all models were RDF descriptors. RDF (radial distribution
function) descriptors as a similar parameter were emerged
in all equations for this study data set, which are based on
the distances distribution in the geometrical representation
of a molecule and constitute a radial distribution function
code. Additionally, the RDF descriptors can be restricted to
specific atom types or distance ranges to represent specific
information in a certain three-dimensional structure space,
e.g., to describe steric hindrance or structure/activity
properties of a molecule, and also these descriptors display
valuable information, for example, about bond distances,
ring types, planar and non-planar systems and atom types.
Topological indices were other important descriptors.
These indices help to differentiate the molecules according
mostly to their size, degree of branching, flexibility and
overall shape (Puzyn et al., 2010).
Fig. 3 Plots of cross-validated predicted values of anti-platelet
aggregation activity by MLR against the experimental values for
different aggregation inducers: (3A) AA, (3B) collagen and (3C) ADP
higher anti-platelet aggregation activity in 3-indole
derivatives.
Conclusion
In general, N-acylhydrazones showed different activity
profiles against the platelet aggregations induced by ADP,
AA and collagen. Furthermore, the QSAR study demon-
strated that different descriptors such as constitutional,
topological and geometrical play important role in anti-
platelet aggregation activity of the synthesized compounds
against mentioned inducers.
We have designed and synthesized thirty-one indole N-
acylhydrazone derivatives 2(a–m) and 3(a–r). It was found
3-indole substituted derivatives are more active than other
compounds in platelet aggregation inhibitory effect.
In addition, comparing the activity of compounds
against platelet aggregation induced by AA shows that
compounds 2(a–m) have less molecular diameters than the
compounds 3(a–r) and increasing in path/walk indices
decreases IC50 values according to the Equation. However,
in collagen model, LP1 with positive sign and also PW4 as
path/walk index with negative sign, demonstrating the
branching and increasing in molecular diameter (shape
decides its nature of binding in the active site), result in
Acknowledgments The authors gratefully acknowledged the
financial support from Research Council of Tehran University of
Medical Sciences.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
123

Med Chem Res
Ethical approval All procedures performed in studies involving
human participants were in accordance with the ethical standards of
institutional and/or national research committee and with the 1964
Helsinki Declaration and its later amendments or comparable ethical
standards.
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