364 Journal of Natural Products, 2009, Vol. 72, No. 3
O’Donnell et al.
Table 5. Short-Term Cytotoxicity Data for Compounds 1, 2, and Cisplatin (IC50) in Human Breast, Lung, and Colorectal Cancer Cell
Lines and Normal Human Fibroblast Cell Linea,b
selectivity index
human breast cancer (MCF7) human lung cancer (A549) human colorectal cancer (HT29) lung fibroblast (WI38) (WI38IC50/ MCF7 IC50)
1
2
0.35
0.39
0.76
0.22
0.78
2.47
1.84
0.8
0.89
1
2.3
2.3
1.4
c
cisplatin
3.27
a Data expressed in µM. b Cells were exposed to compounds for 96 h and stained with sulforhodamine B. c Not determined.
adapted as follows. The appropriate thiol (2.5 mmol) was dissolved in
water (5 mL) containing NaOH (0.10 g, 2.5 mmol, 1 equiv). S-Methyl
methanethiosulfonate (0.315 g, 2.5 mmol, 1 equiv) was then added,
and the solution stirred for 1 h at room temperature. The cloudy
suspension formed was extracted with dichloromethane (20 mL). The
resulting dichloromethane solution was then dried with anhydrous
sodium sulfate, filtered, and concentrated under reduced pressure to
afford the pure disulfide.
mented with 10% (v/v) oleic acid-albumin-dextrose-catalase (OADC;
Difco) and 0.05% Tween 80 with occasional stirring until the
midexponential phase (OD600 of 0.6) was attained. For the quality
control of the mycobacterial cultures, they were stained each time with
a modified Ziehl-Neelsen staining protocol using a Tb-color kit, Bund
Deutscher Hebammen Laboratory (Karlsruhe, Germany), followed by
bright field microscopy.
To measure anaerobic cidal activity of the compounds, M. tuber-
culosis H37Rv (ATCC27294) was cultured in a self-generated oxygen-
depletion model as described by Wayne28 using 19.5 × 145 mm tubes
with a magnetic stirrer. Tubes were sealed with Teflon-lined caps and
subsequently with paraplast and incubated for 3 weeks at 37 °C on a
magnetic stirrer. The tubes were opened in an anaerobic chamber and
diluted 10-fold into anaerobic Dubos medium, and 1 mL volumes were
treated with various concentrations of the compound in 24-well plates.
Control cultures were treated with DMSO. Positive and negative drug
controls were metronidazole and isoniazid, respectively. The plates were
sealed in anaerobic bags and incubated for 7 days at 37 °C. Serial
dilutions were subsequently plated on 7H11 Middlebrook agar to
monitor bacterial survival.
2-(Methyldithio)pyridine (4): pale yellow oil; UV (MeOH) λmax
(log ε) 283.0 (3.66), 237.5 (3.93), 201.0 (3.82) nm; IR (film) νmax 1571,
1559, 1444, 1415, 1307, 1274, 1143, 1112, 1082, 1042, 985, 953, 755,
716 cm-1; 1H NMR (500 MHz, methanol-d4) δ 8.39 (1H, dt, J ) 5.2,
4.8 Hz), 7.77 (2H, m), 7.17 (1H, dt, J ) 9.6, 8.4 Hz), 2.47 (3H, s); 13
C
NMR (125 MHz, methanol-d4) 161.2 (s), 150.6 (d), 139.2 (d), 122.3
(d), 120.8 (d), 23.4 (q); HRESIMS m/z 158.0103 [M + H]+ (calcd for
C6H7NS2 158.0098).
2-(Methyldithio)pyrimidine (5): yellow oil; UV (MeOH) λmax (log
ε) 238.5 (3.94) nm; IR (film) νmax 1558, 1546, 1372, 1189, 1166, 955,
1
800, 770, 741 cm-1; H (500 MHz, methanol-d4) δ 8.66 (2H, d, J )
4.8 Hz), 7.26 (t, J ) 5.2, 4.8 Hz), 2.54 s (3H, s); 13C NMR (125 MHz,
methanol-d4) δ 172.4 (s), 159.4 (d), 119.6 (d), 23.0 (q); HRESIMS
m/z 159.0051 [M + H]+ (calc for C5H6N2S2 159.0051).
For the spot-culture growth inhibition assay,20 bacilli in the culture
were serially diluted in order to give a final concentration of 103 cells/
mL. Then, 5 µL of the diluted culture was spotted onto 5 mL of
Middlebrook 7H10 agar medium, supplemented with 10% (v/v) OADC
in a six-well plate containing various concentrations of compounds 1
and 2, and incubated at 37 °C for 14 days. A well with no compound
as a positive control was also used. MIC was determined as the lowest
concentration at which there is no growth.
Sulforhodamine B Short-Term Cytotoxicity Assay. Short-term
growth inhibition was measured using the SRB assay as described
previously.29 Briefly, cells were seeded (4000 cells/wells) into the wells
of 96-well plates in DMEM and incubated overnight at 37 °C and 5%
CO2 to allow the cells to attach. Subsequently, cells were exposed to
freshly made solutions of compounds at increasing concentrations of
0.1 to 25 µM in quadruplicate and incubated for a further 96 h.
Following this, the cells were fixed with ice-cold trichloroacetic acid
(TCA) (10% w/v) for 30 min and stained with 0.4% SRB dissolved in
1% acetic acid for 15 min. All incubations were carried out at room
temperature. The IC50 value, the concentration required to inhibit cell
growth by 50%, was determined from the mean absorbance at 540 nm
for each drug concentration expressed as a percentage of the control
for untreated well absorbance.
Microarray Analysis. M. tuberculosis H37Rv (ATCC 27294) was
grown in 7H9 Middlebrook medium supplemented with 0.2% glycerol,
0.5% bovine serum albumin fraction V, 0.05% Tween 80, 0.08% NaCl,
and 0.2% glucose to an OD650nm of 0.3 and was treated with either 2,
5, or 10 µg/mL of compound 1 or an equivalent amount of DMSO for
6 h before harvesting cells for RNA isolation. RNA was isolated and
labeled cDNA synthesized, and microarray hybridizations and microar-
ray analysis were performed as described by Boshoff et al.30 Genes
that were predictive for various treatment groups were determined by
a class prediction software developed by Tibshirani et al.31
Maximum Tolerated Dose (MTD). C57BL/6 female mice were
orally administered (by gavage) a single dose of drug at 30, 100, or
300 mg/kg, using three mice per dose. Mice were observed postad-
ministration at 4 and 6 h and then twice daily for the duration of the
study (1 week).
2-(Methyldithio)quinoline (6): yellow oil; UV (MeOH) λmax (log
ε) 334.0 (3.84), 325.0 (3.79), 250.5 (4.39), 212.0 (4.62) nm; IR (film)
υ
max 1615, 1582, 1553, 1492, 1447, 1291, 1137, 1103, 1087, 952, 939,
851, 815, 778, 744 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.12 (1H, d,
J ) 8.8 Hz), 8.02 (1H, d, J ) 8.4 Hz), 7.87 (1H, d, J ) 8.8 Hz), 7.79
(1H, d, J ) 8.4 Hz), 7.71 (1H, t, J ) 8.4 Hz), 7.50 (1H, t, J ) 8.0 Hz),
2.56 (3H, s); 13C NMR (125 MHz, CDCl3) δ 160.4 (s), 148.3 (s), 137.1
(d), 130.3 (d), 128.5 (d), 127.9 (d), 126.4 (s), 126.2 (d), 117.3 (d),
23.5 (q); HRESIMS m/z 208.0256 [M + H]+ (calcd for C10H10NS2
208.0255).
2-(Methyldithio)benzene (7): pale yellow oil; UV (MeOH) λmax
(log ε) 239.0 (3.56), 205.0 (3.56) nm; IR (film) νmax 1576, 1475, 1437,
1
1305, 1136, 1067, 1022, 951, 735, 686 cm-1; H NMR (500 MHz,
CDCl3) δ 7.43 (2H, dd, J ) 5.2, 1.2 Hz), 7.25 (1H, dd, J ) 3.6, 1.2
Hz), 7.00 (2H, dd, J ) 5.2, 3.6 Hz), 2.55 (3H, s); 13C NMR (125 MHz,
CDCl3) δ 137.1 (s), 129.2 (d), 127.7 (d), 127.0 (d), 23.1 (q); ESIMS
m/z 156 [M + H]+ (calcd for C7H8S2 156).
2-(Methyldithio)thiophene (8): orange oil; UV (MeOH) λmax (log
ε) 311.5 (3.80), 240.0 (3.40), 203.5 (3.94) nm; IR (film) νmax 1397,
1327, 1303, 1250, 1214, 1133, 1083, 1023, 987, 950, 846, 742, 699
1
cm-1; H NMR (500 MHz, CDCl3) δ 7.55 (1H, dd, J ) 8.4, 1.2 Hz),
7.35 (1H, t, J ) 8.0 Hz), 7.26 (1H, bd, J ) 7.2 Hz), 2.46 (3H, s); 13
C
NMR (125 MHz, CDCl3) δ 136.6 (s), 134.2 (d), 131.0 (d), 127.8 (d),
23.3 (q); ESIMS m/z 142 [M + H]+ (calcd for C6H6S2 142).
Antibacterial Assay with Fast-Growing Mycobacterium species
and Staphylococcus aureus. Mycobacterium species were acquired
from the NCTC, Salisbury, U.K. Strains were grown on Columbia blood
agar (Oxoid) supplemented with 7% defibrinated horse blood (Oxoid)
and incubated for 72 h at 37 °C prior to minimum inhibitory
concentration (MIC) determination. S. aureus strains were the generous
gift of Dr. Edet Udo (XU212), Prof. Glenn W. Kaatz (SA1199B), and
Dr. Paul Stapleton (EMRSA-15). Antibacterial assays were performed
as previously described.5,27 Ethambutol and isoniazid were used as
positive controls for the mycobacteria, and norfloxacin and oxacillin
were used as positive controls for the staphylococci.
Determination of MIC Value in M. boWis BCG and in M.
tuberculosis Using Spot-Culture Growth Inhibition Assays. M. boVis
BCG was cultured at 37 °C in an incubator with rotation at two
revolutions per minute in Middlebrook 7H9 broth supplemented with
10% (v/v) albumin-dextrose-catalase (ADC; Difco) and 0.05% Tween
80 until the midexponential phase (OD600 of 1.0). M. tuberculosis
H37Rv was grown at 37 °C in an incubator as standing culture in 30
mL unbreakable universals containing Middlebrook 7H9 broth supple-
Evaluation of in Vivo Efficacy against M. tuberculosis. Eight- to
10-week-old female specific-pathogen-free C57BL/6-Ifngtm1ts mice
(gamma interferon gene-disrupted [GKO] mice) were purchased from
Jackson Laboratories, Bar Harbor, ME. Mice were infected via low-
dose aerosol exposure to M. tuberculosis Erdman using a Middlebrook
aerosol generation device (Glas-Col Inc., Terre Haute, IN), and the
short-course mouse model was performed as described previously. One
day postinfection, three mice were sacrificed to verify the uptake of