# 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

Authors: Khalid A. Khan; Hassan M. Faidallah; Abdullah M. Asiri
Journal: Journal of Chemistry
 (2013)

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## Abstract

Several Schiff bases2 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.


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## Body

## 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±0.4% 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 compounds2–5.
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 (δ/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, 3.75d
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
aSolution in CDCl3; b(2CH2, 4H); c12.3 (broad singlet, OH); dOCH3.Table 3
1H and 13C NMR spectral data (δ/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
5.482.35c
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
5.513.73d
2.90
6.72–7.19 (8H)
11.08
48.656.0d
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
2.922.30c
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
2.902.31c
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
2.882.34c
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
3.86e
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
aSolution 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 ketone1 (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-diamine2c 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 of10×103 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 (100-50-25-12.5-6.25-3.125-1.56-0.78 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 40μL 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 cancerMCF 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
aLC50: lethal concentration of the compound which causes death of 50% of the cells in 24 h (μg/mL).
bTotally inactive against this cell line. cPositive control cytotoxic agent.


#### 2.2.2. Procedure for Antimicrobial Activity

The preliminary anti-microbial activities of compounds2–5 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 albicansand 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 derivatives2–5.
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 Table6.Table 6
Antibacterial and antifungal data of Schiff bases and their acyl derivatives (2–5) using UV (366) light.
Compound
Zone of inhibition in mmAntibacterial activity
Zone of inhibition in mmAntifungal 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


## 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±0.4% 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 compounds2–5.
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 (δ/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, 3.75d
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
aSolution in CDCl3; b(2CH2, 4H); c12.3 (broad singlet, OH); dOCH3.Table 3
1H and 13C NMR spectral data (δ/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
5.482.35c
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
5.513.73d
2.90
6.72–7.19 (8H)
11.08
48.656.0d
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
2.922.30c
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
2.902.31c
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
2.882.34c
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
3.86e
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
aSolution 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 ketone1 (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-diamine2c 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.


## 2.1.1. 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 ketone1 (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-diamine2c 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 of10×103 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 (100-50-25-12.5-6.25-3.125-1.56-0.78 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 40μL 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 cancerMCF 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
aLC50: lethal concentration of the compound which causes death of 50% of the cells in 24 h (μg/mL).
bTotally inactive against this cell line. cPositive control cytotoxic agent.


### 2.2.2. Procedure for Antimicrobial Activity

The preliminary anti-microbial activities of compounds2–5 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 albicansand 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 derivatives2–5.
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 Table6.Table 6
Antibacterial and antifungal data of Schiff bases and their acyl derivatives (2–5) using UV (366) light.
Compound
Zone of inhibition in mmAntibacterial activity
Zone of inhibition in mmAntifungal 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


## 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 of10×103 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 (100-50-25-12.5-6.25-3.125-1.56-0.78 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 40μL 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 cancerMCF 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
aLC50: lethal concentration of the compound which causes death of 50% of the cells in 24 h (μg/mL).
bTotally inactive against this cell line. cPositive control cytotoxic agent.


## 2.2.2. Procedure for Antimicrobial Activity

The preliminary anti-microbial activities of compounds2–5 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 albicansand 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 derivatives2–5.
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 Table6.Table 6
Antibacterial and antifungal data of Schiff bases and their acyl derivatives (2–5) using UV (366) light.
Compound
Zone of inhibition in mmAntibacterial activity
Zone of inhibition in mmAntifungal 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.Their1H 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.X-ray crystal structures of2e, 2f, 3c, and 5a.
(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)


### 3.2. Biological Evaluation

#### 3.2.1. In Vitro MTT Cytotoxicity Assay

Twenty-one analogs2a–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 compounds2b, 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

Compounds2–5 were screened in vitrofor 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 coliand 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.


## 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.Their1H 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.X-ray crystal structures of2e, 2f, 3c, and 5a.
(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)


## 3.2. Biological Evaluation

### 3.2.1. In Vitro MTT Cytotoxicity Assay

Twenty-one analogs2a–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 compounds2b, 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

Compounds2–5 were screened in vitrofor 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 coliand 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.


## 3.2.1. In Vitro MTT Cytotoxicity Assay

Twenty-one analogs2a–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 compounds2b, 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

Compounds2–5 were screened in vitrofor 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 coliand 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.



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*Source: 478635-2013-09-08.xml*