Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 593–600 © 2013 Informa UK, Ltd. ISSN 1475-6366 print/ISSN 1475-6374 online DOI: 10.3109/14756366.2012.663365
research Article
Synthesis of novel carbazole chalcones as radical scavenger, antimicrobial and cancer chemopreventive agents Babasaheb P. Bandgar1, Laxman K. Adsul1, Shrikant V. Lonikar1, Hemant V. Chavan1, Sadanand N. Shringare1, Sachin A. Patil1, Shivkumar S. Jalde1, Basawaraj A. Koti1, Nagesh A. Dhole2, Rajesh N. Gacche2, and Amol Shirfule3 Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur, Maharashtra, Biochemistry Research Laboratory, School of Life Sciences, S.R.T.M. University, Nanded, Maharashtra, and 3 Food and Drug Toxicology Research Centre, National Institute of Nutrition, Hyderabad, Andhra Pradesh, India 1 2
Abstract A series of novel carbazole chalcones has been synthesised and evaluated for radical scavenging activity, cytotoxicity and antimicrobial activities. Compounds 12m, 12o and 12c exhibited good 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, compounds 12e, 12m and 12d were excellent hydroxyl radical scavengers and compounds 12a, 12e, 12g, 12n and 12m have shown inhibition of oxidative DNA damage induced by 2,2′-azobis (2-amidinopropane hydrochloride). Compounds 12j, 12i, 12n, 12c, 12m and 12e were most active against the selected cancer cell lines. Compounds 12a, 12e and 12m showed good antibacterial activity and compounds 12h and 12m have shown good antifungal activity. All the compounds were subjected for absorption, distribution, metabolism and excretion (ADME) predictions by computational method and found that these molecules could be considered as potential candidates for oral drug development. Keywords: Chalcones, carbazole, anticancer, antimicrobial, antioxidant, Lipinski rule
Introduction
to malignancy. Damage to DNA by oxidative stress has been widely accepted as a major cause of cancer4. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions have been observed in various tumours. The discovery of the role of free radicals in cancer, diabetes, cardiovascular diseases, autoimmuno diseases, neurodegenerative disorders, aging and other diseases has lead to new medical insight5,6. Minimum oxidative damage may be an important approach to the primary prevention or treatment of these diseases. Antioxidants may stop the free radical formation or interrupt an oxidising chain reaction. This has attracted a great deal of research interest in therapeutic antioxidantbased drug formulations. The development of synthetic compounds capable of scavenging free radicals has been a great success. Carbazole and its derivatives are the important class of heterocyclic compounds endowed with various
Antioxidant substances prevent or inhibit the natural phenomena of oxidation. In the recent years, “antioxidant research” has attracted several researchers because of its broad applications in the management and amelioration of a variety of human ailments1,2. The formation of reactive oxygen species (ROS) is characteristic of aerobic organisms that can normally defend themselves against these highly reactive species using enzymes like superoxide dismutase and glutathione peroxidase, and naturally occurring antioxidants such as α-tocopherol (vitamin E), ascorbic acid (vitamin C) and β-carotene3. However, in pathophysiological conditions, the excessive production of ROS overwhelms the natural antioxidant defence mechanisms. This imbalance is termed as oxidative stress. High levels of free radicals can cause damage to biomolecules such as lipids, proteins, enzymes and DNA in cells and tissues, resulting in mutations that can lead
Address for Correspondence: Babasaheb P. Bandgar, Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, Maharashtra, India. Tel/Fax: 0091-217-2351300. E-mail:
[email protected] (Received 08 December 2011; revised 28 January 2012; accepted 31 January 2012)
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594 B. Bandgar et al. pharmacological activities such as anticancer, antimicrobial, antiviral, anti-inflammatory and antioxidant activity7. Most of carbazole heterocycles are of natural and synthetic origins and have wide spread distribution in terrestrial plants and micro-organisms. From the bioassay-guided approach, various carbazole alkaloids have been isolated and are subjected for biological evaluation. Naturally occurring carbazole derivatives, murrayafoline A 1 (Figure 1), carbazomycin A and B 2, have shown potent antimicrobial activity8. Ellipticine 3, a pyridocarbazole, shows potent antiproliferative activity; rebaccamycin 4, an indolocarbazole derivative, acts as an antibiotic and shows inhibition of topoisomerase I and carazostatin 5 is a potent natural antioxidant9–11. The isolation of these molecules spurred the research to synthesise these molecules and its analogues to optimise these natural lead molecules into a clinical molecule. Among the various biological activities exhibited by carbazole derivatives of the particular interest is anticancer activity12. The majority of carbazole derivatives exert cytotoxicity via interaction with DNA. Because these derivatives possess structural features such as flat polycyclic and aromatic chromophore, the optimal size for intercalating chromophore is built by a tricyclic or tetracyclic ring system. Therefore, tricyclic planar carbazole chromophore is predestined for intercalation into DNA. In addition to the DNA binding, carbazole derivatives present antiproliferative effect by
other mechanisms, such as inhibition of angiogenesis13, inhibition of cyclin-dependent kinase 414, inhibition of microtubulin15, inhibition of check point kinase 116 and inhibition of topoisomerase I and II17,18. Carvedilol 6, an antihypertensive drug, acts as a nonspecific β-adrenergic antagonist19. Carprofen 7, a nonsteroidal anti-inflammatory drug, is a selective COX-2 inhibitor20. In continuation of our studies in synthesising various biologically active compounds21, in this study, we have synthesised and characterised the novel carbazole chalcones and evaluated for antioxidant, cytotoxicity and antimicrobial activities. We also subjected all synthesised compounds for ADME predictions by computational method.
Results and discussion Chemistry Chalcone is a unique template that is associated with several biological activities. The biological activities of chalcones are equally wide ranging. In fact, not many structural templates can claim association with a diverse range of pharmacological activities such as antitumor, anti-inflammatory, antiplasmodial, immunosuppression and antioxidant activities22,23. With the great biological importance of carbazole derivatives, we synthesised a series of carbazole chalcones from
Figure 1. Biologically active natural and synthetic carbazole derivatives.
Journal of Enzyme Inhibition and Medicinal Chemistry
Synthesis of novel carbazole chalcones 595 3-formyl-9-methylcarbazole and various acetophenones (Scheme 1). Carbazole (9) on methylation with methyl iodide gave 9-methyl carbazole (10), which on Vilsmeier–Haack formylation gave 3-formyl-9-methylcarbazole (11). This 3-formyl-9-methylcarbazole (11) on Claisen–Schimdt condensation with various aromatic and heteroaromatic acetophenones in aqueous sodium hydroxide afforded titled compound (12a-o) in good to excellent yields (Table 3, Supplementary information). All the synthesised compounds were characterised by IR, 1 H NMR, 13C NMR, CHN analysis and MS .
Biological evaluation All the synthesised compounds were evaluated for DPPH radical scavenging activity (Table 1). The DPPH radical scavenging method is a well-known method for determining antioxidant activity24,25. Among the tested chalcones, compounds 12m, 12o, 12c, 12g, 12n and 12i have
Scheme 1. Reagents and conditions: (i) DMF, NaH, CH3I, rt, 3 h; (ii) DMF, POCl3, 80°C, 4 h; (iii) Substituted acetophenones, NaOH, ethanol, rt, 24–36 h.
shown good radical scavenging activity (89.53%–82.30%) as compared with the standard ascorbic acid (95.76%). Whereas, compounds 12f, 12h, 12k, 12l, 12a and 12e have shown moderate DPPH radical scavenging activity (79.03%–70.76%). The structure activity relationship (SAR) study reveals electron-withdrawing substituents on B ring increases DPPH radical scavenging activity, while electron-donating substituents decreases the activity with the exception of compound 12c. Replacement of aromatic B ring by heteroaromatic ring enhances the DPPH radical scavenging activity. Hydroxyl radical is known to react with all components of DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone26. Hydroxyl radical scavenging ability of the synthesised chalcones is presented in Table 1. The results reveal that compounds 12e, 12m and 12d have shown excellent hydroxyl radical scavenging activity (91.72%–87.59%) as compared with the standard ascorbic acid (88.34%). While compounds 12a, 12j, 12l, 12g, 12i and 12n have shown good hydroxyl radical scavenging activity (86.84%–71.42%) and compounds 12k, 12h, 12f and 12o are moderate OH radical scavengers (69.54%–54.51%). In general, an electron-withdrawing substituent on B ring increases hydroxyl radical scavenging activity, whereas electron-donating substituent on B ring reduces the activity except in case of 12d. Replacing aromatic B ring of 12a by heterocyclic ring, in particular thiophene, decreases the OH radical scavenging activity. The highest occupied molecular orbital (HOMO), a parameter representing the molecular electron-donating ability, is also a good index predicting antioxidant activity27. The compounds possessing higher energy of HOMO (EHOMO) and
Table 1. Radical scavenging activity of carbazole chalcones. DPPH radical scavenging activity Inhibition (%) IC50 (μg/mL) Compounds R 12a H 72.69 70.15 12b 4-OCH3 18.65 ND 12c 3,4-OCH3 83.84 52.49 12d 2,4,6-OCH3 61.92 ND 12e 4-F 70.76 ND 12f 2,4-F 79.03 67.50 12g 3,4-F 83.07 60.45 12h 4-Cl 76.34 68.15 12i 2,4-Cl 82.30 61.45 12j 3,4-Cl 59.61 ND 12k 2,5-Cl 75.57 ND 12l 4-Br 73.65 ND 12m 4-NO2 89.53 47.10 12n 2-Thiophene 82.88 60.45 12o 2-pyridine 87.50 50.65 — 95.76 45.94 Ascorbic acida The results summarised for inhibition (%) are mean values of two parallel experiments. Inhibition (%) activity tested at 100 μg. IC50 values represent mean ± standard deviation from three different experiments. ND, Not determined. a Standard. © 2013 Informa UK, Ltd.
Hydroxyl radical scavenging activity Inhibition (%) IC50 (μg/mL) 86.84 32.33 69.54 87.59 91.72 61.65 78.19 63.90 74.06 83.83 63.90 79.32 88.34 71.42 54.51 88.34
51.10 ND ND 38.60 35.15 ND 56.10 ND ND 54.34 ND 56.15 36.63 ND ND 48.45
596 B. Bandgar et al. energy of lowest unoccupied molecular orbital (ELUMO) were found to be effective agents for stabilising the DPPH and hydroxyl radicals; the antioxidant effect of carvedilol mainly resides in carbazole moiety28 and N-ethyl carbazole derivatives acts as electron donors29; all these properties will explain the high antioxidant activity of the compounds under investigation. The IC50 values for radical scavenging activities of the tested compounds are listed in Table 1. The compounds 12m, 12o and 12c exhibited low IC50 values (47.10–52.49 μg/mL) for DPPH radical scavenging activity. However, the tested compounds did not show DPPH radical scavenging activity as effective as the standard ascorbic acid (IC50 = 45.94 μg/mL). In addition, IC50 values for hydroxyl radical scavenging activity, compounds 12e, 12m and 12d, have shown low IC50 values (35.15–38.60 μg/mL) as compared with the standard ascorbic acid (IC50 = 48.45 μg/mL). IC50 value is defined as the concentration of the sample causing 50% decrease in absorbance; a lower IC50 value would reflect greater radical scavenging activity of the sample. Permanent modification of genetic material resulting from oxidative damage incidents represents the first step involved in mutagenesis, carcinogenesis and aging. In fact, it is well established that free radical-mediated DNA damage occurs in various cancer tissues. Till date, more than 100 products have been identified from the oxidation of DNA. ROS-induced DNA damage involves single- or double-stranded DNA breaks, purine, pyrimidine or deoxyribose modifications and DNA cross links. DNA damage can result either in arrest or induction of transcription, induction of signal transduction pathways, replication errors and genomic instability, all of which are associated with carcinogenesis30,31. In case of plasmid, damage of DNA results in a cleavage of one of the phosphodiester chains of the supercoiled DNA and produces a relaxed open circular form. Further cleavage near the first breakage results in linear double-stranded DNA molecules. The formation of circular form of DNA is indicative of single-strand breaks and the formation of linear form of DNA is indicative of double-strand breaks32. We have evaluated antioxidant activity of novel carbazole chalcones against 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH)-induced plasmid pUC18 DNA strand breakage using agarose gel electrophoresis analysis (Figure 2)33. Compounds 12a, 12e, 12g, 12n and 12m have shown inhibition of AAPH-induced DNA breakage. The DNA protection is in the order 12g > 12e > 12n > 12a > 12m. These results confirm the antioxidant activity exhibited by these compounds by DPPH, OH methods as well as AAPH-induced DNA damage. Cytotoxicity of the synthesised compounds was tested against selected three cancer cell lines such as Hep 3BPN7 (liver cancer cell line), HL60 P58 (leukaemia cancer cell line), HeLa-B75 (cervix cancer cell line) and one normal cell line PN-15C-12 (a normal chang liver cell line). The results obtained are summarised in Table 2. Compounds 12j, 12i, 12n, 12c, 12m and 12e were found to be most
Figure 2. Agarose gel electrophoresis pattern of pUC 18 DNA strand breakage by 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH) and inhibited by carbazole chalcones. Supercoiled plasmid DNA was incubated with AAPH and/or carbazole chalcones in phosphate-buffered saline (pH 7.4) at 37°C for 1 h. Lane 1: DNA strand breakage induced by AAPH at 10 mM; Lane 2: control super coiled DNA; Lane 3: compound 12g; Lane 4: 12a; Lane 5: 12e; Lane 6: 12n and lane 7: 12m.
effective against selected cell lines. These compounds inhibit the proliferation of selected three cancer cell lines with highest selectivity (3.85–2.06) than the normal cell lines. Whereas compounds 12f, 12k and 12o showed preferential cytotoxicity against the Hep 3BPN7 and HL60 P58. The SAR studies with respect to cytotoxicity revealed that compounds with electron-withdrawing groups on aromatic ring B show higher cytotoxicity than compounds containing electron-donating groups with the exception of compound 12c. Position of substituents on aromatic ring B influences the cytotoxicity; compound with monofluoro substituent (12e) shows cytotoxicity against all the three cancer cell lines. However, compounds with difluoro substituent (12f and 12g) were cytotoxic to only liver and leukaemia cell line. The bioisosteric replacement of aromatic ring (12a) by heteroaromatic ring (12n) increases cytotoxicity. Cancer chemoprevention is regarded as a promising avenue for cancer control and has become increasingly important in recent years. Tumourigenesis is a multistep process that begins with cellular transformation, progresses to hyperproliferation and culminates in the acquisition of invasive potential, angiogenic properties and establishment of metastatic lesions. Chemoprevention was described as the use of natural or synthetic chemicals allowing suppression, retardation or inversion of carcinogenesis. Substantial evidence exists to support the role of both oxygen and organic-free radical intermediates in the bimolecular interactions, which contribute to the initiation, promotion and/or progression stages of chemical carcinogenesis34. There are many reports on the synthetic chalcones as chemopreventive agents. First report on the chalcones as chemopreventive agents by Anto et al.,35 described that compounds with good antioxidant activity showed good antitumor activity. In the present case, compounds 12m, 12e and 12a exhibited Journal of Enzyme Inhibition and Medicinal Chemistry
Synthesis of novel carbazole chalcones 597 Table 2. Cytotoxicity of carbazole chalcones. Compounds Hep 3BPN7 SRHep 3BPN7
HL60 P58
SRHL60 P58
HeLa B75
SRHeLa B75
PN-15C-12
SRaverage
12a 70.33 1.63 57.73 1.34 75.44 1.75 42.95 1.57 12b 63.40 3.56 62.52 3.51 71.26 4.01 17.78 3.69 12c 72.80 2.37 59.32 1.93 73.31 2.39 30.69 2.23 12d 65.60 2.27 49.07 1.70 78.39 2.72 28.85 2.23 12e 69.40 2.12 56.80 1.73 77.09 2.35 32.80 2.06 12f 62.40 2.92 57.89 2.58 62.27 2.78 22.39 2.76 12g 61.53 3.16 55.79 2.86 56.31 2.89 19.49 2.97 12h 67.86 1.81 60.50 1.61 80.58 2.15 37.54 1.85 12i 66.93 2.94 55.12 2.42 76.74 3.37 22.79 2.90 12j 65.10 3.86 51.17 3.03 78.73 4.67 16.86 3.85 12k 61.40 4.36 66.63 4.73 56.31 3.99 14.09 4.36 12l 70.73 1.61 64.28 1.46 75.85 1.73 43.87 1.60 12m 69.46 2.18 58.31 1.83 74.89 2.35 31.88 2.12 12n 70.60 2.54 59.07 2.12 73.89 2.66 27.79 2.44 12o 67.66 2.28 62.85 2.12 65.36 2.20 29.64 2.20 Methotrexate 64.60 1.20 54.08 1.00 78.66 1.46 53.85 1.22 The results are the mean values of n = 2. SR = Inhibition of cancer cell line (%)/Inhibition of normal cell line (%). Hep 3BPN7, liver cancer cell line; HL60 P58, leukaemia cancer cell line; HeLa-B75, cervix cancer cell line; PN-15C-12, normal liver cell line, compounds tested at 100 μg; SR, selectivity ratio.
good antioxidant activity, and these compounds also presented significant cytotoxicity, which reinforces the idea of designing antioxidant-based cancer chemoprevention agents and these compounds could be considered as chemopreventive agents. The newly synthesised compounds were screened for their antibacterial activity against two Gram-negative strains Escherichia coli (NCIM 2065), Proteus vulgaris (NCIM 2813) and one Gram-positive strain Staphylococcus aureus (NCIM 2120) (Table 4, Supplementary information). Most of the compounds are active against P. vulgaris and E. coli, some of the compounds are inactive against S. aureus. Results show that compounds 12a, 12e and 12m inhibited all the three bacterial growth. Whereas compounds 12b, 12g and 12h inhibited P. vulgaris and E. coli selectively, but compounds 12c and 12o are active against S. aureus. The SAR study indicates that the presence of electron-withdrawing groups on B ring is necessary for antibacterial activity; furthermore, replacement of aromatic B ring by heteroaromatic ring slightly decreases the antibacterial activity. Pathogenic yeasts of the genus Candida can cause life-threatening infections in immuno-deficient patients. This is because of the higher number of patients with malignancies treated with intensive immunosuppressive therapy regimens as well as their improved survival from formerly fatal bacterial infections and the rising number of patients undergoing allogenic hematopoietic stem cell or organ transplantation. There is an urgent need for novel fungicidal compounds with low toxicity. Synthesised compounds were screened for antifungal activity against Candida albicans (NCIM 3100) using fluconazole as standard, and the results are given in Table 4, Supplementary information. The antifungal screening data revealed that compounds 12h and 12m showed © 2013 Informa UK, Ltd.
good antifungal activity, while the rest of the compounds were inactive against C. albicans. About 30% of oral drugs fail in development due to poor pharmacokinetics36. Among the pharmacokinetic properties, a low and highly variable bioavailability is indeed the main reason for stopping further development of the drug. An in-silico model for predicting oral bioavailability is very important, both in the early stage of drug discovery to select the most promising compounds for further optimisation and in the later stage to identify candidates for further clinical development. In-silico prediction of oral bioavailability is pioneered by Lipinski37 “rule of five”; if two out of the following parameters are out of the range, then poor absorption or permeability is possible: the molecular weight is over 500, hydrogen bond donors are more than 5, the calculated octanol–water partition coefficient is over 5 (c Log P), hydrogen bond acceptors are more than 10. Drug likeness can be deduced as a delicate balance among the molecular properties of compound that directly influence its pharmacodynamics and pharmacokinetics and ultimately affect their absorption, distribution, metabolism and excretion in human body like a drug38. Thus, to obtain a good oral drug candidate, we have subjected a series of novel carbazole chalcones to the prediction of Lipinski rule of five and other properties and results are shown in Table 5 (Supplementary information). The results revealed that all the compounds satisfy the Lipinski rule of five. Compounds 12h, 12i, 12j, 12k and 12l have c Log P value more than 5; however, these compounds have molecular weight, hydrogen bond donor, hydrogen bond acceptor within the range of Lipinski rule of five, and these compounds have low polar surface area (PSA). Hence, these compounds do not pose absorption and permeation difficulty.
598 B. Bandgar et al. Hydrogen bonding capacity has also been identified as an important parameter for drug permeability. The results (Table 5, Supplementary information) reveal that compounds under study possessing less number of hydrogen bond donor and hydrogen bond acceptor; PSA provides good correlation with experimental transport data. It has been successfully applied for the prediction of intestinal absorption. PSA equal to or less than 140 will have high probability of good oral bioavailability39. Compound having low PSA could be a good oral drug candidate. Navia et al.,40 has postulated the desirability of molecular flexibility for membrane permeation. Molecular flexibility is measured by number of rotatable bonds. Compounds with 10 or fewer rotatable bonds will have probability of good oral bioavailability. The compounds under study have the number of rotatable bonds less than 10. Overall, all the synthesised carbazole chalcones meet the requirement of the Lipinski rule and other physicochemical properties, and may considered as good candidates for oral drug development.
Conclusion In summary, we have synthesised a series of novel carbazole chalcones and evaluated for cytotoxicity, antioxidant and antimicrobial activities. ADME prediction revealed that the compounds could be considered as good candidates for drug development. Compounds 12m, 12o and 12c exhibited good DPPH radical scavenging activity, compounds 12e, 12m and 12d were excellent hydroxyl radical scavengers, compounds 12g, 12e, 12n, 12a and 12m inhibited the oxidative DNA damage induced by AAPH. Compounds 12j, 12i, 12n, 12c, 12m and 12e have shown promising cytotoxicity against selected cancer cell lines, and they were highly selective to cancer cell line than the normal cell line; SAR study revealed that electron-withdrawing groups increase cytotoxicity. Compounds 12a, 12e and 12m showed good antibacterial activity and compounds 12h and 12m have shown good antifungal activity. Overall, compound 12m, 12e and 12a can be considered as novel carbazole chalcones as lead compounds for the development of novel chemopreventive agents.
and were used as supplied unless otherwise stated. Thin layer chromatography (TLC) was performed on silica gel-coated plates for monitoring the reactions.
Synthesis of 9-methyl carbazole (10) Carbazole 9 (4.1 g, 25 mmol) was dissolved in 50 mL DMF and NaH (1.8 g, 75 mmol) was added to it. The solution was stirred for 10 min and methyl iodide (7.05 g, 50 mmol) was added with constant stirring and stirring continued at room temperature for 3 h. After completion of reaction (TLC), the reaction mass was poured over crushed ice. The obtained pale yellow solid of 9-methyl carbazole 10 was filtered, washed with water and dried. Thus, the 9-methyl carbazole 10 (mp: 97°C, yield 94%) obtained was sufficiently pure and used further without purification.
Synthesis of 3-formyl-9-methyl carbazole (11) 9-methyl carbazole 10 (1.81 g, 10 mmol) was dissolved in dry DMF (20 mL) under anhydrous condition. It was cooled to 0°C, and POCl3 (1.87 mL) was added drop wise for 30 min and stirring continued for 4 h at 80°C. After completion of reaction (TLC), reaction mass was poured over crushed ice (50 g), basified with NaOH, extracted with chloroform and dried over anhydrous Na2SO4. Organic layer was concentrated and purified through silica gel column using chloroform as eluting solvent to yield product 11 (1.15 g). Yield: 55%; off white solid; mp: 76°C–78°C; IR (cm−1): 3035, 2704, 1685, 1626, 1591, 1570, 1050, 750; 1H NMR (300 MHz, DMSO-d6, δ in ppm): 3.91 (s, 3H, NCH3), 7.35 (t, 1H, J = 8.2 Hz, ArH), 7.40–7.60 (m, 3H, ArH), 7.98 (d, 1H, J = 8.2 Hz, ArH), 8.17 (d, 1H, J = 8.2 Hz, ArH), 8.60 (s, 1H, ArH), 10.1 (s, 1H, −CHO); m/z = 210.20 (m + 1); Anal. Calcd for C14H11NO: C, 80.36; H, 5.30; N, 6.69%. Found: C, 80.34; H, 5.31; N, 6.71%.
Synthesis of chalcones (13a–o)
General
A mixture of 3-formyl-9-methyl carbazole 7 (1 mmol) and substituted acetophenones (1 mmol) was dissolved in 20 mL ethanol. To this mixture, sodium hydroxide (20%, 1 mL) was added at 0°C–10°C. The reaction mixture was stirred at room temperature for 24–36 h. Then, this reaction mixture was poured over crushed ice and stirred. The solid thus obtained was filtered, washed with water and dried. The solid products were purified by recrystallisation using methanol to afford pure compounds 12a-o.
Melting points were recorded in open capillaries with electrical melting point apparatus and were uncorrected. IR spectra (KBr disks) were recorded using a Perkin–Elmer 237 spectrophotometer. 1H NMR (300 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker Avance spectrometer using DMSO-d6 as solvent. Mass spectra were recorded on a Shimadzu LCMS-QP 1000 EX. Elemental microanalysis was performed on Carlo Erba 1108 (CHN) Analyser. All the reagents and solvents used were of analytical grade
3-(9-Methyl-9H-carbazol-3-yl)-1-phenyl-propenone (12a) MP: 148°C–149°C; IR (cm−1): 3062, 2933, 2780, 1699, 1644, 1279, 1050, 750; 1H NMR (300 MHz, DMSO-d6, δ in ppm): 3.93 (s, 3H, NCH3), 7.27 (t, 1H, J = 7.5 and 7.8 Hz, ArH), 7.47–7.72 (m, 6H, ArH and CH=CH), 7.96 (s, 2H, ArH), 8.02 (d, 1H, J = 8.7 Hz, ArH), 8.15–8.26 (m, 3H, ArH), 8.74 (s, 1H, ArH); 13C NMR (100 MHz, DMSO-d6, δ in ppm): 189.8, 144.4, 139.5, 138.2, 137.0, 135.2, 131.1, 130.2, 128.5, 127.0, 125.5, 122.0, 120.6, 111.3, 109.8, 29.3; m/z = 312 (m
Experimental
Journal of Enzyme Inhibition and Medicinal Chemistry
Synthesis of novel carbazole chalcones 599 + 1); Anal. Calcd for C22H17NO: C, 84.86; H, 5.50; N, 4.50%. Found: C, 84.89; H, 5.53; N, 4.47%.
DPPH radical scavenging activity The DPPH radical scavenging activity of chalcones was measured according to the procedure described by Blois with minor modification41. The reaction mixture contained test compound with equal volume of DPPH radical (10−4 M in absolute ethanol) solution. After 20 min reaction time, the absorbance was recorded at 517 nm using UV–visible spectrophotometer. Ascorbic acid was used as a standard antioxidant agent. For determination of IC50 value, various concentrations of test compounds were taken, and the concentration required for 50% decrease in the absorbance of a control solution of DPPH was expressed as IC50.
Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was determined by the earlier reported method42. The reaction cocktail contained 60 μL of 1 mM FeCl3, 90 μL of 1 mM 1,10-phenanthroline, 2.4 mL of 0.2 M phosphate buffer (pH 7.8), 150 μL of 0.17 M H2O2 and 1.5 mL of test compound in DMSO. Reaction mixture was kept at room temperature for 5 min incubation, and absorbance was measured at 560 nm using spectrophotometer. Ascorbic acid was used as a reference compound.
Inhibition of AAPH-induced oxidative DNA damage The inhibition of AAPH-induced oxidative DNA damage by novel carbazole chalcones was assessed by measuring the conversion of super coiled pUC 18 plasmid DNA to open circular forms by gel electrophoresis33. pUC 18 DNA (2 μL) was incubated with test compounds (8 μL) at 100 μg concentration, and AAPH in phosphate-buffered saline (PBS) (8 μL) at pH 7.4 and 37°C for 1 h. After incubation, the sample (10 μL) was mixed with 2 μL of gel-loading buffer (0.13% bromophenol blue and 30% [W/V] glycerol) and immediately loaded onto prestained 0.8% agarose gel with ethidium bromide (10 μg/mL) and the gel electrophoresis was performed for 1 h. Clear fluorescent plasmid DNA bands were observed and photographed under gel documentation system. (UVItec Cambridge, UK).
Cytotoxicity of novel carbazole chalcones The cytotoxicity assay was performed as per the earlier reported method with slight modification43. The cells were harvested (2.5–3 × 104 cells/well) and inoculated in 96-well microtiter plate. The cells were washed with PBS and the cultured cells were then incubated in with and without test compound (100 μg). After 72 h incubation, the medium is aspirated. About 10 μL of MTT solution (5 mg/mL in PBS, pH 7.2) was added to each well, and the plates were incubated for 4 h at 37°C. After incubation, 100 μL of DMSO (
