€
C.B. Rodl et al. / European Journal of Medicinal Chemistry 84 (2014) 302e311
309
resulting solid, N-(benzo[d][1,3]dioxol-5-ylcarbamothioyl)benza-
5.2.3. Cell preparation
Human PMNL were freshly isolated from leukocyte concentrates
mide (D, Scheme 2), was collected and washed with H2O, followed
by cold acetone, yield: 3.64 g (80%); 1H NMR (250 MHz, DMSO-d6)
obtained at Stadtische Kliniken Frankfurt Hochst (Frankfurt, Ger-
many). Leukocyte concentrates were prepared by centrifugation at
4000 ꢁ g for 20 min at RT. PMNLs were immediately isolated by
dextran sedimentation, centrifugation on Nycoprep cushions (PAA
Laboratories, Linz, Austria), and hypotonic lysis of erythrocytes as
described [63]. Cells were finally re-suspended in phosphate-
buffered saline, pH 7.4 (PBS) containing 1 mg/mL glucose (purity
of > 96e97%). Human Platelets were freshly isolated from platelet
concentrates obtained at the Deutsche Blutspendedienst (Frank-
furt, Germany). Platelets were re-suspended in PBS, pH 5.4 and
centrifuged at 1849 ꢁ g for 15 min at room temperature (RT). The
cells were re-suspended in PBS/NaCl (PBS, pH 5.4 and 0.9% NaCl, 1:1
dilution) and centrifuged at 1849 ꢁ g for 10 min at RT. Finally,
platelets were re-suspended in PBS pH 5.4.
€
€
d
¼ 12.64 (s, 1H, -CS-NH-Ph-OH), 11.44 (s, 1H, eCOeNHe), 8.03 (d,
J ¼ 7.7 Hz, 2H, 2H,6H-Ph), 7.70e7.63 (m, 3H, 3H,4H,5H-Ph), 6.63 (d,
1H, J ¼ 8.2 Hz, 7H-benzo[d][1,3]dioxol), 6.07 (s, 2H, -CH2-), 6.03(s,
1H, 6H-benzo[d][1,3]dioxol), 5.89 (d, J ¼ 8.4 Hz, 1H, 6H-benzo[d]
[1,3]dioxol); m/z ¼ 301.1 [MþH]þ [48].
5.1.1.3. 1-(Benzo[d][1,3]dioxol-5-yl)thiourea (category E, Scheme 2).
A
solution of 1.00 g (3.33 mmol) N-(benzo[d][1,3]dioxol-5-
ylcarbamothioyl)benzamide (D, Scheme 2) in 5 ml aqueous so-
dium hydroxide (2 M) was heated to 100 ꢀC for 1 h. The precipi-
tating solid, 1-(benzo[d][1,3]dioxol-5-yl)thiourea (E, Scheme 2),
was collected and washed with H2O, yield: 529 mg (81%). 1H NMR
(250 MHz, DMSO-d6)
d
¼ 9.51 (s, 1H, -NH-), 7.32 (br s, 2H, -NH2),
6.99 (s, 1H, 4H-benzo[d][1,3]dioxol), 6.86 (d, 1H, J ¼ 8.2 Hz, 7H-
benzo[d][1,3]dioxol), 6.68 (d, 1H, 6H-benzo[d][1,3]dioxol), 6.06 (s,
2H, -CH2-); m/z ¼ 197.2 [MþH]þ [48].
5.2.4. Determination of 5-LO product formation in intact cells
For whole-cell assay freshly isolated PMNL (5 ꢁ 106) were re-
suspended in 1 mL PBS, pH 7.4, containing 1 mg/mL glucose and
1 mM CaCl2 (PGC). After pre-incubation with the test compounds
for 15 min at 37 ꢀC, 5-LO product formation was stimulated by the
5.1.1.4. N-(Benzo[d][1,3]dioxol-5-yl)-4-(4-chlorophenyl)thiazol-2-
amine hydrobromide (category F, Scheme 2) e Compound 24.
A solution of 150 mg (0.64 mmol) 2-bromo-1-(4-chlorophenyl)-
ethanone (B, Scheme 2) and 126 mg (0.64 mmol) 1-(benzo[d][1,3]
dioxol-5-yl)thiourea (E, Scheme 2) in 2 ml ethanol was heated to
80 ꢀC for 30 min under microwave irradiation. The precipitated
white hydrobromide salt was collected, washed with H2O and cold
ethanol, yield: 182 mg (69%). 1H NMR (250 MHz, DMSO-d6)
addition of calcium ionophore A23187 (2.5
(20
M). After 10 min at 37 ꢀC, the reaction was stopped with the
addition of methanol (1 mL) [64]. HCl (30 L, 1 N), prostaglandin B1
(200 ng) and PBS (500 L) were added and 5-LO metabolites were
mM) and exogenous AA
m
m
m
extracted and analyzed by HPLC as described [65]. 5-LO product
formation was determined as nanograms of 5-LO products per
106 cells, which includes leukotriene B4 (LTB4) and its all-trans
isomers, and 5-H(P)ETE (5(S)-hydro(pero)xy-6-trans-8,11,14-cis-
eicosatetraenoic acid). Cysteinyl LTs C4, D4 and E4 were not detec-
ted, and oxidation products of LTB4 were not determined. Each
compound was tested at least three times, and the mean S.E. were
calculated. The direct 5-LO inhibitor BWA4C was used as control at
d
¼ 10.17 (s, 1H, eNHe), 7.90 (d, J ¼ 8.5, 2H, 2H, 6H-Ph-Cl),
7.50e7.46 (m, 3H, 3H,5H-Ph-Cl þ 5H-thiazole), 7.36 (s, 1H, 4H-
benzo[d][1,3]dioxol), 7.05 (dd, 1H, 4J ¼ 2.3 Hz, 3J ¼ 8.5 Hz, 6H-benzo
[d][1,3]dioxol), 6.89 (d, 1H, J ¼ 8.4 Hz, 7H-benzo[d][1,3]dioxol), 5.99
(s, 2H, eCH2e); 13C NMR (63 MHz, DMSO-d6)
d
¼ 163.6,148.6, 147.3,
141.6, 135.8, 133.3, 131.9, 128.6,127.2, 109.6,108.3, 103.3, 100.8, 99.5;
m/z ¼ 331.2 [MþH]þ; anal. calcd. for C16H11ClN2O2S$HBr: C (46.68)
H (2.94) N (6.80) S (7.79); found: C (46.63) H (3.02) N (6.87) S (7.75);
mp ¼ 251.0 ꢀC [48].
a concentration of 0.08
the literature.
mM resulting in an inhibition comparable to
5.2.5. Determination of 12-LO, COX-1 and 15-LO1 product
formation in intact cells
Determination of 15-LO1 product formation was performed in
PMNL preparations as described for 5-LO product formation in
intact cells. For determination of 12-LO and COX-1 product for-
mation 1 ꢁ108 freshly isolated platelets were re-suspended in 1 ml
of PGC buffer and were pre-incubated with the test compounds or
vehicle (DMSO) at the indicated concentrations for 15 min at 37 ꢀC.
12-LO and COX-1 product formation was stimulated by addition of
5.2. Biological assays
5.2.1. Materials
AA, calcium ionophore A23187, BWA4C, DMSO, Albumin from
bovine serum (fatty acid free), IL1b and trypan blue solution were
purchased from SigmaeAldrich (Munich, Germany). Rev-5901 and
zileuton were purchased from Cayman Chemical (Ann Arbor, USA).
HPLC solvents were purchased from Merck (Darmstadt, Germany).
RPMI 1660 and DMEM medium was purchased from Gibco/Invi-
trogen (Paisley, UK). Penicillin and streptomycin were purchased
from PAA laboratory GmbH (Pasching, Austria). Fetal calf serum
(FCS) was purchased from Biochrom AG (Berlin, Germany). Fresh
10 m
M AA. After 10 min at 37 ꢀC, the reaction was stopped with 1 ml
of ice-cold methanol. 12-LO products include 12(S)-hydro(pero)xy-
6-trans-8,11,14-cis-eicosatetraenoic acid (12-H(p)ETE) and elute as
one major peak. COX-1 product formation was measured as 12-
hxdroxy-hepta-decatrienoic acid (12-HHT). 15-LO1 products,
generated from 15-LO1 expressing eosinophils present in the PMNL
preparations, were 15(S)-hydro(pero)xy-5,8,11-cis-13-trans-eico-
satetraenoic acid, which elute as one major peak as well. Data
(mean S.E.; n ꢂ 3) are expressed as percentage of control (DMSO).
For 12- and 15-LO1, the known lipoxygenase inhibitor NDGA
€
blood cell concentrates were provided by Stadtische Kliniken
€
Frankfurt-Hochst and Deutscher Blutspendedienst (Frankfurt,
Germany).
5.2.2. Cell culture
(nordihydroguaiaretic acid, 100 mM) was used as control; for inhi-
The human leukemic monocyte cells U937 and human cervix
carcinoma cells HeLa were purchased from Deutsche Sammlung für
Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Ger-
many). U973 cells were maintained in RPMI 1660 and HeLa cells
were grown in DMEM (Dulbecco's modified Eagle's medium).
bition of COX-1 product formation acetylsalicylic acid (100
used.
mM) was
5.2.6. Determination of COX-2 derived PGE2 in supernatants of
HeLa cells
Complete culture media contained 10% FCS, 100
mg/mL strepto-
HeLa cells were seeded in 24-well plates at a density of
mycin and 100 U/mL penicillin. Cells were cultured at 37 ꢀC in an
atmosphere containing 5% CO2.
3 ꢁ 104 cells/well. After 24 h incubation COX-2 expression was
stimulated with culture media containing 1 ng/ml IL1b and 5 ng/ml