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Journal of Chemistry
Volume 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

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 [1719], 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.

tab1
Table 1: Physical and analytical data of compounds 25.
tab2
Table 2: 1H and 13C NMR spectral data ( /ppm)a of compound series 2.
tab3
Table 3: 1H and 13C NMR spectral data ( /ppm)a of compounds 35.
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.

tab4
Table 4: Cytotoxic effects (LC50; μg/mL)a of the active compounds (25) on some human tumor cell lines using the MTT assay.
2.2.2. Procedure for Antimicrobial Activity

The preliminary anti-microbial activities of compounds 25 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.

tab5
Table 5: Antibacterial and antifungal data of Schiff bases and their acyl derivatives 25.
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.

tab6
Table 6: Antibacterial and antifungal data of Schiff bases and their acyl derivatives (25) using UV (366) light.

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 [2226]. 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).

478635.sch.001
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 [2830] (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).

478635.sch.002
Scheme 2: Cyclization of Schiff bases to trifluoromethyl-chromanone.
fig1
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 2ah, 2j, 3ac, 3ei, 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, 3ac, 3ei, 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 25 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.

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