Journal of the Iranian Chemical Society
seeded into each well of a 96-well cell culture plate. After
12 h of incubation at 37 °C, the test compound (100 μM)
was added. After incubated for 48 h, cells were subjected
to the MTS assay. Compounds with a growth inhibition rate
of 50% were further evaluated at concentrations of 0.064,
0.32, 1.6, 8, 40 and 100 (μM) in triplicate, with cisplatin and
paclitaxel (Sigma, St. Louis, MO, USA) as positive controls.
The IC50 value of each compound was calculated with Reed
and Muench’s method [13]. The results are given in Table 4.
Experimental section
Materials and measurements
Unless otherwise stated, 2-hydro-6-methyl-nicotinonitrile
and 2-cyanopyridine, S-leucinol, S-phenylalaninol, (R)/(S)
-1-phenylethylamine and dimethyl dichloride silane were
purchased from Acros, Aldrich or Fluka, USA. Flash col-
umn chromatography was performed using Merck silica gel
(60, particle size 0.02–0.03 mm). 1H and 13C NMR spectra
were recorded using Bruker AM-500 and Bruker AM-600
spectrometers. Chemical shifts are reported in ppm (δ) with
the solvent relative to tetramethylsilane (TMS) employed
as the internal standard (residual CHCl3, δH 7.26 ppm;
CDCl3, c 77 ppm). The following abbreviations were used
to designate multiplicities: s = singlet, d = doublet, t = tri-
plet, m = multiplet. Infrared spectra were recorded on a
Mattson Galaxy Series FTIR 3000 spectrometer; peaks are
reported in cm−1. Elemental analyses were performed on an
Elemental Analyzer AE-3000. High-resolution mass spec-
tra (HRMS) were obtained on a Micro GCT-MS equipped
with an EI ion source. Optical rotations were measured on
a WZZ-1 automatic polarimeter with a 2-cm cell recorded
at the sodium D-line.
Synthesis of compounds 1–5
2‑(4S‑Isobutyl‑4,5‑dihydro‑oxazol‑2‑yl)‑pyridine zinc,
complex (1)
Dry ZnCl2 1.5603 g (11.45 mmol), 2-cyanopyridine
1.0018 g (7.47 mmol) and l-leucinol 2.0789 g (17.7 mmol)
were added under anhydrous and oxygen-free conditions to
a dry 100-mL Schlenk fask. They were dissolved in 40 mL
of dry chlorobenzene, and the reaction mixture was refuxed
for 60 h. The solvent was removed under reduced pressure,
and the residue was dissolved in 15 mL of H2O and extracted
with 10 × 3 mL of dichloromethane. The solvent was
removed under vacuum, giving a crude red oil. Further puri-
fcation was performed on silica gel (petroleum ether/dichlo-
romethane 1/4). Colorless crystals were obtained in 58%
yield. [a]5D= + 20.83° (c = 0.288, CH3OH): δH (600 MHz,
DMSO-d6, 27 °C) 8.80–8.81 (m, 1H), 8.20–8.24 (m, 1H),
8.04–8.06 (m, 1H), 7.87–7.90 (m, 1H), 5.05–5.09 (m, 1H),
4.58–4.70 (m, 1H), 4.54 (t, J=8.6 Hz, 1H), 1.96–2.00 (m,
2H), 1.52–1.56 (m, 1H), 1.00–1.04 (dd, J=6.3 Hz, 6.4 Hz,
6H); δC (125 MHz, CDCl3) 166.1, 149.9, 141.3, 140.5,
129.8, 124.1, 78.4, 62.2, 44.1, 25.5, 22.7, 22.2. IR: 3445,
3071, 2957, 2870, 1652, 1592, 1493, 1474, 1442, 1404,
1389, 1298, 1280, 1160, 10,921, 1050, 1019, 941, 833, 753,
664, 636, 459; Elemental analysis for C24H32Cl4N4O2Zn2:
Found: C: 42.51, H: 5.10, N: 8.02%; Calculated: 42.32, H:
4.74, N: 8.23%.
X‑ray analyses
X-ray crystal data were collected on a Bruker SMART dif-
fractometer equipped with graphite monochromatic MoKα
radiation (λ=0.7103 Ǻ). The structure was solved by full-
matrix least squares on F2 using the SHELXTL program.
All non-H atoms were refned with anisotropic thermal
parameters. All hydrogen atoms were located theoretically
and refned with riding model position parameters and fxed
isotropic thermal parameters. Crystallographic parameters
Cytotoxicity assay
Leucinol hydrochloride, compound 2
The human tumor cell lines SMMC-7721 (Liver cancer) was
used in the cytotoxic assay. These cell lines were obtained
from ATCC (Manassas, VA, USA). Cells were cultured in
RMPI-1640 or DMEM (Biological Industries, Kibbutz Beit
Haemek, Israel) supplemented with 10% fetal bovine serum
(Biological Industries) at 37 °C in a humidifed atmosphere
with 5% CO2. The cytotoxicity assay was evaluated by the
MTS (Promega, Madison, WI, USA) assay.
This was prepared using the same procedure described
above for compound 1. Further purifcation was performed
on silica gel (petroleum ether/dichloromethane 1/9) to obtain
the colorless crystals of second component, yield: 15%;
m.p.: 68–70 °C, [a]D5 =−10.2° (c 0.098, CH3OH); 1H NMR
(500 MHz, CDCl3) 7.90 (s, 1H), 5.27 (s, 1H), 3.54 (s, 1H),
3.04 (s, 1H), 1.66 (s, 1H), 1.35 (s, 2H), 0.84 (s, 6H); 13C
NMR (75 MHz, CDCl3) 62.7, 52.9, 39.7,25.5, 24.4, 23.9;
calculated: C, 46.90; H, 10.50; N, 9.12%; found: C, 46.68;
H, 10.66; N, 8.98%; IR (KBr): 3415, 3291, 3196, 3062,
2929, 2855, 1586, 1535, 1497, 1454, 1378, 1266, 1195,
1156, 1056, 1011, 901, 846, 760.
The cytotoxicity assay was evaluated by the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Pro-
mega, Madison, WI, USA) assay [12]. Briefy, cells were
1 3