T. Bodill et al. / Bioorg. Med. Chem. 21 (2013) 4332–4341
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4.1.1. 4-Chloro-N-(3-hydroxyphenyl)butanamide 9f
5.71 (2H, s, 2 ꢁ OH), 6.61 (1H, d, J = 8.2 Hz, 40-H), 6.89 (1H, d,
J = 7.6 Hz, 60-H), 7.11 (1H, t, J = 8.2 Hz, 50-H), 7.38 (1H, s, 20-H),
7.52 (1H, s, OH) and 8.82 (1H, s, NH); dC/ppm (100 MHz; DMSO-
d6) 20.7 (d, JP–C = 142.5 Hz, CH2P), 27.7 (d, JP–C = 3.7 Hz, CH2CO),
107.1 (C-20), 111.0 (C-40), 111.8 (C-60), 129.8 (C-50), 139.5 (C-10),
158.1 (C-30) and 169.3 (d, JPꢀC = 15.4 Hz, C@O).
To a stirred solution of 3-aminophenol (1.50 g, 14.0 mmol) in
THF (30 mL) under nitrogen was added NaH (60% dispersion in
mineral oil; 0.60 g, 24 mmol) in small portions to permit controlled
evolution of hydrogen. 4-Chlorobutanoyl chloride (1.18 mL,
14.0 mmol) was then added through a septum and the resulting
solution was stirred for ca. 6 h. The solvent was evaporated in va-
cuo and the residue dissolved in EtOAc (2 ꢁ 50 mL). The organic
solution was washed sequentially with satd aq NaHCO3
(2 ꢁ 100 mL), water (2 ꢁ 100 mL) and brine (2 ꢁ 100 mL). The
aqueous washings were extracted with EtOAc and the combined
organic solutions were dried (anhyd MgSO4). Evaporation of the
solvent in vacuo afforded 4-chloro-N-(3-hydroxyphenyl)butana-
mide 9f as a brown solid (2.15 g, 71%) mp 88–90 °C; (found: M+,
4.1.4. Sodium hydrogen [N-(3-hydroxyphenyl)carbamoyl]-
ethylphosphonate 17f
[N-(3-Hydroxyphenyl)carbamoyl]ethylphosphonic acid 14f
(0.15 g, 0.61 mmol) was treated with
a solution of NaOH
(1.1 mol) in EtOH (0.58 mL) and the mixture was stirred for
30 min. The solvent was removed in vacuo and the residue chro-
matographed [reverse-phase column chromatography; elution
with H2O/MeOH (1:1)] to yield sodium hydrogen [N-(3-hydroxy-
213.05711 C10H12ClNO2 requires: M+, 213.05566);
m
/cmꢀ1 3176
(OH) and 1662 (C@O); dH/ppm (400 MHz; CDCl3) 2.17 (2H, m,
CH2CH2Cl), 2.73 (2H, t, J = 6.8 Hz, CH2CO), 3.59 (2H, t, J = 6.4 Hz,
CH2Cl), 6.45 (1H, dd, J = 6.0 and 2.0 Hz, 40-H), 6.51 (1H, dd, J = 6.0
and 2.4 Hz, 60-H), 6.97 (1H, t, J = 8.0 Hz, 50-H), 7.48 (1H, t,
J = 1.2 Hz, 20-H), 7.65 (1H, s, OH) and 7.69 (1H, s, NH); dC/ppm
(100 MHz; CDCl3) 26.8 (CH2CH2Cl), 33.1 (CH2CO), 44.2 (CH2Cl),
106.4 (C-20), 109.4 (C-40), 111.2 (C-60), 129.7 (C-50), 139.8 (C-10),
158.2 (C-30) and 166.7 (C@O).
phenyl)carbamoyl]ethylphosphonate 17f as
a grey semi-solid
(0.12 g, 89%);
m
/cmꢀ1 3267 (OH), 1671 (C@O) and 1231 (P@O);
dH/ppm (400 MHz; D2O) 2.13 (2H, m, CH2P), 2.73 (2H, m, CH2CO),
6.59 (1H, d, J = 8.2 Hz, 40-H), 6.90 (1H, d, J = 7.6 Hz, 60-H), 7.12
(1H, t, J = 8.2 Hz, 50-H) and 7.35 (1H, s, 20-H); dC/ppm (100 MHz;
D2O) 20.6 (d, JP–C = 142.6 Hz, CH2P), 28.0 (d, JP–C = 3.6 Hz, CH2CO),
107.2 (C-20), 110.8 (C-40), 111.7 (C-60), 129.5 (C-50), 139.8 (C-10),
158.2 (C-30) and 169.5 (d, JPꢀC = 15.2 Hz, C@O).
4.1.2. Diethyl [N-(3-hydroxyphenyl)carbamoyl]ethylphospho-
nate 11f
4.2. Saturation transfer difference (STD) experiments
Triethyl phosphite (0.86 mL, 5.0 mmol) was added through a
septum to 3-chloro-N-(3-hydroxyphenyl)propanamide 8f (0.50 g,
2.5 mmol) under nitrogen in an oven-dried round-bottomed flask
equipped with a reflux condenser, and the resulting mixture was
refluxed for ca. 9 h during which time the reaction was monitored
by TLC. The cooled mixture was then stirred with hexane (20 mL)
for ca. 30 min followed by decantation of the hexane layer to re-
move the excess triethyl phosphite; this was repeated three times.
The crude product was purified by flash chromatography [on silica
gel; elution with hexane/EtOAc (3:1)], and subsequent evaporation
of the solvent in vacuo afforded diethyl [N-(3-hydroxyphenyl)car-
bamoyl]-ethylphosphonate 11f as a dark brown oil (0.29 g, 58%);
The STD experiments, conducted on a
Bruker Avance II⁄
600 MHz NMR spectrometer, were run for the different sets of li-
gands as follows: EcDXR, stored in sodium phosphate buffer (pH
7.0), was freeze-dried and re-suspended in D2O to make a final
concentration of 20
lM. Each set of ligands was dissolved in the
protein solution to give a final ligand concentration of 800
lM
and thus a protein: ligand molar ratio of 1:40. The STD experiment
was carried out using parameters optimized in a previous study in
our group.9 The saturating on-resonance and off-resonance pulses
were set at frequencies of 0.73 and 20 ppm, respectively, while cy-
cling between the on- and off-resonance phases was used to re-
duce the effects of changes in temperature or magnetic field
homogeneity. A 3-9-19 water suppression pulse was applied at
4.7 ppm and 6000 scans were acquired. The on- and off-resonance
spectra were subtracted from each other and processed using Bru-
ker Topspin 2.1 software.
(Found: M+, 301.11247 C13H20NO5P requires: M+, 301.10791);
m/
cmꢀ1 3261 (OH), 1671 (C@O), 1232 (P@O) and 1024 (P–OEt); dH/
ppm (400 MHz; CDCl3) 1.29 (6H, t, J = 6.8 Hz, 2 ꢁ CH3), 2.13 (2H,
m, CH2P), 2.68 (2H, m, CH2CO), 4.06 (4H, m, 2 ꢁ OCH2), 6.60 (1H,
d, J = 8.4 Hz, 40-H), 6.91 (1H, d, J = 7.6 Hz, 60-H), 7.08 (1H, t,
J = 8.0 Hz, 50-H), 7.36 (1H, s, 20-H), 8.44 (1H, s, OH) and 8.92 (1H,
s, NH); dC (100 MHz; CDCl3) 16.2 (d, JP–C = 6.0 Hz, 2 ꢁ CH3), 20.8
(d, JP–C = 142.5 Hz, CH2P), 27.6 (d, JP–C = 3.7 Hz, CH2CO), 62.2 (d,
JP–C = 6.6 Hz, 2 ꢁ OCH2), 107.2 (C-20), 111.1 (C-40), 111.7 (C-60),
129.4 (C-50), 139.1 (C-10), 157.4 (C-30) and 169.5 (d, JP–C = 15.6 Hz,
C@O); dP/ppm (162 MHz; CDCl3) 24.1 (P@O).
4.3. Expression and purification of EcDXR and PfDXR
EcDXR was expressed and purified according to standard pro-
cedures.25,24 In brief, XL-1 Blue competent cells were trans-
formed with EcDXR plasmid DNA. IPTG was used to induce
expression of the recombinant EcDXR gene. The enzyme was
then purified by a combination of Ni2+ affinity- and size exclu-
sion-chromatography. The protein was stored at ꢀ20 °C in a so-
dium phosphate buffer of pH 7. PfDXR purification was
undertaken in a similar manner to that of the EcDXR. However,
expression of PfDXR was undertaken using a codon harmonised
coding region, under conditions of strongly controlled transcrip-
tion. This was achieved by heterologous co-expression using the
lac repressor protein using Escherichia coli M15[pREP4] compe-
tent cells.26
4.1.3. [N-(3-Hydroxyphenyl)carbamoyl]ethylphosphonic acid
14f
Trimethylsilyl bromide (0.22 mL, 1.7 mmol) was added to
diethyl [N-(3-hydroxyphenyl)carbamoyl]ethyl-phosphonate 11f
(0.25 g, 0.83 mmol) in CH3CN (3 mL) and the mixture was heated
in the microwave apparatus set to deliver 100 W of power, with
a reaction temperature of 60 °C and reaction time of 10 min. After
completion, the mixture was cooled to room temperature, treated
with a 95:5 MeOH/H2O mixture and stirred for 30 min. The solvent
was removed in vacuo and the residue chromatographed [prepara-
tive layer chromatography; elution with hexane/EtOAc/MeOH
(1:1:1)] to yield [N-(3-hydroxyphenyl)carbamoyl]ethylphosphonic
acid 14f as a brown viscous liquid (0.12 g, 61%); (found: C, 44.27;
H, 4.98; N, 5.73%. C9H12NO5P requires C, 44.09; H, 4.93; N,
4.4. DXR inhibition assay
As described previously,10 preliminary screening of the ability
of the ligands to inhibit EcDXR and PfDXR was conducted using
an enzyme assay based on the spectrophotometric measurement
of the conversion of NADPH to NADP which occurs when DOXP 1
is converted to MEP by DXR.
5.71%);
(400 MHz; DMSO-d6) 2.12 (2H, m, CH2P), 2.70 (2H, m, CH2CO),
m
/cmꢀ1 3212 (OH), 1682 (C@O) and 1230 (P@O); dH/ppm