E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
6507
1H); 7.66 (t, J = 7.4 Hz, 1H); 7.33 (t, J = 7.6 Hz, 1H); 2.74 (q,
J = 7.3 Hz, 2H); 1.31 (t, J = 7.2 Hz, 3H).
phate-buffered saline (pH 7.2) and swabbed across fresh TSA
plates.
4.6.16. Isopropyl o-nitrophenyl disulfide (9c)
4.7.2. Antimicrobial testing: Kirby–Bauer method
Chromatographed on silica with 3% dichloromethane in hex-
anes as eluent. Isolated 13.7 mg (60%) as a yellow oil. 1H NMR
(400 MHz, CDCl3): d 8.28 (d, J = 8.8 Hz, 1H); 8.23 (d, J = 8.8 Hz,
1H); 7.64 (t, J = 7.9 Hz, 1H); 7.31 (t, J = 7.6 Hz, 1H); 3.05 (s,
J = 6.8 Hz, 1H); 1.31 (d, J = 6.8 Hz, 6H).
Sterile saline (5 ml) was inoculated with a swab of bacteria, then
the concentration was adjusted to 0.5 McFarland standard. Bacterial
solution was then streaked across a TSA plate to give an even lawn of
bacteria. Sterile pipet tips (1–30 ll) were used to drill 6 mm wells
into the agar plate, then 20 ll of 1 mg/ml drug in DMSO was added
to the well. Plates were inoculated overnight at 37 °C.
4.6.17. Butyl o-nitrophenyl disulfide (9d)
Chromatographed on silica with 3% dichloromethane in hex-
anes as eluent. Isolated 12.9 mg (54%) as a yellow oil. 1H NMR
(400 MHz, CDCl3): d 8.29 (d, J = 8.0 Hz, 1H); 8.22 (d, J = 8.8 Hz,
1H); 7.64 (t, J = 7.8 Hz, 1H); 7.31 (t, J = 8.0 Hz, 1H); 2.86–2.79 (m,
1H); 1.75–1.60 (m, 1H); 1.58–1.50 (m, 1H); 1.28 (d, J = 7.2 Hz,
3H); 0.98 (t, J = 7.8 Hz, 3H).
4.7.3. Agar dilution minimal inhibitory concentration (MIC)
assay
A stock solution of 1 mg/ml of each disulfide was prepared in
DMSO. Freshly prepared Mueller-Hinton agar (1.5 ml) was added
to each well of a 24-well plate, and to each well was then added
a specified amount of the disulfide test solution in order to a give
a final drug concentration of 256 mg/ml down to 0.125 lg/ml.
One well within each series received 0.1 ml of DMSO as a blank.
The contents of each well were thoroughly stirred to evenly dis-
tribute the antimicrobial solution within the agar. Once the agar
completely solidified, 10 ll of Mueller-Hinton broth saline contain-
ing 0.5 McFarland standard of the test bacteria was pipetted on top
of each well and the plates were then incubated for 24 h at 37 °C.
Bacterial growth was assessed by visual observation of growth.
4.6.18. Methyl m-nitrophenyl disulfide (10a)
Isolated 7.6 mg (38%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.39 (s, 1H); 8.04 (d, J = 6.4Hz, 1H); 7.79 (d, J = 8.0 Hz,
1H); 7.49 (t, J = 8.0 Hz, 1H); 2.47 (s, 3H).
4.6.19. Ethyl m-nitrophenyl disulfide (10b)
Isolated 7.7 mg (36%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.41 (s, 1H); 8.02 (d, J = 7.2 Hz, 1H); 7.80 (d, J = 7.2 Hz,
1H); 7.47 (t, J = 7.2 Hz, 1H); 2.78 (q, J = 7.3 Hz, 2H); 1.32 (t,
J = 7.2 Hz, 3H).
4.7.4. FabH enzyme assay
FabH assays were carried out using a standard coupled trichlo-
roacetic acid precipitation assay which determines the rate of for-
mation of radiolabeled 3-ketoacyl ACP from malonyl ACP and
radiolabeled acetyl-CoA. In this coupled assay Streptomyces glau-
scescens FabD is used to generate the malonyl ACP substrate from
malonyl-CoA and Streptomyces glauscescens ACP. For inhibition
studies, A standard 20 ll reaction mixture of ecFabH (2 pmol of
monomer, 100 nM) and test compound (0.02 nmol, 1 lM for com-
pounds 15a and 15b; 0.1 nmol, 5 lM for compounds 8a–f) in
50 mM sodium phosphate buffer (pH 7.4) was incubated at room
4.6.20. Isopropyl m-nitrophenyl disulfide (10c)
Isolated 17.0 mg (75%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.41 (s, 1H); 8.01 (d, J = 8.0 Hz, 1H); 7.80 (d, J = 7.2 Hz,
1H); 7.46 (t, J = 7.2 Hz, 1H); 3.09 (septet, J = 6.8 Hz, 1H); 1.30 (d,
J = 6.8 Hz, 6H).
4.6.21. Butyl m-nitrophenyl disulfide (10d)
Isolated 12.6 mg (52%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.42 (s, 1H) 8.00 (d, J = 8.0 Hz, 1H); 7.80 (d, J = 8.0 Hz,
1H); 7.45 (t, J = 8.0 Hz, 1H); 2.88–2.83 (m, 1H); 1.73–1.68 (m, 1H);
1.57–1.50 (m, 1H); 1.28 (d, J = 7.2 Hz, 3H); 0.97 (t, J = 7.2 Hz, 3H).
temperature for 15 min prior to addition to
a solution of
[1-14C]acetyl-CoA (0.8 nmol, 40 lM) and MACP (0.2 nmol,
10 lM). The inhibition was measured using standard FabH as-
say.51 The % inhibition was determined by measuring FabH activ-
ity in the presence of test compound relative to a negative control
that has no inhibitor (100% activity). The assay was run in
duplicate.
4.6.22. Methyl 4-nitrobenzyl disulfide (11)
Prepared according to the general procedure outlined above for
the aryl–alkyl disulfides. Following chromatography (3:1 hexanes/
CH2Cl2), 3.1 mg (5%) was obtained as a pale yellow solid. 1H NMR
(200 MHz, CDCl3): d 8.20 (d, J = 8.7 Hz, 2H); 7.51 (d, J = 8.6 Hz,
2H); 3.94 (s, 2H); 2.15 (s, 3H).
Acknowledgment
We thank the NIH for supporting these studies through research
Grant R01 AI51351.
4.6.23. Methyl 2-(p-nitrophenethyl) disulfide (12)
Obtained 9.7 mg (15%) as a pale yellow oil. 1H NMR (200 MHz,
CDCl3): d 8.17 (d, J = 8.0 Hz, 2H); 7.37 (d, J = 8.0 Hz, 2H); 3.19–
2.85 (m, 4H); 2.43 (s, 3H). MS (EI) m/z: 229 (M+).
References and notes
1. Turos, E.; Konaklieva, M. I.; Ren, R. X.-F.; Shi, H.; Gonzalez, J.; Dickey, S.; Lim, D.
Tetrahedron 2000, 56, 5571.
4.7. General microbiological methods
2. Long, T. E.; Turos, E. Curr. Med. Chem. Anti-Infect. Agents 2002, 1, 251.
3. Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.; Shim, J.-Y.; Dickey, S.; Lim, D.
V.; Cannons, A. Bioorg. Med. Chem. Lett. 2002, 12, 2229.
4. Coates, C.; Long, T. E.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg. Med. Chem. 2003,
11, 193.
5. Long, T. E.; Turos, E.; Konaklieva, M. I.; Blum, A. L.; Amry, A.; Baker, E. A.;
Suwandi, L. S.; McCain, M. D.; Rahman, M. F.; Dickey, S.; Lim, D. V. Bioorg. Med.
Chem. 2003, 11, 1859.
6. Turos, E.; Coates, C.; Shim, J.-Y.; Wang, Y.; Leslie, J. M.; Long, T. E.; Reddy, G. S.
K.; Ortiz, A.; Culbreath, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg.
Med. Chem. 2005, 13, 6289.
7. Heldreth, B.; Long, T. E.; Jang, S.; Reddy, G. S. K.; Turos, E.; Dickey, S.; Lim, D. V.
Bioorg. Med. Chem. 2006, 14, 3775.
Staphylococcus aureus (ATCC 25923) and MRSA (ATCC 43300)
were purchased from ATCC sources. Bacillus bacteria from Sterne
spore vaccine was purchased from Colorado Serum Co., Denver, CO.
4.7.1. Culture preparation
From a freezer stock in tryptic soy broth (Difco Laboratories, De-
troit, MI) and 20% glycerol, a culture of each microorganism was
transferred with a sterile Dacron swab to TrypticaseÒ Soy Agar
(TSA) plates (Becton-Dickinson Laboratories, Cockeysville, MD),
streaked for isolation, and incubated at 37 °C for 24 h. A 108 stan-
dardized cell count suspension was then made in sterile phos-
8. Turos, E.; Long, T. E.; Heldreth, B.; Leslie, J. M.; Reddy, G. S. K.; Wang, Y.; Coates,
C.; Konaklieva, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg. Med.
Chem. Lett. 2006, 16, 2084.