Open Access

Ammonium chloride catalyzed synthesis of novel Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitriles and evaluation of their antimicrobial and anti-breast cancer activities

  • Hossa F. Al-Shareef1,
  • Heba A. Elhady1, 2,
  • Amany H. Aboellil3 and
  • Essam M. Hussein1, 4Email author
SpringerPlus20165:887

https://doi.org/10.1186/s40064-016-2458-0

Received: 6 April 2016

Accepted: 27 May 2016

Published: 24 June 2016

Abstract

Background

Indolinone and spiro-indoline derivatives have been employed in the preparation of different important therapeutic compounds required for treatment of anticonvulsants, antibacterial, Antitubercular, and anticancer activities. Schiff bases have been found to possess various pharmacological activities such as antitubercular, plant growth inhibiting, insecticsidal, central nerve system depressant, antibacterial, anticancer, anti-inflammatory, and antimicrobial. Mannich bases have a variety of biological activities such as antibacterial and antifungal activities.

Results

In this study, a green, rapid and efficient protocol for the synthesis of a new series of Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives using ammonium chloride as a very inexpensive and readily available reagent. The prepared compounds were assessed in vitro for their antimicrobial activity. Also, the cytotoxic activity of the prepared compounds was assessed in vitro against human cells line MCF7 breast cancer.

Conclusion

Good activity was distinguished for Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitriles, with some members recorded higher antimicrobial and anti-breast cancer activities.

Keywords

Ammonium chlorideSchiff basesSpiro[indoline-3,4′-pyran]-3′-carbonitrilesAntimicrobialAnti-breast cancer

Background

The development of eco-friendly and environmentally benign catalytic systems is one of the main themes of modern organic synthesis. Ammonium chloride (NH4Cl) is a very inexpensive and readily available catalyst; it has been reported as a catalyst for the synthesis of various heterocyclic compounds (Shaabani et al. 2003; Dabiri et al. 2009; Fortenberrya et al. 2013; Foroughifarab et al. 2011; Maleki and Salehabadi 2010; Shaabani et al. 2008; Hussein 2015). There are many bioactive molecules which possess various heteroatoms such as nitrogen, sulfur and oxygen, always taken the attention of chemists over the years mainly because of their biological significance. Pyrano derivatives have well-known biological effects, such as analgesic and anti-inflammatory activities (El-Zohry et al. 2008a). Indolinone and spiro-indoline derivatives have possessed broad-spectrum therapeutic activities such as anticonvulsants (Ragavendran et al. 2007; Azam et al. 2009; Sridhar et al. 2002), antibacterial (Rahman et al. 2004; Singh and Luntha 2009; Olomola and Bada 2009), Antitubercular (Sriram et al. 2005), and anticancer activities (Vine et al. 2007; Solomon et al. 2009; Wee et al. 2009). Schiff bases are important compounds owing to their wide range of biological activities and industrial applications. They have been found to possess various pharmacological activities such as antitubercular (Kascheres 2003), plant growth inhibiting (Simunek and Machácek 2010), insecticsidal (Shams et al. 2011; Boyd 1988), central nerve system (CNS) depressant (Drews 2000), antibacterial (Maren 1976; Supuran and Scozzafava 2000), anticancer (Simunek et al. 2007), anti-inflammatory, and antimicrobial (Abbate et al. 2004; Abdel-Mohsen and Hussein 2014). Moreover, Mannich bases are reported to show a variety of biological activities, such as antibacterial and antifungal activities (Singare and Ingle 1976; Huneck et al. 1993; Hussein et al. 2015a). Based on these prior observations, we postulated that a Schiff base containing both indoline and pyran pharmacophores could be very effective for antimicrobial and anticancer activity. In this paper and as a consequence of our previous work on the green synthesis of different spiroheterocyclic (Hussein 2013; Hussein and El-Khawaga 2012; Hussein 2012; El-Zohry et al. 2008b, c, 2009), and bioactive heterocyclic compounds (Hussein et al. 2015b; Hussein and Abdel-Monem 2011), we investigated a novel green and efficient protocol that was developed for the synthesis of some Schiff bases (5al) by the condensation of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives (3ac) with aromatic aldehydes (4ad) using ammonium chloride (10 mol%) in refluxing ethanol as shown in Scheme 2 and Table 1. The antimicrobial and cytotoxic properties of the prepared compounds were screened.
Table 1

Synthesis of the Schiff bases 5al using NH4Cl (10 mol%)

Entry

Producta

R1, R2

R3

Yieldb (%)

1

5a

(C6H5)2

2-OH

92

2

5b

(C6H5)2

4-OCH3

82

3

5c

(C6H5)2

4-Cl

78

4

5d

(C6H5)2

4-NO2

75

5

5e

(C2H5)2

2-OH

88

6

5f

(C2H5)2

4-OCH3

84

7

5g

(C2H5)2

4-Cl

80

8

5h

(C2H5)2

4-NO2

77

9

5i

1-Piperidinyl

2-OH

90

10

5j

1-Piperidinyl

4-OCH3

86

11

5k

1-Piperidinyl

4-Cl

84

12

5l

1-Piperidinyl

4-NO2

74

aReaction conditions: spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives 3ac (10 mmol), aromatic aldehydes 4ad (10 mmol), and NH4Cl (10 mol%) in 10 mL ethanol/reflux, 2 h

bIsolated yields

Results and discussion

Chemistry

Synthesis of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives (3a–c)

The spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives 3ac described in this study were prepared as outlined in Scheme 1. The isatin Mannich bases 2ac were prepared by condensing the active hydrogen atom of istain with formaldehyde and secondary amine namely diphenylamine, diethylamine, and piperidine in ethanol at room temperature as previously reported procedure (Solomon et al. 2009). Compounds 3ac were obtained in good yield via three-component condensation of 2ac, malononitrile and ethyl acetoacetate in refluxed ethanol in presence of catalytic amount of piperidine.
Scheme 1

Synthesis of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives 3ac. Reagents and conditions: a Mannich reaction (HCHO and secondary amine in ethanol, rt, 2–4 h, b malononitrile and ethyl acetoacetate in ethanol/piperidine (one drop), reflux, 5–6 h

Synthesis of target compounds

The Schiff bases 5al were obtained by the condensation of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives 3ac with aromatic aldehydes 4ad using ammonium chloride (10 mol%) in refluxing ethanol (Scheme 2; Table 1).
Scheme 2

Synthesis of Schiff bases 5al

To find out the suitable conditions for the reaction, a series of experiments were performed with the standard reaction of ethyl 2′-amino-3′-cyano-1-((diphenylamino)methyl)-6′-methyl-2-oxospiro[indoline-3,4′-pyran]-5′-carboxylate (3a), salicylaldehyde (4a) as a model reaction (Table 2; Scheme 3).
Table 2

The effect of reaction condition on the synthesis of 5a

Entry

Solventa

Catalystb

Yieldc (%)

1

AcOH

47

2

MeOH

40

3

EtOH

44

4

DMF

AcOH

52

5

EtOH

AcOH

61

6

EtOH

Et3N

71

7

EtOH

Piperidine

71

8

Dioxane

NH4Cl

73

9

DMF

NH4Cl

75

10

MeOH

NH4Cl

87

11

EtOH

NH4Cl

92

The reaction was carried out with ethyl 2′-amino-3′-cyano-1-((diphenylamino)methyl)-6′-methyl-2-oxospiro[indoline-3,4′-pyran]-5′-carboxylate (3a) (10 mmol) and 2-hydroxybenzaldehydes (4a) (10 mmol)

a10 mL solvent/reflux, 2 h

b10 mol%

cIsolated yields

Effect of the reaction conditions

In our initial study, we tried to optimize the model procedure mentioned above by detecting the efficiency of different reaction conditions in the absence and presence of catalysts, such as AcOH, MeOH, EtOH, DMF/AcOH, EtOH/AcOH, EtOH/Et3N, EtOH/piperidine, dioxane/NH4Cl, DMF/NH4Cl, MeOH/NH4Cl, and EtOH/NH4Cl (Scheme 3).
Scheme 3

Model reaction

In each case, the reactants (10 mmol) were allowed together in 10 mL solvent at reflux temperature for 2 h. In the absence of catalyst, the reaction proceeded with comparatively lower reaction yield (Table 2, entries 1–3). DMF/AcOH, EtOH/AcOH, EtOH/Et3N and EtOH/piperidine can push the reaction towards the formation of product in yields of 52, 61, 71, and 71 %, respectively (Table 2, entries 4–7). In the presence of ammonium chloride (NH4Cl) the reaction was possible and the product (5a) was obtained in good yields. Ammonium chloride was used in different reaction media such as dioxane, DMF, methanol and ethanol (Table 2, entries 8–11). The best results were obtained when NH4Cl was used as catalyst in ethanol as reaction medium, which provided a yield of 92 %.

Evaluation of catalytic activity of ammonium chloride

To determine the appropriate concentration of the catalyst used, we investigated the model reaction at different concentrations of NH4Cl (5, 10, 15, 20, and 25 mol%). The product was formed in 80, 92, 92, 89, and 85 % yield, respectively (Table 3). This indicates that 10 mol% NH4Cl is sufficient to carry out the reaction smoothly.
Table 3

Evaluation of catalytic activity of NH4Cl in the synthesis of 5a

Entry

Amount of NH4Cl (mol%)

Yielda (%)

1

5

80

2

10

92

3

15

92

4

20

89

5

25

85

The reaction was carried out with ethyl 2′-amino-3′-cyano-1-((diphenylamino)methyl)-6′-methyl-2-oxospiro[indoline-3,4′-pyran]-5′-carboxylate (3a) (10 mmol) and 2-hydroxybenzaldehydes (4a) (10 mmol), and NH4Cl in 10 mL ethanol at refluxing temperature/2 h

aIsolated yield of (5a)

The structures of the isolated new products 5a–l were deduced by analyzing their physical and spectroscopic data, such as the data obtained using IR, 1H NMR, and 13C NMR spectroscopy. Taking 5a as an example, broad absorption band at 3356 cm−1 for OH group, sharp absorption band at 2210 cm−1 for CN group, and two absorption band at 1735, 1620 cm−1 for two C=O groups were observed in the IR spectrum with absence of absorption bands at 3350, 3260 cm−1 which corresponding to NH2 group. The 1H NMR spectrum showed the presence of triplet and quartet signals at 1.28, and 3.85 for ethyl protons, as well as, four singlet signals at δ = 2.28, 5.30, 8.15, and 10.38 ppm for the methyl, methylene, methane, and OH protons, respectively. In the 13C NMR spectrum, the quaternary spiro carbon typically appeared at δ = 48.9 ppm. The nitrile and two carbonyl carbons resonated at 117.4, 164.4, and 178.5 ppm, respectively.

Biological activity

Antimicrobial activity

In view of biological significance, it was studied the synthesized some spiro-indoline derivatives as previous, to get the activities of the potent compounds and evaluated their potential in vitro as antibacterial, antifungal and antitumor activities.

Antimicrobial activities of all the synthesized Schiff bases 5al were done by cup-plate agar diffusion method. The compounds were prepared in DMSO and evaluated them for their in vitro antibacterial and antifungal activities against Bacillus subtilis and Fusarium moniliforme respectively. The bacterial isolate was grown on nutrient agar (37 °C, 24 h) the fungus was grown on potato dextrose agar plates (26 °C, 48–72 h). The results were noted by the presence of clear zone of inhibition around the active compounds (Table 4).
Table 4

Biological activities of the synthesized spiro-indoline derivatives 3ac and 5al

Compounds

R1, R2

R3

IC50

Inhibition zone (mm)

B. subtilis

Inhibition zone (mm)

F. moniliforme

3a

(C6H5)2

>50

12 ± 2

Nill

3b

(C2H5)2

>50

24 ± 2

15 ± 2

3c

Piperidinyl

18.8

19 ± 4

2 ± 1

5a

(C6H5)2

2-OH

44.5

20 ± 2

12 ± 1

5b

(C6H5)2

4-OCH3

23.9

Nill

Nill

5c

(C6H5)2

4-Cl

>50

24 ± 2

10 ± 2

5d

(C6H5)2

4-NO2

25.0

18 ± 3

6 ± 1

5e

(C2H5)2

2-OH

25.0

18 ± 1

4 ± 1

5f

(C2H5)2

4-OCH3

>50

Nill

Nill

5g

(C2H5)2

4-Cl

11.9

Nill

Nill

5h

(C2H5)2

4-NO2

20.9

25 ± 3

10 ± 1

5i

Piperidinyl

2-OH

>50

19 ± 3

Nill

5j

Piperidinyl

4-OCH3

18.8

16 ± 2

4 ± 2

5k

Piperidinyl

4-Cl

>50

12 ± 1

Nill

5l

Piperidinyl

4-NO2

38.3

4 ± 1

Nill

All the synthesized compounds 3ac and 5al were tested for in vitro antibacterial activity by inhibition zone method against the reference compound amoxicillin (20 mm). It has been observed that all the compounds tested showed mild to moderate activity against tested bacterium but 5f, 5g and 3a. The antifungal activity of the compounds was studied with F. Moniliforme. The results are summarized in Table 4. Fluconazole has been used as reference for inhibitory activity (18 mm) against fungi and some tested compounds showed lesser activity to standard against the tested fungi. While the others showed no antifungal activities against the fungus.

In vitro anticancer activity

Antitumor activities were found moderate effective as screened for in vitro cytotoxicity activity against human cancer cells line MCF7 breast cancer (Table 5). Although the positive impact of each of the synthesized compound conducted toxicity in cells, some lost IC50 in the concentrations used. Viewing of the results, the IC50 required was higher than that of the reference compound (3.8 µg/mL) used in the analysis. There are no significant differences between the results of the synthetic chemical compounds compared to the reference compound, where statistically significant differences is the numerical value, and therefore all synthesized compounds located with reference drug in one hand.
Table 5

Cytotoxicity activity of spiro-indoline derivatives 3ac and 5al at different concentrations (0.0, 5.0, 12.5, 25 and 50 µg/mL) of some synthesized compounds and reference drug against human cancer cells line MCF7 breast cancer

CONC

DOX

3a

5a

5b

5c

5d

3b

5e

5f

5g

5h

3c

5i

5j

5k

5l

0.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

5.0

0.361

0.836

0.758

0.867

0.930

0.977

0.786

0.911

0.764

0.853

0.978

0.799

0.974

0.688

0.824

0.906

12.5

0.385

0.738

0.479

0.633

0.837

0.740

0.542

0.876

0.643

0.728

0.669

0.642

0.752

0.637

0.588

0.803

25.0

0.332

0.653

0.340

0.498

0.698

0.619

0.462

0.467

0.547

0.560

0.628

0.437

0.829

0.498

0.414

0.725

50.0

0.299

0.631

0.353

0.502

0.442

0.619

0.444

0.416

0.608

0.453

0.564

0.526

0.772

0.479

0.368

0.829

SD

0.024

0.009

0.013

0.025

0.020

0.016

0.034

0.039

0.120

0.022

0.020

0.025

0.010

0.013

0.058

0.009

It is worth mentioning, that the curve of the compound 5c only showed clear a straight line. A high concentration compared to the control has shown. Theoretically an expectation of the IC50 may be located on the curve. Statistically the range of lethal concentrations IC50 may be at about 70 µg/mL, concentration that’s when kills ninety percent of the living cells.

The biological activities of 3ac and its derivatives 5al were summarized in (Fig. 1). Only cell toxicity of 3a did not record (>50 µg/mL) and antimicrobial activities of 5b and antifungal activity of 5c were not detected. The compounds 3b and 5f just showed IC50 exceed 50 µg/mL but antimicrobial activities did not detect by 5g and 5f only. IC50 did not show in the tested concentrations of 5i and 5k. Also antifungal activities did not record in 5i, 5k and 5l as shown in (Fig. 1).
Fig. 1

Biological activities of spiro-indoline derivatives of 3ac and 5al

Conclusion

The authors have developed a green, rapid and efficient protocol for the synthesis of a new series of Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives using ammonium chloride as a very inexpensive and readily available reagent. The prepared compounds were assessed in vitro for their antibacterial activity against B. subtilis as well as antifungal activity against F. moniliforme. Also, the cytotoxic activity of the prepared compounds was assessed in vitro against human cells line MCF7 breast cancer.

Experimental

Chemistry

General methods

The IR spectra of the synthesized compounds were taken on a Shimazu FT spectrometer with a device of singly perturbed internal reflection. 1HNMR spectra (in DMSO-d6) were recorded on Bruker Ac-400 ultra-shield NMR spectrometer at 400 MHz, using TMS as internal standard. The 13C NMR (100 MHz) spectra were run in dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were obtained on a Joel JMSD-300 spectrometer operating at 70 eV. The elemental analysis was carried out on a perkin-Elmer C, H, N analyzer. Melting points were determined in open capillaries on a Gallenkemp melting point apparatus and are uncorrected.
  • Synthesis of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives 3ac

General procedure

A mixture of 1-((diphenylamino)methyl)indoline-2,3-dione (2a) (3.28 g, 10 mmol) and malononitrile (0.66 g, 10 mmol) was dissolved in 20 mL absolute ethanol and stirred for 30 min. Then ethyl acetoacetate (1.30 g, 10 mmol) was added in the presence of piperidine (one drop) and the reaction mixture was heated under reflux with stirring for 6 h. Then cooled and the formed crystals was collected by filtration. Dried and recrystallized for a proper solvent.

Ethyl-6-amino-5-cyano-1′-((diphenylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (3a)

White crystals (ethanol), yield 75 %, mp 225–227 °C. IR (KBr): 3260, 3150 (NH2), 2185 (CN), 1724 (C=O), 1670 (C=O). 1H NMR: δ = 1.25 (t, 3H, CH3), 2.29 (s, 3H, CH3), 3.98–4.00 (q, 2H, CH2), 5.41 (s, 2H, CH2), 6.80 (s, 2H, NH2, D2O-exchangeable), 6.78–7.71 (m, 14H, Ar–H) ppm. 13C NMR: δ = 13.6 (CH3), 18.6 (CH3), 49.0 (C-spiro), 56.57, 60.3 (CH2), 76.7 (CH2), 100.4, 118.5 (CN), 121.5, 121.9, 123.0, 123.4, 125.1, 128.6, 128.7, 142.1, 151.0, 156.5, 159.2, 164.6 (C=O), 166.7 (C=O) ppm. MS: m/z (%) = 506.05 (M+, 45), 169.11 (100). Anal. Calcd. For C30H26N4O4 (506.55): C, 71.13; H, 5.17; N, 11.06. Found: C, 71.17; H, 5.08; N, 10.89.

Ethyl-6-amino-5-cyano-1′-((diethylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (3b)

As pale yellow crystals (dioxane), yield 90 %, mp 140–142 °C. IR (KBr): 3270, 3190 (NH2), 2190 (CN), 1722 (C=O), 1660, (C=O). 1H NMR: δ = 1.10 (t, 6H, CH3), 1.22 (t, 3H, CH3), 1.72 (s, 3H, CH3), 2.48 (q, 4H, 2CH2), 4.18–4.20 (q, 2H, OCH2), 4.39 (s, 2H, CH2), 6.79 (s, 2H, NH2, D2O-exchangeable), 6.92–7.76 (m, 4H, Ar–H) ppm. MS: m/z (%) = 410.19 (M+, 23), 133 (100). Anal. Calcd. For C22H26N4O4 (410.47): C, 64.37; H, 6.38; N, 13.65. Found: C, 64.42; H, 6.37; N, 13.59.

Ethyl-6-amino-5-cyano-1′-(piperidin-1-ylmethyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (3c)
As pale yellow crystals (ethanol), yield 87 %, mp 189–190 °C. IR (KBr): 3240, 3100 (NH2), 2170 (CN), 1715 (C=O), 1665 (C=O). 1H NMR: δ = 1.30 (t, 3H, CH3), 1.57–1.59 (m, 6H, 3CH2), 1.74 (s, 3H, CH3), 2.60 (t, 4H, 2CH2), 4.19–4.21 (q, 2H, CH2), 4.31 (s, 2H, CH2), 6.85 (s, 2H, NH2, D2O-exchangeable), 6.87–7.26 (m, 4H, Ar–H) ppm. 13C NMR: δ = 13.9 (CH3), 14.1 (CH3), 26.0, 26.3, 48.1 (C-spiro), 52.8 (C-pipredine), 53.9, 61.6 (CH2), 76.8 (CH2), 106.1, 120.0 (CN), 122.9, 123.8, 126.5, 127.5, 131.2, 138.0, 151.5, 156.2, 165.44 (C=O), 167.3 (C=O). MS: m/z (%) = 422.15 (M+, 23), 142 (100). Anal. Calcd. For C23H26N4O4 (422.48): C, 65.39; H, 6.20; N, 13.26. Found: C, 65.36; H, 6.18; N, 13.19.
  • General procedure for the synthesis of the Schiff bases 5a–l

General procedure

To a solution of spiro[indoline-3,4′-pyran]-3′-carbonitrile derivative 3a (0.51 g, 1 mmol) in absolute ethanol (10 mL), corresponding aromatic aldehyde (1 mmol) was added. Then NH4Cl (5.35 mg, 10 mol %) was added and the reaction mixture was refluxed for 2 h (monitored by TLC). After completion of the reaction, cold water (15–25 mL) was added to the reaction mixture. The solid product was filtered, washed with cold water, dried, and recrystallized from proper solvents.

Ethyl-6-(2-hydroxybenzylidenamino)-5-cyano-1′-((diphenylamino)methyl)-2methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5a)

As yellow crystals (ethanol), mp 215–217 °C. IR (KBr): 3356 (br. OH), 2210 (CN), 1735 (C=O), 1620 (C=O). 1H NMR (DMSO-d6): δ = 0.85 (t, 3H, CH3), 2.53 (s, 3H, CH3), 3.80 (s, 2H, CH2), 3.82–3.85 (q, 2H, CH2), 6.80–7.21 (m, 18H, Ar–H), 10.27 (s, 1H, OH), 10.38 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 12.9 (CH3), 18.5 (CH3), 48.9 (C-spiro), 60.2 (CH2), 74.9 (CH2), 104.6, 111.0, 117.4 (CN), 123.3, 125.7, 127.6, 127.9, 128.4, 129.0, 131.1, 131.6, 134.5, 142.1, 144.2, 158.4, 158.9, 163.7 (N=CH), 164.4 (C=O), 178.5 (C=O). MS: m/z (%) = 610.08 (M+, 10), 262.10 (100). Anal. Calcd. For C37H30N4O5 (610.66): C, 72.77; H, 4.95; N, 9.17. Found: C, 72.82; H, 4.76; N, 9.14.

Ethyl-6-(4-methoxybenzylidenamino)-5-cyano-1′-((diphenylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5b)

As pale yellow crystals (ethanol), mp 212–214 °C. IR (KBr): 2180 (CN), 1740 (C=O), 1625 (C=O). 1H NMR (DMSO-d6): δ = 0.80 (t, 3H, CH3), 2.33 (s, 3H, CH3), 3.32 (s, 3H, OCH3), 3.81–3.83 (q, 2H, CH2), 3.89 (s, 2H, CH2), 6.80–7.90 (m, 18H, Ar–H), 10.38 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 12.9 (CH3), 18.5 (CH3), 48.9 (C-spiro), 55.6 (CH3), 56.6, 60.2 (CH2), 75.4 (CH2), 104.6, 117.4 (CN), 119.3, 121.5, 122.9, 123.8, 125.1, 127.9, 128.4, 129.1, 130.3, 131.7, 134.5, 142.1 (C-aromatic), 158.3 (C-pyrane), 164.6 (N=CH), 169.4 (C=O), 178.5(C=O). MS: m/z (%) = 624.20 (M+, 13), 252.51 (100). Anal. Calcd. For C38H32N4O5 (624.68): C, 73.06; H, 5.16; N, 8.97. Found: C, 72.91; H, 4.90; N, 9.02.

Ethyl-6-(4-chlorobenzylidenamino)-5-cyano-1′-((diphenylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5c)

As pale brown crystals (ethanol), mp 230–232 °C. IR (KBr): 2180 (CN), 1742 (C=O), 1635 (C=O). 1H NMR (DMSO-d6): δ = 0.80 (t, 3H, CH3), 2.33 (s, 3H, CH3), 3.80 (s, 2H, CH2), 3.82–3.85 (q, 2H, CH2), 6.80–7.71 (m, 18H, Ar–H), 10.38 (s, 1H, N=CH) ppm. MS: m/z (%) = 628.61 (M+, 16), 262.11 (100). Anal. Calcd. For C37H29ClN4O4 (629.10): C, 70.64; H, 4.65; Cl, 5.64; N, 8.91. Found: C, 70.75; H, 4.48; Cl, 5.50; N, 8.93.

Ethyl-6-(4-nitrobenzylidenamino)-5-cyano-1′-((diphenylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5d)

As pale yellow crystals (ethanol), mp 235–237 °C. IR (KBr): 2200 (CN), 1740 (C=O), 1630 (C=O). 1H NMR (DMSO-d6): δ = 0.80 (t, 3H, CH3), 2.30 (s, 3H, CH3), 3.32 (s, 2H, CH2), 3.81–3.83 (q, 2H, CH2), 6.80–8.24 (m, 18H, Ar–H), 10.37 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 12.90 (CH3), 18.46 (CH3), 48.91 (C-spiro), 61.12 (CH2), 75.48 (CH2), 104.64, 117.35 (CN), 118.45, 121.74, 123.35, 125.75, 127.95, 128.52, 129.21, 131.77, 134.47, 142.00, 158.87 (C-pyrane), 163.65 (N=CH), 166.41 (C=O), 178.45 (C=O). MS: m/z (%) = 639.20 (M+, 13), 169.69 (100). Anal. Calcd. For C37H29N5O6 (639.66): C, 69.47; H, 4.57; N, 10.95. Found: C, 69.44; H, 4.48; N, 10.81.

Ethyl-6-(2-hydroxybenzylidenamino)-5-cyano-1′-((diethylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5e)

As pale yellow crystals (ethanol), mp 125–127 °C. IR (KBr): 3414 (br. OH), 2191 (CN), 1724 (C=O), 1620 (C=O). 1H NMR (DMSO-d6): δ = 1.15 (t, 6H, 2CH3), 1.24 (t, 3H, CH3), 2.27 (s, 3H, CH3), 2.39 (q, 4H, 2CH2), 3.86 (s, 2H, CH2), 4.32–4.39 (m, 4H, 2CH2), 6.84–7.80 (m, 8H, Ar–H), 9.85 (s, 1H, N=CH), 12.50 (s, 1H, OH, exchangeable with D2O) ppm. 13C NMR (DMSO-d6): δ = 13.8 (2CH3), 14.1 (CH3), 14.2 (CH3), 49.0 (C spiro), 61.4 (2CH2), 61.6 (CH2), 67.7 (CH2), 100.2, 110.9 (CN), 123.1, 123.4, 123.7, 124.3, 125.7, 127.7, 129.2, 130.4, 131.2, 134.6, 140.1, 147.8, 162.2, 166.3 (N=CH), 167.3 (C=O), 170.3 (C=O). MS: m/z (%) = 514.08 (M+, 41), 262.14 (100). Anal. Calcd. For C29H30N4O5 (514.57): C, 67.69; H, 5.88; N, 10.89. Found: C, 67.42; H, 5.62; N, 11.01.

Ethyl-6-(4-methoxybenzylidenamino)-5-cyano-1′-((diethylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5f)

As pale yellow crystals (petroleum ether 60–80), mp 70–72 °C. IR (KBr): 2160 (CN), 1720 (C=O), 1650 (C=O). 1H NMR (DMSO-d6): δ = 0.84–1.24 (m, 6H, 2CH3), 1.41 (t, 3H, CH3), 2.83 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.19–4.39 (m, 8H, 3CH2), 6.85–7.83 (m, 8H, Ar–H), 9.87 (s, 1H, N=CH) ppm. MS: m/z (%) = 528.21 (M+, 20), 234.05 (100). Anal. Calcd. For C30H32N4O5 (528.60): C, 68.17; H, 6.10; N, 10.60. Found: C, 68.21; H, 6.01; N, 10.56.

Ethyl-6-(4-chlorobenzylidenamino)-5-cyano-1′-((diethylamino)methyl)-2-methyl-2′-one-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5g)

As pale yellow crystals (diethyl ether), mp 90–92 °C. IR (KBr): 2190 (CN), 1730 (C=O), 1655 (C=O). 1H NMR (DMSO-d6): δ = 0.91–1.32 (m, 6H, 2CH3), 1.39 (t, 3H, CH3), 2.83 (s, 3H, CH3), 4.19–4.39 (m, 6H, 3CH2), 5.51 (s, 2H, CH2), 7.03–7.65 (m, 8H, Ar–H), 9.88 (s, 1H, N=CH) ppm. MS: m/z (%) = 532.09 (M+, 31), 146.00 (100). Anal. Calcd. For C29H29ClN4O4 (533.02): C, 65.35; H, 5.48; Cl, 6.65; N, 10.51. Found: C, 65.45; H, 5.73; Cl, 6.61; N, 10.47.

Ethyl-6-(4-nitrobezylidenamino)-5-cyano-1′-((diethylamino)methyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5h)

As pale yellow crystals (diethyl ether), m.p 85–87 °C. IR (KBr): 2170 (CN), 1724 (C=O), 1650 (C=O). 1H NMR (DMSO-d6): δ = 0.88–1.31 (m, 6H, 2CH3), 1.41 (t, 3H, CH3), 2.83 (s, 3H, CH3), 4.21–4.36 (m, 6H, 3CH2), 4.38 (s, 2H, CH2), 7.12–7.85 (m, 8H, Ar–H), 9.89 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 13.9 (2CH3), 14.2 (CH3), 15.1 (CH3), 49.1 (C spiro), 61.3 (2CH2), 61.6 (CH2), 76.7 (CH2), 100.1, 117.3 (CN), 123.1, 123.4, 123.7, 124.3, 125.7, 127.7, 129.2, 130.4, 131.2, 134.6, 147.9, 162.2, 166.0 (N=CH), 167.3 (C=O), 170.0 (C=O). MS: m/z (%) = 543.01 (M+, 15), 234 (100). Anal. Calcd. For C29H29N5O6 (543.57): C, 64.08; H, 5.38; N, 12.88. Found: C, 64.04; H, 5.64; N, 12.84.

Ethyl-6-(2-hydroxybenzylidenamino)-5-cyano-1′-(piperidin-1-ylmethyl)-2methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5i)

As pale yellow crystals (diethyl ether), mp 110–112 °C. IR (KBr): 3455 (br. OH), 2210 (CN), 1753 (C=O), 1634 (C=O). 1H NMR (DMSO-d6): δ = 090–1.13 (m, 6H, 3CH2), 1.16 (t, 3H, CH3), 2.67 (t, 4H, 2CH2), 3.46 (s, 3H, CH3), 3.64 (s, 2H, CH2), 4.15–4.31 (q, 2H, CH2), 6.81–7.87 (m, 8H, Ar–H), 9.96 (s, 1H, N=CH), 10.51 (s, 1H, OH) ppm. 13C NMR (DMSO-d6): δ = 13.9 (CH3), 14.1 (CH3), 26.2, 26.6, 52.7 (CH2-piperidine), 49.0 (C-spiro), 53.9, 61.1, 61.6 (CH2), 76.7 (CH2), 109.2, 117.6 (CN), 119.8, 120.1, 121.5, 123.0, 123.8, 124.6, 126.5, 131.1, 133.7, 137.0, 140.6 (C-aromatic), 156.9, 163.5 (N=CH), 165.4 (C=O), 167.2 (C=O). MS: m/z (%) = 526.09 (M+, 5), 234.22 (100). Anal. Calcd. For C30H30N4O5 (526.58): C, 68.43; H, 5.74; N, 10.64. Found: C, 68.40; H, 5.71; N, 10.36.

Ethyl-6-(4-methoxybenzylidenamino)-5-cyano-1′-(piperidin-1-ylmethyl)-2-methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5j)

As brown crystals (diethyl ether), mp 114–117 °C. IR (KBr): 2210 (CN), 1742 (C=O), 1631 (C=O). 1H NMR (DMSO-d6): δ = 0.90–1.13 (m, 6H, 3CH2), 1.16 (t, 3H, CH3), 2.67 (t, 4H, 2CH2), 3.46 (s, 3H, CH3), 3.64 (s, 2H, CH2), 3.75 (s, 3H, OCH3), 4.15–4.30 (q, 2H, CH2), 6.86–7.89 (m, 8H, Ar–H), 9.95 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 14.0 (CH3), 14.1 (CH3), 25.9, 26.1 (CH2), 48.4 (C-spiro), 52.1 (CH2), 53.4 (CH3), 55.8, 61.6 (CH2), 76.8 (CH2), 106.0, 120.0 (CN), 121.1, 122.6, 123.3, 124.8, 125.9, 126.3, 128.3, 129.7, 131.9, 156.2, 164.3, 164.7 (N=CH), 167.5 (C=O), 169.9 (C=O). MS: m/z (%) = 540.20 (M+, 31), 299 (100). Anal. Calcd. For C31H32N4O5 (540.61): C, 68.87; H, 5.97; N, 10.36. Found: C, 68.63; H, 5.69; N, 10.32.

Ethyl-6-(4-chlorobenzylidenamino)-5-cyano-1′-(piperidin-1-ylmethyl)-2methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5k)

As pale yellow crystals (diethyl ether), mp 140–142 °C. IR (KBr): 2219 (CN), 1725 (C=O), 1622 (C=O). 1H NMR (DMSO-d6): δ = 1.13–1.17 (m, 6H, 3CH2), 1.25 (t, 3H, CH3), 2.65 (t, 4H, 2CH2), 3.46 (s, 3H, CH3), 3.65 (s, 2H, CH2), 4.15–4.31 (q, 2H, CH2), 6.81–7.85 (m, 8H, Ar–H), 9.96 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6): δ = 14.1 (CH3), 14.2 (CH3), 26.3, 26.6 (CH2), 49.1 (C-spiro), 53.9 (CH2), 61.3, 61.6 (CH2), 76.7 (CH2), 106.2, 120.0 (CN), 121.5, 123.0, 124.5, 125.7, 126.5, 128.3, 128.7, 130.9, 131.1, 134.7 (C-aromatic), 156.2, 163.7, 165.7 (N=CH), 167.4 (C=O), 174.1 (C=O). MS: m/z (%) = 544.10 (M+, 11), 261.15 (100). Anal. Calcd. For C30H29ClN4O4 (544.03): C, 66.11; H, 5.36; Cl, 6.50; N, 10.28. Found: C, 66.13; H, 5.30; Cl, 6.47; N, 10.32.

Ethyl-6-(4-nitrobenzylidenamino)-5-cyano-1′-(piperidin-1-ylmethyl)-2methyl-2′-oxo-4H-spiro[pyran-4,3′-indoline]-3-carboxylate (5l)

As pale brown crystals (ethanol), mp 160–162 °C. IR (KBr): 2210 (CN), 1732 (C=O), 1630 (C=O). 1H NMR (DMSO-d6): δ = 1.15–1.17 (m, 6H, 3CH2), 1.28 (t, 3H, CH3), 2.15 (t, 4H, 2CH2), 3.58 (s, 3H, CH3), 4.15–4.35 (q, 2H, CH2), 5.50 (s, 2H, CH2), 6.98–7.37 (m, 8H, Ar–H), 8.39 (s, 1H, N=CH) ppm. MS: m/z (%) = 555.12 (M+, 25), 205 (100). Anal. Calcd. For C30H29N5O6 (555.58): C, 64.85; H, 5.26; N, 12.61. Found: C, 64.82; H, 5.28; N, 12.59.

Biological screening

Antibacterial activity

The newly synthesized spiro-indoline derivatives 3ac and 5al were screened for their antibacterial activity against bacterial isolate namely B. subtilis by inhibition zone method against the reference compound amoxicillin (20 mm). The bacterial subcultures (18–24 h grown) were added to sterilize nutrient agar medium and shaken thoroughly to ensure uniform distribution of organism throughout the medium. In sterilized Petri dishes containing about 20 mL of the medium, wells were made with a sterile cork borer and were filled with 0.1 mL of respective solution. Then, the Petri dishes were kept for incubation in an inverted position for 24–48 h at 37 °C in an incubator. When growth inhibition zones were developed, diameter (in mm) was measured and compared with that of amoxicillin.

Antifungal activity

The newly synthesized spiro-indoline derivatives 3ac and 5al were screened for their antifungal activity against fungus F. moniliforme at the concentration levels of 50 µg/mL by inhibition zone method. Fluconazole has been used as reference for inhibitory activity (18 mm) against fungi. To the sterilized potato dextrose agar medium, subculture of fungus were added and shaken thoroughly to ensure uniform distribution and incubated for 72 h. Then, this was poured into sterilized and labeled Petri dishes and allowed to solidify. Wells were made in each plate by a cork borer. Each well was filled with 0.1 mL of test solution and the other with respective concentrations of standard dilutions. The plates were left 2–3 h for diffusion and incubated at 37 °C for 24 h. The diameter of the zones of growth inhibition was measured and compared with that of standard. The solutions of required concentration (50 μg/mL) of test compounds were prepared by dissolving the compounds in DMSO.

Anticancer activity

Breast cancer cell line (MCF7) as human tumor was used in this study. The cytotoxicity was measured in vitro for the newly synthesized compounds assay using the method of Philip et al. (1990). The in vitro anticancer.

Screening was done by the pharmacology unit at Pharmacology unit, Cancer biology department, the National Cancer Institute, Cairo University. Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the compound(s) to allow attachment of cell to the wall of the plate. Tested compounds were dissolved in dimethyl sulfoxide (DMSO). Different concentrations of the compound under test (0.0, 5.0, 12.5, 25.0 and 50.0 µg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C and in atmosphere of 5 % CO2. After 48 h. cells were fixed, washed and stained for 30 min with 0.4 % (W/V) SRB dissolved in 1 % acetic acid. Excess unbound dye was removed by four washes with 1 % acetic acid and attached stain was recovered with Tris–EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve for breast tumor cell line after the specified time. The molar concentration required for 50 % inhibition of cell viability (IC50) was calculated and compared to the reference drug Doxorubicin (CAS, 25316-40-9). The surviving fractions were expressed as means and the results are given in Table 5.

Declarations

Authors’ contributions

HFA-S analyzed the data and shared in experimental section; HAE analyzed the data, shared in the experimental section and shared in writing the manuscript; AHA performed the biological activity and shared in writing manuscript. EMH designed the research, shared in the experimental work, shared in writing the manuscript, and revised the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Department of Chemistry, Faculty of Applied Sciences, Umm Al-Qura University
(2)
Department of Chemistry, Faculty of Science (Girl’s), Al-Azhar University
(3)
Department of Botany and Microbiology, Faculty of Science, Cairo University
(4)
Department of Chemistry, Faculty of Science, Assiut University

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