jurnal kimed

Synthesis, antibacterial activity against MRSA,and in vitro cytotoxic activity against HeLa cell linesof novel 3-a-carboxy ethyl-5-benzylidene rhodaninederivatives

Kaveri Sundaram Subban Ravi

Received: 19 November 2012 / Accepted: 29 April 2013 / Published online: 24 May 2013

Springer Science+Business Media Dordrecht 2013

Abstract A series of novel 3-a-carboxy ethyl-5-benzylidene rhodanines, 3ah,has been accomplished by Knoevenagel condensation with 3-a-carboxy ethyl rho-danine and various substituted aromatic aldehydes. All the synthesized compounds

3ah were confirmed by spectroscopic techniques. The cytotoxic studies of com-pounds 3ah were performed against human cervical cancer cell line (HeLa) byMTT assay. Further, the compounds 3ah were also screened for their antibacterialactivity against methicillin-resistant Staphylococcus aureus (MRSA). SAR study

was carried out for the in vitro cytotoxic studies against HeLa cell lines and anti-

bacterial activity against MRSA. The results suggested that this series of compounds

could serve as the basis for the development of novel anticancer and antimicrobial

agents.

Keywords 3-a-Carboxyethylrhodanine MTT assays MRSA Antibacterial activity

Introduction

Chemotherapy is considered to be the mainstay of cancer treatments while

traditional cytotoxic chemotherapeutics are not satisfactory due to diverse toxic side

effects. It is necessary to develop novel anticancer agents with high potency and low

toxicity. The rhodanine scaffold is present in many classes of biologically active

compounds [1]. In our ongoing investigations on rhodanines [2, 3], we have

prepared 5-isopropylidene-3-ethyl rhodanine (I) and 5-benzylidene-3-ethyl rhoda-

nine (II) and have tested for the cytotoxicity properties against leukemic CEM cells

by tryptone blue, MTT, LDH, and cell cytometry assay. The results were compared

K. Sundaram S. Ravi (&)Department of Chemistry, Karpagam University, Coimbatore 641021, Tamil Nadu, India

e-mail: [email protected]

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Res Chem Intermed (2015) 41:10111021

DOI 10.1007/s11164-013-1251-8

IkaPutriNurhayatiHighlight

with their O and N counterparts of 5-isopropylidene-3-ethyl rhodanine, oxazolid-

inedione, and hydantoin, respectively. The rhodanine derivatives (I, II) are found to

be more potent than the corresponding oxazolidenediones and hydantoins. They

showed a dose- and time-dependent cytotoxic potential. The results suggested that

the rhodanine affects the cell viability by inhibiting cell division, probably by

interfering with DNA replication and inducing apoptosis. FACS analysis indicated

that it interfered with cell division by inducing S phase arrest followed by apoptosis.

It was further observed that the accumulation of cells at the GO phase indicated

fragmentation of the DNA and the gel electrophoresis of the treated CEM cells

confirmed the activation of cell death pathways upon treatment with rhodanine.

Further, it was ROS generation which could be an intermediate step in rhodanine-

induced cytotoxicity. So the rhodanine scaffold proved suitable for structure activity

relationship (SAR) exploration with other cell lines and synthetic modification. It is

known that the thioxo group in the rhodanine molecule is a carboxylic acid

bioisostor by size with low electro negativity and possesses the ability to build

hydrogen bonds [4] (Fig. 1).

S

NO SC2 H5

S

NO S

CH

C2H5

S

NH

S O

I II III

Inspired by this, a series of 5-benzylidene derivatives of 3-a-carboxy ethylrhodanine 2 were prepared and screened for cytotoxic activity by MTT assay againstHeLa cell lines. Rhodanine derivatives 3ah were prepared for exploration of SARaround the 3- and 5- positions of the rhodanine scaffold leading to the development

of the detected SAR data and the identification of the potent analog. Several hybrid

compounds possessing chalcones and rhodanine-3-acetic acid moieties were

synthesized and tested for their antibacterial activity [5]. This class of compounds

exhibited high potency against the Gram-positive bacteria Staphylococcus aureus

but did not inhibit the growth of the Gram-negative bacteria Escherichia coli. In

another similar study [6], a series of rhodanine compounds containing various

substituents at the N-3 and C-5 positions were synthesized and their in vitro activity

against a panel of clinically relevant MRSA was reported. This prompted us to study

the antibacterial activity of the synthesized compounds 3ah.

Chemistry

The 3-a-carboxy ethyl rhodanine was prepared by the reaction of dl-alanine andcarbon disulphide as summarized in Scheme 1. Compounds 3ah (Table 1) wereprepared by Knoevenagel condensation [7] of 3-a-carboxy ethyl rhodanine (2) withdifferent substituted aromatic aldehydes. The structure of the desired compounds

was determined by UV, IR, 1H-NMR, 13C-NMR, and elemental analysis.

Compounds 3ag have a benzylidene rhodanine moiety and compound 3h has a

1012 K. Sundaram, S. Ravi

123



furalidine moiety. All the compounds 3ah were analyzed as a mixture ofenantiomers. In the IR spectrum of compound 2, the presence of a broad band in theregion 2,5503,400 cm-1 for the hydroxy group and a strong band at 1,729 cm-1

for the C=O group indicated the presence of the carboxylic acid function in the

molecule. In the 1H-NMR spectra, the hydroxy proton appeared at d 5.27 as amultiplet. Further, the doublet signal at d 1.43 (3H, J = 7.2 Hz, H-10), a quateretsignal at d 3.80 for (1H, H-30), and a doublet at d 4.219 (2H, J = 2.0 Hz, H-5) werethe characteristic signals of compound 2. It was confirmed by its 13C-NMRspectra by exhibiting six signals at d 203.10 (C-2), 173.75 (C-4), 169.83 (C-30),54.31 (C-20), 34.71 (C-5), and at 13.73 (C-10). In compound 3b, the signal at d 4.219disappeared. In compound 3b, the benzylidene proton H-6 appeared at d 7.80 as asinglet and the aromatic protons appeared as a pair of doublets, each integrating for

two protons at d 7.64 (d, 2H, J = 8.8 Hz, H-300 and 500) and 7.15 (d, J = 8.8 Hz, 2H,H-200 and 600). The methoxy proton H-700 resonated as a singlet at d 3.85 and thearomatic proton signals appeared between d 6.0 and 8.0, and in the 13C-NMRspectra, the peak at d 34.71 disappeared and a new peak at 117.97 was generated. Inaddition to the signals of the rhodanine moiety, a bunch of signals appeared between

d 100 and 160 for the benzylidine moiety. The two intense signals at d 115.14 and125.32, each for two carbon atoms, were attributed to the pair of carbons, C-300 and500 and C-200 and 600, respectively. The two quarternary carbon atoms of the aromaticring appeared at d 132.95 (C-100) and 161.64 (C-400). The methoxy carbon resonatedat d 55.43 (C-700) and the C-6 carbon atom exhibited at d 135.00.

Results and discussion

In the present study, we report the synthesis of eight new rhodanine derivatives (3ah).The N-3 position of the rhodanine moiety is substituted with an optically active amino

acid substituent. The constituent amino acid providing a potential hydrogen bond

donor and acceptor is important for activity [8]. To start with, the compound 2 wastested for its anti-cancer activity using MTT assay against HeLa cell lines. The results

showed that compound 2 was potent against HeLa cell lines with an IC50 value of200 ll. Further, the results showed that cell viability was affected even at lowerconcentrations of the tested compounds.

In an attempt to study the SAR against the human cervical cancer cell line HeLa,

all the synthesized compounds 3ah were tested against HeLa by MTT assay fortheir cytotoxic activity. The medium without the sample of the HeLa cell lines

served as control. The % cell inhibition was determined. To compare the cytotoxic

activity among the compounds 3ah, a 10 lg/ml concentration solution wasprepared and the percentage inhibition of the HeLa cells was determined; the results

are reported in Table 2. From Table 2, it can be observed that all the compounds

3ah exhibited cytotoxicity and showed inhibition of the HeLa cell line growth evenat 10 lg/ml concentration. Table 2 also shows that the introduction of a benzylidenemoiety (3ag) or a furalidene moiety (3h) increases the cytotoxic activity. Thepreliminary SAR suggested that substituted benzylidene on the C-5 position of the

rhodanine ring is essential for the anti-cancer activity against HeLa cell lines. The

Novel 3-a-carboxy ethyl-5-benzylidene rhodanine derivatives 1013

123

Table 1 Synthesis of compounds 3ah

S. no. Compounds R Yield (%) Melting point (C)

1 3a H 75 216

2 3b 4-OCH3 70 224

3 3c 4-Cl 74 226

4 3d 2-NO2 70 228

5 3e 3-NO2 77 230

6 3f 4-NO2 80 226

7 3g 4-CHO 68 220

8 3h

O

65 204

H3CHCNH2

COOHa, b

S

NHC

CH3

COOH

S O

S

NHC

CH3

COOH

S Oc 1

24

5

1'

2'3'

31''

2'' 3''4''

5''6'' R

S

NS O

HC COOHCH3

O CHOd

S

NS O

HC COOHCH3

O

1'

2' 3'

12 3

4

5

1''2''

3'' 4''

5''

6

6

3a: R= H3b: R= OCH33c: R= 4-Cl3d: R= 2-NO23e: R= 3-NO23f : R= 4-NO23g: R= 4-CHO

3h

1 2 3a-g

Scheme 1 Synthesis of compounds 3a-h. a NaOH, CS2, water, 3 h stirring b ClCH2COONa, dil. HClc RCHO (ag), Anhydrous CH3COONa, glacial acetic acid, 6 h reflux d Anhydrous CH3COONa, glacialacetic acid, 6 h reflux


123

substituents are polar in nature which may induce H-bonding. At 10 lg/mlconcentration level, compound 2 showed only 14.28 % inhibition of the HeLa cellswhere as compounds 3ah showed from 28 to 52 % inhibition. This may be due tothe substituents being polar in nature, which may enhance the H-bonding between

the rhodanine moiety and the target cells. Further, the compounds without any

substituent in the benzylidene moiety showed a fairly good activity and exhibited an

inhibition of 42.86 %. However, there is a marginal increase in the synthesized

compounds when it is substituted with a methoxy group (inhibition 52 %) and by an

aldehyde group in the para position (inhibition 45.72 %). Both the compounds, due

to the extended conjugation, would have a better interaction between the

compounds and the HeLa cells. A benzylidene system may exist with the

electron-withdrawing groups, like 4-Cl, 2-NO2, 3-NO2 and 4-NO2, showing 41.4,

28.52, 35.7 and 40.0 % inhibition, respectively, while the activity is found to be

less. Among the nitro group in different positions of the benzylidene ring system,

the para nitro compound showed higher activity. This suggests that conjugation of

the double bonds with the benzylidene moiety may be an important factor to be

considered for the activity.

Antibacterial activity is of particular importance due to the rise of drug-resistant

bacteria and the paucity of new agents currently in development [912]. In one of

the published reports on rhodanine, it identifies the key requirements for

antibacterial activity to be the NH at the 3-position, a heteroatom at the 1-position.

and a substituted phenyl group at the 5-position. A series of rhodanine compounds

containing various substituents at the N-3 and C-5 positions were synthesized and

their antibacterial activity was reported [5, 6]. But so far, to the best of our

knowledge, no studies on the antibacterial activity have been carried out with 3-a-carboxy ethyl rhodanines. This motivated us to study the SAR with respect to the

antibacterial activity of the compounds 3ah. The antibacterial activity was alsoevaluated against methicillin-resistant S. aureus MTCC84 by measuring the zone of

inhibition in diameter and minimum inhibitory concentration (MIC) (Table 3). Two

samples, 3d and 3g, have been found to exhibit growth inhibition activity against thetest organism with a MIC of 20 lg and all other compounds (3a, 3b, 3c, and 3f, butnot 3h) showed an MIC value of 25 lg. Compounds with NO2 in the ortho positionof the aromatic ring system exhibited good antibacterial activity against MRSA. The

Table 2 in vitro cytotoxicactivity by MTT assay for the

compounds 2 and 3ah

Compounds % of inhibition with

10 lg of compounds

2 14.28

3a 42.85

3b 52.00

3c 41.40

3d 28.52

3e 35.70

3f 40.00

3g 45.72

3h 42.85


123





reason may be that the nitro and aldehydes groups, being highly polar, may polarize

the bacteria to form a strong bond with them. In vitro antimicrobial activity was

evaluated using the MIC and zone of inhibition methods against Gram-positive and

Gram-negative strains (including multidrug-resistant S. aureus MTCC84). The

Gram-positive bacteria S. aureus MTCC430, Bacillus cereus MTCC441 and the

Gram-negative bacteria E. coli MTCC724 were used for the present study. All the

compounds are highly active against E. coli with a zone of inhibition value of

1224 mm. The structures of the examined compounds 3ag were modified byintroducing substitutents (OCH3, 4-Cl, 2-NO2, 4-NO2, and CHO) into various

positions of the benzene ring. The presence of additional substituents in various

positions of the aromatic or hetero aromatic rings of biologically active compounds

changes the electron density distribution, and the spatial structure molecule changes

in these parameters may change the drug transport in the body and any match

between the receptor and the drug, thereby altering the strength of the drug action.

All the synthesized compounds were active against the Gram-negative bacteria

except 3g and 3h. In 3g, an aldehyde group is present, and in 3h, it contains athiophene ring instead of the benzene ring. Further, compounds 3d and 3f are activeagainst S. aureus and 3a and 3h are active against B. cereus. The antibacterialactivity of [5] rhodanine-3-acetic acid moiety with a similar type of compounds and

a chalcone system in the C-5 position was determined against Gram-positive and

Gram-negative bacteria. It has been reported that compounds with an electron-

withdonating group showed lower activity than the halogens. Furthermore,

the position of the substituent on the phenyl ring significantly influenced the

antibacterial substituents in the para position showing higher activity than the other

positions. In the present study, we have prepared the rhodanine compounds with

electron-donating groups. Our results also coincide with the results of Chen et al. [5]

that substituents in the para position performed well when compared with the other

substituents. One more observed difference is that compounds 3ag are activeagainst E. coli, whereas the compunds reported in the literature are not active

against it. This may be due to the introduction of an a-methyl ethanoic moiety in the

Table 3 Antibacterial activity against methicillin-resistant Staphylococcus aureus MTCC84 (MRSA) byzone of inhibition and MIC for the compounds 2 and 3ah

Compounds Zone of inhibition (mm) MIC

100 ll 150 ll 200 ll 250 ll (lg/ml)

2 12 25

3a 16 25

3b 14 25

3c 11 25

3d 22 24 20

3e 14 25

3f 12 25

3g 10 12 20

3h [25


123

nitrogen atom. This suggests that compounds possessing rhodanine-3-a-methylpropionic acid moieties may present greater antibacterial properties. These results

suggested that further development of such compounds may be of interest.

Conclusion

Eight novel 3-a-carboxy ethyl-5-benzylidene rhodanines, 3ah, have been synthe-sized and evaluated for their antibacterial activity against E. coli, S. aureus, and B.

cereus, including MRSA by zone of inhibition and MIC method and anticancer

activity against HeLa cell lines by MTT assay. Compounds 3b and 3g are morepotent with respect to anticancer activity. All the compounds possess antibacterial

activity; howevers compounds 3b, 3c, 3d, 3f, and 3g showed a good antibacterialactivity against the tested organisms including MRSA. The results suggested that

further development of such compounds with rhodanine moiety may be of interest.

Experimental protocols

Synthesis

Melting points were determined in a XT-5 digital melting point instrument and are

uncorrected. IR spectra were recorded on a Shimadzu 360 FT-IR spectrometer. 1H

NMR and 13C NMR spectra were measured at 400 and 125 MHz, respectively, on a

Bruker-400 spectrometer using TMS as internal standard and DMSO-d6 as solvent.

MS spectra were obtained on a Shimadzu MS instrument. Elemental analyses were

determined using a PerkinElmer 240C Elemental Analyzer.

General procedure for synthesis of 3-a-carboxy ethyl rhodanine (2)

Compound 2 was prepared by the method reported [11]. In a round-bottomed flaskequipped with a magnetic stirrer, 8.902 g (100 mmol) of dl-alanine were dissolved

in 14 ml of 40 % aqueous sodium hydroxide solution (wt/vol) and 28 ml of water.

The solution was cooled to room temperature (25 C) and 7.61 g (100 mmol) ofcarbon disulfide were added with stirring which was continued for 3 h. To this was

then added 9.45 g (100 mmol) of chloroacetic acid which had been neutralized with

saturated sodium carbonate solution, and stirring was continued for 3 h. The

reaction mixture was made acidic with dilute HCl until the pH was 1.0, and refluxed

overnight. The reaction mixture was neutralized with saturated sodium carbonate

solution and again acidified with dilute HCl. The product formed was washed with

water, dried, and recrystallized with ethanol.

2-(4-oxo-2-thioxo thiozolidine-3-yl) propanoic acid (2)

UV (MeOH) kmax (nm): 294.50, 260.50; IR (KBr)cm-1 : 2,5003,400 (br. band,

COOH) and 1,729 (C=O); 1H NMR(DMSO-d6): d 1.439(d, J = 7.2 HZ, 3H, H-10),


123

4.219(s, 2H, H-5), 5.279(q, J = 7.2 HZ, 1H, H-20), 3.80 (s, 1H, H-30); 13C

NMR(DMSO-d6): d 13.73 (C-10), 34.71 (C-5), 54.31 (C-20), 169.83 (C-30), 173.75(C-4) and 203.10 (C-2). Anal. Calcd. for C6H7NO3S2: C, 35.11; H, 3.44; N, 6.82;

found: C, 35.08; H, 3.46; N, 6.83.

General procedure for the preparation of compounds (3ah)

To a solution of compound 2 (0.001 mol) and anhydrous sodium acetate (0.001 mol)in glacial acetic acid, were added the respective aldehydes (3ah).The mixture wasstirred under reflux for 46 h and then poured into ice-cold water. The precipitate

was filtered and washed with water. The dried product was recrystallized with

ethanol. The yield, melting point, and spectral data of each compound are given

below.

2-(5-benzylidene-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3a)

UV (MeOH) kmax (nm): 375.50, 272.50; IR (KBr)cm-1 : 2,5003,400 (br. Band,

COOH), 1,724 (C=O), and 1,589 (C=C); 1H NMR (DMSO-d6): d 1.56 (d, J = 7.2HZ, 3H, H-1

0), 5.62 (q, J = 7.2 HZ, 1H, H-20), 3.30 (s, 1H, H-30), 7.55 (m, 3H, H-300,400, 500), 7.65 (dd, J = 7.0 HZ, 2.0 HZ, 2H, H-100, 600), 7.84 (s, 1H, H-6);

13C NMR

(DMSO-d6): d 13.38 (C-10), 52.81 (C-20), 121.46 (C-5), 129.52 (C-200, 600), 130.67(C-300, 500), 131.14 (C-400), 132.84 (C-100), 133.65 (C-6), 166.18 (C-30), 169.48 (C-4)and 192.98 (C-2). Anal. Calcd. for C13H11NO3S2: C, 53.22; H, 3.78; N, 4.77; found:

C, 53.23; H, 3.75; N, 4.81.

2-(5-(4-methoxy benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3b)

UV (MeOH) kmax (nm): 392.00, 289.00; IR (KBr)cm-1: 2,5003,400 (br. Band,

COOH), 1,743 (C=O), 1,697 (C=O), 1,585 (C=C), 1,172 (CO); 1H-NMR (DMSO-

d6): d 1.55 (d, J = 7.2 HZ, 3H, H-10), 3.85 (s, 3H, H-700), 5.62 (q, J = 7.2 HZ, 1H,H-20), 7.15 (d, J = 8.8 HZ, 2H, H-200, 600), 7.64 (d, J = 8.8 HZ, 2H, H-300, 500), 7.80(s. 1H, H-6); 13C-NMR (DMSO-d6): d 13.32 (C-10), 52.66 (C-20), 55.53 (C-700),115.14 (C-300, 500), 117.97 (C-5), 125.32 (C-200, 600), 132.95 (C-100), 133.80 (C-6),161.64 (C-400), 166.19 (C-30), 169.48 (C-4) and 192.68 (C-2). Anal. Calcd. forC14H13NO4S2: C, 52.00; H, 4.05; N, 4.33; found: C, 52.04; H, 4.03; N, 4.31.

2-(5-(4-chloro benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3c)

UV (MeOH) kmax (nm): 379.00; IR (KBr)cm-1 : 2,5003,400 (br. band, COOH),

1,770 (C=O), 1,693 (C=O), 1,577 (C=C), 655 (CCl); 1H-NMR (DMSO-d6): d 1.55(d, J = 7.2 HZ, 3H, H-1

0), 5.59 (q, J = 7.2 HZ, 1H, H-20), 7.67 (m, 4H, H-200, 300, 500,600), 7.83 (s, 1H, H-6); 13C-NMR (DMSO-d6): d 13.32 (C-10), 52.85 (C-20), 122.40(C-5), 129.51 (C-200, 600), 131.67 (C-100), 132.09 (C-400), 132.19 (C-300, 500), 135.70(C-6), 166.05 (C-30), 169.36 (C-4) and 192.60 (C-2). Anal. Calcd. forC13H10ClNO3S2: C, 47.63; H, 3.07; N, 4.27; found: C, 47.67; H, 3.06; N, 4.26.


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2-(5-(2-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3d)


COOH), 1,712 (C=O), 1,600 (C=C), 1,346 (NO); 1H-NMR (DMSO-d6): d 1.57 (D,J = 7.2 HZ, 3H, H-1

0), 5.60 (q, J = 7.2 HZ, 1H, H-20), 3.20 (s, 1H, H-30), 7.76 (m,2H, H-400, 500), 7.92 (m, 1H, H-600), 8.07 (s, 1H, H-6), 8.25 (d, J = 7.2 HZ, 1H,H-300); 13C-NMR (DMSO-d6): d 13.41 (C-10), 53.12 (C-20), 125.59 (C-300), 126.08(C-5), 128.63 (C-400), 129.50 (C-600), 130.08 (C-100), 131.51 (C-500), 134.72 (C-6),147.85 (C-200), 165.43 (C-30), 169.35 (C-4) and 193.14 (C-2). Anal. Calcd. forC13H10N2O5S2: C, 46.15; H, 2.98; N, 8.28; found: C, 46.13; H, 3.00; N, 8.25.

2-(5-(3-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3e)

UV (MeOH) kmax (nm): 370.00, 362.50, 264.00; IR (KBr)cm-1 : 2,5003,400 (br.

Band, COOH), 1,716 (C=O), 1,604 (C=C), 1,350 (NO); 1H-NMR (DMSO-d6): d 1.56(d, J = 7.2 HZ, 3H, H-1

0), 3.40 (s, 1H, H-30), 5.61 (q, J = 7.2 HZ, 1H, H-20), 7.85 (t,J = 8.00 HZ, 1H, H-5

00), 8.00 (s, 1H, H-6), 8.06 (d, J = 8.00 HZ, 1H, H-600), 8.34 (d,1H, H-400), 8.52 (s, 1H, H-200); 13C-NMR (DMSO-d6):d 13.41 (C-10), 53.11 (C-20),124.47 (C-5), 124.96 (C-400), 125.12 (C-200), 130.94 (C-500), 131.06 (C-600), 134.48 (C-100), 135.72 (C-6), 148.32 (C-300), 165.98 (C-30), 169.37 (C-4) and 192.43 (C-2). Anal.Calcd. for C13H10N2O5S2: C, 46.15; H, 2.98; N, 8.28; found: C, 46.16; H, 2.96; N, 8.31.

2-(5-(4-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3f)

UV (MeOH) kmax (nm): 376.50, 280.50; IR (KBr)cm-1: 2,5003,400 (br. band,

COOH), 1,712 (C=O), 1,593 (C=C), 1,342 (NO); 1H-NMR (DMSO-d6): d 1.57 (d,J = 6.8 HZ, 3H, H-1

0), 5.62 (q, J = 7.2 HZ, 1H, H-20), 7.88 (t, J = 8.8 HZ, H-6, 200,600), 8.37 (d, J = 9.2 HZ, 2H, H-300, 500);

13C-NMR (DMSO-d6): d 18.58 (C-10),58.37 (C-20), 129.57 (C-200, 600), 131.03 (C-5), 135.76 (C-100), 136.69 (C-300, 500),144.08 (C-6), 152.96 (C-400), 171.26 (C-30), 174.71 (C-4) and 197.77 (C-2). Anal.Calcd. for C13H10N2O5S2: C, 46.15; H, 2.98; N, 8.28; found: C, 46.18; H, 2.99; N,

8.25.

2-(5-(4-formyl benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3g)

UV (MeOH) kmax (nm): 441.00, 416.50, 384.50, 278.50; IR (KBr)cm-1 :

2,5003,400 (br, band, COOH), 1,701 (C=O), 1,604 (C=C); 1H-NMR (DMSO-

d6): d 1.56 (d, J = 7.2 HZ, 3H, H-10), 5.59 (q, J = 7.2 HZ, 1H, H-20), 7.87 (m, 3H,H-6, 200, 600), 8.06 (d, J = 8.4 HZ, 2H, H-300, 500), 10.06 (s, 1H, H-700);

13C-NMR

(DMSO-d6): d 13.33 (C-10), 53.00 (C-20), 130.11 (200, 600), 131.00 (300, 500), 131.37(C-5), 131.68 (C-400), 136.65 (C-100), 138.03 (C-6), 166.02 (C-30), 169.32 (C-4) and192.53 (C-2, 700). Anal. Calcd. for C14H11NO4S2: C, 52.32; H, 3.45; N, 4.36; found:C, 52.28; H, 3.47; N, 4.35.


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2-(5-(furan-2-yl methylene)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3h)


COOH), 1,700 (C=O), 1,608 (C=C); 1H-NMR (DMSO-d6): d 1.53 (d, J = 7.2 HZ,3H, H-10), 5.59 (q, J = 7.2 HZ, 1H, H-20), 6.80 (dd J = 3.6 HZ, 1.6 HZ, H-400), 7.26(d, J = 3.6 HZ, H-3

00), 7.67 (s, 1H, H-6), 3.20 (s,1H, H-30); 13C-NMR (DMSO-d6):d13.87 (C-10), 52.84 (C-20), 114.02 (C-400), 118.03 (C-5), 119.24 (C-300), 120.81 (C-6), 148.81 (C-500), 149.43 (C-200), 165.84 (C-30), 169.46 (C-4) and 193.68 (C-2).Anal. Calcd. for C11H9NO4S2: C, 46.63; H, 3.20; N, 4.94; found: C, 46.66; H, 3.19;

N, 4.98.

MTT assay for cytotoxicity screening

The human cervical cancer cell line was obtained from National Centre for Cell

Science (NCCS), Pune, and grown in Eagles minimum essential medium

containing 10 % fetal bovine serum (FBS). Cells were maintained at 37 C, 5 %CO2, 95 % air, and 100 % relative humidity. Maintenance cultures were passaged

weekly, and the culture medium was changed twice a week. The monolayer cells

were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single

cell suspensions, and viable cells were counted using a hemocytometer and diluted

with medium containing 5 % FBS to give the final density of 1 9 105 cells/ml. One

hundred microlitres per well of cell suspension were seeded into 96-well plates at a

plating density of 10,000 cells/well and incubated to allow for cell attachment at

37 C, 5 % CO2, 95 % air, and 100 % relative humidity. After 24 h, the cells weretreated with serial concentrations of the test samples. They were initially dissolved

in neat dimethylsulfoxide (DMSO) to prepare the stock (200 mM) and stored frozen

prior to use. At the time of drug addition, an aliquot of frozen concentrate was

thawed and diluted to twice the desired final maximum test concentration with

serum-free medium. An dditional four, 10-fold serial dilutions were made to provide

a total of five drug concentrations. Aliquots of 100 ll of these different drugdilutions were added to the appropriate wells already containing 100 ll of medium,resulting in the required final drug concentrations. Following the drug addition, the

plates were incubated for an additional 48 h at 37 C, 5 % CO2, 95 % air, and100 % relative humidity. The medium containing no samples served as control and

triplicates were maintained for all concentrations. After the 48 h incubation, 15 llof MTT (5 mg/ml) in phosphate buffer saline (PBS) was added to each well and

incubated at 37 C for 4 h. The medium with MTT was then flicked off and theformed Formosan crystals were solubilized in 100 ll of DMSO and then measuredfor absorbance at 570 nm using a micro-plate reader. The percentage cell inhibition

was determined using the following formula.

% Cell inhibition 100 Abs sample =Abs control 100A nonlinear regression graph was plotted between % cell inhibition and log10concentration, and IC50 was determined using Graph Pad Prism software.


123

Antibacterial activity

The bacterial strain methicillin-resistant S. aureus MTCC84 (Tables 3, 4) was

inoculated in the nutrient broth under aseptic conditions and incubated at 37 C for18 h. After the incubation period, the test bacterial was swabbed on the nutrient agar

plate using a sterile cotton swab. In each of these plates, wells were cut out using a

sterile cork borer. All the synthesized compounds were dissolved in DMSO with four

different concentrations (100, 150, 200, and 250 ll) and used for the activity. Then, thePetri dishes were incubated at 37 C for 14 h. The antibacterial activity was evaluatedby measuring the diameter of the zone of inhibition in millimeters. The same method

was carried out for E. coli MTCC724, S. aureus MTCC430, and B. cereus MTCC441.

Acknowledgments We thank Karpagam University for providing the Karpagam University ResearchFellowship.

References

1. T. Tomasic, L.P. Masic, Curr. Med. Chem. 16(13), 1596 (2009)2. S. Ravi, K.K. Chiruvella, K. Rajesh, V. Prabhu, S.C. Raghavan, Eur. J. Med. Chem. 45, 2748 (2010)3. B.T. Moorthy, S. Ravi, M. Srivastava, K.K. Chiruvella, H. Hamlal, O. Joy, S.C. Raghavan, Bioorg.

Med. Chem. Lett. 20, 6297 (2010)4. G.A. Patani, E.J. Lavoie, Chem. Rev. 96, 3147 (1996)5. Z.H. Chen, C.J. Zheng, L.P. Sun, H.R. Piao, Eur. J. Med. Chem. 45, 5739 (2010)6. D. Hardej, C.R. Ashby Jr, N.S. Khadtare, S.S. Kulkarni, S. Singh, T.T. Talele, Eur. J. Med. Chem. 45,

5827 (2010)

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10. F.X. Liu, C.J. Zheng, L.P. Sun, X.K. Liu, H.R. Piao, Eur. J. Med. Chem. 46, 3469 (2011)11. N.S. Habib, S.M. Ridal, E.A.M. Badaweyl, H.T.Y. Fahmyl, H.A. Ghozlanz, Eur. J. Med. Chem. 32,

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Table 4 Antibacterial activity of compounds 2 and 3ah against human pathogens

Compounds Zone of inhibition (mm)

E. coli MTCC724

Gram-negative

S. aurues MTCC430

Gram-positive

B. cereus MTCC441

Gram-positive

2 Mild Mild

3a 12 4 6

3b 16 4 Mild

3c 24 Mild

3d 10 6

3e 14 Mild Mild

3f 18 10

3g 4 4

3h 6 4 6


123

Synthesis, antibacterial activity against MRSA, and in vitro cytotoxic activity against HeLa cell lines of novel 3- alpha -carboxy ethyl-5-benzylidene rhodanine derivativesAbstractIntroductionChemistryResults and discussionConclusionExperimental protocolsSynthesisGeneral procedure for synthesis of 3- alpha -carboxy ethyl rhodanine (2)2-(4-oxo-2-thioxo thiozolidine-3-yl) propanoic acid (2)General procedure for the preparation of compounds (3a--h)2-(5-benzylidene-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3a)2-(5-(4-methoxy benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3b)2-(5-(4-chloro benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3c)2-(5-(2-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3d)2-(5-(3-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3e)2-(5-(4-nitrobenzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3f)2-(5-(4-formyl benzylidine)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3g)2-(5-(furan-2-yl methylene)-4-oxo-2-thioxothiozolidene-3-yl) propanoic acid (3h)

MTT assay for cytotoxicity screeningAntibacterial activity

AcknowledgmentsReferences

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