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Original article
13 (
1
); 377-392
doi:
10.1016/j.arabjc.2017.05.004

Diazenyl schiff bases: Synthesis, spectral analysis, antimicrobial studies and cytotoxic activity on human colorectal carcinoma cell line (HCT-116)

, , , , ,
a Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India
b Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
c Collaborative Drug Discovery Research (CDDR) Group, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor Darul Ehsan, Malaysia
d Department of Pharmacology and Toxicology, College of Pharmacy, Qassim University, Buraidah 51452, Saudi Arabia
e Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

⁎Corresponding author. naru2000us@yahoo.com (Balasubramanian Narasimhan)

Received: , Accepted: ,
© The Author(s) 2017
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

A series of diazenyl schiff bases have been synthesized by reaction of salicylaldehyde containing azo dyes with various substituted aniline derivatives in the presence of acetic acid as catalyst. The structures of diazenyl derivatives were determined by FTIR, UV–vis, 1H NMR, 13C NMR, CHN analysis, fluorimetric and mass spectroscopic studies. The synthesized derivatives were screened for their in vitro antimicrobial activity against various Gram-positive (S. aureus, B. subtilis, B. cereus), Gram-negative (S. typhi, S. enterica, E. coli, P. aeruginosa) bacterial and fungal (C. albicans, A. niger and A. fumigatus) strains, using cefadroxil (antibacterial) and fluconazole (antifungal) as standard drugs. The diazenyl schiff bases were also screened for their cytotoxicity against human colorectal carcinoma cell line (HCT-116) using 5-fluorouracil as standard drug by Sulforhodamine-B Stain (SRB) assay. The schiff bases exhibited significant activity toward both Gram-positive, Gram-negative bacterial and fungal strains. Most of the synthesized derivatives showed high activity against S. enterica. 4-((2,5-Dichlorophenyl)diazenyl)-2-((3-bromophenylimino)methyl)phenol (SBN-40) was found to be very active against S. aureus, B. cereus and E. coli, with MIC = 0.69 (µM/ml × 102). The compound 4-((2-bromophenyl)diazenyl)-2-((4-nitrophenylimino)methyl)phenol (SBN-13) possessed comparable activity (IC50 = 7.5 µg/ml) to the standard drug 5-fluorouracil (IC50 = 3.0 µg/ml) against human colorectal carcinoma cell line (HCT-116).

Keywords

Diazenyl
Schiff base
Antimicrobial
Anticancer
Fluorescence
Bathochromic
1

1 Introduction

The number of life-threatening infectious diseases is increasing day by day due to the emergence of antibiotic resistance crisis which led to the threat of bioterrorism (Sengupta et al., 2013; Ventola, 2015). The discovery of penicillin, the first true antibiotic in 1928 had opened the door for the treatment of infectious diseases. Since then, the antibiotics have revolutionized modern medicine and saved the life of the millions of people (Kardos and Demain, 2011; Davies and Davies, 2010). However, in the late 1950s, the bulk production and overuse of penicillin led to antimicrobial resistance (AMR). The other emerged classes of antibiotics also exhibited the similar pattern from discovery, through increasing utilization to resistance (Morgan et al., 2011). The pharmaceutical industry had introduced several new antibiotics to overcome the drug resistance problem, from the late 1960s through the early 1980s, but afterward, the antibiotic pipeline began to dry up and fewer new drugs were introduced. As a result, in 2016, many decades after the first antibiotic introduced for the treatment of patients, the microbial infections have yet again become a threat (Woon and Fisher, 2016). So, in the present scenario, there is emergence need to discover new antibiotics or drugs required to combat the global spread of drug-resistant microbial pathogens and emerging infections (Bueno, 2016).

Human colorectal cancer (CRC), representing the third most common cancer worldwide and second malignity as the cause of death, is a prominent global health problem (Ferlay et al., 2015). Approximately 20–25% of patients have metastatic CRC (mCRC) at diagnosis, and 35–50% of patients develop mCRC after surgical treatment of locoregional disease (Boleij et al., 2016). Several chemotherapeutics agents including 5-fluorouracil, oxaliplatin, irinotecan, and more recently targeted biological agents, such as panitumumab, cetuximab, bevacizumab and regorafenib are used in different combinations, depending on the tumor characteristics and drug availability, which lead to variable survival rates in different patients (Lai et al., 2016; Troiani et al., 2013). However, developed resistance to both chemotherapy and molecularly targeted agents is the major challenge in the treatment of mCRC. So, there is a need to develop new agents and identify new targets for treatment of mCRC (Moriarity et al., 2016).

In the past, diazenyl derivatives have gained a lot of interest due to their potential therapeutic effects as antineoplastic, antiviral, antidiabetic, antiseptic, antifungal and antimicrobial agents (Awale et al., 2013; Zhu et al., 2013; Sahoo and Paidesetty, 2015). These compounds show their effect by inhibition of DNA, RNA and protein synthesis, by targeting enzymes such as GST, AGT, CDKs or targeting receptors such as tyrosine kinase, EGFR, etc. (Shridhar et al., 2016). These are derivatives of diazene (diimide), HN⚌NH, where both hydrogens are substituted by different aromatic or heteroaromatic groups. The biological activity of these derivatives depends upon their oxidation-reduction behavior (Escobar et al., 2016). The sulfonamide drug, prontosil was the first diazenyl based chemotherapeutic agent used for the cure of bacterial infections in human systemically (Brown, 2007). Similarly, dacarbazine (diazenyl compound) was approved for treatment of cancer in the late 1970s (Sahoo and Paidesetty, 2016). Several diazenyl derivatives have been synthesized in the past and evaluated for antimicrobial and anticancer potentials. The majority of them have shown promising antimicrobial activities and significant cytotoxic activities, even against several drug-resistant cell lines (Kaur et al., 2016). In view of these facts and in continuation of our studies related to the development of antimicrobial and anticancer agents (Deep et al., 2016a, 2016b), this study is focused on the synthesis of some new diazenyl schiff bases with various acceptor and donor groups, analysis of their spectral characteristics, evaluation of antimicrobial activity and cytotoxicity on HCT-116 cell line.

2

2 Materials and methods

The analytical grade chemicals required for synthesis and activity procured from Merck chemicals (India). Media for antimicrobial activity were obtained from Hi-media Laboratories. Microbial type cell cultures (MTCC) for antimicrobial activity were procured from Institute of Microbial Technology and Gene bank (IMTECH), Chandigarh. All raw materials and reagents used for analysis were 99% pure. Melting points were determined on labtech melting point equipment by an open glass capillary method and are uncorrected. Infrared (IR) spectra of the synthesized compounds were recorded on Bruker 12060280, spectrophotometer using KBr pellet method and expressed in cm−1. The nuclear magnetic resonance (NMR) spectra 1H NMR and 13C NMR spectra were recorded in deuterated CDCl3 on spectrometer (Model: Bruker Avance II 400 NMR) at a frequency of 400 MHz downfield to tetramethylsilane standard and chemical shifts were recorded as δ (parts per million) and s, d, t, q, m, dd, td, dt and tt are designated as singlet, doublet, triplet, quartet, multiplet, doublet of doublets, triplet of doublets, doublet of triplets and triplet of triplets respectively. Coupling constants (J) were reported in hertz (Hz). Electronic absorption studies were carried out using the dilute solution of the synthesized compounds in dichloromethane (DCM) and dimethyl sulphoxide (DMSO) on double beam UV–vis spectrophotometer (Model: UV 3000+, LabIndia), the absorption maxima (λmax) measured in nm. Fluorimetric data of synthesized compounds were determined with spectrofluorophotometer (Model: RF5301PC, Shimadzu, Japan). The mass spectra of synthesized derivatives were taken by mass spectrometer (Model: Advion expression CMS, USA) using atmospheric pressure chemical ionization as the ion source. Elemental analysis for synthesized diazenyl derivatives was performed on CHNN/CHNS/O analyzer (Flash EA1112N series, Thermo finnigan, Italy). The progress of the reaction was monitored by TLC performed on 0.25 mm precoated silica gel plates (60 F254) procured from Merck, and the spots were visualized in iodine chamber. The synthesized compounds were purified by column chromatography on silica gel 100–200 mesh size and recrystallization techniques.

2.1

2.1 General procedure for synthesis of diazenyl schiff bases (SBN 1-SBN 43)

The starting material azo dyes (1–17) for the synthesis of diazenyl schiff bases were prepared by diazotization of various substituted anilines with sodium nitrite and hydrochloric acid followed by coupling with salicylaldehyde in the presence of alkaline solution at 0 °C by reported procedures (Yahyazadeh and Azimi, 2013; Arabahmadi, 2016). The diazenyl schiff bases (SBN1-SBN43) were synthesized by reaction of salicylaldehyde azo dyes (5 mmol) with mono or di-substituted anilines (5 mmol) in ethanol/DMSO as the solvent and few drops of acetic acid according to the reported procedure (Halve et al., 2009) (Fig. 1). The reaction mixture was refluxed for 7–8 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction volume was concentrated to half and kept in the refrigerator overnight for precipitation of schiff bases. The synthesized derivatives were purified by column chromatography and recrystallization techniques. The structures of synthesized derivatives were established by UV–vis, IR, NMR, fluorimetric, mass and CHN/O/S analysis.

Synthetic scheme for synthesis of diazenyl derivatives (SBN1-SBN-43).
Figure 1 Synthetic scheme for synthesis of diazenyl derivatives (SBN1-SBN-43).

2.1.1

2.1.1 Analytical data

5-((4-Fluorophenyl)diazenyl)-2-hydroxybenzaldehyde (1): Yellow powder (1.7 g, 70%): Rf = 0.72 (ethyl acetate/hexane 2:3); mp: 120–125 °C; IR (KBr): 3456.44br (OH), 3163.26 (Ar—H), 1666.56vs (C⚌O), 1481.33s (N⚌N), 1396.46 (C—H), 1280.73 (O—H), 1157.29 (C—O), 902.88 (C—F), 850.46 (CH bending), cm−1; APCI-MS m/z found for C13H9FN2O2: 244 (M+), 245 (M+1)+.

5-((2-Fluorophenyl)diazenyl)-2-hydroxybenzaldehyde (2): Yellow powder (2.3 g, 95%): Rf = 0.80 (ethyl acetate/hexane 2:3); mp: 115–120 °C; IR (KBr): 3487.30br (OH), 3186.40 (Ar—H), 2870.08 (aldehydic CH), 1666vs (CO), 1543.05 (C⚌C), 1481.33s (N⚌N), 1381.03 (CH), 1280.67 (O—H), 1195.87 (C—O), 1095.57 (C—F), 840.96 and 756.10 (CH), 702.09 cm−1; APCI-MS m/z found for C13H9FN2O2: 244 (M+), 245 (M+1)+.

5-((2-Bromophenyl)diazenyl)-2-hydroxybenzaldehyde (3): Dark brown powder (2.8 g, 93%): Rf = 0.70 (ethyl acetate/hexane 1:3); mp: 110–115 °C; IR (KBr): 3441.01br (OH), 3055.24 (Ar—H), 2839.22 (aldehydic CH), 1668vs (C⚌O), 1573.91 (C⚌C), 1481.33s (N⚌N), 1373.22 (CH), 1280.73vs (O—H), 1103.26 (C—O), 702.09 (C—Br), 663.51 (CH) cm−1; APCI-MS m/z found for C13H9BrN2O2: 303 (M+), 304 (M+1)+.

5-((2,4-Dimethyl)diazenyl)-2-hydroxybenzaldehyde (4): Dark red powder (2.4 g, 95%): Rf = 0.65 (ethyl acetate/hexane 1:1); mp: 70–75 °C; IR (KBr): 3309.85br (OH), 2916.37 (aliphatic C—H), 2854.65 (aldehydic C—H), 1667vs (C⚌O), 1573.91 (C⚌C), 1481.33s (N⚌N), 1373.32 (CH3), 1237.73 (OH), 1172.72 (C—N), 1149.57 (C—O), 1033.85, 902.69 and 686.66 (CH) cm−1; APCI-MS m/z found for C15H14N2O2: 254 (M+), 255 (M+1)+.

5-((2-Chloro-4-nitrophenyl)diazenyl)-2-hydroxybenzaldehyde (5): Maroon red powder (2.29 g, 75%): Rf = 0.74 (ethyl acetate/hexane 2:3); mp: 80–75 °C; IR (KBr): 3329.99br (OH), 2862.36 (aldehydic C—H), 1666.50vs (C⚌O), 1581.63 (C⚌C), 1519.91 (NO2), 1481.33 (N⚌N), 1342.46 (NO2), 1195.87 (C—N), 1049.28 (C—O), 879.54 (C—Cl), 748.38 and 686.66 (CH) cm−1; APCI-MS m/z found for C13H8ClN3O4: 305 (M+), 306 (M+1)+.

5-((4-Chloro-3-nitrophenyl)diazenyl)-2-hydroxybenzaldehyde (6): Yellow powder (2.81 g, 92%): Rf = 0.58 (ethyl acetate/pet ether 1:4); mp: 130–135 °C; IR (KBr): 3310.12br (OH), 2840.36 (aldehydic C—H), 1667vs (C⚌O), 1573.63 (C⚌C), 1519.91 (NO2), 1481.33 (N⚌N), 1352.46 (NO2), 1186.87 (C—N), 1048.28 (C—O), 865.23 (C—Cl), 676.24 (CH bend) cm−1; APCI-MS m/z found for C13H8ClN3O4: 305 (M+), 306 (M+1)+.

5-((4-Chloro-2-nitrophenyl)diazenyl)-2-hydroxybenzaldehyde (7): Red powder (1.37 g, 45%): Rf = 0.53 (ethyl acetate/pet ether 1:4); mp: 80–85 °C; IR (KBr): 3325.28br (OH), 3070.68 (Ar—H) 2862.36 (aldehydic CH), 1651.07 (C⚌O), 1573.91 (C⚌C), 1527.62 (NO2), 1473.62 (N⚌N), 1373.32 (NO2), 1280.73 (O—H), 1226.73, 1149.57 m (C—N), 1095.57s (C—O) 918.12, 833.25 (C—Cl), 763.81(CH) cm−1; APCI-MS m/z found for C13H8ClN3O4: 305 (M+), 306 (M+1)+.

5-((4-Bromophenyl)diazenyl)-2-hydroxybenzaldehyde (8): Yellow powder (2.7 g, 91%): Rf = 0.71 (ethyl acetate/pet ether 1:2); mp: 170–175 °C; IR (KBr): 3394.72br (OH), 2870.08s (aldehydic CH), 1681.93s (C⚌O), 1573.91s (C⚌C), 1481.33s (N⚌N), 1381.03 (CH), 1280.73 (O—H), 1149.57 (C—N), 1064.71 (C—O), 833.25 (C—Br), 717.52 and 655.80 (CH) cm−1; APCI-MS m/z found for C13H9BrN2O2: 305 (M+), 306 (M+1)+.

2-Hydroxy-5-(phenyldiazenyl)benzaldehyde (9): Brown powder (2.1 g, 95%); Rf = 0.83 (ethyl acetate/pet ether 1:2); mp: 175–179 °C; IR (KBr): 3310br (OH), 1666.21vs (C⚌O), 1527.62s (C⚌C), 1481.33s (N⚌N), 1350.17 (CH), 825.53 and 756.10 (CH) cm−1; APCI-MS m/z found for C13H10N2O2: 226 (M+), 227 (M+1)+.

4-((3-Formyl-4-hydroxyphenyl)diazenyl)benzoic acid (10): Yellow powder (1.62 g, 60%): Rf = 0.52 (ethyl acetate/pet ether 1:2); mp: 285–290 °C; IR (KBr): 3441.01br (OH), 2947.23 (C—H), 2862.36 (aldehydic CH), 1813.09 (C⚌O), 1667s (C⚌O), 1573.91s (C⚌C), 1481.33vs (N⚌N), 1427.32vs, 1373.32 (C—H), 1288.45 (O—H), 1157.29 (C—O), 1103.28 (C—O) 902.69, 864.11, 840.96, 771.53, 709.80 and 663.51 (CH) cm−1; APCI-MS m/z found for C14H10N2O4: 270 (M+), 271 (M+1)+.

4-((3-Formyl-4-hydroxyphenyl)diazenyl)benzenesulfonamide (11): Yellow powder (1.7 g, 56%): Rf = 0.55 (ethyl acetate/pet ether 1:2); mp: 300–305 °C; IR (KBr): 3363.86br (OH), 3263.56 (N—H), 3070.68 (Ar—H), 2862.36 (aldehydic C—H), 2121.70, 1666.50s (C⚌O), 1581.63s (C⚌C), 1481.33s (N⚌N), 1334.74 (SO2), 1280.73s (OH), 1157.29 (C—N), 1087.86 (C—O), 902.69, 840.96, 671.23 (CH) cm−1; APCI-MS m/z found for C13H11N3O4S: 305 (M+), 306 (M+1)+.

5-((3-Bromophenyl)diazenyl)-2-hydroxybenzaldehyde (12): Yellow powder (2.9 g, 95%): Rf = 0.80 (ethyl acetate/pet ether 1:4); mp: 95–100 °C; IR (KBr): 3441.01br (OH), 3309.85, 3055.24 (Ar—H), 2870.08 (aldehydic CH), 1651.07s (C⚌O), 1573.91vs (C⚌C), 1481.33s (N⚌N), 1381.03s (CH), 1095.57 (C—O), 825.53, 771.53, 732.95vs (C—Br), 671.23 (CH) cm−1; APCI-MS m/z found for C13H9BrN2O2: 305 (M+), 306 (M+1)+.

4-((3-Formyl-4-hydroxyphenyl)diazenyl)benzenesulfonic acid (13): Orange crystals (1.16 g, 38%): Rf = 0.81 (ethyl acetate/pet ether 1:4); mp: 310–315 °C; IR (KBr): 3471.87br (OH), 3054.24 (Ar—H), 2870.08 (aldehydic C—H), 1658.78s (C⚌O), 1573.91s (C⚌C), 1481.33vs (N⚌N), 1373.32s (SO), 1288.45s (OH), 1203.58 (OH), 1178.71 (SO), 1033.85 (C—O), 840.96, 740.67, 666.66, 632.65 cm−1; APCI-MS m/z found for C13H10N2O5S: 305 (M+), 306 (M+1)+.

5-((4-Chlorophenyl)diazenyl)-2-hydroxybenzaldehyde (14): Yellow powder (2.2 g, 85%); Rf = 0.42 (ethyl acetate/pet ether 1:4); mp: 130–135 °C; IR (KBr): 3232.70br (OH), 3055.24 (Ar—H), 2870.08 (aldehydic-CH), 1666.50 (CO), 1573.91 (C⚌C), 1473.62 (N⚌N), 1381.03, 1280.73, 1157.29, 1087.85 (C—O), 902.69, 833.25, 725.23 (CH bend) cm−1; APCI-MS m/z found for C13H9ClN2O2: 260 (M+), 261 (M+1)+.

5-((3-Chlorophenyl)diazenyl)-2-hydroxybenzaldehyde (15): Red powder (2.29 g, 88%): Rf = 0.46 (ethyl acetate/pet ether 1:4); mp: 115–120 °C; IR (KBr): 3310br (OH), 1666.21s (C⚌O), 1527.62s (C⚌C), 1350.17 (CH), 825.53 (C—Cl), 756.10 (CH) cm−1; APCI-MS m/z found for C13H9ClN2O2: 260 (M+), 261 (M+1)+.

5-((2,5-Dichlorophenyl)diazenyl)-2-hydroxybenzaldehyde (16): Red powder (2.45 g, 83%), Rf = 0.48 (ethyl acetate/pet ether-1:4); mp: 285–290 °C; IR (KBr): 3433.29br (OH), 3387.00, 3086.11s (Ar—H), 2854.65s and 2777.50 (aldehydic CH), 1651.07 (CO), 1519.91s (C⚌C), 1450.47 (N⚌N), 1373.32 (CH), 1180.44 (C—N), 1056.99 (C—O), 941.26, 840.96s (C—Cl), 810.10, 748.36, 686.66 (CH) cm−1; APCI-MS m/z found for C13H8Cl2N2O2: 294 (M+), 295 (M+1)+.

5-((2,4-Dinitrophenyl)diazenyl)-2-hydroxybenzaldehyde (17): Dark maroon powder (2.72 g, 72%): Rf = 0.48 (ethyl acetate/pet ether 1:3); mp: 135–140 °C; IR (KBr): 3470.01br (OH), 3387.00, 3109.25 (Ar—H), 2854.65s and 2746.63 (aldehydic CH), 1666.50 (C⚌O), 1559.34 (NO2), 1481.76s (N⚌N), 1419.61s (NO2), 1018.41 (C—O), 981.12, 833.25 (C—Cl), 709.80 (CH) cm−1; APCI-MS m/z found for C13H8N4O6: 316 (M+), 317 (M+1)+.

4-((3-Bromophenyl)diazenyl)-2-((4-chlorophenylimino)methyl)phenol (SBN-1): Dark orange powder (1.14 g, 55%): Rf = 0.56 (Hexane/chloroform/ethyl acetate 5:2:1); mp: 155–160 °C; 1H NMR (400 MHz, CDCl3): 7.14 (d, J = 4 Hz, 2H), 7.35–7.40 (m, 4H), 7.44 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 5.4 Hz, 1H), 8.03 (dd, J1 = 8 Hz, J2 = 1.6 Hz, 2H), 8.72 (s, 1H), 13.69 (s, 1H); 13C NMR (400 MHz, CDCl3):164.32, 162.30, 153.48, 146.27, 145.37, 133.21, 133.10, 130.44, 129.70, 128.50, 127.72, 124.37, 123.14, 122.77, 122.50, 119.459, 118.79, 118.34 ppm; IR (KBr): 3448.72 (OH), 2916.37 (C—H), 2854.65 (aldehydic C—H), 1612.49s (CH⚌N), 1566.20s (C⚌C), 1481s (N⚌N), 1465.90, 1404.18, 1288.45s (OH), 1095.57s (C—O), 964.41, 879.54, 828s (C—Cl), 632.65 (C—Br), 509.21 (CH) cm−1; APCI-MS m/z found for C19H13BrClN3O: 414 (M+), 416 (M+2)+; Anal. calcd for C19H13BrClN3O: C 55.0, H 3.2, N 10.1, O 3.9, found: C 55.0, H 3.2, N 10.5 O, 3.8.

4-((2-Chloro-4-nitrophenyl)diazenyl)-2-((2-fluorophenylimino)methyl)phenol (SBN-9): Red powder (1.34 g, 67%): Rf = 0.85 (ethyl acetate/hexane 2:3); mp: 200–205 °C; 1H NMR (400 MHz, CDCl3):7.30 (m, 6H), 7.80 (s, 1H), 8.18 (m, 2H), 8.45 (s, 1H), 8.75 (s, 1H), 14.06 (s, 1H); IR (KBr): 3353.18 (OH), 3160.27 (Ar—H), 1612.17s (CH⚌N), 1572.80s (C⚌C), 1512.43s (NO2), 1425.80s (N⚌N), 1340.26 (NO2), 1296 (O—H), 1048.45s (C—F), 880.73, 830.34s (C—Cl), 735.80 (CH) cm−1; APCI-MS m/z found for C19H12ClFN4O3: 399 (M+), 401 (M+2)+; Anal. calcd for C19H12ClFN4O3: C 57.2, H 3.0, N 14.05, O 12.0, found: C 57.1, H 3.1, N 14.35, O 11.9.

4-((3-Bromophenyl)diazenyl)-2-((2-methoxyphenylimino)methyl)phenol (SBN-12): Maroon shining crystals (1.02 g, 50%): Rf = 0.45 (ethyl acetate/hexane 3:7); mp: 110–115 °C; 1H NMR (400 MHz, CDCl3): 3.85 (s, 3H), 6.95 (dd, J1 = 5.72 Hz, J2 = 0.96 Hz, 1H), 6.98 (td, J1 = 5.04 Hz, J2 = 0.8 Hz, 1H), 7.07 (d, J = 5.72 Hz, 1H), 7.23–7.26 (m, 2H), 7.34 (t, J = 5.24 Hz, 1H), 7.78 (d, J = 5.28 Hz, 1H), 7.81 (d, J = 4.72 Hz, 1H), 7.95–7.98 (m, 3H), 8.74 (s, 1H), 14.81 (s, 1H); 13C NMR (400 MHz, CDCl3): 166.420, 160.579, 153.566, 152.931, 144.775, 135.168, 132.946, 130.411, 128.826, 128.543, 127.304, 124.306, 123.115, 122.729, 121.045, 119.459, 118.960, 118.811, 111.920, 55.872 ppm; IR (KBr): 3308.53 (OH), 3207.11 (C—H), 1612s (CH⚌N), 1573.98s (C⚌C), 1475.86s (N⚌N), 1391.76 (CH3), 1343.60, 1284.84 (OH), 1191.35 (C—N), 1091.20 (C—O), 996.47, 829.94 (C—Br), 732.70 (CH) cm−1. APCI-MS m/z found for C20H16BrN3O2: 410 (M+), 412 (M+2)+; Anal. calcd for C20H16BrN3O2: C 58.55, H 3.9, N 10.2, O 7.8, found: C 58.7, H 4.0, N 10.3, O 7.9.

4-((2-Bromophenyl)diazenyl)-2-((4-nitrophenylimino)methyl)phenol (SBN-13): Light Orange crystals (1.10 g, 52%): Rf = 0.64 (ethyl acetate/hexane 3:7); mp: 130–135 °C; 1H NMR (400 MHz, CDCl3): 7.18 (d, J = 5.88 Hz, 1H), 7.26–7.43 (m, 4H), 7.67 (dd, J1 = 5.32 Hz, J2 = 1.12 Hz, 1H), 7.74–7.76 (m, 1H), 8.12–8.16 (m, 2H), 8.32 (dd, J1 = 4.52 Hz, J2 = 1.4 Hz, 2H), 8.77 (s, 1H), 13.14 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.99, 164.31, 153.44, 149.41, 146.41, 145.93, 133.79, 131.73, 129.36, 128.87, 128.02, 125.72, 125.30, 121.99, 118.56, 118.49, 117.71, 117.77 ppm; IR (KBr): 3345.34 (OH), 3057.11 (Ar—H), 1612s (CH⚌N), 1586.20s (C⚌C), 1519.17s (NO2) 1489.05s (N⚌N), 1284.84 (OH), 1103.28s (C—O), 756.10s (C—Br), 732.70 (CH) cm−1; APCI-MS m/z found for C19H13BrN4O3: 425 (M+), 427 (M+2)+; Anal. calcd for C19H13BrN4O3: C 53.7, H 3.1, N 13.2, O 11.3, found: C 53.7, H 3.2, N 13.2, O 11.15.

4-((4-Chloro-3-nitrophenyl)diazenyl)-2-((2-methylphenylimino)methyl)phenol (SBN-14): Maroon crystals (1.05 g, 58%): Rf = 0.74 (ethyl acetate/hexane 1:3); mp: 140–145 °C; 1H NMR (400 MHz, CDCl3): 2.43 (s, 3H), 7.14 (d, J = 5.92 Hz, 1H), 7.16–7.31 (m, 4H), 7.67 (d, J = 5.64 Hz, 1H), 8.02–8.08 (m, 3H), 8.36 (d, J = 1.42 Hz, 1H), 8.69 (s, 1H), 14.39 (s, 1H); 13C NMR (400 MHz, CDCl3): 165.79, 161.1, 151.10, 148.50, 146.21, 144.93, 132.48, 132.45, 130.97, 129.27, 128.01, 127.57, 127.45, 127.35, 127.21, 119.04, 118.69, 118.64, 117.63, 18.24 ppm; IR (KBr): 3310 (OH), 1620.21 (CH⚌N), 1527.62 (NO2), 1350.17 (NO2), 1288.45 (OH), 1103.28 (C—O), 910.44, 825.53s (C—Cl), 756.10 (CH) cm−1; APCI-MS m/z found for C20H15ClN4O3: 395 (M+), 397 (M+2)+; Anal. calcd for C20H15ClN4O3: C 60.8, H 3.8, N 14.2, O 12.2, found: C 60.9, H 3.9, N 14.5, O 12.2.

4-((4-Chlorophenyl)diazenyl)-2-((2,4-dimethylphenylimino)methyl)phenol (SBN-15): Orange crystals, yield (0.94 g, 52%): Rf = 0.67 (ethyl acetate/hexane 1:3); mp: 135–140 °C; 1H NMR (400 MHz, CDCl3): 2.35 (s, 3H), 2.40 (s, 3H), 7.08–7.12 (m, 4H), 7.45–7.48 (m, 2H), 7.82–7.84 (m, 2H), 7.99–8.02 (m, 2H), 8.67 (s, 1H), 14.32 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.67, 160.48, 150.98, 145.30, 143.88, 137.40, 136.32, 132.46, 131.68, 129.32, 127.95, 127.68, 127.29, 123.85, 119.09, 118.26, 117.26, 21.04, 18.20 ppm; IR (KBr): 3448.72br (OH), 2916.37 (aliphatic C—H), 2854.65 (CH), 1620.21s (CH⚌N), 1489.05 (N⚌N), 1350.17s (CH3), 1288.45s (OH), 1195.87, 1080.14 (C—O), 972.12, 810.10 (C—Cl) cm−1; APCI-MS m/z found for C21H18ClN3O: 364 (M+), 366 (M+2)+; Anal. calcd for C21H18ClN3O: C 69.3, H 5.0, N 11.55, O 4.4, found: C 69.55, H 5.2, N 11.8, O 4.6.

4-((3-Chlorophenyl)diazenyl)-2-((2,6-dimethylphenylimino)methyl)phenol (SBN-18): Orange crystals (0.87 g, 48%): Rf = 0.67 (hexane/ethyl acetate/CHCl3 8:1:1); mp: 62–65 °C; 1H NMR (400 MHz, CDCl3): 2.28 (s, 6H), 7.04–7.17 (m, 4H), 7.39–7.45 (m, 2H), 7.79 (dt, J1 = 5.04 Hz, J2 = 1.04 Hz, 1H), 7.87 (t, J = 1.32 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 8.05 (dd, J1 = 5.88 Hz, J2 = 1.6 Hz, 1H), 8.45 (s, 1H), 13.78 (s, 1H); 13C NMR (400 MHz, CDCl3):166.42, 164.65, 153.44, 147.45, 145.27, 135.15, 130.29, 130.14, 128.52, 128.38, 128.20, 125.41, 122.18, 121.55, 118.49, 118.40, 18.56, 17.63 ppm; IR (KBr): 3340 (OH), 3070.68 (Ar—H), 2954.95 (aliphatic C—H), 2808.36 (aldehydic CH), 1620.21s (CH⚌N), 1557.39 (C⚌C), 1473.62 (N⚌N), 1342.46 (CH), 1288.45 (OH), 1195.87, 1111.00 (C—O), 825.53 (C—Cl), 786.96 (CH) cm−1; APCI-MS m/z found for C21H18ClN3O: 364 (M+), 366 (M+2)+; Anal. calcd for C21H18ClN3O: C 69.3, H 5.0, N 11.55, O 4.4, found: C 69.4, H 5.1, N 11.6, O 4.5.

4-((2,5-Dichlorophenyl)diazenyl)-2-((4-(methylthio)phenylimino)methyl)phenol (SBN-20): Red powder (1.10 g, 53%): Rf = 0.27 (hexane/chloroform 4:1); mp: 125–130 °C; 1H NMR (400 MHz, CDCl3): 2.51 (s, 3H), 7.11 (d, J = 5.76 Hz, 1H), 7.25–7.31 (m, 5 H), 7.45 (d, J = 5.68 Hz, 1H), 7.68 (d, J = 1.68 Hz, 1H) 8.02–8.04 (m, 2H), 8.71 (s, 1H), 14.06 (s, 1H); 13C NMR (400 MHz, CDCl3): 165.09, 160.67, 148.89, 145.54, 144.30, 138.32, 133.44, 133.20, 131.54, 130.73, 128.62, 128.12, 127.33, 121.72, 118.95, 118.42, 117.74, 15.91 ppm; IR (KBr): 3448.72 (OH), 3086.11 (Ar—H), 2985.81 (aliphatic C—H), 1612.49s (CH⚌N), 1589.34 (C⚌C), 1465.90s (N⚌N), 1350.17 (CH), 1288.45s (OH), 1103.28 (C—O), 879.54, 817.82s (C—Cl), 740.67 (CH bending) cm−1; APCI-MS m/z found for C20H15Cl2N3OS: 416 (M+), 418 (M+2)+; Anal. calcd for C20H15Cl2N3OS: C 57.7, H 3.6, N 10.1, O 3.8, S 7.7, found: C 57.9, H 3.7, N 10.2, O 3.9, S 7.8.

4-((2,5-Dichlorophenyl)diazenyl)-2-((2,4-dimethylphenylimino)methyl)phenol (SBN-21): Pink fluffy crystals (1.19 g, 60%): Rf = 0.62 (hexane/ethyl acetate 7:3); mp: 141–145 °C; 1H NMR (400 MHz, CDCl3): 2.36 (s, 3H), 2.41 (s, 3H), 7.09–7.14 (m, 4H), 7.32 (d, J1 = 5.70 Hz, J2 = 1.70 Hz, 1H), 7.47 (d, J = 5.72 Hz, 1H), 7.71 (d, J = 1.68 Hz, 1H), 8.04 (dd, J1 = 7.0 Hz, J2 = 1.5 Hz, 2H), 8.70 (s, 1H), 14.51 (s, 1H); 13C NMR (400 MHz, CDCl3): 165.42, 160.33, 149.01, 145.43, 143.61, 137.52, 133.45, 133.14, 132.45, 131.69, 131.50, 130.66, 128.71, 127.97, 127.70, 119.06, 118.53, 117.79, 117.28, 21.04, 18.19 ppm; IR (KBr): 3471.87br (OH), 3140.11, 3086.11 (Ar—H), 2916.37 (aliphatic C—H), 2862.36 (C—H), 1620.21vs (CH⚌N), 1481.21s (N⚌N), 1350.17 (CH3), 1288.45 (OH), 1149.57 (C—N), 1103.28 (C—O), 1049.28, 817.82 (C—Cl) cm−1; APCI-MS m/z found for C22H18Cl2N2O: 398 (M+), 400 (M+2)+; Anal. calcd for C22H18Cl2N2O: C 66.5, H 4.6, N 7.0, O 4.0, found: C 66.8, H 4.8 N 7.3 O 4.3.

4-((3-Chlorophenyl)diazenyl)-2-((2,4-dimethylphenylimino)methyl)phenol (SBN-25): Red powder (0.78 g, 43%): Rf = 0.58 (hexane/ethyl acetate 7:3); mp: 120–125 °C; 1H NMR (400 MHz, CDCl3): 2.34 (s, 3H), 2.39 (s, 3H), 7.07–7.12 (m, 4H), 7.38–7.44 (m, 2H), 7.77 (dt, J1 = 5.08 Hz, J2 = 1.04 Hz, 1H), 7.85 (t, J = 1.28 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.99–8.00 (m, 1H), 8.65 (s, 1H), 14.36 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.95, 160.35, 153.46, 145.18, 143.75, 137.43, 135.11, 132.47, 131.69, 130.18, 130.10, 128.25, 127.70, 127.30, 122.20, 121.52, 119.06, 118.31, 117.23, 21.10, 18.21 ppm; IR (KBr): 3309.85br (OH), 3062.96 (Ar—H), 2970.38 (aliphatic C—H), 2916.37 (C—H), 2854.65 (CH), 1612.49s (CH⚌N), 1458.18 (N⚌N), 1350.17s (CH3), 1288.45s (OH), 1188.15, 1141.86 (C—O), 1095.57, 864.11 (C—Cl), 786.96 and 678.94 (CH) cm−1; APCI-MS m/z found for C22H19ClN2O: 364 (M+), 366 (M+2)+; Anal. calcd for C22H19ClN2O: C 72.8, H 5.3, N 7.7, O 4.4, found: C 73.0, H 5.4, N 7.9, O 4.5.

2-((2,5-Dichlorophenylimino)methyl)-4-((2,4-dimethylphenyl)diazenyl)phenol (SBN-26): Red powder (1.29 g, 65%): Rf = 0.63 (hexane/ethyl acetate 7:3); mp: 105–110 °C; 1H NMR (400 MHz, CDCl3): 2.32 (s, 3H), 2.64 (s, 3H), 7.01 (dd, J1 = 5.46 Hz, J2 = 1.38 Hz, 1H), 7.06–7.08 (m, 2H), 7.13 (dd, J1 = 5.64 Hz, J2 = 1.56 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 5.64 Hz, 1H), 7.52 (d, J = 5.4 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 5.88 Hz, J2 = 1.56 Hz, 1H), 8.55 (s, 1H), 13.34 (s, 1H); 13C NMR (400 MHz, CDCl3): 163.55, 163.23, 148.46, 146.05, 145.483, 141.09, 138.02, 133.41, 131.86, 131.05, 128.40, 128.21, 128.01, 127.82, 127.24, 119.22, 118.41, 118.25,115.23, 21.42, 17.57 ppm; IR (KBr): 3417.86 (OH), 3140.11 and 3086.11 (Ar—H), 2916.37 (aliphatic C—H), 2826.36 (CH), 1666.50s (CH⚌N), 1573.92s (C⚌C), 1481s (N⚌N), 1419.61, 1381.03 (CH3), 1280.73 (OH), 1165.00 (C—N), 1087.85s (C—O), 833.25vs (C—Cl), 725.23s (CH) cm−1; APCI-MS m/z found for C22H18Cl2N2O: 398 (M+), 400 (M+2)+; Anal. calcd for C22H18Cl2N2O: C 66.5, H 4.6, N 7.05, O 4.0, found: C 66.7, H 4.8, N 7.3, O 4.0.

2-((4-Fluorophenylimino)methyl)-4-((2,4-dimethylphenyl)diazenyl)phenol (SBN-28): Brown granules (1.18 g, 68%): Rf = 0.71 (hexane/ethyl acetate 2:1); mp: 95–100 °C; 1H NMR (400 MHz, CDCl3): 2.33 (s, 3H), 2.65 (s, 3H), 7.01 (dd, J1 = 5.48 Hz, J2 = 1.28 Hz, 1H), 7.03–7.25 (m, 6H), 7.54 (d, J = 5.44 Hz, 1H), 7.91 (d, J = 1.6 Hz, 1H), 8.02–8.04 (dd, J1 = 5.84 Hz, J2 = 1.56 Hz, 1H), 8.58 (s, 1H), 13.59 (s, 1H); 13C NMR (400 MHz, CDCl3): 163.38, 162.67, 161.89, 161.04, 148.58, 146.08, 143.99, 140.97, 137.90, 131.86, 128.04, 127.25, 127.16, 122.77, 122.71, 118.75, 118.01, 116.38, 116.23, 115.26, 21.43, 17.54 ppm; IR (KBr): 3370.68 (OH), 3033.18 (Ar—H), 1621.12s (CH⚌N), 1573.34s (C⚌C), 1480.33s (N⚌N), 1340.09 (CH3), 1275.73 (OH), 1209.01, 1190.57 (C—O), 1021 (C—F), 860.11, 825.25, 760.81 cm−1; APCI-MS m/z found for C22H19FN2O: 347 (M+), 348 (M+1)+; Anal. Calcd for C22H19FN2O: C 76.3, H 5.5, N 8.1, O 4.6, found: C 76.6, H 5.7, N 8.3, O 4.7.

2-((2,6-Dimethylphenylimino)methyl)-4-((2-fluorophenyl)diazenyl)phenol (SBN-29): Orange crystals (1.11 g, 64%): Rf = 0.90 (hexane/ethyl acetate 3:2); mp: 70–75 °C; 1H NMR (400 MHz, CDCl3): 2.12 (s, 6H), 6.92–7.15 (m, 6H), 7.29 (ddd, J1 = 5.6 Hz, J2 = 4.5 Hz, J3 = 1.24 Hz, 1H), 7.64 (dd, J1 = ‘5.24 Hz, J2 = 1.2 Hz, 1H), 7.91 (d, J = 1.6 Hz, 1H), 7.98 (dd, J1 = 5.92 Hz, J2 = 1.6 Hz, 1H), 8.33 (s, 1H), 13.67 (s, 1H); 13C NMR (400 MHz, CDCl3):166.53, 164.59, 160.76, 159.06, 147.49, 145.80, 140.62, 131.99, 128.37, 127.73, 125.40, 124.34, 118.50, 118.33, 117.76, 117.11, 116.97, 18.57 ppm; IR (KBr): 3371.57 (OH), 3024.28 (Ar—H), 2893.22 (aliphatic C—H), 1620.21 (CH⚌N), 1589.34 (C⚌C), 1481.33s (N⚌N), 1342.36 (CH3), 1280.73 (O—H), 1219.01, 1195.87 (C—O), 1095.57 (C—F), 864.11, 833.25, 763.81 (CH) cm−1; APCI-MS m/z found for C22H19FN2O 347 (M+), 348 (M+1)+; Anal. calcd for C22H19FN2O: C 76.3, H 5.5, N 8.1, O 4.6, found: C 76.5, H 5.7, N 8.4, O 4.8.

4-((3-Bromophenyl)diazenyl)-2-((2,5-dichlorophenylimino)methyl)phenol (SBN-32): Orange crystals (1.55 g, 69%): Rf = 0.68 (hexane/ethyl acetate/CHCl3 4:1:1); mp: 150–154 °C; 1H NMR (400 MHz, CDCl3): 7.21 (d, J = 5.96 Hz, 1H), 7.40–7.42 (m, 2H), 7.59 (d, J = 5.2 Hz, 1H), 7.85 (d, J = 5.2 Hz, 1H), 8.12–8.13 (m, 3H), 8.27 (d, J = 1.6 Hz, 1H), 8.43 (s, 1H), 8.79 (s, 1H), 13.09 (s, 1H); 13C NMR (400 MHz, CDCl3): 165.42, 164.48, 133.46, 130.49, 129.34, 128.91, 125.87, 124.41, 123.41, 123.18, 122.87, 119.93, 118.82, 118.41 ppm; IR (KBr): 3348.42 (OH), 3170.97 and 3078.39 (Ar—H), 2985.81 (aliphatic C—H), 2893.22, 1620.21s (CH⚌N), 1566.20s (C⚌C), 1485.90s (N⚌N), 1357.89 (CH), 1288.45s (O—H), 1195.87 (C—N), 1103.28 (C—O), 925.83, 810.10 (C—Cl), 678.94s (C—Br), 601.79, 540.07 (CH) cm−1; APCI-MS m/z found for C20H13BrCl2N2O: 449 (M+), 450 (M+1)+; Anal. calcd for C20H13BrCl2N2O: C 53.6, H 2.9, N 6.25, O 3.6, found: C 54.0, H 3.2, N 6.45, O 3.7.

4-((3-Bromophenyl)diazenyl)-2-((2-chloro-4-nitrophenylimino)methyl)phenol (SBN-33): Orange crystal (1.49 g, 65%): Rf = 0.68 (hexane/ethyl acetate 3:2); mp: 140–145 °C; 1H NMR (400 MHz, CDCl3): 7.21 (d, J = 5.96 Hz, 1H), 7.42(dd, J1 = 6.36 Hz, J2 = 3.6 Hz, 2H), 7.60 (d, J = 5.2 Hz, 1H), 7.85 (d, J = 5.2 Hz, 1H), 8.12–8.13 (m, 3H), 8.27 (d, J = 1.6 Hz, 1H), 8.43 (s, 1H), 8.79 (s, 1H), 13.09 (s, 1H); 13C NMR (400 MHz, CDCl3): 165.42, 164.48, 133.46, 130.49, 129.34, 128.91, 125.87, 124.41, 123.41, 123.18, 122.87, 119.93, 118.82, 118.41 ppm; IR (KBr): 3209.55 (OH), 3078.39 (Ar—H), 2970.36 and 2908.65 (CH), 2846.43 (CH), 1612.49 (CH⚌N), 1589.34 (C⚌C), 1512.19 (NO2), 1473.62 (N⚌N), 1334.74 (NO2), 1188.15 (C—N), 1141.86 (C—O), 1041.56, 979.84, 894.97, 825.53s (C—Cl), 786.96s (C—Br), 678.94 (CH) cm−1; APCI-MS m/z found for C20H13BrClN3O3: 460 (M+), 461 (M+1)+; Anal. calcd for C20H13BrClN3O3: C 52.4, H 2.9, N 9.2, O 10.5, found: C 52.5, H 3.0, N 9.45, O 10.6.

4-((2-Chloro-4-nitrophenyl)diazenyl)-2-((2-methoxyphenylimino)methyl)phenol (SBN-34): Dark chocolate brown powder (1.48 g, 72%): Rf = 0.62 (hexane/ethyl acetate 7:3); mp: 165–170 °C; 1H NMR (400 MHz, CDCl3): 6.82–6.93 (m, 3H), 7.16 (d, J = 2.8 Hz, 1H), 7.35 (t, J = 5.6 Hz, 1H), 7.79 (d, J = 3.2 Hz, 1H), 8.13 (m, 2H), 8.20 (s, 1H), 8.43 (s, 1H), 8.76 (s, 1H), 14.24 (s, 1H); 13C NMR (400 MHz, CDCl3): 163.70, 131.15, 128.98, 127.96, 124.34, 119.49, 118.52, 117.71 ppm; IR (KBr): 3352.62 (OH), 3248.21, 2912.64 (aliphatic C—H), 1612.23s (CH⚌N), 1482.33s (N⚌N), 1423.24s (NO2), 1369.32s (NO2), 1142.11 (C—N), 1095.66 (C—O), 885.23, 830.16 (C—Cl), 765.48 (CH) cm−1; APCI-MS m/z for C21H16ClN3O4: 411 (M+), 413 (M+2)+; Anal. calcd for C21H16ClN3O4: C 61.5, H 3.9, N 10.25, O 15.6, found: C 61.7, H 4.0, N 10.4, O 15.85.

4-((2-Chloro-4-nitrophenyl)diazenyl)-2-((4-fluorophenylimino)methyl)phenol (SBN-35): Dark chocolate brown powder (1.49 g 75%): Rf = 0.62 (hexane/ethyl acetate 3:2); mp: 195–200 °C; 1H NMR (400 MHz, CDCl3): 7.25–7.27 (m, 9H), 7.81 (s, 1H), 8.19 (s, 1H), 8.45 (s, 1H), 14.21 (s, 1H). IR (KBr): 3452.63 (OH), 3082.21 (Ar—H), 1621.96vs (CH⚌N), 1573.85s (NO2), 1423.71s (N⚌N), 1342.52 (NO2), 1296.59 (OH), 1179.78 (C—N), 1107.76 (C—O), 1049.45 (C—F), 888.84, 831.47 (C—Cl), 739.81 (CH) cm−1; APCI-MS m/z for C19H12ClFN4O3: 399 (M+), 401 (M+2)+; Anal. calcd for C19H12ClFN4O3: C 57.2, H 3.0, N 14.05, O 12.0, found: C 57.45, H 3.2, N 14.35, O 12.2.

4-((2-Chloro-4-nitrophenyl)diazenyl)-2-((2,6-dimethylphenylimino)methyl)phenol (SBN-36): Dark Maroon powder (1.51 g, 74%): Rf = 0.71 (hexane/ethyl acetate 3:2); mp: 150–155 °C; 1H NMR (400 MHz, CDCl3): 2.44 (s, 6H), 7.06–7.08 (m, 1H), 7.13 (d, J = 5.0 Hz, 2H), 7.20 (d, J = 5.96 Hz, 1H), 7.79 (dd, J1 = 5.96 Hz, J2 = 0.96 Hz, 1H), 8.09 (s, 1H), 8.15 (d, J = 6.0 Hz, 1H), 8.19 (d, J = 5.96 Hz, 1H), 8.43 (s, 1H), 8.50 (s, 1H), 14.10 (s, 1H); 13C NMR (400 MHz, CDCl3): 166.28, 166.23, 152.26, 148.26, 147.03, 145.79, 134.98, 129.51, 128.55, 128.52, 128.39, 126.19, 125.61, 122.66, 118.90, 118.52, 118.28, 18.54 ppm; IR (KBr): 3348br (OH), 3078 (Ar—H), 2939 (aliphatic C—H), 1612s (CH⚌N), 1496s (N⚌N), 1427s (NO2), 1373 (NO2), 1149s (C—N), 1095 (C—O), 833s (C—Cl), 702 (CH) cm−1; APCI-MS m/z found for C22H18ClN3O3: 408 (M+), 409 (M+1)+; Anal. calcd for C22H18ClN3O3: C 64.8, H 4.45, N 10.3, O 11.8, found: C 64.8, H 4.55, N 10.5, O 11.8.

2-((2,5-Dichlorophenylimino)methyl)-4-((2-fluorophenyl)diazenyl)phenol (SBN-37): Brown powder (1.59 g, 82%): Rf = 0.48 (hexane/ethyl acetate 7:3); mp: 160–165 °C; 1H NMR (400 MHz, CDCl3): 7.13–7.45 (m, 7H), 7.77 (s, 1H), 8.12 (s, 1H), 8.23 (d, J = 5.84 Hz, 1H), 8.76 (s, 1H), 13.47 (s, 1H); 13C NMR (400 MHz, CDCl3): 163.70, 131.15, 128.98, 127.96, 124.34, 119.49, 118.52, 117.71 ppm; IR (KBr): 3232.70br (OH), 3032.90 (Ar—H), 1666.50s (CH⚌N), 1573.93s (C⚌C), 1481.30s (N⚌N) 1381.03 (CH), 1280.73s (OH), 1157.29 (C—O), 902.60s (C—F), 840.96vs (C—Cl), 717.52 and 578.64 (CH) cm−1; APCI-MS m/z for C22H18ClN3O3: 388 (M+), 390 (M+2)+; Anal. calcd for C22H18ClN3O3: C 62.0, H 3.4, N 7.2, O 4.1, found: C 62.25, H 3.65, N 7.3, O 4.2.

2-((2,4-Dimethylphenylimino)methyl)-4-((2-fluorophenyl)diazenyl)phenol (SBN-38): Brown powder (1.21 g, 70%): Rf = 0.53 (hexane/ethyl acetate 7:3); mp: 95–100 °C; 1H NMR (400 MHz, CDCl3): 2.31 (s, 3H), 2.36 (s, 3H), 7.03–7.05 (m, 3H), 7.08 (d, J = 5.56 Hz, 1H), 7.16–7.24 (m, 2H), 7.38 (q, J1 = 4.48 Hz, J2 = 1.2 Hz, 1H), 7.72 (td, J1 = 5.2 Hz, J2 = 1.6 Hz, 1H), 7.99–8.02 (m, 2H), 8.60 (s, 1H), 14.35 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.88, 160.36, 159.05, 145.70, 143.71, 140.65, 137.40, 132.50, 131.91, 131.68, 128.01, 127.71, 124.32, 119.05, 118.21, 117.73, 117.21, 117.07, 116.93, 21.05, 18.22 ppm; IR (KBr): 3363.86 (OH), 2916.37 (aliphatic C—H), 2854.65 (C—H), 1603.99s (CH⚌N), 1481.59s (N⚌N), 1349.52 (CH3), 1209.27, 1152.03 (C—N), 1103.28 (C—O), 1096.59 (C—F), 972.12, 817.86vs, 757.29 (CH) cm−1; APCI-MS m/z for C21H18FN3O: 347 (M+), 348 (M+1)+; Anal. calcd for C21H18FN3O: C 72.6, H 5.2, N 12.1, O 4.6, found: C 72.6, H 5.25, N 12.15, O 4.8.

(4-((2,5-Dichlorophenyl)diazenyl)-2-((2-methoxyphenylimino)methyl)phenol (SBN-39): Red powder (1.56 g, 78%): Rf = 0.63 (hexane/ethyl acetate 7:3); mp: 140–145 °C; 1H NMR (400 MHz, CDCl3): 3.93 (s, 3H), 7.01 (dd, J1 = 5.48 Hz, J2 = 0.8 Hz, 1H), 7.03 (td, J1 = 5.08 Hz, J2 = 0.8 Hz, 1H), 7.11 (d, J = 5.76 Hz, 1H), 7.28–7.32 (m, 3H), 7.46 (d, J = 5.68 Hz, 1H), 7.70 (d, J = 1.64 Hz, 1H), 8.05–8.07 (m, 2H), 8.81 (s, 1H), 14.96 (s, 1H); 13C NMR (400 MHz, CDCl3): 167.25, 164.60, 160.52, 152.90, 149.05, 145.10, 134.99, 133.52, 133.05, 131.48, 130.54, 129.39, 128.61, 127.95, 121.08, 119.44, 119.28, 118.76, 117.77, 111.94, 55.90 ppm; IR (KBr): 3387 (OH), 3062 (Ar—H), 2893 (aliphatic C—H), 1612.49s (CH⚌N), 1458.18s (N⚌N), 1350.17 (CH3), 1288.45 (OH), 1249.87 (C—N), 1103.28 (C—O), 1026.13, 887.26, 817.82 (C—Cl), 756.10 and 547.78 (CH) cm−1; APCI-MS m/z for C20H15Cl2N3O2: 400 (M+), 401 (M+1)+; Anal. calcd for C20H15Cl2N3O2: C 60.0, H 3.8, N 10.5, O 8.0, found: C 60.3, H 3.65, N 10.6, O 8.1.

4-((2,5-Dichlorophenyl)diazenyl)-2-((3-bromophenylimino)methyl)phenol (SBN-40): Orange powder (1.0 g, 45%); Rf = 0.73 (hexane/ethyl acetate 7:3); mp: 135–140 °C; 1H NMR (400 MHz, CDCl3): 7.15 (d, J = 5.76 Hz, 1H), 7.26–7.48 (m, 6H), 7.71 (d, J = 1.52 Hz, 1H), 8.09 (d, J = 5.28 Hz, 2H), 8.72 (s, 1H), 13.60 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.83, 163.05, 149.11, 148.91, 145.69, 133.47, 133.28, 131.55, 130.85, 130.83, 130.30, 128.90, 128.65, 124.23, 123.19, 120.23, 118.73, 118.511, 117.77 ppm; IR (KBr): 3201.83, 3047.53 (Ar—H), 1620.21s (CH⚌N), 1566.20s (C⚌C), 1465.90 (N⚌N), 1350.17 (CH), 1288.45 (OH), 1141.86 (C—N), 1056.99 (C—O), 979.84, 894.97s (C—Cl), 810.10, 779.24, 601.79s (C—Br), 462.92 cm−1; APCI-MS m/z found for C19H12BrCl2N3O: 449 (M+), 451 (M+1)+; Anal. calcd for C19H12BrCl2N3O: C 50.8, H 2.7, N 9.4, O 3.6, found: C 50.7, H 2.8 N 9.3, O 3.6.

4-((2,5-Dichlorophenyl)diazenyl)-2-((4-fluorophenylimino)methyl)phenol (SBN-41): Orange powder (1.16 g, 60%): Rf = 0.81 (hexane/ethyl acetate 6:3); mp: 155–160 °C; 1H NMR (400 MHz, CDCl3): 7.15–7.26 (m, 5H), 7.48 (d, J = 4.6 Hz, 1H), 7.71 (s, 1H), 8.11 (m, 2H), 8.73 (s, 1H), 8.77 (s, 1H), 13.87 (s, 1H); 13C NMR (400 MHz, CDCl3): 164.85, 161.68, 148.97, 145.67, 143.77, 133.47, 133.20, 131.53, 130.79, 128.66, 128.30, 122.78, 122.73, 118.89, 118.44, 117.78, 116.50, 116.35, 99.98 ppm; IR (KBr): 3300 (OH), 3050 (Ar—H), 2848.93 (CH), 1620.21s (CH⚌N), 1566.20s (C⚌C), 1350.17 (CH), 1288.45 (OH), 1195.87 (C—N), 1103.28 (C—O), 941.26 (C—F), 887.26 (C—Cl), 802.39, 732.95 and 601.79 (CH), cm−1; APCI-MS m/z for C19H12Cl2FN3O: 387.9 (M+), 388.9 (M+1)+; Anal. calcd. for C19H12Cl2FN3O: C 58.8, H 3.1, N 10.8, O 4.1, found: C 58.7, H 3.2, N 10.84, O 4.1.

2-((3-Bromophenylimino)methyl)-4-((2,4-dimethylphenyl)diazenyl)phenol (SBN-42): Orange powder (1.18 g, 58%): Rf = 0.86 (hexane/ethyl acetate 7:3); mp: 90–95 °C; 1H NMR (400 MHz, CDCl3): 2.37 (s, 3H), 2.68 (s, 3H), 7.06 (dd, J1 = 5.48 Hz, J2 = 1.24 Hz, 1H), 7.12–7.14 (m, 2H), 7.23–7.25 (m, 1H), 7.30 (t, J = 5.44 Hz, 1H), 7.43–7.47 (m, 2H), 7.56 (d, J = 5.44 Hz, 1H), 7.99–8.03 (m, 2H), 8.69 (s, 1H), 13.36 (s, 1H); 13CNMR (400 MHz, CDCl3): 163.39, 163.37, 149.43, 148.60, 146.17, 141.05, 137.92, 131.85, 130.78, 130.08, 128.14, 127.68, 127.24, 124.16, 123.13, 120.29, 118.62, 118.12, 115.25, 21.41, 17.53 ppm; IR (KBr): 3371.57br (OH), 2916.37 (aliphatic C—H), 2854.65 (CH), 1620.21s (CH⚌N), 1573.91s (C⚌C), 1481.33s (N⚌N), 1360.1 (CH3), 1280.73s (C—O), 1186.15 (C—N), 1095.57 (C—O), 879.54, 794.67 (C—Br), 663.51 (CH) cm−1; APCI-MS m/z for C21H18BrN3O: 408 (M+), 410 (M+2)+; Anal. calcd for C21H18BrN3O: C 61.8, H 4.4, N 10.3, O 3.9, found: C 61.9, H 4.4, N 10.2 O, 4.0.

4-((4-Bromophenyl)diazenyl)-2-((2,6-dimethylphenylimino)methyl)phenol (SBN-43): Orange powder (1.04 g, 51%): Rf = 0.88 (hexane/ethyl acetate 7:3). 1H NMR (CDCl3, 400 MHz): 3.85 (s, 3H), 6.96 (dd, J1 = 5.72 Hz, J2 = 0.96 Hz, 1H), 6.98 (td, J1 = 5.04 Hz, J2 = 0.8 Hz, 1H), 7.07 (d, J = 5.72 Hz, 1H), 7.23–7.26 (m, 2H), 7.34 (t, J = 5.24 Hz, 1H), 7.60–7.76 (m, 2H), 7.94–7.98 (m, 3H), 8.05 (dd, J1 = 5.92 Hz, J2 = 1.6 Hz, 1H), 8.74 (s, 1H), 14.82 (s, 1H); 13C NMR (CDCl3): 166.44, 164.45, 162.62, 151.30, 147.46, 145.36, 132.31, 128.50, 128.35, 127.97, 127.96, 125.38, 124.84, 124.11, 118.48, 118.35, 36.51, 31.47, 18.52 ppm; IR (KBr): 3332.42br (OH), 3038.47 (Ar—H), 2913.68 (C—H) 2854.65 (C—H), 1611.06vs (CH⚌N), 1573.91(C⚌C), 1473.52s (N⚌N), 1342.46s (CH3), 1284.76vs (OH), 1171.59 (C—N), 1103.28 (C—O), 824.61vs (C—Br), 767.25 (CH) cm−1; APCI-MS m/z found for C21H18BrN3O: 408 (M+), 410 (M+2)+; Anal. calcd for C21H18BrN3O: C 61.8, H 4.4, N 10.3, O 3.9, found: C 61.85, H 4.65, N 10.35, O 4.0.

2.2

2.2 Antimicrobial screening

The antimicrobial potential of synthesized compounds was evaluated by determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) or MFC (minimum fungicidal concentration).

2.2.1

2.2.1 Determination of MIC

The synthesized diazenyl derivatives were screened for in vitro antimicrobial activity by serial broth dilution method (Cappucino and Sherman, 1999), using cefadroxil (antibacterial) and fluconazole (antifungal) as standard drugs. The microbial strains screened were Gram-positive bacteria: Bacillus subtilis MTCC 2063, Bacillus cereus MTCC 1305, Staphylococcus aureus MTCC 2901, Gram-negative bacteria: Escherichia coli MTCC 1652, Pseudomonas aeruginosa MTCC 1688, Salmonella typhi MTCC 3216, Salmonella enterica MTCC 733 and fungal strains: Candida albicans MTCC 227, Aspergillus niger MTCC 8189 and Aspergillus fumigatus MTCC 2584. The stock solutions of 100 µg/ml concentration for standard and test drugs were prepared in DMSO. The bacterial and fungal inoculums were prepared from fresh pure cultures. The 100 µL of inoculum was added to different test-tubes containing serial dilutions (50, 25, 12.5, 6.25, 3.12 and 1.56 µg/ml) of test and standard compounds in nutrient broth and Sabouraud dextrose broth to give final inoculum size of 5∗105 colony forming units (cfu) ml−1 under aseptic conditions. The bacterial cultures were incubated for a time of 24 h at 37 ± 2 °C. The incubation time for C. albicans was 48 h at 37 ± 2 °C and for A. niger and A. fumigatus were 7 d at 25 ± 2 °C. The results of antimicrobial activity were recorded in terms of MIC.

2.2.2

2.2.2 Determination of MBC/MFC

After MIC determination, the screening was further proceeded for determination of MBC and MFC. Aliquot of 100 µL of culture from each tube with no growth in MIC experiments was transferred to sterilized petri plates containing fresh nutrient agar and Sabouraud dextrose agar medium. The petri plates were incubated at 37 ± 2 °C/24 h for bacterial and 25 ± 2 °C/7 days for fungal growth and analyzed visually. The MBC and MFC were reported as the minimum concentration of compounds in aliquots showing no visual growth after incubation.

2.3

2.3 In vitro cytotoxicity activity

The in vitro cytotoxicity screening of the synthesized diazenyl derivatives was assessed on HCT-116 (human colorectal carcinoma) cell line by Sulforhodamine-B (SRB) assay (Skehan et al., 1990) which based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic acid-fixed cells. The results of anticancer activity were expressed as IC50 (amount of drug necessary to reduce the cell viability by 50%) and compared with the standard anticancer drug 5-fluorouracil. The 7500 cells/well were allowed to attach for a period of 72 h to the walls of the 96-multititre plates before treatment with the test compounds. The solution of standard and test compounds was prepared in DMSO and appropriate volume makeup was done with saline. Monolayer cells were then incubated at 37 °C for 72 h with different concentrations (6.25, 12.5, 25, 50 and 100 µg/mL) of the test compounds in an atmosphere of 5% carbon dioxide. After fixing with trichloroacetic acid for an hour followed by washing with water, the cells were stained with 0.4% w/v solution of pink colored amino xanthine dye, Sulforhodamine-B, in acetic acid for half an hour. The cultures were washed with 1% acetic acid 3–4 times to get rid of the excess stain. Tris–EDTA buffer (pH = 10) was used to recover the attached stain. The color intensity was measured using ELISA reader at 570 nm for calculating the % cell viability given by Eq. (1). The experiment was performed in triplicate to report the cytotoxicity of the compounds.

(1)
% cell viability = [ Abs . sample - Abs . blank / Abs . control - Abs . blank ] × 100

3

3 Results and discussion

3.1

3.1 Synthesis

The various mono and di-substituted aniline derivatives were first diazotized in the presence of sodium nitrite and hydrochloric acid, followed by coupling with salicylaldehyde in the presence of alkaline solution to afford mono and di-substituted 5-aryldiazenylsalicylaldehyde derivatives (1–17) in good yields. Further the reaction of these azo dyes with various mono and di-substituted aniline derivatives afforded 25 diazenyl schiff bases (SBN1-SBN43), which were purified by recrystallization from suitable solvents and column chromatography.

3.2

3.2 UV–vis absorption spectra

The UV–vis spectrum of synthesized dyes and diazenyl schiff bases were taken in solvents DCM and DMSO at room temperature at conc. of 1 × 10−5 M. The characteristics UV–vis absorption bands of azo dyes and diazenyl schiff bases are presented in Figs. 2–4 and Tables 1 and 2 respectively. The UV–vis spectra of synthesized derivatives showed two main absorption maxima (λmax), one ranging from 235 to 295 nm and other ranging from 350 to 575 nm depending on the polarity of the solvent. The first λmax for all synthesized derivatives extending from 235 to 295 nm was the result of π-π∗ electronic transition, due to the presence of C⚌C bond particular to benzene ring and other aromatic nuclei in the derivatives. The second λmax in the range from 350 to 575 nm was due to π-π∗ electronic transitions of the azo linkages (N⚌N) present in the derivatives. The π-π∗ transition for azo linkage (N⚌N) of diazenyl schiff bases was more red-shifted as compared to the related azo linkage in dyes. This bathochromic shift was due to intramolecular H-bonding between phenolic OH and C⚌N groups of schiff base, resulted in greater negative charge density on the OH group. As it can be seen, the absorption spectra of the synthesized derivatives in DMSO solution were more red-shifted as compared to the spectra in DCM solutions, signifying comparatively high interaction between the DMSO and synthesized derivatives as compared to DCM. This positive solvatochromism can be explained on the basis of different polarities of the ground and excited states of synthesized derivatives result from the effect of change of excited state dipole moment, change in the H-bonding strength, and/or due to excited state protonation.

UV–vis spectra of dyes (1–17) in DCM and DMSO at conc. of 1 × 10−5 M.
Figure 2 UV–vis spectra of dyes (1–17) in DCM and DMSO at conc. of 1 × 10−5 M.
UV–vis spectra of diazenyl schiff bases (SBN1-SBN43) in DCM at conc. of 1 × 10−5 M.
Figure 3 UV–vis spectra of diazenyl schiff bases (SBN1-SBN43) in DCM at conc. of 1 × 10−5 M.
UV–vis spectra of diazenyl schiff bases (SBN1-SBN43) in DMSO at conc. of 1 × 10−5 M.
Figure 4 UV–vis spectra of diazenyl schiff bases (SBN1-SBN43) in DMSO at conc. of 1 × 10−5 M.
Table 1 Absorption maxima for dyes in DCM and DMSO at conc. of 1 × 10−5 M.
Dyes DCM(λmax) DMSO(λmax)
1 242, 326, 334, 366 285, 390
2 256, 330, 348 285, 399
3 252, 328, 344 285, 392
4 240, 334, 370 285, 416
5 240, 330, 376 285, 470
6 252, 328, 334, 356 285, 435
7 240, 336, 356, 372 285, 495
8 240, 330, 346 285, 385
9 – 285, 465
10 – 285, 410
11 252, 338, 346, 352 285, 410
12 240, 330, 372 285, 463
13 – 285, 435
15 240, 330 285, 495
16 240, 330, 372 285, 495
17 252, 324, 372 285, 440, 575
Table 2 Absorption maxima for diazenyl derivatives in DCM and DMSO at conc. of 1 × 10−5 M.
S. No. Compound DCM(λmax) DMSO(λmax) S. No. Compound DCM(λmax) DMSO(λmax)
1 SBN-1 255, 420 285, 420 14 SBN-32 250, 405 285, 435
2 SBN-9 255, 445 285, 470 15 SBN-33 255, 410 285, 415
3 SBN-12 255, 475 285, 475 16 SBN-34 250, 480 285, 525
4 SBN-13 250, 415 285, 430 17 SBN-35 255, 470 285, 480
5 SBN-14 255, 425 285, 460 18 SBN-36 255, 440 285, 500
6 SBN-15 250, 425 290, 465 19 SBN-37 250, 465 285, 465
7 SBN-18 250, 420 285, 425 20 SBN-38 250, 440 285, 420
8 SBN-20 255, 470 285, 485 21 SBN-39 255, 480 285, 490
9 SBN-21 250, 395 285, 485 22 SBN-40 255, 445 285, 473
10 SBN-25 255, 430 285, 450 23 SBN-41 255, 420 285, 505
11 SBN-26 255, 470 295, 480 24 SBN-42 255, 420 285, 425
12 SBN-28 250, 460 420, 481 25 SBN-43 250, 420 285, 477
13 SBN-29 255, 430 285, 460

3.3

3.3 IR absorption spectra

The IR spectra of synthesized azo dyes and diazenyl schiff bases were determined by KBr pellet method. The all synthesized compounds showed phenolic O—H stretch in the range of 3200–3470 cm−1. The C⚌O peak due to aldehydic group appeared at 1651–1675 cm−1 in spectra of most of the azo dyes. The spectra of the synthesized azo dyes also showed absorption bands due to Ar—H stretch at 3055–3190 cm−1, C⚌C stretch at 1519–1581 cm−1, C⚌C bend at 700–750 cm−1 and Ar—O— stretch at 1100–1280 cm−1. The azo linkage (N⚌N) was confirmed by the presence of absorption band at the 1450–1481 cm−1. The IR spectrum of dyes containing methyl group showed absorption band at 2850–3000 cm−1 due to aliphatic C—H stretch. The NO2 group stretch in dye 5, 6, 7 and 17 was confirmed by the presence of two strong bands at 1519–1527 cm−1 and 1341–1372 cm−1. The C-X (halogen) absorption frequency band appeared in the range of 620–880 cm−1. The IR spectrum of the diazenyl schiff bases showed characteristics peaks at 1610–1635 (CH⚌N stretch), complete disappearance of C⚌O peak (1666 cm−1) present in azo derivatives, 1419–1491 (N⚌N stretch), and all other peaks present in azo dyes.

3.4

3.4 NMR spectra

1H NMR: The NMR spectrum of the all azo dyes generally showed aldehydic proton peak at 9.5–10.3 ppm. But this peak disappeared in diazenyl schiff bases which showed a singlet at δ 8.3–8.8 indicating the presence of CH⚌N proton. The most of azo dyes showed phenolic proton at δ 11.0–11.5 while this value shifted to δ 13–15 in diazenyl derivatives. Most other aromatic protons came in the range of δ 6.5–8.7. The aldehyde containing ring protons H8, H11 and H12 (Fig. 5) appeared as d peaks at 7.8–8.4 ppm, d at 6.9.0–7.5 ppm and dd at 7.50–7.95 ppm respectively in most of the diazenyl derivatives. The spectra of derivatives SBN 29, SBN 37 and SBN 38 having fluoro substituent at 2nd position in phenyl ring showed td at 7.12 ppm for H3, q at 7.3 ppm for H4, td at 7.14 ppm for H5 and td at 7.64 ppm for H6 protons since the fluorine nucleus also couples to the H3, H4, H5 and H6 protons. The spectra of SBN 29 are presented in Fig. 6. Similarly for SBN 28, 35, 41 having fluoro substituent at p-position of the ring, which also couple with the other proton nucleus of the ring resulted in dd of H15, H19 at 7.23 ppm and H16, H18 at 7.02 ppm. The spectra of SBN-28 are presented in Fig. 7. The NMR spectrum of the SBN 12, SBN 34 and SBN 39 showed a singlet at δ 3.86–3.92 indicating the presence of three —OCH3 protons. The NMR spectrum of the SBN 14 showed a singlet at δ 2.43, indicating the presence of single methyl group. The signals of two CH3 groups in SBN 15, SBN 21, SBN 25, SBN 26, SBN 28, SBN 38 and SBN 42 appeared as singlet at δ 2.30–2.36 and δ 2.36–2.70 respectively while in SBN 18, SBN 29, SBN 36 and SBN 43 these two CH3 protons appeared at δ 2.10–2.28. The protons of -SCH3 were obtained as singlet at δ 2.52 in SBN 20.

General structure of Schiff base.
Figure 5 General structure of Schiff base.
1H NMR spectrum of SBN-29.
Figure 6 1H NMR spectrum of SBN-29.
1H NMR spectrum of SBN-28.
Figure 7 1H NMR spectrum of SBN-28.

13C Spectra: The azo compounds generally showed aldehydic carbon in the range of 185–188 ppm. This aldehydic carbon peak disappeared in diazenyl derivatives and CH⚌N carbon appeared in the range of 163–166.5 ppm. The diazenyl derivatives showed phenolic carbons at 160.2–165.5 ppm. The other carbon peaks in 13C NMR spectra of diazenyl derivatives appeared at 130.0–139 ppm (C8), at 115–118 ppm (C11) and at 124–128 ppm (C12). The SBN 28, 35, 41 showed doublet at 161.88 ppm (C17-F), C15, C19 d at 116.38 ppm, C16, C18 d at 122.76 ppm, C14 d at 143.98 ppm. 13C NMR spectrum of SBN-28 is shown in Fig. 8. Similarly 13C NMR spectra of SBN 29, SBN 37, and SBN 38, the F-substituted at second position also couples to the nucleus of other carbon atoms of the aromatic ring resulted in doublets at C1, C2, C3, C4, C5, C6 at the 140.6, 159.05, 117.0, 131.9, 128.3, 124.3 ppm respectively. In SBN 12, SBN 34, SBN 39 —OCH3 carbon peak appeared at 55.87 ppm. In SBN 14 CH3 carbon peak appeared at 18 ppm while in SBN 15, SBN 21, SBN 25, SBN 26, SBN 28, SBN 38 and SBN 42, aliphatic methyl carbon peaks appeared at 17–18 ppm and 21 ppm. In SBN 18, SBN 29, SBN 36, SBN 43 both methyl carbons appeared at single shift value of 18 ppm. In SBN 20 —SCH3 peak appeared at 15 ppm.

13C NMR spectrum of SBN-28.
Figure 8 13C NMR spectrum of SBN-28.

3.5

3.5 Fluorimetric data

The fluorimetric data of synthesized azo dyes were taken in DMSO at a concentration of 10−4 M (Fig. 9). Most of the dyes showed emission peak around 570–595 nm (Table 3). The chloro-substituted dyes showed very weak emission around 630–640 nm. The dye 8 was found to be highly fluorescent. The fluorimetric data of synthesized diazenyl derivatives were taken in DCM at a concentration of 10−4 M. The synthesized diazenyl derivatives showed emission at 610–650 nm with excitation around 540–595 nm (Fig. 9 and Table 4).

Fluorescence spectra of dyes and diazenyl schiff bases at conc. of 1 × 10−4 M.
Figure 9 Fluorescence spectra of dyes and diazenyl schiff bases at conc. of 1 × 10−4 M.
Table 3 Fluorimetric data for dyes in DMSO at conc. of 1 × 10−4 M.
Dye DMSO
(λmax) 		Dye DMSO
(λmax)
1 560 8 537
2 565 9 560
3 565 10 569
4 563 11 560
5 574 12 564
6 564 15 640
7 591 16 633
Table 4 Fluorimetric data for diazenyl schiff bases in DCM at conc. of 1 × 10−4 M.
Comp. No Excitation
(λmax) 		Emission
(λmax) 		Comp. No Excitation
(λmax) 		Emission
(λmax)
SBN-1 565 649 SBN-29 564 643
SBN-9 590 648 SBN-32 566 652
SBN-12 591 645 SBN-34 591 610
SBN-13 568 649 SBN-36 590 647
SBN-14 570 651 SBN-38 563 648
SBN-18 564 654 SBN-39 591 653
SBN-20 579 649 SBN-40 569 647
SBN-21 570 651 SBN-41 568 650
SBN-25 591 656 SBN-42 569 611
SBN-26 563 651 SBN-43 561 643
SBN-28 549 590

3.6

3.6 Mass spectroscopy and elemental analysis

The synthesis of azo dyes and diazenyl schiff bases was finally confirmed by molecular weight determination on Advion expression compact mass spectrometer using APCI mode as ion source. Most of the azo dyes and diazenyl schiff bases showed M+ (molecular ion peak), (M++1), (M++2) in positive chemical ionization and M+, (M+−1), (M+−2) in negative chemical ionization mode. The elemental analysis confirmed the synthesis of diazenyl derivatives. The % of C, H, N, O and S in the synthesized derivatives was found to be within defined limits.

3.7

3.7 Microbiology results

The serial broth dilution method was used for antimicrobial evaluation of the synthesized diazenyl compounds against standards cefadroxil and fluconazole. The MIC and MBC/MFC values of compounds and standards have been given in Tables 5 and 6 respectively. The results revealed that the diazenyl schiff bases were highly efficient antibacterial and antifungal agents in comparison with the standard drugs. The highest activity was found for SBN 18 with MIC = 1.72 (µM/ml × 102) against B. subtilis, SBN 26 with MIC and MBC both = 0.78 (µM/ml × 102) against B. cereus, SBN 20 and SBN 21 with MIC and MBC of 0.75 (µM/ml × 102) against S. typhi, SBN 39 with MIC and MBC of 0.78 (µM/ml × 102) against P. aeruginosa. SBN 40 exhibited the highest activity against S. aureus, B. cereus, S. enterica and E. coli with MIC = 0.69 (µM/ml × 102). Most of synthesized diazenyl schiff bases, found to be very active against S. enterica (MIC ranges from 0.35 to 0.86 (µM/ml × 102)), with highest activity of SBN 32 with MIC = 0.35 (µM/ml × 102). For fungal strains, SBN 33 showed maximum activity against C. albicans and A. niger having MIC values of 1.36 and 2.72 (µM/ml × 102) respectively. SBN 34 and SBN 36 were found to be most active against A. fumigatus with MIC and MFC of 0.76 and 3.0 (µM/ml × 102) respectively. From SAR studies it was found that substitution of the electron withdrawing groups on the aromatic ring A is necessary for activity. The presence of bromine or chlorine at the ring A enhances the antimicrobial activity. The compounds with di-halogen substituted in the same ring are more active as compared to the mono-halogenated derivatives and this activity enhanced further by substitution of third halogen in another aromatic ring.

Table 5 Antimicrobial activity of diazenyl schiff bases.
Compound MIC (µM/ml × 102)
B. subtilis B. cereus S. aureus E. coli S. typhi S. enterica P. aeruginosa C. albicans A. niger A. fumigatus
SBN-1 3.02 1.51 3.02 3.02 1.51 0.75 1.51 3.02 6.04 3.01
SBN-9 3.14 3.14 6.28 3.14 3.14 1.57 1.57 1.57 3.14 1.57
SBN-12 2.75 1.37 1.37 1.37 1.37 0.76 1.37 2.75 2.75 6.10
SBN-13 2.94 1.47 1.47 1.47 1.47 0.73 1.47 1.47 2.94 2.94
SBN-14 3.17 1.59 3.17 3.17 1.59 3.16 1.59 3.17 3.17 1.58
SBN-15 3.44 0.86 1.72 1.72 0.86 0.86 1.72 13.77 3.44 1.72
SBN-18 1.72 1.72 1.72 0.86 0.86 0.86 1.72 3.44 3.44 3.43
SBN-20 3.00 0.75 1.50 1.50 0.75 0.75 1.50 3.00 3.00 3.00
SBN-21 3.14 0.78 0.78 1.57 0.78 0.78 1.57 3.14 3.14 3.14
SBN-25 3.44 1.72 3.44 1.72 3.44 0.43 3.44 1.72 3.44 3.43
SBN-26 3.14 0.78 0.78 1.57 1.57 0.78 1.57 3.14 6.28 3.14
SBN-28 3.60 0.90 0.90 0.90 0.90 0.45 0.90 3.60 3.60 1.80
SBN-29 3.60 3.60 3.60 3.60 3.60 0.90 3.60 3.60 3.60 3.60
SBN-32 2.78 1.39 1.39 1.39 1.39 0.35 1.39 1.39 2.78 2.78
SBN-33 2.72 0.68 1.36 1.36 1.36 0.68 1.36 1.36 2.72 1.36
SBN-34 3.05 0.76 1.52 3.05 3.05 1.52 1.52 3.05 3.05 0.76
SBN-35 3.14 3.14 3.14 3.14 1.57 1.57 3.14 3.14 3.14 1.57
SBN-36 3.06 3.06 3.06 3.06 1.53 1.53 3.06 3.06 3.06 0.76
SBN-37 3.22 3.22 3.22 3.22 3.22 0.80 3.22 3.22 3.22 3.22
SBN-38 3.60 3.60 0.90 1.80 3.60 1.80 1.80 14.41 3.60 7.20
SBN-39 3.13 0.78 0.78 0.78 1.56 0.78 0.78 1.56 3.13 3.13
SBN-40 2.78 0.69 0.69 0.69 1.39 0.69 1.39 2.78 2.78 2.78
SBN-41 3.22 1.61 1.61 1.61 3.22 0.80 1.61 3.22 3.22 3.22
SBN-42 3.06 0.76 0.76 0.76 1.53 0.38 1.53 1.53 3.06 1.53
SBN-43 3.06 0.76 1.53 1.53 3.06 1.53 1.53 1.53 3.06 3.06
Cefadroxil 3.45 1.72 3.45 3.45 3.45 1.72 3.45 – – –
Fluconazole – – – – – – – 4.08 8.16 8.16

NOTE: More active compounds presented in bold.

Table 6 MBC/MFC of synthesized diazenyl schiff bases.
Compound MBC/MFC (µM/ml × 102)
B. subtilis B. cereus S. aureus E. coli S. typhi S. enterica P. aeruginosa C. albicans A. niger A. fumigatus
SBN-1 12.08 3.02 6.04 12.08 1.50 0.75 1.51 12.08 12.05 12.05
SBN-9 6.28 3.14 6.28 >12.56 >12.56 6.27 >12.56 3.14 12.53 12.53
SBN-12 2.75 >10.99 2.75 2.75 2.75 0.76 1.37 2.75 6.10 12.20
SBN-13 5.88 1.47 1.47 5.88 2.94 0.73 2.94 1.47 5.88 11.76
SBN-14 6.35 1.59 12.69 3.17 >12.69 >12.66 >12.69 3.17 12.66 12.66
SBN-15 3.44 0.86 3.44 3.44 1.72 1.72 1.72 – 13.74 6.87
SBN-18 6.89 1.72 1.72 1.72 3.44 3.43 1.72 3.44 6.87 13.74
SBN-20 6.01 1.50 3.00 3.00 0.75 1.50 1.50 3.00 3.00 12.02
SBN-21 6.28 1.57 12.56 3.14 0.78 0.78 1.57 3.14 12.56 12.56
SBN-25 6.89 1.72 3.44 3.44 >13.77 0.86 >13.77 3.44 13.74 13.74
SBN-26 6.28 0.78 3.14 3.14 3.14 0.78 1.57 6.28 6.28 12.56
SBN-28 7.20 14.41 3.60 1.80 0.90 0.90 0.90 3.60 14.41 7.20
SBN-29 7.20 3.60 3.60 >14.41 >14.41 1.80 >14.41 3.60 14.41 14.41
SBN-32 5.57 1.39 1.39 2.78 1.39 1.39 >11.14 2.78 11.14 11.14
SBN-33 5.45 >10.89 2.72 2.72 1.36 0.68 1.36 2.72 5.43 10.87
SBN-34 6.10 1.52 3.05 3.05 3.05 3.04 >12.20 3.05 3.04 3.04
SBN-35 6.28 6.28 3.14 3.14 3.14 6.28 >12.56 3.14 12.56 6.28
SBN-36 6.13 12.25 3.06 3.06 >12.25 1.53 >12.25 3.06 12.25 3.06
SBN-37 12.89 3.22 3.22 6.44 >12.89 1.61 12.89 3.22 12.89 6.44
SBN-38 7.20 7.20 3.60 14.41 >14.41 3.60 3.60 7.20 14.41 14.41
SBN-39 6.25 >10.89 1.56 3.13 1.56 1.56 0.78 3.13 6.25 12.50
SBN-40 5.57 >11.14 1.39 1.39 1.39 1.39 5.57 2.78 2.78 11.14
SBN-41 6.44 >12.89 1.61 3.22 12.89 1.61 >12.89 3.22 6.44 12.89
SBN-42 6.13 1.53 1.53 3.06 6.13 0.76 3.06 12.25 12.25 3.06
SBN-43 12.25 1.53 3.06 12.25 12.25 6.13 1.53 3.06 3.06 6.13
Cefadroxil 3.45 3.45 6.88 6.88 3.45 1.72 – – – –
Fluconazole – – – – – – – 4.08 16.34 16.34

3.8

3.8 Cytotoxic activity

The diazenyl compounds were screened for cytotoxicity on HCT-116 cell line by SRB assay and the results were compared with cytotoxicity of 5-FU as a standard drug. The relationship between % cell viability and log concentration of diazenyl compounds was plotted to get the survival curve for HCT-116 cell line and IC50 values were calculated from survival curve plots (Table 7). Among the synthesized compounds, SBN 13 (Fig. 10) showed the maximum cytotoxicity IC50 = 7.5 µg/ml followed by SBN 26 and SBN 28 both of which possessed IC50 = 14.0 µg/ml against HCT-116 cell line in comparison with the 5-FU (IC50 = 3.0 µg/ml).

Table 7 The IC50 values of diazenyl schiff bases using SRB assay on HCT 116 cells.
Compound IC50 (µg/ml)
SBN-1 95
SBN-9 >100
SBN-12 50
SBN-13 7.5
SBN-14 62
SBN-15 >100
SBN-18 20
SBN-20 50
SBN-21 100
SBN-25 40
SBN-26 14
SBN-28 14
SBN-29 22.5
SBN-32 85
SBN-33 100
SBN-34 48
SBN-35 >100
SBN-36 50
SBN-37 >100
SBN-38 50
SBN-39 40
SBN-40 50
SBN-41 42.3
SBN-42 31.8
SBN-43 46.2
5-FU 3
SBN-13.
Figure 10 SBN-13.

From structural analysis, it seems that the NO2 group at p-position at ring C as in SBN 13 (IC50 = 7.5 µg/ml) is essential for cytotoxicity. Replacement of this group by chloro group as in SBN 1 (IC50 = 95 µg/ml) significantly decreased the activity. SBN 21 (IC50 = 95 µg/ml) and SBN-26 (IC50 = 14 µg/ml), both having same type and number of substituents (two chloro and two methyl groups) but with altered position of substituents at rings A and C, had shown significantly different cytotoxicities. This suggests that the presence of two electron donating groups at ring A could increase the activity. The substitution of two electron withdrawing groups at ring A resulted in decreased activity. The compounds with three substituted halogens atoms (SBN-32 IC50 = 85 µg/ml; SBN 37 IC50 = >100 µg/ml) or three substituted electron withdrawing groups (SBN 9 = >100 µg/ml; SBN 35 IC50 = >100 µg/ml) were found to be less cytotoxic.

4

4 Conclusion

In the present investigation, new diazenyl schiff bases of salicylaldehyde containing azo dyes with various donor or acceptor groups on aromatic rings were synthesized. The diazenyl derivatives were characterized by various spectroscopic means and evaluated for their in vitro activity against different microbial strains and human colorectal carcinoma cell line (HCT-116). The results revealed that most of the synthesized derivatives have shown significant antimicrobial activities. SBN-40 showed the highest activity against various Gram positive and Gram negative bacterial strains. The tri-halogen substituted derivatives were found to have significant antimicrobial activities, particularly the compounds having two substituted chloro groups at the same ring. The diazenyl derivative SBN-13 possessed significant cytotoxicity IC50 = 7.5 µg/ml against HCT-116. So, these diazenyl derivatives surely hold a greater promise in discovering potent antimicrobial agents and can be explored further for their mechanistic details of action on different microbial strains.

Acknowledgment

The authors indebtedly acknowledge the University Grant Commission for providing SRF award to first author, H. Kaur via award letter no. F.25-1/2013-14(BSR)/7-344/2011(BSR). The authors also want to thank Principal, PGIMS Rohtak, for fluorimetric analysis.

References

  1. , . Synthesis, spectral characterization, thermal analysis and electrochemistry properties of Ni(II) complexes derived from azo dyes. J. Therm. Anal. Calorim.. 2016;123:595-605.
    [Google Scholar]
  2. , , , . Synthesis, spectral properties and applications of some mordant and disperse mono azo dyes derived from 2-amino-1,3-benzothiazole. Res. J. Chem. Sci.. 2013;3:81-87.
    [Google Scholar]
  3. , , , . RAS testing practices and RAS mutation prevalence among patients with metastatic colorectal cancer: results from a Europe-wide survey of pathology centres. BMC Cancer. 2016;16:825.
    [Google Scholar]
  4. , . Resistance to ocular antibiotics: an overview. Clin. Exp. Optom.. 2007;90:258-262.
    [Google Scholar]
  5. , . Antimicrobial adjuvants drug discovery, the challenge of avoid the resistance and recover the susceptibility of multi-drug resistant strains. J. Microb. Biochem. Technol.. 2016;8:169-176.
    [Google Scholar]
  6. , , . Microbiology: A Laboratory Mannual. California: Addison Wesley Longman Inc.; . p. :263.
  7. , , . Origins and evolution of antibiotic resistance. MMBR. 2010;74:417-433.
    [Google Scholar]
  8. , , , . Synthesis, antimicrobial and anticancer evaluation of 2-azetidinones clubbed with quinazolinone. Pharm. Chem. J.. 2016;50:24-28.
    [Google Scholar]
  9. , , , . 4-Thiazolidinone derivatives: synthesis, antimicrobial, anticancer evaluation and QSAR studies. RSC Adv.. 2016;111:109485-109494.
    [Google Scholar]
  10. , , , . Synthesis and characterization of new diazenecarboxamide ligands using a selective adduct formation with B(C6F5)3. J. Braz. Chem. Soc.. 2016;27:194-201.
    [Google Scholar]
  11. , , , . Cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. Int. J. Cancer. 2015;136:E359-E386.
    [Google Scholar]
  12. , , , . 4-phenyldiazenyl 2-(phenylimino methyl) phenols; synthesis and in-vitro biological evaluation as potential antibacterial agents. J. Enzyme Inhib. Med. Chem.. 2009;24:176-180.
    [Google Scholar]
  13. , , . Penicillin, the medicine with the greatest impact on therapeutic outcomes. Appl. Microbiol. Biotechnol.. 2011;92:677-687.
    [Google Scholar]
  14. , , , . Diazenyl derivatives and their complexes as anticancer agents. Anticancer Agents Med. Chem.. 2016;16:1240-1265.
    [Google Scholar]
  15. , , , . Palliative chemotherapy in advanced colorectal cancer patients 80 years of age and older. Curr. Oncol.. 2016;23:144-153.
    [Google Scholar]
  16. , , , . Non-prescription antimicrobial use worldwide: a systematic review. Lancet Infect. Dis.. 2011;11:692-701.
    [Google Scholar]
  17. , , , . Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol.. 2016;8:276-293.
    [Google Scholar]
  18. , , . Biological evaluation and spectral characterization of 4-hydroxy coumarin analogues. J. Taibah. Univ. Med. Sci.. 2015;10:306-319.
    [Google Scholar]
  19. , , . Medicinal interest of azo based organic compounds: a review. Asian J. Pharm. Clin. Res.. 2016;9:33-39.
    [Google Scholar]
  20. , , , . The multifaceted roles of antibiotics and antibiotic resistance in nature. Front. Microbiol.. 2013;4:47.
    [Google Scholar]
  21. , , , . Synthesis and biological activities of bis alkyl 1,3,4-oxadiazole incorporated azo dye derivatives. Arab. J. Chem.. 2016;9:S1643-S1648.
    [Google Scholar]
  22. , , , . New colorimetric cytotoxicity assay for anticancer-drug screening. J. Nat. Cancer Inst.. 1990;82:1107-1112.
    [Google Scholar]
  23. , , , . Targeted approach to metastatic colorectal cancer: what comes beyond epidermal growth factor receptor antibodies and bevacizumab? Ther. Adv. Med. Oncol.. 2013;5:51-72.
    [Google Scholar]
  24. , . The antibiotic resistance crisis Part 1: causes and threats. Pharmacy Therapeutice. 2015;40:277-283.
    [Google Scholar]
  25. , , . Antimicrobial agents–optimising the ecological balance. BMC Med.. 2016;14:114.
    [Google Scholar]
  26. , , . Synthesis of some unsymmetrical new schiff bases from azo dyes synthesis of some unsymmetrical new schiff bases from azo dyes. Eur. Chem. Bull.. 2013;2:453-455.
    [Google Scholar]
  27. , , , . Hypoxia-selective O6-alkylguanine-DNA alkyltransferase inhibitors: design, synthesis and evaluation of 6-(Benzyloxy)-2-(aryldiazenyl)-9H-purines as prodrugs of O6-Benzylguanine. J. Med. Chem.. 2013;56:1355-1359.
    [Google Scholar]

Appendix A

Supplementary material

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2017.05.004.

Appendix A

Supplementary material

Supplementary Fig. 1

Supplementary Fig. 2

Supplementary Fig. 3

Supplementary data 1

Supplementary data 1

Supplementary data 2

Supplementary data 2

Supplementary data 3

Supplementary data 3

Supplementary data 4

Supplementary data 4


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