MEDICINAL CHEMISTRY RESEARCH
Med Chem Res (2013) 22:758–767 DOI 10.1007/s00044-012-0071-5
ORIGINAL RESEARCH
Synthesis, characterization, anticancer, and antioxidant activity of some new thiazolidin-4-ones in MCF-7 cells Arun M. Isloor • Dhanya Sunil • Prakash Shetty • Shridhar Malladi • K. S. R. Pai • Naseer Maliyakkl
Received: 17 December 2011 / Accepted: 19 April 2012 / Published online: 4 May 2012 Ó Springer Science+Business Media, LLC 2012
Abstract There are limited studies centring on the potential of thiazolidin-4-ones as anticancer agents. In this study, a new series of 2-(3-substituted-1H-pyrazol-4-yl)-3(3-substituted-5-sulfanyl-1,2,4-triazol-4-yl)-1,3-thiazolidin4-one (4a–o) have been synthesized by cyclo-condensation reaction of 5-substituted-4-[(3-substituted-1H-pyrazol-4ylmethylidene)amino]-2H-1,2,4-triazole-3-thione (3a–o) and thioglycolic acid. The structures of all the synthesized compounds were confirmed by elemental analysis, spectral techniques like IR, 1H NMR, and mass spectroscopy. Few compounds exhibited dose-dependent cytotoxic effect in MTT assay in human breast cancer (MCF-7) cells. Apoptotic degradation of DNA due to action of potent thiazolidin-4-ones was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).
A. M. Isloor (&) S. Malladi Medicinal Chemistry Laboratory, Department of Chemistry, National Institute of Technology, Surathkal, Mangalore 575 025, India e-mail:
[email protected] D. Sunil Department of Chemistry, Manipal Institute of Technology, Manipal University, Manipal 576 104, India P. Shetty Department of Printing and Media Engineering, Manipal Institute of Technology, Manipal University, Manipal 576 104, Karnataka, India K. S. R. Pai N. Maliyakkl Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576 104, India
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A concentration-dependent increase in tail length and olive tail moment was observed when treated with thiazolidin-4ones. In vitro antioxidant studies like DPPH and ABTSfree radical scavenging assays-indicated moderate activity of thiazolidin-4-ones. Keywords Thiazolidin-4-one Cytotoxicity DNA fragmentation Antioxidant activity
Introduction Cancer, characterized by uncontrolled, rapid, and pathological proliferation of abnormal cells, is the second leading cause of human death after cardiovascular diseases in developing as well as advanced countries (Bandgar et al., 2010). Although there are many therapeutic strategies including chemotherapy and radiotherapy, high-systemic toxicity, and drug resistance limit the successful outcomes in most cases. Breast cancer is one of the oldest known forms of tumors in humans. Globally, breast cancer is the most common cancer in women, after skin cancer, representing 16 % of all female cancers (Porter, 2008). Since 1970s, the number of cases has significantly increased worldwide, partly attributed to this lifestyle. The treatment of breast cancer starts with surgery, and then chemotherapy, radiation, or they are given in combinations. One of the most common treatments is cyclophosphamide plus doxorubicin; these drugs damage DNA in the cancer, but also cause serious side effects in normal cells (Lee et al., 2002). Damage to the heart muscle is the most dangerous complication of doxorubicin. Therefore novel diagnosis, treatment, and prevention approaches are immediately required for breast cancer therapy. The development of effective,
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selective, and less toxic anticancer agents remains an important and challenging goal in medicinal chemistry. In recent years, numerous 1,2,4-triazole derivatives have been found to be associated with antimicrobial (Isloor et al., 2009; Demirbas et al., 2004; Malladi et al. 2011a, b; Swamy et al., 2006; Palekar et al., 2009), anti-inflammatory (Malladi et al. 2011a), analgesic (Mathew et al., 2007), and anticancer (Holla et al., 2002, Ibrahim 2009, Padmavathi et al., 2009) properties. AK-2123 (Sanazol), a nitrotriazole hypoxic cell sensitizer has apparently improved results in head and neck cancers, uterine cervical cancers, and other solid tumors when added to radical radiotherapy (Dobrowsky et al., 2005). Further, existing literature indicates 1,2-pyrazole derivatives to possess various biological activities (Chandrakantha et al., 2011; Vijesh et al., 2010; Vijesh et al. 2011a, b). Non-steroidal aromatase inhibitors obtained from triazole derivatives are currently used in the treatment of breast cancer (Brueggemeier et al., 2004). Thiazolidinones were reported to exhibit several biological properties (Amir and Azam, 2004; Hafez and El-Gazzar, 2009; Siddiqui et al., 2007; Selvi and Rajendran, 2007; Wilson et al., 2008). Reactive oxygen species (ROS)-like superoxide anion, hydroxyl radical, and hydrogen peroxide attack various biological molecules like proteins, enzymes, DNA, and other biomolecules. Oxidative stress conditions give rise to a number of inflammatory, metabolic disorders, cellular aging, and cancer (Bandgar et al., 2009, 2010). Antioxidants inhibit oxidative damage induced by free radicals and ROS. Thus, antioxidant therapy has also gained immense significance in the treatment of cancer. In view of the wide spectrum of medicinal applications of triazole, pyrazole, and thiazolidinone derivatives and in continuation of our research on new anticancer molecules, we hereby report the synthesis, characterization, anticancer, and antioxidant activities of some new thiazolidin-4-ones.
Results and discussion
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substituted-1H-pyrazol-4-ylmethylidene)amino]-2H-1,2,4triazole-3-thiones (Schiff bases) (3a–o) (Sunil et al., 2011). The new 2-(3-substituted-1H-pyrazol-4-yl)-3-(3substituted-5-sulfanyl-1,2,4-triazol-4-yl)-1,3-thiazolidin-4ones (4a–o) were synthesized by the cyclo-condensation of the Schiff bases with thioglycolic acid in dimethyl formamide in presence of anhydrous zinc chloride (Selvi and Rajendran, 2007). The reaction sequence has been presented in Scheme 1 and the plausible mechanism for the formation of thiazolidinone has been given in Scheme 2. The structures of the newly synthesized compounds were established by spectral data and elemental analysis. Thin layer chromatography was conducted on 0.25 9 10-3 m silica gel plates to monitor the progress and to check the purity of the compounds. A 1:1 mixture of ethyl acetate and petroleum ether solution was used as the eluent. Visualization was made by using iodine vapors. The IR spectra in KBr pellets were recorded using Schimadzu FTIR 8400S spectrophotometer. 1H NMR spectra were recorded in deuterated dimethyl sulphoxide in AV500 NMR spectrometer using tetramethyl silane as internal standard. The mass spectra were recorded in a Schimadzu GCMS-QP5050 mass spectrometer. The elemental analysis was done in Flash thermo 1112 series CHN analyser. Melting points were determined by open capillary method and were uncorrected. IR spectra of the newly synthesized thiazolidinones displayed a strong absorption band due to C=O stretching at 1,730 cm-1. The 1H NMR spectra showed a singlet at 5.05 ppm due to the methylene protons in thiazolidinone ring. The singlet corresponding to the tertiary methine protons of thiazolidinone ring was observed at 5.35 ppm. The O-CH2 protons resonated at 5.63 ppm and were observed as a singlet. The pyrazole C–H protons resonated at 6.92 ppm. Singlet at 12.20 ppm is due to pyrazole NH, the broad singlet at 13.78 ppm was due to the S–H protons. The aromatic protons were seen resonating between 7.1 and 7.7 ppm. The mass spectra of all newly synthesized thiazolidin-4-ones showed molecular ion peaks which were in accordance with their respective molecular masses.
Chemistry Pharmacology 3-substituted-4-amino-5-mercapto-1,2,4-triazoles (1) were synthesized through multistep reaction of substituted phenols (Sunil et al., 2011). 3-substituted-1H-pyrazole carbaldehydes (2) were prepared using semicarbazones by Vilsmeier Haack reaction (Vijesh et al., 2011a, b) using phosphorous oxychloride and dimethyl formamide followed by hydrolysis. Amino-condensation of 3-substituted4-amino-5-mercapto-1,2,4-triazoles (1) with different 3-substituted-1H-pyrazole carbaldehydes (2) in absolute ethanol medium in the presence of catalytic amount of concentrated sulphuric acid yielded 5-substituted-4-[(3-
Anticancer activity Evaluation of anticancer activity was carried out in human breast cancer (MCF-7) cells, purchased from National Cancer Center for Cell Science, Pune, India. The cells were cultured in Dulbecco’s modified Eagles medium (DMEM)containing 10 % fetal bovine serum (FBS) at 37 °C in an atmosphere containing 5 % CO2. Unless otherwise mentioned, all the chemicals used in this study were from Sigma-Aldrich, USA.
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N R O
R1
N N N H 2N
N
1a-e
SH
N N
O Conc. H2SO4
+
SH
CHO
N
R
R1
EtOH
N H
N
N H
2a-c 3a-o
SHCH2COOH
N N
R O
SH
N O
N
NH N
S R1 4a-o
R = Phenyl, 2-methylphenyl, 4-methylphenyl, 1-naphthyl, 2-naphthyl R1 = 4-chlorophenyl, 4-flurophenyl, 4-methoxyphenyl Scheme 1 Synthetic route for the triazolothiazolidinones Scheme 2 Plausible mechanism for the formation of thiazolidinone
Pyz
N
Tzl
Pyz
Pyz S
S Step-1
Step-2 Tzl
SH
C
NH
N
-H2O
Tzl
O O
OH
OH
O
N
N
R1 SH N
N O R
123
N H
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MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (Mosmann, 1983; Kavitha et al., 2009) Ten of the randomly selected synthesized thiazolidin-4ones were screened for their anticancer activities at Manipal College of Pharmaceutical Sciences, Manipal University, India. The stock solution of thiazolidin-4-ones to determine their cytotoxic effect were prepared in 10 % dimethyl sulphoxide (DMSO) and further diluted in phosphate buffer saline (PBS) before use. The final concentration of DMSO in the solution was less than 0.1 %. Cells were seeded in duplicates in 96-well plates at 1 9 104 cells/well. After 24 h, the thiazolidin-4-ones were added at a concentration of 0.1, 1, 10, and 100 lg/mL and incubated for another 24 h. 5 9 10-3 mol of MTT reagent was added and incubated for additional 4 h. The purple formazan crystals were then dissolved in 0.1 mL of hydrochloric acid (0.4 N):isopropanol (1:24). Cells grown in culture media with appropriate concentration of DMSO were used as control. Doxorubicin was used as the standard drug. The optical density of each well was measured at 570 nm in an ELISA plate reader. The results were reported as percentage survival of the cells when compared to that of the untreated control cells ± standard deviation (Table 1). The IC50 for the standard drug doxorubicin was found to be 18 lg/mL. Among the ten new thiazolidin-4-ones screened for cytotoxicity in MTT assay, 4b, 4d, and 4f exhibited excellent cytotoxicity with IC50 values of 30, 22, and 29 lg/mL. The methyl group in the ortho position probably might have increased the activity of 4d and 4f. The presence of methyl groups increases the lipophilicity, which can drastically modify the bioavailability of the compound and thus its efficacy. 4j, 4l, and 4m with the bulky naphthyl groups displayed least cytotoxic activity.
Comet assay (Single cell gel electrophoresis) (Kavitha et al., 2009; Singh, 2000) To assess the genotoxic effect of the selected thiazolidin-4ones comet assay was performed in MCF-7 cells. The three thiazolidin-4-ones, 4b, 4d, and 4f which showed good cytotoxicity in the MTT assay were selected for this study. MCF-7 (1 9 106) cells were treated with three different concentrations, 10, 25, and 50 lg/mL of thiazolidin-4-ones for 24 h. The cells were then washed and 200 lL of cell suspension in low melting agarose (LMA) was layered on to the labelled slides precoated with agarose (1.5 %). The slides were placed on ice for 10 min and submerged in lysis buffer (2.5 % NaCl, 100 mM EDTA, 10 mM Tris, 10 % DMSO, and 1 % Troton X—100) at pH 10 at 4 °C for more than 1 h. The slides were then equilibrated in alkaline buffer (30 mM NaOH, 1 mM EDTA) at pH 13 at 4 °C, electrophoresed at 0.86 V/cm at 4 °C, neutralized, washed, and dried. At the time of capturing the images, the slides were stained with ethidium bromide (EtBr, 150 lL 1X) and cover slips were placed over them. For visualization of DNA-damage EtBr-stained slides were observed under 209 objectives of a fluorescent microscope (Olympus BX-51, Japan). The images of 50–100 randomly selected cells were captured per slide using a CCD camera. The images of comet assay for control, cells treated with doxorubicin (0.1 lM, 54 lg/mL), 4b (50 lg/mL), 4d (50 lg/mL), and 4f (50 lg/mL) are shown in Fig. 1. Slides were analysed for parameters like tail length (TL), and olive tail moment (OTM) using image analyzer CASP software version 1.2.2. The results of the assay for TL and OTM are shown in Figs. 2 and 3, respectively. head extent 2 Olive tail moment ¼ Tail length %Tail DNA
Tail length ¼ Tail extent þ
Table 1 Cytotoxic activity data of thiazolidin-4-ones in MTT assay Vehicle control (% survival)
0.1 lg/mL (% survival)
1 lg/mL (% survival)
4b
100 ± 5.99
65.92 ± 4.74
69.43 ± 8.41
4c
100 ± 7.53
Compound no.
100 ± 10.45
10 lg/mL (% survival)
100 lg/mL (% survival)
IC50 lg/mL
76.47 ± 4.65
33.60 ± 1.77
30
100 ± 5.10
96.80 ± 1.8
46.45 ± 6.50
85
67.57 ± 3.4
4d
100 ± 5.90
70.0 ± 4.60
80.7 ± 5.0
13.2 ± 1.40
22
4e
100 ± 5.90
81.4 ± 6.20
83.1 ± 4.60
92.5 ± 5.70
62.1 ± 8.40
[100
4f 4h
100 ± 7.50 100 ± 5.99
81.5 ± 10.4 80.84 ± 2.6
87.0 ± 5.40 74.66 ± 4.7
84.7 ± 1.70 87.5 ± 8.50
25.4 ± 1.80 71.4 ± 8.70
29 [100
95.38 ± 9.4
4i
100 ± 5.90
95.05 ± 4.8
4j
100 ± 2.09
100.8 ± 2.1
70.5 ± 3.70
90.2 ± 5.2 60.3 ± 7.90
43.67 ± 5.5 51.8 ± 10.3
78 100
4m
100 ± 7.50
99.7 ± 10.2
100 ± 3.70
81.4 ± 3.08
50.7 ± 4.30
100
4n
100 ± 7.50
100 ± 7.50
100 ± 3.60
99.85 ± 2.54
96.5 ± 5.40
[100
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Fig. 1 Detection of DNA damage in MCF-7 cells. Treated cells (24 h) were layered over agarose gel, lysed, electrophoresed in alkaline buffer and stained with propidium iodide. Control cells were treated with DMSO alone. The DNA fragmentation resulting in a comet-like appearance is seen control and in cells treated with doxorubicin, 4b, 4d, and 4f
In the comet assay, the images of cells treated with doxorubicin, 4b, 4d, and 4f showed the formation of comets. No comet pattern was observed in the control cells. There was dose-dependent increase in tail length and OTM when treated with 4b, 4d, and 4f. 4d presented maximum apoptotic DNA damage among the three thiazolidin-4-ones studied, which was in accordance with its maximum cytotoxicity as seen in MTT assay. None of the thiazolidin-4-ones exhibited apoptotic DNA damage to the extent of doxorubicin. The quantified increase in DNA damage suggested that all three thiazolidin-4-ones induced dose-dependent fragmentation of chromosomal DNA leading to apoptosis. Antioxidant studies DPPH radical scavenging assay (Sreejayan and Rao, 1996; Vaijanathappa et al., 2008) The DPPH (1,1-diphenyl-2-picryl hydrazide) antioxidant assay is based on the ability of DPPH, a stable free radical,
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to decolorize in the presence of antioxidants. DPPH radical scavenging is mediated by transfer of hydrogen atom. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for visible deep purple color. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance. 100 lL of various concentrations of 4b, 4d, and 4f were added to respective wells of a 96-well micro plate. Equal amount of DPPH was also added to each well to make up a final volume of 200 lL. After 20 min incubation in the dark, the ability of the three thazolidin-4-ones to scavenge the free radical DPPH, was measured by recording the absorbance at 517 nm using an ELISA plate recorder. Experiment was performed in triplicates and average values were considered. An equal amount of methanol and DPPH was added to the control wells. Vitamin C (Ascorbic acid), the antioxidant most made known for its potential benefits to cancer patients, was used as the standard. Results of the DPPH assay are shown in Table 2.
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400
763 Table 2 Results of antioxidant studies of thiazolidin-4-ones
A a
Compounds
IC50 (in lg/mL) DPPH
300
Tail length
a,b a,b
a,b
a,b a,b
a,b
200
a,b
Asc. A
18.74 ± 1.16
31.53 ± 2.85
4b
27.16 ± 2.15
143.67 ± 5.70
4d
31.52 ± 1.98
73.03 ± 3.91
4f
46.95 ± 2.42
165.78 ± 6.44
Asc. A Ascorbic acid (standard)
a,b
a,b
ABTS
100
ABTS radical scavenging assay (Sreejayan and Rao, 1996; Vaijanathappa et al., 2008)
0 Sham Control
DOX (0.1 µM)
4b (10 µg/ml)
4b (25 µg/mL)
4b (50 µg/mL)
4d (10 µg/mL)
4d (25 µg/mL)
4d (50 µg/mL)
4f (10 µg/mL)
4f (25 µg/mL)
4f (50 µg/mL)
Fig. 2 Graph comparing the effect of thiazolidin-4-ones on the tail length in comet assay. There was a concentration-dependent increase in the apoptotic DNA fragmentation and hence an increase in tail length in all three thiazolidin-4-ones. The extent of damage caused by doxorubicin (0.1 lM) was more when compared to the three thiazolidinon-4-ones, 4b, 4d, and 4f. 4d caused maximum DNA damage in the comet assay
100
B a
% scavenging of ABTS Absorbance of control Absorbance of test sample ¼ Absorbance of control
Olive Tail Moment
80
a,b
60
a,b a,b
a,b a,b
b
b
20
0 Sham Control
DOX (0.1 µM)
4b (10 µg/ml)
4b (25 µg/mL)
4b (50 µg/mL)
4d (10 µg/mL)
4d (25 µg/mL)
4d (50 µg/mL)
4f (10 µg/mL)
4f (25 µg/mL)
4f (50 µg/mL)
Fig. 3 Graph relating the effect of thiazolidin-4-ones on the olive tail moment in comet assay. There was a concentration-dependent increase in the OTM in all three thiazolidin-4-ones. The extent of damage caused by doxorubicin (0.1 lM) was more when compared to the three thiazolidinon-4-ones, 4b, 4d, and 4f. 4d exhibited maximum OTM in the comet assay
% scavenging of DPPH Absorbance of control Absorbance of test ¼ Absorbance of control 100
100
a,b
40
a,b
ABTS is chemically 2,2-azino bis 3-ethyl benzo-thiazoline6-sulphonic acid. The reduction of this radical by 4b, 4d, and 4f was measured at 690 nm. The electron transfer capability of the three thiazolidin-4-ones was studied using ABTS radical scavenging assay. In a 96-welled microtitre plate, 40 lL of the thiazolidin-4-ones/ascorbic acid, 200 lL of methanol, and 30 lL of ABTS solution were added. The plate was then incubated at 37 °C for 20 min after which the absorbance was measured at 690 nm using an ELISA plate reader. Sample blank and control were also taken. The experiment was performed in triplicates and average values were considered. The results of ABTS assay is shown in Table 2.
In DPPH and ABTS assay, none of the three thiazolidin4-ones were as potent as the standard compound ascorbic acid. 4d was found to show moderate antioxidant properties with comparatively low IC50 values. As the IC50 value of 4d in DPPH assay was lower than that in ABTS assay, the mechanism of antioxidant action is presumed to be through hydrogen transfer.
Conclusion In this study, a series of fifteen new thiazolidin-4-ones were synthesized by the cyclo-condensation reaction between Schiff bases and thioglycollic acid. The different spectral techniques and the elemental analysis confirmed the structure of the compounds. Three thiazolidin-4-ones, 4b, 4d, and 4f exhibited excellent cytotoxic activity in MCF-7 cell lines with low IC50 values. The comet assay showed the extent of apoptotic DNA damage. 4d exhibited
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maximum DNA fragmentation and presented moderate antioxidant properties in DPPH and ABTS assays. The chlorine atom might have added to the potency of 4d as it produces simultaneously an increase in lipophilicity and an electron attracting effect. The presence of chlorine atom and a methyl group in the ortho position has drastically increased the anticancer activity of 4d, as compared to the bulky naphthyl groups.
Experimental General procedure for the synthesis of 2-(3-substituted1H-pyrazol-4-yl)-3-(3-substituted-5-sulfanyl-1,2,4triazol-4-yl)-1,3-thiazolidin-4-one (4a–o) 0.01 mol of 5-substituted-4-(3-substituted-1H-pyrazol-4-yl methylidene) amino]-2H-1,2,4-triazole-3-thione and 0.01 mol of thioglycolic acid in dimethyl formamide were allowed to undergo nucleophilic cyclo-condensation under reflux in presence of 0.05 g ZnCl2 for 24 h. Excess of the solvent was distilled off and the reaction mixture was poured into ice cold water. The solid separated was collected, washed with NaHCO3 solution and water. The precipitate of 2-(3-substituted-1H-pyrazol-4-yl)-3-(3-substituted-5-sulfanyl-1,2,4-triazol-4-yl)-1,3-thiazolidin-4-one obtainedwas dried and recrystallized from chloroform to petroleum ether mixture. 2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(phenoxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4a) Yellow solid (75 %); m.p. 162–164 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,185 (Ar. C–H str.), 1,731 (C=O of thiazolidinone), 1,498 (Ar.C=C str.), 1016, 1245 (Ar.C–O–C), 608 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 5.01 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.94 (s, 1H, pyrazole C–H), d 7.1–7.8 (m, 9H, Ar.H), d 11.8 (s, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 484 (M?); Anal. Calcd. for C21H17ClN6O2S2: C, 52.01; H, 3.51; N, 17.34; found: C, 52.15; H, 3.36; N, 17.49. 2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(phenoxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4b) Yellow solid (67 %); m.p. 164–166 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,180 (Ar. C–H str.), 1,732 (C=O of thiazolidinone), 1,508 (Ar.C=C str.), 1018, 1240 (Ar.C–O–C), 610 ([C–S–C\str.); 1H
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NMR (400 MHz, DMSO-d6): d 5.02 (s, 2H, CH2 of thiazolidinone ring), d 5.31 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.94 (1H, pyrazole C–H), d 7.1–7.9 (m, 9H, Ar.H), d 11.68 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 468 (M?); Anal. Calcd. for C21H17FN6O2S2: C, 53.85; H, 3.63; N, 17.95; found: C, 54.01; H, 3.54; N, 17.85. 3-(3-mercapto-5-(phenoxymethyl)-4H-1,2,4-triazol-4-yl)2-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (4c) Yellow solid (65 %); m.p. 138–140 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3090 (Ar. C–H str.), 2950, 2860 (methyl C–H str.), 1,729 (C=O of thiazolidinone), 1,507 (Ar.C=C str.), 1018, 1249 (Ar.C–O–C), 610 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 3.27 (s, 3H, OCH3), d 5.02 (s, 2H, CH2 of thiazolidinone ring), d 5.32 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.95 (1H, pyrazole C–H), d 7.1–7.8 (m, 9H, Ar.H), d 11.81 (brs, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 480 (M?); Anal. Calcd. for C22H20N6O3S2: C, 55.00; H, 4.17; N, 17.50; found: C, 55.19; H, 4.01; N, 17.29. 2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(o-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4d) Yellow solid (73 %); m.p. 156–158 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,190 (Ar. C–H str.), 2950, 2860 (methyl C–H str.), 1,733 (C=O of thiazolidinone), 1,506 (Ar.C=C str.), 1018, 1244 (Ar.C–O–C), 608 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.25 (s, 3H, CH3), d 5.02 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.6 (s, 2H, OCH2), d 6.95 (1H, pyrazole C–H), d 7.1–7.9 (m, 8H, Ar.H), d 11.68 (s, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 498 (M?); Anal. Calcd. for C22H19 ClN6O2S2: C, 52.96; H, 3.81; N, 16.85; found: C, 53.08; H, 3.75; N, 17.07. 2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(o-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4e) Yellow solid (71 %); m.p. 142–144 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,185 (Ar. C–H str.), 2950, 2862 (methyl C–H str.), 1,730 (C=O of thiazolidinone), 1,506 (Ar.C=C str.), 1019, 1248 (Ar.C–O– C), 605 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.25 (s, 3H, CH3), d 5.02 (s, 2H, CH2 of thiazolidinone ring), d 5.34 (s, 1H, CH of thiazolidinone ring), d 5.61 (s, 2H, OCH2), d 6.94 (1H, pyrazole C–H), d 7.1–7.9 (m, 8H,
Med Chem Res (2013) 22:758–767
Ar.H), d 11.90 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 482 (M?); Anal. Calcd. for C22H19 FN6O2S2: C, 54.77; H, 3.94; N, 17.43; found: C, 54.89; H, 3.81; N, 17.52. 3-(3-mercapto-5-(o-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)2-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (4f) Yellow solid (70 %); m.p. 174–176 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,170 (Ar. C–H str.), 2960, 2890 (methyl C–H str.), 1,740 (C=O of thiazolidinone), 1,505 (Ar.C=C str.), 1017, 1292 (Ar.C–O– C), 600 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.22 (s, 3H, CH3), d 3.29 (s, 3H, OCH3), d 5.03 (s, 2H, CH2 of thiazolidinone ring), d 5.31 (s, 1H, CH of thiazolidinone ring), d 5.61 (s, 2H, OCH2), d 6.84 (1H, pyrazole C–H), d 7.1–7.8 (m, 8H, Ar.H), d 11.55 (s, 1H, Pyrazole N–H), 13.80 (brs, 1H, SH); MS (m/z): 494 (M?); Anal. Calcd. for C23H22N6O3S2: C, 55.87; H, 4.45; N, 17.00; found: C, 56.01; H, 4.31; N, 17.13. 2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(p-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4g) Yellow solid (71 %); m.p. 182–184 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,147 (Ar. C–H str.), 2930, 2860 (methyl C–H str.), 1,737 (C=O of thiazolidinone), 1,510 (Ar.C = C str.), 1021, 1238 (Ar.C–O– C), 610 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.23 (s, 3H, CH3), d 5.01 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.6 (s, 2H, OCH2), d 6.95 (1H, pyrazole C–H), d 7.1–7.8 (m, 8H, Ar.H), d 11.68 (s, 1H, Pyrazole N–H), d 13.81 (brs, 1H, SH); MS (m/z): 498 (M?); Anal. Calcd. for C22H19 ClN6O2S2: C, 52.96; H, 3.81; N, 16.85; found: C, 52.84; H, 3.77; N, 16.93. 2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5(p-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)thiazolidin-4-one (4h) Yellow solid (71 %); m.p. 174–176 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,160 (Ar. C–H str.), 2850, 2930 (methyl C–H str.), 1,732 (C=O of thiazolidinone), 1,507 (Ar.C=C str.), 1011, 1242 (Ar.C–O–C), 608 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.25 (s, 3H, CH3), d 5.05 (s, 2H, CH2 of thiazolidinone ring), d 5.35 (s, 1H, CH of thiazolidinone ring), d 5.63 (s, 2H, OCH2), d 6.92 (1H, pyrazole C–H), d 7.1–7.7 (m, 8H, Ar.H), d 11.65 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 482 (M?); Anal. analysis Calcd. for
765
C22H19FN6O2S2: C, 54.77; H, 3.94; N, 17.43; found: C, 54.67; H, 4.05; N, 17.34. 3-(3-mercapto-5-(p-tolyloxymethyl)-4H-1,2,4-triazol-4-yl)2-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (4i) Yellow solid (68 %), m.p. 160–162 °C, IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,185 (Ar. C–H str.), 2960, 2850 (methyl C–H str.), 1,730 (C=O of thiazolidinone), 1,505 (Ar.C=C str.), 1019, 1250 (Ar.C–O– C), 612 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 2.23 (s, 3H, CH3), d 3.32 (s, 3H, OCH3), d 5.01 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.61 (s, 2H, OCH2), d 6.96 (1H, pyrazole C–H), d 7.1–7.8 (m, 8H, Ar.H), d 11.65 (s, 1H, Pyrazole N–H), d 13.80 (brs, 1H, SH); MS (m/z): 494 (M?); Anal. Calcd. for C23H22N6O3S2: C, 55.87; H, 4.45; N, 17.00; found: C, 55.72; H, 4.29; N, 16.87. 2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5((naphthalen-1-yloxy)methyl)-4H-1,2,4-triazol-4yl)thiazolidin-4-one (4j) Yellow solid (71 %); m.p. 176–178 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,112 (Ar. C–H str.), 1,734 (C=O of thiazolidinone), 1,504 (Ar.C=C str.), 1018, 1245 (Ar.C–O–C), 825 (Ar.C–H def.), 730, 820, 842 (naphthalene C–H def.), 608 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 5.03 (s, 2H, CH2 of thiazolidinone ring), d 5.34 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.92 (1H, pyrazole C–H), d 7.1–8.1 (m, 11H, Ar.H), d 11.62 (s, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 534 (M?); Anal. Calcd. for C25H19ClN6O2S2: C, 56.13; H, 3.55; N, 15.72; found: C, 56.25; H, 3.45; N, 15.60. 2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5((naphthalen-1-yloxy)methyl)-4H-1,2,4-triazol-4yl)thiazolidin-4-one (4k) Yellow solid (68 %); m.p. 180–182 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,095 (Ar. C–H str.), 1,732 (C=O of thiazolidinone), 1,508 (Ar.C=C str.), 1019, 1248 (Ar.C–O–C), 824 (Ar.C–H def.), 750, 830, 844 (naphthalene C–H def.), 610 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 5.01 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.94 (1H, pyrazole C–H), d 7.1–7.9 (m, 11H, Ar.H), d 11.75 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 518 (M?); Anal. Calcd. for C25H19FN6O2S2: C, 57.92; H, 3.67; N, 16.22; found: C, 58.03; H, 3.74; N, 16.35.
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Med Chem Res (2013) 22:758–767
3-(3-mercapto-5-((naphthalen-1-yloxy)methyl)-4H-1,2,4triazol-4-yl)-2-(3-(4-methoxyphenyl)-1H-pyrazol-4yl)thiazolidin-4-one (4l)
3-(3-mercapto-5-((naphthalen-2-yloxy)methyl)-4H-1,2,4triazol-4-yl)-2-(3-(4-methoxyphenyl)-1H-pyrazol-4yl)thiazolidin-4-one (4o)
Yellow solid (72 %); m.p. 145 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,118 (Ar. C–H str.), 1,733 (C=O of thiazolidinone), 2950, 2860 (methyl C–H str.), 1,505 (Ar.C=C str.), 1015, 1242 (Ar.C–O–C), 820 (Ar.C–H def.), 752, 828, 840 (naphthalene C–H def.), 609 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 3.29 (s, 3H, OCH3), d 5.03 (s, 2H, CH2 of thiazolidinone ring), d 5.32 (s, 1H, CH of thiazolidinone ring), d 5.62 (s, 2H, OCH2), d 6.93 (1H, pyrazole C–H), d 7.1–8.1 (m, 11H, Ar.H), d 11.45 (s, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 530 (M?); Anal. Calcd. for C26H22N6O3S2: C, 58.87; H, 4.15; N,15.85; found: C, 58.67; H, 4.22; N, 15.79.
Yellow solid (66 %); m.p. 203–205 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,170 (Ar. C–H str.), 2950, 2860 (methyl C–H str.), 1,733 (C=O of thiazolidinone), 1,505 (Ar.C = C str.), 1019, 1246 (Ar.C– O–C), 820 (Ar.C–H def.), 752, 825, 846 (naphthalene C–H def.), 608 ([C–S–C\str.); 1H NMR (400 MHz, DMSOd6): d 3.28 (s, 3H, OCH3), d 5.04 (s, 2H, CH2 of thiazolidinone ring), d 5.33 (s, 1H, CH of thiazolidinone ring), d 5.61 (s, 2H, OCH2), d 6.95 (1H, pyrazole C–H), d 7.1–8.1 (m, 11H, Ar.H), d 11.65 (s, 1H, Pyrazole N–H), d 13.79 (brs, 1H, SH); MS (m/z): 530 (M?); Anal. Calcd. for C26H22N6O3S2: C, 58.87; H, 4.15; N, 15.85; found: C, 59.02; H, 3.98; N, 15.99.
2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5((naphthalen-2-yloxy)methyl)-4H-1,2,4-triazol-4yl)thiazolidin-4-one (4m)
Acknowledgments The authors are thankful to the Head-SIF, Indian Institute of Science, Bangalore for providing NMR and mass spectral data. AMI is thanks Department of Atomic Energy, Board for Research in Nuclear Sciences, Government of India for the ‘Young Scientist’ award.
Yellow solid (69 %); m.p. 217–219 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,110 (Ar. C–H str.), 1,735 (C=O of thiazolidinone), 1,507 (Ar.C=C str.), 1018, 1240 (Ar.C–O–C), 820 (Ar.C–H def.), 750, 820, 840 (naphthalene C–H def.), 606 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 5.03 (s, 2H, CH2 of thiazolidinone ring), d 5.35 (s, 1H, CH of thiazolidinone ring), d 5.63 (s, 2H, OCH2), d 6.93 (1H, pyrazole C–H), d 7.1–8.1 (m, 11H, Ar.H), d 11.64 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 534 (M?); Anal. Calcd. for C25H19ClN6O2S2: C, 56.13; H, 3.55; N, 15.72; found: C, 55.97; H, 3.49; N, 15.63.
2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-3-(3-mercapto-5((naphthalen-2-yloxy)methyl)-4H-1,2,4-triazol-4yl)thiazolidin-4-one (4n) Yellow solid (70 %); m.p. 218–220 °C; IR (KBr, mmax cm-1): 3,100–3,200 (pyrazole N–H str.), 3,195 (Ar. C–H str.), 1,731 (C=O of thiazolidinone), 1,504 (Ar.C=C str.), 1019, 1244 (Ar.C–O–C), 825 (Ar.C–H def.), 752, 820, 845 (naphthalene C–H def.), 610 ([C–S–C\str.); 1H NMR (400 MHz, DMSO-d6): d 5.02 (s, 2H, CH2 of thiazolidinone ring), d 5.32 (s, 1H, CH of thiazolidinone ring), d 5.63 (s, 2H, OCH2), d 6.95 (1H, pyrazole C–H), d 7.1–7.9 (m, 11H, Ar.H), d 11.78 (s, 1H, Pyrazole N–H), d 13.78 (brs, 1H, SH); MS (m/z): 518 (M?); Anal. Calcd. for C25H19FN6O2S2: C, 57.92; H, 3.67; N, 16.22; found: C, 57.82; H, 3.53; N, 15.99.
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References Amir M, Azam F (2004) Synthesis and biological evaluation of some 4-thiazolidinones. Indian J Heterocycl Chem 14:119–122 Bandgar PB, Gawande SS, Bodade RG, Gawande NM, Khobragade CN (2009) Synthesis and biological evaluation of a novel series of pyrazole chalcones as anti-inflammatory, antioxidant and antimicrobial agents. Bioorg Med Chem 17:8168–8173 Bandgar PB, Gawande SS, Bodade RG, Totre JV, Khobragade CN (2010) Synthesis and biological evaluation of simple methoxylated chalcones as anticancer, anti-inflammatory and antioxidant agents. Bioorg Med Chem 18:1364–1370 Brueggemeier RW, John CH, Edgar SD (2004) Aromatase inhibitors in the treatment of breast cancer. Endocrine Rev 26:331–345 Chandrakantha B, Isloor AM, Shetty P, Isloor S, Malladi S, Fun HK (2011) Synthesis, characterization and antimicrobial activity of novel ethyl 1-(N-substituted)-5-phenyl-1H-pyrazole-4-carboxylate derivatives. Med Chem Res. doi:10.1007/s00044-011-9796-9 Demirbas N, Karaoglu SA, Demirbas A, Sanack K (2004) Synthesis and antimicrobial activities of some new 1-(5-phenylamino[1,3,4]thiadiazol-2-yl)methyl-5-oxo-[1,2,4]triazole and 1-(4-phenyl-5-thioxo-[1,2,4] triazole-3-yl)methyl-5-oxo[1,2,4]triazole derivatives. Eur J Med Chem 39:793–804 Dobrowsky W, Huigol NG, Jayatilake RS, Kizilbash NI, Okkan S, Kagiya TV, Tatsuzaki H (2005) AK-2123 (Sanazol) as a radiation sensitizer in the treatment of stage III cancer cervix: initial results of an IAEA multicentre randomized trial. J Cancer Res Ther 1:75–78 Hafez HN, El-Gazzar ABA (2009) Synthesis and antitumor activity of substituted triazolo[4,3-a]pyrimidin-6-sulfonamide with an incorporated thiazolidinone moiety. Bioorg Med Chem Lett 19:4143–4147 Holla BS, Poojary KN, Rao BS, Shivananda MK (2002) New bisaminomercaptotriazoles and bis-triazolothiadiazoles as possible anticancer agents. Eur J Med Chem 37:511–517
Med Chem Res (2013) 22:758–767 Ibrahim DA (2009) Synthesis and biological evaluation of 3,6disubstituted [1,2,4] triazolo[3,4-b][1,3,4]thiadiazole derivatives as a novel class of potential anti-tumor agents. Eur J Med Chem 44:2776–2781 Isloor AM, Kalluraya B, Shetty P (2009) Regioselective reaction: synthesis, characterization and pharmacological studies of some new Mannich bases derived from 1,2,4-triazoles. Eur J Med Chem 44:3784–3787 Kavitha CV, Nambiar M, Ananda Kumar CS, Choudhari B, Muniyappa K, Rangappa KS, Raghavan SC (2009) Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells. Biochem Pharmacol 77:348–363 Lee T, Lau T, Ng I (2002) Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother Pharmacol 49:78–86 Malladi S, Isloor AM, Isloor S, Akhila DS (2011a) Synthesis, characterization and ntibacterial activity of some new pyrazole based Schiff bases. Arabian J Chem. doi:10.1016/j.arabjc. 2011.10.009 Malladi S, Isloor AM, Shetty P, Fun HK, Telkar S, Mahmood R, Isloor N (2011b) Synthesis and anti-inflammatory evaluation of some new 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles bearing pyrazole moiety. Med Chem Res. doi:10.1007/ s00044-011-9865-0 Mathew V, Keshavayya J, Vaidya VP, Giles D (2007) Studies on synthesis and pharmacological activities of 3,6-disubstituted1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and their dihydro analogues. Eur J Med Chem 42:823–840 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Immunol Methods 65:55–63 Padmavathi V, Reddy GS, Padmaja A, Kondaiah P, Ali-Shazia (2009) Synthesis, antimicrobial and cytotoxic activities of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles. Eur J Med Chem 44:2106–2112 Palekar VS, Damle AJ, Shukla SR (2009) Synthesis and antibacterial activity of some novel bis-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and bis-4-thiazolidinone derivatives from terephthalic dihydrazide. Eur J Med Chem 44:5112–5116 Porter P (2008) Westernizing women’s risks? Breast cancer in lowerincome countries. N Engl J Med 358:213–216
767 Selvi G, Rajendran SP (2007) Synthesis of 2-[30 (20 -chloroquinolinyl)3-carbethoxymethyl]-4-thiazolidinones. Indian J Het Chem 17: 201–202 Siddiqui N, Deepanjali, Md Arshad F, Rana A (2007) Synthesis and anticonvulsant screening of 2-(substituted aryl)-3-(4H-1,2,4triazol-4-yl)-1,3-thiazolidinon-4-ones. Indian J Het Chem 16: 403–404 Singh NP (2000) Microgels for estimation of DNA strand breaks, DNA protein cross links and apoptosis. Mutation Res 455: 111–127 Sreejayan N, Rao MN (1996) Free radical scavenging activity of curcuminoids. Drug Res 46:169–171 Sunil D, Isloor AM, Shetty P, Chandrakantha B, Satyamoorthy K (2011) Synthesis, characterization and in vitro cytotoxic properties of some new Schiff and Mannich bases in Hep G2 cells. Med Chem Res 20:1024–1032 Swamy SN, Basappa, Priya BS, Prabhuswamy B, Doreswamy BH, Prasad JS, Rangappa KS (2006) Synthesis of pharmaceutically important condensed heterocyclic 4,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives as antimicrobials. Eur J Med Chem 41:531–538 Vaijanathappa J, Badami S, Bhojraj S (2008) In vitro antioxidant activity of Enicostemmaaxillare. J Health Sci 54:524–528 Vijesh AM, Isloor AM, Prabhu V, Ahmad S, Malladi S (2010) Synthesis, characterization and anti-microbial studies of some novel 2,4-disubstituted thiazoles. Eur J Med Chem 45: 5460–5464 Vijesh AM, Isloor AM, Telkar S, Peethambar SK, Rai S, Isloor N (2011a) Synthesis, characterization and antimicrobial studies of some new pyrazole incorporated imidazole derivatives. Eur J Med Chem 46:3531–3536 Vijesh AM, Isloor AM, Peethambar SK, Shivananda KN, Arulmoli T, Isloor NA (2011b) Hantzsch reaction: synthesis and characterization of some new 1,4-dihydropyridine derivatives as potent antimicrobial and antioxidant agents. Eur J Med Chem 46:5591–5597 Wilson C, Gomes CRB, Vellasco WT (2008) Chemistry and biological activities of 1,3-thiazolidin-4-ones. Mini-Rev Org Chem 5:336–344
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