Synthesis and in vitro anticancer, antibacterial, and

Res Chem Intermed (2016) 42:6543–6555 DOI 10.1007/s11164-016-2479-x

Synthesis and in vitro anticancer, antibacterial, and antioxidant studies of unsymmetrical Schiff base derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl] benzene-1,3-diol Ikechukwu P. Ejidike1 • Peter A. Ajibade1

Received: 22 December 2015 / Accepted: 5 February 2016 / Published online: 4 March 2016 Springer Science+Business Media Dordrecht 2016

Abstract Schiff bases such as 2-hydroxy-1-(4-hydroxyphenyl)ethanone (DHAP) and its derivatives have attracted attention because they are useful in design and development of novel organic compounds for potential pharmaceutical applications. In this work, a series of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) Schiff bases were synthesized by reaction of ethylenediamine, DHAP, and appropriate aldehyde moieties. The compositions of the prepared compounds were established using elemental analysis and Fourier-transform infrared (FTIR) and ultraviolet–visible (UV–Vis) spectroscopies. The compounds were screened against three Gram-positive and three Gram-negative bacteria, and the results compared with standard drugs ciprofloxacin and amoxicillin. Compounds 4g, 4h were found to have higher activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) value of 2.5 mg/mL, while compounds 4f and 4h inhibited Escherichia coli with MIC values of 2.5 and 5 mg/mL, respectively. The IC50 values of compounds 4a–h for scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical ranged from 2.63 ± 0.79 to 3.85 ± 0.83 lM with good correlation coefficient of R2 = 0.957–0.994. In vitro anticancer screening of the compounds showed that compounds 4f, 4h, and parthenolide efficiently affected cell viability of cancer cell line MCF-7 with IC50 values of 4.10 ± 1.32, 4.01 ± 2.26, and 0.44 ± 2.02 lM, respectively. Keywords

DHAP Antioxidant Schiff base ligands Antibacterial Anticancer

& Peter A. Ajibade [email protected] 1

Department of Chemistry, Faculty of Science and Agriculture, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa

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I. P. Ejidike, P. A. Ajibade

Introduction Design and development of novel organic compounds with potential biological activities and reduced side-effects has attracted great interest from researchers, especially design and synthesis of Schiff bases for medicinal applications [1]. Schiff bases are considered ‘‘fortunate ligands’’ [2, 3] due to their ease of preparation via condensation reactions between aldehydes and imines. Schiff bases were first reported when Hugo Schiff reacted amine and carbonyl base compounds [4, 5], bearing azomethine ([C=N–) functional group, and have been established as versatile pharmacophores for design and development of various biologically active compounds [1]. The carbon–nitrogen double-bond group in these compounds has been revealed to be pivotal to their biological properties [6, 7]. Due to the existence of a lone pair of electrons on the nitrogen atom, the electron-donating character of the double bond, and the low electronegativity of nitrogen, the N of the azomethine group ([C=N) acts as a good donor site, making Schiff bases biologically active [8– 10] for application as antifungal, antiradical, antimalarial, antiviral, anticancer, antibacterial, antiinflammatory, and antipyretic agents [9, 11]. The activities of free oxygen radicals have been of great importance in experimental trials in recent years [12]. Beneficial effects of reactive oxygen species (ROS) are evident in the physiological activity of cellular reactions to anoxia, as a means of resistance against infectious diseases. In contrast to this benefit, recent evidence [13] suggests that radicals, generated during any biological–organic oxidation–reduction process, can prompt oxidative impairment in various components of the body and can also lead to mutant formation [14]. Schiff base ligands containing different donor atoms show broad biological activities due to their various bonding modes to metal ions. Such interactions yield a fascinating chain of ligands whose properties can be tailored to give different compounds by varying substituents, which may lead to variation in the ultimate donating atoms [8]. The potential dianionic chelating abilities of the tridentate-ONO and tetradentateONNO Schiff base category with a number of organometallic moieties have been reported [15, 16]. With respect to the aforementioned properties of Schiff bases and 2-hydroxy-1-(4-hydroxyphenyl)ethanone derivatives, we report herein synthesis and spectroscopic characterization of (ONN) Schiff base derivatives of 4-[(1E)-N-(2aminoethyl)ethanimidoyl]benzene-1,3-diol and their bacteriological, anticancer, and radical-scavenging activities.

Experimental Materials and methods Chemicals and solvents of analar grade were used. Ethylenediamine, benzaldehyde, benzoyl chloride, and ascorbic acid were obtained from Merck, acetanilide, acetophenone, and vanillin (4-hydroxy-3-methoxybenzaldehyde) from BDH, and 4-methoxybenzaldehyde, 20 ,40 -dihydroxyacetophenone, veratraldehyde (3,4dimethoxybenzaldehyde), and formaldehyde from Sigma Aldrich. 1,1-Diphenyl-2-

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Synthesis and in vitro anticancer, antibacterial, and…

6545

picrylhydrazyl (DPPH) and rutin hydrate were obtained from Sigma Chemical Co. A PerkinElmer Spectrum 2000 FTIR spectrometer was used to obtain IR spectra in the range from 4000 to 400 cm-1 from KBr pellets. Elemental analysis was carried out using a PerkinElmer elemental analyzer. A PerkinElmer Lambda-25 UV–Vis spectrometer was used for absorption spectral measurements in the range of 200–800 nm. Melting points were obtained using a Stuart melting point device (SMP 11). General synthesis for derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl] benzene-1,3-diol Synthetic procedure for compounds 4a–e Ethylenediamine (15 mmol) in 20 mL ethanol was carefully added to ethanol solution (30 mL) of 20 ,40 -dihydroxyacetophenone (15 mmol), stirred for 60 min at room temperature, followed by dropwise addition for 10–15 min of appropriate aldehyde (3a–e) (15 mmol) in 20 mL ethanol at room temperature. The resulting mixture was refluxed with stirring for 3–4 h, and left to stir further for 22–24 h at room temperature to give derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–e) (Schemes 1, 2) as crystalline solid after suction filtration, washed with ethanol. The crude product was recrystallized from warm ethanol. The products were dried in vacuum at 50 C overnight to give the target molecule in good to excellent yields (65.7–88.8 %). Synthetic procedure for compounds 4f–h Ethylenediamine (15 mmol) in 20 mL ethanol was carefully added to ethanol solution (30 mL) of 20 ,40 -dihydroxyacetophenone (15 mmol), stirred for 60 min at room temperature, followed by dropwise addition for 10–15 min of appropriate aldehyde (3f–h) (15 mmol) dissolved in 30 mL ethanol and stirred for 120 min. The mixture was left standing with continuous stirring for approximately 36 h without heat, giving the derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3diol (4f–h) (Schemes 1, 2) as crystalline solid after suction filtration, washed with ethanol. The crude product was recrystallized from warm ethanol. The products

H3 C

NH2

O OH

H 2N

NH2

+

N

OH

Ethanol Stirred, -H2O

H3C

OH 1a

1b

OH 2a-h

Scheme 1 Synthesis of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol

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I. P. Ejidike, P. A. Ajibade H

H3C N

N H

OH

H3C

4a

N

HO

H3C N

Cl

OH

H

Cl

OH

4b

O H

O

4h

HO

3a

O

HO H

OCH3 O H3C N

N

H

HO

H3C

O

OH

2a-h

H

3c

4c HO

3f O

OH

H3C

O

CH3

HN

OCH3

N

3d

N

CH3

H3C N

H OH

H3CO

OCH3

N

OH O

OH

OH 4g

H3C

N

N H

3b

NH2

OCH3

3g

OCH3

H

3h OCH3

OCH3

N H

N

CH3 OH

N H

4d

3e

HO

OH H3C

4f HO

NH N

N CH3

OH 4e HO

Scheme 2 Synthesis of derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol

were dried in vacuum at 50 C overnight to give the target molecule in good to excellent yields (64.2–73.8 %). 4-{(1E)-N-[2-(methylideneamino)ethyl]ethanimidoyl}benzene-1,3-diol (4a) Darkish-yellow solid; yield 2.75 g (88.8 %); decomp. temp. 218–219 C; IR (KBr) cm-1: 1254 (C–O), 1551 (C=C), 1608 (C=N), 2985 (C–H)alip., 3044 (C–H)arom., 3470 (Ar–OH); UV–Vis (DMF): kmax. 319, 386; Found for C11H14N2O2 (%):C 63.98, H 7.02, N 13.49; Anal. Calcd. (%): C 64.06, H 6.84, N 13.58. 4-[(1E)-N-{2-[(Z)-benzylideneamino]ethyl}ethanimidoyl]benzene-1,3-diol (4b) Dark-yellow solid; yield 3.03 g (71.5 %); decomp. temp. 233–234 C; IR (KBr) cm-1: 1266 (C–O), 1586 (C=C), 1613 (C=N), 2929 (C–H)alip., 3081 (C–H)arom., 3473 (Ar–OH); UV–Vis (DMF): kmax. 276, 308, 378 nm; Found for C17H18N2O2 (%): C 72.17, H 6.31, N 9.86; Anal. Calcd. (%): C 72.32, H 6.43, N 9.92. 4-[(1E)-N-{2-[(Z)-(4-methoxybenzylidene)amino]ethyl}ethanimidoyl]benzene-1,3-diol (4c) Yellowish-brown solid; yield 3.08 g (65.7 %); decomp. temp. 235–236 C; IR (KBr) cm-1: 1244 (C–O–C), 1267 (C–O), 1590 (C=C), 1615 (C=N), 2989 (C– H)alip., 3083 (C–H)arom., 3471 (Ar–OH); UV–Vis (DMF): kmax. 275, 306, 384 nm;

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Found for C18H20N2O3 (%): C 69.16, H 6.34, N 8.91; Anal. Calcd. (%): C 69.21, H 6.45, N 8.97. 4-[(1E)-N-{2-[(Z)-(1-phenylethylidene)amino]ethyl}ethanimidoyl]benzene-1,3-diol (4d) Pale-yellow solid; yield 3.01 g (67.6 %); decomp. temp. 247–248 C; IR (KBr) cm-1: 1267 (C–O), 1585 (C=C), 1614 (C=N), 2928 (C-H)alip., 3022 (CH)arom., 3472 (Ar-OH); UV–Vis (DMF): kmax. 276, 307, 386 nm; Found for C18H20N2O2 (%): C 72.98, H 6.71, N 9.40; Anal. Calcd. (%): C 72.95, H 6.80, N 9.45. (1Z)-N0 -(2-{(E)-[1-(2,4-dihydroxyphenyl)ethylidene]amino}ethyl)-N-phenylethanimidamide (4e) Golden-yellow solid; yield 3.22 g (69.0 %); decomp. temp. 251–252 C; IR (KBr) cm-1: 1267 (C–O), 1585 (C=C), 1616 (C=N), 2981 (C– H)alip., 3035 (C–H)arom., 3244 (N–H), 3477 (Ar–OH); UV–Vis (DMF): kmax. 275, 308, 383 nm; Found for C18H21N3O2 (%): C 69.39, H 6.76, N 13.41; Anal. Calcd. (%): C 69.43, H 6.80, N 13.49. 4-[(1E)-N-{2-[(Z)-(4-hydroxy-3-methoxybenzylidene)amino]ethyl}ethanimidoyl]benzene-1,3-diol (4f) Brownish-red solid; yield 3.64 g (73.8 %); decomp. temp. 209–210 C; IR (KBr) cm-1: 1244 (C–O–C), 1288 (C–O), 1597 (C=C), 1619 (C=N), 2962 (C–H)alip., 3061 (C–H)arom., 3476 (Ar–OH); UV–Vis (DMF): kmax. 274, 303, 388 nm; Found for C18H20N2O4 (%): C 65.79, H 6.11, N 8.45; Anal. Calcd. (%): C 65.84, H 6.14, N 8.53. 4-[(1E)-N-{2-[(Z)-(3,4-dimethoxybenzylidene)amino]ethyl}ethanimidoyl]benzene1,3-diol (4g) Brownish-yellow solid; yield 3.39 g (66.0 %); decomp. temp. 222–223 C; IR (KBr) cm-1: 1243 (C–O–C), 1268 (C–O), 1597 (C=C), 1616 (C=N), 2926 (C–H)alip., 3027 (C–H)arom., 3475 (Ar–OH); UV–Vis (DMF): kmax. 274, 305, 382 nm; Found for C18H20N2O4 (%): C 66.61, H 6.42, N 8.09; Anal. Calcd. (%): C 66.65, H 6.48, N 8.18. N-(2-{(E)-[1-(2,4-dihydroxyphenyl)ethylidene]amino}ethyl)benzenecarboximidoyl chloride (4h) Brownish-yellow solid; yield 3.05 g (64.2 %); decomp. temp. 234–235 C; IR (KBr) cm-1: 753 (C–Cl), 1287 (C–O), 1584 (C=C), 1606 (C=N), 2929 (C–H)alip., 3061 (C–H)arom., 3462 (Ar–OH); UV–Vis (DMF): kmax. 275, 306, 380 nm; Found for C17H17ClN2O2 (%): C 64.44, H 5.36, N 8.76; Anal. Calcd. (%): C 64.46, H 5.41, N 8.84. In vitro antimicrobial studies The synthesized derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3diol (4a–h) were tested to assess their growth inhibitory action against six reference strains of bacteria by agar diffusion method [17] using Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 29212), and Bacillus cereus (ATCC 10702) as Gram-positive bacteria and Pseudomonas aeruginosa (ATCC 19582), Escherichia coli (ATCC 25922), and Shigella flexneri (KZN) as Gram-negative bacteria with ciprofloxacin and amoxicillin as standard antibacterial agents.

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Subculturing of bacterial isolates on nutrient agar (SAARCHEM, Gauteng SA) plates was achieved by incubating at 37 C for 24 h. Bacterial cells from nutrient agar plates were incubated in 50 mL nutrient broth in a 250-mL flask at 37 C for 16 h, enhanced with vigorous shaking. The culture was diluted with fresh media after incubation to obtain D600nm of 0.1. Then, 100 lL of cell culture was added onto the plate and spread into a bacterial lawn using a sterile glass spreader. Agar dilution method as contained in NCCLS [18] was used to determine the minimum inhibitory concentration (MIC) of compounds 4a–h and standard drugs as previously described [19]. Antioxidant assay Scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical The antioxidant activity of derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) was determined using stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent following a method reported previously [19]. DMF solution (1 mL) of compound 4a–h at various concentrations (100, 200, 300, 400, and 500 lg/mL) was thoroughly mixed with 1 mL methanolic solution of 0.4 mM DPPH and allowed to interact for about 30 min in the dark. Reduction in absorption of the solutions was measured spectrophotometrically at 517 nm against control. The following equation was used to obtain the percentage scavenged DPPH radical: Percentage scavenging activity ¼

Absorbance of control Absorbance of sample 100: Absorbance of control

Cell viability assay The sulforhodamine B (SRB) assay was used for in vitro anticancer study of compounds 4f, 4h, as previously described [20]. Human renal cancer cell line TK10, human melanoma cancer cell line UACC-62, and human breast cancer cell line MCF-7 were cultured at 37 C with 95 % air/5 % CO2 and 100 % relative humidity in Roswell Park Memorial Institute (RPMI) medium, supplemented with 5 % fetal bovine serum (FBS), 50 lg mL-1 gentamicin, and 2 mmol/L L-glutamine, as described [21]. Passages 3–19 were inoculated into 96-well microtiter plates at plating densities of 7000–10,000 cells/well and incubated for 24 h. Treatment of the cells with solutions of compounds in DMSO was done after 24 h, diluted in medium to yield five concentrations (0.01, 0.1, 0, 10, and 100 lM); cells with no drug/ sample were used as control, and the blank comprised complete medium with no cells. The standard used for this study was parthenolide. Incubation of plates for 48 h was followed by addition of the compounds. Viable cells were fixed to the bottom of each well with cold 50 % trichloroacetic acid, washed, dried, and dyed by SRB. Unbound dye was detached, while 10 mmol/L Tris base was used for extraction of protein-bound dye, and its optical density was determined using a multiwell spectrophotometer at wavelength of 540 nm. The IC50 value was

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calculated as the concentration for 50 % cell growth inhibition using absorbance values plotted against concentration of compounds 4f, h. To ensure the quality of immunocytochemical assays such as the sulforhodamine B (SRB) assay, the Z0 factor coefficient was adapted.

Results and discussion Synthesis and characterization of Schiff base ligands We describe herein the synthesis of various (ONN) Schiff base derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) by simple condensation reaction of ethanimidoylbenzene-1,3-diol (2a–h) (Scheme 1) and substituted aromatic aldehydes (3a–h) in equimolar condition, as shown in Scheme 2. The derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) were synthesized as golden-darkish yellow solids, with the exception of 4-[(1E)-N-{2[(Z)-(4-hydroxy-3-methoxybenzylidene)amino]ethyl}ethanimidoyl]benzene-1,3-diol (4f) that was brownish red. Data from elemental analyses were found to be in conformity with the assigned structure. FTIR spectra revealed presence of stretching vibrations due to –OH hydroxyl group at 3470–3477 cm-1, C=N azomethine group (1608–1619 cm-1), and –C–O group ranging from 1254 to 1288 cm-1 [22, 23]. The electronic spectra of compounds 4a–h were recorded in DMF as solvent. Spectra of the compounds exhibit three main absorption bands at about 274–276, 303–319, and 380–388 nm. The first and second band ranges are attributable to conjugation within the ring p–p* and imino p–p* transition, respectively. The third band range in the spectra (378–396 nm) is assigned to intraligand n–p* transitions [5, 23]. The derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) were screened against some pathogenic bacteria, and their scavenging potential for DPPH radical was assessed, while the SBR assay was used to investigate the anticancer activities of 4f, 4h, and parthenolide against three cancer cell lines. In vitro antimicrobial studies The newly synthesized derivatives of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) were checked for their antimicrobial potential against some bacterial strains: Staphylococcus aureus, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Shigella flexneri, and Escherichia coli. The MIC results are summarized in Table 1. The antimicrobial activity of the derivatives matched the standard drugs amoxicillin and ciprofloxacin. Close examination of the results indicates that the analogues of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3diol (4a–h) exhibited various ranges of minimum inhibition depending upon the substituent on the ring structure. The compounds showed moderate to good biological activity against the bacterial strains. Compound 4f showed excellent activity against both Gram-positive and Gram-negative bacterial strains as compared with the standard drugs amoxicillin and ciprofloxacin. Compounds 4g, 4h were found to have excellent activity against S. aureus with MIC value of

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Table 1 Minimum inhibitory concentration (MIC, aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) Compound

Gram-positive bacteria S. faecalis

B. cereus

mg/mL)

of

analogues

of

4-[(1E)-N-(2-

Gram-negative bacteria S. aureus

P. aeruginosa

S. flexneri

E. coli –

4a

–

–

5

–

10

4b

10

–

5

5

–

–

4c

–

10

5

–

10

–

4d

–

5

–

–

5

–

4e

–

–

10

5

–

–

4f

5

1.25

5

5

1.25

2.5

4g

10

5

2.5

–

5

–

4h

10

–

2.5

–

5

5

Amoxicillina

1.250

0.312

1.250

1.250

1.250

0.625

Ciprofloxacina

0.312

0.312

0.312

0.312

0.312

0.312

a

Standards; ‘‘–’’ represents ‘‘inactive’’

2.5 mg/mL. The compounds showed minimum inhibition within the considered range of concentrations against S. flexneri, except for 4e, which was inactive. Furthermore, compounds 4f and 4h exhibited inhibition of E. coli with MIC values of 2.5 and 5 mg/mL, respectively (Table 1). The presence of different functional groups on the benzene ring selectively resulted in bioactivity against strains at low concentrations, showing higher activity against S. aureus, S. flexneri, B. cereus, and E. coli. Cell wall synthesis disruption can be related to normal cell process blockage due to hydrogen-bond formation via cell constituent active centers with [CH=N– [24]. Furthermore, the variation in the efficacy of derivatives 4a–h partly depends on the impermeability or ribosome modifications of the microbial cells [25]. The particle size of the compounds also partly affects their antimicrobial activity, because nanosized particles exhibit increased antimicrobial activity [26]. Antioxidant assay 1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable organic radical that is extensively used to ascertain the capability of biological reagents/compounds with free radicalscavenging potential or hydrogen donors, thus expressing the magnitude of their antioxidation ability [19]. The antioxidant activities of the newly synthesized compounds and the standards (ascorbic acid and rutin) were assessed on the basis of the free radical-scavenging ability as measured using the DPPH free radical activity [27]. The changes or trend of inhibition in the DPPH radical-scavenging ability as percentage inhibition of the analogues of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) is presented in Fig. 1, being concentration dependent. The percent inhibition results for the samples and standards are also expressed as IC50 values (the amount of antioxidant necessary to reduce the initial DPPH concentration by 50 %).

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DPPH radical scavenging activity

80 70

DPPH % Inhibitor

60 50 40 30 20 10 0 100

200

300

400

500

Concentration (μg/mL) 4a

4b

4c

4d

4e

4f

4g

4h

Vit. C

Run

Fig. 1 Free radical-scavenging activity in DPPH system

It is observed from Fig. 1 that, at the highest concentration (500 lg/mL), 4a exhibited the highest inhibition of DPPH radical while 4f had the lowest free radical-scavenging potential among the synthesized compounds studied. It is worthy of note that the synthesized compounds exhibited higher inhibition of DPPH at the lowest concentration (100 lg/mL) than rutin (standard), while at 200 lg/mL all other compounds showed higher percentage inhibition than rutin (standard) except for 4f. IC50 and the corresponding R2 (correlation coefficient) values of the tested compounds are listed in Table 2. The IC50 values of compounds 4a–h for DPPH Table 2 Influence of investigated compounds and standard drugs on DPPHa free radicals

(n = 3, X ± SEM), IC50— inhibitory concentration; the IC50 value is the concentration of the tested compound for which the percent inhibition was 50 %. R2 = correlation coefficient a

Standards

Compound

DPPH radical-scavenging activity Equation

IC50 (lM)

R2

4a

y = 18.42x ? 1.48

2.63 ± 0.79

0.974

4b

y = 15.85x ? 1.59

3.05 ± 0.54

0.962

4c

y = 16.42x ? 0.85

2.99 ± 0.93

0.988

4d

y = 16.46x ? 0.65

3.00 ± 0.52

0.992

4e

y = 15.75x ? 1.68

3.07 ± 0.63

0.957

4f

y = 12.77x ? 0.85

3.85 ± 0.83

0.989

4g

y = 15.57x ? 0.67

3.17 ± 0.63

0.987

4h

y = 15.64x ? 0.94

3.13 ± 0.55

0.985

Vit. Ca

y = 25.08x ? 1.83

1.92 ± 1.07

0.978

Rutina

y = 19.05x ? 1.99

2.52 ± 1.60

0.798

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radical ranged from 2.63 ± 0.79 to 3.85 ± 0.83 lM, while the standards vitamin C and rutin showed IC50 values of 1.92 ± 1.07 and 2.52 ± 1.60 lM, respectively. The order of antioxidant activity of the analogues of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol can be given as 4a [ 4c [ 4d [ 4b [ 4e [ 4h [ 4g [ 4f (Table 2). The observed differential scavenging activities of the synthesized compounds against DPPH radical could be due to the presence of different substituent on the ring structure [28]; also, the reaction of DPPH with compounds containing hydroxyl groups such as phenols is reversible, resulting in low readings for antioxidant activity [29]. Although the results of the DPPH free radical-scavenging assay for the synthesized compounds were lower than for the reference drugs ascorbic acid and rutin, the moderate inhibition exhibited on DPPH radical suggests that the analogues of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4a–h) have potent antioxidant ability for scavenging free radicals. These compounds could be used as a potent source for developing cancer chemotherapy and therapeutic agents for treating stress-induced pathological conditions. Anticancer screening Analogues of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol (4f, 4h) were selected and tested in vitro for their anticancer activity against human renal cancer cell line TK-10, human melanoma cancer cell line UACC-62, and human breast cancer cell line MCF-7 based on their antimicrobial properties against the screened bacterial strains in this study. Figure 2a–c shows the concentration-dependent inhibitory effect of the compounds on the percentage cell survival at 48 h of exposure in culture medium. In Fig. 2a–c, it can be observed that compounds 4f, 4h showed major cytotoxic activity (*100 lM) in the survival range of 55–80 %. Compound 4h showed higher inhibition of cell proliferation at 100 lM concentration against human renal cancer cell line TK-10 and human melanoma cancer cell line UACC-62 than 4f, with cell survival of 58.52 and 75.92 % respectively (Fig. 2a, b), while 4f gave better cell proliferation inhibition than 4h toward human breast cancer cell line MCF-7 with cell survival of 53.76 % (Fig. 2c). Parthenolide showed strong levels of anticancer effect against all cell lines, in line with previous reports [30, 31]. Table 3 presents the IC50 values obtained from nonlinear regression analysis of dose–response data for the compounds tested. These results demonstrate that treatment with compounds 4f, 4h, and parthenolide efficiently affected cell viability of MCF-7 cells with IC50 values of 4.10 ± 1.32, 4.01 ± 2.26, and 0.44 ± 2.02 lM, respectively (Fig. 2c; Table 3). The nature of substituents (hydroxyl, alkyl, methoxy, chloride) and the bridging spacer ethylenediamine play critical roles in determining the cytotoxicity of 4f, 4h [32]. The antimicrobial, antioxidant, and anticancer evaluations provide some insight into the structure–activity relationship of the synthesized compounds. According to the nature of the substituent, hydroxyl, methoxy, chloride, azomethine group ([C=N), and alkyl substitution (such as methyl and phenyl) could be responsible for the increased activity and observed biological properties of the active compounds.

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% Cell Viability

(a)

100 80 60 40 20 0 0.01 0.1 1 10 100

Concentration (μM)

Parthenolide 4f 4h

% Cell Viability

(b)

100 80 60 40 20 0 0.01 0.1 1 10 100

Concentration (μM)

Parthenolide 4f 4h

(c)

% Cell Viability

Fig. 2 In vitro anticancer screening of compounds 4h, 4f, and parthenolide for a human renal cancer cell line TK-10, b human melanoma cancer cell line UACC-62, and c human breast cancer cell line MCF-7 after culture for 48 h

6553

100 80 60 40 20 0 0.01 0.1 1 10 Concentration (μM)

100

Parthenolide 4f 4h

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Table 3 IC50 values (lM) of compounds 4f, 4h, and parthenolide against tumor cell lines Compound

Molecular formula

Anticancer activity, IC50 (lM) 48 h TK-10

UACC-62

MCF-7

4f

C18H20N2O4

24.12 ± 3.13

10.31 ± 0.59

4.10 ± 1.32

4h

C17H17ClN2O2

4.48 ± 2.49

8.48 ± 0.94

4.01 ± 2.26

Parthenolidea

C15H20O3

0.50 ± 1.43

0.89 ± 2.18

0.44 ± 2.02

a

Standard; cells were treated with various concentrations of compounds required to inhibit 50 % of culture growth when exposed for 48 h (giving IC50 values). Each value represents the mean ± standard deviation (SD) of three independent experiments

Conclusion We present synthesis of 4-[(1E)-N-(2-aminoethyl)ethanimidoyl]benzene-1,3-diol derivatives. All synthesized compounds were characterized using elemental analysis and FTIR and UV–Vis spectroscopies. The ethanimidoylbenzene-1,3-diol derivatives were screened for their antimicrobial activity against three Gram-positive and three Gram-negative bacterial strains; compound 4f showed enhanced activities against both Gram-positive and Gram-negative bacterial strains as compared with standard drugs amoxicillin and ciprofloxacin. Antiradical screening of the compounds against DPPH free radical revealed that 4a exhibited the highest inhibition of DPPH radical with IC50 value of 2.63 ± 0.79 lM. In vitro cytotoxicity screening indicated that compounds 4f, 4h, and parthenolide efficiently affected the cell viability of MCF-7 cells with IC50 values of 4.10 ± 1.32, 4.01 ± 2.26, and 0.44 ± 2.02 lM. The results obtained from the biological studies suggest that these compounds might serve as leads for developing novel chemotherapeutic agents for treating stress-induced pathological conditions associated with radical generation and disease control. Acknowledgments The authors would like to extend their sincere appreciation to NRF-Sasol Inzalo Foundation for funding this research. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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