synthesis and biological activities of some benzofuran...
TRANSCRIPT
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
138
Synthesis and biological activities of some benzofuran
derivatives with 4-thiazolidinone moiety
A B S T R A C T
Benzofurans are very interesting heterocycles, which are available in
nature and show a wide range of pharmacological activities. Hence, different
benzofurans having 4-thiazolidinones have been synthesized for the purpose
of pharmacological evaluation. The desired compounds were prepared using
simple starting materials like salicylaldehyde and ethyl bromoacetate. The
reaction between these compounds in the presence of potassium carbonate
yielded the ethyl benzofuran-2-carboxylate. This on reaction with hydrazine
hydrate resulted in the formation of the corresponding hydrazide. Further, on
condensation with different aldehydes gave the corresponding schiff bases.
Then, these schiff bases on condensation and cyclization with mercapto acetic
acid in presence of anhydrous ZnCl2 yielded the corresponding
4-thiazolidinone derivatives. These compounds have been characterized by
spectral methods. All the newly synthesized compounds have been screened for
biological activities viz., anti-inflammatory, analgesic and anti-microbial.
Finally, the interaction of the most active compound with HSA has been
investigated.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
139
INTRODUCTION
Natural products play important roles in both drug discovery and chemical
biology. In fact, many approved therapeutics as well as drug candidates are
derived from natural sources [1, 2]. Benzofurans, often found in naturally
occurring and synthetic compounds, are attractive to chemists for their
biological activities and roles in plant defense systems. The hydroxylated
benzofuran cicerfuran (I) was first obtained from the roots of a wild species of
chickpea, Cicer bijugum, and reported to be a major factor in the defense
system against Fusarium wilt.
These benzofurans belong to one of the most studied structural units in both
synthetic and medicinal chemistry. They are widely rooted in many
biologically interesting natural products and synthetic analogues [3]. Some of
the compounds containing benzofuran moieties are known to act as analgesic
compounds (eg. BRL 37959, II) with low gastric irritancy and some others as
potential anti-cancer agents (III and IV) [4].
Benzofuran skeletons are the common motifs in natural products,
agrochemicals and pharmaceuticals [5]. Literature survey has revealed that
much effort has been devoted to the synthesis of 2-substituted and
2,3-disubstituted benzofurans, but the synthetic route for the substitution only
I
III
IV
II
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
140
at 3rd
position of benzofuran ring has not been explored much. Substituted
benzofurans find applications as anti-oxidants, brightening agents and in a
variety of fields of chemistry and agriculture [6]. Some of the benzofurans are
also used in the treatment of asthama, rheumatism and ulcers [7].
Thiazolidinones are the derivatives of thiazolidine which belong to an
important class of heterocyclic compounds containing sulfur and nitrogen in a
five membered ring with a carbonyl group. A large amount of research work
on thiazolidinones has been carried out in the past. The nucleus is known to be
important since it gives derivatives of different biological activities [8]. The
structures of 4-thiazolidinones are widely studied for their pharmacological
properties [9, 10]. These derivatives are well-known for their
antimycobacterial (V) [11, 12] and anti-fungal (VI) [13, 14] activities.
Some other 4-thiazolidinone derivatives are useful for their
anti-tuberculosis (V, VII) [11, 15], anti-inflammatory (VIII, IX) [16, 17] and
anti-HIV (X, XI, XII) [18, 19] activities.
V
VI
VII
VIII
IX
X
XI
XII
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
141
The imino group (C=N), containing compounds typically known as schiff
bases, form a significant class of compounds in medicinal and pharmaceutical
chemistry with several biological applications [20]. Schiff bases also play a
major role in bioinorganic chemistry as they exhibit remarkable biological
activities. These are useful due to their pharmacological applications. They
find applications in biology and many of their ramifications have been used as
antifebrile, pain and anti-rheumatism agents [21]. Schiff bases show good
anti-microbial and pharmacological activities [22]. In recent years, schiff bases
are reported to exhibit broad-spectrum of chemotherapeutic properties such as
anti-viral (XIII) [23, 24], anti-tubercular (XIV) [25, 26], antifungal (XV) [27]
and antibacterial activities (XVI) [28, 29].
In view of the above, we have planned to synthesize some schiff bases of
benzofuran and the benzofuran derivatives containing thiazolidinone moiety.
The newly synthesized compounds were characterized by IR, NMR and mass
spectral data and tested for their pharmacological activities viz.,
anti-inflammatory and analgesic and anti-microbial activities like antibacterial
XV
XIII
XIV
XVI
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
142
and anti-fungal. The compound 5a has shown good anti-inflammatory activity.
So, this compound has been selected to carryout the binding studies with
protein (HSA) at different temperatures (ie., at 295, 306, and 313 K) by
fluorescence technique.
EXPERIMENTAL
General
Melting points were determined in open capillaries and are uncorrected.
The chemical shifts in NMR spectra are reported in parts per million (ppm, δ)
and peak multiplicities are designated as: s for singlet; d for doublet; dd for
doublet of doublet; t for triplet and m for multiplet. TLC was performed on
precoated sheets of silica gel 60 F254. The synthetic route for the synthesis of
schiff bases and thiazolidinones is outlined in Scheme 1.
Scheme 1
5, 6 ― R
a ― CH3
b
c
d
e
f
g
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
143
Procedures
Synthesis of ethyl benzofuran-2-carboxylate (3) [30]
The mixture of salicylaldehyde (3.5 g, 0.029 M), K2CO3 (7.5 g, 0.054 M),
and ethyl bromoacetate (7.64 g, 0.046 M) in ethyl alcohol (15 mL) was
refluxed for 6 h. After the completion of the reaction, the reaction mixture was
diluted with ethyl acetate, washed with water, 1N NaOH and brine, dried over
anhydrous sodium sulphate and concentrated under reduced pressure to get the
title compound as colourless oil. Yield: 81%, BP: 276-278 0C.
Synthesis of benzofuran-2-carbohydrazide (4) [31]
The mixture of hydrazine hydrate (15 mL) and ethyl benzofuran-2-
carboxylate (0.01 M) in ethanol was stirred at 0-5 0C for 30 min. The reaction
was monitored by TLC (hexane:ethyl acetate, 4:6). Then, the reaction mixture
was stirred at room temperature to obtain benzofuran-2-carbohydrazide as a
colourless solid product. This was filtered and washed with ethanol and dried.
Yield: 78%, MP: 190-192 0C.
General procedure for the synthesis of schiff base (5a-g) [32]
The mixture containing compound (4) and different aldehydes was refluxed
in ethyl alcohol for about 2 h in the presence of a catalytic amount of
hydrochloric acid. The separated solid product (schiff base) was filtered,
washed with ethanol and dried.
General procedure for the synthesis of N-(4-oxothiazolidin-3-
yl)benzofuran-2-carboxamide derivatives (6a-g)
An equimolar mixture of schiff bases (4a-e) and thioacetic acid, and the
catalytic amount of anhydrous ZnCl2 was taken in DMF and refluxed for 6-8 h.
The excess DMF was distilled off and the reaction mixture was poured into ice
cold water. Then, the separated solid was filtered and dried to obtain
N-(4-oxothiazolidin-3-yl) benzofuran-2-carboxamide derivatives.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
144
RESULTS AND DISCUSSION
Salicylaldehyde and ethyl bromoacetate were used as starting compounds
for the synthesis of ethyl benzofuran-2-carboxylate (3) following the reported
method [30]. Then, the benzofurn-2-carbohydrazide (4) was prepared by
stirring the mixture of ethyl benzofuran-2-carboxylate and hydrazine hydrate
for about 30 min [31]. Then, the condensation of benzofuran-2-carbohydrazide
with different aldehydes furnished the different schiff bases (5a-g) [32], which
upon reaction with thioacetic acid in the presence of catalytic amount of zinc
chloride yielded N-(4-oxothiazolidin-3-yl)benzofuran-2-carboxamide
derivatives (6a-g). The purity of the synthesized compounds was checked by
TLC and elemental analyses (Table 1) and the compounds of this study were
characterized by IR, NMR and mass spectral data.
In IR spectra of compounds 5a-g, the absorption band at 3300 cm-1
due to
-NH2 group was found to be absent. Further, the presence of an absorption
band at ~ 1600 cm-1
due to C=N group indicated the formation of schiff bases
(5a-g). Absence of 1H NMR peaks at ~ δ 2.0 ppm due to -NH2 group also
confirmed this. The IR spectra of compounds, 6a-g showed an absorption band
at ~ 1700 cm-1
due to C=O group. 1H NMR spectra of 6a-g exhibited the
downfield singlet ~ δ 8.2 ppm onwards indicating the presence of azomethane
protons. 6a-g also exhibited a singlet at ~ δ 4.0 ppm due to (S-CH2-C) and
another singlet at ~ δ 6.0 ppm due to (N-CH-S) thereby revealing the formation
of 1,3-thiazolidin-4-one ring. All the newly synthesized compounds have
shown the molecular ion peaks at the expected molecular weight in their mass
spectra. The IR, NMR and mass spectra of some representative compounds are
given in Fig. numbers 1-16. The Physical properties and CHN analysis data
(both calculated and found) are given in Table 1. The important IR, 1H NMR
and mass spectral data of newly synthesized compounds are given below:
IR, 1H NMR and mass Spectral interpretation
(E)-N'-ethylidenebenzofuran-2-carbohydrazide (5a).
IR (KBr, cm-1
): 3318 (-NH stretching for amide group), 1656 (-C=O stretching
for amide carbonyl), 1578 (-C=N stretching), 2892 (Alkyl CH stretching).
1H NMR (300 MHz CDCl3, δ ppm): 1.15 (d, CH3), 9.90 (q, CH), 7.00-7.60
(m, Ar), 8.04 (s, NH). m/z: 203 (M+1).
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
145
(E)-N'-benzylidenebenzofuran-2-carbohydrazide (5b).
IR (KBr, cm-1
): 3380 (-NH stretching for amide group), 1674 (-C=O stretching
for amide carbonyl), 1566 (-C=N stretching), 2882 (Alkyl CH stretching).
1H NMR (300 MHz CDCl3, δ ppm): 7.00-7.60 (m, Ar), 8.18 (s, NH), 8.74
(s, CH). m/z: 265 (M+1).
(E)-N'-((E)-3-phenylallylidene)benzofuran-2-carbohydrazide (5c).
IR (KBr, cm-1
): 3338 (-NH stretching for amide group), 1671 (-C=O stretching
for amide carbonyl), 1568 (-C=N stretching), 1628 (C=C stretching). 1H NMR
(300 MHz CDCl3, δ ppm): 6.71 (d, CH), 7.02 (s, CH), 7.17 (d, CH), 7.81
(s, NH), 7.40-8.50 (m, Ar). m/z: 290 (M+).
(E)-N'-(3-hydroxybenzylidene)benzofuran-2-carbohydrazide (5d).
IR (KBr, cm-1
): 3372 (-NH stretching for amide group), 1670 (-C=O stretching
for amide carbonyl), 1560 (-C=N stretching), 3408 (Phenol O-H stretching).
1H NMR (300 MHz CDCl3, δ ppm): 5.11 (s, OH), 7.00-7.50 (m, Ar), 8.11
(s, NH), 8.78 (s, CH). m/z: 281 (M+1).
(E)-N'-(3,4-dimethoxybenzylidene)benzofuran-2-carbohydrazide (5e).
IR (KBr, cm-1
): 3328 (-NH stretching for amide group), 1692 (-C=O stretching
for amide carbonyl), 1556 (-C=N stretching), 2886 (Alkyl CH stretching).
1H NMR (300 MHz DMSO-d6, δ ppm): 3.79 (s, CH3), 6.90-8.00 (m, Ar), 8.11
(s, NH), 9.72 (s, CH). m/z: 325 (M+1).
(E)-N'-(4-methoxybenzylidene)benzofuran-2-carbohydrazide (5f).
IR (KBr, cm-1
): 3342 (-NH stretching for amide group), 1678 (-C=O stretching
for amide carbonyl), 1562 (-C=N stretching), 2893 (Alkyl CH stretching).
1H NMR (300 MHz DMSO-d6, δ ppm): 3.91 (s, CH3), 7.00-7.70 (m, Ar), 8.08
(s, NH), 8.86 (s, CH). m/z: 294 (M+).
(E)-N'-(4-hydroxy-3-methoxybenzylidene)benzofuran-2-carbohydrazide
(5g).
IR (KBr, cm-1
): 3351 (-NH stretching for amide group), 1675 (-C=O stretching
for amide carbonyl), 1558 (-C=N stretching), 3380 (Phenol OH stretching),
2886 (Alkyl CH Stretching). 1H NMR (300 MHz DMSO-d6, δ ppm): 3.69
(s, CH3), 5.28 (s, OH), 7.00-7.80 (m, Ar), 7.97 (s, NH), 8.54 (s, CH). m/z: 311
(M+1).
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
146
N-(2-methyl-4-oxothiazolidin-3-yl)benzofuran-2-carboxamide (6a).
IR (KBr, cm-1
): 3342 (-NH stretching for amide group), 1668 (-C=O stretching
for amide carbonyl), 2878 (Alkyl CH stretching). 1H NMR (300 MHz
DMSO-d6, δ ppm): 1.43 (d, CH3), 3.66 (s, CH2), 4.45 (q, CH), 7.00-7.40
(m, Ar), 8.28 (s, NH). m/z: 276 (M+).
N-(4-oxo-2-phenylthiazolidin-3-yl)benzofuran-2-carboxamide (6b).
IR (KBr, cm-1
): 3354 (-NH stretching for amide group), 1685 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ / ppm): 3.66 (s, CH2), 5.11
(s, CH), 7.00-8.30 (m, Ar), 8.74 (s, NH). m/z: 339 (M+1).
(E)-N-(4-oxo-2-styrylthiazolidin-3-yl)benzofuran-2-carboxamide (6c).
IR (KBr, cm-1
): 3362 (-NH stretching for amide group), 1665 (-C=O stretching
for amide carbonyl), 1634 (C=C stretching). 1H NMR (300 MHz CDCl3,
δ ppm): 4.22 (s, CH2), 5.18 (d, CH), 6.32 (d, Th CH), 7.00 (dd, CH), 7.00-8.00
(m, Ar), 8.72 (s, NH). m/z: 364 (M+).
N-(2-(2-hydroxyphenyl)-4-oxothiazolidin-3-yl)benzofuran-2-carboxamide
(6d).
IR (KBr, cm-1
): 3361 (-NH stretching for amide group), 1681 (-C=O stretching
for amide carbonyl), 3379 (Phenol OH stretching). 1H NMR (300 MHz CDCl3,
δ ppm): 3.74 (s, CH2), 4.88 (s, OH), 5.41 (s, CH), 7.00-8.40 (m, Ar), 8.79
(s, NH). m/z: 355 (M+1).
N-(2-(3,4-dimethoxyphenyl)-4-oxothiazolidin-3-yl)benzofuran-2-
carboxamide (6e).
IR (KBr, cm-1
): 3367 (-NH stretching for amide group), 1674 (-C=O stretching
for amide carbonyl), 2879 (Alkyl CH Stretching). 1H NMR (300 MHz CDCl3,
δ ppm): 3.78 (s, CH2), 4.08 (s, CH3), 5.72 (s, CH), 6.90-8.00 (m, Ar), 8.72
(s, NH). m/z: 399 (M+1).
N-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)benzofuran-2-carboxamide
(6f).
IR (KBr, cm-1
): 3359 (-NH stretching for amide group), 1678 (-C=O stretching
for amide carbonyl), 2886 (Alkyl CH stretching). 1H NMR (300 MHz
DMSO-d6, δ ppm): 3.71 (s, CH2), 4.15 (s, CH3), 5.41 (s, CH), 7.00-8.10
(m, Ar), 8.57 (s, NH). m/z: 369 (M+1).
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
147
N-(2-(4-hydroxy-3-methoxyphenyl)-4-oxothiazolidin-3-yl)benzofuran-2-
carboxamide (6g).
IR (KBr, cm-1
): 3364 (-NH stretching for amide group), 1682 (-C=O stretching
for amide carbonyl), 3381 (Phenol OH stretching), 2891 (Alkyl CH stretching).
1H NMR (300 MHz DMSO-d6, δ ppm): 3.82 (s, CH2), 4.02 (s, CH3), 5.41
(s, OH), 5.82 (s, CH) 7.00-8.00 (m, Ar), 8.22 (s, NH). m/z: 385 (M+1).
BIOLOGICAL ACTIVITIES (In vivo models)
Anti-inflammatory activity
The newly synthesized compounds (5a-g and 6a-g) were screened for their
anti-inflammatory activity by carrageenin induced rat paw oedema method
[33]. For this, the rats were weighed, numbered and divided into control,
standard, and different test groups consisting of six animals in each group. All
the animals were made to fast overnight with free access to water before
experiment. In all groups, acute inflammation was produced by subplanter
injection of 0.1 mL of freshly prepared 1% suspension of carrageenin in the
right hind paw of rats and paw volume was measured plethysmometrically at 0
h and 6 h after administering carrageenin injection. The test compounds (100
mg/kg body weight) were administered orally while standard group was treated
with indomethacin (20 mg/kg body weight) orally 1 h before by injection and
control group received only vehicle. Mean difference in paw volume was
measured (typical experimental set up is shown below) and percentage
inhibition was calculated using the following equation:
% Inhibition of oedema = Vc – Vt
x 100 Vc
where Vt is the mean paw volume of test group and Vc is the mean paw volume
of control group.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
148
Among the compounds tested for their anti-inflammatory activity, all the
compounds exhibited marked anti-inflammatory activity when compared to
control group. The compound 5a, which is a schiff base exhibited promising
activity while the remaining compounds have shown moderate to low
anti-inflammatory activity compared with the standard drug, indomethacin. In
this activity, the standard drug and other test compounds have shown their
actions in the first half an hour. The results of anti-inflammatory activity are
given in Table 2.
Analgesic activity
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
149
The newly synthesized compounds (5a-g and 6a-g) were screened for their
analgesic activity by tail flick method in rats [33]. The typical experimental set
up used is shown above. The reaction time was measured at the end of 0, 30,
60, 120 and 240 min after the administration of the compound and the standard
drug, tramadol hydrochloride.
The animals were weighed, numbered and divided into different groups, like
control, standard and test groups containing six animals in each group. The
animals were made to fast overnight with free access of water before
experiment. The basal reaction time to the radiant heat for the selected animals
was checked by placing the tip (last 1-2 cm) of the tail on the radiant heat
source. The tail withdrawal time from the radiant heat (flicking response) was
taken as the end point. The test compounds (100 mg/kg body weight) and
standard drug (20 mg/kg body weight) were administered orally while the
control group received vehicle only. Then the reaction time (flicking response
time) was noted at different time intervals from 0 to 240 min. As the reaction
time reached 10 s, it was considered maximum analgesia and the tail was
removed from the source of heat to avoid the tissue damage. The percentage of
increase in reaction time (index of analgesia) at each time interval was
calculated.
The results (Table 3) of the screening of the analgesic activity revealed that
all the tested compounds exhibited moderate to good analgesic activity
compared to that of the reference drug. The compound, 5c (at 60th
min)
exhibited the most significant analgesic activity; the compounds, 5a, 5d and 6c
showed the significant activity; the compound, 5e displayed moderately
significant activity while the rest exhibited low activity.
Anti-microbial activity
The compounds (5a-g and 6a-g) were also tested for their anti-microbial
activities like anti-bacterial and anti-fungal [34, 35] by agar diffusion methods.
Both anti-bacterial and anti-fungal activities were examined at different
concentrations ranging from 25 to 800 µg and identified the MIC.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
150
Microbial cultures of bacteria; Escherichia coli (E. coli), Staphylococcus
aureus (S. aureus), Bacillus subtilis (B. subtilis), Salmonella typhi (S. typhi),
and fungus; Aspergillus niger (A. niger), Candida albicans (C. albicans),
Aspergillus flavus (A. flavus), Neurospora crassa (N. crassa) were obtained
from Imtech, Chandigarh and were used for their respective anti-microbial
activities. All the bacteria were maintained in NB media at 37 0C and all the
fungi were maintained in PDA media at 28 0C.
1. Anti-bacterial studies
The media used for this activity is NB which contained 10 g peptone, 10 g
sodium chloride, 5 g yeast extract and 20 g agar. All these are added to
1000 mL of distilled water and prepared the medium. Initially, the stock
cultures of bacteria were revived by inoculating in broth media and grown at
37 0C for 18 h. The agar plates containing the above media were prepared and
wells were made in the plates. Each plate was inoculated with 18 h old cultures
(100 μl, 10-4
CFU) and spread evenly on the plate. After 20 min, the wells
were filled with the test compounds of different concentrations. The control
wells with gentamycin were also prepared. All the plates were incubated at
37 0C for 24 h and the diameter of inhibition zone was recorded. The results of
MIC are given in Table 4.
The results of anti-bacterial studies revealed that all the tested compounds
exhibited moderate to good anti-bacterial activity against all the tested bacterial
strains compared with that of gentamycin. The compound 6c exhibited very
good activity against the bacteria B. subtilis at a MIC of 25 µg and has shown
good activity against the remaining three microorganisms (E. coli, S. aureus
and S. typhi) at a MIC of 100 µg, whereas the moderate activity was observed
for the compounds 5e and 5g against E. coli; 5e, 6b and 6d against B. subtilis,
and 5e and 6f against S. typhi with a MIC of 400 µg. The compounds, 5c and
6f against E. coli, the compounds 5a, 5c, 5e, 6a, 6e and 6f against S. aureus,
the compounds 5a-d, 5f, 6a and 6e against B. subtilis and the compounds 5b,
5c and 6d against S. typhi have shown low activity at a MIC of 800 µg. Rest of
the compounds have shown no activity against all the four organisms.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
151
2. Anti-fungal studies
The PDA was prepared by boiling 250 g of peeled potato (20 min). Later
this was squeezed and filtered. To this filtrate, 20 g dextrose was added and the
volume was made up to 1000 ml with distilled water.
Initially, the stock cultures of fungi were revived by inoculating in broth
media and grown at 27 0C for 48 h. The agar plates of the above media were
prepared and wells were made in the plates. Each plate was inoculated with
48 h old cultures (100 μl, 10-4
CFU) and spread evenly on plates. After 20 min,
the wells were filled with test compounds of different concentrations. The
control plates with antibiotic (amphotericin) were also prepared. All the plates
were incubated at 27 0C for 48 h and the diameter of inhibition zone were
noted. The results of MIC are given in Table 5. The results of anti-fungal
activity revealed that all the tested compounds showed the moderate activity
against all the tested fungi compared to that of amphotericin.
The compounds 5e and 6b have shown moderate activity at a MIC of
400 µg and the compounds 5b, 5d, 6b, 6d, 6e and 6g have shown least activity
at a MIC of 800 µg, whereas the remaining compounds did not show the
activity (> 800 µg) against the fungi C. albicans. The compounds 6c, 6d and
6f have shown moderate activity at a MIC of 400 µg and the compounds 5e, 5f,
6a, 6b and 6e have exhibited least activity at a MIC of 800 µg, whereas the
remaining compounds have not shown the activity (> 800 µg) against the fungi
A. niger. The compound 6c has shown very good activity (MIC 400 µg,
standard also) and the compounds 5e, 6b, 6e and 6f exhibited moderate activity
at a MIC of 800 µg, while the remaining compounds showed low activity
(> 800 µg) against A. flavus. Finally, the compound 6c has shown very good
activity (MIC 400 µg, standard also) and the compounds 6b, 6d and 6f have
shown moderate activity at a MIC of 800 µg, while that of the remaining
compounds have shown lesser activity (> 800 µg) against the fungi N. cressa.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
152
Binding studies
Among the compounds screened for their biological activities, the
compound 5a has shown good anti-inflammatory activity. Hence, the binding
studies of this compound with HSA have been carried out using fluorescence
technique. For this, we have recorded the fluorescence intensity of HSA in the
presence and absence of 5a (Fig. 17). It was evident from the figure that the
compound 5a has quenched the fluorescence of HSA. From this quenching
data, the binding parameters viz.,Stern-Volmer quenching constant (KSV),
binding constant (K) and the number of molecules that bind to protein (n) at
different temperatures (viz., 295, 306 and 313 K) have been evaluated. The
values of KSV are given in Table 6. It was noticed that these values increased
with increase in temperature indicating the presence of dynamic type of
quenching mechanism in the interaction process.
Using equation 4 (Page number 33), the values of K and n were obtained
and are shown in Table 6. The values K (of the order of 106) indicated that
there was strong binding between 5a and HSA. The values of n close to unity
indicated that the binding ratio between 5a and HSA was 1:1.
In order to know the binding forces that are operating between 5a and HSA,
the thermodynamic parameters viz., change in enthalpy (ΔH0), change in
entropy (ΔS0) and Gibb’s free energy change (ΔG
0) were determined and
presented in Table 6. In this case, both the values of ΔH0 and ΔS
0 were
noticed to be positive thereby revealing that the hydrophobic forces played a
major role in the interaction process of 5a-HSA system.
The binding site for 5a on protein was located based on displacement
experiments using different site markers such as phenyl butazone, ibuprofen
and digitoxin for site I, II and III, respectively. The binding constant values of
5a-HSA system were calculated in the presence of these site probes and found
to be 4.34 x 105, 5.02 x 10
6 and 4.99 x 10
6 M
-1, respectively. Significant
decrease in the binding constant value was noticed in the presence of phenyl
butazone while it remained almost same to that of 5a-HSA system
(5.01 x 106 M
-1) in the absence of any site probe. This revealed that the site I
located in hydrophobic pocket of the sub-domain IIA was the main binding site
for 5a on protein.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
153
CONCLUSIONS
Some benzofuran derivatives consisting of thiazolidinone moieties have
been synthesized for the first time and characterized by IR, NMR and mass
spectral data. The compounds were subjected to biological activities like
anti-inflammatory, analgesic and anti-microbial. Among all the compounds, 5a
has shown good anti-inflammatory activity while the compounds 5c and 5e
have exhibited good analgesic activity. Binding of compound 5a with HSA
has also been investigated and the results revealed the presence of dynamic
quenching mechanism.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
154
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Chapter 7 (Sec. A) Benzofurans with thiazolidinones
156
Fig. 1. IR Spectrum of 5a.
Fig. 2. IR spectrum of 5d.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
157
Fig. 3. IR Spectrum of 6c.
Fig. 4. IR spectrum of 6f.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
158
Fig. 5. 1H NMR Spectrum of 5b.
Fig. 6. 1H NMR Spectrum of 5d.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
159
Fig. 7. 1H NMR Spectrum of 5e.
Fig. 8. 1H NMR Spectrum of 6a.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
160
Fig. 9. 1H NMR Spectrum of 6b.
Fig. 10. 1H NMR Spectrum of 6c.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
161
Fig. 11. 1H NMR Spectrum of 6d.
Fig. 12. 1H NMR Spectrum of 6e.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
162
Fig. 13. 1H NMR Spectrum of 6f.
Fig. 14. Mass spectrum of 5a.
O
HN
O
NS
O
OH3C
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
163
Fig. 15. Mass Spectrum of 6a.
Fig. 16. Mass Spectrum of 6c.
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
164
0
50
100
150
200
290 310 330 350 370 390
Wavelength, nm
Inte
nsit
y
Fig. 17. Fluorescence spectra of (a) HSA (5 μM) in the presence of 5a.
5a concentration was kept at (1) 0, (2) 1.25, (3) 2.50, (4) 3.75, (5) 5.00,
(6) 6.25, (7) 7.50, (8) 8.75, (9) 10.00 and (10) 11.25 µM of 5a-HSA
system.
1
10
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
165
Table 1.
Physical and analytical data of the compounds, 5a-g and 6a-g.
Comp. Mol. formula
(MW) MP
(0C)
Yield
(%)
Calculated % (Found)
C H N
5a C11H10N2O2
(202.21) 184 86.96
65.34
(65.29)
4.98
(4.95)
13.85
(13.88)
5b C16H12N2O2
(264.28) 197 88.50
72.72
(72.73)
4.58
(4.62)
10.60
(10.55)
5c C18H14N2O2
(290.32) 228 76.82
74.47
(74.50)
4.86
(4.85)
9.65
(9.71)
5d C16H12N2O3
(280.28) 281 76.50
68.56
(68.51)
4.32
(4.30)
9.99
(9.97)
5e C18H16N2O4
(324.33) 293 65.25
66.66
(66.69)
4.97
(4.99)
8.64
(8.67)
5f C17H14N2O3
(294.30) 288 68.10
69.38
(69.41)
4.79
(4.85)
9.52
(9.51)
5g C17H14N2O4
(310.30) 306 68.65
65.80
(65.85)
4.55
(4.52)
9.03
(9.05)
6a C13H12N2O3S
(276.31) 259 72.12
56.51
(56.54)
4.38
(4.36)
10.14
(10.12)
6b C18H14N2O3S
(338.38) 297 68.40
63.89
(63.94)
4.17
(4.18)
8.28
(8.25)
6c C20H16N2O3S
(364.42) 316 62.85
65.92
(65.95)
4.43
(4.40)
7.69
(7.70)
6d C18H14N2O4S
(354.38) 322 65.35
61.01
(61.05)
3.98
(3.95)
7.90
(7.86)
6e C20H18N2O5S
(398.43) 338 58.90
60.29
(60.25)
4.55
(4.56)
7.03
(7.01)
6f C19H16N2O4S
(368.41) 331 58.50
61.94
(61.96)
4.38
(4.42)
7.60
(7.62)
6g C11H10N2O2
(384.41) 305 57.25
59.37
(59.39)
4.20
(4.26)
7.29
(7.26)
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
166
Table 2.
The results of anti-inflammatory activity of compounds, 5a-g and 6a-g.
Name of the
Compound
Dose
mg/kg
Percentage of inhibition
Time in min
0 30 60 120 240 360
Control -- -- -- -- -- -- --
Indomethacin 20 11.11 33.33 26.66 15.50 4.44 11.11
5a 100 00 44.44 44.44 88.88 33.33 11.11
5b 100 11.11 44.44 55.55 55.55 33.33 22.22
5c 100 4.44 22.22 22.22 22.22 6.66 17.77
5d 100 11.11 37.00 44.00 26.66 22.00 00
5e 100 00 55.55 33.33 6.66 4.44 00
5f 100 00 4.44 6.66 11.11 6.66 4.44
5g 100 6.66 12.26 22.22 22.22 11.11 4.44
6a 100 11.11 33.33 36.33 22.22 11.11 00
6b 100 00 4.44 6.66 11.11 6.66 4.44
6c 100 4.44 11.11 22.22 33.33 11.11 6.66
6d 100 4.44 6.66 6.66 11.11 4.44 00
6e 100 11.11 22.22 44.44 22.22 12.22 6.66
6f 100 6.66 11.11 11.11 22.22 6.66 4.44
6g 100 00 6.66 11.11 11.11 4.44 4.44
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
167
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
168
Table 4.
The MIC results (in µg) of anti-bacterial activity of compounds 5a-g and 6a-g.
Compound
Bacterial strains
E. coli S. aureus B. subtilis S. typhi
Gentamycin < 25 < 25 25 25
5a >800 800 800 >800
5b >800 >800 800 800
5c 800 800 800 800
5d >800 >800 800 >800
5e 400 800 400 400
5f >800 >800 800 >800
5g 400 >800 >800 >800
6a >800 800 800 >800
6b >800 >800 400 >800
6c 100 100 25 100
6d >800 >800 400 800
6e >800 800 800 >800
6f 800 800 >800 400
6g >800 >800 >800 >800
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
169
Table 5.
The MIC results (in µg) of anti-fungal activity of compounds 5a-g and 6a-g.
Compound
Fungi
C. albicans A. niger A. flavus N. cressa
Amphotericin 50 100 400 400
5a >800 >800 >800 >800
5b 800 >800 >800 >800
5c >800 >800 >800 >800
5d 800 >800 >800 >800
5e 400 800 800 >800
5f >800 800 >800 >800
5g >800 >800 >800 >800
6a >800 800 >800 >800
6b 800 800 800 800
6c 400 400 400 400
6d 800 400 >800 800
6e 800 800 800 >800
6f >800 400 800 800
6g 800 >800 >800 >800
Chapter 7 (Sec. A) Benzofurans with thiazolidinones
170
Table 6.
Binding and thermodynamic parameters for 5a-HSA system at different
temperatures.
Temp
(K)
KSV
(M-1
)
K
(M-1
) n
ΔG0
(kJ mol-1
)
ΔH0
(kJ mol-1
)
ΔS0
(J mol-1
K-1
)
295 1.75 x 105 1.74 x 10
6 1.21 - 35.24
70.11 357.21 306 2.05 x 105 5.01 x 10
6 1.29 - 39.25
313 2.82 x 105 8.93 x 10
6 1.31 - 41.66
Chapter 7 (Sec. B) Benzofurans with heterocycles
171
Synthesis, characterization, binding and biological activities of
benzofuran compounds containing heterocyclic moieties
A B S T R A C T
Some benzofuran derivatives containing thiazole and pyridine moieties
have been synthesized and characterized by IR, NMR and mass spectral
methods. These compounds were screened for biological activities. Further,
the mechanism of binding of the most active compound with HSA has been
studied.
Chapter 7 (Sec. B) Benzofurans with heterocycles
172
INTRODUCTION
Small and simple heteroaromatics often have surprisingly complex
biological properties and belong to one of the most important classes of
compounds in medicinal chemistry [1, 2]. For instance, amines containing five
membered heteroaryl groups such as furans, thiophenes, thiazoles, pyrazoles
etc are usually found in natural products and drugs [3-5]. Among these,
1, 3-thiazoles constitute an important class of S, N containing heterocycles [6].
Thiazole is an important scaffold in heterocyclic chemistry and 1,3-thiazole
ring is present in several pharmacologically active compounds [7-9]. Thiazole
derivatives find applications as bacteriostatics and antibiotics (I) [10-12].
Imidazo[2,1-b]thiazoles are reported to possess fungicidal and anti-histaminic
activities (II) [13]. Much interest in thiazoles and their derivatives is attributed
to their biological significance as constituents of biomolecules including
antibiotics (III) [14]. The thiazolyl group is also of great importance in
treating biological systems. Compounds of this functional group showed
anti-microbial [15, 16], anti-tumour [17, 18], analgesic [15, 16, 19],
anti-inflammatory and anti-pyretic [16, 20] properties. Further, some synthetic
thiazoles exhibited a wide range of biological activities such as anti-tumor,
anti-filarial (IV), anti-bacterial, anthelmintic, anti-fungal and anti-inflammatory
(V) [21].
Poly-substituted pyridines are the important class of compounds owing to
their abundance in biologically important natural compounds and their
usefulness as synthetic intermediates in organic synthesis [22]. Many naturally
I
II
III
IV
V
Chapter 7 (Sec. B) Benzofurans with heterocycles
173
occurring and synthetic compounds containing the pyridine scaffold possesses
interesting pharmacological properties [23]. Among these compounds,
2-amino-3-cyanopyridines have been identified as IKK-β-inhibitors (VI) [24].
Besides, they are important and useful intermediates in preparing variety of
heterocyclic compounds [25, 26]. Recently, several thieno[3,2-b]pyridines
have shown important biological activities like antitumor and antiangiogenesis
or dual activity, essentially by acting as inhibitors of tyrosine kinase receptors
VII [27], VIII [28] and IX [29] or non receptor X [30] which have been
implicated in the growth and progression of various human cancers, and,
therefore, have been crucial in the development of anticancer drugs.
Benzyl-alkyl-ammonium salts and pyridinium salts are the cationic
surfactants, which are most popularly used as disinfectants (XI, XII) [31, 32].
The pyridinium salts are effective against a number of microorganisms. These
are used to wound healing and in the treatment of urological infections (XI)
[31]. Cetylpyridinium chloride controls supragingival plaque and gingivitis
(XIII) [31, 33]. Moreover, it also has direct anti-inflammatory activity and
inhibits action on several matrix metalloproteinase proteins which cause
inflammation. The pyridine derivatives are also used as disinfectants for eating
and drinking utensils and food processing equipments. Moreover, their
anti-microbial activity has also been employed in dairy industry for sanitization
of milk cans and milk machines [34, 35]. 2-substituted-imidazo[4,5-
b]pyridines possess different chemical and pharmacological features (XIV)
VII
VIII
IX
X
VI
Chapter 7 (Sec. B) Benzofurans with heterocycles
174
[36, 37], which impart them diverse biological properties like anti-cancer (XV,
XVI, XVII) [38, 39], anti-viral (XVIII, XIX) [40, 41], anti-mitotic (XX) [42],
anti-inflammatory (XXI) [43] and tuberculostatic [44] activity.
In view of biological importance, we have synthesized some benzofuran
derivatives containing thiazole and pyridine moieties. The newly synthesized
compounds were characterized by IR, NMR and mass spectral data. Further,
these compounds were screened for anti-inflammatory, analgesic, anti-bacterial
and anti-fungal activities.
Synthesis of benzofuran derivatives containing thiazole and pyridine
moieties is shown in Schemes 1 and 2.
XI
XII
XIII XIV
XV
XVI
XVII
XVIII
XIX
XX
XXI
Chapter 7 (Sec. B) Benzofurans with heterocycles
175
Scheme 1
R1 = Br, Cl, NO2, CH3
R2 = Br, Cl, NO2, OCH3
Scheme 2
-R =
Chapter 7 (Sec. B) Benzofurans with heterocycles
176
EXPERIMENTAL
Procedures
Synthesis of ethyl benzofuran-2-carboxylate (3) [45]
Synthesis of the above title compound has been outlined in Section A on
page number 143.
Synthesis of benzofuran-2-carboxylic acid (4)
Ethyl benzofuran-2-carboxylate (5.7 g, 0.03 M) in ethyl alcohol (20 mL)
was refluxed for 3 h in the presence of dilute hydrochloric acid to get
benzofuran-2-carboxylic acid as a white coloured solid. This compound was
filtered and washed with ethyl alcohol and dried. Yield: 86%, MP: 138-140 0C.
Synthesis of benzofuran-2-carbonyl chloride (5)
The mixture of benzofuran-2-carboxylic acid (1 eq) and thionyl chloride
(2.5 eq) were heated for about 2-3 h in an oil bath at about 100 0C. After
completion of the reaction, the excess thionyl chloride was removed under
vacuum (below 55 0C) to get the required benzofuran-2-carbonyl chloride.
This was further used for coupling it with different amines.
General procedure for coupling of benzofuran-2-carbonyl chloride with
different amines (6a-l)
Sodium hydride (1 eq) is activated by washing it with hexane twice.
Then, the activated sodium hydride was stirred at room temperature in a freshly
dried THF for about 15-20 min. Later, different heterocyclic amines were
added to the above suspension and stirred it again for about 15-20 min at room
temperature. Then, the benzofuran-2-carbonyl chloride which was prepared in
the earlier stage was added slowly by maintaining the reaction mixture at about
10-15 0C. The contents were stirred for about 2-3 h at room temperature.
Chapter 7 (Sec. B) Benzofurans with heterocycles
177
General procedure for the preparation of different substituted heterocyclic
amines (a-j).
The different substituted heterocyclic amines were prepared by the
addition of resublimed iodine (1 eq) to different substituted acetophenones and
thiourea (2 eq). Then, the mixture was heated overnight in an oil bath at
100 0C. After cooling, the reaction mixture was triturated with diethyl ether
(ca. 50 mL) to remove any unreacted iodine and acetophenone. The solid
residue was put in cold distilled water (200 mL) and treated with 25% aqueous
ammonium hydroxide (pH 9-10). The precipitated thiazole was collected and
purified by crystallization from hot ethanol.
RESULTS AND DISCUSSION
Salicylaldehyde and ethyl bromoacetate were used as starting materials for
the synthesis of ethyl benzofuran-2-carboxylate (3) by refluxing them in the
presence of potassium carbonate for 6 h following the reported method [45].
Then, compound (3) on refluxing in the presence of dilute hydrochloric acid for
about 3 h yielded white colored solid, benzofuran-2-carboxylic acid (4). This
compound on refluxing with thionyl chloride in dichloromethane gave the
corresponding acid chloride, benzofuran-2-carbonyl chloride (5). This on
condensation with different heterocyclic amines like 4-phenylthiazol-2-amine
derivatives and amino pyridines yielded the expected final compounds (6a-l).
4-phenylthiazol-2-amine derivatives were synthesized by the reaction of
different acetophenones with thiourea in the presence of sublimed iodine at
100 0C on an oil bath, overnight, by following the reported method [46]. The
progress and completion of all the reactions was monitored by TLC. Further,
the purity of the synthesized compounds was examined by TLC and elemental
analyses (Table 1) and the compounds were confirmed by IR, NMR and mass
spectral data.
The IR spectra of compounds 6a-l showed the presence of bands at ~ 3400
and 1700 cm-1
which are attributed to -NH and -C=O group of amide bond
Chapter 7 (Sec. B) Benzofurans with heterocycles
178
respectively, confirming the formation of amide bond in compounds 6a-l.
Further, a broad band at ~ 3000 cm-1
which corresponds to -OH group of
benzofuran-2-carboxylic acid was found to be absent in IR spectra of
compounds 6a-l. This revealed that the benzofuran-2-carboxylic acid was
converted into its acid chloride before the formation of title compounds 6a-l.
The 1H NMR spectra showed a downfield singlet at ~ δ 9.0 ppm which was
attributed to -NH protons. All the aromatic protons were observed at
~ δ 7.0-8.0 ppm. Further, the -OH proton at ~ δ 11.0 ppm were absent in
1H NMR spectra of compounds 6a-l which confirmed the coupling of
benzofuran-2-carboxylic acid with different substituted amines.
The IR, NMR and mass spectra of some representative compounds are
given in Fig. Nos. 1-12. The physical properties and CHN analysis data (both
calculated and found) are given in Table 1 and the IR, NMR and mass spectral
interpretation of newly synthesized molecule is given in detailed below:
IR, NMR and mass spectral data
N-(4-phenylthiazol-2-yl)benzofuran-2-carboxamide (6a).
IR (KBr, cm-1
): 3388 (-NH stretching for amide group), 1652 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 7.00-8.00 (m, Ar),
9.13 (s, NH). m/z: 320 (M+).
N-(4-(4-bromophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6b).
IR (KBr, cm-1
): 3380 (-NH stretching for amide group), 1694 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz DMSO-d6, δ ppm): 7.00-8.00 (m, Ar),
9.18 (s, NH). m/z: 398 (M+).
N-(4-(2-bromophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6c).
IR (KBr, cm-1
): 3358 (-NH stretching for amide group), 1691 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz DMSO-d6, δ ppm): 7.00-8.00 (m, Ar),
9.21 (s, NH). m/z: 398 (M+).
Chapter 7 (Sec. B) Benzofurans with heterocycles
179
N-(4-(4-chlorophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6d).
IR (KBr, cm-1
): 3378 (-NH stretching for amide group), 1690 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz DMSO-d6, δ ppm): 7.00-8.00 (m, Ar),
9.41 (s, NH). m/z: 355 (M+1).
N-(4-(2-chlorophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6e).
IR (KBr, cm-1
): 3348 (-NH stretching for amide group), 1696 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 7.00-8.00 (m, Ar),
9.06 (s, NH). m/z: 355 (M+1).
N-(4-(4-nitrophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6f).
IR (KBr, cm-1
): 3332 (-NH stretching for amide group), 1676 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz DMSO-d6, δ ppm): 7.00-8.00
(m, Ar), 9.26 (s, NH). m/z: 366 (M+1).
N-(4-(2-nitrophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6g).
IR (KBr, cm-1
): 3356 (-NH stretching for amide group), 1669 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz DMSO-d6, δ ppm): 7.00-8.00 (m, Ar),
9.34 (s, NH). m/z: 365 (M+).
N-(4-(4-methoxyphenyl)thiazol-2-yl)benzofuran-2-carboxamide (6h).
IR (KBr, cm-1
): 3332 (-NH stretching for amide group), 1662 (-C=O stretching
for amide carbonyl), 2873 (Alkyl CH Stretching). 1H NMR (300 MHz
DMSO-d6, δ ppm): 3.92 (S, CH3), 7.00-8.00 (m, Ar), 9.29 (s, NH). m/z: 351
(M+1).
N-(4-(m-tolyl)thiazol-2-yl)benzofuran-2-carboxamide (6i).
IR (KBr, cm-1
): 3359 (-NH stretching for amide group), 1689 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 2.16 (S, CH3),
7.00-8.00 (m, Ar), 9.12 (s, NH). m/z: 335 (M+1).
N-(4-(o-tolyl)thiazol-2-yl)benzofuran-2-carboxamide (6j).
IR (KBr, cm-1
): 3342 (-NH stretching for amide group), 1654 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 2.53 (S, CH3),
7.00-8.00 (m, Ar), 9.24 (s, NH). m/z: 335 (M+1).
N-(pyridin-2-yl)benzofuran-2-carboxamide (6k).
IR (KBr, cm-1
): 3369 (-NH stretching for amide group), 1671 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 7.00-8.00 (m, Ar),
9.17 (s, NH). m/z: 238 (M+).
Chapter 7 (Sec. B) Benzofurans with heterocycles
180
N-(pyridin-3-yl)benzofuran-2-carboxamide (6l).
IR (KBr, cm-1
): 3362 (-NH stretching for amide group), 1676 (-C=O stretching
for amide carbonyl). 1H NMR (300 MHz CDCl3, δ ppm): 7.00-8.00 (m, Ar),
8.83 (s, Py-H), 9.38 (s, NH). m/z: 239 (M+1).
BIOLOGICAL ACTIVITIES (In vivo models)
Anti-inflammatory activity
Anti-inflammatory activity of the newly synthesized compounds (6a-l) was
investigated by carrageenin induced rat paw oedema method [47] as outlined in
Section A (Page number 147). All the compounds have shown marked
anti-inflammatory activity compared to control group. Among these
compounds, the compound 6c has exhibited promising anti-inflammatory
activity compared to the standard drug, indomethacin. The compounds 6a, 6d,
6e and 6g have showed moderate activity while the remaining compounds
exhibited low anti-inflammatory activity compared to the standard drug. All
the compounds have initiated their action at 30th
min and the compounds, 6a,
6c, 6d, 6e and 6g have exhibited higher activity at 60th
min. The corresponding
results are presented in Table 2.
Analgesic activity
The newly synthesized compounds were screened for analgesic activity by
tail flick method [47] and the results are given in Table 3. All the compounds
exhibited comparable analgesic activity compared to the control group.
However, the compound, 6b has shown the most significant analgesic activity
at 30th
min. The compounds, 6a, 6d, 6e and 6j have exhibited the significant
analgesic activity compared to the standard drug, tramadol hydrochloride.
Anti-microbial study
Anti-microbial (bacterial and fungal) activity of all the newly synthesized
compounds (6a-l) was analysed by cup plate method [48, 49]. These activities
Chapter 7 (Sec. B) Benzofurans with heterocycles
181
were analysed using six different concentrations (ranging from 25 to 800 µg)
and estimated the MIC for these compounds. The microbial cultures like
bacterial and fungal strains used in the study are given in Section A of this
chapter. These bacterial cultures were maintained in NB media at 37 0C and all
the fungal cultures were maintained in PDA media at 28 0C.
1. Anti-bacterial activity
The anti-bacterial activity was carried out following the procedure
mentioned in Section A of this chapter. The results of the activity are given in
Table 4. The results indicated that all the compounds showed higher activity.
Among all the compounds, the compound 6j (against the bacteria E. coli), the
compounds 6a, 6e, 6g, 6j and 6k (against S. aureus), the compounds 6e, 6i and
6j (against B. subtilis) and 6g (against S. typhi) exhibited moderate
anti-bacterial activity compared to the standard drug, gentamycin with MIC
values of 400 µg. However, rest of the compounds showed low activity.
2. Anti-fungal activity
The agar diffusion method was used to screen the anti-fungal activity of
newly synthesized compounds (Procedure is given in Section A). The MIC
results of the anti-fungal activity are shown in Table 5. It was noticed from the
Table that the compound, 6j exhibited better anti-fungal activity against the
fungi N. cressa whereas the compounds 6g (against A. albicans), 6a, 6c, 6e
and 6k (against A. niger) and the compound, 6f (against A. flavus) exhibited
moderate anti-fungal activity. The rest of the compounds showed low activity
against all the four fungal strains. These results were compared with the
standard anti-fungal drug, amphotericin.
Binding studies
Since the compound 6c exhibited good anti-inflammatory activity among
the tested compounds, we have investigated the mechanism of binding of
compound 6c with HSA. For this, we have employed spectrofluorescence
technique. The fluorescence intensity of HSA was recorded in the presence of
Chapter 7 (Sec. B) Benzofurans with heterocycles
182
increasing amounts of 6c (Fig. 13). The compound showed concentration
dependent quenching thereby indicating the interaction between 6c and HSA.
In order to know the quenching mechanism, the binding studies were
performed at 295, 306 and 313 K. The values of KSV were obtained from the
slopes of the plot (not shown) of F0/F vs [Q] and the corresponding values are
incorporated in Table 6. It was observed from the table that the KSV values
increased with increase in temperature indicating the presence of dynamic
quenching mechanism in the interaction between 6c and HSA.
From the equation 4 given on page number 33 and from the intercepts and
slopes of the plot of log [(F0-F)/F] vs log [Q], the values of K and n were
calculated and the corresponding data are tabulated in Table 6. The values of n
were found to be close to unity indicating that one molecule of the compound
6c bound to one molecule of HSA.
Thermodynamic parameters viz., ΔH0, ΔS
0 and ΔG
0 were determined in
order to propose the binding forces that were operating in the interaction
between 6c and HSA using the van’t Hoff’s equation and Gibbs-Helmholtz
equations shown on page number 34. The corresponding results are
incorporated in Table 6. The positive values of both ΔH0 and ΔS
0 indicated
that the hydrophobic forces played a chief role in the interaction of 6c with
HSA. The negative ΔG0 values indicated the spontaneity of the interaction
process.
The exact location of the binding site for the compound on HSA was also
identified by competitive experiments. For this, the fluorescence data were
obtained in the presence of site competitors viz., phenyl butazone for site I,
ibuprofen for site II and digitoxin for site III and calculated the binding
constant values. The corresponding results are indicated in Table 7. Based on
these results, the site I located in the hydrophobic pocket of the sub-domain IIA
was proposed as the binding site for the compound 6c in protein.
Chapter 7 (Sec. B) Benzofurans with heterocycles
183
CONCLUSIONS
The synthetic route for the preparation of some benzofuran derivatives is
reported for the first time. Among the newly synthesized compounds, the
compound 6c has shown promising anti-inflammatory activity and the
compounds 6b and 6e have shown good analgesic activity. Mechanism of
interaction between compound 6c and HSA is also investigated by spectro
fluorescence technique.
Chapter 7 (Sec. B) Benzofurans with heterocycles
184
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Chapter 7 (Sec. B) Benzofurans with heterocycles
188
Fig. 1. IR Spectrum of 6a.
Fig. 2. IR Spectrum of 6b.
Chapter 7 (Sec. B) Benzofurans with heterocycles
189
Fig. 3. IR Spectrum of 6k.
Fig. 4. 1H NMR Spectrum of 6a.
Chapter 7 (Sec. B) Benzofurans with heterocycles
190
Fig. 5. 1H NMR Spectrum of 6b.
Fig. 6. 1H NMR Spectrum of 6c.
Chapter 7 (Sec. B) Benzofurans with heterocycles
191
Fig. 7. 1H NMR Spectrum of 6d.
Fig. 8. 1H NMR Spectrum of 6e.
Chapter 7 (Sec. B) Benzofurans with heterocycles
192
Fig. 9. 1H NMR Spectrum of 6j.
Fig. 10. 1H NMR Spectrum of 6k.
Chapter 7 (Sec. B) Benzofurans with heterocycles
193
Fig. 11. Mass Spectrum of 6a.
Fig. 12. Mass Spectrum of 6k.
Chapter 7 (Sec. B) Benzofurans with heterocycles
194
0
50
100
150
200
290 330 370 410 450 490
Wavelenght, nm
Inte
nsit
y
Fig. 13. Fluorescence spectra of (a) HSA (5 μM) in the presence of 6c.
6c concentration was kept at (1) 0, (2) 1.25, (3) 2.50, (4) 3.75, (5) 5.00,
(6) 6.25, (7) 7.50, (8) 8.75, (9) 10.00, (10) 11.25 and (10) 12.50 µM
6c-HSA system.
1
11
Chapter 7 (Sec. B) Benzofurans with heterocycles
195
Table 1.
Physical and analytical data of the compounds, 6a-l.
Compound Mol. formula
(MW)
MP
(0C)
Yield
(%)
Calculated % (Found)
C H N
6a C18H12N2O2S
(320.06) 259 52.50
67.48
(67.42)
3.78
(3.85)
8.74
(8.78)
6b C18H11BrN2O2S
(397.97) 281 52.90
54.15
(54.13)
2.78
(2.72)
7.02
(7.05)
6c C18H11BrN2O2S
(397.97) 273 56.42
54.15
(54.19)
2.78
(2.75)
7.02
(7.08)
6d C18H11ClN2O2S
(354.02) 292 54.50
60.93
(60.91)
3.12
(3.10)
7.90
(7.97)
6e C18H11ClN2O2S
(354.02) 273 55.25
60.93
(60.89)
3.12
(3.16)
7.90
(7.97)
6f C18H11N3O4S
(365.05) 269 58.10
59.17
(59.21)
3.03
(3.05)
11.50
(11.51)
6g C18H11N3O4S
(365.05) 301 52.65
59.17
(59.15)
3.03
(3.09)
11.50
(11.45)
6h C19H14N2O3S
(350.07) 269 52.12
65.13
(65.14)
4.03
(4.06)
7.99
(7.82)
6i C19H14N2O2S
(334.08) 296 48.40
68.24
(68.29)
4.22
(4.28)
8.38
(8.35)
6j C19H14N2O2S
(334.08) 308 42.95
68.24
(68.25)
4.22
(4.20)
8.38
(8.45)
6k C14H10N2O4
(238.07) 272 45.35
70.58
(70.55)
4.23
(4.28)
11.76
(11.79)
6l C14H10N2O4
(238.07) 268 48.90
70.58
(70.55)
4.23
(4.26)
11.76
(11.71)
Chapter 7 (Sec. B) Benzofurans with heterocycles
196
Table 2.
The results of anti-inflammatory activity of compounds, 6a-l.
Name of the
Compound
Dose
mg/kg
Percentage of inhibition
Time in min
0 30 60 120 240 360
Control -- -- -- -- -- -- --
Indomethacin 20 11.11 33.33 26.66 15.50 4.44 11.11
6a 100 11.11 37.77 55.55 44.44 37.30 26.66
6b 100 17.77 33.33 26.66 26.66 22.22 4.44
6c 100 11.11 44.44 65.55 44.44 37.30 22.22
6d 100 4.44 33.33 44.44 33.33 33.33 11.11
6e 100 4.44 37.77 48.88 44.44 44.44 4.44
6f 100 4.44 22.22 22.22 22.22 6.66 17.77
6g 100 11.11 22.22 44.44 22.22 12.22 6.66
6h 100 11.11 33.33 22.22 22.22 11.11 00
6i 100 11.11 33.33 26.66 15.50 11.11 4.44
6j 100 6.66 12.26 22.22 22.22 11.11 4.44
6k 100 00 4.44 6.66 11.11 6.66 4.44
6l 100 11.11 33.33 36.33 22.22 11.11 00
Chapter 7 (Sec. B) Benzofurans with heterocycles
197
Chapter 7 (Sec. B) Benzofurans with heterocycles
198
Table 4.
The MIC results (in µg) of anti-bacterial activity of compounds, 6a-l.
Compound
Bacterial strains
E. coli S. aureus B. subtilis S. typhi
Gentamycin < 25 < 25 25 25
6a 800 400 800 800
6b >800 800 800 800
6c 800 800 >800 >800
6d >800 >800 800 >800
6e 800 400 400 800
6f >800 800 800 >800
6g 800 400 >800 400
6h >800 800 800 >800
6i >800 >800 400 >800
6j 400 400 400 800
6k >800 400 800 >800
6l >800 >800 >800 800
Chapter 7 (Sec. B) Benzofurans with heterocycles
199
Table 5.
The MIC results (in µg) of anti-fungal activity of compounds, 6a-l.
Compound
Fungi
C. albicans A. niger A. flavus N. cressa
Amphotericin 50 100 400 400
6a 800 400 800 >800
6b 800 >800 800 >800
6c >800 400 800 >800
6d 800 800 >800 >800
6e 800 400 800 800
6f 800 >800 400 >800
6g 400 >800 >800 >800
6h >800 800 >800 800
6i 800 800 800 >800
6j >800 800 800 100
6k >800 400 800 800
6l >800 >800 >800 >800
Chapter 7 (Sec. B) Benzofurans with heterocycles
200
Table 6.
Binding and thermodynamic parameters for 6c-HSA system at different
temperatures.
Temp
(K)
KSV
(M-1
)
K
(M-1
) n
ΔG0
(kJ mol-1
)
ΔH0
(kJ mol-1
)
ΔS0
(J mol-1
K-1
)
295 5.01 x 104 1.37 x 10
4 0.88 - 23.36
72.85 324.87 306 6.65 x 104 2.26 x 10
4 0.90 - 25.50
313 6.92 x 104 8.37 x 10
4 1.02 - 29.50
Table 7.
Comparison of binding constant of 6c-HSA before and after the addition of site
probes.
System K without the
site probe (M-1
)
K with
warfarin (M-1
)
K with
ibuprofen (M-1
)
K with digitoxin
(M-1
)
6c-
HSA 2.26 x 10
4 4.77 x 10
3 2.25 x 10
4 2.26 x 10
4