Results 5a–m and 6a–f in addition to the intermediates

Results and discussion

Chemistry

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

The Preparation of ultimate compounds 5a–m and 6a–f
in addition to the intermediates 1–4 is illustrated in Scheme 1. Cyclohexanone
was reacted with potassium cyanide and aniline in glacial acetic acid to produce
the nitrile compound 1 which was hydrolyzed using sulfuric acid at room
temperature and producing the amidic compound 3.

The
penultimate intermediate 4 was achieved by acetylation and subsequently,
reaction with different amines to afford 5a-m or reaction with
formaldehyde and different amines to afford 6a-m.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Reagents and conditions: i) Cyclohexanone, Potassium cyanide, acetic acid glacial, r.t, 24
h   ii) H2SO4, r.t,  48 h, iii) ClCOCH2Cl, CHCl3,
r.t , 24 h, iv) ethanol, appropriate amine, 12 h, reflux  v) HCHO, ethanol, appropriate amine, 12 h,
reflux.

Biological Evaluation

Antiproliferation assay

The antitumor activity for compounds 5a-m and 6a-f
were evaluated against the four cancer cell lines HepG2 (liver), MCF-7
(breast), A549 (lung) and Caco-2 (colorectal). Results are illustrated in (Table
1). Most of the compounds are selective and having potential cytotoxicity towards
MCF-7 adenocarcinoma with IC50 values range 3.25-36.8 ?M as compared
with doxorubicin (IC50 value of 6.77 ?M). Compound 5i showed
the most potent biological activity with IC50 value = 3.25 ?M.

Additionally, compound 5i showed high potential
activity toward human HepG2 hepatocellular carcinoma with IC50 11.5
?M, A549 lung adenocarcinoma with IC50 6.95 ?M and Caco-2 colorectal
adenocarcinoma with IC50 8.98 ?M as compared with doxorubicin (3.07,
0.887, 2.78 ?M respectively). Examining selectivity, the test compounds showed
high activity against MCF-7.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table1. Antiproliferative activity for compounds 5a-m
and 6a f.

 
Compound

MCF-7
IC50
?M

HepG2
IC50
?M

A549
IC50
?M

Caco-2
IC50
?M

5a

26.15

50.2

35.4

30.21

5b

3.68

10.61

7.98

5.46

5c

7.21

15.02

9.97

14.12

5d

28.41

60.3

42.7

52.40

5e

31.22

56.4

40.6

50.22

5f

20.08

43.32

39.74

4.33

5g

9.13

33.88

30.3

25.43

5h

22.68

58.3

31.29

47.23

5i

3.25

11.5

6.95

8.98

5j

13.59

31.28

15.64

22.34

5k

5.48

20.71

14.59

13.66

5l

           4.58

12.08

8.51

11.25

5m

6.48

13.82

11.83

10.53

6a

            36.8

6b

23.51

16.46

31.21

28.67

6c

6d

17.05

15.91

26.22

39.52

6e

26.13

21.74

       42.09

51.43

6f

22.89

17.54

31.6

44.12

Doxorubicin

6.77

3.07

0.887

2.78

2.     
(-): No activity

Cell cycle analysis and apoptosis
induction

    Compound 5i was
the most potent against MCF-7 cancer cell line. Consequently, we examined its
effect on the cell cycle progression using BD FASCCalibur after treatment with
3.25 ?M of 5i for 48 h. Then, cell was stained with an annexin V-FITC
antibody and propidium iodide by FACS (Table 2). For the cell cycle, compound 5i
revealed induction of apoptosis at pre G1 phase and arresting at G2/M phase.

 

 

 

 

Table 2.  Cell cycle analysis
for control and compound 5i at concentration 3.25 ?M for 48 h on MCF-7 cell
line.

Sample data

         Result

%G0-G1

%S

% G2-M

% Apoptosis

Comment

5i

7.66

24.77

46.17

21.4

PreG1 apoptosis &cell growth [email protected]/M

Cont. MCF-7

71.7

22.45

5.23

0.62

Control pattern

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig.2.
Graphical view of % of DNA content in G0/G1, S, and G2/M phase for control and
compound 5i

 

 

 

 

 

 

 

 

Fig.3. Cell cycle analysis and apoptotic assay
graphs for control and compound 5i at concentration 3.25 ?M for 48 h on MCF-7 cell line