SARs in PAF
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2153
temperature, the mixture was refluxed for 4 h. After adding
EtOH (2 mL), the solution was washed twice with water, dried
(MgSO4), filtered and concentrated in vacuo. The residue was
chromatographed on a silica gel column using MeOH/CH2Cl2,
0.5:99.5, v/v as eluent to yield 2.44 g (49%) of the amide 11:
Rf 0.36 (CH2Cl2/MeOH, 95:5, v/v); IR (ν cm-1) 3383 (NH), 2955
magglutinin (PHA)-P-activated umbilical blood mononuclear
cells (UBMC). The viral stock was ultracentrifugated to
eliminate soluble factors such as cytokines, resuspended in
RPMI 1640 and titrated using PHA-P-activated PBMC. Fifty
percent (50%) tissue culture infectious doses (TCID50) were
calculated using Ka¨rber’s formula.31
5. An tivir a l Assa y. One million MDMs were pretreated 1
h in the presence of different concentrations of compounds and
infected with 10 000 TCID50 (0.01 moi, multiplicity of infection)
of the HIV-1/Ba-L strain. After 24 h, MDM were washed once
to eliminate excess virus and fed with fresh medium A. Twice
a week, supernatants were removed and stored at -20 °C in
order to measure viral replication by RT activity dosage. Cell
culture medium and drugs were then renewed, and cells were
microscopically observed to assess possible drug-induced cy-
totoxicity. Moreover, at the end of the culture, cytotoxicity was
confirmed using neutral red assay and, for PMS 601, by testing
until 1 mM dose.
1
(ArCH), 1746 (CdO), 1603 (ArCdC); H NMR δ 7.2 (m, 10H,
ArH), 6.4 (s, 1H, NH), 3.8 (m, 2H, CH2NCdO), 3.3 (m, 5H,
CH2Ph and piperazine CH), 2.7-2.2 (m, 6H, piperazine CH2),
1.2 (m, 9H, CH3).
2-(2,2-Dim eth ylpr opion ylam in om eth yl)piper azin e, Hy-
d r och lor id e (12). To a solution of amide 11 (2.44 g, 6.43
mmol) in EtOH (40 mL) and 12 N HCl (1 mL) was added 100
mg Pd/C, and this mixture was warmed (40 °C) with stirring
under hydrogen atmosphere. After disappearance of the start-
ing material (3 h) as shown by TLC, the suspension was
filtered and the catalyst washed several times with EtOH and
H2O. The solvents were evaporated and the residue crystal-
lized in MeOH/ether to give 1.2 g (68.6%) of 12: mp 278.6 °C;
Rf 0.13 (CHCl3/MeOH/NH4OH, 80:20:2, v/v/v).
6. Dosa ge of Vir a l Rep lica tion . HIV replication was
assessed by the dosage of RT activity in cell culture superna-
tants, as described elsewhere.32
2-(2,2-Dim eth ylp r op ion yla m in om eth yl)-1,4-d i(3,4,5-tr i-
m eth oxyben zoyl)p ip er a zin e (8). To a solution of diamine
hydrochloride 12 (1.2 g, 4.4 mmol) in Et3N (3 mL, 41 mmol)
and CH2Cl2 (50 mL) was added dropwise 3,4,5-trimethoxy-
benzoyl chloride (2.33 g, 10.1 mmol) in CH2Cl2 (30 mL). After
stirring overnight at room temperature, EtOH (2 mL) was
added and the solution was washed twice with water, dried
(MgSO4) and concentrated in vacuo. The residue was chro-
matographed on a silica gel column using 2% MeOH in CH2-
Cl2 as eluent to yield 0.9 g (34.6%) of 9: mp 146.5 °C; Rf 0.29
(CHCl3/MeOH, 95:5, v/v); IR (ν cm-1) 3391 (NH), 1740 (CdO
7. Da ta An a lysis. All experiments were repeated with cells
isolated from a second blood donor. Results were expressed
as the mean of RT activity ( standard deviation (SD). Fifty
percent (50%) inhibition concentrations (IC50) were calculated
using cumulative RT activity and microcomputer software (J .
and T. C. Chou, Biosoft, Cambridge, U.K.). Fifty percent (50%)
cytotoxic concentrations (CC50) were determined using the
same microcomputer software and DO values (neutral red
staining assay).
Ack n ow led gm en t. The authors thank the Centre
de Transfusion Sanguine des Arme´es (CTSA, Clamart,
France), the Service de Cytaphe´re`se de l’Hoˆpital Saint-
Louis (Paris, France), and the Maternite´ de Sainte-
Fe´licite´ (Paris, France) as well as N. Boggetto and Prof.
M. Reboud (Institut J acques Monod, CNRS-Universite´s
Paris 6 et 7, Paris, France) for the antiprotease assay.
This work was supported by the Scientific Council of
the Universite´ Paris 7 - Denis Diderot (Paris, France),
the Agence Nationale de Recherches sur le SIDA
(ANRS, Paris, France), the Fondation pour la Recherche
Me´dicale-Sidaction (Paris, France), the Institut de
Formation Supe´rieure Biome´dicale (IFSBM, Villejuif,
France), the Association Claude Bernard (ARC, Paris,
France), the Association Naturalia et Biologia (NEB,
Paris, France), and the Association pour la Recherche
en Neurovirologie (ARN, Griselles, France).
1
amide), 1608 (ArCdO), 1584 (ArCdC); H NMR δ 6.6 (s, 4H,
ArH), 6.4 (s, 1H, NH), 4.5 (m, 4H, CH2NCdO), 3.7 (s, 18H,
CH3O), 3.6 (m, 2H, CH2N), 3.3 (m, 3H, CH2 and CHNCdO),
1.1 (m, 9H, CH3).
Biologica l Meth od s. 1. P la telet Aggr ega tion . The inhi-
bition of platelet aggregation was determined using platelet-
rich plasma (PRP) of New Zealand rabbits by the method of
Cazenave et al.27 Blood samples were collected from auricular
artery into a citrate buffer (3.8%, pH 7.4), and PRP was
obtained by centrifugation for 15 min at 1200 rpm. The
antagonists were solubilized in EtOH at concentrations from
10-2 to 10-7 M and added to the incubated and stirred PRP
for 2 min before PAF (2.5 nM) challenge. Platelet aggregation
induced by PAF in the presence of the antagonists was
monitored by continuous recording of light transmission in a
dual-channel recorder (Cronolog Coultronics apparatus) and
was compared to a control aggregation induced by PAF alone.
The drug concentration required to produce 50% inhibition
(IC50) was calculated from dose-response curves (number of
determinations: 5-6).
Refer en ces
2. Mon ocyte-Der ived Ma cr op h a ge Isola tion . Human
peripheral blood mononuclear cells (PBMC) were obtained
from healthy HIV-, HCV-, HBV-seronegative blood donor by
Ficoll-Hypaque density gradient centrifugation (MSL 2000,
Eurobio, Les Ulis, France). Monocytes were isolated from
PBMC by countercurrent centrifugal elutriation as previously
described with an enrichment degree g95%.28,29 Freshly
isolated human monocytes were resuspended in medium A:
RPMI 1640 medium (Roche Diagnostics, Meylan, France)
supplemented with 10% heat-inactivated (56 °C for 30 min)
fetal calf serum (FCS) (Roche Diagnostics), 2 mM L-glutamine
(Roche Diagnostics), and 1% tri-antibiotic mixture (penicillin,
streptomycin, neomycin, PSN; Life Technologies, Grand Island,
NY). Cells were placed at 1 million cells/well of 48-well plates
(Becton Dickinson Labware, Lincoln Park, NJ ) and maintained
7 days in humidified 5% CO2 atmosphere to allow their
differentiation in MDM.
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3. Dr u gs. Drugs were solubilized in RPMI 1640 or dimethyl
sulfoxide (DMSO, Sigma) and conserved at -80 °C. Dilutions
were performed in cell culture medium.
4. Vir u s. MDM were infected with the reference macroph-
age-tropic HIV-1/Ba-L strain.30 In the Service de Neurovirolo-
gie, this virus was amplified in vitro using human phytohe-