A. Luniwal et al. / Bioorg. Med. Chem. 20 (2012) 2950–2956
2955
commercially available molecules was examined. This library was
assembled from chemical structures that can potentially maintain
the key binding interactions that are present between ASADH and
its substrates (i.e., aspartyl phosphate and aspartyl b-semialde-
hyde). Each of these chosen molecules have at least one negative
charge-bearing functionality to support an electrostatic interaction
with one of the active site arginyl residues, and an appropriately
positioned second functional group to bridge across to the other
active site arginyl residue (Fig. 3).
4.3.2. General procedure for N-alkylation of phthalimides
N-Methylation of phthalimide derivative M11 was obtained by
nucleophilic displacement of iodide from alkyl iodide by deproto-
nated phthalimide. A mixture of appropriate phthalimide, iodoal-
kane, and potassium carbonate in DMF was stirred for 6–10 h at
70–110 °C. After completion, the mixture was poured into an ice/
water mixture. The aqueous phase was extracted with dichloro-
methane. The combined organic phase was washed with 0.1 HCl,
brine and was dried over anhydrous sodium sulfate. The desired
N-alkylated product was isolated using flash column
chromatography.
4
.3. Synthesis
Chemical reactions were conducted under nitrogen in anhy-
drous solvents unless stated otherwise. Anhydrous solvents were
purchased from commercial sources and were used without addi-
4
1
6
8
.3.2.1. 4-Nitro-N-methylphthalimide (M11m).
= 0.54 (EtOAc/hexanes (1:1)); 1H NMR ((CD
3 2
)
Mp = 163–
70 °C; TLC R
00 MHz) d 8.57 (1H, dd, J = 1.8, 8.4 Hz), 8.52 (1H, d, J = 1.2 Hz),
f
O,
tional purification except for: (i) Acetone (Me
2
CO) which was fur-
13
.12 (1H, d, J = 8.4 Hz), 3.15 (3H, s); C NMR ((CD
3
)
2
O, 150 MHz)
ther dried over 3 Å molecular sieves; and (ii) tetrahydrofuran (THF)
which was further distilled under nitrogen over sodium-benzophe-
none. All other reagents obtained from commercial suppliers were
used without further purification. Thin-layer chromatography
d 167.1, 166.8, 137.7, 134.6, 130.0, 125.0, 118.4, 24.4.
4.3.2.2. 4-Nitro-N-ethylphthalimide
120 °C; TLC R
600 MHz) d 8.62 (2H, m), 8.04 (1H, d, J = 8.4 Hz), 3.81 (2H, q,
(M11e).
Mp = 117–
1
f
3
= 0.66 (EtOAc/hexanes (1:1)); H NMR (CDCl ,
(
TLC) was done on 250 lm fluorescent TLC plates (Baker-flex, Silica
Gel IB-F from VWR International, LLC) and visualized by using UV
light or iodine vapor. Normal-phase flash and gravity column chro-
matography were performed using silica gel (200–425 mesh 60 Å
pore size) and ACS grade solvents. Melting points were determined
on an Electrothermal digital melting point apparatus and are
uncorrected. NMR spectra were recorded on either a Varian Ino-
va-600 spectrometer at 600 MHz, or a Unity-400 spectrometer at
1
3
J = 7.2 Hz), 1.31 (3H, t, J = 7.2 Hz); C NMR (CDCl
3
, 150 MHz) d
1
66.0, 165.7, 151.6, 136.6, 133.6, 129.1, 124.3, 118.5, 33.6, 13.7.
4
.3.3. General procedure for N-alkylation of benzimidazolinone
Additionally, the 5-nitro-2-benzimidazolinone was N-alkylated
through a sequential deprotonation and nucleophilic displacement
maneuver. The benzimidazolinone was first deprotonated by using
sodium hydride, which then performed a nucleophilic displacement
of the iodo group upon addition of the respective alkyl iodides
4
00 MHz. Peak locations were referenced using either tetramethyl-
silane (TMS) or residual nondeuterated solvent as an internal stan-
1
3
dard. C NMR chemical shifts are reported to the first decimal
place unless peaks are very close wherein for such instances values
are reported to a second decimal place.
(Scheme 2). To a mixture of 60% NaH in DMF, a solution of nitro-
benzimidazolinone in DMF was added under inert atmosphere.
The resulting mixture was stirred at rt for 30 min. To this mixture
appropriate iodoalkane was added. The reaction mixture was stir-
red at rt for 6–8 h. After completion, the reaction was quenched
with 0.1 N HCl. The aqueous phase was extracted with ethyl acetate.
The combined organic phase was washed with 5% sodium bicarbon-
ate, brine and was dried over anhydrous sodium sulfate. The desired
product was purified using flash column chromatography.
4
.3.1. General esterification procedure
Synthesis of methyl ester derivatives of M6, M7, and M17 was
carried out under catalytic esterification conditions as depicted in
Scheme 1. To a solution of the appropriate carboxylic acid in
methanol few drops of conc. sulfuric acid were added. Subse-
quently, the reaction mixture was refluxed for 6–10 h. After com-
pletion, the volatile solvents were evaporated under reduced
pressure and the residue was dissolved in water/ethyl acetate
mixture. The aqueous phase was extracted with ethyl acetate
and the combined organic phase was washed with 5% sodium
bicarbonate, brine, and was dried over anhydrous sodium sulfate.
After filtration the desired ester was obtained using flash column
chromatography.
4
.3.3.1.
4-Nitro-N,N-dimethylbenzimidazolinone
Mp = 200–204 °C; TLC = 0.27 (EtOAc/hexanes
2:1)) H NMR (CDCl , 600 MHz) d 8.13 (1H, dd, J = 1.8, 8.4 Hz),
.83 (1H, d, J = 1.8 Hz), 7.03 (1H, d, J = 8.4 Hz), 3.50 (3H, s), 3.49
(
M14m).
R
f
1
(
7
3
1
3
(
3H, s); C NMR (CDCl
3
, 100 MHz) d 154.7, 142.6, 135.0, 129.9,
1
18.4, 106.4, 103.2, 27.6, 27.5.
4
.3.3.2. 4-Nitro-N,N-diethylbenzimidazolinone (M14e).
Mp
4
.3.1.1. 5-Nitropyridine-2-methylcarboxylate (M6m).
= 0.5 (EtOAc/hexanes (1:1)); 1H NMR (CDCl
3
,
Mp =
1
= 134–138 °C, TLC R
4
7.03 (1H, d, J = 8.4 Hz), 3.98 (4H, m), 1.36 (6H, m); C NMR (CDCl
1
3
f
= 0.5 (EtOAc/hexanes (1:1)); H NMR (CDCl
3
,
1
6
56–159 °C; TLC R
00 MHz) d 8.53 (1H, s), 8.66 (1H, dd, J = 1.8, 8.4 Hz), 8.56 (1H, d,
f
00 MHz) d 8.08 (1H, dd, J = 2.0, 8.4 Hz), 7.89 (1H, d, J = 2.0 Hz),
1
3
13
3
,
J = 8.4 Hz), 4.08 (3H, s);
1
3
C NMR (CDCl , 150 MHz) d 153.8,
00 MHz) d 153.7, 142.3, 134.2, 129.0, 118.1, 106.4, 103.2, 36.36,
6.31, 13.5.
52.1, 153.6, 145.1, 138.4, 125.5, 53.6; [M+Na]/Z = 205.3.
4
1
6
.3.1.2. Pyridine-2,5-dimethylcarboxylate (M7m).
62–166 °C; TLC R
= 0.33 (EtOAc/hexanes (1:1); 1H NMR (CDCl
00 MHz) d 8.31 (1H, s), 8.45 (1H, dd, J = 1.8, 7.8 Hz), 8.22 (1H, d,
Mp =
4.4. Enzymatic assay
f
3
,
13
J = 1.8 Hz), 4.05 (3H, s), 4.00 (3H, s); C NMR (CDCl
64.9, 164.8, 151.8, 150.7, 138.3, 126.6, 124.7, 53.2, 52.3;
M+Na]/Z = 218.3.
3
, 150 MHz) d
The ASADHs fromS. pneumoniae andV. cholerae were cloned, ex-
pressed, and purified following our published procedures.26 After
concentrating, the enzyme was stored at À20 °C in 50 mM HEPES
1
[
(
pH 7) containing 1 mM EDTA and dithiothreitol (DTT). ASADH
4
.3.1.3. 5-[[(4-Nitrophenyl)amino]carbonyl]-1,3-benzenedim-
ethylcarboxylate (M17m). Mp = 225–228 °C; TLC R = 0.84
, 600 MHz) d 8.54 (1H, s), 8.52 (2H, m),
generates an aldehyde from an acyl phosphate by reductive
dephosphorylation as shown in Scheme 3. This is a reversible reac-
tion and, because of instability of aspartyl phosphate, the reverse
reaction is followed by monitoring the increase in the absorbance
of NADPH at 340 nm.
f
1
(
EtOAc); H NMR (CDCl
3
1
3
8
.38 (2H, d, J = 8.4 Hz), 8.08 (2H, d, J = 8.4 Hz), 3.37 (3H, s);
C
3
NMR (CDCl , 150 MHz) d 131.5, 128.3, 125.3, 125.2, 124.1, 89.2, 52.8.