Antiamoebic coumarins from the root bark of Adina cordifolia and their new thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.06.003

In continuation of our search for potential antiamoebic agents from folklore Indian medicinal plants, we found that the benzene and ethyl acetate extracts from the root bark of Adina cordifolia exhibited strong antiamoebic activity with IC₅₀ values of 2.92 and 2.50 μg/ml, respectively. Bioassay-guided fractionation of benzene and ethyl acetate extracts led to the isolation of 7-hydroxycoumarin (umbelliferone 1) and 7-β-D-glucosylcoumarin (skimmin 2), respectively. Umbelliferone 1 was converted into 7-acetoxycoumarin 1a, which on treatment with aluminium chloride afforded 7-hydroxy-8-acetylcoumarin 2a. A new series of thiosemicarbazones 3a-e of 7-hydroxy-8-acetylcoumarin with different thiosemicarbazides were synthesized. Umbelliferone was also converted into its methoxy derivative (7-methoxycoumarin 4). Subsequently, all the compounds were assessed for antiamoebic activity against HM1:IMMS strain of Entamoeba histolytica. Umbelliferone and skimmin were found to possess a very good activity with IC₅₀ values of 6.38 and 4.35 μM/ml, respectively. The activity drastically increased on converting compound 2a into its thiosemicarbazone derivatives 3a-e with IC₅₀ values ranging between 1.06 and 4.46 μM/ml. Compounds 3b,c and e with IC₅₀ values of 1.49, 1.56 and 1.06 μM/ml, respectively, exhibited even higher antiamoebic activity than the standard drug metronidazole (IC₅₀ = 2.62 μg/ml). The activity of 7-methoxycoumarin (IC₅₀ = 8.92 μM/ml) was less than umbelliferone. Compounds 3b, c and e were tested for toxicity using H9c2 cardiac myoblasts cell line. The compounds exhibit >80% viability at 3.125-200 μg/ml. It is apparent from these results that umbelliferone and skimmin may be a useful lead for the development of new antiamoebic drugs.

Full text of DOI:10.1016/j.ejmech.2008.06.003

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 2252e2259
http://www.elsevier.com/locate/ejmech
Short communication
Antiamoebic coumarins from the root bark of Adina cordifolia
and their new thiosemicarbazone derivatives
*
Prince Firdoos Iqbal, Abdul Roouf Bhat, Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 6 March 2008; received in revised form 31 May 2008; accepted 6 June 2008
Available online 18 June 2008
Abstract
In continuation of our search for potential antiamoebic agents from folklore Indian medicinal plants, we found that the benzene and ethyl
acetate extracts from the root bark of Adina cordifolia exhibited strong antiamoebic activity with IC50 values of 2.92 and 2.50 mg/ml,
respectively. Bioassay-guided fractionation of benzene and ethyl acetate extracts led to the isolation of 7-hydroxycoumarin (umbelliferone 1)
and 7-b-D-glucosylcoumarin (skimmin 2), respectively. Umbelliferone 1 was converted into 7-acetoxycoumarin 1a, which on treatment with
aluminium chloride afforded 7-hydroxy-8-acetylcoumarin 2a. A new series of thiosemicarbazones 3aee of 7-hydroxy-8-acetylcoumarin with
different thiosemicarbazides were synthesized. Umbelliferone was also converted into its methoxy derivative (7-methoxycoumarin 4).
Subsequently, all the compounds were assessed for antiamoebic activity against HM1:IMMS strain of Entamoeba histolytica. Umbelliferone
and skimmin were found to possess a very good activity with IC50 values of 6.38 and 4.35 mM/ml, respectively. The activity drastically increased
on converting compound 2a into its thiosemicarbazone derivatives 3aee with IC50 values ranging between 1.06 and 4.46 mM/ml. Compounds
3b,c and e with IC50 values of 1.49, 1.56 and 1.06 mM/ml, respectively, exhibited even higher antiamoebic activity than the standard drug
metronidazole (IC50 ¼ 2.62 mg/ml). The activity of 7-methoxycoumarin (IC50 ¼ 8.92 mM/ml) was less than umbelliferone. Compounds 3b, c
and e were tested for toxicity using H9c2 cardiac myoblasts cell line. The compounds exhibit >80% viability at 3.125e200 mg/ml. It is apparent
from these results that umbelliferone and skimmin may be a useful lead for the development of new antiamoebic drugs.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Adina cordifolia; Umbelliferone; Skimmin; Thiosemicarbazones; Entamoeba histolytica; MTT assay
1. Introduction
recent years there has been a rise in the number of reports with
amoebic brain abscess [3e7]. Metronidazole is known to be
a highly effective amoebicide and is considered to be the
drug of choice for the treatment of amoebiasis, but this drug
has been shown to be mutagenic in a microbiological system
and carcinogenic to rodents [8e10]. The treatment with
metronidazole is known to be associated with several side ef-
fects that include nausea, vomiting, dry mouth, metallic taste,
abdominal pain, headache and diarrhoea or constipation may
also occur [11]. In addition, the drug recipients exhibit lower
immune response both cell and humoral mediated [12].
Moreover, there is possibility of drug resistance as demon-
strated in other protozoan parasites [13]. Some recent reports
also have described the in vitro generation of strains resistant
to metronidazole [14,15]. In spite of the tremendous use of
metronidazole, currently used therapies for treating disease
Amoebiasis, a disease caused by ingestion of contaminated
food or water containing cysts of Entamoeba histolytica
remains one of the major threats to public health in most parts
of the globe. It infects over 50 million people per annum
leading to 50,000e100,000 deaths annually, making it the
second leading cause of death among parasitic diseases [1].
Though ubiquitous in distribution, this parasite is more
prevalent in tropical and subtropical regions with poor sanitary
conditions and contaminated drinking water [2]. Moreover, in
* Corresponding author. Tel.: þ91 11 2698 1717/3253; fax: þ91 11 2698
0229/1232.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.003

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
2253
caused by Entamoeba provide inadequate protection and
hence continuous research for discovering and developing
newer antiamoebic agents even today is required so that the
limitation as presented by available therapeutic agents can
be minimized and more safer, effective antiamoebic drugs or
vaccines can come into existence.
During the last 50 years a number of compounds have been
isolated from plants and/or synthesized that have been screened
in vitro against E. histolytica as a result of which numerous
antiamoebic compounds have been identified. In developing
countries many people rely on traditional preparations from
various medicinal plants to treat this disease because these
products are safe, widely available at low cost and easy to
access. Thus, scientific validation and in vitro and/or in vivo
evaluation of these traditional remedies are needed to prove
their claimed effectiveness against the disease.
NMR and DEPT spectra were recorded on Bruker AVANCE
400 spectrometer using DMSO-d6 as solvent with TMS as
internal standard. ESI-MS was recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer. Splitting
patterns are designated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet. Chemical shift values are given in parts
per million. Precoated aluminium sheets (Merck silica gel
60 F254) were used for thin-layer chromatography (TLC) and
spots were visualized under UV light. Analytical grade
solvents were purchased from Merck (India).
3. Results and discussion
3.1. Identification of isolated compounds
n-Hexane, benzene, chloroform, ethyl acetate, acetone and
n-butanol extracts from the stem bark of A. cordifolia were
subjected to in vitro antiamoebic activity against HM1:IMSS
strain of E. histolytica using metronidazole (IC50 ¼ 0.45 mg/
ml) as a reference drug. Results indicated that the benzene
and ethyl acetate extracts (IC50 ¼ 2.92 and 2.50 mg/ml,
respectively) were the most active while the remaining extracts
showed moderate activity against E. histolytica (Table 1).
Therefore, benzene and ethyl acetate extracts were selected
for the isolation of active constituents and were fractionated
by column chromatography, which led to the isolation of
umbelliferone 1 and skimmin 2 (Fig. 1). The structures of
these compounds were elucidated by spectroscopic methods
Adina cordifolia (Roxb.) Hook.f.ex Brandis (Rubiaceae),
Syn. Haldinia cordifolia (Roxb.) Ridsd. is found scattered in
deciduous forests throughout the greater part of India,
ascending to an altitude of 900 m in the sub-Himalayan tract.
It is also common in the forests of South India. The plant A.
cordifolia has been used in oriental medicine since ancient
times as an essential component of various antiseptic and
febrifuge prescriptions [16]. The bark is acrid and bitter and
is used in biliousness [17]. The roots are used as an astringent
in dysentery [17]. The A. cordifolia stem has been evaluated
for its antiulcer potential and enzyme assay-guided fraction-
ation of the chloroform extract yielded 7-hydroxycoumarin
which showed interesting Hþ/Kþ ATPase inhibitory activity
[18]. Antifertility properties of the leaf extract of A. cordifolia
have been also examined [19].
Keeping in view the medicinal importance of A. cordifolia
and as a part of our programme to explore naturally occurring
bioactive compounds from Indian folklore medicinal plants for
the treatment of amoebic dysentery, we have investigated the
root bark of A. cordifolia, which is traditionally used in
folklore medicine for the treatment of dysentery in different
parts of India, especially in Pauri Garhwal region of
Uttrakhand [20]. In this study the in vitro antiamoebic activity
of the extracts, isolated compounds and their derivatives from
the root bark of A. cordifolia are reported.
1
(IR, H NMR, 13C NMR, DEPT and ESI-MS) and compared
with the literature data [18,21].
These isolated compounds were then assessed in vitro for
antiamoebic activity and it was found that umbelliferone
(IC50 ¼ 1.04 mg/ml) and skimmin (IC50 ¼ 1.35 mg/ml)
exhibited better activity than their corresponding extracts. To
the best of our knowledge, this is the first report that umbelli-
ferone and skimmin inhibit the growth of E. histolytica.
3.2. Pharmaco-modulation of umbelliferone
Umbelliferone (7-hydroxycoumarin) 1 was initially reacted
with acetic anhydride to give 7-acetoxycoumarin 1a, which
was subjected to Fries rearrangement by treating with alumin-
ium chloride to give 7-hydroxy-8-acetylcoumarin 2a as the
main product [22,23]. Various thiosemicarbazides aee were
prepared according to the literature procedure [24]. A series
2. Chemistry
2.1. Plant material
The stem bark of A. cordifolia was collected from the Hills
of Pauri Garhwal district, Uttrakhand, India. The plant
material was authenticated by Professor Tasneem Fatima,
Department of Bioscience, Jamia Millia Islamia, New Delhi,
India. A voucher specimen (TFU-537) has been deposited in
the herbarium of the Department of Bioscience.
Table 1
In vitro antiamoebic activity of the extracts HM1:IMSS strain of E. histolytica
Extract
IC50 (mg/ml)
SDa
n-Hexane
Benzene
Chloroform
Ethyl acetate
Acetone
n-Butanol
a
37.1
2.92
20.33
2.5
0.53
0.32
0.21
0.18
0.25
0.1
2.2. Analytical material and methods
42.02
16.04
IR spectra were recorded on PerkineElmer model 1600
1
FT-IR RX1 spectrophotometer as KBr discs. H NMR, 13C
Standard deviation.

2254
P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
O
O
R
A characteristic signal for acetyl (C]O) appeared at d 169 and
a signal at d 19.7 revealed the presence of methyl group.
The formation of compound 2a was confirmed by its IR spec-
trum which showed the presence of OH group absorption around
3400 cmꢀ1 andusualstrongbandswererecordedinthespectrum
at 1680e1780 cmꢀ1 (C]O, lactone). The structure was further
confirmed by 1H NMR spectrum, which showed the presence of
a signal at d 12.3 due to (OH) proton and a singlet at d 3.1 due to
(COCH3)protons.
1. R = OH (Umbelliferone)
2. R = OGlc (Skimmin)
Fig. 1. Structure of isolated compounds.
of thiosemicarbazones 3aee of 7-hydroxy-8-acetylcoumarin
with different thiosemicarbazides were synthesized [25]
(Scheme 1). Umbelliferone was also allowed to react with
dimethyl sulphate and potassium carbonate in dry acetone to
prepare its methyl derivative
(Scheme 2). All compounds 1a, 2a, 3aee, 4 were crystallized
The structure of the compounds 3aee was confirmed by the
characteristic IR bands observed at 3380 and 3233e3115 cmꢀ1
assigned to n(OH) and n(NH), respectively. A band at
1726 cmꢀ1 was assigned to n(C]O) of the lactone moiety.
This band remained unchanged in the final products, indicating
that the lactone oxygen was not involved in thiosemicarbazone
formation. The appearance of n(C]S) at 1110e1160 cm
n(C]N) at 1610e1680 cmꢀ1 further confirmed the formation
4
(7-methoxycoumarin)
from appropriate solvents and were characterized by elec-
1
tronic, IR, H and 13C NMR spectra.
ꢀ1 and
The formation of compound 1a was confirmed by IR
spectrum which showed the absence of OH group absorption
around 3400 cmꢀ1, while strong bands were recorded in the
spectrum at 1680e1780 cmꢀ1 (C]O, lactone), and
1620 cmꢀ1, 1565 cmꢀ1 (C]C, aromatic). The 1H NMR
spectrum showed the absence of a broad singlet at d 8.40 due
to OH group and the presence of a singlet at d 2.02 due to CH3
group. The structure was further supported by 13C NMR spectra.
1
of the final products 3aee. The H NMR spectrum of 3aee
gave signals at d 11.58 and 10.07 assigned to (NHeC]S)
and (OH) protons, respectively, whereas a signal at d 8.36
may be assigned to the (NHR) proton. The coumarin ring
protons appeared as doublets centered at d 6.12 and 7.98 and
the methyl proton signal at d 2.31. The formation of 3aee
was further confirmed by 13C NMR spectra. A characteristic
KOH + S=C=S+ HR
ClCH2COONa
S
O
O
O
O
OH
1
i
NaOOCCH2
S
R
HCl
OCOCH3
1a
S
ii
HOOCCH
H2NNH
S
R
2
H2NNH2.H2O
S
O
O
OH
COCH3
R
a-e
2a
iii
O
O
OH
H3C
NNHCSR
3a-e
iii: Propan-1-ol/reflux/24 h
i: Ac2O reflux/5 h, ii: AlCl3/125-127°C/2 h,
NH
N
NH
N
Where R = 3a NH2, 3b
,
3e
3d
,
,
3c
Scheme 1. Synthesis of thiosemicarbazone derivatives of umbelliferone.

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
2255
Palladium (II) complexes of 5-nitrothiophene-2-carboxal-
dehyde thiosemicarbazones substituted with adamantamine
showed better activity whereas when substituted with p-
toluidine and N-methyl benzyl amine showed less activity
than metronidazole [28].
Dry acetone
(CH3)2SO4 /K2CO3
O
O
OH
O
O
OCH3
4
1
Scheme 2. Methylation of umbelliferone.
In the present work the coumarin thiosemicarbazone
derivatives substituted with adamantamine, p-benzyl piperi-
dine and N-methyl benzyl amine showed better activity
whereas when substituted with p-toluidine showed less activity
than metronidazole. These results thus indicated that the
antiamoebic activity enhances by the introduction of adamant-
amine, p-benzyl piperidine and N-methyl benzyl amine.
Toxicity profile. To ensure the toxicity of the compounds
3b, c and e with better IC50 values than metronidazole, they
were tested against H9c2 cardiac myoblasts. A subconfluent
population of H9c2 cells was treated with increasing
concentrations of compounds 3b, c and e, and the number of
viable cells was measured after 48 h by MTT cell viability
assay. The concentration range of compounds 3b, c and e
was 3.125e200 mg/ml. Fig. 2 depicted that compounds 3b, c
and e exhibited >80% viability at the concentration range of
3.125e200 mg/ml.
signal for (C]S) appeared at 172 and a signal at d 166 showed
the presence of (C]N) of thiosemicarbazone.
The formation of compound 4 was confirmed by IR
spectrum, which revealed the absence of (OH) band around
3400 cmꢀ1
,
while strong absorption band at 1680e
1683 cmꢀ1 was assigned to the coumarin n(C]O). The H
NMR spectrum of compound 4 showed the absence of a broad
singlet at d 8.40 due to OH group, while the presence of a sin-
glet at d 3.25 due to CH3 group showed the conversion of OH
into OCH3. The formation of compound 4 was further
supported by the appearance of a signal due to (OCH3) at
d 50 in 13C NMR spectrum.
1
All the compounds 1a, 2a, 3aee, 4 were then assessed for
in vitro antiamoebic activity and it was found that the activity
of compounds 3aee with IC50 values of 4.46, 1.49, 1.56, 3.17
and 1.06 mM/ml, respectively, was lower than that of the
parent compound
1
(IC50 ¼ 6.38 mM/ml). The activity
drastically increased on converting compound 2a into its
thiosemicarbazone derivatives 3aee with IC50 values ranging
between 1.06 and 4.46 mM/ml. Compounds 3b, c, and e with
IC50 values of 1.49, 1.56, and 1.06 mM/ml, respectively,
exhibited even higher antiamoebic activity than the standard
drug metronidazole (IC50 ¼ 2.63 mM/ml). The activity of 7-
methoxycoumarin (IC50 ¼ 8.92 mM/ml) was less than umbelli-
ferone (Table 2).
4. Experimental
4.1. Extraction and isolation of umbelliferone and
skimmin from A. cordifolia
Dried and well-ground root bark (4 kg) was extracted by
refluxing with methanol (5 ꢁ 10 L, 3 h each). The insoluble
material was removed by filtration and the extract was
concentrated under reduced pressure. The dried, crude
methanolic extract (1.5 kg, 37.5%) was then fractionated
successively by refluxing with n-hexane, benzene, chloroform,
ethyl acetate, acetone and n-butanol (3 ꢁ 2.5 L, 3 h each) to
give the hexane (20 g, 1.33%), benzene (8 g, 0.53%), chloro-
form (15 g, 1%), ethyl acetate (13 g, 0.86%), acetone (25 g,
1.66%) and n-butanol (23 g, 1.35%) extracts, respectively.
In our previous work regarding the antiamoebic activity of
thiosemicarbazone derivatives by using different amines, we
herein give a comparative study of the present work with the
previous one. The 3,7-dimethyl-pyrazolo [3,4e] [1,2,4]
triazine-4-yl thiosemicarbazides derivatives substituted with
adamantamine showed better activity whereas when
substituted with p-toluidine showed less activity than
metronidazole [26]. Similarly 1-N-substituted thiocarbamoyl-
3,5-diphenyl-2-pyrazoline derivatives substituted with
adamantamine showed better activity than metronidazole [27].
Table 2
In vitro antiamoebic activity of the isolated compounds and their thiosemicar-
bazone derivatives against HM1:IMSS strain of E. histolytica
Compound
IC50 (mM/mL)
SDa
Umbelliferone (1)
Skimmin (2)
6.380
4.15
13.08
6.51
4.46
1.49
1.56
3.17
1.06
8.92
2.63
0.30
0.11
2.3
1a
2a
2.1
0.9
3a
3b
0.27
0.16
0.19
0.2
3c
3d
3e
4
Metronidazole
0.3
0.08
a
Standard deviation.
Fig. 2. -

2256
P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
The benzene and ethyl acetate extracts showed inhibition of E.
histolytica and were selected for the isolation of active
constituents.
(OeC]O), 160.95 (C-2), 156.73 (C-7), 152.63 (C-9),
144.95 (C-4), 126.42 (C-5), 119.86 (C-6), 117.11 (C-10),
113.5 (C-3), 106.90 (C-8), 19.79 (CH3); ESI-MS m/z: 205
[M þ H]þ.
The benzene extract (8 g) was chromatographed over a sil-
ica gel column (60e120 mesh) eluting with C6H6eEtOAc
mixtures of increasing polarity. Upon concentration, the
fractions eluted with 9.5:0.5 (v/v) C6H6eEtOAc gave a cream
coloured solid powder, which was identified as umbelliferone
1 (3 g, 37.5%). The ethyl acetate extract (13 g) was chromato-
graphed over a column of silica gel (60e120 mesh), eluting
with CHCl3eMeOH mixtures of increasing polarity. The
fractions eluted with 9.5:0.5 (v/v) CHCl3eMeOH upon
concentration gave a white powder, identified as skimmin 2
(2.2 g, 16.9%).
4.3. Synthesis of 7-hydroxy-8-acetylcoumarin 2a
(Fries rearrangement)
A mixture of 7-acetoxycoumarin (24.5 mmol), anhydrous
aluminium chloride (74.9 mmol) and anhydrous sodium
chloride (34.4 mmol) was heated at 170 ꢂC for 1.5 h. The
mixture was allowed to cool and dil. HCl (50 ml) was added.
The reaction mixture was refluxed for 10 min, diluted with
water (200 ml) and extracted three times (100 ml) with ethyl
acetate, dried over sodium sulphate. The solvent was
evaporated and solid residue was subjected to column
chromatography and eluted with CHCl3 yielded 7-hydroxy-
8-acetylcoumarin (2a) free from the 6-isomer (7-hydroxy-6-
acetylcoumarin). Yield: 60%; m.p.: 180 ꢂC; light brown
crystals; IR nmax cmꢀ1: 3185 (Ar-H), 2890 (C-H), 1785
(C]O, lactone), 1640, 1555 (C]C, aromatic); 1H NMR
(DMSO-d6) d (ppm): 12.76 (s, 1H, OH), 7.76 (d, 1H, H-4,
J ¼ 9.5 Hz), 7.52 (d, 1H, H-5, J ¼ 8.6 Hz), 6.78 (d, 1H, H-6,
J ¼ 8.6 Hz), 6.32 (d, 1H, H-3, J ¼ 9.5 Hz), 2.95 (s, 3H,
CH3); 13C NMR (DMSO-d6) d (ppm): 195.50 (C]O), 161.4
(C-2), 159.9 (C-7), 152.31 (C-9), 144.60 (C-4), 136.42 (C-
5), 120.21 (C-8), 116.80 (C-10), 113.71 (C-3), 111.51 (C-6),
27.90 (CH3); ESI-MS m/z: 205 [M þ H]þ.
4.1.1. Umbelliferone 1
M.p.: 234e237 ꢂC; cream coloured solid; UV lmax nm:
1
296, 256, 226; IR nmax cmꢀ1: 3195, 1680, 1603, 1567; H
NMR (DMSO-d6) d (ppm): 7.70 (d, 1H, H-4, J ¼ 9.5 Hz),
7.40 (d, 1H, H-5, J ¼ 10.3 Hz), 6.83 (d, 1H, H-8, J ¼ 2 Hz
due to m-coupling with HC-6), 6.78 (dd, 1H, H-6,
J ¼ 10.3 Hz, 2 Hz), 6.20 (d, 1H, H-3, J ¼ 9.5 Hz); 13C NMR
(DMSO-d6) d (ppm): 160.93 (C-2), 161.74 (C-7), 155.94 (C-
9), 144.95 (C-4), 130.14 (C-5), 113.57 (C-3), 111.84 (C-10),
111.72 (C-6), 102.61 (C-8); ESI-MS m/z: 163 [M þ H]þ.
4.1.2. Skimmin 2
M.p.: 221e222 ꢂC; white powder; UV lmax nm: 294, 257,
224; IR nmax cmꢀ1: 3370, 1683, 1615, 1562; 1H NMR
(DMSO-d6) d (ppm): 7.80 (d, 1H, H-4, J ¼ 9.5 Hz), 7.52 (d,
1H, H-5, J ¼ 10 Hz), 6.71 (d, 1H, H-8, J ¼ 2 Hz due to m-
coupling with HC-6), 6.55 (dd, 1H, H-6, J ¼ 10 Hz), 6.45 (d,
1H, H-3, J ¼ 9.5 Hz), 4.80 (d, 1H, H-10, J ¼ 7.6 Hz), 3.87
(dd, 1H, H-6b0, J ¼ 12.0, 1.0 Hz), 3.68 (dd, 1H, H-6a0,
J ¼ 12.0, 5.0 Hz), 3.60 (t, 1H, H-30, J ¼ 8.5 Hz), 3.42 (dd,
1H, H-40, J ¼ 9.5, 8.5 Hz), 3.30e3.41 (m, 1H, H-50), 3.29
(dd, 1H, H-20, J ¼ 9.0, 7.5 Hz); 13C NMR (DMSO-d6)
d (ppm): 160.73 (C-7), 160.67 (C-2), 155.48 (C-9), 144.73
(C-4), 129.90 (C-5), 114.11 (C-3), 113.73 (C-10), 113.60
(C-6), 103.60 (C-8), 100.40 (C-10), 77.58 (C-50), 76.35 (C-
30), 73.57 (C-20), 70.07 (C-40), 61.09 (C-60); ESI-MS m/z:
325[M þ H]þ, 347[M þ Na]þ.
4.4. General procedure for the synthesis of substituted
thiosemicarbazides aee
4.4.1. Preparation of substituted thioglycolic acid
Carbondisulphide(1 equiv.)wasaddeddropwisetoasolution
of primary or secondary amines (1 equiv.) containing potassium
hydroxide (1 equiv.) in water:ethanol (1:3) mixture. The temper-
ature of the reaction was maintained below 10 ꢂC. Sodium chlor-
oacetate (1 equiv.) was added and the reaction mixture was left
overnight at room temperature. Addition of conc. hydrochloric
acid (pH w 1) precipitated substituted thioglycolic acid, which
was crystallized by appropriate solvent.
4.4.2. Conversion of thioglycolic acid into
thiosemicarbazides aee
4.2. Synthesis of 7-acetoxycoumarin 1a
A solution of thioglycolic acid (1 equiv.) in water (15 ml)
containing sodium hydroxide (1 equiv.) and hydrazine hydrate
(1 equiv.) was refluxed for 2 h with continuous stirring. The
compound separated out during the reaction or on cooling at
0 ꢂC for 12 h. The product was filtered and crystallized from
suitable solvent.
Umbelliferone (0.005 mol) was refluxed with acetic
anhydride for 5 h and the reaction mixture was cooled, poured
onto the ice and left overnight. The precipitate was filtered,
dried and crystallized by acetone to afford the acetoxy product
7-acetoxycoumarin 1a. Yield: 88%; m.p.: 140 ꢂC; white
powder; IR nmax cmꢀ1: 3184 (Ar-H), 2910 (C-H), 1780
(C]O, lactone), 1636, 1560 (C]C, aromatic), 1215 (CeO);
4.5. Preparation of thiosemicarbazones of 7-hydroxy-8-
acetylcoumarin 3aee
1H NMR (DMSO-d6)
d
(ppm): 7.90 (d, 1H, H-4,
J ¼ 8.8 Hz), 7.50 (d, 1H, H-5, J ¼ 9.5 Hz), 7.02 (d, 1H, H-8,
J ¼ 2.2 Hz due to m-coupling with HC-6), 6.95 (dd, 1H, H-
6, J ¼ 9.5 Hz, J ¼ 2.2 Hz), 6.10 (d, 1H, H-3, J ¼ 8.8 Hz),
2.95 (s, 3-H, CH3); 13C NMR (DMSO-d6) d (ppm): 172.00
A mixture of 7-hydroxy-8-acetylcoumarin 2a (5 mmol) and
the corresponding thiosemicarbazides, in the molar ratio 1:1
was refluxed in 1-propanol (20 ml) for 24 h. The mixture

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
2257
was allowed to cool and the resulting precipitate was filtered
and subsequently dried to afford the pure products 3aee.
H, 4.4; N, 11.3%. IR nmax cmꢀ1: 3390 (OeH), 3154 (NeH),
1720 (C]O), 1660 (C]N), 1123 (C]S); 1H NMR
(DMSO-d6) d (ppm): 11.85 (s, 1H, HNeC]S), 10.03 (s,
1H, OH), 8.62 (s, 1H, NHR), 7.91 (d, 1H, H-4, J ¼ 9.5 Hz),
7.62 (d, 1H, H-5, J ¼ 8.2 Hz), 7.50 (d, 2H, J ¼ 7.8 Hz, Ph),
7.07 (d, 2H, J ¼ 7.8 Hz, Ph), 6.38 (d, 1H, H-6, J ¼ 8.2 Hz),
6.11 (d, 1H, H-3, J ¼ 9.5 Hz), 2.35 (s, 3H, CH3), 2.22 (s,
3H, CH3); 13C NMR (DMSO-d6) d (ppm): 182.51 (C]S),
172.41 (C]N), 160.23 (C]O), 159.80 (C-7), 152.42 (C-9),
146.31, 128.81, 126.22, 114.20 (Ph), 144.30 (C-4), 130.91
(C-5), 122.50 (C-8), 116.21 (C-10), 113.40 (C-3), 110.90
(C-6), 25.31 (CH3), 16.80 (CH3); ESI-MS m/z: 368 [M þ H]þ.
4.5.1. 7-Hydroxy-8-acetylcoumarin-N(4)
thiosemicarbazone 3a
Yield: 55%; m.p.: 195 ꢂC; brown solid; calculated for
C12H11O3N3S: C, 51.9; H, 3.9; N, 15.2%. Found: C, 52.2;
H, 3.7; N, 15.1%. IR nmax cmꢀ1: 3380 (OeH), 3195 (NeH),
1726 (C]O), 1665 (C]N), 1145 (C]S); 1H NMR
(DMSO-d6) d (ppm): 12.18 (s, 1H, HNeC]S), 10.21 (s,
1H, OH), 8.36 (s, 2H, NHR), 7.57 (d, 1H, H-4, J ¼ 9.3 Hz),
7.21 (d, 1H, H-5, J ¼ 8.4 Hz), 6.45 (d, 1H, H-6, J ¼ 8.4 Hz),
6.12 (d, 1H, H-3, J ¼ 9.3 Hz), 2.23 (s, 3H, CH3); 13C NMR
(DMSO-d6) d (ppm): 180.6 (C]S), 170.21 (C]N), 160.90
(C]O), 158.51 (C-7), 151.42 (C-9), 144.43 (C-4), 133.91
(C-5), 121.50 (C-8), 117.57 (C-10), 113.32 (C-3), 112.70
(C-6), 18.41 (CH3). ESI-MS m/z: 278 [M þ H]þ.
4.5.5. 7-Hydroxy-8-acetylcoumarin-N(4,4)methylbenzyl
thiosemicarbazone 3e
Yield: 36%; m.p.: 187 ꢂC; white solid; calculated for
C20H19O3N3S: C, 62.9; H, 4.9; N, 11.0%. Found: C, 63.2;
H, 4.7; N, 10.9%. IR nmax cmꢀ1: 3405 (OeH), 3216 (NeH),
1734 (C]O), 1613 (C]N), 1121 (C]S); 1H NMR
(DMSO-d6) d (ppm): 12.13 (s, 1H, HNeC]S), 10.22 (s,
1H, OH), 7.79 (d, 1H, H-4, J ¼ 9.6 Hz), 7.31 (d, 1H, H-5,
J ¼ 8.8 Hz), 7.04e7.24 (m, 5H, Ph), 6.42 (d, 1H, H-6,
J ¼ 8.8 Hz), 6.11 (d, 1H, H-3, J ¼ 9.6 Hz), 5.21 (s, 2H,
CH2), 3.18 (s, 3H, CH3), 2.12 (s, 3H, CH3); 13C NMR
(DMSO-d6) d (ppm): 180.20 (C]S), 169.51 (C]N), 160.80
(C]O), 159.31 (C-7), 153.41 (C-9), 144.62 (C-4), 140.51,
129.51, 126.12 (Ph), 132.90 (C-5), 121.71 (C-8), 117.54 (C-
10), 112.51 (C-3), 108.42 (C-6), 59.00 (CH2), 38.82 (CH3),
16.41 (CH3); ESI-MS m/z: 382 [M þ H]þ.
4.5.2. 7-Hydroxy-8-acetylcoumarin-N(4)adamentamine
thiosemicarbazone 3b
Yield: 43%; m.p.: 210 ꢂC; brown solid; calculated for
C22H25O3N3S: C, 64.2; H, 6.1; N, 10.2%. Found: C, 64.5;
H, 6.2; N, 10.1%. IR nmax cmꢀ1: 3410 (OeH), 3205 (NeH),
1722 (C]O), 1676 (C]N), 1145 (C]S); 1H NMR
(DMSO-d6) d (ppm): 12.51 (s, 1H, HNeC]S), 10.05 (s,
1H, OH), 8.06 (s, 1H, NHR), 7.35 (d, 1H, H-4, J ¼ 9.2 Hz),
7.11 (d, 1H, H-5, J ¼ 8.3 Hz), 6.52 (d, 1H, H-6, J ¼ 8.3 Hz),
6.22 (d, 1H, H-3, J ¼ 9.2 Hz), 2.22 (s, 3H, CH3), 1.03e2.31
(m, 15H, adamentyl ring); 13C NMR (DMSO-d6) d (ppm):
179.80 (C]S), 168.71 (C]N), 161.30 (C]O), 158.70 (C-
7), 152.31 (C-9), 144.62 (C-4), 130.91 (C-5), 120.11 (C-8),
116.20 (C-10), 112.51 (C-3), 110.21 (C-6), 25e47 (adamentyl
ring), 16.70 (CH3); ESI-MS m/z: 412 [M þ H]þ.
4.6. Preparation of 7-methoxycoumarin 4
Umbelliferone (200 mg) was refluxed in dry acetone
(50 ml) with dimethyl sulphate (0.5 ml) and anhydrous potas-
sium carbonate (5 g) for 72 h till it did not give any colour
with FeCl3. The reaction mixture was then allowed to cool
at room temperature and filtered off insoluble potassium
carbonate, which was washed several times with small
portions of dry acetone. The washings and filtrate were
combined and evaporated to dryness. A cream coloured solid
was left which was washed with petroleum ether and then
with water. Yield: 80%; m.p.: 90e92 ꢂC; cream coloured
crystals; IR nmax cmꢀ1: 3165 (Ar-H), 2890 (C-H), 1680
4.5.3. 7-Hydroxy-8-acetylcoumarin-N(4)4-benzylpiperidine
thiosemicarbazone 3c
Yield: 40%; m.p.: 160 ꢂC; red solid; calculated for
C24H25O3N3S: C, 66.2; H, 5.7; N, 9.6%. Found: C, 66.5; H,
5.4; N, 9.3%. IR nmax cmꢀ1: 3385 (OeH), 3190 (NeH),
1740 (C]O), 1650 (C]N), 1077 (C]S); 1H NMR
(DMSO-d6) d (ppm): 11.95 (s, 1H, HNeC]S), 10.90 (s,
1H, OH), 7.36 (d, 1H, H-4, J ¼ 8.8 Hz), 7.12 (d, 1H, H-5,
J ¼ 8.2 Hz), 7.02e7.75 (m, 5H, Ph), 6.33 (d, 1H, H-6,
J ¼ 8.2 Hz), 6.21 (d, 1H, H-3, J ¼ 8.8 Hz), 2.68e3.16 (m,
4H, eNCH2), 2.63 (d, 2H, CH2, J ¼ 7.3 Hz), 2.13 (s, 3H,
CH3), 1.60e1.86 (m, 1H, CH), 1.32e1.87 (m, 4H, CH2);
13C NMR (DMSO-d6) d (ppm): 179.30 (C]S), 169.21
(C]N), 160.60 (C]O), 159.51 (C-7), 150.00 (C-9), 144.22
(C-4), 132.51 (C-5), 140.41, 128.90, 124.72 (Ph), 118.00 (C-
8), 116.61 (C-10), 112.55 (C-3), 108.93 (C-6), 52.62
(2 ꢁ CH2), 44.33 (CH2), 33.12 (CH), 29.80 (2 ꢁ CH2), 17.72
(CH3); ESI-MS m/z: 437 [M þ H]þ.
1
(C]O), 1603, 1555 (C]C, aromatic); H NMR (DMSO-d6)
d (ppm): 7.82 (d, 1H, H-4, J ¼ 9.5 Hz), 7.46 (d, 1H, H-5,
J ¼ 9.2 Hz), 6.94 (m, 1H, H-8, J ¼ 2.0 Hz), 6.53 (m, 1H, H-
6, J ¼ 10.11 Hz), 6.12 (d, 1H, H-3, J ¼ 9.5 Hz), 3.25 (s, 3H,
CH3); 13C NMR (DMSO-d6) d (ppm): 160.91 (C-2), 158.30
(C-7), 151.32 (C-9), 144.35 (C-4), 127.28 (C-5), 115.50 (C-
10), 112.51 (C-3), 111.20 (C-6), 107.91 (C-8), 57.23 (CH3);
ESI-MS m/z: 177 [M þ H]þ.
4.7. In vitro antiamoebic assay
4.5.4. 7-Hydroxy-8-acetylcoumarin-N(4)p-toluidine
thiosemicarbazone 3d
Yield: 43%; m.p.: 173 ꢂC; white solid; calculated for
C19H17O3N3S: C, 62.1; H, 4.6; N, 11.4%. Found: C, 62.3;
E. histolytica trophozoites were cultured in wells of 96-well
microtiter plate by using Diamond TYIS-33 growth medium
[29]. All the extracts and isolated compounds/derivatives

2258
P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
were screened in vitro for antiamoebic activity against
HM1:IMSS strain of E. histolytica by microdilution method
[30]. All the extracts (10 mg) and compounds/derivatives
(1 mg) were dissolved in DMSO (40 ml, level at which no in-
hibition of amoeba occurs) [31,32]. The stock solutions of the
extracts and the compounds/derivatives were prepared freshly
before use at a concentration of 10 and 1 mg/ml, respectively.
Twofold serial dilutions were made in the wells of 96-well mi-
crotiter plate (costar). Each test includes metronidazole as
a standard amoebicidal drug, control wells (culture medium
plus amoebae) and a blank (culture medium only). All the ex-
periments were carried out in triplicate at each concentration
level and repeated thrice. The amoebal suspension was pre-
pared from a confluent culture by pouring off the medium at
37 ꢂC and adding 5 ml of fresh medium, chilling the culture
tube on ice to detach the organisms from the side of the flask.
The suspension was diluted to 105 organisms/ml by adding
fresh medium and 170 ml of this suspension was added to
the test and control wells in the plate so that the wells were
completely filled (total volume, 340 ml). An inoculum of
1.7 ꢁ 104 organisms/well was chosen so that confluent, but
not excessive growth, took place in control wells. Plates
were sealed and gassed for 10 min with nitrogen before incu-
bation at 37 ꢂC for 72 h. After incubation, the growth of
amoeba in the plate was checked with a low power micro-
scope. The culture medium was removed by inverting the plate
and shaking gently. Plate was then immediately washed with
sodium chloride solution (0.9%) at 37 ꢂC. This procedure
was completed quickly and the plate was not allowed to
cool in order to prevent the detachment of amoebae. The plate
was allowed to dry at room temperature and the amoebae were
fixed with methanol and when dried, stained with (0.5%) aque-
ous eosin for 15 min. The stained plate was washed once with
tap water, then twice with distilled water and allowed to dry. A
200 ml portion of 0.1 N sodium hydroxide solution was added
to each well to dissolve the protein and release the dye. The
optical density of the resulting solution in each well was deter-
mined at 490 nm with a microplate reader. The % inhibition of
amoebal growth was calculated from the optical densities of
the control and test wells and plotted against the logarithm
of the dose of the drug tested. Linear regression analysis
was used to determine the best fitting line from which the
IC50 value was found. The IC50 values in mg/ml and mM/ml
are reported in Table 1 and Table 2, respectively.
were dissolved with acid isopropanol and the absorbance
measured at 570 nm. All assays were performed in triplicate.
Percent viability was defined as the relative absorbance of
treated versus untreated control cells. Plates were analyzed in
an ELISA plate reader (Labsystems Multiskan RC, Helsinki,
Finland) at 570 nm with a reference wavelength of 655 nm.
Acknowledgements
This work was supported by Department of Science and
Technology (Grant no. Vll-PRDSF/44/2004-05/TT), New
Delhi, India.
References
[1] S.L. Stanly Jr., Lancet 361 (2003) 1025e1034.
[2] J. Acker, D. Mirelman, Exp. Parasitol. 110 (2005) 170e172.
[3] S. Campbell, Semin. Neurol. 13 (1993) 153e160.
[4] A.A. Shah, H. Shaikh, M. Karim, J. Neurol. Neurosurg. Psychiatr. 57
(1994) 240e241.
[5] K. Ohnishi, M. Murata, Epidemiol. Infect. 119 (1997) 363e367.
[6] C. Sundaram, B.C. Prasad, G. Bhaskar, V. Lakshmi, J.M. Murthy, J.
Assoc. Physicians India 52 (2004) 251e252.
[7] K. Ohnishi, M. Murata, H. Kojima, N. Takemura, T. Tsuchida,
H. Tachibana, Am. J. Trop. Med. Hyg. 51 (1994) 180e182.
[8] M.C. Conde-Bonfil, C. De la Mora-Zerpa, un desafio vigente, Salud
Publica Mex. 34 (1992) 335e341.
[9] R. Cedillo-Rivera, A. Tapia-Contreras, J. Torres, O. Munoz, Arch. Med.
Res. 28 (1997) 295e297.
[10] K. Kapoor, M. Chandra, D. Nag, J.K. Paliwal, R.C. Gupta, R.C. Saxena,
Int. J. Clin. Pharmacol. Res. 19 (1999) 83e88.
[11] S.C. Swetman, Martindale, The Complete Drug Reference, 33rd ed.
Pharmaceutical Press, London, 2002, p. 594.
[12] A. Saxena, S. Chugh, V.K. Vinayak, Indian J. Med. Res. 81 (1985)
387e390.
[13] M. Foley, L. Tilley, Pharmacol. Ther. 79 (1998) 55e87.
[14] E. Orozco, C. Lopez, C. Gomez, D.G. Perez, L. Marchat, C. Banuelos,
D.M. Delgadillo, Parasitol. Int. 51 (2002) 353e359.
[15] N.A. Samarawickrema, D.M. Brown, J.A. Upcroft, N. Thammapalerd,
P. Upcroft, J. Antimicrob. Chemother. 40 (1997) 833e840.
[16] R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian Medicinal
Plants, C.S.I.R. Publication, New Delhi, 1956, p. 7.
[17] Y.R. Chadha, The Wealth of India: Raw Materials, vol. 1, C.S.I.R., New
Delhi, 1985, pp. 82e84.
[18] V.R.R. Kasinadhuni, G. Rajashekhar, R. Rajagopalan, V.M. Sharma,
C.V. Krishna, P. Sairam, G.S. Prasad, S. Sadhukhan, G.G. Rao, Fitotera-
pia 70 (1999) 93e95.
[19] M. Sabir, M.K. Razdan, Indian J. Physiol. Pharmacol. 14 (1970)
209e210.
MTT assay. H9c2 rat cardiac myoblasts were cultured and
maintained as monolayer in Dulbecco’s modified Eagle’s
medium (DMEM), high glucose, supplemented with 10% fetal
bovine serum (heat inactivated), 100 units/ml penicillin,
100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B, at
37 ꢂC in humidified incubator with 5% CO2 [33]. Cells were in-
cubated with different concentrations of metronidazole and
compounds 3b, c and e, for 48 h at 37 ꢂC in 5% CO2
humidified incubator together with untreated control sample.
At appropriate time points, cells were washed in PBS, treated
with 600 ml MTT solution (5 mg/ml, tetrazolium salt) and
incubated for 45 min at 37 ꢂC. After 45 min of incubation at
37 ꢂC, the cell supernatants were discarded, MTT crystals
[20] R.D. Gaur, Flora of District Garhwal, North West Himalaya (with Eth-
nobotanical Notes), Transmedia Bhandari Bag, Srinagar Uttrakhand,
1999.
[21] Y. Asheervadam, P.S. Rao, R.D.H. Murray, Fitoterapia
231e234.
[22] M.I. Nabila, Phosphorus Sulfur Silicon 181 (2006) 1773e1784.
4 (1986)
[23] F. Dall’Acqua, D. Vedaldi, F. Bordin, F. Baccichetti, F. Carlassare,
M. Tamaro, P. Rodighiero, G. Pastrorini, A. Guiotto, G. Recchia,
M. Cristofolini, J. Med. Chem. 26 (1983) 870e876.
[24] D.G. O’Sullivan, P.W. Sadler, C. Webley, Chemotherapia 7 (1963)
17e24.
[25] A. Kotali, I.S. Lafazanis, P.A. Harris, Tetrahedron Lett. 48 (2007)
7181e7183.
[26] S. Singh, K. husain, F. Athar, A. Azam, Eur. J. Pharm. Sci. 25 (2005)
255e262.

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 2252e2259
2259
[27] A. Budakoti, M. Abid, A. Azam, Eur. J. Med. Chem. 41 (2006) 63e70.
[28] N. Bharti, Shailendra, S. Sharma, F. Naqvi, A. Azam, Bioorg. Med.
Chem. 11 (2003) 2923e2929.
[31] F.D. Gillin, D.S. Reiner, M. Suffness, Antimicrob. Agents Chemother. 22
(1982) 342e345.
[32] A.T. Keene, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta Med. 52
(1986) 278e284.
[29] L.S. Diamond, D.R. Harlow, C.C.R. Cunnick, Trans. R. Soc. Trop. Med.
Hyg. 72 (1978) 431e432.
[30] C.W. Wright, M.J. O’Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob.
Agents Chemother. 32 (1988) 1725e1729.
[33] M.K. Gupta, T.V. Neelakantan, M. Sanghamitra, R.K. Tyagi, A. Dinda,
S. Mualik, C.K. Mukhopadhyay, S.K. Goswami, Antioxid Redox Signal
8 (2006) 1081e1093.