Synthesis, characterization and screening of antimicrobial, antituberculosis, antiviral and anticancer activity of novel 1,3-thiazolidine-4-ones derived from 1-[2-(benzoylamino)-4- (methylthio)butyryl]-4-alkyl/arylalkyl thiosemicarbazides †

1,3-Thiazolidine-4-ones have been known to possess miscellaneous pharmacological activities such as antibacterial, antimycobacterial, antiviral, anticancer and anticonvulsant. Therefore, we synthesized some novel 1-[2-(benzoylamino)-4-(methylthio)butyryl]-4-alkyl/aryl alkyl thiosemicarbazides, N -{1-[[2-(3-Alkyl/arylalkyl-4-oxo-1,3-thiazolidin-2-ylidene) hydrazino


Introduction
The enhanced prevalence of infectious diseases threatens world population.Although tuberculosis appeared as a curable disease for years, it is regaining importance due to the multidrug resistance. 1,2Worldwide statistics on tuberculosis surprisingly reveals that, nearly one-third of the world's population is infected with tuberculosis, with approximately eight million new patients every year. 3A major issue is the increase of multi-drug resistant tuberculosis (MDRTB) giving rise to the disease expensive and incurable especially in immunodeficient subjects such as AIDS patients. 4Hence, there is an increased demand to develop new antituberculosis agents effective against pathogens resistant to current treatment.
Human immunodeficiency virus type 1 (HIV-1) has been recognized to be responsible for transmission and progress of acquired immune deficiency syndrome (AIDS). 5There are many targets for anti -HIV drug development due to the unique nature of the replication of HIV-1.Of these target macromolecules, reverse transcriptase (RT) plays an essential role in the replicative cycle of HIV-1. 6A key step for the replication and spread of the virus requires reverse transcription of the retroviral RNA to proviral DNA by the enzyme reverse transcriptase (RT). 7,8NRTIs bind in a noncompetetive way to a unique region on the enzyme, namely nonsubstrate-binding site, resulting in alteration of its function and therefore achieving supression of HIV-1 replication with high selectivity. 9Although combined use of nucleoside inhibitors of RT (NRTIs), non-nucleoside RT inhibitors (NNRTIs) and protease inhibitors (PIs) in the treatment of HIV infections may provide a dramatic recovery in most HIV-infected persons, resistance to the currently used agents remains as a clinical problem. 5Consequenlty, there is still a need for novel and safe antiviral agents with potent and selective action which are also effective against mutant strains of HIV.Nevirapine, etravirine and efavirenz are representative examples of NNRTIs used in clinical practice today. 10Ongoing studies for discovery of new antiviral agents then gave rise to novel types NNRTIs which include tetrahydroimidazobenzodiazepinthione (TIBO) compounds 11 , phenethylthiazolylthiourea (PETT) derivatives 11 , and 1H,3H-thiazolo [3,4-a]benzimidazoles (TBZs). 12ecent studies on molecular modification of the latter (TBZs) revealed that, dismantling of the imidazole nucleus leading to the design of new 1,3-thiazolidin-4-one derivatives, maintained the molecular requirements for enzyme inhibition. 12 literature search revealed that, 4-thiazolidinone derivatives may exhibit antibacterial [13][14] , antituberculosis, [15][16][17] antiviral 9,12,[18][19][20][21][22][23] and anticancer [24][25][26][27] properties.According to Andres et al. 13 , 4-thiazolidinones may be considered as phosphate bioisosteres and therefore inhibit the bacterial enzyme MurB which is involved in the biosynthesis of peptidoglycan layer of the cell wall. 13In addition, some thiazolidinones were recently reported as novel inhibitors of mycobacterial rhamnose synthetic enzymes. 4This new approach is believed to be selective as rhamnose which is not found in humans, has been shown to be essential for mycobacterial cell wall synthesis. 4ased on these observations, we have designed and synthesized a number of 2,3-disubstituted 1,3-thiazolidin-4-ones 8-11 as well as their 5 Structures of the synthesized compounds were elucidated by the use of their UV, IR, 1 H-NMR, 13 C-NMR, HR-EI and HR-FAB Mass spectral data.The presence of the thiazolidinone pharmacophore as reported in previous studies, led us to investigate their antimicrobial, antimycobacterial and antiviral properties.The synthesized compounds were characterized by their IR, 1 H-NMR, 13 C-NMR, HR-EI and HR-FAB Mass Spectral data.The IR spectra of compound 1 was characterized by the presence of a new C=O absorption band at 1637 cm -1 .IR spectral data of compound 2 was also confirmed on the basis of IR spectral data in literature. 28The band at 1650 cm -1 was attributed to the C=O streching band of hydrazide.The bands at 3315 cm -1 and 3267 cm -1 were assigned to NH 2 bands of hydrazide and they did not occur in the IR spectra of compounds 4-7.Absorption bands at 1212-1247 cm -1 , which were attributed to the C=S streching vibrations observed in the IR spectra of compounds 4-7.0][31] The Ar-Cl and Ar-F absorption bands were observed in the IR spectra of the compounds 12-27.The exhibited chemical shifts obtained from 1 H-NMR spectra of compounds 4-7 supported the proposed structures of the compounds.Resonances assigned to the N 1 -H, N 2 -H, N 4 -H protons of thiosemicarbazides 4-7 were detected at 10.05-10.12,9.23-9.35,7.47-8.943][34] Other NH proton of thiosemicarbazides 4-7 which belong to benzoylamino group were detected at 7.68-8.94ppm.Absence of resonances assigned to the N 1 -H, N 2 -H, N 4 -H protons of thiosemicarbazides 4-7 and the detected signals at about 3.65-3.97and 3.74-4.11ppm attributed to the CH 2 protons at the 5 th position of the 1,3thiazolidine-4-one ring supported the exact structures of 8-11.The endocyclic -CH 2 -protons are used to be detected as a singlet peak with an integration of two protons but in our work they were detected as two singlet peaks with an integration of two protons.The -NH-N=C< protons of 1,3thiazolidin-4-ones 8-11 were detected as two singlet peaks at 7.71-8.00ppm and 9.15-9.35ppm.The signals at 7.02-7.34were attributed to Ar-CO-NH. 1 H-NMR spectra of compounds 8-11 revealed the presence of two isomers as concluded from the -CONHN, -H 3 C-S-CH 2 -CH, endocyclic -CH 2 protons.As an appraisal the first singlet peak belongs to E isomer and the second one belongs to Z isomer and this may be explained on the basis of the difference in the relative stability of E and Z isomers formed due to the rotational restriction about the exocyclic N=C bond. 35Compound 8 was selected as prototype and its 13 C-NMR spectrum was used for further support.Detecting -H 3 C-S-CH 2 -CH, -H 3 C-S-CH 2 -CH, endocyclic -CH 2 , both of the C=O and some of the aromatic C atoms as two peaks in stead of one provided confirmatory evidence for geometric isomerism.

Chemistry
The representative example compound 8 was fragmented via three prominent pathways to afford fragments at m/z 306.0809, m/z 145.0303 and m/z 232.0955 based on thioether moiety, -CONH bond cleavage, benzoyl cation as base peak and methylidene sulfonium cation, respectively, in accordance with literature. 35,36Compounds 9-11 were fragmented via quasimolecular ion by HR-FAB 37 .
The purity of compounds 4-27 was demonstrated by TLC and HPLC.There were no spots or peaks accounting for the starting compounds ; but for some compounds we observed more than one peak with the same UV spectra.By using HPLC-UV/DAD peak homogeneity was proved.Resonances assigned to the endocyclic -CH 2 -protons at the 5 th position of the 1,3-thiazolidine-4one ring were not detected and instead of the mentioned proton's signal, two peaks with an integration of one proton symbolizing the methine proton of >C=CH-Ar moiety was observed between 7.23-8.11ppm. 38Considering the aromatic region of 1 H-NMR spectra of compounds 12-27 displayed signals of aromatic protons of arylidene segment as an evidence for the supposed structures.For further evidence to prove the geometric isomerism as a reason of rotational restriction of -N=C< and >C=CH-Ar bonds 13 C-NMR spectra of compound 13 was observed. 13C-NMR data of the representative compounds 8 and 13, which were obtained using DEPT technique at 100 MHz, have also supported the carbon framework by discrimination of CH 3 , CH 2 , CH and quarternary carbons (C q ) (see experimental section).On the other hand, high accuracy between experimental and calculated 13 C chemical shifts were also observed (Table 1).Calculations of the 13 C-NMR chemical shifts were performed using ACD/CNMR Predictor software available online at ACD/I-Lab Interactive Laboratory website (http://www.acdlabs.com/ilab).
Resonances due to the R 1 and R 2 groups of the compounds were recorded at expected values.The benzylic protons of compound 11 and compounds 23-27 were observed at 5.01-5.07ppm and 4.80-5.03ppm, respectively, and the benzylic protons of compound 11 could easily be differentiated from endocyclic CH 2 protons at the 5 th position of the 1,3-thiazolidine-4-one ring.
The representative example compound 14 fragmented via two prominent pathways to afford fragments by -CONH bond cleavage followed by dissociation of the thioether moiety and -NHN bond cleavage.

Biological Activity
In view of the antimycobacterial and antiviral activity ascertained for similar 4-thiazolidinones, the synthesized compounds were evaluated for their antibacterial, antituberculosis and antiviral effects.Compounds 24-26 exhibited marginal activity (MIC = 250 µg/ml) against Staphylococcus aureus ATCC 29213, Bacillus subtilis A57 and Candida albicans A177.Ofloxacin, sulbactam + cefoperazone and/or netilmicin were used as positive reference standards to determine the sensitivity of one strain/isolate in each microbial species tested.
In vitro evaluation of antimycobacterial activity against M.tuberculosis H37Rv was carried out at the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), National Institute of Allergy and Infectious Diseases, Southern Research Institute, Birmingham, Alabama, USA.Primary screen was conducted for all of the synthesized compounds 1-27 at 6.25 µg/ml against M.tuberculosis H37Rv in BACTEC 12B medium using the BACTEC 460 radiometric system.Rifampicin was used as the standard in the anti-tuberculosis activity screening.The most active derivative was identified as 27 with 90% inhibition of mycobacterial growth at 6.25 µg/ml.The remaining compounds showed marginal or no effect at this concentration.
Antiviral activities of the synthesized compounds were screened against various types of viruses in HEL, HeLa and Vero cell cultures.Anti-HIV and cytotoxicity data were also obtained with the compounds using the strains HIV-1(III B ) and HIV-2(ROD) in an MT-4/MTT based assay.The compounds were also evaluated for in vitro antiviral activity against herpes simplex virus [HSV-1 (strain KOS), thymidine kinase deficient (TK -) strain of HSV-1 resistant to acyclovir (ACV R ), HSV-2 (G)], vaccinia virus (VV) and vesicular stomatitis virus (VSV) in HEL cell cultures ; VSV, Coxsackie virus B4 and respiratory syncytial virus (RSV) in HeLa cell cultures ; Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus in Vero cell cultures.Acyclovir, Brivudin, (S)-DHPA, Ganciclovir and Ribavirin were used as standard drugs for comparison of the test compounds.For anti-HIV screening, nevirapine, delaviridine, efavirenz and zidovudine were used as the reference drugs.None of the tested compounds showed antiviral activity at subtoxic doses whereas some of them exhibited remarkable cytotoxic potential.
Compounds 8, 13, 14, 23, 25 were evaluated for their anticancer activity against 60 human tumoral cell lines derived from nine different cancer types (non-small cell lung, colon, breast, ovarian, renal, prostate, CNS, leukemia and melanoma) by the National Cancer Institute (NCI).They did not exhibit anticancer activity having GI 50 values at a high concentration.Therefore, these compounds were not selected for further testing.

Experimental Section
General Procedures.All melting points (ºC, uncorrected) were determined using Büchi 530 melting point apparatus.Infrared spectra were recorded in KBr disc using BIO-RAD FTS-135, FT-IR spectrometer and expressed in wavenumber ν (cm -1 ).NMR spectra were obtained on a Bruker AVANC-DPX 400 NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13 C-DEPT, the chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) using CDCl 3 , CDCl 3 -acetone and CDCl 3 -DMSO as solvent.High resolution electron impact and fast atom bombardment mass spectra were recorded on a Jeol JMS-700 instrument.The liquid chromatographic system, used in the present study, consisted of an Agilent technologies 1100 series instrument equipped with a quaternary solvent delivery system and a model Agilent series G-1315 A photodiode array detector.A Rheodyne syringe loading sample injector with a 50 µl sample loop was used for the injection of analytes.Chromatographic data were collected and processed using Agilent Chemstation Plus software.The separation was performed at ambient temperature, on a reversed phase NovoPak -C18 column (150 x 3.9 mm ; 5 µm particle size).All experiments were employed in the isocratic mode.The mobile phase was prepared by mixing methanol, acetonitrile and distilled water (50:10:40, v/v/v).This phase was filtered through a 0.45 µm membrane and degassed by ultrasonication, prior to use.Solvent delivery was employed at a flow rate of 1.0 ml.min -1 .Detection of the analytes was carried out at 210 nm.

Antibacterial assay
Newly synthesized compounds were individually tested against laboratory strains of totally 64 microbial culture isolates of 56 bacteria and 7 fungi and 1 yeast species which described previously 17 were tested by using disc-diffusion, micro-well dilution assay 41,42 and MIC agar dilution assay 43 .Microorganisms were provided by the Department of Biology, Faculty of Art and Science, and at Atatürk University, Erzurum, Turkey.The identity of the microorganisms used in this study was confirmed by Microbial Identification System in Biotechnology Application and Research Center at Atatürk University.

Antiviral assay
Evaluation of the antiviral activity of the compounds against HIV-1 strain III B and HIV-2 strain (ROD) in MT-4 cells was performed using the MTT assay as previously described 44 .HIV-1(IIIB) 45 or HIV-2 (ROD) 46 stock (50 µl) at 100-300 CCID 50 (cell culture infectious dose) or culture medium was added to either the infected or mock-infected wells of the microtiter tray.Mock-infected cells were used to evaluate the effect of test compound on uninfected cells in order to assess the cytotoxicity of the test compound.Exponentially growing MT-4 cells 47 were centrifuged for 5 minutes at 1000 rpm and the supernatant was discarded.The MT-4 cells were resuspended at 6 x 10 5 cells/ml, and 50-µl volumes were transferred to the microtiter tray wells.Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically by the MTT assay.[50]

Antimycobacterial assay
The primary screen was conducted at 6.25 µg mL −1 against M. tuberculosis H37Rv in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA).
Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. 51ompounds effecting <90% inhibition in the primary screen (MIC > 6.25 µg mL −1 ) were not further evaluated.Compounds demonstrating at least 90% inhibition in the primary screen were re-tested at lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration (MIC) in the MABA.The MIC was defined as the lowest concentration inhibiting 99% of the inoculum.

Anticancer assay
A total of 60 human tumor cell lines derived from nine cancer types (non-small cell lung, colon, breast, ovarian, leukemia, renal, prostate, CNS, melanoma) formed the basis of this test.3] Density of the inoculum depended on the types of tumor cells and their growth characteristics. 54These cells are then preincubated on the microtiter plate for 24h before adding the compounds.These were tested starting from DMSO solutions at five different concentrations (10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 M).After an incubation of the chemical agent for 48h with the tumor cell lines, sulforhodamine B (SRB) protein assay was used to estimate cell viability and growth.The cytotoxic effects are evaluated and the assay results and doseresponse parameters were calculated as described. 55