Pharmacophore of Leflunomide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2015
and using the same procedure as for compound 8c: mp 125-
127 °C dec; 1H NMR (DMSO-d6) δ 12.78 (s, 1H), 7.81 (m, 2H),
7.75 (m, 2H), 2.64 (m, 4H), 1.95 (m, 2H); 13C NMR (DMSO-d6)
δ 197.0, 169.9, 140.4, 126.6, 125.1, 124.4, 121.4, 103.4, 34.4,
18.9. Anal. (C14H12F3NO3) C, H, F, N.
activity was expressed as IC50, the concentration of compound
that exerted 50% inhibition of proliferation with respect to that
of untreated control cultures. All the experiments were
repeated at least three times.
In h ibition of Lym p h ocyte Activa tion An tigen s Ex-
p r ession . Human peripheral blood mononuclear cells (PBMC)
from healthy donors were separated by Ficoll-Hypaque density-
gradient centrifugation. The cells recovered were resuspended
in culture medium (CM): RPMI 1640 supplemented with 10%
fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin,
and 100 µg/mL streptomycin.
N-(4-(Tr iflu or om et h yl)p h en yl)-2-a cet oxyb en za m id e
(10). Acetylsalicylic acid (10 g, 55 mmol) was added to thionyl
chloride (8.1 mL, 110 mmol), and the suspension was refluxed
until complete dissolution of the acid. The yellow solution was
then cooled at room temperature, and thionyl chloride was
evaporated under reduced pressure. Crude acyl chloride was
dissolved in chloroform, and the solvent was removed under
reduced pressure to give a reddish oil. This procedure was
repeated three times. The oil was dissolved in dichlo-
romethane (100 mL) and the resulting solution slowly added
at 0 °C to a solution of 4-(trifluoromethyl)aniline (6.3 mL, 50
mmol) and triethylamine (23 mL, 166 mmol) in dichlo-
romethane (100 mL). The mixture was stirred overnight at
room temperature, and then the organic solution was washed
successively with 1 N HCl (2 × 200 mL), 1 N NaOH (200 mL),
and water (200 mL). The organic phase was dried with
anhydrous sodium sulfate and evaporated under reduced
pressure. The crude product was purified by flash chroma-
tography on silica gel (eluent n-hexane-ethyl acetate, 8:2),
giving 13 g (80%) of pure 10 as a white solid: mp 210-212
PBMC, suspended at 106 cells/mL in CM, were incubated
in 24-well plates (Costar, Cambridge, MA) with the tested
compounds and stimulated with optimal concentration of the
monoclonal antibody OKT3 (Ortho Diagnostic Inc., Raritan,
NJ ) for 24 h at 37 °C and 5% CO2. After incubation,
immunofluorescence staining procedure for CD2, CD25, and
CD71 antigens and flow cytometry analysis were performed
as previously described.25 The experiments were repeated at
least twice, and the difference in IC50 values was less than
10%.
An tigen -in d u ced P a w Ed em a In Th e Mou se. Groups
of four to eight female BALB/c mice (20-22 g; Charles River,
Calco, Italy) were immunized by two sc injections on days 0
and 7 of keyhole limpet hemocyanin (KLH, 100 µg/mouse;
Sigma Chemicals, St. Louis, MO) emulsified in 0.2 mL
complete Freund’s adjuvant (CFA, Sigma Chemicals, St. Louis,
MO). Nonimmunized mice (Control) were treated with CFA
alone. On day 14 immunised mice were challenged in the hind
footpad with 20 µg of KLH suspended in physiological solution.
Eight hours after antigen challenge the edema was quantified
by measuring the dorsal-ventral thickness of the footpad with
a scientific micrometer (Borletti, Italy). To evaluate the
immunosuppressive activity, the compounds were adminis-
tered daily from day 0 until day 13. 11, dissolved in DMSO,
was administered ip; leflunomide, suspended in carboxymeth-
ylcellulose, was administered po; cyclosporin A was dissolved
in physiological solution containing 18% ethyl alcohol and 2%
Tween 80 and administered ip. The potencies of the com-
pounds were expressed as percentage of inhibition of edema
formation calculated as follows:
1
°C; H NMR (DMSO-d6) δ 10.74 (s, 1H), 7.97 (d, 2H), 7.74 (d,
3H), 7.63 (t, 1H), 7.45 (t,1H), 7.30 (d,1H), 2.22 (s,3H). Anal.
(C16H12F3NO3) C, H, F, N.
N-(4-(Tr iflu or om et h yl)p h en yl)-2-h yd r oxyb en za m id e
(11). Imidazole (960 mg, 14 mmol) was added to 10 (13. g,
40 mmol) in methanol (300 mL) and the solution stirred at
room temperature for 2 h. The white precipitate was filtered
and dried, giving 7.3 g (65%) of pure 11 as a white solid: mp
212-214 °C; 1H NMR (DMSO-d6) δ 11.51 (s, 1H), 10.67 (s, 1H),
7.98 (m, 2H), 7.93 (dd, 1H), 7.75 (m, 2H), 7.47 (m, 1H), 7.02
(d, 1H), 7.00 (t, 1H); 13C NMR (DMSO-d6) δ 166.9, 158.1, 142.3,
134.0, 129.6, 126.3, 124.6, 124.2, 120.8, 119.5, 118.5, 117.4.
Anal. (C14H10F3NO2) C, H, F, N.
Con figu r a tion a l a n d Con for m a tion a l Stu d ies. 2, 3a ,b,
and 8a -c were studied with a systematic search involving all
the torsions with 30° steps. Tripos60 force field as imple-
mented in Sybyl6.2 (Tripos) was used without the electrostatic
component. The systematic search was set up to filter only
the conformers having potential energy within 20 kcal/mol of
the minimum. Each conformer was then submitted to geom-
etry optimization through semiempirical calculations (AM1
hamiltonian with keywords: MMOK GNORM)0). Geometri-
cal isomers and/or conformers of each compound with the
lower heats of formation were compared. The lowest energy
conformers from each compound were then fitted using as
reference atoms the six-membered H-bond pattern depicted
in Figure 3.
100 - (thickness of immunized animals treated with
compounds - thickness of naive animals)/
(thickness of immunized animals -
thickness of naive animals) × 100
Statistical analysis was carried out by unpaired T-test with
Welch correction using GraphPad 2.01 software (Intuitive
Software for Science Inc., San Diego, CA).
Refer en ces
Mixed Lym p h ocyte Rea ction (MLR) Assa y. Bidirec-
tional MLR using murine cells was adopted. Splenocytes were
prepared from BALB/c and DBA/2 mice (18-20 g) (Charles
River, Calco, Italy). Briefly, single-cell suspensions were
isolated from spleen by Ficoll-Hypaque (Biochrom, Berlin,
Germany) density gradient centrifugation; 5 × 104 splenocytes
of each preparation were cocultured for 96 h in triplicate in
96-well round-bottom plates (Nunc, Roskilde, Denmark) in the
absence or presence of serial dilutions of the tested compounds.
All compounds were dissolved in dimethyl sulfoxide (DMSO)
to a final 20 mM concentration and then diluted in the culture
medium used for the cell assay. To take into account a possible
interfering effect of DMSO, each experiment included a control
in which the DMSO concentration was made equal to those
in compound-treated cultures. Culture medium was RPMI
1640 (Biochrom, Berlin, Germany), supplemented with 5% of
fetal bovine serum (Hyclone Laboratories Inc., Logan, UT), 50
µM 2â-mercaptoethanol, 2 mM L-glutamine, 0.1 mM nones-
sential amino acids, 1 mM pyruvate sodium, 100 IU/mL
penicillin, and 100 µg/mL streptomycin. During the last 18 h
of culture, cells were pulsed with 1 µCi of [3H]thymidine
(Amersham, UK) and then harvested onto glass fiber filters
by means of a cell harvester. Radioactivity, proportional to
proliferation, was measured with a â-counter. Inhibitory
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