Introduction

The β-lactam (or azetidin-2-one) scaffold is an important strained heterocyclic compound due to its diverse spectrum of applications in the field of chemistry, biology as well as in pharmaceutical products. This four-membered ring system is a key structural unit in a large number of pharmacologically active compounds such as the penicillins and cephalosporins, and has been identified as crucial for their bioactivity. Owing to the increasing bacterial resistance against classic antibacterials, it is necessary to search for new types of β-lactam antibiotics and β-lactamase inhibitors (Piens et al. 2016; Hosseyni and Jarrahpour 2018). Although β-lactams have been initially introduced for their renowned antibacterial properties (Chavan and Pai 2007; Bhat et al. 2011; Jarrahpour and Zarei 2006; Cerić et al. 2010), they have since moved beyond this, showing a significant use in different therapeutic areas such as inhibition of HIV-1 protease (Kamath and Ojima 2012) and acyl coenzyme A cholesterol transferases (Indrani et al. 2017) and have also been exhibited antidiabetic activity (Galletti and Giacomini 2011), antimalarial activity (Alborz et al. 2018), anticancer activity (Salunkhe and Piste 2014), antioxidant activity (Nagarajan et al. 2012), anti-inflammatory (Bhati and Kumar 2008) and antifungal activity (Jarrahpour et al. 2017). β-Lactams are key materials for the preparation of various heterocyclic compounds having biological and medicinal significance, such as the anticancer drug Taxol (Indrani et al. 2017). Furthermore, 2-azetidinones have gained an important place in organic chemistry as versatile building blocks, serving as intermediates for the synthesis of many acyclic and cyclic nitrogen compounds (Alcaide et al. 2007; D’hooghe et al. 2010; Afzal et al. 2015). Various azacyclic compounds have been used as antimicrobial agents. Among these, substituted quinolines are prominent in the pharmaceutical sciences (Mishra et al. 2007) as antimalarial (Bawa et al. 2010), antitumor, anti-inflammatory, antibacterial (Kumar et al. 2009; Ahmed and Daneshtalab 2012; El-Gamal et al. 2015), antiviral (Shipra et al. 2015), antioxidant and cytotoxic activity (Ramachandran et al. 2012) agents. Some specific examples, 3,4-dihydro-2H-thiopyrano[2,3-b]quinolone (A) shows mGlu1 receptor antagonist activity (Zhong et al. 2011), N′-((2-chlorobenzo[h]quinolin-3-yl)methylene)methanesulfonohydrazide (B) possesses antibacterial activity (Baluja and Chanda 2017), ethyl 4-ethoxy-2-((ethoxymethyl)thio)benzo[h]quinoline-3-carboxylate (C) has cytotoxic activity against several cancer cell lines (Bawa et al. 2010), and N-methyl-2-(4-(2-oxo-1,2-dihydrobenzo[h]quinolin-3-yl)benzylidene)-hydrazine-1-carbothioamide (D) showed antioxidant activity (Ramachandran et al. 2012) (Fig. 1).

Fig. 1
figure 1

Structures of some known quinoline derivatives with diverse biological activities

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2-Mercaptobenzothiazoles are a second important class of bioactive and industrially important organic compounds. These compounds have been reported for their antimicrobial, antifungal, anti-inflammatory, antitumor, antiulcer and chemoprotective activity (Herrera Cano et al. 2015; Wang et al. 2011; Cressier et al. 2009; Zhilitskaya et al. 2017; Huang and Yang 2006). With the intention to explore the utility of hybrid compounds, we decided to prepare structurally unique β-lactams bearing 2-mercaptobenzothiazole and benzoquinoline, as a means to explore their potential medicinal properties (Raj et al. 2014; Vandekerckhove and D’hooghe 2013; Meunier 2007; Morphy and Rankovic 2006) (Fig. 2).

Fig. 2
figure 2

Structures of two 2-mercaptobenzothiazole derivatives with diverse biological activities

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Results and discussion

Chemistry

In this study, thirteen novel cis-β-lactam hybrids of 2-mercaptobenzothiazole and benzoquinoline were synthesized by the Staudinger reaction (Rajamäki et al. 2016) (Scheme 1). In the first step, a mixture of 1-naphthylamine (1) and acetic anhydride (2) in methanol containing a small drop of acetic acid as a catalyst afforded N-(naphthalen-1-yl)acetamide (3). Then, N-(naphthalen-1-yl)acetamide (3) was added to a mixture of Vilsmeier-Haack reagent (prepared by dropwise addition of POCl3 in ice-cooled of DMF) to prepare benzo[h]quinoline-3-carbaldehyde (4) by a reported procedure (Srivastava and Singh 2005). The chloro group in compound 4 was replaced by 2-mercaptobenzothiazole (5) to yield 2-(benzo[d]thiazol-2-ylthio)benzo[h]quinoline-3-carbaldehyde (6) in the presence of NaH in DMSO in 80% yield. The structure of the product 6 was confirmed by its spectroscopic data and elemental analysis. The IR spectrum of compound 6 showed the characteristic band at υ 1683 cm−1 for the aldehyde carbonyl group. Its 1H NMR spectrum displayed a singlet for the aldehyde carbonyl group at δ = 10.42 ppm. Treatment of 6 with aniline derivatives 7a-d generated the corresponding imines 8a-d. The IR spectrum of 8a showed the characteristic band at υ 1592 cm−1 corresponding to the imine (CH=N). The 1H NMR spectrum of 8a revealed the signal of an imine proton at δ = 8.89. The E geometry of the imines reflects its thermodynamic preference under thermal reaction conditions. Next, imine derivatives 8a-d and various phenoxyacetic acid derivatives 9a-d were treated in the presence of triethylamine and tosyl chloride, in molar ratios of 1:1.5:5:1.5 in anhydrous CH2Cl2 at room temperature, to give the cis-β-lactams 10a-m in yields varying from 75–90% (Scheme 1).

Scheme 1
scheme 1

Synthesis of 2-mercaptobenzothiazole β-lactams 10a-m

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To confirm the cis structure of the newly synthesized 2-azetidinones, X-ray single crystal analysis was done on 10a (Fig. 3). Both bond angles and bond lengths were normal and analogous with those reported for related compounds (Westrip 2010; Çelik et al. 2015). Crystal data, data collection and structure refinement details are presented in supplementary material.

Fig. 3
figure 3

ORTEP diagram of diastereomer 10a

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Assignment of the structures of the reaction products was based on their spectral data. The 1H NMR spectrum of 10a showed characteristic signals at 5.75 ppm [1H, d, J = 5.0 Hz, H-4 β-lactam] and 6.06 ppm [1H, d, J = 5.0 Hz, H-3 β-lactam]. The IR spectrum of 10a identified the presence of a β-lactam carbonyl group stretching frequency at υ 1753 cm−1. The cis stereoisomers 10a-m were assigned on the basis of the observed coupling constant between β-lactam ring hydrogens H-3 and H-4 (J3,4 < 3.0 Hz for the trans and J3,4 > 4.0 Hz for the cis stereoisomer) (Islami et al. 2010; Bandyopadhyay et al. 2012). Elemental and mass spectral data of compounds 10a-m provided additional support for the proposed structures.

Biological activities

Antibacterial testing

All the newly synthesized β-lactams bearing 2-mercaptobenzothiazole and benzoquinoline, 10a-m were evaluated for in vitro antimicrobial activity against six common bacteria (Table 1). Five of the compounds, 8a, 8b, 8c, 10k and 10m, have respectable bioactivities against one or more bacterial species, with minimum inhibitory concentrations (MICs) below 50 µg/ml. Judging from the structures of these five compounds, the unsubstituted phenyl group on the lactam enhances antibacterial activity relative to those carrying substituents. The strongest antibacterial activity was observed for 10k and 10m, with MIC values of 20 µg/ml for P. aeruginosa (a Gram-negative bacterium). These β-lactams have a phenoxy group on their position 3 and 10K has a p-N,N-dimethylaminophenyl group and 10m has a p-methoxyphenyl group on position 1. It is interesting to note that the Schiff bases 8a-c also are moderately bioactive, particularly towards P. aeruginosa and S. aureus. Compounds 8a and 8b have the most potent anti-pseudomonas and staphylococcal activity, with MIC values of 42 µg/ml. The mode of antibacterial action has not yet been identified.

Table 1 Antimicrobial activities of 6, 8a-d, and 10a-m
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The basis for antibacterial activity, and in particular, effective inhibition of the Gram-negative microbes, for these compounds is not yet known and would have to be investigated further. Most likely, these compounds do not act upon the penicillin-binding proteins since they do not carry a requisite ionizable functional group off the β-lactam ring for binding. Therefore, the finding that some of the lactams, and imine precursors, possess discernible antibacterial activity is notable.

Mammalian cell toxicity and hemolytic activity

Since antibacterial compounds should have minimum toxic effects on human tissue in order to be useful medicinally, the five best antibacterial compounds (8a-c, 10k, and 10m) were evaluated against a human breast cancer cell line (MCF-7) and human red blood cells. As shown in Table 2, each of these compounds showed negligible cytotoxicity against the MCF-7 cells after 72 h of exposure. At 200 µM, over 75% of the cells were alive while at 50 µM more than 90% of the cells were viable. These low cytotoxicity effects on eukaryotic cells support the potential application of the compounds as viable antibacterial agents, and certainly suggest the need for further investigation.

Table 2 Cell viability percentage of MCF-7 cells treated with some selected compounds at 50 and 200 µM (final concentrations) for 72 h
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A study of the in vitro hemolysis of human red blood cells of these compounds was conducted at an elevated compound concentration of 200 µM. As shown in Table 3, all five compounds revealed less than 10% hemolysis. In particular, lactam 10m induces only 3.51% hemolysis at 124 µg/ml.

Table 3 Hemolysis percentage of human red blood cells treated with the selected compounds at 200 µM final concentration
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Conclusion

In this study, for the first time cis-β-lactams 10 bearing 2-mercaptobenzothiazole and benzoquinoline moieties as ring substituents have been synthesized and evaluated for potential in vitro antibacterial activity. The β-lactam derivatives 10k and 10m as well as their synthetic precursors, Schiff bases 8a, 8b and 8c, showed reasonably good antibacterial activity against either the Gram-negatives E. coli and P. aeruginosa or the Gram-positive S. aureus. These five compounds showed very low cytotoxicity towards MCF-7 human cells at or above the bacterial minimum inhibitory concentration. These results suggest a selection of previously unexplored antibacterial candidates for further investigation to assess their full antimicrobial potential and molecular mechanism of action.

Materials and methods

General

All needed chemicals were purchased from Aldrich, Fluka, Merck and Acros chemical companies and used without further purification. CH2Cl2 and Et3N were dried before to use by distillation over CaH2. All products were characterized by comparison of FT-IR 8300 spectrophotometer using potassium bromide pellets (ʋ in cm−1). The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were run in CDCl3 using a Bruker Avance DPX instrument. Chemical shifts were reported in parts per million (δ) downfield from tetramethylsilane. Coupling constants (J) are reported in hertz (Hz). Splitting patterns are indicated as s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, dd: doublet of doublet. Melting points were recorded on a Buchi 510 melting point apparatus in open capillary tubes. Elemental analyses were run on a Thermo Finnigan Flash EA-1112 series. The mass spectra were recorded on a Shimadzu GC-MS QP 1000 EX instrument. Buchi 510 melting point apparatus. X-ray data were collected on a Bruker D8 VENTURE diffractometer. Thin-layer chromatography was carried out on silica gel 254.

General procedure for the synthesis of 2-(benzo[d]thiazol-2-ylthio)benzo[h]quinoline-3-carbaldehyde (6)

A mixture of 1-naphthylamine (1) (1.00 mmol) and acetic anhydride (2) (5 ml) in methanol (20 ml) containing a small drop of acetic acid as a catalyst was refluxed in a hot water bath. The crude product was isolated by evaporation, and crystallized from methanol. N-(naphthalen-1-yl)acetamide (3) (1.00 mmol) was added to a mixture of Vilsmeier-Haack reagent (prepared by dropwise addition of 6.5 ml of POCl3 in ice-cooled 2 ml of DMF) and refluxed for 16 h. The reaction mixture was poured into ice and kept overnight, followed by neutralization using solid sodium bicarbonate. The crude product 4 was isolated and crystallized from ethanol. To a mixture of benzo[d]thiazole-2-thiol (5) (11 mmol), NaH (20 mmol), and DMSO (20 mL) was added 2-chloro-3-formylquinolines (4) (10 mmol) and the mixture was heated at 90 °C for the given time. Then the reaction was quenched with water (60 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic layer was washed with brine solution, dried over anhydrous Na2SO4, and concentrated in vacuo. 2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinoline-3-carbaldehyde (6) was obtained by flash column chromatography on silica (n-hexane/EtOAc, 10:1/6:1, v/v).

2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinoline-3-carbaldehyde (6)

Yellow solid; Mp. 180–182 °C; IR (KBr, cm−1): 1683 (CO aldehyde); 1H-NMR (400 MHz, CDCl3): 7.52–7.60 (3H, m, ArH), 7.71–7.77 (2H, m, ArH), 7.86 (1H, d, J = 5.2 Hz, ArH), 7.90 (1H, d, J = 4.5 Hz, ArH), 7.99 (1H, d, J = 4.7 Hz, ArH), 8.16 (1H, d, J = 4.7 Hz, ArH), 8.63 (1H, s, ArH), 8.77 (1H, d, J = 5.0 Hz, ArH), 10.42 (1H, s, CHO); 13C-NMR (100 MHz, CDCl3) δ 189.7, 160.6, 154.8, 152.8, 148.7, 141.8, 137.2, 135.2, 130.1, 130.0, 128.9, 127.9, 127.6, 127.3, 126.3, 125.9, 125.5, 124.9, 123.9, 123.2, 121.2; GC-MS m/z = 372 [M+]; Analysis calculated for C21H12N2OS2: C, 67.72; H, 3.25; N, 7.52; S, 17.22%. Found: C, 67.25; H, 2.93; N, 7.43; S, 16.97%.

General procedure for preparation of Schiff bases 8a-d

A mixture of compound 6 (1.00 mmol) and one of the aniline derivatives 7a-d (1.00 mmol) was refluxed in ethanol and 2–3 drops of acetic acid for an appropriate time. The mixture was then cooled to room temperature, filtered and evaporated under reduced pressure. The precipitate was then recrystallized from ethanol to give purified Schiff bases 8a-d.

1-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-N-(4-ethoxyphenyl)methanimine (8a)

Yellow solid; Mp. 183–185 °C; IR (KBr, cm−1): 1592 (CH = N); 1H-NMR (400 MHz, CDCl3): 1.47 (3H, t, J = 4.2 Hz, CH3), 4.07 (2H, q, J = 4.2 Hz, OCH2), 6.96 (2H, d, J = 5.0 Hz, ArH), 7.37 (2H, d, J = 5.2 Hz, ArH), 7.45 (1H, t, J = 4.5 Hz, ArH), 7.53 (1H, d, J = 5.0 Hz, ArH), 7.57 (1H, d, J = 5.0 Hz, ArH), 7.56–7.67 (2H, m, ArH), 7.75 (1H, d, J = 5.2 Hz, ArH), 7.84 (1H, d, J = 4.7 Hz, ArH), 7.92 (1H, d, J = 4.7 Hz, ArH), 8.11 (1H, d, J = 5.0 Hz, ArH), 8.58 (1H, s, ArH), 8.89 (1H, s, CH = N), 8.98 (1H, d, J = 5.0 Hz, ArH); 13C-NMR (100 MHz, CDCl3) δ 163.2, 158.5, 153.2, 152.7, 152.6, 146.8, 143.2, 137.1, 136.5, 134.4, 130.4, 129.1, 129.0, 128.4, 127.8, 127.3, 126.1, 125.5, 125.1, 125.0, 124.7, 122.7, 121.1, 115.0, 63.7, 14.9; GC-MS m/z = 491 [M+]; Analysis calculated for C29H21N3OS2: C, 70.85; H, 4.31; N, 8.55; S, 13.04%. Found: C, 70.58; H, 4.25; N, 8.15, S, 12.65%.

1-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-N-(4-methoxyphenyl)methanimine (8b)

Cream solid; Mp. 189–191 °C; IR (KBr, cm−1): 1591 (CH = N); 1H-NMR (400 MHz, CDCl3): 3.86 (3H, s, OCH3), 6.97 (2H, d, J = 5.5 Hz, ArH), 7.37 (2H, d, J = 5.2 Hz, ArH), 7.45 (1H, t, J = 4.7 Hz, ArH), 7.53 (1H, d, J = 5.0 Hz, ArH), 7.58 (1H, d, J = 5.0 Hz, ArH), 7.64–7.67 (2H, m, ArH), 7.75 (1H, d, J = 5.5 Hz, ArH), 7.84 (1H, d, J = 5.0 Hz, ArH), 7.92 (1H, d, J = 4.7 Hz, ArH), 8.11 (1H, d, J = 5.0 Hz, ArH), 8.59 (1H, s, ArH), 8.89 (1H, s, CH = N), 8.98 (1H, d, J = 5.0 Hz, ArH); 13C-NMR (100 MHz, CDCl3) δ 163.2, 159.1, 153.2, 152.8, 152.6, 146.8, 143.4, 137.1, 136.5, 134.4, 130.4, 129.0, 128.5, 127.8, 127.3, 126.1, 125.5, 125.1, 125.0, 124.7, 122.7, 121.1, 114.5, 55.5; GC-MS m/z = 477 [M+]; Analysis calculated for C28H19N3OS2: C, 70.42; H, 4.01; N, 8.80; S, 13.43%. Found: C, 70.51; H, 4.33; N, 8.15, S, 12.98%.

4-(((2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)methylene)amino)-N,N-dimethylaniline (8c)

Green solid; Mp. 199–201 °C; IR (KBr, cm−1): 1585 (CH = N); 1H-NMR (400 MHz, CDCl3): 3.02 (6H, s, NCH3), 6.77 (2H, d, J = 5.2 Hz, ArH), 7.41–7.46 (3H, m, ArH), 7.52–7.61 (2H, m, ArH), 7.66 (1H, d, J = 4.7 Hz, ArH), 7.70 (1H, d, J = 5.5 Hz, ArH), 7.78 (1H, d, J = 5.5 Hz, ArH), 7.86 (1H, d, J = 5.0 Hz, ArH), 7.92 (1H, d, J = 5.0 Hz, ArH), 8.11 (1H, d, J = 5.0 Hz, ArH), 8.64 (1H, s, ArH), 8.96 (1H, s, CH = N), 9.03 (1H, d, J = 5.0 Hz, ArH); 13C-NMR (100 MHz, CDCl3) δ 163.6, 153.0, 152.6, 150.1, 149.6, 146.5, 139.2, 136.5, 136.4, 134.3, 130.5, 129.7, 128.8, 128.4, 127.8, 127.3, 126.1, 125.4, 125.1, 125.0, 124.9, 122.9, 122.6, 121.1, 112.5, 40.5; GC-MS m/z = 490 [M+]; Analysis calculated for C29H22N4S2: C, 70.99; H, 4.52; N, 11.42; S, 13.07%. Found: C, 70.64; H, 4.41; N, 11.15, S, 12.82%.

4-(((2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)methylene)amino)-N,N-diethylaniline (8d)

Brown solid; Mp. 210–212 °C; IR (KBr, cm−1): 1588 (CH = N); 1H-NMR (400 MHz, CDCl3): 1.24 (6H, t, J = 4.5 Hz, CH3), 3.45 (4H, d, J = 4.5 Hz, NCH2), 6.76 (2H, d, J = 5.5 Hz, ArH), 7.43–7.47 (3H, m, ArH), 7.54 (1H, t, J = 4.5 Hz, ArH), 7.61 (1H, t, J = 4.5 Hz, ArH), 7.70 (1H, t, J = 4.5 Hz, ArH), 7.77 (1H, d, J = 5.5 Hz, ArH), 7.84 (1H, d, J = 5.7 Hz, ArH), 7.92 (2H, t, J = 5.5 Hz, ArH), 8.10 (1H, d, J = 5.0 Hz, ArH), 8.75 (1H, s, ArH), 9.05–9.06 (2H, m, CH = N, ArH); 13C-NMR (100 MHz, CDCl3) δ 163.6, 153.1, 152.7, 148.8, 147.6, 146.6, 138.3, 136.5, 136.2, 134.3, 130.6, 130.0, 128.9, 128.4, 127.8, 127.3, 126.1, 125.4, 125.2, 125.1, 125.0, 123.2, 122.6, 121.1, 111.8, 44.6, 12.7; GC-MS m/z = 518 [M+]; Analysis calculated for C31H26N4S2: C, 71.78; H, 5.05; N, 10.80; S, 12.36%. Found: C, 71.52; H, 5.06; N, 10.72, S, 11.88%.

General procedure for the synthesis of β-lactams of 2-mercaptobenzothiazole and benzoquinoline, 10a-m

A mixture of Schiff bases 8a-d (1.00 mmol), triethylamine (5.00 mmol), one of the substituted acetic acids 9a-d (1.50 mmol) and tosyl chloride (1.50 mmol) in dry CH2Cl2 (15 mL) was stirred overnight at room temperature. Then it was washed with 1 N HCl (20 mL), saturated NaHCO3 (20 mL) and brine (20 mL). The organic layer was dried (Na2SO4), filtered and the solvent was evaporated to give crude products 10a-m that was purified by recrystallization from ethanol.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-1-(4-ethoxyphenyl)-3-phenoxyazetidin-2-one (10a)

White solid; Mp. 198–200 °C; IR (KBr, cm−1): 1753 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.37 (3H, t, J = 7.0 Hz, CH3), 3.96 (2H, q, J = 7.0 Hz, OCH2), 5.75 (1H, d, J = 5.0 Hz, H-4), 6.06 (1H, d, J = 5.0 Hz, H-3), 6.83 (5H, d, J = 8.5 Hz, ArH), 7.06 (2H, t, J = 8.2 Hz, ArH), 7.36–7.41 (3H, m, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.63 (1H, d, J = 9.0 Hz, ArH), 7.68–7.75 (2H, m, ArH), 7.83 (2H, d, J = 8.5 Hz, ArH) 7.89–7.93 (1H, m, ArH), 7.98 (1H, d, J = 8.0 Hz, ArH), 8.19 (1H, s, ArH), 9.22–9.25 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.6, 162.5, 156.8, 156.2, 152.5, 150.9, 146.7, 136.0, 135.8, 134.0, 130.4, 129.8, 129.4, 129.1, 129.0, 127.9, 127.8, 127.6, 126.3, 125.5, 125.2, 125.1, 124.9, 122.6, 122.4, 121.1, 118.8, 115.9, 115.2, 81.7, 63.7, 58.2, 14.8; GC-MS m/z = 625 [M+]; Analysis calculated for C37H27N3O3S2: C, 71.02; H, 4.35; N, 6.72; S, 10.25. Found: C, 70.51; H, 4.33; N, 7.15, S, 10.58%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-1-(4-(diethylamino)phenyl)-3-phenoxyazetidin-2-one (10b)

Yellow solid; Mp. 184–186 °C; IR (KBr, cm−1): 1751 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.09 (6H, t, J = 7.0 Hz, CH3), 3.27 (4H, q, J = 7.0 Hz, OCH2), 5.71 (1H, d, J = 5.0 Hz, H-4), 6.02 (1H, d, J = 5.0 Hz, H-3), 6.55 (2H, d, J = 9.0 Hz, ArH), 6.77–6.86 (3H, m, ArH), 7.04 (2H, t, J = 7.5 Hz, ArH), 7.28 (2H, t, J = 7.5 Hz, ArH), 7.38 (1H, t, J = 7.2 Hz, ArH), 7.48 (1H, t, J = 7.2 Hz, ArH), 7.64 (1H, d, J = 8.7 Hz, ArH), 7.68–7.72 (2H, m, ArH), 7.82 (2H, d, J = 8.5 Hz, ArH), 7.88–7.92 (1H, m, ArH), 7.99 (1H, d, J = 8.5 Hz, ArH), 8.24 (1H, s, ArH), 9.22–9.25 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.8, 162.0, 156.9, 152.5, 150.9, 146.6, 145.4, 136.2, 135.9, 134.0, 130.5, 129.4, 128.9, 128.2, 127.9, 127.5, 126.3, 125.6, 125.5, 125.2, 125.1, 125.0, 122.5, 122.4, 121.1, 119.1, 115.9, 112.0, 81.5, 58.1, 44.4, 12.5; GC-MS m/z = 652 [M+]; Analysis calculated for C39H32N4O2S2: C, 71.75; H, 4.94; N, 8.58; S, 9.82%. Found: C, 71.28; H, 5.05; N, 9.04; S, 9.67%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(4-chlorophenoxy)-1-(4-(dimethylamino)phenyl)azetidin-2-one (10c)

White solid; Mp. 190–192 °C; IR (KBr, cm−1): 1760 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 2.86 (6H, s, NCH3), 5.67 (1H, d, J = 5.0 Hz, H-4), 6.02 (1H, d, J = 5.0 Hz, H-3), 6.63 (2H, d, J = 9.0 Hz, ArH), 6.78 (2H, d, J = 9.0 Hz, ArH), 6.97 (2H, d, J = 9.2 Hz, ArH), 7.32 (2H, d, J = 7.7 Hz, ArH), 7.41 (1H, d, J = 6.5 Hz, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 9.0 Hz, ArH), 7.71–7.74 (2H, m, ArH), 7.83 (2H, d, J = 9.0 Hz, ArH), 7.91 (1H, t, J1 = 5.7 Hz, ArH), 7.98 (1H, d, J = 8.0 Hz, ArH), 8.18 (1H, s, ArH), 9.23–9.27 (1H, m ArH); 13C-NMR (100 MHz, CDCl3) δ 162.5, 161.7, 155.4, 152.5, 150.8, 148.1, 146.7, 136.1, 135.8, 134.0, 130.4, 129.3, 129.1, 129.0, 127.9, 127.8, 127.7, 127.6, 126.3, 125.5, 125.1, 125.0, 122.4, 121.1, 118.7, 117.3, 112.8, 81.6, 58.0, 40.6; GC-MS m/z = 660 [M+, 37Cl], 658 [M+, 35Cl]; Analysis calculated for C37H27ClN4O2S2: C, 67.41; H, 4.13; N, 8.50; S, 9.73%. Found: C, 66.29; H, 4.08; N, 8.56; S, 10.11%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(4-chlorophenoxy)-1-(4-ethoxyphenyl)azetidin-2-one (10d)

Cream solid; Mp. 205–207 °C; IR (KBr, cm−1): 1761 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.37 (3H, t, J = 6.7 Hz, CH3), 3.96 (2H, q, J = 6.7 Hz, OCH2), 5.68 (1H, d, J = 5.0 Hz, H-4), 6.04 (1H, d, J = 5.0 Hz, H-3), 6.77 (2H, d, J = 9.0 Hz, ArH), 6.83 (2H, d, J = 9.0 Hz, ArH), 6.99 (2H, d, J = 9.0 Hz, ArH), 7.35–7.42 (3H, m, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 8.7 Hz, ArH), 7.71–7.75 (2H, m, ArH), 7.84 (2H, d, J = 8.7 Hz, ArH), 7.90–7.93 (1H, m, ArH), 7.97 (1H, d, J = 8.0 Hz, ArH), 8.15 (1H, s, ArH), 9.23–9.27 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.4, 162.1, 156.3, 155.3 152.5, 150.7, 146.7, 135.9, 135.8, 134.1, 130.4, 129.7, 129.3, 129.2, 129.1, 128.0, 127.8, 127.7, 127.6, 126.4, 125.5, 125.2, 125.1, 124.9, 122.4, 121.1, 118.8, 117.3, 115.2, 81.7, 63.7, 58.2, 14.8; GC-MS m/z = 661 [M+, 37Cl], 659 [M+, 35Cl]; Analysis calculated for C37H26ClN3O3S2: C, 67.31; H, 3.97; N, 6.36; S, 9.71%. Found: C, 67.07; H, 3.83; N, 6.28; S, 10.15%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(2,4-dichlorophenoxy)-1-(4-ethoxyphenyl)azetidin-2-one (10e)

White solid; Mp. 200–202 °C; IR (KBr, cm−1): 1755 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.37 (3H, t, J = 6.7 Hz, CH3), 3.96 (2H, q, J = 6.7 Hz, OCH2), 5.72 (1H, d, J = 5.0 Hz, H-4), 6.08 (1H, d, J = 5.0 Hz, H-3), 6.83 (2H, d, J = 9.0 Hz, ArH), 7.00–7.05 (1H, m, ArH), 7.18–7.26 (2H, m, ArH), 7.36–7.41 (3H, m, ArH), 7.48 (1H, t, J = 7.7 Hz, ArH), 7.63 (1H, d, J = 8.7 Hz, ArH), 7.71–7.75 (2H, m, ArH), 7.84 (2H, d, J = 8.8 Hz, ArH), 7.90–7.97 (2H, m, ArH), 8.20 (1H, s, ArH), 9.26–9.29 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.4, 161.5, 156.3, 152.4, 151.1, 150.7, 146.8, 136.1, 135.9, 134.1, 130.5, 129.9, 129.6, 129.1, 129.0, 127.9, 127.7, 127.6, 127.3, 126.3, 125.5, 125.2, 125.1, 124.9, 123.9, 122.3, 121.1, 118.8, 116.3, 115.2, 81.7, 63.7, 58.0, 14.8; GC-MS m/z = 695 [M+, 37Cl], 693 [M+, 35Cl]; Analysis calculated for C37H25Cl2N3O3S2: C, 63.98; H, 3.63; N, 6.05; S, 9.23%. Found: C, 63.38; H, 3.55; N, 5.98; S, 9.71%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-1-(4-ethoxyphenyl)-3-(naphthalen-2-yloxy)azetidin-2-one (10f)

White solid; Mp. 170–172 °C; IR (KBr, cm−1): 1760 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.37 (3H, t, J = 7.0 Hz, CH3), 3.96 (2H, q, J = 7.0 Hz, OCH2), 5.89 (1H, d, J = 4.7 Hz, H-4), 6.15 (1H, d, J = 4.7 Hz, H-3), 6.82 (3H, d, J = 8.2 Hz, ArH), 7.13–7.21 (2H, m, ArH), 7.30–7.73 (12H, m, ArH), 7.83 (1H, d, J = 8.7 Hz, ArH), 7.88–7.96 (2H, m, ArH), 8.24 (1H, s, ArH), 9.17 (1H, d, J = 7.8 Hz, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.7, 162.2, 156.2, 154.4, 152.5, 150.9, 146.7, 136.1, 135.7, 134.0, 133.8, 130.4, 129.7, 129.6, 129.5, 129.1, 129.0, 127.9, 127.6, 127.5, 127.4, 127.0, 126.3, 126.2, 125.4, 125.1, 124.9, 124.8, 124.3, 122.3, 121.0, 118.8, 118.0, 115.2, 109.5, 81.5, 63.7, 58.3, 14.8; GC-MS m/z = 675 [M+]; Analysis calculated for C41H29N3O3S2: C, 72.87; H, 4.33; N, 6.22; S, 9.49%. Found: C, 72.48; H, 4.23; N, 6.20; S, 9.71%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(4-chlorophenoxy)-1-(4-methoxyphenyl)azetidin-2-one (10g)

White solid; Mp. 192–194 °C; IR (KBr, cm−1): 1760 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 3.75 (3H, s, CH3), 5.68 (1H, d, J = 5.0 Hz, H-4), 6.05 (1H, d, J = 5.0 Hz, H-3), 6.77 (2H, d J = 8.7 Hz, ArH), 6.83 (2H, d J = 9.0 Hz, ArH), 6.99 (2H, d J = 8.7 Hz, ArH), 7.36–7.42 (3H, m, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 8.7 Hz, ArH), 7.69–7.77 (2H, m, ArH), 7.84 (2H, d, J = 8.5 Hz, ArH), 7.90–7.93 (1H, m, ArH), 7.97 (1H, d, J = 8.0 Hz, ArH), 8.15 (1H, s, ArH), 9.23–9.27 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.4, 162.1, 156.9, 155.3, 152.5, 150.7, 146.7, 135.9, 135.8, 134.1, 130.4, 129.8, 129.3, 129.2, 129.1, 128.0, 127.7, 127.6, 126.4, 125.5, 125.2, 125.1, 124.8, 122.4, 121.1, 118.8, 117.3, 114.7, 81.7, 58.2, 55.5; GC-MS m/z = 647 [M+, 37Cl], 645 [M+, 35Cl]; Analysis calculated for C36H24ClN3O3S2: C, 66.92; H, 3.74; N, 6.50; S, 9.92%. Found: C, 66.88; H, 3.53; N, 6.39; S, 10.21%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(2,4-dichlorophenoxy)-1-(4-methoxyphenyl)azetidin-2-one (10h)

White solid; Mp. 204–206 °C; IR (KBr, cm−1): 1755 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 3.75 (3H, s, CH3), 5.72 (1H, d, J = 5.0 Hz, H-4), 6.07 (1H, d, J = 5.0 Hz, H-3), 6.83 (2H, d, J = 9.0 Hz, ArH), 6.99–7.04 (2H, m, ArH), 7.19 (1H, d, J = 8.5 Hz, ArH), 7.35–7.40 (3H, m, ArH) 7.48 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 8.7 Hz, ArH), 7.71–7.76 (2H, m, ArH), 7.83 (2H, d, J = 8.7 Hz, ArH), 7.89–7.97 (2H, m, ArH), 8.20 (1H, s, ArH), 9.25–9.29 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.4, 161.5, 156.9, 152.4, 151.1, 150.7, 146.8, 136.2, 135.9, 134.1, 130.5, 129.9, 129.7, 129.2, 129.0, 127.9, 127.7, 127.6, 127.3, 126.3, 125.5, 125.2, 125.1, 124.9, 124.0, 122.4, 121.1, 118.9, 116.3, 114.7, 81.7, 58.0, 55.5; GC-MS m/z = 681 [M+, 37Cl], 679 [M+, 35Cl]; Analysis calculated for C36H23Cl2N3O3S2: C, 63.53; H, 3.41; N, 6.17; S, 9.42%. Found: C, 63.18; H, 3.35; N, 6.32; S, 9.71%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(4-chlorophenoxy)-1-(4-(diethylamino)phenyl)azetidin-2-one (10i)

Yellow solid; Mp. 183–185 °C; IR (KBr, cm−1): 1748 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.10 (6H, t, J = 7.0 Hz, CH3), 3.28 (4H, q, J = 7.0 Hz, NCH2), 5.65 (1H, d, J = 5.0 Hz, H-4), 6.00 (1H, d, J = 5.0 Hz, H-3), 6.56 (2H, d, J = 9.0 Hz, ArH), 6.77 (2H, d, J = 9.0 Hz, ArH), 6.97 (2H, d, J = 9.0 Hz, ArH), 7.29 (2H, d, J = 9.0 Hz, ArH), 7.39 (1H, t, J = 7.2 Hz, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.64 (1H, d, J = 8.7 Hz, ArH), 7.70–7.74 (2H, m, ArH), 7.83 (2H, d, J = 8.7 Hz, ArH), 7.89–7.93 (1H, m, ArH), 7.98 (1H, d, J = 8.0 Hz, ArH), 8.21 (1H, s, ArH), 9.23–9.27 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.5, 161.6, 155.4, 152.5, 150.9, 146.6, 145.4, 136.1, 135.8, 134.0, 130.4,129.5, 129.3, 129.0, 128.0, 127.6, 127.5, 126.3, 125.5, 125.4, 125.3, 125.1, 125.0, 122.4, 121.1, 119.1, 117.3, 112.0, 81.5, 58.0, 44.4, 12.5; GC-MS m/z = 688 [M+, 37Cl], 686 [M+, 35Cl]; Analysis calculated for C39H31ClN4O2S2: C, 68.16; H, 4.55; N, 8.15; S, 9.33%. Found: C, 68.08; H, 4.38; N, 8.25; S, 9.53%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(2,4-dichlorophenoxy)-1-(4-(diethylamino)phenyl)azetidin-2-one (10j)

Cream solid; Mp. 178–180 °C; IR (KBr, cm−1): 1757 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 1.10 (6H, t, J = 7.0 Hz, CH3), 3.28 (4H, q, J = 7.0 Hz, NCH2), 5.69 (1H, d, J = 5.0 Hz, H-4), 6.03 (1H, d, J = 5.0 Hz, H-3), 6.56 (2H, d, J = 9.0 Hz, ArH), 7.01 (2H, d, J = 10.5 Hz ArH), 7.21 (1H, d, J = 8.7 Hz, ArH), 7.29 (2H, d, J = 9.0 Hz ArH), 7.38 (1H, t, J = 7.5 Hz, ArH), 7.48 (1H, t, J = 7.7 Hz, ArH), 7.65 (1H, d, J = 8.7 Hz, ArH), 7.70–7.74 (2H, m, ArH), 7.83 (2H, d, J = 8.7 Hz, ArH), 7.90–7.93 (1H, m, ArH), 7.97 (1H, d, J = 8.2 Hz, ArH), 8.25 (1H, s, ArH) 9.26–9.30 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.5, 160.9, 152.4, 151.2, 150.8, 146.7, 145.5, 136.3, 135.9, 134.1, 130.5, 129.8, 129.0, 128.0, 127.7, 127.6, 127.5, 127.4, 126.3, 125.5, 125.2, 125.1, 123.9, 122.4, 121.1, 119.1, 116.3, 112.0, 81.5, 57.7, 44.4, 12.5; GC-MS m/z = 722 [M+, 37Cl], 720 [M+, 35Cl]; Analysis calculated for C39H30Cl2N4O2S2: C, 64.90; H, 4.19; N, 7.76; S, 8.88%. Found: C, 64.66; H, 4.07; N, 7.62; S, 9.10%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-1-(4-(dimethylamino)phenyl)-3-phenoxyazetidin-2-one (10k)

Cream solid; Mp. 196–198 °C; IR (KBr, cm−1): 1740 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 2.88 (6H, s, NCH3), 5.73 (1H, d, J = 4.7 Hz, H-4), 6.03 (1H, d, J = 4.7 Hz, H-3), 6.62 (2H, d, J = 8.7 Hz, ArH), 6.78–6.86 (3H, m, ArH), 7.05 (2H, t, J = 8.0 Hz, ArH), 7.33 (2H, d, J = 9.0 Hz, ArH), 7.40 (1H, d, J = 7.7 Hz, ArH), 7.49 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 9.0 Hz, ArH), 7.69–7.73 (2H, m, ArH), 7.82 (1H, d, J = 9.5 Hz, ArH), 7.88–7.92 (2H, m, ArH), 7.99 (1H, d, J = 8.0 Hz, ArH), 8.21 (1H, s, ArH), 9.22–9.25 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.8, 162.1, 156.9, 152.5, 150.9, 148.0, 146.6, 136.2, 135.8, 134.0, 130.4, 129.4, 128.9, 128.0, 127.9, 127.5, 126.5, 126.3, 125.5, 125.1, 125.0, 122.5, 122.4, 121.1, 118.8, 115.9, 112.8, 81.5, 58.1, 40.6; GC-MS m/z = 624 [M+]; Analysis calculated for C37H28N4O2S2: C, 71.13; H, 4.52; N, 8.97; S, 10.26%. Found: C, 71.02.; H, 4.19; N, 8.62; S, 10.51%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-3-(2,4-dichlorophenoxy)-1-(4-(dimethylamino)phenyl)azetidin-2-one (10l)

Cream solid; Mp. 221–223 °C; IR (KBr, cm−1): 1751 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 2.89 (6H, s, NCH3), 5.70 (1H, d, J = 5.0 Hz, H-4), 6.04 (1H, d, J = 5.0 Hz, H-3), 6.62 (2H, d, J = 8.7 Hz, ArH), 7.01 (2H, d, J = 9.7 Hz, ArH), 7.21 (1H, d, J = 8.5 Hz, ArH), 7.32 (2H, d, J = 9.0 Hz, ArH), 7.39 (1H, d, J = 7.7 Hz, ArH), 7.48 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 8.7 Hz, ArH), 7.70–7.74 (2H, m, ArH), 7.82 (2H, d, J = 9.5 Hz, ArH), 7.89–7.93 (1H, m, ArH), 7.97 (1H, d, J = 8.0 Hz, ArH), 8.23 (1H, s, ArH), 9.26–9.29 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.5, 161.1, 152.4, 151.1, 150.8, 148.1, 146.7, 136.2, 135.9, 134.1, 130.5, 129.8, 129.0, 128.9, 127.9, 127.5, 126.3, 126.2, 125.5, 125.2, 125.1, 123.9, 122.3, 121.1, 118.8, 116.3, 112.8, 81.5, 125.0, 57.8, 40.5; GC-MS m/z = 694 [M+, 37Cl], 692 [M+, 35Cl]; Analysis calculated for C37H26Cl2N4O2S2: C, 64.07; H, 3.78; N, 8.08; S, 9.24%. Found: C, 63.98; H, 3.65; N, 7.92; S, 9.69%.

4-(2-(Benzo[d]thiazol-2-ylthio)benzo[h]quinolin-3-yl)-1-(4-methoxyphenyl)-3-phenoxyazetidin-2-one (10m)

White solid; Mp. 207–209 °C; IR (KBr, cm−1): 1762 (CO β-lactam); 1H-NMR (250 MHz, CDCl3): 3.74 (3H, s, CH3), 5.74 (1H, d, J = 4.7 Hz, H-4), 6.05 (1H, d, J = 4.7 Hz, H-3), 6.80–6.86 (5H, m, ArH), 7.06 (2H, t, J = 7.7 Hz, ArH), 7.37–7.40 (3H, m, ArH), 7.48 (1H, t, J = 7.7 Hz, ArH), 7.62 (1H, d, J = 9.0 Hz, ArH), 7.69–7.73 (2H, m, ArH), 7.82 (2H, d, J = 8.5 Hz, ArH), 7.88–7.92 (1H, m, ArH), 7.97 (1H, d, J = 8.0 Hz, ArH), 8.18 (1H, s, ArH), 9.21–9.25 (1H, m, ArH); 13C-NMR (100 MHz, CDCl3) δ 162.7, 162.5, 156.8, 152.5, 150.9, 146.7, 136.0, 135.8, 134.0, 130.4, 129.9, 129.4, 129.1, 129.0, 128.0, 127.8, 127.6, 126.3, 125.5, 125.1, 124.9, 122.6, 122.4, 121.1, 118.8, 115.9, 114.6, 81.7, 58.2, 55.4; Analysis calculated for C36H25N3O3S2: C, 70.68; H, 4.12; N, 6.87; S, 10.48%. Found: C, 70.48; H, 4.09; N, 6.62; S, 10.87%.

General procedure for MIC activity assay

E. coli ATCC 25922, P. aeruginosa ATCC 9027, S. aureus ATCC 25923, S. typhi ATCC 7251, E. faecalis ATCC 29212 and B. subtilis ATCC 6051 were used in this study to test the compounds for antibacterial activity. These bacteria were cultivated on Brain Heart Infusion (BHI) plates overnight. Several colonies were suspended into normal saline and adjusted to OD600nm = 0.07 (0.5 McFarland unit). The inoculum solution was made by preparing a 1:100 dilution of 0.5 McFarland normal saline suspension using BHI broth and 100 mL of this suspension was added to 100 mL of BHI broth containing various concentrations of test antibiotics. The test antibiotic solution was serially diluted 2-fold by automatic pipette in a 96-well microtiter format. After inoculation with bacterial strains (final density was approximately 5 × 105 CFU/mL), the microtiter plates were incubated at 37 °C for 18 h. MIC was determined as the lowest concentration of the test compound in which the absorbance at 600 nm is less than or equal to 0.025 (Letavic et al. 2002).

Mammalian cytotoxicity

MCF-7 (a breast cancer cell line) cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C in DMEM high medium supplemented with 100 U. mL−1 penicillin, 100 μg mL−1 streptomycin and 10% fetal bovine serum. Cell viability assay was performed using the MTT method (Riss et al. 2013). Briefly 1 × 104 cells were seeded in 96 well flat-bottom plates and incubated at 37 oC for 24 h in the presence of 5% CO2, then the cells exposed to the synthesized compounds at final concentration of 50 and 200 μM for a period of 72 h. Afterward, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well to achieve a final concentration of 0.45 mg/ml and incubated at 37 °C for another 4 h. Finally, equal volume of solubilization solution (40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid and 16 % sodium dodecyl sulfate (SDS), pH ̴ 4.7) was added to each well to dissolve formazan crystals and absorbance recorded at 570 nm using SPECTROstarNano (BMG Labtech, Germany) microplate reader. Absorbance at 690 nm was also measured to omit the turbidity. Cell viability percentage calculated as follow:

$${\mathrm{A}}_{\mathrm{T}} \,=\, {\mathrm{A}}_{570} - {\mathrm{A}}_{690}$$
$${\mathrm{Cell}}\,{\mathrm{viability}}\,\% \,=\, \left[ {{\mathrm{A}}_{\mathrm{T}}\,{\mathrm{sample}}/{\mathrm{A}}_{\mathrm{T}}\,{\mathrm{control}}} \right] \times 100$$

Hemolytic activity

Fresh human red blood cells (RBC) were washed with phosphate buffer saline (PBS) until the upper phase was clear after centrifugation. The pellet was resuspended in PBS to reach an OD600 of 24.0. Stock solutions of the synthesized compounds (1000×) were prepared in DMSO and diluted in RBC suspension to achieve a final concentration of 200 µM. DMSO and Triton X-100 (1% final concentration) were used as negative and positive controls, respectively. After one hour of incubation at 37 °C, the cells were centrifuged at 1000 × g for 10 min (Ling et al. 2015). The absorbance of supernatants at A450nm was measured and hemolysis percentage was calculated as follow:

$$\begin{array}{ccccc}\\ {\mathrm{Hemolysis}}\,{\mathrm{percentage}} = & \left[ {\left( {{\mathrm{Absorbance}}\,{\mathrm{of}}\,{\mathrm{sample}}-{\mathrm{Absorbance}}\,{\mathrm{of}}\,{\mathrm{negative}}\,{\mathrm{control}}} \right)} \right.\cr \\ & \left. { \div \left( {{\mathrm{Absorbance}}\,{\mathrm{of}}\,{\mathrm{positive}}\,{\mathrm{control}}-{\mathrm{Absorbance}}\,{\mathrm{of}}\,{\mathrm{negative}}\,{\mathrm{control}}} \right)} \right] \times 100\\ \end{array}$$

Both hemolysis and cytotoxicity experiments were performed triplicate.