preliminary evaluation of thiosemicarbazones and...

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Preliminary evaluation of thiosemicarbazones and

hydrazones towards antimicrobial, antitubercular

and anticancer activities.

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Introduction

Antimicrobial activity

The use of antimicrobial agents is critical to the successful treatment of infectious

diseases. The innovative research for antibiotics has improved mankind’s health status by

confining life threatening infections. Although there are numerous classes of drugs that

are routinely used to treat infections in humans, there are several reasons why the

discovery and development of new antimicrobial agents are important. The dramatically

rising prevalence of multi-drug resistant microbial infections has become a serious health

care problem. This increased resistance has limited the selection of antimicrobials that

may be used to treat specific organisms. Consequently, the search for new antimicrobial

agents always remains as an important and challenging task for medicinal chemists.

Antibacterial activity

The resistance of bacteria against antimicrobial agents has become a widespread

medical problem especially as nosocomial pathogens [1]. Treatment options for these

infections are often limited, especially in debilitated and immune compromised patients

[2, 3]. In the last decade, there has been a reemergence of Gram-positive bacteria, in

particular Staphylococcus aureus, which is considered to be one of the main causes of

nosocomial infections [4, 5, 6]. The infectious disease caused by MRSA (methicillin-

resistant Staphylococcus aureus) has been a concern all over the world [7, 8]. This

situation becomes worse and more threatening regarding the ease of transmission of the

pathogen among individuals, resulting in the dissemination of MRSA [9] because these

bacteria show a multidrug-resistant phenotype, that is, resistance not only to methicillin

but also to several other drugs except glycopeptides, such as vancomycin [10]. However,

the widespread use of glycopeptides in the past has led to the emergence of glycopeptides

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resistant organisms and, consequently, there are recommendations to restrict the use of

these agents [11]. In 1998 the first clinical high-level VRSA (vancomycin-resistant

Staphylococcus aureus) was isolated [13].

The emergence of serious staphylococcal infections with reduced susceptibility to

vancomycin highlights the need for more antimicrobial therapeutic alternatives with

increased potency or enhanced bactericidal activity against MRSA, VISA (vancomycin-

intermediate S. aureus) [14, 15] and VRSA. Although potent antistaphylococcal drugs are

available, this infection continues to present significant morbidity and mortality rates [16,

17].

Considering the increased incidence of severe disseminated infections produced

by bacteria in immune compromised hosts, there is an emerging need for new

antibacterial agents with potent activity.

Antituberculosis activity

Tuberculosis (TB) remains the leading cause of mortality because of a bacterial

pathogen, Mycobacterium tuberculosis. The statistics indicate that 3 million people

worldwide die annually from complication of TB1 and about 8 million of new cases each

year, 95% of which occur in developing countries [18]. There are increased numbers of

reports of multi-drug resistant mycobacteria throughout the world [19].

Drugs such as isoniazid (INH) and rifampicin have historically been successful in

the treatment of TB infections. In recent history, however, poor compliance with the

prolonged and complicated chemotherapeutic regimens currently used to treat the disease,

in conjunction with the advent of the AIDS epidemic and the increased mobility of human

populations, has led to the emergence of numerous multidrug-resistant Mycobacterium

tuberculosis strain [20]. Extended length of treatment and adverse side effects of the

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drugs used for treatment contribute to the lack of compliance, justifying the need for the

development of more effective compounds for its treatment. Thus, new drugs are

necessary to overcome the current problems of therapy.

Antifungal activity

Significant impacts by infections caused by pathogenic fungi upon human health

have been confirmed by epidemiological studies in recent years [21, 22]. Although the

genus Candida is present as commensal flora in the majority of healthy people, at the

same time it is a common fungus responsible for opportunistic infections and it can

become pathogenous because of predisposing conditions related to the host, like

immunological compromise (AIDS, anti-cancer therapy, and transplants), excessive

prophylaxis with antimicrobial agents, and extended use of invasive catheters [23]. Large-

scale surveillance for fungal infections has demonstrated an increasing incidence of drug-

resistant fungal pathogens. As a matter of fact, a significant number of fungi exhibited

primary resistance to amphotericin B or was less susceptible to triazoles [24].

Furthermore, as a consequence of the toxicity of the currently used polyene antifungal

drugs, which leads to interrupt the therapy, and the emergence of candidal species

resistant to azole-based agents, there is an urgent need for developing alternative drug

therapies [25].

In addition, microorganisms are constantly changing, finding new places to live

and new ways to survive, and adapting to new situations. During this process, harmless

organisms may turn deadly or deadly strains may move from their normal host to humans.

With the continuing discovery of new infectious diseases and the development of new

disease processes of existing pathogens (i.e., necrotizing fasciitis caused by Streptococcus

pyogenes), it is important to continue to find anti-infective agents that can be used to treat

these infections [26, 27].

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Anticancer activity

Cancer is one of the leading causes of death worldwide. Most of the small-

molecular anticancer drugs currently in clinical use including cisplatin, doxorubicin and

paclitaxel are inherently associated with lack of tumor selectivity and short blood

circulation time, which cause various toxic side effects [28, 29, 30, 31, 32]. In particular,

cisplatin has unfavorable pharmacokinetic profile [33] and all platinum based anticancer

drugs with a low molecular weight have a short half life in the blood circulation system

[34].

In the search for anticancer drugs, many compounds with different structures have

been tested. Even though some of them exhibit therapeutical properties and are widely

used, the hunt for new substances especially those with improved efficiency and no side

effects is still an important research goal.

Development of novel classes of drugs, drugs with fewer side effects, and drugs

with shorter lengths of treatment are key in continuing the fight against infectious disease.

The suggested essential role of the Schiff base linkage in certain biochemical reactions

and the fact that the hydrazino and thiosemicarbazone groups frequently confers activity

upon a given structure makes these compounds of considerable interest, and should serve

as a fruitful field for further biological investigations. A careful perusal of literature

revealed that hydrazone [35, 36, 37, 38, 39, 40, 41, 42, 43] and thiosemicarbazone [44,

45, 46, 47, 48, 49, 50, 51] based chemical entities possess diverse biological activities

including antibacterial, antifungal, antitubercular and anticancer and activities.

Prompted by above observations it seemed worthwhile to investigate if the

presence of hydrazone or thiosemicarbazone moieties on the arms of the s-triaizne or

cyclotriphosphazene based star shaped architectures can also give rise to a new class of

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biologically active compounds. The goal of this chapter was to test the new star shaped

molecules bearing multiple copies of hydrazone or thiosemicarbazone for their

antibacterial, antifungal, antitubercular and anticancer activities.

As a preliminary evaluation, target star shaped molecules were assayed for their in

vitro antibacterial activity against bacterial strains such as Staphylococcus aureus,

Escherichia coli, and fungal strains Candida Albicans, Aspergillus niger using a two-fold

serial dilution method. We also present the preliminary results concerning the in vitro

antituberculour and anticancer activity of these compounds.

This chapter accounts for the preliminary results of biological activities of

multifunctional thiosemicarbazones and hydrazones designed during the present study.

This chapter is divided into Section A and Section B.

Section A accounts for the study of anti-microbial activity and also encompasses the anti-

mycobacterial activity. Muller-Hinton broth dilution method was used to evaluate the

anti-microbial and anti-tubercular activities.

Section B presents the study of antiproliferative activity by MTT assay.

Chapter 6A

197

Section 6A : Antimicrobial and antimycobacterial activity of

thiosemicarbazones and hydrazones.

I. Antimicrobial activity

The tri arm star shaped synthesized thiosemicarbazone derivatives 5a-g, 8a-g

and hexa arm star shaped 11a-g, 13a-g were tested for their antimicrobial activity. The tri

arm star shaped hydrazones 15a-k and 16a-k were screened for the inhibition of

microbial growth using a Muller-Hinton broth dilution method in duplicate [52]. The

compounds were tested at different concentrations and minimum inhibitory concentration

(MIC) was determined.

Antimicrobial activity Protocol

Muller-Hinton broth dilution method involves the preparation of growth media

which is as follows.

The suspension of Peptone (5 g), sodium chloride (5 g), beef extract (1.5 g) in

1000 mL distilled water was boiled to dissolve all the ingredients completely. The pH of

this solution was adjusted to 7.4 ± 0.2 at 25 oC and sterilized by autoclaving at 15 lb

pressure (121 oC) for 15 minutes. One day prior to the test, bacterial and fungal strains

were made overnight in the sterile nutrient broth and incubated at 37 oC. Sample solutions

of test compounds and standards Streptomycin and Fluconozole were prepared by

dissolving 2.5 mmol of sample in 1 mL of DMSO and diluted to 10 mL with the broth

media to achieve 250 μM concentrations.

Serial dilution broth susceptibility method was adopted as a reference method.

Serial dilutions of test compounds are made in broth, after which a standardized micro-

organism suspension (100 μL) is added to 10 test tubes containing 100 μL of test

compounds. Quantities of test compounds are serially diluted to attain the final

Chapter 6A

198

concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.6, 0.8, 0.4, 0.2 and 0.1 μM. One of the

test tubes is kept as control, which is free from antibiotics or test compounds. Each of the

10 test tubes is incubated with a suspension of microorganism to be tested and incubated

at 35 oC for 18 hours. At the end of the incubation period, the tubes are visually examined

for the turbidity.

Result and Discussion

Thiosemicarbazone and hydrazone derivatives have been synthesized by the

condensation of aromatic thiosemicarbazides and hydrazides with respective aldehyde

and ketones of 1,3,5-triazine and cyclotriphospazene cores. Structures of the synthesized

products are presented in Figure 6.1, 6.2, 6.3, 6.4, 6.5 and 6.6. The synthesis and

characterization of thiosemicarbazone compounds have been discussed in chapter 2 and

that of hydrazone derivatives is presented in chapter 3.

Chapter 6A

199

N

N

N

O

OO

N

NHN

SHN

NH

NH

S

N

HN

HN

S

H

H

H

R

R

R

N

N

N

O

OO

N

NHN

SHN

NH

NH

S

N

HN

HN

S

CH3

CH3

H3C

R

R

R

Cl

Br

F

R

a

b

c

d

OCH3

CH3

e

f

g

OH

Figure 6.1 structure of compounds 5a-g

Figure 6.2 structure of compounds 8a-g

Chapter 6A

200

N

PN

P

NP

OO

O

O

OO

NN

N

N

N

N

HN

NH

NH

NH

HN

HN

HN S HN

S

HN

S

NHS

NH

S

NH

S

H

H

H

H

H

H

RR

R

R

R

R

Figure 6.3 Structure of compounds 11a-g

N

PN

P

NP

OO

O

O

OO

NN

N

N

N

N

HN

NH

NH

NH

HN

HN

HN S HN

S

HN

S

NHS

NH

S

NH

S

H3C

CH3

CH3

CH3

CH3

H3C

RR

R

R

R

R

Figure 6.4 Structure of compounds 13a-g

Cl

Br

F

R

a

b

c

d

OCH3

CH3

e

f

g

OH

Chapter 6A

201

R

a

b

c Cl

d F

e

f

g

h NO2

i

j

O

k

S

OCH3

OH

Br

CH3

N

N

N

N

O

OO

N

NHN

OR

NH

R

O

N

HN R

O

H

H

H

N

N

N

O

OO

N

NHN

OR

NH

R

O

N

HN R

O

CH3

CH3

H3C

Figure 6.5 Structure of compounds 15a-k

Figure 6.6 Structure of compounds 16a-k

A. Antibacterial activity

The star shaped thiosemicarbazones and hydrazones were tested against skin

disease causing Gram (+) bacterium like Staphylococcus aureus (SA) and Gram (-)

strain Escherichia coli (EC). The results were compared with standard antibacterial drug

Streptomycin.

Chapter 6A

202

i. Antibacterial activity of tri arm star shaped thiosemicarbazones 5a-g & 8a-g

Results of MIC of 5a-g & 8a-g are summarized in table Table 6.1. In the series

5a-g (Figure 6.1), the compound 5c with p-chloro substituent has shown highest activity

against SA (MIC 1.6 µM) as compared to other compounds, but is found to be moderately

active against EC (MIC 6.25 µM). The compound 5e possessing p-hydroxy substituent

and 5g with p-methyl substituent exhibited less activity against SA but are moderately

active (MIC 3.125 µM) against EC.

Table 6.1: In vitro antibacterial activity of the thiosemicarbazones 5a-g, 8a-g and

standard drug (MIC in µM).

Compound SA EC Compound SA EC

5a 50 25 8a 3.125 6.25

5b 25 ….. 8b 1.6 25

5c 1.6 6.25 8c 1.6 50

5d 6.25 12.5 8d 25 3.125

5e 50 3.125 8e 25 25

5f 12.5 6.25 8f 12.5 6.25

5g 100 3.125 8g 3.125 6.25

Streptomycin 0.8 0.8 Streptomycin 0.8 0.8

SA= Staphylococcus aureus; EC= Escherichia coli

In the series 8a-g (Figure 6.2), the p-bromo substituted 8b and 8c with p-chloro

substituent have shown highest activity against SA (MIC 1.6 µM) but their activity

against EC strain is less (MIC 25 µM and 50 µM respectively). The 8d bearing p-Flouro

substitution demonstrated better activity against EC. Compounds 8a (p-H substituent), 8f

(p-OCH3 substituent) and 8g (p-CH3 substituent) have shown moderate activity (MIC

6.25 µM) against EC.

Among all the tested compounds in the series 5a-g & 8a-g, 5c and 8c with p-

chloro substituent demonstrated highest activity and are found to be equipotent against

Chapter 6A

203

SA. Compounds 5e (p –OH substituent), 5g (p-OCH3 substituent) and 8d (p-F substituent)

are found to be moderately active and equipotent against the EC. However the activity of

these compounds is less as compared to the standard drug used in the study. The activity

of 5a-g against SA and EC is represented in the form of a bar chart in Figure 6.7, while

that of 8a-g is presented in Figure 6.8.

Figure 6.7 In vitro antibacterial activity of the compounds 5a-g along with the standard

drug streptomycine.


drug streptomycine

Chapter 6A

204

ii. Antibacterial activity of hexa arm star shaped thiosemicarbazones 11a-g & 13a-g

Results of MIC of 11a-g & 13a-g are summarized in table Table 6.2 and are

compared with standard antimicrobial drug streptomycine.

In the series 11a-g (Figure 6.3), the compound 11d with p-fluoro substituent

exhibited highest activity (MIC 1.6 µM) against SA but is found to be less potent against

EC (MIC 6.25 µM). The compounds 11c with p-chloro substituent and 11f bearing

methoxy group at the para position show good activity against SA (MIC 3.125 µM). The

compound 11c (p-Cl substituent) is also found to be equally active against EC (MIC

3.125 µM), but 11f with p-methoxy substituent possessed highest activity against EC

(MIC 1.6 µM).

Among the series 13a-g, the compounds 13b (p-Br substituent), 13c (p-Cl

substituent) and 13e (p-OH substituent) have shown highest activity against SA (MIC 1.6

µM) but their activity against EC strain is poor. In fact none of the compound in the

series 13a-g have shown any significant activity against EC.

Table 6.2: In vitro antibacterial activity of the thiosemicarbazones 11a-g, 13a-g and



11a 25 12.5 13a 50 12.5

11b 12.5 25 13b 1.6 25

11c 3.125 3.125 13c 1.6 50

11d 1.6 6.25 13d 12.5 12.5

11e 12.5 12.5 13e 1.6 100

11f 3.125 1.6 13f 25 12.5

11g 100 50 13g 50 25



Among all the tested compounds, 11d (p-fluoro substituent) and 13b (p-bromo

substituent), 13c (p-chloro substituent), 13e (p–hydroxy substituent) demonstrated very

good activity against SA. The compounds 11f (p–methoxy substituent) is found to be

Chapter 6A

205

significantly active against the EC. In comparison with the standard drug, the activity of

these compound is less. Figure 6.9 represents the activity of 11a-g. While Figure 6.10

presents the activity of 13a-g against SA & EC in the form of a bar chart.


drug streptomycine


drug streptomycine

Chapter 6A

206

iii. Antibacterial activity of tri arm star shaped hydrazones 15a-k and 16a-k

Results of MIC of 15a-k & 13a-k are summarized in table Table 6.3

Table 6.3: In vitro antibacterial activity of the hydrazones 15a-k, 16a-k and



15a 12.5 25 16a 12.5 12.5

15b 1.6 12.5 16b 25 3.125

15c 1.6 1.6 16c 1.6 6.25

15d 6.25 1.6 16d 12.5 1.6

15e 3.125 3.125 16e 3.125 6.25

15f 25 25 16f 6.25 25

15g 6.25 6.25 16g 6.25 25

15h 12.5 6.25 16h 12.5 50

15i 6.25 12.5 16i 12.5 3.125

15j 12.5 12.5 16j 6.25 12.5

15k 12.5 6.25 16k 6.25 12.5



In the series 15a-k, the compounds 15b (p-bromo substituent) and 15c (p-chloro

substituent) have shown highest activity against SA (MIC 1.6 µM) as compared to other

compounds. The compound 15c (p-chloro substituent) is also found to be equally active

against EC (MIC 1.6 µM), but the compound 15b (p-bromo substituent) has shown less

activity (MIC 12.5 µM) against EC. 15d with p-fluoro substitution also exhibited better

activity against EC.

In the series 16a-k, the compounds 16c (p-chloro substituent) has shown highest

activity against SA (MIC 1.6 µM) but its activity against EC strain is less (MIC 6.25

µM). Compound 16d with p-fluoro substitution has shown highest activity against EC

(MIC 1.6 µM) but found to be less active against SA strain is less (MIC 12.5 µM).

Chapter 6A

207

Figure 6.11 In vitro antibacterial activity of the compounds 15a-k along with the

standard drug streptomycine

Figure 6.12 In vitro antibacterial activity of the compounds 16a-k along with the

standard drug streptomycine

Among all the tested compounds, 15b (p-bromo substituent), 15c and 16c

possessing p-chloro substituent demonstrated highest activity and are found to be

equipotent against SA. Compounds 15c (p-chloro substituent), 15d and 16d with p-fluoro

substituent exhibited better activity against EC and are found to be equally active.

However the activity of these compounds is less as compared to the standard drug used in

Chapter 6A

208

the study. The activity of 15a-k against SA and EC is represented in the form of bar chart

in Figure 6.11, while that of 16a-k is presented in Figure 6.12.

B. Antifungal activity

Candida Albicans (CA) and Aspergillus niger (AN) are the fungal strains used to

demonstrate the antifungal potency of the synthesized star shaped compounds. The results

of inhibition were compared with standard antifunagal drug Fluconazole. Muller-Hinton

broth dilution method in duplicate [52] was used to study the antifungal activty. The

compounds were tested at different concentrations and minimum inhibitory concentration

(MIC) was determined.

i. Antifungal activity of tri arm star shaped thiosemicarbazones 5a-g & 8a-g

The results are summarized in Table 6.4 and represented in the form of bar chart

in Figure 6.13 (5a-g) and 6.14 (8a-g). Among the compounds of 5a-g series, 5c (p-Cl

substituent) and 5e (p–OH substituent) are more active against both CA and AN (MIC 0.4

µM). In the 8a-g series, 8b (p-Br substituent) and 8c (p-Cl substituent) show lower MIC

(0.2 µM) values against both CA and AN. It is interesting to note that the activity of 8b &

8c is nearly equal to that of standard. 8b and 8c can be the potent antifungal agents

against CA & AN and attracts further indepth study to prove their efficacy as drug.

Table 6.4: In vitro antifungal activity of the thiosemicarbazones 5a-g, 8a-g and


Compounds CA AN Compounds CA AN

5a 3.125 3.125 8a 0.8 0.8

5b 1.6 0.8 8b 0.2 0.2

5c 0.4 0.4 8c 0.2 0.2

5d 3.125 1.6 8d - 1.6

5e 0.4 0.4 8e 0.4 0.8

5f 1.6 0.8 8f 1.6 3.125

5g 6.25 1.6 8g 0.8 6.25

Fluconazole 0.1 0.1 Fluconazole 0.1 0.1

CA= Candida albicans, AN=Aspergillus niger

Chapter 6A

209

Figure 6.13 In vitro antifungal activity of the compounds 5a-g along with the standard

drug Flucanazole


drug Flucanazole

Chapter 6A

210

ii. Antifungal activity of hexa arm star shaped thiosemicarbazones 11a-g & 13 a-g

The results are summarized in Table 6.5 and represented in the form of bar chart in

Figure 6.15 (11a-g) and 6.16 (13a-g). Among the compounds of 11a-g series, 11b (p-Br

substituent) and 11c (p-Cl substituent) are more active against CA as well as AN

demonstrating lower MIC (0.2 µM) values, while in the 13a-g series, 13b (p-Br

substituent), 13c (p-Cl substituent), 13e (p –OH substituent) show lower MIC (0.2 µM)

values against CA. 13c is the potent compound against AN strain. The activity exhibited

by some of the compounds is comparable to the standard antifungal drug in the study

Table 6.5: In vitro antifungal activity of the thiosemicarbazones 11a-g, 13a-g and


Compounds CA AN Compounds CA AN

11a 1.6 0.8 13a 0.8 3.125

11b 0.2 0.2 13b 0.2 0.8

11c 0.2 0.2 13c 0.2 0.4

11d 0.4 0.4 13d 0.4 1.6

11e 0.8 0.8 13e 0.2 0.8

11f 0.4 0.8 13f 0.8 0.8

11g 0.8 0.4 13g 1.6 1.6

Fluconazole 0.1 0.1 Fluconazole 0.1 0.1

CA= Candida albicans, AN=Aspergillus niger

Chapter 6A

211


drug Flucanazole


drug Flucanazole

Chapter 6A

212

iii. Antifungal activity of tri arm star shaped hydrazones 15a-k & 16a-k

Among the compounds of 15a-k series, 15a (p-H substituent), 15b (p-Br substituent)

and 15c (p-Cl substituent) are more active (MIC 0.8 µM) against CA. While in the 16a-k

series, 16b (p-Br substituent) and 16c (p-Cl substituent) have shown lower MIC (0.8 µM)

values. However activity exhibited by these compounds is slightly less compared to the

standard antifungal drug used in the study. The results are summarized in Table 6.6 and

represented in the form of bar chart in Figure 6.17 (15a-k) and 6.18 (16a-k).

Table 6.6: In vitro antifungal activity of the compounds 15a-k, 16a-k and standard

drug (MIC in µM).

Compounds CA Compounds CA

15a 0.8 16a 6.25

15b 0.8 16b 0.8

15c 0.8 16c 0.8

15d 6.25 16d 1.6

15e 3.13 16e 25

15f 12.5 16f 6.25

15g 1.6 16g 12.5

15h 25 16h 25

15i 12.5 16i 3.13

15j 6.25 16j 12.5

15k 3.13 16k 1.6

Fluconazole 0.2 Fluconazole 0.2

CA= Candida albicans

Chapter 6A

213

Figure 6.17 In vitro antifungal activity of the compounds 15a-k along with the standard

drug Flucanazole

Figure 6.18 In vitro antifungal activity of the compounds 16a-k along with the standard

drug Flucanazole

C. Anti-tubercular activity

The anti-mycobacterial activity of synthesized thiosemicarbazones and

hydrazones were assessed against M. tuberculosis H37Rv at several µM concentrations.

The Minimum Inhibitory Concentrations (MIC) of compounds was compared with

Isoniazid, the standard anti-TB drug.

Chapter 6A

214

Protocol followed

Test compounds were evaluated for in vitro anti-mycobacterial activity. The MICs

were determined and interpreted for Mycobacterium tuberculosis H37Rv according to the

procedure of the approved micro dilution reference method of antimicrobial susceptibility

testing [53]. Compounds were taken at concentrations of 100, 50, 25, 12.5, 6.25, 3.125,

1.6, 0.8, 0.4, 0.2 and 0.1 μM in DMSO. M. tuberculosis H37Rv strain was used in Middle

brook 7H9 (culture media, used to grow pure cultures of mycobacteria ) broth which was

inoculated with standard as well test compounds and incubated at 37 °C for 4 weeks. The

bottles were inspected for growth, twice a week for a period of three weeks. Readings

were taken at the end of 4 weeks. The appearance of turbidity was considered as growth

and indicates resistance to the compound. The growth was confirmed by making a smear

from each bottle and performing a Ziehl-Neelsen stain. Test compounds were compared

with isoniazid, used as the standard.

Results and discussion

i. Anti-tubercular activity of tri arm star shaped thiosemicarbazones 5a-g and 8a-g


in Figures 6.19 and 6.20.

Among the compounds studied 8b (p-Br substituent) has shown comparatively

good inhibition (MIC 6.25 µM), while 5b (p-Br substituent), 5c (p-Cl substituent) and 8c

(p-Cl substituent) have shown moderate inhibition (MIC 12.5 µM). All the tested

compounds have shown poor inhibition compared to the standard used.

Chapter 6A

215

Table 6.7: In vitro anti-mycobacterial activity of the compounds 5a-g, 8a-g and


Compound

M. tuberculosis H37RV

Compound


5a 25 8a 50

5b 12.5 8b 6.25

5c 12.5 8c 12.5

5d 25 8d 50

5e 25 8e 25

5f 50 8f 25 5g 25 8g 50

INH 1.6 INH 1.6

Figure 6.19 In vitro anti-mycobacterial activity of the compounds 5a-g along with the

standard drug Isoniazide



Chapter 6A

216

ii. Anti-tubercular activity of hexa arm star shaped thiosemicarbazones 11a-g &

13a-g

The activity results are summarized in Table 6.8 and represented in the form of

bar chart in Figure 6.21 and 6.22.

All the tested compounds have shown poor inhibition compared to the standard

used. Among the compounds studied 11b (p-Br substituent) and 11c (p-Cl substituent)

exhibited moderate activity (MIC 12.5 µM).

Table 6.8: In vitro anti-mycobacterial activity of the compounds 11a-g, 13a-g and


Compound

M. tuberculosis

H37RV

Compound

M. tuberculosis

H37RV

11a 25 13a 100

11b 12.5 13b 25

11c 12.5 13c 25

11d 25 13d 50

11e 25 13e 25

11f 50 13f 50

11g 25 13g 50

INH 1.6 INH 1.6

Chapter 6A

217





Chapter 6A

218

iii. Anti-tubercular activity of tri arm star shaped hydrazones 15a-k & 16a-k


in Figure 6.23 (15a-k) and 6.24 (16a-k).

Among the compounds studied 16e demonstrated better activity (MIC 3.125 µM),

Compounds 15c, 15d, 15j, and 16g, 16i, 16j have shown moderate activity (MIC 6.25

µM) and are equipotent. The activity of the compounds studies is less compared to the

standard drug used.

Table 6.9: In vitro anti-mycobacterial activity of the compounds 15a-k, 16a-k and


Compound


Compound


15a 25 16a 12.5

15b 12.5 16b 25

15c 6.25 16c 50

15d 6.25 16d 12.5

15e 50 16e 3.125

15f 12.5 16f 12.5

15g 12.5 16g 6.25

15h 25 16h 12.5

15i 50 16i 6.25

15j 6.25 16j 6.25

15k 12.5 16k 25

INH 1.6 INH 1.6

Chapter 6A

219

Figure 6.23 In vitro anti-mycobacterial activity of the compounds 15a-k along with the


Figure 6.24 In vitro anti-mycobacterial activity of the compounds 16a-k along with the


Chapter 6B

220

Section 6B: Antiproliferative activity of thiosemicarbazones and

hydrazones

Anticancer activity

The antiproliferative activities of triazine thiosemicarbazones (5a-g) and

cyclotriphosphazene thiosemicarbazones (8a-g) are assayed against breast cancer (MCF-

7) and ovarian cancer (PA-1) cell lines. Triazine hydrazones 15a-k and 16a-k were tested

against human liver carcinoma (HepG2) and human cervix carcinoma (HeLa) cell lines.

The synthesis and characterization of these compounds have been discussed in chapter 2

and 3.

Protocol followed

Under the sterile condition, breast cancer cells (MCF7), ovarian cancer cells (PA-

1), Human liver carcinoma cell line (HepG2) and Human cervix carcinoma cell line

(HeLa) were grown in MEM supplemented with 10% fetal calf serum (FCS).

Cytotoxicity of compounds 5a-g, 11a-g at 50 and 100 μM concentration and 15a-k, 16a-

k at 100 μM concentration was tested against cancer cells using MTT assay [54, 55].

Each compound was initially solubilized in dimethyl sulfoxide (DMSO), however, each

final dilution contained less than 1% DMSO.

The cells at approximately 80% confluence were selected for trypsinization. The

cells were harvested by removing the medium and then 1 mL of trypsin-EDTA (200 mg/L

for EDTA, 500 mg/L for trypsin in a ratio (1:250) was added and incubated at 37°C for

about 5 minutes. The cells were detached from the plate and collected in a centrifuge tube

and centrifuged at 1000 rpm for 5 minutes. Cell number was determined using

hemocytometer and the cells at 5x104 cells/mL were seeded in 96 well plates and grown

for 24 h at 37 °C and 5% CO2.

Chapter 6B

221

After 24 h incubation, the cells were treated with the newly synthesized

compounds and then incubated for further 48 h at 37 °C. 5 µL of PBS solution of MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 5 mg/mL] was added to

each well, and incubation was continued for another 4 h. the resultant formazan blue

crystals were dissolved in 100 μL SDS (10% in 0.1N HCl) and the optical density (OD)

was measured at 570 nm (reference filter 690 nm) using a microplate reader. Triplicate

readings were obtained for each sample and optical density values of the three wells were

averaged for one sample. The results were recorded and expressed as % inhibition in cell

growth of ovarian cancer cell line (PA-1) and breast cancer (MCF-7) cell line, Human

liver carcinoma cell line (HepG2) and Human cervix carcinoma cell line (HeLa)

compared with the blank control.

Cell lines

Cancer remains the leading cause of death in the World and as a result there is a

pressing need for novel and effective treatments. Despite major breakthroughs in many

areas of modern medicine over the past 100 years, the successful treatment of cancer

remains a significant challenge. We have used different cancer cell lines for our study.

MCF-7 cell line

MCF-7 is a breast cancer cell line isolated in 1970 [56]. MCF-7 cells are useful for in

vitro breast cancer studies because the cell line has retained several ideal characteristics

particular to the mammary epithelium. The MCF-7 cell line is an estrogen receptor (ER)

positive control cell line. It is the second leading cause of cancer death in women after

lung cancer [57]. Approximately 1.2 million cases of breast cancer are diagnosed around

the world each year [58].

PA-1 cell line

PA-1 is an ovarian cancer cell line is derived from epithelial organ of the ovary

[59]. Ovarian cancer is diagnosed in nearly a quarter of a million women globally each

Chapter 6B

222

year. It is the eighth most common cancer in women and the seventh leading cause of

cancer death among women, responsible for approximately 140,000 deaths each year

[60]. It has the highest mortality rate of all gynecological cancers. Most of the ovarian

cancers are diagnosed at advanced stage where the patients have developed a wide spread

disease[59]. Although there are many chemotherapeutic drugs which have shown high

response rate after surgery but patients have poor survivalance (25%) which further

decreases to 5% when patients diagnosed at stage III and IV [59]

HePG2 cell line

Hep G2 is a perpetual cell line which was derived from the liver tissue [61]. Liver

cancer is one of the leading causes of worldwide cancer mortality, with an estimated 1

million deaths annually and 5-year survival rates of less than 5% [62]. Liver cancer

incidence of 4 to 15 per 100,000 has been reported in Western countries, as compared

with 120 per 100,000 in Asia and Africa [63]. Previous research has revealed that liver

cancer is largely refractory to chemotherapy because of tumor heterogeneity and the

development of multidrug resistance phenotypes [64]. Thus, up to now, availability of

treatments for liver cancer remains unsatisfactory [65] because liver cancer cells are

present p53 gene mutations and tend to be more aggressive and extremely resist to

chemotherapy [66].

HeLA cell line

HeLa derived from cervical cancer cells. It is the oldest and most commonly used

human cell line [67]. Cervical cancer is the third most common cancer in women,

worldwide, with 530,000 new cases diagnosed in 2008 [60].Virtually all cervical cancers

are associated with human papilloma viruses (HPV). More than 85% of these cases and

deaths occur in developing countries. India, the second most populous country in the

world, accounts for 27% (77,100) of the total cervical cancer deaths [60].

Chapter 6B

223

Results and discussion

i. Antiproliferative activity of star shaped thiosemicarbazones 5a-g & 11a-g

The thiosemicarbazones 5a-g and 11a-g were tested for their in vitro

antiproliferative activity against breast cancer (MCF-7) and ovarian cancer (PA-1) cell

lines at 50 and 100 μM concentration by using MTT assay. The results presented in

Figure. 6.25 to 6.28 indicate that the compounds exhibited moderate antiproliferative

activity against the cell lines tested. Since compounds behaved differently in relation to

the different cell lines, proper structural activity relationship could not be drawn.

Figure 6.25 Anticancer activity of 5a-g by MTT assay against MCF-7 cell line.

Figure 6.26 Anticancer activity of 5a-g by MTT assay against PA-1 cell line.

Chapter 6B

224

Figure 6.27 Anticancer activity of 11a-g by MTT assay against MCF-7 cell line.

Figure 6.28 Anticancer activity of 11a-g by MTT assay against PA-1 cell line.

In case of antiproliferative activity of 5a-g against MCF-7 cells, the compound 5b

bearing p-bromo substituent exhibited highest activity followed by compounds 5c and 5d

with p-chloro and p-fluoro groups respectively. In all the cases the % inhibition is found

to be more at 100 μM concentration. Triazine thiosemicarbazone derivatives were not

sufficiently effective against the PA-1 cell lines.

Chapter 6B

225

In case of the 11a-g series, the compounds 11b bearing p-bromo substitution

exhibited highest against both MCF-7 and PA-1 cell lines. Compounds 11c and 11d with

p-chloro and p-Fluoro benzene ring respectively are moderately active against MCF-7

cells. Compounds 11c and 11g with p-chloro and p-methyle substituents respectively at

100 μM concentration show moderate activity against PA-1. It is interesting to note that

the compounds 11b and 11c exhibited very good activity against both MCF-7 and PA-1

cell lines (Table 6.10 and 6.11)

Table 6.10: Anticancer activity of 5a-g test compounds by MTT assay (%

Inhibition)

Compounds MCF 7 PA 1

50 µmol 100 µmol 50 µmol 100 µmol

5a 17.20 23.58 15.10 19.96

5b 81.25 85.87 44.16 56.17

5c 65.17 73.34 46.19 66.40

5d 56.26 63.43 39.31 52.64

5e 15.75 29.71 49.93 55.01

5f 52.24 59.37 51.85 54.73

5g 32.12 44.28 44.44 47.74

Table 6.11:Anticancer activity of 11a-g test compounds by MTT assay (%

Inhibition)

Compounds MCF-7 PA-1

50 µmol 100 µmol 50 µmol 100 µmol

11a 58.28 63.11 54.4 61.88

11b 90.1 96.94 83.64 86.38

11c 78.08 87.74 71.91 73.02

11d 67.00 84.6 63.8 60.18

11e 67.18 70.86 59.64 66.48

11f 73.25 78.96 57.81 57.88

11g 53.40 63.03 67.9 68.31

Chapter 6B

226

ii. Antiproliferative activity of star shaped hydrazones 15a-k &16a-k

The compounds synthesized were tested for their in vitro antiproliferative activity

against human liver carcinoma (HepG2) and human cervix carcinoma (HeLa) cell lines.

The result presented in Figure. 6.29 and 6.30 indicates that the compounds exhibited

moderate antiproliferative activity against the cell lines tested. The compounds behaved

differently in relation to the different cell lines.

Figure 6.29 Anticancer activity of 15a-k by MTT assay.

Figure 6.30 Anticancer activity of 16a-k by MTT assay.

Chapter 6B

227

In case of the antiproliferative activity of 15a-k against for HepG2 cells, the

compound 15h bearing p-nitro substituent exhibited highest activity followed by

compounds 15g with p-methyl groups. Trizine hydrazone derivatives were not

sufficiently effective against the HeLa cell lines. It is interesting to note that the

compound 15h with p-nitro substituent exhibited least activity against HeLa cells but

highest activity against the HepG2 cells.

In case of the 16a-k series, the compound 16f bearing p-methoxy groups at the

periphery exhibited highest antiproliferative activity against HepG2 cells. These

hydrazone derivatives were found to be less active against HeLa cell lines. It is

interesting to note that the compound 16f with p-methoxy substituent exhibited highest

activity against the HepG2 cells, but very less activity against HeLa cells.

The present compounds were not sufficiently affective against HeLa cell lines but

exhibited reasonably moderate in vitro cytotoxicity against HepG2 cell lines (Table

6.12).

Table 6.12: Anticancer activity of 15a-k and 16a-k test compounds by MTT assay (% Inhibition).

Compounds Cell lines

Compounds Cell lines

Hep-G2 HeLa Hep-G2 HeLa

15a 26.31 13.23 16a 18.24 16.24

15b 8.42 19.46 16b 10.72 25.38

15c 6.41 28.36 16c 8.24 30.22

15d 4.32 18.19 16d 10.26 14.11

15e 36.24 16.45 16e 32.56 15.62

15f 34.69 9.87 16f 50.48 8.92

15g 52.34 21.39 16g 36.92 20.22

15h 58.91 8.36 16h 29.33 6.45

15i 30.77 23.35 16i 15.46 23.67

15j 17.46 12.51 16j 22.32 10.23

15k 23.78 14.39 16k 16.57 19.52

Chapter 6B

228

Chapter 6

228

Conclusion

All the newly synthesized triazine and cyclotriphosphazene based star shaped

thiosemicarbazone and hydrazone derivatives were assayed in vitro for antibacterial

activity against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-

negative), and the antifungal activity was evaluated against Candida albicans and

Aspergillus niger strains. The MIC values were determined by the twofold serial dilution

technique in Mueller–Hinton broth for the antibacterial and antifungal assay, respectively.

streptomycin and flucanozole were used as standards.

The compounds were also tested for their in vitro antimycobacterial activity

against H37RV. The Minimum Inhibitory Concentrations (MIC) of compounds was

compared with Isoniazid as standard drug. An examination of the in vitro antiproliferative

activity profile of new star shaped molecules have been undertaken against breast cancer

(MCF-7), ovarian cancer (PA-1) cell lines and human liver carcinoma (HepG2), human

cervix carcinoma (HeLa) cell lines.

Investigations on antibacterial activities indicate that star shaped compounds

bearing thiosemicarbazone functionalitie on their arms are more potent than those with

hydrazone units. Among the compounds tested 5c, 8b, 8c, 11d, 13b, 13c, 15b, 15c and

16c have shown highest activity and are equipotent against Staphylococcus aureus (SA)

bacterial strain, while the compounds 11f, 15c, 15d, and 16d are found to be highly

active against Escherichia coli (EC) and their activity is equal. The activity of these

compounds is less compared to the standard used.

Among the compounds synthesized 8b, 8c, 11b, 11c, 13b, 13c and 13e have

shown highest and equal potent antifungal activity against CA and 8b, 8c, 11b, 11c are

equally effective against AN also .

Chapter 6

229

All the tested compounds have shown poor anti tubercular activity compared to

the standard used. The thiosemicarbazone/hydrazone derivatives showed higher activity

against tested cancer cell lines. In case of the 5a-g and 11a-g, antiproliferative activity for

MCF-7 cell lines, the thiosemicarbazone compound 5b and 11b exhibited highest activity

and for PA-1 cell lines the compounds 5c and 11b exhibited better activity at higher

concentration.

In case of the 15a-g and 16a-g antiproliferative activity for HepG2 cell lines, the

hydrazone compound 15h and 16f exhibited highest activity and for HeLa cell lines the

compounds 15c and 16c exhibited highest activity. Overall, thiosemicarbazone

derivatives were more effective than hydrazone derivatives against tested cell lines.

The present compounds exhibited comparatively better antimicrobial activity

against SA, EC, CA and poor antimycobatarial activity against H37Rv. In case of the

antiproliferative activity, the tested compounds have shown moderate activity against

MCF-7 and PA-1 cell lines. Antiproliferative activity against HeLa cell lines is less but

reasonably moderate against HepG2 cell lines.

Few of the synthesized compounds, particularly 8b, 8c, 11b and 11c which have

shown the antifungal activity comparable to the standard drugs in the market, are the

better candidate for further indepth study.

Chapter 6

230

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