Journal of Chemistry

Volume 2013 (2013), Article ID 478635, 9 pages

http://dx.doi.org/10.1155/2013/478635

Research Article

Synthesis and Biological Evaluation of New N,N′-Bis(1-substituted-ethylidene)-ethane1,2-diamine and Their Acetyl and Trifluoroacetyl Derivatives as Cytotoxic and Antimicrobial Agents

Khalid A. Khan,¹ Hassan M. Faidallah,¹ and Abdullah M. Asiri1,2

¹Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

²Center of Excellence for Advanced Materials Research, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

Received 27 May 2013; Accepted 1 August 2013

Academic Editor: Svetlana Ibric

Copyright © 2013 Khalid A. Khan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Several Schiff bases 2 have been prepared by the condensation of various methyl ketones 1 with 1,2-ethylenediamine. Subsequent acylation of these Schiff bases with trifluoroacetic anhydride in THF yields the corresponding trifluoroacetyl derivatives 3. However, o-hydroxy- and o-methoxy-substituted derivatives yielded substituted-trifluoromethylchroman-4-ones. Additionally, Schiff bases when reacted with acetic anhydride afforded the C-acetylated derivatives 4. The structures of the isolated compounds were fully determined by spectral methods. Preliminary biological screening of the prepared compounds revealed significant cytotoxic and antimicrobial activities.

1. Introduction

Schiff bases and their complexes have been studied for their interesting and important properties, for example, their ability to reversibly bind with oxygen [1], their catalytic activity in hydrogenation of olefins [2], their photochromic properties [3, 4], and their complexing ability towards some toxic metals [5]. The high affinity for the chelation of the Schiff bases towards the transition metal ions is utilized in preparing their solid complexes.

Recently there has been a considerable interest in the chemistry of hydrazine and hydrazone compounds because of their potential pharmacological applications [6, 7]. They also form basic units in certain dyes [8]. The presence of azomethine and sulfonamide functional group is responsible for antimicrobial activity, which can be altered depending upon the type of substituent present on the aromatic rings [9]. In azomethine derivatives, the C=N linkage is essential for biological activity owing to their excellent chelating properties. Several azomethines were reported to possess remarkable antibacterial, antifungal, anticancer, and diuretic activities [10, 11].

Fluorine plays a significant role in altering the physico-chemical characteristics and thereby influencing the electronic, lipophilic, and steric parameters of the organic molecules. Such modulation can profoundly affect the physical and pharmacokinetic properties of drug molecules. Schiff’s base of ethylenediamine with 2-furaldehyde and thiophenecarboxaldehyde has been reported [12] but its trifluoroacetyl analogs have not been prepared yet. Trifluoroacetyl groups have been recognized as general binding elements for biological targets with polar active sites. It has also been shown that selective organofluorine interactions with protein residues can be used to substantially enhance protein-ligand binding affinity and selectivity [13]. Compounds containing the trifluoroacetyl group were found to show tumor specific cytotoxic [14] and antimicrobial [15] activities. However, these residues are fully hydrated in aqueous solution, and these hydrates, as part of biomimetic ligands, are good entities to bind to the active sites [16]. In an extension of our previous study, in the area of synthesis of drugs, semidrugs, and bioactive compounds [17–19], we report the synthesis, characterization, and bacterial growth inhibitory and anticancer activities of trifluoroacetyl derivatives of the Schiff bases of ethylenediamine. An attempt has also been made to compare the antibacterial and anticancer activities of fluorinated and the corresponding nonfluorinated analogs.

2. Experimental

2.1. Chemistry

Melting points were determined in open glass capillaries, on a Gallenkamp melting point apparatus, and were uncorrected. The infrared (IR) spectra were recorded on Perkin-Elmer 297 infrared spectrophotometer, using the NaCl plate technique. Elemental analyses were performed at the Microanalytical Unit, Faculty of Science, King Abdul-Aziz University, Jeddah, Saudi Arabia, and the found values were within of the theoretical values (Table 1). The ¹H-NMR spectra were recorded on a Varian EM 360 spectrometer, using tetramethylsilane as the internal standard (Chemical shifts in δ, ppm). Splitting patterns were designated as follows: s: singlet, d: doublet, and m: multiplet (Tables 2 and 3). Followup of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminium sheets (Type 60 F254, Merck), and the spots were detected by the exposure to UV-lamp at λ 254.

Table 1: Physical and analytical data of compounds 2–5.

Compd.
R
Yield
m.p.
Mol. formula
Calc. %
Found %

(%)
(°C)
C
H
N
C
H
N

2a
C₆H₅
80
95–98
C₁₈H₂₀N₂
81.78
7.63
10.60
81.80
7.61
10.62

2b
4-BrC₆H₄
86
192–194
C₁₈H₁₈Br₂N₂
51.21
4.30
6.64
51.22
4.38
6.60

2c
2-HOC₆H₄
92
190–192
C₁₈H₂₀O₂N₂
72.95
6.80
9.45
73.04
6.88
9.48

2d
2-CH₃OC₆H
76
90–92
C₂₀H₂₄N₂O₂
74.04
7.46
8.64
74.12
7.50
8.62

2e
2-Thienyl
84
142-143
C₁₄H₁₆N₂S₂
60.83
5.83
10.13
60.90
5.91
10.21

2f
3-Thienyl
82
132-133
C₁₄H₁₆N₂S₂
60.83
5.83
10.13
60.88
5.87
10.19

2g
2-Furyl
74
113–115
C₁₄H₁₆N₂O₂
68.83
6.60
11.47
68.85
6.62
11.40

2h
2-Pyridyl
72
98–100
C₁₆H₁₈N₄
72.15
6.81
21.04
72.21
6.73
21.12

2i
4-CH₃C₆H₄
88
110–112
C₂₀H₂₄N₂
82.15
8.27
9.58
82.06
8.32
9.61

2j
4-CH₃OC₆H₄
86
148–150
C₂₀H₂₄N₂O₂
74.04
7.46
8.64
74.12
7.51
8.77

2k
3,4-(CH₃O)₂C₆H₄
82
108–110
C₂₂H₂₈N₂O₄
68.73
7.34
7.29
68.81
7.48
7.33

3a
C₆H₅
72
252–254
C₂₂H₁₈F₆N₂O₂
57.90
3.98
6.14
58.03
4.02
6.19

3b
4-BrC₆H₄
76
180–182
C₂₂H₁₆Br₂F₆N₂O₂
43.02
2.63
4.56
43.11
2.64
4.60

3c
2-Thienyl
74
158-159
C₁₈H₁₄F₆N₂O₂S₂
46.15
3.01
5.98
46.18
3.12
5.82

3d
3-Thienyl
72
163–165
C₁₈H₁₄F₆N₂O₂S₂
46.15
3.01
5.98
46.14
3.04
6.01

3e
2-Furyl
69
180–182
C₁₈H₁₄F₆N₂O₄
49.55
3.23
6.42
49.65
3.28
6.47

3f
2-Pyridyl
67
196–198
C₂₀H₁₆F₆N₄O₂
52.41
3.52
12.22
52.52
3.61
12.18

3g
4-CH₃C₆H₄
78
188–190
C₂₄H₂₂F₆N₂O₂
59.50
4.58
5.78
59.55
4.56
5.81

3h
4-CH₃OC₆H₄
74
196–198
C₂₄H₂₂F₆N₂O₄
55.82
4.29
5.42
55.90
4.38
5.60

3i
3,4-(CH₃O)₂C₆H₄
70
205–208
C₂₆H₂₆F₆N₂O₆
54.17
4.55
19.77
54.28
4.60
19.91

4a
C₆H₅
76
150–152
C₂₂H₂₄N₂O₂
75.83
6.94
8.04
75.94
7.10
8.13

4b
4-BrC₆H₄
78
169–171
C₂₂H₂₂Br₂N₂O₂
52.20
4.38
5.53
52.28
4.42
5.58

4c
2-Furyl
58
184–186
C₁₈H₂₀N₂O₄
65.84
6.14
8.53
65.93
6.22
8.46

5a
2-OHC₆H₄
69
204–206
C₁₀H₇F₃O₃
51.74
3.04
—
51.82
3.12
—

5b
2-CH₃OC₆H₄
66
105–107
C₁₁H₉F₃O₃
53.67
3.68
—
53.77
3.70
—

Table 2: 1H and ¹³C NMR spectral data (/ppm)a of compound series 2.

Compd.
R
1H NMR
13C NMR

CH₃ (s)
CH₂ (s)b
Ar-H (m)
CH₃
CH₂
C=N
Ar-C

2a
C₆H₅
2.32
3.94
7.62–7.29 (10H)
14.6
48.2
164.8
128.5, 129.1, 130.6, 137.3

2b
4-BrC₆H₄
2.29
3.91
7.46–7.51 (8H)
14.5
48.4
164.6
125.3, 131.3, 132.0, 136.5

2c
2-OHC₆H₄
2.36
3.82
6.77–7.51 (8H)c
14.9
48.1
164.5
115.9, 121.2, 124.6, 130.6, 132.3, 157.8

2d
2-CH₃OC₆H
2.38, 3.73d
3.79
6.80–7.51 (8H)
14.8, 56.0d
47.9
164.2
114.2, 120.9, 122.8, 130.1, 131.8, 162.5

2e
2-Thienyl
2.30
3.50
6.98–7.32 (6H)
15.3
48.3
164.7
126.4, 127.6

2f
3-Thienyl
2.32
3.48
7.02–7.28 (6H)
15.2
48.2
164.8
126.8, 127.9, 130.2

2g
2-Furyl
2.39
3.39
6.51–7.43 (6H)
15.4
45.3
164.6
110.4, 143.6

2h
2-Pyridyl
2.19
3.19
7.63–8.83 (8H)
14.6
48.4
164.4
124.3, 125.7, 135.1, 149.8, 158.6

2i
4-CH₃C₆H₄
2.33, 2.41
3.89
7.09–7.51 (8H)
14.6, 20.7
48.3
164.6
128.9, 129.3, 134.3, 140.0

2j
4-CH₃OC₆H₄
2.30, 3.75 d
3.92
6.81–7.54 (8H)
14.7, 56.0d
48.0
164.6
114.4, 129.6, 130.3, 163.9

2k
3,4-(CH₃O)₂C₆H₄
2.36, 3.73d 3.80d
3.91
6.69–7.02 (6H)
14.4, 56.3d, 56.4d
48.2
164.8
115.4, 115.8, 122.6, 130.8, 147.8, 150.1

Solution in CDCl₃; b(2CH₂, 4H); c12.3 (broad singlet, OH); dOCH₃.

Table 3: 1H and ¹³C NMR spectral data (/ppm)a of compounds 3–5.

Compd.
R
1H NMR
13C NMR

CH= (s)
CH₂ (s)b
Ar-H (m)
Others NH or OH
CH₂
CH= & =C–NH
CF₃
Ar-C
C=O

3a
C₆H₅
5.50
2.91
7.14–7.32 (10H)
10.91
48.9
99.6, 160.8
132.4
126.2, 127.6, 128.3, 134.8
195.8

3b
4-BrC₆H₄
5.46
2.83
7.19–7.42 (8H)
11.02
48.7
98.8, 161.4
132.1
122.3, 128.5, 131.6, 134.2
196.0

3c
2-Thienyl
5.46
2.90
7.31–7.68 (6H)
10.89
48.3
104.5, 161.5
132.2
126.2, 127.4, 130.3, 137.0
196.5

3d
3-Thienyl
5.44
2.92
7.01–7.42 (6H)
10.93
48.5
104.5, 161.3
132.1
120.2, 125.8, 126.1, 135.0
196.4

3e
2-Furyl
5.42
2.94
6.70–7.86 (6H)
10.95
46.2
104.6, 161.3
131.6
111.6, 112.5, 145.2, 155.1
196.3

3f
2-Pyridyl
6.08
2.91
6.50–8.66 (8H)
11.10
48.9
104.3, 165.0
132.2
121.3, 122.2, 149,6, 155.7
196.5

3g
4-CH₃C₆H₄
5.48 

2.35c
2.93
7.01–7.20 (8H)
10.88
48.8,  

20.9c
98.9, 161.3
132.1
126.1, 129.3, 131.1, 136.6
196.3

3h
4-CH₃OC₆H₄
5.51 

3.73d
2.90
6.72–7.19 (8H)
11.08
48.6 

56.0d
96.1, 160.8
132.0
114.0, 127.3, 127.6, 161.2
196.5

3i
3,4-(CH₃O)₂C₆H₄
5.62
2.90
6.61–6.75 (6H)
11.12
48.9, 56.1d 

56.3d
98.6, 161.5
132.1
112.8, 115.0, 119.5, 128.2, 146.8, 147.6
196.4

4a
C₆H₅
5.46
2.92 

2.30c
7.14–7.33 (8H)
10.82
48.7
98.6, 161.3
24.8c
126.4, 127.7, 128.2, 134.8
196.7

4b
4-BrC₆H₄
5.44
2.90 

2.31c
7.19–7.39 (8H)
10.76
48.8
98.7, 161.6
24.9c
122.2, 128.5, 131.6, 134.0
196.4

4c
2-Furyl
5.38
2.88 

2.34c
6.68–7.72 (6H)
10.82
46.7
102.3, 160.2
24.5c
112.0, 112.9, 144.2, 156.3
195.9

5a
2-OHC₆H₄

3.86 e
6.85–7.78 (4H)
4.02
35.1
105.0f
123.4
114.1, 120.0, 123.1, 129.2,

133.5, 158.9
197.6

5b
2-CH₃OC₆H₄

3.91e 

3.42d
6.90–7.82 (4H)

32.6, 45.2d
102.3f
125.6
114.3, 121.3, 124.0, 130.1,

132.8, 156.4
198.2

Solution in CDCl₃; b(2CH₂, 4H); cCH₃; dOCH₃; eH-3 (m, 2H); f(C-2).

2.1.1. N,N′-Bis(1-substituted-ethylidene)ethane-1,2-diamine (2)

A mixture of 1,2-diaminoethane (0.6 g, 10 mmol) and the appropriate ketone 1 (20 mmol) in dry benzene (50 mL) was refluxed using Dean-Stark trap until no more water was collected (about 2 h). Benzene was then removed under reduced pressure, and the residue was treated with methanol. The solid which separated out was recrystallized from ethanol as needles.

2.1.2. 1,1,1-Trifluoro-4-substituted-4-[(2-{[4,4,4-trifluoro-3-oxo-1-substituted-but-1-en-1-yl]amino}-ethyl)amino]but-3-en-2-one (3)

A mixture of the N,N′-Bis(1-substituted ethylidene)ethane-1,2-diamine (10 mmol) in THF (30 mL) and trifluoroacetic anhydride (25 mmol) was refluxed for 2 h. The solid which separated on cooling was recrystallized from ethanol as needles.

2.1.3. 4-Substituted 4-[2-(1-Substituted-3-oxo-but-1-enylamino)-ethylamino]-but-3-en-2-one (4)

A mixture of the N,N′-Bis[1-substituted-ethylidene]ethane-1,2-diamine (10 mmol) in THF (30 mL) and acetic anhydride (25 mmol) was refluxed for 2 h. The solid which separated on cooling was recrystallized from ethanol as needles.

2.1.4. 2-Substituted 2-Trifluoromethylchroman-4-one (5)

A mixture of the N,N′-Bis(1-substituted-ethylidene)ethane-1,2-diamine 2c and 2d (10 mmol) in THF (30 mL) and trifluoroacetic anhydride (25 mmol) was refluxed for 2 h. The solid which separated on cooling was recrystallized from ethanol as needles.

2.2. Biological Evaluation

2.2.1. In Vitro MTT Cytotoxicity Assay

All procedures and screening techniques were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Stanford, ME, USA). Cells were batch cultured for 10 days then seeded at concentration of  cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37°C for 24 h under 5% CO₂ using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of the test compounds to give a final concentration of ( μg/mL). DMSO was employed as a vehicle for the dissolution of the tested compounds and its final concentration on the cells was less than 0.2%. Cells were suspended in RPMI 1640 medium (for HepG2 and HT29 cell lines) and DMEM (for MCF 7 cell line), 1% antibiotic-antimycotic mixture (10,000 IU/mL penicillin potassium, 10,000 μg/mL streptomycin sulfate and 25 μg/mL amphotericin B), and 1% L-glutamine in 96-well flat bottom microplate at 37°C under 5% CO₂.

After 48 h of incubation, the medium was aspirated, and 40 μL of MTT salt (2.5 μg/mL) was added to each well and incubated for further 4 h at 37°C under 5% CO₂. To stop the reaction and dissolve the formed crystals, 200 μL of 10% sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37°C. The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, CA, USA) at 595 nm and a reference wavelength of 620 nm. Doxorubicin is used as a positive control cytotoxic agent. The cytotoxicity assay data can be found in Table 4.

Table 4: Cytotoxic effects (LC₅₀; μg/mL)a of the active compounds (2–5) on some human tumor cell lines using the MTT assay.

Compound no.
Human colon carcinoma HT29
Human hepatocellular carcinoma HepG2
Human breast cancer

MCF 7

2b
41.2
39.4
—

2e
35.8
47.3
51.6

2g
74.3
—b
—

2h
61.4
47.2
—

2j
40.3
—
54.2

3a
14.6
25.8
32.4

3b
9.6
23.5
3.2

3c
12.9
31.8
9.7

3e
19.1
26.3
34.2

3f
8.4
20.7
2.1

3g
17.9
29.4
28.2

3h
8.8
21.6
6.4

3i
11.8
25.2
30.1

4b
55.1
48.7
64.9

5a
19.8
37.2
46.4

5b
20.5
28.1
48.9

Doxorubicinc
21.1
1.69
2.14

LC₅₀: lethal concentration of the compound which causes death of 50% of the cells in 24 h (μg/mL).

Totally inactive against this cell line. cPositive control cytotoxic agent.

2.2.2. Procedure for Antimicrobial Activity

The preliminary anti-microbial activities of compounds 2–5 were measured in a concentration of 50 mg/L by disc diffusion method [20, 21]. The prepared compounds were tested for their antimicrobial activity against two types of bacterium, Staphylococcus aureus (ATCC 25923) as an example of Gram positive bacteria and Escherichia coli (ATCC 25922) as Gram negative bacteria, and the antifungal activity was tested using the pathogenic yeast strains Candida albicans and Aspergillus niger. DMSO was used as a solvent control, and the standard drugs used were Ampicillin and Griseofulvin. The disc diffusion method was performed using Muller-Hinton agar (Hi-Media) medium. The inhibition zones were measured in mm at the end of an incubation period of 24 h at 37°C for bacteria and 72 h at 24°C for fungi. The zone of inhibition in mm is expressed in Table 5.

Table 5: Antibacterial and antifungal data of Schiff bases and their acyl derivatives 2–5.

Compound
Zone of inhibition in mm

Escherichia coli
Staphylococcus aureus
Aspergillus niger
Candida albicans

2a
12
14
13
12

2b
17
17
15
15

2c
15
15
14
16

2d
14
16
13
15

2e
15
17
16
14

2f
16
15
14
15

2g
18
16
15
14

2h
19
18
18
17

2i
13
15
14
12

2j
15
14
16
11

2k
16
13
14
10

3a
24
25
26
25

3b
31
29
28
29

3c
27
26
26
26

3d
28
27
28
25

3e
27
25
26
27

3f
30
28
29
28

3g
27
26
25
24

3h
29
27
26
25

3i
28
24
25
26

4a
19
16
20
18

4b
22
20
21
20

4c
17
16
15
16

5a
26
25
24
22

5b
27
24
25
21

Ampicillin
32
28
—
—

Griseofulvin
—
—
31
29

2.2.3. Procedure for Antimicrobial Activity Using UV (366 nm) Light

This test was performed as mentioned before but the Petri dishes containing microorganisms and the impregnated disks were exposed to UV light (366) for 3 h before incubation. The obtained data is shown in Table 6.

Table 6: Antibacterial and antifungal data of Schiff bases and their acyl derivatives (2–5) using UV (366) light.

Compound
Zone of inhibition in mm

Antibacterial activity
Zone of inhibition in mm

Antifungal activity

Escherichia coli
Staphylococcus aureus
Aspergillus niger
Candida albicans

2a
13
—
14
13

2b
18
—
—
16

2c
—
17
—
—

2d
15
—
—
16

2e
16
—
—
16

2f
17
16
15
16

2g
19
17
17
15

2h
20
—
—
—

2i
14
16
—
—

2j
—
—
17
13

2k
—
14
—
—

3a
25
26
—
26

3b
—
—
—
—

3c
—
—
27
27

3d
29
28
29
—

3e
28
—
—
28

3f
—
—
—
—

3g
28
27
—
—

3h
30
28
26
—

3i
—
26
—
—

4a
20
17
21
19

4b
—
21
—
21

4c
—
17
—
—

5a
27
26
—
—

5b
—
25
—
—

Ampicillin
32
28
—
—

Griseofulvin
—
—
31
29

3. Results and Discussion

3.1. Chemistry

The synthesis of Schiff bases, reaction involving amine and carbonyl compound, and their metal complexes are well known [22–26]. In general, 1,2-ethylenediamine was reacted with various methyl aryl ketones to give the Schiff bases 2a–k [27] in moderate to good yields (Scheme 1).

Scheme 1: Synthesis of Schiff bases of 1,2-diamine. i: appropriate ketone (20 mmol) 1,2-diaminoethane (10 mmol, 0.6 g), dry benzene (50 mL), and reflux using Dean-Stark trap ~2 h; ii: N,N′-bis(1-substituted-ethylidene)-ethane-1,2-diamine (10 mmol), THF (30 mL), (CF₃CO)O (25 mmol), and reflux 2 h; iii: N,N′-bis(1-substituted-ethylidene)ethane-1,2-diamine (10 mmol), THF (30 mL), (CF₃CO)O (25 mmol), and reflux 2 h.

The IR spectra showed a characteristic C=N absorption bands at 1618–1622 cm⁻¹. Their ¹H NMR spectra exhibited, beside the aromatic and methyl protons, a singlet of four-proton intensity at δ 2.19–3.94 for the 2CH₂ (Table 2). Acylation of Schiff bases 2 with trifluoroacetic anhydride in THF yielded the corresponding trifluoroacetyl derivatives 3a–i. The IR spectra exhibited, beside the absorption bands corresponding to the NH group at 3216–3288 cm⁻¹, a characteristic carbonyl band at 1672–1675 cm⁻¹.

Their ¹H NMR spectra displayed signals due to aromatic and aliphatic protons but lacked signals characteristic of methyl group of the corresponding Schiff bases (Table 3). The structures of the Schiff bases 2 and the trifluoroacetyl derivatives 3 were further confirmed from their ¹³C NMR spectra (Table 3) as well as their X-ray crystallography [28–30] (Figure 1). However, reaction of the Schiff bases 2 with acetic anhydride afforded the corresponding acetyl derivatives 4. The IR of 4a–c showed a carbonyl band at 1664–1666 cm⁻¹ beside the absorption bands of the NH group at 3335–3372 cm⁻¹. Their ¹H NMR spectra exhibited, beside the aromatic and aliphatic protons, a singlet at δ 2.30-2.31 for the CH₃ group (Table 2). The structures were further confirmed from their ¹³CNMR spectra data (Table 3). Moreover, reaction of the Schiff bases 2c and 2d with trifluoroacetic anhydride in THF afforded the corresponding trifluoromethylchroman-4-one derivatives 5a and 5b instead of the expected trifluoroacetyl derivatives. A possible mechanism for the reaction is shown in Scheme 2. The IR spectra of chromanone derivatives 5 exhibited a strong carbonyl band at 1668–1670 cm⁻¹, while their ¹H NMR spectra displayed beside the aromatic protons a singlet of two-proton intensity at δ 3.86–3.91 for H-3 (Table 3). The structures of 5 were further confirmed from their ¹³CNMR spectra as well as X-ray crystallography [31] (Figure 1).

Scheme 2: Cyclization of Schiff bases to trifluoromethyl-chromanone.

(a) N,N′-Bis[1-(thiophen-2-yl)ethylidene]ethane-1,2-diamine (2e)

(b) N,N′-Bis[(E)-1-(thiophen-3-yl)ethylidene]ethane-1,2-diamine (2f)

(c) 1,1,1-Trifluoro-4-(thiophen-2-yl)-4-[(2-{[4,4,4-trifluoro-3-oxo-1-(thiophen-2-yl)but-1-en-1-yl]amino}ethyl)amino]but-3-en-2-one (3c)

(d) 2-Hydroxy-2-trifluoro-methyl-3,4-dihydro-2H-1-benzopyran-4-one (5a)

Figure 1: X-ray crystal structures of 2e, 2f, 3c, and 5a.

3.2. Biological Evaluation

3.2.1. In Vitro MTT Cytotoxicity Assay

Twenty-one analogs 2a–h, 2j, 3a–c, 3e–i, 4a,b, and 5a, 5b were selected to be evaluated for their in vitro cytotoxic effect via the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method [32, 33] against a panel of three human tumor cell lines, namely, Caucasian breast adenocarcinoma MCF7, hepatocellular carcinoma HepG2, and colon carcinoma HT29. The results are presented in Table 4 as LC₅₀ (μg/mL) which is the lethal concentration of the compound which causes death of 50% of the cells in 24 h.

The obtained data revealed that the three tested human tumor cell lines exhibited variable degree of sensitivity profiles towards sixteen of the tested compounds 2b, 2e, 2g, 2h, 2j, 3a–c, 3e–i, 4b, 5a, and 5b. Among these, the human colon carcinoma HT29 cell line showed pronounced sensitivity against fluorinated compounds 3b, 3f, and 3h with LC₅₀ values of 9.6, 8.4, and 8.8 μg/mL, respectively. In addition, a significant cytotoxic activity was displayed by compounds 3c and 3i against the same cell line (12.9 and 11.8 μg/mL). Meanwhile, compounds 3e, 3 g, 5a and 5b revealed moderate cytotoxicity profile against colon carcinoma HT29 with LC₅₀ values 19.7, 17.9, 19.8 and 20.5 μg/mL, respectively. Nonfluorinated compounds 2b, 2e, 2h, and 4b were able to exhibit mild activity against the same cell line with LC₅₀ values range of 35.8–61.4 μg/mL. Low activity was displayed by compounds 2g at LC₅₀ value 74.3 μg/mL. However, the growth of the human hepatocellular carcinoma HepG2 cell line was found to be moderately inhibited by fourteen of the active compounds 2b, 2e, 2j, 3a, 3b, 3c, 3e, 3f–i, 4b, 5a, and 5b with LC₅₀ values range of 20.7–48.7 μg/mL. Among these, the highest cytotoxic activity was displayed by fluoro analogs 3b, 3e, 3f, 3h, and 3i which were almost equally potent (LC₅₀ values 23.5, 26.3, 20.7, 21.6, and 25.2 μg/mL, resp.). On the other hand, human breast cancer MCF 7 was proved to be the least sensitive among the cell lines tested as it was affected by only seven of the test compounds. However, an outstanding growth inhibition potential was shown by compounds 3b, 3c, 3f and 3h as evidenced from their LC₅₀ values (3.2, 9.7, 2.1, and 6.4 μg/mL, resp.). The remaining eight active compounds 2d, 3a, 3e, 3g, 3i, 4b, 5a, and 5b showed mild to moderate activity against the same cell line with LC₅₀ values range of 28.2–64.9 μg/mL (Table 4). Further interpretation of the results revealed that, compounds 3b, 3c, 3e and 3f, and 5a showed considerable broad spectrum of cytotoxic activity against the three tested human tumor cell lines. In particular, compound 3f proved to be the most active member in this study with a broad spectrum of activity against all the tested cell lines, with special effectiveness against the human colon carcinoma HT29 and human breast cancer MCF 7 cell lines (LC₅₀ values 8.4 and 2.1 μg/mL, resp.) (Table 4). A close examination of the structure of the active compounds showed that the presence of trifluoroacetyl moiety with 2-pyridyl nucleus in diamine skeleton is the most favorable substituent when compared with the nonfluorinated analogs. The presence of trifluoroacetyl group in the diamine skeleton (as in compounds 3 and 5) is responsible for the high activity displayed by these analogs. The Schiff bases without the trifluoroacetyl moiety resulted in moderate to weakly active compounds (2 and 4b), whereas other compounds are completely inactive.

3.2.2. Antimicrobial Activity

Compounds 2–5 were screened in vitro for their antimicrobial and antifungal activities against Escherichia coli, Staphylococcus aureus, Aspergillus niger, and Candida albicans. The zones of inhibition formed for the compounds against bacteria and fungi are summarized in Table 5. The overall results evidently suggest that compounds containing trifluoroacetyl substituent exhibited relatively better antimicrobial and antifungal activities than all the nonfluorinated analogs indicating a positive role of fluorine substitution in the present series. Compounds 3 and 5 were, however, significantly active when compared with rest of the series. Furthermore, after using UV-Visible light, most of the tested compounds showed an additional activity especially towards Escherichia coli and Candida albicans (Table 6). All test data in Tables 5 and 6 were of average values from triplicate runs, and the test compounds showed reduced antimicrobial activities when compared with their respective standards.

4. Conclusions

In this paper, several new N,N′-bis(1-substituted ethylidene)ethane-1,2-diamines were synthesized by the condensation of 1,2-ethylenediamine with the appropriate methyl ketone. Acetylating these Schiff bases with trifluoroacetic anhydride or acetic anhydride afforded the corresponding acetyl derivatives. Structures of the prepared compound were confirmed by elemental analysis, IR, ¹H, ¹³C NMR spectral analysis, and X-ray crystallography. Preliminary biological testing of these compounds revealed that trifluoroacetyl derivatives exhibited significant cytotoxic and antimicrobial activities. Further, the incorporation of trifluoroacetyl group is justified by a comparative study with the nonfluorinated analogs. The fluorinated analogs were found to be more active than their nonfluorinated counterparts.

Acknowledgments

The authors are deeply thankful to the authorities of the Bioassay-Cell Culture Laboratory, Drug Discovery Unit, National Research Centre, Cairo, Egypt, for their efforts in performing the MTT cytotoxicity assay. The authors would like to thank Ms. Samina Shamim, English Language Institute, King Abdulaziz University, for reviewing the language and grammar of the paper.

References

T. Suzuki, Y. Suzuki, K. Hachisu, and H. Shinozaki, “Photo-reversible oxygen binding of a cobalt schiff-base complex with an axially coordinating photo-isomerizable polymer ligand,” Polymers for Advanced Technologies, vol. 22, no. 8, pp. 1278–1282, 2011.

S. Alexander, V. Udayakumar, and V. Gayathri, “Hydrogenation of olefins by polymer-bound palladium(II) Schiff base catalyst,” Journal of Molecular Catalysis A, vol. 314, no. 1-2, pp. 21–27, 2009.

E. Hadjoudis, A. Rontoyianni, K. Ambroziak, T. Dziembowska, and I. M. Mavridis, “Photochromism and thermochromism of solid trans-N,N-bis-(salicylidene)-1,2-cyclohexanediamines and trans-N,N-bis-(2-hydroxy-naphylidene)-1,2-cyclohexanediamine,” Journal of Photochemistry and Photobiology A, vol. 162, no. 2-3, pp. 521–530, 2004.

Y. Chen and D. X. Zeng, “Study on photochromic diarylethene with phenolic schiff base: preparation and photochromism of diarylethene with benzoxazole,” Journal of Organic Chemistry, vol. 69, no. 15, pp. 5037–5040, 2004.

O. Kocyigit, A. N. Kursunlu, and E. Guler, “Complexation properties and synthesis of a novel Schiff base with triphenylene nucleus,” Journal of Hazardous Materials, vol. 183, no. 1-3, pp. 334–340, 2010.

X. Ran, L. Wang, D. Cao, Y. Lin, and J. Hao, “Synthesis, characterization and in vitro biological activity of cobalt(II), copper(II) and zinc(II) Schiff base complexes derived from salicylaldehyde and D,L-selenomethionine,” Applied Organometallic Chemistry, vol. 25, no. 1, pp. 9–15, 2011.

A. Prakash and D. Adhikari, “Application of Schiff bases and their metal complexes: a review,” International Journal of ChemTech Research, vol. 3, no. 4, pp. 1891–1896, 2011.

H. Karaer and I. E. Gümrükçüoǧlu, “Synthesis and spectral characterisation of novel azo-azomethine dyes,” Turkish Journal of Chemistry, vol. 23, no. 1, pp. 67–71, 1999.

S. Kumar, M. S. Niranjan, K. C. Chaluvaraju, C. M. Jamakhandi, and D. Kadadevar, “Synthesis and antimicrobial study of some Schiff bases of sulfonamides,” Journal of Current Pharmaceutical Research, vol. 1, no. 1, pp. 39–42, 2010.

C. T. Supuran, M. Barboiu, C. Luca, E. Pop, M. E. Brewster, and A. Dinculescu, “Carbonic anhydrase activators. Part 14. Syntheses of mono and bis pyridinium salt derivatives of 2-amino-5-(2-aminoethyl)- and 2-amino-5-(3-aminopropyl)-1,3,4-thiadiazole and their interaction with isozyme II,” European Journal of Medicinal Chemistry, vol. 31, no. 7-8, pp. 597–606, 1996.

A. M. Asiri and S. A. Khan, “Synthesis and anti-bacterial activities of some novel schiff bases derived from aminophenazone,” Molecules, vol. 15, no. 10, pp. 6850–6858, 2010.

C. A. Bolos, G. S. Nikolov, L. Ekateriniadou, A. Kortsaris, and D. A. Kyriakidis, “Structure-activity relationships for some diamine, triamine and Schiff base derivatives and their copper(II) complexes,” Metal-Based Drugs, vol. 5, no. 6, pp. 323–332, 1998.

J. A. Olsen, D. W. Banner, P. Seiler et al., “Fluorine interactions at the thrombin active site: protein backbone fragments H–Cα–C=O comprise a favorable C-F environment and interactions of C–F with electrophiles,” ChemBioChem, vol. 5, no. 5, pp. 666–675, 2004.

H. Hatano, F. Takekawa, K. Hashimoto et al., “Tumor-specific cytotoxic activity of 1,2,3,4-tetrahydroisoquinoline derivatives against human oral squamous cell carcinoma cell lines,” Anticancer Research, vol. 29, no. 8, pp. 3079–3086, 2009.

A. A. Bekhit, H. M. Abdel-Rahman, and A. A. Guemei, “Synthesis and biological evaluation of some hydroxypyrazole derivatives as anti-inflammatory-antimicrobial agents,” Archiv der Pharmazie, vol. 339, no. 2, pp. 81–87, 2006.

C. Mattos, C. R. Bellamacina, E. Peisach et al., “Multiple solvent crystal structures: probing binding sites, plasticity and hydration,” Journal of Molecular Biology, vol. 357, no. 5, pp. 1471–1482, 2006.

H. M. Faidallah, K. A. Khan, and A. M. Asiri, “Synthesis and biological evaluation of new 3- trifluoromethylpyrazolesulfonyl-urea and thiourea derivatives as antidiabetic and antimicrobial agents,” Journal of Fluorine Chemistry, vol. 132, no. 2, pp. 131–137, 2011.

H. M. Faidallah, K. A. Khan, and A. M. Asiri, “Synthesis and biological evaluation of new 3,5-di(trifluoromethyl)-1,2,4- triazolesulfonylurea and thiourea derivatives as antidiabetic and antimicrobial agents,” Journal of Fluorine Chemistry, vol. 132, no. 11, pp. 870–877, 2011.

H. M. Faidallah and K. A. Khan, “Synthesis and biological evaluation of new barbituric and thiobarbituric acid fluoro analogs of benzenesulfonamides as antidiabetic and antibacterial agents,” Journal of Fluorine Chemistry, vol. 142, pp. 96–104, 2012.

W. R. Kirkpatrick, T. M. Turner, A. W. Fothergill et al., “Fluconazole disk diffusion susceptibility testing of Candida species,” Journal of Clinical Microbiology, vol. 36, no. 11, pp. 3429–3432, 1998.

T. Premkumar and S. Govindarajan, “Antimicrobial study of pyrazine, pyrazole and imidazole carboxylic acids and their hydrazinium salts,” World Journal of Microbiology and Biotechnology, vol. 21, no. 4, pp. 479–480, 2005.

M. R. Bermejo, M. I. Fernández, E. Gómez-Fórneas et al., “Self-assembly of dimeric MnIII-Schiff-base complexes tuned by perchlorate anions,” European Journal of Inorganic Chemistry, no. 24, pp. 3789–3797, 2007.

A. Pui, C. Dobrota, and J.-P. Mahy, “Electrochemical and spectroscopic characterization and catalytic activity of Co(II) complexes of tetra-chloro-R-Salen ([tClSalen=bis(3,5-di-chloro- α-R salicylidene)ethylenediamine]) and tetra-chloro-R-Salophen ([tClSalophen=bis(3,5-di-chloro-α-R salicylidene)-1,2-phenylenediamine]), R=H, CH₃, CH₂–CH₃,” Journal of Coordination Chemistry, vol. 60, no. 5, pp. 581–595, 2007.

S. R. Doctrow, K. Huffman, C. B. Marcus et al., “Salen-manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: structure-activity relationship studies,” Journal of Medicinal Chemistry, vol. 45, no. 20, pp. 4549–4558, 2002.

C. S. Higham, D. P. Dowling, J. L. Shaw, A. Cetin, C. J. Ziegler, and J. R. Farrell, “Multidentate aminophenol ligands prepared with Mannich condensations,” Tetrahedron Letters, vol. 47, no. 26, pp. 4419–4423, 2006.

U. Casellato, S. Tamburini, P. Tomasin, and P. A. Vigato, “Cyclic and acyclic compartmental Schiff bases, their reduced analogues and related mononuclear and heterodinuclear complexes,” Inorganica Chimica Acta, vol. 357, no. 14, pp. 4191–4207, 2004.

B. N. Ghose, “Schiff base complexes of boron: reaction of boron acetate with dibasic quadridentate Schiff bases,” Synthesis and Reactivity in Inorganic and Metal-Organic Chemistry, vol. 16, no. 10, pp. 1383–1393, 1986.

A. M. Asiri, A. O. Al-Youbi, H. M. Faidallah, K. A. Alamry, and S. W. Ng, “N,N′-Bis[1-(thiophen-2-yl)ethylidene]ethane-1,2-diamine,” Acta Crystallographica Section E, vol. 67, no. 9, article o2465, 2011.

A. M. Asiri, A. O. Al-Youbi, H. M. Faidallah, and S. W. Ng, “1,1,1-Trifluoro-4-(thiophen-2-yl)-4-[(2-{[4,4,4-trifluoro-3-oxo-1- (thiophen-2-yl)but-1-en-1-yl]amino}ethyl)amino]but-3-en-2-one,” Acta Crystallographica Section E, vol. 67, no. 10, pp. o2659–o2660, 2011.

A. M. Asiri, H. M. Faidallah, K. A. Khan, S. W. Ng, and E. R. T. Tiekink, “N,N′-Bis[(E)-1-(thio-phen-3-yl)ethyl-idene]ethane-1,2-diamine,” Acta Crystallographica Section E, vol. 68, no. 4, article o1026, 2012.

A. M. Asiri, H. M. Faidallah, K. A. Alamry, S. W. Ng, and E. R. T. Tiekink, “2-Hydroxy-2-trifluoro-methyl-3, 4-dihydro-2H-1-benzopyran-4-one,” Acta Crystallographica Section E, vol. E68, no. 7, article o2299, 2012.

T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55–63, 1983.

F. Denizot and R. Lang, “Rapid colorimetric assay for cell growth and survival—Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability,” Journal of Immunological Methods, vol. 89, no. 2, pp. 271–277, 1986.