ACS Infectious Diseases
Article
resultant crude oil was dissolved in EtOAc, washed with water
(2×) and brine (2×), dried over anhydrous magnesium sulfate,
filtered, and concentrated under reduced pressure using rotary
evaporation. The crude mixture was purified by prep-HPLC
(C18-silica, 0% B to 95% B over 17 min, then 95% B to 100%
B over 8 min) to afford compound 15 in 84% yield (60 mg,
= 6.2 Hz, 2H), 2.54−2.36 (m, 5H), 1.64−1.31 (m, 8H). C
NMR (150 MHz, CDCl3): δ 173.36, 172.54, 171.85, 171.54,
165.46, 165.24, 162.38, 156.41, 151.68, 146.79, 146.62, 136.58,
136.37, 134.14, 129.22, 129.03, 128.75, 128.69, 128.55, 128.52,
128.45, 128.28, 128.21, 128.01, 127.99, 127.65, 127.63, 127.11,
124.43, 124.34, 123.22, 123.14, 122.98, 117.11, 116.99, 116.86,
116.73, 76.46, 76.38, 76.24, 71.28, 71.19, 66.49, 47.33, 45.54,
45.38, 45.05, 43.31, 39.30, 39.23, 38.99, 37.08, 36.39, 32.56,
31.64, 29.70, 28.93, 28.71, 28.64, 27.53, 26.74, 26.65, 26.45,
characterization data.
1
0.05 mmol). H NMR (600 MHz, CDCl3): δ 8.06−7.53 (m,
5H), 7.42−7.27 (m, 12H), 7.26−6.99 (m, 13H), 5.11−4.97
(m, 10H), 4.71 (d, J = 6.5 Hz, 2H), 3.72−3.64 (m, 1H), 3.59−
3.48 (m, 2H), 3.24−2.99 (m, 11H), 2.58−2.35 (m, 5H), 2.01
(s, 3H), 1.50 (m, 4H), 1.43−1.26 (m, 2H). C NMR (150
MHz, CDCl3): δ 177.46, 173.23, 172.72, 172.32, 171.24,
165.68, 165.47, 151.67, 146.81, 146.70, 136.34, 134.29, 129.16,
128.99, 128.77, 128.74, 128.69, 128.60, 128.28, 128.23, 127.63,
127.51, 126.98, 126.88, 124.45, 124.36, 123.19, 123.17, 123.07,
123.00, 117.09, 98.95, 71.27, 68.90, 67.93, 60.42, 53.44, 47.50,
45.51, 45.45, 44.92, 43.49, 39.45, 39.13, 38.98, 37.18, 37.09,
31.92, 31.57, 29.71, 29.36, 29.04, 28.78, 28.71, 28.50, 27.47,
26.58, 26.38, 25.91, 25.58, 25.26, 24.76, 23.77, 22.68, 22.16,
21.65, 21.04, 20.36, 18.75, 14.18, 14.11, 11.42. See Figures S13
Compound 3. To a stirring solution of compound 16 (10
mg, 0.008 mmol) dissolved in 1 mL MeOH under an Ar
atmosphere was added 10% Pd/C (10 mg) as a solid. The flask
was purged with a balloon of H2, and the resultant solution
stirred under an H2 atmosphere (1 atm). Upon confirmation of
reaction completion by LC-MS (∼1 h) the reaction mixture
was filtered, and the solution was concentrated under reduced
pressure via rotary evaporation. The resulting crude mixture
was purified by prep-HPLC (C18-silica, 0% B to 95% B over
17 min, then 95% B to 100% B over 8 min) to afford
1
Compound 2. To a stirring solution of compound 15 (60
mg, 0.05 mmol) dissolved in 2 mL of MeOH under an Ar
atmosphere was added 10% Pd/C (10 mg) as a solid in one
portion. The flask was purged with a balloon of H2, and the
resultant solution was stirred under an H2 atmosphere (1 atm).
Upon confirmation of reaction completion by LC-MS (∼1 h)
the reaction mixture was filtered, and the solution was
concentrated under reduced pressure using rotary evaporation.
The resulting crude mixture was purified by prep-HPLC (C18-
silica, 0% B to 95% B over 17 min, then 95% B to 100% B over
8 min) to afford compound 2 in 24% yield (8.6 mg, 0.01
mmol). 1H NMR (600 MHz, CD3OD): δ 7.25−7.18 (m, 2H),
6.95−6.89 (m, 2H), 6.75−6.66 (m, 2H), 3.50−3.43 (m, 6H),
3.13 (d, J = 6.8 Hz, 2H), 2.68 (dt, J = 18.4, 6.6 Hz, 2H), 2.54−
2.45 (m, 2H), 1.98 (q, J = 8.0, 7.5 Hz, 1H), 1.93 (s, 3H),
1.86−1.79 (m, 2H), 1.76−1.57 (m, 6H), 1.51−1.43 (m, 4H).
C NMR (150 MHz, CD3OD): δ 173.34, 172.46, 171.77,
170.17, 148.88, 145.95, 118.15, 117.17, 115.33, 48.42, 45.27,
42.91, 38.93, 38.86, 38.60, 38.39, 36.55, 36.07, 32.66, 31.38,
30.53, 28.60, 28.52, 27.86, 27.04, 26.25, 25.58, 24.61, 23.80,
23.48, 22.67, 22.20, 21.12, 18.79. HR-MS (m/z): [M + H]+
calcd. C32H45N5O10: 660.3166, found 660.3234. See Figures
S15−S18 for characterization data and purity analysis (>95%
by LC-MS).
compound 3 in 78% yield (4.2 mg, 0.006 mmol). H NMR
(600 MHz, DMSO): δ 7.42−6.56 (m, 6H), 3.38−3.09 (m,
3H), 3.06−2.90 (m, 3H), 2.72−2.64 (m, 1H), 2.42−2.16 (m,
2H), 2.03−1.63 (m, 1H), 1.60−1.10 (m, 16H). C NMR (150
MHz, DMSO): δ 173.21, 171.71, 163.80, 156.46, 150.05,
146.68, 137.60, 128.80, 128.17, 119.17, 118.53, 118.07, 117.52,
115.36, 65.65, 52.25, 51.81, 38.87, 36.98, 34.62, 32.91, 31.55,
30.99, 29.48, 29.24, 26.63, 25.27, 24.16, 23.24, 22.48, 17.28,
14.45, 12.81. HR-MS (m/z): [M + H]+ calcd. C33H48N6O10:
ization data and purity analysis (>90% by LC-MS).
Preparation of Fe(III) and Ga(III) Siderophore Com-
plexes. The Fe(III) complexes of Fim, Acb, and compounds
1−3 were prepared by treatment with Fe(acac)3 in MeOH,
respectively. Samples were concentrated under reduced
pressure using rotary evaporation, and the resulting residue
was triturated with Et2O to remove excess acac ligand.
Stoichiometric Fe(acac)3 was used to prepare 1:1 metal:ligand
complexes with Fim and compounds 1−3.11,41 We refer to
these metal complexes as Fim-Fe, 1-Fe, 2-Fe, and 3-Fe. A 2:1
ratio of Acb:Fe(acac)3 was used to form the corresponding
[Fe(Acb)2] complex, referred to simply as Acb-Fe. The
Ga(III) complexes of compounds 1−3 (1-Ga, 2-Ga, and 3-
Ga) were prepared analogously using stoichiometric Ga-
(acac)3. All siderophore metal chelation complexes were
prepared fresh directly before biological or biochemical studies.
A. baumannii Growth Studies with Metal-free Side-
rophores and Ferric Iron Complexes. Stock solutions of
each test compound (1, 1-Fe, 2, 2-Fe, 3, and 3-Fe) were
prepared in DMSO at a concentration of 10 mM. A 96-well
plate was filled with 50 μL of M9-succinate minimal medium
per well. The DMSO stock solutions were diluted in sterile
M9-succinate minimal medium providing a working concen-
tration of 250 μM test compound. A 50 μL aliquot of this M9-
succinate solution was added to the first row of a 96-well plate
preloaded with 50 μL of sterile M9-succinate medium in each
well. Each test compound was serially diluted 2-fold down the
row of each 96-well plate. An inoculum was made by adding
100 μL of 0.5 McFarland standard of A. baumannii ATCC
17978 prepared from a fresh overnight culture to 4.0 mL of
sterile M9-succinate minimal media supplemented with 350
μM 2,2′-dipyridyl. Plates were inoculated by adding 50 μL of
Compound 16. Activated zinc (60 mg, 0.9 mmol) was
added to a stirring solution of compound 13 (44 mg, 0.04
mmol) in a 1:1 mixture of THF:AcOH (10 mL total volume)
at rt under an Ar atmosphere. Cbz-beta-alanine-N-hydrox-
ysuccinimide ester (113 mg, 0.35 mmol) was added to the
mixture slowly. Upon reaction completion (as determined by
LC-MS, ∼ 3 h) the mixture was filtered to remove insoluble
zinc salts and the solvent removed under reduced pressure
using rotary evaporation. The resultant crude oil was dissolved
in EtOAc, washed with water (2×) and brine (2×), dried over
anhydrous magnesium sulfate, filtered, and concentrated under
reduced pressure using rotary evaporation. The crude mixture
was purified by prep-HPLC (C18-silica, 0% B to 95% B over
17 min, then 95% B to 100% B over 8 min) to afford
1
compound 16 in 19% yield (10 mg, 0.008 mmol). H NMR
(600 MHz, CDCl3): δ 8.09−7.88 (m, 3H), 7.75−7.59 (m,
2H), 7.48−7.28 (m, 30H), 7.19−7.06 (m, 4H), 5.18−5.02 (m,
12H), 4.73 (d, J = 6.7 Hz, 2H), 3.29−3.03 (m, 11H), 2.60 (d, J
J
ACS Infect. Dis. XXXX, XXX, XXX−XXX