606
J. Quiroga et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 603–606
Ho); 13C-NMR (100 MHz, CDCl3): d 106.7 (C-29), 116.6 (CN), 126.6 (C-
59), 127.6 (C-39), 128.2 (C-49), 129.3 (Co), 130.3 (Cm), 131.8 (Cp), 136.4
(Ci), 137.9 (=CH), 138.9 (C-2). Anal. Calcd. for C13H8ClNS 6ꢂ H2O:
C, 61.30; H, 3.56; N, 5.50. Found: C, 61.37; H, 3.51; N, 5.41.
with MOPS (Sigma). The starting inocula, prepared according to
reported procedures [13] were 16104 to 56104 CFU/mL. Micro-
titers trays were incubated at 358C for yeasts and hialophypho-
mycetes and at 28–308C for dermatophyte strains in a moist,
dark chamber. MIC values were recorded at 48 h for yeasts, and
at a time according to the control fungus growth, for the rest of
the fungi. The susceptibilities of the standard drugs amphoteri-
cin B (Sigma), ketoconazole (Janssen Pharmaceutica, Beerse, Bel-
gium) and terbinafine (Novartis, Buenos Aires, Argentina) were
defined as the lowest concentration of drug which resulted in
total inhibition of fungal growth.
For the assay, compound stock solutions were two-fold diluted
with RPMI from 250–0.98 lg/mL (final volume = 100 lL) and a
final DMSO concentration =1%. A volume of 100 lL of inoculum
suspension was added to each well with the exception of the ster-
ility control where sterile water was added to the well instead.
The MIC was defined as the minimum inhibitory concentration
of the compounds which resulted in total inhibition of the fun-
gal growth.
Z-3-(4-Chlorophenyl)-2-(3-thienyl)acrylonitrile 8a
This compound was obtained according to general procedure as
white solid. Mp. 107–1088C: Yield 50%. MS EI (30 eV) m/z (%): 247/
245 (24/64) [M+], 211 (17), 210 (100) [M+-Cl], 209 (48), 177 (8), 166
1
(13). H-NMR (400 MHz, CDCl3): d 7.36 (dd, 1H, H-59), 7.38–7.46
(m, 4H, =CH, H-49, Hm), 7.61, (dd, 1H, H-29), 7.78 (d, 2H, Ho); 13C-
NMR (100 MHz, CDCl3): d 107.4 (C-19), 117.6 (CN), 121.6 (C-29),
124.0 (C-39), 124.1 (C-49), 129.3 (Cm), 130.3 (Co), 132.1 (Cp), 136.3 (Ci),
138.8 (=CH). Anal. Calcd. for C13H8ClNS 6ꢂ H2O: C, 61.30; H, 3.56;
N, 5.50. Found: C, 61.75; H, 3.55; N, 5.37.
Compounds 7b–k and 8b–e were prepared by adopting the
same procedure.
Cytotoxicity assays
The cytotoxic activity was determined according to the method
of Monks et al. [12]. Briefly, cells breast (MCF-7), lung (H-460); and
central nervous system (SF-268), (obtained from U.S. National
Cancer Institute) were counted, diluted with fresh medium, and
added to 96-well microtiter plates (100 lL/well) containing test
materials (1 mg in 100 lL in DMSO). Test plates were incubated
for 2 days at 378C in a 5% CO2 incubator. All treatments were per-
formed in duplicate. After the incubation periods, cells were
fixed by addition of 50 lL of cold 50% aqueous TCA solution (48C
for 60 min.), washed 4-5 times with tap water and air-dried. The
fixed cells were stained with 100 lL sulforhodamine B (SRB) (0.4%
wt/vol. in 1% acetic acid) for 15 min. Free SRB solution was then
removed by rinsing with 1% acetic acid (65). The plates were
then air-dried, the bound dye was solubilized with 100 lL of
10 mM tris-base, and absorbance was determined at 515 nm
using an ELISA plate reader (Model ELX-800, Bio-Tek Instruments,
Inc., Winooski, VT, USA). Finally, the absorbance values obtained
with each of the treatment procedures were averaged, and the
averaged value obtained with the zero-day control was sub-
tracted measuring in this way the relative cell growth or inviabil-
ity in treated and untreated cells. From the curves, growth inhib-
ition (or growth stimulation) and 50% inhibition of growth (GI50)
was calculated. Adriamycin was used as the reference compound.
References
[1] S. Naruto, H. Mizuta, T. Yoshida, H. Uno, et al., Chem.
Pharm. Bull. 1983, 31, 3022–3032.
[2] V. S. Parmar, A. Kumar, A. K. Prasad, S. K. Singh, et al.,
Bioorg. Med. Chem. 1988, 25, 911–914.
[3] S. A. Shiba, Phosphorus Sulfur 1996, 114, 29–37.
[4] P. Sanna, A. Carta, M. E. R. Nikookar, Eur. J. Med. Chem.
2000, 35, 535–543.
[5] P. Sanna, A. Carta, G. Paglietti, Heterocycles 1999, 51,
2171–2181.
[6] K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, et al., J.
Med. Chem. 1998, 41, 3022–3032.
[7] F. Saczewski, P. Reszka, M. Gdaniec, R. Grunert, P. I. Bed-
narski, J. Med. Chem. 2004, 47, 3438–3449.
[8] Z. Brzozowski, F. Saczewski, Eur. J. Med. Chem. 2002, 37,
709–720.
[9] V. N. Sonar, S. Parkin, P. A. Crooks, Acta Cryst. 2004, C60,
217–218.
Antifungal assays
Microorganisms and Media
[10] J. G. Stuart, M. J. Quast, G. E. Martin, V. M. Lynch, et al., J.
Heterocyclic. Chem. 1986, 23, 1215–1234.
For the antifungal evaluation, strains from the American Type
Culture Collection (ATCC), (Rockville, MD, USA) and CEREMIC
(C), (Centro de Referencia en Micologꢃa, Facultad de Ciencias Bio-
quꢃmicas y Farmacꢀuticas, Suipacha 531-(2000)-Rosario, Argen-
tina) were used: Candida albicans ATCC10231, C. tropicalis C131,
Saccharomyces cerevisiae ATCC9763, Cryptococcus neoformans
ATCC32264, Aspergillus flavus ATCC9170, A. fumigatus
ATTC26934, A. niger ATCC9029, Trichophyton rubrum C110, T.
mentagrophytes ATCC9972, and Microsporum gypseum C115.
[11] D. Cobo, J. Quiroga, J. Cobo, J. N. Low, C. Glidewell, Acta
Cryst. 2005, E61, 3639–3641; D. Cobo, J. Cobo, J. N. Low,
C. Glidewell, Acta Cryst., 2006, E62, 5179–5180; D. Cobo,
J. Quiroga, J. M. de la Torre, J. Cobo, et al., Acta Cryst.
2006, C62, 550–553.
[12] A. Monks, D. A. Scudiero, G. S. Johnson, K. D. Pauli, E. A.
Sausville, Anticancer Drug Design 1997, 12, 533–541; A.
Monks, D. Scudiero, P. Skehan, R. Shoemaker, et al., J.
Nat. Cancer Inst. 1991, 83, 757–766.
[13] NCCLS, National Committee for Clinical and Laboratory
Standards, 2002, Method M27-A2, Second Edition,
Wayne, Pa Vol. 22(15), pp. 1–29; NCCLS, National Com-
mittee for Clinical and Laboratory Standards, 2002,
Method M-38A, Second Edition, Wayne, Pa Vol. 22 (15),
1–27.
Antifungal susceptibility testing
The minimal inhibitory concentration (MIC) of each extract was
determined by using broth microdilution techniques following
the guidelines of CLSI (formerly NCCLS) for yeasts and filamen-
tous fungi (M27-A2 and M38-A) [13]. MIC values were determined
in RPMI-1640 (Sigma, St Louis, MO, USA) buffered to a pH 7.0
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim