Design, Synthesis, Molecular Docking and Biological Activity
151
166 – 168°C; IR spectrum, n , cm-1: 1662.68 (C=O),
culture medium and MCF-7 cells were cultured in RPMI
1640 medium supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin and incubated at 37°C
max
1323,159, 1234.19(C–N), 655.07 (C–Cl). 1H NMR spectrum
(chloroform-d d, ppm: 8.16 (d, J = 9.5 Hz, arom. 2H.), 6.86
3)
in humidified CO atmosphere.
(d, J = 9.6 Hz, arom. 2H), 6.22 (s, 1H, CHCl ), 4.04 – 3.75
2
2
(m, 4H, CH NCH ), 3.67 – 3.40 (m, 4H, CH NCH ). 13C
The cytotoxic activity of synthesized compounds was
checked by MTT assay as described previously [30 – 32].
Briefly, the cells were harvested and plated in 96-well micro-
plates at a density of 1 ´ 104 cells per well in 100 mL com-
plete culture medium (containing FBS and antibiotics). After
24 h incubation, each cell was treated with different concen-
trations of each compound (from 2 ´ 10 – 4 to 1 ´ 10 – 7 M) in
triplicate. Various concentrations of DCA were also used as
positive control. Three untreated wells were considered as
negative controls. After 72h, media were completely re-
moved and replaced with 100 mL media containing
0.5 mg/mL MTT solution and incubated for 3 – 4 h.. Then,
MTT containing medium was discarded, 150 mL dimethyl-
sulfoxide was added to each well so as to dissolve formazan
crystals, and the plates were incubated for another 3 h. After
30 min, the absorbance of individual wells at 570 nm was
measured by Bio-Rad Model 680 microplate reader. Data
were processed and expressed as 50% inhibitory concentra-
2
2
2
2
NMR spectrum (chloroform-d ) d, ppm: 162.22, 154.26,
3
139.44, 125.91, 113.28, 77.21, 65.57, 46.87, 46.59, 45.51,
42.57.
1-(4-Acetylpiperazin-1-yl)-2,2-dichloroethan-1-one
(f6). White solid; yield, 0.415 g (58%); m.p. 115 – 116°C; IR
spectrum, n , cm-1: 1637.11 (CH -C=O), 1670.95 (C=O),
max
3
1
1435.99 (bending CH ), 1245 (C–N). H NMR spectrum
2
(chloroform-d ) d, ppm: 6.20 (s, 1H, CHCl ), 4.19 – 3.17 (m,
3
2
8H, 2 NCH CH N), 2.13 (s, 3H, CH ). 13C NMR (chloro-
2
2
3
form-d ) d, ppm: 169.20, 162.39, 65.73, 65.43, 46.26, 46.10,
3
45.59, 43.11, 40.89, 40.73, 21.30.
2,2-Dichloro-1-(piperidin-1-yl)ethan-1-one (f7). White
solid; yield, 0.358 g (61%); m.p. 41 – 42°C; IR spectrum,
cm-1: 1667.04 (C=O), 1443 (bending CH ),
2
n
,
max
1245(C–N), 648.45 (C–Cl). 1H NMR spectrum (chloro-
form-d ) d, ppm: 6.22 (s, 1H, CHCl ), 3.96 – 3.23 (m, 4H,
3
2
CH NCH ), 1.93 – 1.25 (m, 6H, CH CH CH ). 13C NMR
2
2
2
2
2
tions (IC ). Each experiment was independently repeated
50
spectrum (chloroform-d ) d, ppm: 161.82, 65.78, 47.4, 44.38,
3
three times. The results are shown in Table 2, where all val-
25.85, 25.30, 24.08.
ues are presented as mean ± SEM.
2,2-Dichloro-1-(4-methylpiperidin-1-yl)ethan-1-one
(f8). White solid; yield, 0.567 g (90%); m.p. 44 – 45°C. IR
spectrum, v , cm-1: 1661.96(C=O), 2932(stretching CH ),
3. RESULTS AND DISCUSSION
max
2
1
1451 (bending CH ), 1253 (C–N), 654 (C–Cl). H NMR
2
In this study, nine piperazine and piperidine analogs of
DCA were synthesized. In order to determine the binding
spectrum (chloroform-d ) d, ppm: 6.22 (s, 1H, CHCl ), 4.45
3
2
(d, J = 13.5 Hz, 1H axial, NCH), 4.13 (d, J = 13.6 Hz, 1H ax-
ial, NCH), 3.09 (t, J = 12.1 Hz, 1H equtorial, NCH), 2.68 (t,
J = 11.8 Hz, 1H equtorial, NCH,), 1.69 (t, J = 12.3 Hz, 4H,
2CH ), 1.23 (ddd, J = 17.0, 12.6, 6.3 Hz, 1H, CHCH ), 0.95
2
3
TABLE 1. Docking Binding Energy (kcal/mol) of Synthesized
(d, J = 6.2 Hz, 3H, CH ). 13C NMR spectrum (chloro-
Compounds on PDK1-4 Isoenzymes
3
form-d ) d, ppm: 162.24, 66.23, 47.11, 44.19, 34.42, 33.88,
3
31.12, 21.94.
2,2-Dichloro-1-(4-phenylpiperidin-1-yl)ethan-1-one
(f9). White solid; yield, 0.775 g (95%); m.p. 74 – 75°C; IR
spectrum, n , cm-1: 1652 (C=O), 1451.87 (aryl C=C),
max
1
699.78 (C–Cl). H NMR spectrum (chloroform-d ) d, ppm:
3
Compound
Lowest binding energy DGb (kcal/mol)
7.90 – 6.77 (m, arom. 5H), 6.25 (s, 1H, CHCl ), 4.96 – 4.44
2
Receptor
2BU8
–4.27
–5.28
–5.54
–4.07
–4.31
–4.78
–4.81
–5.49
–5.76
–5.78
3D2R
–4.08
–4.19
–4.50
–3.46
–3.71
–4.65
–4.10
–4.68
–4.73
–4.92
1Y8O
–3.87
–5.68
–6.81
–4.68
–6.59
–9.77
–5.21
–5.31
–5.56
–6.85
2Q8H
–4.08
–4.77
–5.67
–4.19
–4.63
–5.31
–4.99
–6.11
–6.48
–6.02
(m, 1H axial, NCH), 4.59 – 4.12 (m, 1H axial, NCH),
3.53 – 3.04 (m, 1H, ph-CH), 2.99 – 2.55 (m, 2H equtorial,
NCH), 2.20 – 1.68 (m, 4H, 2CH ). 13C NMR spectrum (chlo-
DCA
f1
2
roform-d) d, ppm: 162.97,145.62, 129.17, 127.68, 66.89,
f2
48.07, 45.14, 43.32, 34.25, 33.62.
f3
2.3. Biological Activity
f4
f5
The cytotoxicity of all compounds was studied in vitro
by standard 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) assay. Human colon (HT-29) and hu-
man breast (MCF7) cancer cell lines for this test were ob-
tained from National Cell Bank of Iran (NCBI, Pasteur Insti-
tute, Tehran, Iran). HT-29 cell line was cultured in DMEM
f6
f7
f8
f9