合成及抗菌活性的新2 - (Z)((5-(4-Hydroxybenzylidene)-4-Oxo-4,5 Dihydrothiazol-2-Yl)Amino) Acid and its Derivatives

Abstract

A series of (Z)-2-((5-(4-hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol- 2-yl)amino) acid and its derivatives were synthesized from appropriate 2-thioxothiazolidin-4-one using nucleophilic substitution and Knoevenagel condensation. All compounds have been synthesized, characterized and screened for theirantimicrobial activityagainst gram positive bacteria and gram negative bacteria along with Ampicillin and Ciprofloxacin as standard drug. According to results obtained all compound shows good to moderate activity against all strain tested, compatible toAmpicillinbut lower than Ciprofloxacin. Amongst these compounds (6c) shows very good and moderate activity as compare to standards drugs against Gram positive bacteria B. subtilus and S. aureus. Some of the most potent compounds, namely (6b, 6h and 6j) possessed selectively antimicrobial activity.

Keywords

2-Thioxothiazolidin-4-One, Knoevenagel Condensation, Amino Acids, Antimicrobial Activity.

Introduction

Now a day the treatment of bacterial and fungal infections still remains a very challenging therapeutic problem because of emerginginfectious diseasesand the increasing number of multidrug-resistant microbial pathogens. Despite the manyantibioticsandchemotherapeuticsavailable, the emergence of old and newer antibiotic-resistant bacterial strains in the last two decades constitutes a substantial very needful for new classes of antimicrobial agents [1,2,3]. Compounds containing the 2-thioxothiazolidin- 4-one (Rhodanine) and its derivatives have also been reported to exhibit a broad spectrum of biological activities, such asantibacterial[4,5],antidiabetic[6],antifungal[7,8],anticancer[9],antitubercular[10,11],anti-HIV[12-14],antiparasitic[15],hypnotic[16] andantiproliferativeagents [17,18]. The rhodanine has been known for over 50 years, so there have been several attempts to design antimicrobial agents based on this heterocycle. There are various reports available on rhodanine derivatives as antimicrobial agents [19,20,21]. These reports suggested that a chain containing free carboxyl group at rhodanine nucleus was important to the observed levels of antimicrobial activity [22,23]. With this in mind, we initiated a program to synthesized rhodanine derivatives having amino acids chain as antimicrobial agent by preparing hybrid molecules having the similar features of reported potent antimicrobial agents (Figure 1).

In continuation of our work [24-26], on the synthesis of bioactive compounds, we have synthesized some rodanine analogues. In view of the facts mentioned above, rhodanine derivatives were synthesized (Z)-2-((5-(4-hydroxybenzylidene)-4-oxo-4,5- dihydrothiazol-2-yl)amino)amino) acid (6a-l). The compounds (3, 4, 6a-l) were characterized by different spectral analytical techniques and screened for their in vitro antimicrobial activities against pathogenic strains such as B. subtilis, S. aureus, E. coli, C. albicans, A. flavus, A. niger species. The synthesized compounds were characterizeTAAd on the basis of IR, 1H NMR and Mass spectral data [4-7].

Experimental

绕丹宁(2-thioxothiazolidin-4-one) 4-hydroxybenzaldehyde, anhydrous sodium acetate, triethylamine, dichloromethane, iodomethane and various solvents were commercially available. The major chemicals were purchased from Sigma Aldrich and Avra labs. Reaction courses were monitored by TLC on silica gel precoated F254 Merck plates. Developed plates were examined with UV lamps (254 nm). IR spectra were recorded on a FT-IR (Bruker). Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected. 1H NMR spectra (I, ppm) were recorded in DMSO-d6 solutions on a Varian-Mercury 400 MHz spectrometer using tetramethylsilane as the internal reference. Chemical shifts are reported as Ippm units. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). 13C NMR spectra were recorded in DMSO-d6 solutions on a Bruker Avance II 400 spectrometer at 400 MHz using tetramethylsilane as the internal reference. Mass spectra were taken with Micromass-QUATTRO-II of WATER mass spectrometer.

Synthesis of (Z)-5-(4-hydroxybenzylidene)-2-thioxothiazolidin-4-one (3)

In a 100 ml round bottom flask, equimolar amount of rhodanine (1 mmol), anhydrous sodium acetate (1 mmol) was added in glacial acetic acid (5 mL) and 4-hydroxybenzaldehyde was added to the reaction mixture. The mixture was stirred under reflux condition for 6 h. The progress of reaction was monitored by TLC (20% ethyl acetate: n-hexane). After completion of the reaction, the reaction mixture was poured into the ice-cold water. The precipitate was filtered, washed with water (3×10 mL), dried, and purified by recrystalization in ethanol as solvent to give 80% yield.

Synthesis of (Z)-5-(4-hydroxybenzylidene)-2-(methylthio)thiazol-4(5H)-one (4)

In a 100 ml round bottom flask, the compound (3) (1 mmol), triethylamine (1.5 mmol) was added in dichloromethane (5 mL) at room temperature. To the stirred reaction mixture with iodomethane (1.5 mmol) was added after 4 min and stirred for 2 h. at room temperature. The progress of reaction was monitored by TLC (10% chloroform: methanol). After completion of reaction the reaction mixture was concentrated in-vacuo. The residue was washed with water (3×15 mL) to afford the crude product. The crude product was recrystallized using ethanol as solvent to give yield in the range 82%.

Synthesis of (Z)-2-((5-(4-hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino) amino) acid (6a-l)

在50毫升圆底烧瓶中,化合物(4)(1mmol), amino acids (5a-l) (1.5 mmol) with a catalytic amounts of potassium carbonate (K2CO3) (1.5 mmol) were added in ethanol and water (1:1 v/v) and stirred for 30-55 min at room temperature. The progress of reaction was monitored by TLC (10% chloroform: methanol). After completion of reaction the reaction mixture was concentrated in-vacuo. The residue was washed with water (3×15 mL) to afford the crude product. The compounds (6a-l) were recrystallized from ethanol and isolated as yellow solid.

Results and Discussion

Chemistry

The compound (3) was then subjected to a Knoevenagel condensation with the appropriate rhodanine, which had themselves been synthesized using the reported procedure [27,28], to provide new series of target compounds (6a-l). The structures of the desired compounds were confirmed by IR, 1H NMR, 13C NMR, mass spectral analysis. The (Z)-5-(4-hydroxybenzylidene)- 2-thioxothiazolidin-4-one (3) was prepared in prominent good yields via a Knoevenagel condensation between the corresponding heterocyclic cores of rhodanine and 4-hydroxybenzaldehyde (Scheme 1).

2-thioxothiazolidin-4-one化合物syn为基础thesized by reflux with sodium acetate which acts as a base and glacial acetic acid as catalysts. In theory of E and Z geometrical isomers around the exocyclic double bond (CH=C) are possible for 5-benzylidene derivatives (3). The IR spectrum of (Z)-5-(4-hydroxybenzylidene)-2-thioxothiazolidin-4-one (3), showed a strong absorption band at 1672 cm-1 that is due to a carbonyl group. The mass spectrum revealed a molecular ion peak at m/z = 237 (M+H) corresponding to a molecular formula C10H7NO2S2. 1H NMR spectra of compounds (3) show only one signal for the methyne proton in the range I 7.70 ppm, at lower field values than those expected for the E-isomers, which was strongly indicates that the compounds have the Z-configuration. The isolation of single crystals for some compounds permitted us to corroborate the postulated structures including their Z-configuration by single crystal X-ray diffraction analysis [29,30]. Compound (4) was synthesized (Scheme 1) from Compound (3) and the structures of the desired compounds were confirmed by spectral analysis. The IR spectrum of (Z)-5-(4-hydroxybenzylidene)-2-(methylthio)thiazol-4(5H)-one (4), showed a strong absorption band at 1694 cm-1 that is due to a carbonyl group. The mass spectrum revealed a molecular ion peak at m/z = 252 (M+H) corresponding to a molecular formula C11H9NO2S2. 1H NMR spectra of compounds (4) show only one signal for the methyne proton in the range I 7.70 ppm, sulfur attached methyl proton shows the singlet in the range I 2.71 ppm. We synthesized series of novel (Z)-2-((5-(4-hydroxybenzylidene)- 4-oxo-4,5-dihydrothiazol-2-yl) amino) acid (6a-l) (Scheme 2从化合物(4),是一个甲基的位移sulfinyl group by amino acids from the C2 position of the thiazolone ring. The structures of the desired compounds (6a-l) were confirmed by IR, 1H NMR, 13C NMR, mass spectral analysis. The IR spectrum of (Z)-2-((5-(4-hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino) propanoic acid (6a), showed a strong absorption band at 1732 cm-1 that is due to a carbonyl of carboxylic group and 3386 cm-1 due to hydroxyl of carboxylic group. The mass spectrum revealed a molecular ion peak at m/z = 293.05 (M+H) corresponding to a molecular formula C13H12N2O4S. Their 1H NMR spectra revealed the signals of (6a) as a representative example, show one signal for the methyne proton in the range I 7.70 ppm, phenyl ring proton shows the multiplet in the range I 7.30–7.66 ppm, one of the methyl group proton shows doublet in the range of I 1.25–1.42 ppm and adjacent to carboxylic acid proton shows quartet in the range of I 4.51–4.85 ppm, carboxylic acid proton shows singlet in the range of I 14.13 ppm (Table 1).

Antimicrobial activity

All the synthesized compounds (3,4 and 6a-l) were screened for their in-vitro antimicrobial activity against two gram positive bacteria; Bacillus subtilis (NCIM-2063) and Staphylococcus aureus (NCIM-2901), one gram negative bacteria; Escherichia coli (NCIM-2256) and three fungal strains; Candida albicans (NCIM-3471), Aspergillus flavus (NCIM-539) and Aspergillus niger (NCIM-1196). The antibacterial activity of compounds was monitored by observing their Minimum Inhibitory Concentration (MIC, μg/mL) as previously mentioned [31,32,33] by broth dilution method with Ciprofloxacin and Ampicillin as control drugs. While the antifungal study was carried by standard agar dilution method were Fluconazole and Miconazole used as control drugs. Methanol was used as solvent control for both antibacterial and antifungal testing (Table 2).

Synthesized compounds of present series shows variety of antimicrobial activity, ranging from broad spectrum molecule active against the majority of bacterial and fungal strains tested to the narrow spectrum compounds, active only against only one strains. Amongst the series compounds (6b, 6g, 6h and 6j) are found to be most active molecules and they are specific towards the gram positive bacterial, S. aureus and B. subtilis. Compound (6j) is more active (MIC of 5.5 and 6.0 μg/mL against B. subtilis and S. aureus respectively) than both standards used in the experiment, while compound (6g and 6h) (MIC of 7.2 and 10 μg/mL against S. aureus and MIC of 6.0 and 10 μg/mL against B. subtilis) have better activity, but little lower than Ciprofloxacin and comparative level of activity as Ampicillin. The compounds (6b and 6c) have somewhat better activity compare to both the standard drugs, more important is that, they are broad spectrum in nature and shows the activity against majority of bacterial and fungal strains tested which is not shown by any other molecule of the series; both are active against bacteria S. aureus and B. subtilis, and fungus C. albicans, A. flavus and A. niger. On the other hand compounds (6d, 6e and 6f) are found to be narrow spectrum molecules, Remaining compounds of the series (3, 4, 6a, 6i, 6k, and 6l) have very high MIC values and therefore they are inactive as antimicrobial agents. It interesting to find out that bacterium E. coli is resistance to all compounds, suggest that molecules of the series may be inactive against gram negative bacteria.

Structure activity relationship (SAR)

The results of the antimicrobial screening demonstrated some definite and interesting facts about the structural-activity relationship (SAR) of synthesized thiazolone moiety. In majority of cases, dependence of activity profile on structural modifications of the molecule is clear and fascinating. Due to different types of amino acid and variation in activity profile of molecules are also directly attributed with the structural variations. The important highlights of structure-activity relationship are as follows

Effect of alkane chain:

In the present study it is clear that the activity profile of molecule is strongly affected by the branching pattern and chain length of alkane chain. Attachment of methyl group at C2 position on the thiazolone moiety (6a) makes molecule inactive against bacterial and fungal strains may be due to its small size and electron donating effects.

Effect of alkane chain with hydroxyl group:

Compounds (6g and 6l) containing alkane chain with hydroxyl groups with deferent position. Substitution by hydroxymethyl (6g) at C2 position on the thiazolone moiety make the molecule selective active against gram positive bacteria, S. aureus, while substitution by 1-hydroxyethyl (6l), however, make the molecule inactive.

Effect of heterocyclic ring:

Substitution by phenylmethyl (6d) at thiazolone moiety gives the inactive molecule towards S. aureus, B. subtilis, A. niger and C. albicans, not good as standard drugs. While 4-hydroxyphenyl (6k) substitution make the molecule inactive. This observation clearly shows that only phenyl ring gives inactive molecule and addition of hydroxyl group at position 4 causes lose in this activity.

Effect of heterocyclic ring:

The compounds (6j) containing methyl-imidazole ring at thiazolone moiety makes the molecule specifically active towards Gram positive bacteria B. subtilis and S. aureus.

Effect of sulfur containing group:

The compound (6e) with terminal methylthio group is inactive to all strain, but compound (6h) with terminal mercapto group shows specificity active towards Gram positive bacteria B. subtilis.

Conclusion

In Conclusion, the objective of the our present study was to synthesize and investigated the potent antimicrobial activities of some new (Z)-2-((5-(4-hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino) acid with the hope of discovering new structures that could be used as potent antimicrobial agents. Our aim has been verified by the synthesis of thiazolone moiety, different types of amino acids attached at C2 position on thiazolone moiety, which are very effective for the enhanced antimicrobial activities. Our results clearly revealed that the substituted thiazolone exhibited good antimicrobial activity.

Acknowledgement

The authors are thankful to the Head, Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004 (MS), India for providing the laboratory facility.

References

1. TomasicT, Zidar N, Rupnik V, Kovac A, Blanot D, et al.Synthesis and biological evaluation of new glutamic acid-based inhibitors of MurD ligase. Bioorg Med ChemLett.2009; 19: 153–157.
2. Sink R,KovacA,TomasicT,Rupnik V, Boniface A, et al.Synthesis and biological evaluation of N-acylhydrazones as inhibitors of MurC and MurD ligases. Chem Med Chem. 2008; 3: 1362–1370.
3. Mueller-Premru M, Zidar N, Spik, VC, Krope, A, Kikelj D. Benzoxazine series of histidine kinase inhibitors as potential antimicrobial agents with activity against enterococci. Chemotherapy , 2009; 55, 414–417.
4. MoelleringJr RC, Current Treatment Options for Community-Acquired Methicillin-Resistant Staphylococcus aureus Infection. ClinInfectDis, 2008; 46: 1032–1037.
5. GualtieriM, Bastide L, LatouchePV, LeonetteJP. In vitro activity of a new antibacterial rhodanine derivative against Staphylococcus epidermidis biofilms. J. Antimicrob. Chemother, 2006; 58: 778–783.
6. Murugan R, AnbazhaganS, NarayananSS, Synthesis and in vivo antidiabetic activity of novel dispiropyrrolidines through [3 + 2] cycloaddition reactions with thiazolidinedione and rhodanine derivatives. EurJ Med Chem, 2009; 4: 3272–3279.
7. PetrikaiteV,TaraseviciusE,PavilonisA.Newethacridine derivatives as the potential antifungal and antibacterial preparations. Medicina (Kaunas), 2007; 43: 657–663.
8. 8. Sortino M, Delgado P, Juarez S,Quiroga J, AboniaR,et al. Synthesis and antifungal activity of (Z)-5-arylidenerhodanines. BioorgMedChem.2007; 15: 484–494.
9. ChandrappaS,KavithaCV,ShahabuddinMS,VinayaK,Ananda CS, et al. Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl) methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells. BioorgMedChem.2009; 17: 2576–2584.
10. Brooke EW, Davies SG,Mulvaney AW, Okada M,Pompeo F, Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)et al.Bioorg Med Chem. 2003; 13: 2527–2530.
11. MallikarjunaBP,Sastry BS, Suresh Kumar GV,RajendraprasadY,ChandrashekarSM, et al. Synthesis of new 4-isopropylthiazole hydrazide analogs and some derived clubbed triazole, oxadiazole ring systems--a novel class of potential antibacterial, antifungal and antitubercular agents. EurJMedChem.2009; 4: 4739–4746.
12. Balzarini J, Orzeszko B, Maurin JK, Orzeszko A. Synthesis and anti-HIV studies of 2-adamantyl-substituted thiazolidin-4- ones.Eur J Med Chem.2007; 42: 993–1003.
13. MurugesanV,TiwariVS,SaxenaR,TripathiR,ParanjapeR,et al. Lead optimization at C-2 and N-3 positions of thiazolidin-4- ones as HIV-1 non-nucleoside reverse transcriptase inhibitors.Bioorg Med Chem. 2011; 19: 6919–6926.
14. Rawal RK, Tripathi R, Katti SB, Pannecouque C, Clercq ED. Non-nucleoside inhibitors of the hepatitis C virus NS5B RNAdependant RNA polymerase: 2-Aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives. Bioorg Med Chem. 2007; 15: 3134– 3142.
15. Zhang X, Li X, Li D, Qua G, Wang J.Ionic liquid mediated and promoted eco-friendly preparation of thiazolidinone and pyrimidine nucleoside–thiazolidinone hybrids and their antiparasitic activities. Bioorg Med ChemLett.2009; 19: 6280– 6283.
16. VermaA,Saraf SK. 4-thiazolidinone--a biologically active scaffold. EurJ Med Chem. 2008; 43: 897–905.
17. Gouveia FL, de Oliveira RMB, de Oliveira TB, da Silva IM, do Nascimento SC, et al. Synthesis, antimicrobial and cytotoxic activities of some 5-arylidene-4-thioxo-thiazolidine-2-ones. EurJMed Chem. 2009; 44: 2038–2043.
18. OttanaR,CarottiS,MaccariR, LandiniI, ChiricostaG, et al.In vitro antiproliferative activity against human colon cancer cell lines of representative 4-thiazolidinones. Part I. Bioorg Med ChemLett, 2005; 15: 3930–3933.
19. XuLL,Zheng CJ, Sun LP, Miao J, PiaoHR.Synthesis of novel 1,3-diaryl pyrazole derivatives bearing rhodanine-3-fatty acid moieties as potential antibacterial agents. EurJ Med Chem. 2012; 48: 174–178.
20. Sim MM, Ng SB, Buss AD,CrastaSC,Goh KL, et al. BenzylideneRhodanines as Novel Inhibitors of UDP-N-Acetylmuramate/l- Alanine Ligase. BioorgMedChemLett. 2002; 12: 697–699.
21. Jin X,Zheng CJ, Song MX, Wu Y, Sun LP, et al.Synthesis and antimicrobial evaluation of l-phenylalanine-derived C5-substituted rhodanine and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone. EuJ Med Chem. 2012; 56: 203–209.
22. Chen ZH,Zheng CJ, Sun LP,Piao HR. Synthesis of new chalcone derivatives containing a rhodanine-3-acetic acid moiety with potential anti-bacterial activity. EurJMedChem.2010; 45: 5739–5743.
23. Zheng CJ, Song MX, Sun LP, Wu Y, Hong L,etal.Synthesis and biological evaluation of 5-aryloxypyrazole derivatives bearing a rhodanine-3-aromatic acid as potential antimicrobial agents. Bioorg Med ChemLett.2012; 22:7024–7028.
24. PansareDN,MullaNA,PawarCD,ShendeVRShindeDB,One pot three components microwave assisted and conventional synthesis of new 3-(4-chloro-2-hydroxyphenyl)-2-(substituted) thiazolidin-4-one as antimicrobial agents. Bioorg Med Chem Lett.2014; 24: 3569–3573.
25. DarandaleSN,PansareDN,MullaNA,ShindeDB.Green synthesis of tetrahydropyrimidine analogues and evaluation of their antimicrobial activityBioorgMedChemLett. 2013; 23: 2632–2635.
26. DarandaleSN,MullaNA,PansareDN,Sangshetti JN, Shinde DB. A novel amalgamation of 1,2,3-triazoles, piperidines and thieno pyridine rings and evaluation of their antifungal activity.EurJ Med Chem. 2013; 65: 527–532.
27. PansareDN,Shinde DB. A facile synthesis of (Z)-5-(substituted)-2-(methylthio)thiazol-4(5H)-one using microwave irradiation and conventional method. Tetrahedron Lett.2014; 55: 1107–1110.
28. LesykRB,Zimenkovsky BS. 4-Thiazolidones: Centenarian History, Current Status and Perspectives for Modern Organic and Medicinal Chemistry. Curr Org Chem. 2004; 8: 1547–1577.
29. Delgado P,QuirogaJ,Cobo J, Low JN,GlidewellC.Supramolecular structures of four (Z)-5-arylmethylene-2-thioxothiazolidin-4-ones: hydrogen-bonded dimers, chains of rings and sheets. ActaCrystallogr, 2005; 61:4 77–482.
30. Delgado P,QuirogaJ,Cobo J, Low JN,GlidewellC.Three substituted (Z)-5-benzylidene-2-thioxothiazolidin-4-ones: hydrogenbonded dimers that can be effectively isolated or linked into chains either by aromatic [pi]-[pi] stacking interactions or by dipolar carbonyl-carbonyl interactions. ActaCrystallogr, 2006; 62: 382–385.
31. Collins AH, Microbiological Methods, (2nded), 1976. Butterworth: London.
32. Khan ZK. In vitro and in vivo screening techniques for bioactivity screening and evaluation, 1997.ProcInt Workshop UNIDOCDRI, 210.
33. Duraiswamy B, Mishra SK,SubhashiniV,Dhanraj SA, Suresh B.Studies on the antimicrobial potential of Mahonialeschenaultii Takeda root and root bark. Indian J Pharm Sci. 2006; 68: 389–391.