High-Throughput Synthesis of LpxC Inhibitors
A R T I C L E S
General. (Polystyrene)tosyl chloride resin was obtained from
Argonaut Technology. HPLC analysis was performed with a Hewlett-
Packard series 1100 HPLC using an Econosphere C18 5U column (4.6
mm × 250 mm). HPLC conditions were as follows: 25 °C, flow rate
) 1 mL/min, 10 µL injection (as a solution in MeOH and CH3CN),
UV detection (220 nM). Gradient elution A: CH3CN (+0.1% TFA);
B: H2O (+0.1% TFA). T ) 0 min: 25% A, 75% B. T ) 10 min:
65% A, 35% B. T ) 13 min: 25% A, 75% B. NMR analysis was
performed using a Varian INOVA 300 MHz instrument. Direct probe
ionization (DIP) MS analysis was performed on an HP 5988A. FAB-
MS was performed using a JEOL SX102 instrument. Pyridine and CH2-
Cl2 were distilled from CaH2 immediately prior to use.
for C12H18N2O5 + 0.5H2O: C, 51.62; H, 6.86; N, 10.03. Found: C,
52.02; H, 6.54; N, 9.79.
General Procedure (A) for the Synthesis of 11{n} (n ) 1-28,
31-48, 50-61). The acid chloride (0.17 mmol) was dissolved in 0.5
mL of freshly distilled pyridine in a 10-mL scintillation vial. To this
was added a solution of 7 (50 mg, 0.18 mmol) and Et3N (0.17 mmol,
23 µL) in 1.5 mL freshly distilled pyridine. After stirring for 10 min,
the resulting solution was added to a 5-mL Quest 210 reaction vessel
containing 0.27 g (∼0.67 mmol) of polystyrene tosyl chloride resin.
The vessel was capped and placed in a freezer (-15 °C) for 24 h. The
vessel was then inserted into the Quest 210 synthesizer, and the resin
was washed with CH2Cl2 (2×), DMSO (2×), and CH2Cl2/MeOH (1:1,
7×). The resin was then treated with 3 mL of 49:49:2 pyridine:THF:
Et3N and agitated for 24 h at room temperature. The resin was filtered
and washed with CH2Cl2 (2×). The combined organic filtrates were
concentrated overnight on a speedvac (37 °C). The residue was
dissolved in 1 mL of CH2Cl2 and added to the top of a Celite cartridge
saturated with water. The Celite pad was washed with CH2Cl2 (2 × 2
mL), and the combined organic washings were concentrated on a
speedvac. The residue was dissolved in 800 µL CH2Cl2 and treated
with Me2EtSiH (100 µL) and TFA (100 µL). After allowing the reaction
to stand for 1 h, the solution was added to the top of a 1 in. silica pad
in a Pasteur pipet. After allowing the mixture to drain into the silica,
the pipet column was washed twice with 1.5 mL of 3:1 Hex/EtOAc.
The washings were discarded. The hydroxamate product was eluted
into a preweighed vial with 4 × 1.5 mL 2:1 CH2Cl2/EtOH. Concentra-
tion in a speedvac (37 °C) gave the desired oxazoline hydroxamates
11{n}.
O-(2,4-Dimethoxybenzyl)hydroxylamine (4). N-Hydroxyphthal-
imide (17.5 g, 107 mmol) and 2,4-dimethoxybenzyl alcohol (17.5 g,
107 mmol) were stirred in 700 mL of CH2Cl2 at 0 °C. Tributyl
phosphine (40.0 mL, 161 mmol) was added followed (slowly) by
diisoproyl azodicarboxylate (31.9 mL, 161 mmol). The solution was
stirred at room temperature for 24 h. The reaction was concentrated
and recrystallized from 1.5 L of boiling ethanol to give 28.0 g (85%)
of the desired intermediate as white crystals. This white solid was stirred
as a suspension in 800 mL of refluxing ethanol. N-Methylhydrazine
(5.20 mL, 100 mmol) was added, and the mixture was stirred at reflux
for 1 h. The solution was concentrated to remove the ethanol. Ether
(800 mL) was added, and the reaction mixture was allowed to stand at
room temperature for 30 min. The resulting solid was filtered. The
organic solution was concentrated to give 17 g of the desired product
as an oil (100% plus minor amounts of the phthalamide byproduct).
The product could be further purified by repeating the ether treatment.
The product thus obtained was identical to the previously reported
compound.
General Procedure (B) for the Synthesis of 11{n} (n ) 62-74).
Serine derivative 7 (50 mg, 0.19 mmol) and Et3N (26 µL, 0.185 mmol)
were stirred in 2 mL of dry DMF in a 10-mL vial. The acid chloride
(0.185 mmol) was added as a solution in 1 mL of CH2Cl2. After stirring
for 20 min, Burgess’ reagent (57 mg, 0.24 mmol) was added, and the
vial was sealed. The solution was heated in a Discover (CEM)
microwave at 200 W for 10 min (maximum temperature, 85 °C). After
cooling, the solution was poured over EtOAc (10 mL) and washed
twice with water. After drying over MgSO4, the mixture was purified
on a preparative TLC plate (3:1 EtOAc/Hex, UV or PMA stain; see
specific examples for Rf values). The resulting intermediate was
immediately treated with 1.5 mL of hexafluoro2-propanol and 30 µL
of anisole. TFA (50 µL) was added, and the deep red solution was
allowed to stand for 20 min. The resulting reaction mixture was added
slowly to the top of a 10-mL syringe containing 2 mL of polyvinylpy-
ridine above 1.5 mL of silica gel (prewet with CH2Cl2). Slow addition
of the solution was essential because silica gel in the presence of trace
amounts of TFA quickly decomposes the desired product. The column
was washed with 10 mL of CH2Cl2 and 4 mL of 1% EtOH in CH2Cl2.
The washings were discarded. The product was eluted into a preweighed
vial with 10 mL of 20% EtOH in CH2Cl2.
[1-(2,4-Dimethoxybenzyloxycarbamoyl)-2-hydroxy-ethyl]carbam-
ic Acid tert-Butyl Ester (6). Boc-D-Ser-OH (1.2 g, 6.0 mmol) and 4
(1.0 g, 6.0 mmol) were dissolved in 50 mL of CH2Cl2. After stirring
briefly, EDC (ethyldimethylaminopropyl carbodiimide, 1.5 g, 7.8 mmol)
was added, and the solution was stirred overnight. The solution was
washed once with water, dried over MgSO4, filtered, and concentrated.
The desired product could be purified by chromatography (3:2 EtOAc/
Hex, Rf ≈ 0.17) to give 1.3 g (64%) to give 6 as a white solid.
Alternatively, recrystallization of the crude product from ethanol/water
gave 58% of 6; mp 115-116 °C. IR (thin film) 3305, 2982, 1668,
1
1616, 1511, 1292, 1162 cm-1. H NMR (CD3CN) δ 9.17 (1 H, bs),
7.24 (1 H, d, J ) 8.7 Hz), 6.48-6.45 (2 H, m), 5.53 (1 H, bs), 4.91 (2
H, s), 4.11-3.98 (2 H, m), 3.84 (3 H, s), 3.81 (3 H, s), 3.66-3.58 (1
H, m), 3.02 (1 H, bs), 1.43 (9 H, s). 13C NMR (CDCl3) δ 168.66, 161.83,
159.55, 156.12, 132.85, 116.04, 104.34, 98.77, 80.80, 73.53, 62.93,
55.82, 55.65, 53.55, 28.64. FAB MS (M): 370. GCMS (M + H): 371.
HRMS m/z calcd for C13H17N2O6 (M-tert-butanoate): 297.1087, found
297.1085.
2-Amino-N-(2,4-dimethoxybenzyloxy)-3-hydroxypropionamide (7).
Serine derivative 6 (0.80 g, 2.2 mmol) was stirred in 16 mL of CH2Cl2
under N2. After cooling to 0 °C, 2,6-lutidine (1.2 mL, 11 mmol) was
added followed by TMS-triflate (1.6 mL, 8.6 mmol). The solution was
warmed to room temperature and stirred for 1.5 h. Methanol (40 mL)
was added followed by Et3N (8 mL), and the stirring was continued
overnight. The solution was concentrated in a vacuum under gentle
heat. The desired product was isolated by chromatography (10% ) 20%
EtOH in CH2Cl2, Rf ≈ 0.05, stained with PMA) to give 0.44 g (76%
from 6) of 7 as a thick oil that solidified overnight; mp 122-124 °C.
IR (Nujol) 3444, 3217, 1614, 1593, 1509, 1464, 1290 cm-1. 1H NMR
(CD3CN) δ 7.22 (1 H, d, J ) 8.4 Hz), 6.54 (1 H, d, J ) 2.4 Hz), 6.49
(1 H, dd, J ) 2.4, 8.4 Hz), 4.80 (2 H, s), 4.48 (3 H, bs), 3.81 (3 H, s),
3.79 (3 H, s), 3.70-3.58 (2 H, m), 3.52 (1 H, t, J ) 4.6 Hz). 13C NMR
(CD3OD) δ 169.43, 160.74, 158.43, 131.72, 115.96, 104.38, 98.16,
71.28, 64.00, 55.43, 55.21, 54.83. FAB MS (M + H): 271. Anal. Calcd
General Procedure (C) for the Synthesis of 11{n} (n ) 29, 30,
49). Serine derivative 7 (0.050 g, 0.19 mmol) was dissolved in 2 mL
of pyridine. The corresponding benzoic acid (0.19 mmol) was added
followed by EDC (46 mg, 0.24 mmol). The solution was stirred for 10
min and then evaporated under gentle heat (∼55 °C). The residue was
partitioned between EtOAc and H2O (15 mL each), and the organic
layer was washed twice with water. The solution was dried over MgSO4,
filtered, and evaporated. The residue was dissolved in 2 mL of THF
and treated with Burgess’ reagent (57 mg, 0.24 mmol) and then heated
in a microwave reactor (Discover, CEM) to 100 °C at 80 W for 15
min. The residue was concentrated and purified by prep-TLC (3:1
EtOAc/Hex; see specific examples for Rf values.) The resulting product
was dissolved in 1 mL of CH2Cl2 and treated with 100 µL each of
Et3SiH and TFA. The solution was evaporated after standing for 10
min. A few drops of MeOH were added followed (slowly) by 2 mL of
ether. The desired product precipitated as a white or pink solid.
9
J. AM. CHEM. SOC. VOL. 125, NO. 6, 2003 1585