Synthesis and biological activities of some benzofuran...

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.


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


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


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


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


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.


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).


145

(E)-N'-benzylidenebenzofuran-2-carbohydrazide (5b).

IR (KBr, cm-1



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


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


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



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



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


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).


146

N-(2-methyl-4-oxothiazolidin-3-yl)benzofuran-2-carboxamide (6a).

IR (KBr, cm-1


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


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


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


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


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


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).


147

N-(2-(4-hydroxy-3-methoxyphenyl)-4-oxothiazolidin-3-yl)benzofuran-2-

carboxamide (6g).

IR (KBr, cm-1


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.


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


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.


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.


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.


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.


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.


154

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Pharma. Res. 1 (2010) 26.

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(2006) 437.

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testing and interpretation of results. In: Antimicrobial Therapy in

Veterinary Medicine, Eds. Ames, IA, Iowa State University Press, 2000.


156

Fig. 1. IR Spectrum of 5a.

Fig. 2. IR spectrum of 5d.


157

Fig. 3. IR Spectrum of 6c.

Fig. 4. IR spectrum of 6f.


158

Fig. 5. 1H NMR Spectrum of 5b.

Fig. 6. 1H NMR Spectrum of 5d.


159

Fig. 7. 1H NMR Spectrum of 5e.

Fig. 8. 1H NMR Spectrum of 6a.


160


Fig. 10. 1H NMR Spectrum of 6c.


161




162

Fig. 13. 1H NMR Spectrum of 6f.

Fig. 14. Mass spectrum of 5a.

O

HN

O

NS

O

OH3C


163

Fig. 15. Mass Spectrum of 6a.

Fig. 16. Mass Spectrum of 6c.


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


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)


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


167


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


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


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.


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


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


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


175

Scheme 1

R1 = Br, Cl, NO2, CH3

R2 = Br, Cl, NO2, OCH3

Scheme 2

-R =


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.


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


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


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


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



9.21 (s, NH). m/z: 398 (M+).


179

N-(4-(4-chlorophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6d).

IR (KBr, cm-1



9.41 (s, NH). m/z: 355 (M+1).

N-(4-(2-chlorophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6e).

IR (KBr, cm-1



9.06 (s, NH). m/z: 355 (M+1).

N-(4-(4-nitrophenyl)thiazol-2-yl)benzofuran-2-carboxamide (6f).

IR (KBr, cm-1


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



9.34 (s, NH). m/z: 365 (M+).

N-(4-(4-methoxyphenyl)thiazol-2-yl)benzofuran-2-carboxamide (6h).

IR (KBr, cm-1


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


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


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



9.17 (s, NH). m/z: 238 (M+).


180

N-(pyridin-3-yl)benzofuran-2-carboxamide (6l).

IR (KBr, cm-1



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


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


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.


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.


184

REFERENCES

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188

Fig. 1. IR Spectrum of 6a.

Fig. 2. IR Spectrum of 6b.


189

Fig. 3. IR Spectrum of 6k.

Fig. 4. 1H NMR Spectrum of 6a.


190


Fig. 6. 1H NMR Spectrum of 6c.


191




192

Fig. 9. 1H NMR Spectrum of 6j.

Fig. 10. 1H NMR Spectrum of 6k.


193

Fig. 11. Mass Spectrum of 6a.

Fig. 12. Mass Spectrum of 6k.


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


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)


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


197


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


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


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

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