H.S. Cardoso et al. / European Journal of Medicinal Chemistry 41 (2006) 408–416
415
were used without further purification. The strains of Salmo-
nella typhimurium were purchased from Xenometrix (San Die-
go, CA, USA).
was adjusted to alkaline pH with 2N NaOH solution and the
resulting suspension was extracted with diethyl ether. The or-
ganic phase was dried over magnesium sulfate, filtered, then
evaporated to dryness to yield intermediate 4 as a brown oil
1
(13.7 g, 95%); H-NMR data are reported as hydrochloride
5.2. Chemistry
salt. Free base 4 (13 g) was dissolved in ethanol (100 ml),
treated with an excess of diethyl ether saturated with dry, gas-
eous HCl. Recrystallization from diethyl ether/ethanol (8:2,
v/v) provided 5 (13g, 89%) as a white solid; 1H-NMR
(CDCl3) δ 8.36(s, 1H, CONH), 7.81(d,1H, J = 2.9 Hz, ArH),
7.72 (d, 1H, J = 7.9 Hz, ArH), 7.25 (d, 2H, J = 7.9 Hz, ArH),
6.92 (d, 2H, J = 7.9 Hz, ArH), 6.80 (d, 1H, J = 2.9 Hz, ArH),
3.39 (q, 2H, J = 6.9 Hz, CH2NH), 2.34 (s, 3H, CH3), 1.50 (q,
2H, J = 6.9 Hz, CH2CH2NH), 1.31 (m, 2H, J = 6.9 Hz,
CH2CH3), 0.85 ppm (t, 3H, J = 6.9 Hz, CH2CH3). MS m/z
299.2 (M+).
5.2.1. 5-amino-N-butyl-2-(p-tolyloxy)-benzamide (PA31)
1-hydroxybenzotriazole (HOBt) (14.7 g, 109 mmol) and di-
cyclohexylcarbodiimide (DCC) (22.5 g, 109 mmol) were
added to a solution of 2-chloro-5-nitrobenzoic acid 1 (20 g,
99.0 mmol) in anhydrous DMF (150 ml) at 0 °C. The resulting
reaction mixture was stirred for 30 min, and then n-butylamine
(10.8 ml, 109 mmol) was added. The reaction mixture was
stirred at 0 °C for 2 h and stored overnight at room tempera-
ture. N,N′-dicyclohexylurea (DCU) was filtered off, and the
DMF was evaporated in a vacuum. The residue was dissolved
in CH2Cl2, washed consecutively with brine, 1 N NaOH, brine,
1 N HCl and brine. The organic phase was dried over magne-
sium sulfate, filtered, concentrated in vacuum and, then the
residue was purified by chromatography on a silica gel column
(elution with diethyl ether/petroleum ether 7:3 v/v). Crystalli-
zation from diethyl ether/ethanol produced 19.6 g (77%) of
5.3. Instrumentation
Absorbance measurements were performed on a Shimadzu
model 1240 UV-Visible spectrophotometer. The HPLC system
consisted of two Shimadzu LC10AD solvent pumps and a
CTC Analytics PAL System autosampler. Mass spectra were
obtained using Applied Biosystems API 4000 electrospray io-
nization (ESI) spectrometer.
1
pure 2 as a yellow solid, m.p. 134 °C. H-NMR (CDCl3) δ
8.46 (d, 1H, J = 2.9 Hz, ArH), 8.19 (dd, 1H, J = 2.9 Hz,
ArH), 7.59 (d, 1H, J = 7.9 Hz, ArH), 6.23 (br, 1H, CONH),
3.48 (q, 2H, J = 6.9 Hz, CH2NH), 1.89 (q, 2H, J = 6.9 Hz,
CH2CH2NH), 1.44 (m, 2H, J = 7.9 Hz, CH2CH3), 0.98 ppm
(t, 3H, J = 6.9 Hz, CH2CH3). Next, a solution of sodium hy-
dride (60% in mineral oil, 1.87 g, 78.0 mmol) in dry dioxane
(100 ml) was added to p-cresol (5.57 g, 51.0 mmol) in an ice-
water bath under a nitrogen atmosphere. After the evolution of
hydrogen had ceased, the mixture was stirred at 70 °C for 2 h.
Then intermediate 2 (10.0 g, 39.0 mmol) in anhydrous dioxane
(80 ml) was added drop-wise, and the resulting reaction mix-
ture was stirred at 70 °C for 4 h and then overnight at ambient
temperature. The solvent was removed under reduced pressure
and the residue was dissolved in ethyl acetate and washed with
brine. The organic phase was dried over magnesium sulfate,
filtered, concentrated in vacuum and the residue purified by
chromatography on a silica gel column (elution with hexane/
ethylacetate 8:2 v/v) to produce intermediate 3 (15 g, 89%), m.
5.4. Mutagenicity assay (Ames test)
Mutagenicity was assessed in the Ames Salmonella assay
with strains TA98, TA100, TA102, TA1535 and TA1537. Re-
cipes for reagents and media were performed as described by
Mortelmans and Zeiger [18]. Bacteria were grown in nutrient
broth (25 g/l, #2 Oxoid) for 15 h, at 37 °C, and 120 rpm, to
give suspensions of 3 × 108 cells/ml (A550nm = 0.25; McFar-
land scale, bioMérieux, Lyon, France). Test compounds were
dissolved in dimethylsulfoxide (DMSO) to give solutions of
50, 25, 12.5, 6.2, 3.1 and 1.55 mg/ml. Assays without meta-
bolic activation (no S9) were performed mixing 20 μl of each
test substance solution with 500 μl of phosphate buffer (0.1 M,
pH 7.4) and 100 μl of bacteria suspension. After 30 min of
incubation, 2 ml of molten top agar supplemented with traces
of histine and biotine (50 μM each, final concentration) were
added, rapidly vortexed and poured on GM agar plates. As the
top agar hardened, plates were inverted and incubated for 48 h,
at 37 °C. Assays with in vitro metabolism were similarly per-
formed replacing phosphate buffer by an equal volume of
S9mix (10% v/v S9, 4.7 mM NADP, 6 mM D-glucose-6-phos-
phate, 19 mM MgCl2, 36 mM KCl, phosphate buffer 0.1 M
pH 7.4). Rat liver S9 was prepared as described by Ames et
al. [19], except that rats were treated for 3 consecutive days
with intraperitoneal injections of phenobarbital (100 mg/kg)
and β-naphthoflavona (80 mg/kg). Positive controls were per-
formed with strain-specific substances largely used in the
Ames assay [18]. Negative controls were performed with plain
DMSO. Experiments were run in triplicate. The results were
1
p. 77 °C. H-NMR (CDCl3) δ 9.12 (d, 1H, J = 2.9 Hz, ArH),
8.15–8.13(dd, 1H, J = 2.9 Hz, ArH), 7.69 (br, 1H, CONH),
7.29 (d, 2H, J = 7.9 Hz, ArH), 7.02 (d, 2H, J = 7.9 Hz, ArH),
6.82 (d, 2H, J = 7.9 Hz, ArH), 3.51 (q, 2H, J = 6.9 Hz,
CH2NH), 2.40 (s, 3H, CH3), 1.61 (q, 2H, J = 6.9 Hz,
CH2CH2NH), 1.41 (m, 2H, J = 7.9 Hz, CH2CH3), 0.93 ppm
(t, 3H, J = 6.9 Hz, CH2CH3), which was converted to 4 as fol-
lows: A suspension of (50 ml) of NaBH4 3.65 g (96.5 mmol) in
water was added to a suspension of Pd/C (0.59 g) in 40 ml of
water. The resulting reaction mixture was stirred at room tem-
perature under nitrogen for 10 min. Then 3 (15.82 g,
48.2 mmol) in 150 ml of methanol was added drop-wise, and
the mixture was stirred for 30 min. The reaction mixture was
filtered through a Celite. The solution was acidified with 1N
HCl to remove the excess of NaBH4. The acidified solution
Cardoso
