tuned by synthetic or biosynthetic manipulation of the pyrrole
moiety.
Chloropyrrole-2-carboxylic acids (7,15)12,19
Trichloroacetylpyrrole (213 mg, 1 mmol) was dissolved in CHCl3
(5 mL), and cooled on ice. SO2Cl2 (84 mL, 1 mmol) was added
dropwise with stirring. Additional aliquots of SO2Cl2 were added
until no starting material remained. The solution was warmed
to room temperature over an hour. The reaction mixture was
quenched by adding ice, extracted with 50 mM Na2HPO4 (pH
7.0) until neutral, and the organic phase reduced to dryness under
vacuum. The solid residue was dissolved in 2 M NaOH (10 mL)
and stirred for 30 min at room temperature. The aqueous solution
was acidified to pH 1, extracted with Et2O (2 ¥ 20 mL), and
the organic extracts reduced to dryness. The crude product was
purified by HPLC (Zorbax StableBond 4.6 ¥ 250 mm 5 mm column,
5 mL min-1, isocratic MeCN/H2O (30%, 0.05% HCOOH) to yield
the two chlorinated products 7 (100 mg, 67%), and 15 (8.3 mg,
5%) as off-white solids.
Experimental
Auxarthron umbrinum DSM-3193 was obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ). All
media components were purchased from Sigma–Aldrich and
Oxoid. 4-Nitro-pyrrole-2-carboxylic acid (12) was purchased from
Fluorochem. The fluorinated pyrroles 9 and 16 were synthesised
according to literature procedures.13,14,18 All other chemicals were
purchased from Sigma–Aldrich and AGB, except as otherwise
specified. All solvents were HPLC grade and used without further
purification.
HPLC was carried out using a Varian Prostar system
consisting of two solvent delivery modules (210), diode ar-
ray detector (335), autoinjector (410) and fraction collector
(710). A Zorbax StableBond C-18 column, 9.4 ¥ 250 mm, 5
mm particle size, was used in all preparative HPLC separa-
tions. NMR was conducted using Varian Inova 300, 400, and
500 MHz spectrometers, and referenced to residual signals in
the solvent (for DMSO, dH 2.50 and dC 39.5 ppm), and CFCl3
for 19F. UV-vis spectra were recorded on a Thermo-Spectronic
Helios-b spectrophotometer. Low resolution mass spectra were
obtained using a Micromass Quattro mass spectrometer, and high
resolution spectra on a Micromass LCT time-of-flight mass spec-
trometer, both coupled to a Waters Alliance 2695 solvent delivery
system.
4-Chloro- (7) 1H NMR (d6-DMSO, 500 MHz) dH 12.57 (br s),
12.02 (br s), 7.07 (dd, J = 2.9, 1.7), 6.68 (dd, J = 2.3, 1.7); 13C
NMR (d6-DMSO, 125 MHz) dC 161.1, 122.8, 120.9, 113.2, 111.3;
ESI(-)MS m/z 144.2/146.2 (M - H), 100.1/102.1 (M - CO2H).
1
4,5-Dichloro- (15) H NMR (d6-DMSO, 300 MHz) dH 12.88
(br s), 6.80 (s); 13C NMR (d6-DMSO, 125 MHz) dC 160.1, 121.8,
116.4, 113.9, 108.8; ESI(-)MS m/z 178.3/180.3/182.3 (M - H),
134.2/136.2/138.2 (M - CO2H).
Bromopyrrole-2-carboxylic acids (8,13)12,20
Trichloroacetylpyrrole (213 mg, 1 mmol) was dissolved in CHCl3
(5 mL), and cooled on ice. Br2 (60 mL, 1 mmol) was added dropwise
with stirring. The solution was warmed to room temperature over
an hour. Additional aliquots of Br2 (60 mL) were added until MS
revealed that no starting material remained. The reaction mixture
was quenched by adding ice, extracted repeatedly with 50 mM
Na2HPO4 (pH 7.0) until neutral, and the organic phase reduced
to dryness under vacuum. The solid residue was dissolved in 2 M
NaOH (10 mL) and stirred (30 min, rt). The aqueous solution was
acidified to pH 1, extracted with Et2O (2 ¥ 20 mL), and the organic
extracts reduced to dryness. The crude product was purified by
HPLC (Zorbax StableBond 4.6 ¥ 250 mm 5 mm column, 3 mL
min-1, isocratic MeCN/H2O (35%, 0.05% HCOOH)) to yield 13
(8.2 mg, 4.4%) and 8 (123 mg, 66%) as off-white solids.
Cellular viability was assessed 48 h after addition of the
test compound using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) colorimetric assay. The assay is
based on the reduction of the soluble yellow tetrazolium MTT salt,
via the mitochondria of metabolically active cells, leading to the
precipitation of purple water-insoluble formazan crystals. Relative
numbers of viable cells are determined spectrophotometrically
following solubilisation of the formazan crystals in DMSO. Prior
to treatment, cells were seeded at a density of 10,000 cells per
well (For HeLa and A549) in 96-well plates, in a volume of
150 mL of medium per well. After 24 h the medium was aspirated
from cells and replaced with media containing the test compound
at concentrations ranging from 1 ¥ 10-4 M to 0.8 ¥ 10-6 M.
The plates were placed in the incubator for 48 h, after which
50 mL of a fresh sterile filtered solution of MTT (5 mg mL-1)
was added to each well of treated plates. The MTT solution was
prepared using sterile PBS and filter sterilised using a 0.2 mm filter
(Millipore). Plates were then returned to the incubator for 3 h.
The media/MTT solution was aspirated from the well, with care
taken not to dislodge the formazan crystals from the bottom of
each well. The crystals were dissolved following addition of 200 mL
DMSO to each well. Finally, absorbance of the resulting solution
was measured at 570 nm using a Wallac 1420 Multilabel HTS plate
reader (Wallac, MD, USA). Absorbance values in treated plates
were expressed as a percentage of untreated controls in order to
obtain percentage viability values. The percentage viability values
were plotted against the log of test compound concentration and
a sigmoidal log dose curve was calculated by non-linear regression
analysis using Graphpad Prism software version 5.0 for Windows
(Graphpad Software, CA, USA). From these curves, IC50 values
were obtained.
4-Bromo- (8). 1H NMR (d6-DMSO, 300 MHz) dH 12.58 (brs),
12.09 (brs), 7.09 (t, J = 1.5 Hz), 6.73 (br s); 13C NMR (d6-
DMSO, 75 MHz) dC 161.4, 124.3, 123.7, 116.1, 96.0; ESI(-)MS
m/z 188.1/190.1 (M - H), 144.0/146.0 (M - CO2H).
5-Bromo- (13). 1H NMR (d6-DMSO, 300 MHz) dH 12.43 (brs),
6.69 (d, J = 3.6 Hz), 6.18 (d, J = 3.6 Hz); 13C NMR (d6-DMSO,
75 MHz) dC 161.0, 125.3, 116.2, 111.6, 104.3; ESI(-)MS m/z
188.1/190.1 (M - H), 144.0/146.0 (M - CO2H).
4-Iodopyrrole-2-carboxylic acid (10)12,21
Iodine (120 mg, 0.47 mmol) was added in small portions to a
stirred solution of trichloroacetylpyrrole (110 mg, 0.52 mmol) and
AgNO3 (85 mg, 0.50 mmol) in CHCl3 (5 mL) at 0 ◦C. The reaction
mixture was allowed to warm to room temperature and stirred for a
further 2 h. The suspension was then filtered, washed with Na2SO3
solution (5 mL, 5%) and water (5 mL, ¥ 2), dried over MgSO4,
and concentrated under nitrogen. The residue was suspended in
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6306–6311 | 6309
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