Toll-like receptor 2 antagonists. Part 1: Preliminary SAR investigation of novel synthetic phospholipids

Article abstract of DOI:10.1016/j.bmcl.2005.08.080

Novel synthetic phospholipid compound 1 was discovered to be an antagonist of human toll-like receptor 2 (TLR2) signaling. In a preliminary SAR campaign we synthesized several analogues of 1 and found that considerable structural changes could be made wit

Full text of DOI:10.1016/j.bmcl.2005.08.080

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5494–5498
Toll-like receptor 2 antagonists. Part 1: Preliminary
SAR investigation of novel synthetic phospholipids
Mark R. Spyvee,a,* Huiming Zhang,a Lynn D. Hawkinsa and Jesse C. Chowb
aLead Optimization Department, Eisai Research Institute, 100 Research Drive, Wilmington, MA 01887, USA
bTarget Identification and Assay Development Department, Eisai Research Institute, 4 Corporate Drive, Andover, MA 01810, USA
Received 5 July 2005; revised 26 August 2005; accepted 29 August 2005
Abstract—Novel synthetic phospholipid compound 1 was discovered to be an antagonist of human toll-like receptor 2 (TLR2)
signaling. In a preliminary SAR campaign we synthesized several analogues of 1 and found that considerable structural changes
could be made without loss of TLR2 antagonistic activity.
Ó 2005 Elsevier Ltd. All rights reserved.
The innate immune system is the first line of defense in
humans and protects the body against foreign microor-
ganisms, such as bacteria, fungi, viruses, mycoplasma,
and protozoae, resulting in the generation of a multitude
of inflammatory responses in the host. A family of in-
nate immune proteins named the toll-like receptors
(TLRs) can detect cellular components of invading
pathogens and elicit cellular signaling events that lead
to the elevation of proinflammatory mediators, includ-
ing IL-1, IL-6, IL-8, and TNFa.1 For example, TLR2
recognizes lipoproteins, peptidoglycan, lipoteichoic
acid, and zymosan, as well as lipopolysaccharide (LPS)
of Gram-positive bacteria; TLR3 recognizes dsRNA of
viruses; TLR4 recognizes LPS of Gram-negative bacte-
ria; TLR5 recognizes flagellin of motile bacteria;
TLR7 and TLR8 recognize ssRNA; and TLR9 recog-
nizes DNA rich in CpG motifs.1,2 Certain TLRs can
also heterodimerize with one another to provide addi-
tional selectively for ligand recognition; for example,
TLR2 cooperates with TLR1 to recognize Pam3CysSK4
(2), or with TLR6 to recognize lipopeptide (R)-MALP-
2.1 Hyperstimulation of TLR2-expressing immune cells
by microbial products can contribute to pathogen-
induced chronic inflammatory joint disease, Gram-
positive sepsis, and other inflammatory disorders.3,4
In light of the connection between TLR2 and a wide
range of inflammatory disorders, we decided to embark
upon a high throughput screening (HTS) campaign to
identify inhibitors of TLR2 signaling. At the onset of
our investigations there were, to our knowledge, no
reported small molecule TLR2 antagonists. Our HTS
assay system involved measurement of the inhibition
of stimulation of TLR2-transfected Hek293 cells, car-
rying an NF-kB reporter gene, with synthetic
Pam3CysSK4 (2) (Fig. 1).5 Our HTS effort culminated
in the discovery of phospholipid compound
1
(Fig. 2).6 Lead compound 1 had previously been syn-
thesized on an earlier in-house program investigating
TLR4 activity. It interested us that our newly discov-
ered TLR2 antagonist bore structural similarity to
the TLR2 agonist ligand 2 (i.e., a lipophilic compo-
nent, comprising three hydrocarbon chains, attached
through a linker to a lipophobic component). Owing
to the lability of the malonate stereocenter, 1 was pre-
pared and tested as a ca. 1:1 mixture of diastereomers.
In our early investigations, we were eager to test the
stereochemical significance of the two non-epimerizable
asymmetric centers of 1. Consequently, we synthesized
O
NH2
O
NH2
O
R1
S
O
R2
O
HN
O
O
H
H
H
R3
N
N
N
HO
N
H
N
H
O
O
O
O
HO
Keywords: Toll-like receptor; TLR2; TLR2 antagonist; Synthetic
phospholipid.
2 (R1 = R2 = R3
= )
nC15H31
NH2
NH2
*
Corresponding author. Tel.: +1 978 988 7088; fax: +1 978 794
1117; e-mail: mark_spyvee@eisai.com
Figure 1. Pam3CysSK4, 2, a highly potent TLR2 agonist.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.080

M. R. Spyvee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5494–5498
5495
O
O
t
CO2 Bu
CO2H
CO2Allyl
iii-vi
R1
O
i-ii
HO
HN
O
O
O
P
O
O
O
R3
CO2Et
BnO
HO
HO
N
H
AllylO
6
7
8
R2
nC11H23
)
O
OH
nC13H27, R2
O
nC13H27
NH2
1 (R1
=
=
nC7H15, R3
=
HN
vii-viii
HO
O
OH
TBDPSO
O
O
OH
10
Figure 2. Compound 1, a novel TLR2 antagonist.
nC7H15
O
11
nC11H23
nC7H15
nC13H27
ix-x
HN
HO
O
O
diastereomers 3–5 (Table 1). We found that the TLR2
antagonistic potency did not vary greatly between dia-
stereomers, the most potent being 1, with centers b and
c both having the (R)-configuration. The potential
stereochemical consequences to TLR4 activity were
also of interest to us so the diastereomers were tested
in an appropriate assay.7 In this case, we observed a
modest stereochemical significance with respect to
TLR4 antagonistic potency, with the (S,S)-diastereo-
mer being the most potent. It was also noted that with-
out exception these compounds exhibited greater
selectivity for TLR4 over TLR2. In addition to demon-
strating antagonism in Hek293 cells (Table 1), com-
pound 1 also showed antagonistic activity in human
peripheral blood mononuclear cells (PBMCs).8,9
12
nC7H15
O
NHBoc
O
O
nC11H23
xiii-xiv
O
nC13H27
O
NiPr2
NH2
O
HN
O
P
P
O
O
xi-xii
1
9
13
O
nC7H15
Scheme 1. Synthesis of the lead TLR2 antagonist 1. Reagents and
conditions: (i) CH2(CO2Et)CO2 tBu, piperidine, AcOH, PhH, reflux/
Dean and Stark; (ii) H2, Pd/C, MeOH (83% over two steps); (iii)
CH2@CHCH2Br, K2CO3, AcMe (77%); (iv) LiOH, THF, H2O (65%);
(v) CH2@CHCH2Br, K2CO3, nBu4NI, DMF (99%); (vi) TFA, Et3SiH,
n
DCM (96%); (vii) C13H27COCl, NaHCO3, THF (78%); (viii)
n
TBDPSCl, DMAP, NEt3, DCM (67%); (ix) C11H23CO2H, EDCI,
DMAP, DCM (80%); (x) TBAF, AcOH, THF (77%); (xi) 12,
pyridinium trifluoroacetate, DCM, À10 °C then 30% H2O2 (64%);
(xii) TFA, Et3SiH, DCM then NaHCO3; (xiii) 6, EDCI, NEt3, DCM
(41% over two steps); (xiv) Pd(PPh3)4, Ph3SiH, PPh3, THF (69%).
For the synthesis of 1, the requisite left-hand side frag-
ment 6 was prepared via a six-step sequence from com-
merically available 4-benzyloxybenzaldehyde 7 (Scheme
1). This involved a Knoevenagel condensation10 of 7
with ethyl tert-butyl malonate followed by catalytic
hydrogenation to give phenol intermediate 8. Etherifica-
tion of 8 with allyl bromide followed by saponification
of the ethyl ester, subsequent re-esterification with allyl
alcohol, and acid-promoted hydrolysis of the tert-butyl
ester gave intermediate 6. The right-hand fragment 9
was derived from amino diol 1011 over a separate six-
step sequence. This involved myristoylation of 10 with
subsequent selective mono-silyl protection of the pri-
mary hydroxyl to give 11. The remaining secondary
hydroxyl was then lauryolated and the silyl-protecting
group removed to give intermediate 12. Reaction of 12
with phosphorylating reagent 13,11 followed by oxida-
tive workup and subsequent Boc removal, afforded
advanced intermediate 9. Carbodiimide-activated cou-
pling of the left fragment 6 with the right fragment 9,
followed by deallylation, gave final product 1.
For the synthesis of diastereomer 3, the starting alcohol
11 was first subjected to a Mitsunobu12 procedure
(Scheme 2). The resultant inverted alcohol intermediate
was lauroylated and subsequently desilylated to give
alcohol 14. An identical sequence of steps to that used
for 1 was then employed to complete the synthesis of 3
via intermediate 15.
Diastereomers 4 and 5 were synthesized in analogous
fashion to 3 starting from intermediates 1611 and 17,11
respectively (Schemes 3 and 4).
The next phase of our SAR investigations involved
examination of the effects of replacing various linkage
Table 1. Diastereomers of 1
O
O
i-iv
nC11H23
HN
R1
O
v-vi
O
O
O
nC13H27
11
O
P
HN
a
O
O
c
O
R3
HO
N
H
b
HO
O
O
R2
14
OH
nC13H27, R2
O
nC7H15
O
1 (R1
=
=
nC7H15, R3
=
nC11H23
)
O
nC11H23
HO
nC13H27
O
NH2
HN
15
vii-viii
O
P
O
O
O
3
Compound
a
b
c
TLR2 inhibition TLR4 inhibition
IC50, lMa
IC50, lMa
O
nC7H15
1
3
4
5
R/S
R/S
R/S
R/S
R
R
S
S
R
S
S
R
3.07
6.46
0.35
0.16
0.08
0.25b
Scheme 2. Synthesis of the (R/S,R,S)-diastereomer 3. Reagents and
conditions: (i) DEAD, PPh3, 4-NO2(C6H4)CO2H, THF (91%); (ii)
6.90
22.00b
n
NaOMe, MeOH, THF (98%); (iii) C11H23CO2H, EDCI, DMAP,
DCM (96%); (iv) TBAF, AcOH, THF (98%); (v) 13, pyridinium
trifluoroacetate, DCM, MeCN, À10 °C then 30% H2O2; (vi) TFA,
Et3SiH, DCM then NaHCO3; (vii) 6, EDCI, NEt3, DCM (20% over
three steps); (viii) Pd(PPh3)4, Ph3SiH, PPh3, THF (40%).
a Values are means of at least two experiments.
b Value not reliable owing to partial insolubility under assay
conditions.

5496
M. R. Spyvee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5494–5498
O
appear to reduce potency, though only slightly (com-
O
O
nC11H23
pound 27). More interestingly, analogues 28 and 29
showed that when the X3/X4 functionality was changed
from an ether to either an amide or an ester this gave
moderate improvements in potency. The synthetic
routes to the aforementioned linkage analogues are as
described below.
nC13H27
O
nC13H27
HN
HN
i-iv
TBDPSO
OH
HO
O
O
16
18
nC7H15
nC7H15
O
O
nC11H23
nC13H27
O
v-vi
vii-viii
NH2
HN
O
P
4
O
O
O
nC7H15
19
Synthesis of des-carboxy analogue 25 was carried out
according to the route outlined in Scheme 5. Phenol
30 was subjected to silylation, carbonyl reduction, and
subsequent bromination. The intermediate bromide
was then displaced with sodio diethylmalonate, desily-
lated with TBAF, etherified under Mitsunobu condi-
tions,12 and then saponified to give malonic acid
derivative 31. Copper-catalyzed decarboxylation of 31
gave propionic acid derivative 32, which was taken
through a sequence similar to that described earlier to
give 25.
O
Scheme 3. Synthesis of the (R/S,S,S)-diastereomer 4. Reagents and
conditions: (i) DEAD, PPh3, 4-NO2(C6H4)CO2H, THF (77%); (ii)
n
K2CO3, MeOH, THF (98%); (iii) C11H23CO2H, EDCI, DMAP,
DCM (98%); (iv) TBAF, AcOH, THF (94%); (v) 13, HOAc, MeCN,
DCM, H2O2; (vi) TFA, Et3SiH, DCM then NaHCO3; (vii) 6, EDC,
DCM (15% over three steps); (viii) Pd(PPh3)4, Ph3SiH, PPh3, THF
(39%).
O
Ph
nC13H27
N
HN
ii-iii
Glycerol analogue 26 was synthesized according to the
route outlined in Scheme 6. Diol 3311 was first tosylated
to give 34. The sodium salt of commercially available
(S)-2,2-dimethyl-1,3-dioxolane-4-methanol was then
used to displace the tosyl group of 34. Lauroylation fol-
lowed by acidic hydrolysis of the dioxolane gave 35.
Selective mono-silylation of 35 yielded alcohol interme-
diate 36. Conversion of 36 into the glycol analogue 26
was achieved using an identical sequence of steps to that
described earlier.
O
O
OR1
R2O
1 = R2 = H (21)
1 = H, R2 = TBDPS (22)
O
OR1
R
R
R1 = MPM (17)
nC7H15
nC7H15
iv
v
i
R
1 = H (20)
R1 = C(O)nC11H23, R2 = TBDPS (23
O
nC13H27
O
O
C11H23
NH2
HN
vi-viii
ix-x
O
5
O
O
O
P
O
nC7H15
24
Scheme 4. Synthesis of the (R/S,S,R)-diastereomer 5. Reagents and
conditions: (i) CAN, MeCN, H2O (83%); (ii) 4 M HCl, MeOH, 65 °C
then 4 M NaOH, MeOH, 85 °C (98% over two steps); (iii)
nC13H27COCl, NaHCO3, THF; (iv) TBDPSCl, Et3N, DMAP, DCM
(87% over two steps); (v) nC11H23CO2H, EDCI, DMAP, DCM (75%);
(vi) TBAF, AcOH, THF (98%); (vii) 13, HOAc, MeCN, DCM then
H2O2; (viii) TFA, Et3SiH, DCM then NaHCO3; (ix) 6, EDC, DCM
(23% over three steps); (x) Pd(PPh3)4, Ph3SiH, PPh3, THF (40%).
The synthesis of 27 started with a six-step conversion of
diol 3311 into azido alcohol 38 (Scheme 7). This se-
quence involved selective mono-silylation, Mitsunobu
inversion,12 mesylation, followed by azide displacement.
The hydroxyl of 38 was then mesylated and displaced
with (R)-serinol benzimide acetal13 under basic condi-
tions to give intermediate 39. Hydrolysis of the benzim-
idine of 39, myristoylation of the resultant free amino
group, and subsequent oxidative phosphorylation with
13 gave 40. Staudinger reduction14 of 40 followed by
lauroylation, Boc removal, coupling with 6, and subse-
quent treatment with palladium(0) gave 27.
functionalities. Five simple analogues were synthesized
(Table 2). It was found that removal of the X1 carboxyl
group was not detrimental to TLR2 antagonistic poten-
cy, neither was exchanging the X2 NH for O (com-
pounds 25 and 26). Exchanging the X5 O for NH did
Table 2. Biological data for linkage analogues
O
O
nC11H23
X5
X2
nC13H27
X4
O
O
P
X1
O
O
N
H
X3
nC7H15
OH
HO
Compound
X1
X2
X3
X4
X5
TLR2 inhibition IC50, lMa
1
CO2H
H
NH
NH
O
H2
H2
H2
H2
O
O
O
3.07
2.15
1.25
5.20
0.95
0.90
25
26
27
28
29
O
O
CO2H
CO2H
CO2H
CO2H
O
O
NH
NH
NH
O
NH
O
O
NH
O
O
a Values are means of at least two experiments.

M. R. Spyvee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5494–5498
5497
O
nC11H23
CO2H
i-vii
ix-x
O
25
ii
RO
OH
R
RO
O
HO
AllylO
viii
30
R = CO2H (31)
R = H (32)
nC7H15
C7H15
R = H (33)
R = TBDPS (41)
R = TBDPS (42)
R = H (43)
i
iii
Scheme 5. Synthesis of the des-carboxy analogue 25. (i) TBDPSCl,
imidazole, DMF (78%); (ii) NaBH4, AcOH, EtOH (83%); (iii) NBS,
PPh3, DCM, 0 °C (58%); (iv) NaCH(CO2Et)2, DMF, THF; (v) TBAF,
THF (93%); (vi) CH2@CHCH2OH, DEAD, PPh3, THF, 0 °C (84%);
(vii) LiOH, H2O, THF, 70 °C, 2 h (90%); (viii) Cu2O, MeCN, 105 °C
(94%); (ix) 9, EDCI, DMF, NEt3; (x) Pd(PPh3)4, Ph3SiH, PPh3, THF
(55% over two steps).
O
NHBoc
O
nC11H23
vi-ix
nC13H27
HN
X
Y
HO
O
O
O
47
X = OH, Y = OMe (44)
X = OTBS, Y = OMe (45)
X = OTBS, Y = OH (46)
iv
v
O
nC7H15
O
O
O
nC11H23
nC13H27
O
NH2
HN
x-xi
O
P
xii-xiii
O
O
O
28
O
nC11H23
48
OH
O
nC7H15
vi-ix
RO
OH
ii-iv
RO
O
O
nC7H15
Scheme 8. Synthesis of the ester analogue 28. Reagents and condi-
tions: (i) TBDPSCl, iPr2NEt, DMAP, DCM (90%); (ii) nC11H23CO2H,
EDCI, DMAP, DCM (95%); (iii) TBAF, AcOH, THF (82%); (iv)
TBSCl, imidazole, DMF (69%); (v) LiOH, H2O, THF (83%); (vi) 43,
EDCI, DMAP, DCM (58%); (vii) TFA, Et3SiH, DCM then NaHCO3;
nC7H15
R = H (35)
R = TBDPS (36)
R = H (33)
R = Ts (34)
v
i
O
O
nC11H23
x-xi
nC13H27
O
NH2
O
O
26
O
O
O
P
n
(viii) C13H27COCl, NaHCO3, THF, 0 °C (73% over two steps); (ix)
37
O
nC7H15
TBAF, AcOH, THF (43%); (x) 13, 1H-tetrazole, DCM, 0 °C then
oxone, H2O (40%); (xi) TFA, Et3SiH, DCM then NaHCO3; (xii) 6,
HBTU, iPr2NEt, NMP (31% over two steps); (xiii) Pd(PPh3)4, Ph3SiH,
PPh3, THF (60%).
Scheme 6. Synthesis of the glycol analogue 26. Reagents and condi-
tions: (i) TsCl, pyridine, DCM; (ii) (S)-2,2-dimethyl-1,3-dioxolane-4-
methanol, NaH, DMF, THF (60% over two steps); (iii) nC11H23CO2H,
EDCI, DMAP, DCM; (iv) AcOH, H2O (74% over two steps); (v)
TBDPSCl, NEt3, DMAP, DCM (87%); (vi) nC13H27CO2H, EDCI,
DMAP, DCM (38%); (vii) HF, MeCN (70%); (viii) 13, 1H-tetrazole,
DCM, 0 °C then oxone, H2O (88%); (ix) TFA, Et3SiH, DCM then
NaHCO3; (x) 6, EDCI, DMF, NEt3 (79% over two steps); (xi)
Pd(PPh3)4, Ph3SiH, PPh3, THF (83%).
myristoylation and desilylation, gave intermediate 47.
Oxidative phosphorylation of 47 with 13, followed
by Boc removal, gave advanced intermediate 48.
Amine 48 was then converted into ester analogue 28
via the usual procedure described earlier.
Amide analogue 29 was synthesized according to the
route outlined in Scheme 9. Requisite amine 49 was
constructed from mono-protected diol 50 via mesyla-
tion, azidation, and subsequent Staudinger reduc-
tion.14 Coupling of amine 49 with acid 46, followed
by oxidative removal of the PMB protecting group,
lauroylation, Boc removal, myristoylation, and desily-
lation, gave 51. Phosphorylation of 51 with 13 with
oxidative workup, followed by treatment with TFA,
gave advanced amine intermediate 52. Coupling of
Ph
vii-viii
ix-xi
27
HO
R
N
nC7H15
O
O
N3
R = OH (33)
R = N3 (38)
O
39
nC7H15
i-vi
nC13H27
O
HN
xii-xv
O
P
O
O
N3
nC7H15
BocHN
40
O
Scheme 7. Synthesis of the tail amide analogue 27. Reagents and
conditions: (i) TBDPSCl, Et3N, DMAP, DCM; (ii) DEAD, PPh3, 4-
NO2(C6H4)CO2H, PPh3, THF, 0 °C (79%); (iii) K2CO3, MeOH, THF
(77%); (iv) MsCl, Et3N, DMAP, DCM, 0 °C; (v) NaN3, DMF, 140 °C
(70%); (vi) TBAF, THF (99%); (vii) MsCl, Et3N, DMAP, DCM, 0 °C
(90%); (viii) (R)-serinol benzimidine acetal, KOtBu, THF, 0 °C (46%);
(ix) HCl, H2O, MeOH, 90 °C; (x) nC13H27COCl, NaHCO3, THF, 0 °C
(76%); (xi) 13, pyridinium trifluoroacetate, DCM, À10 °C then 30%
H2O2 (46%); (xii) PPh3, H2O, THF; (xiii) nC11H23CO2H, EDCI, HOBt,
DCM (39%); (xiv) TFA, Et3SiH, DCM then NaHCO3; (xv) 6, EDCI,
NEt3, DMF (69% over two steps); (xvi) Pd(PPh3)4, Ph3SiH, PPh3,
THF (71%).
O
nC13H27
O
nC11H23
x-xi
iv-ix
HN
R
OPMB
nC7H15
H
HO
N
O
51
O
nC7H15
R = OH (50)
R = NH2 (49)
i-iii
O
nC13H27
O
nC11H23
NH2
O
HN
xii-xiii
H
29
O
O
N
O
P
52
O
O
nC7H15
Ester analogue 28 was synthesized according to the
route outlined in Scheme 8. The first step was a selec-
tive silylation of the primary hydroxyl of diol 3311 to
give 41. Lauroylation of the secondary hydroxyl of 41
yielded 42, which was subsequently desilylated to give
alcohol 43. Silylation of N-Boc (S)-serine methyl ester
44 gave 45, which was saponified to give acid interme-
diate 46. Esterification of the acid fragment 46 with
alcohol 43, followed by Boc removal, and subsequent
Scheme 9. Synthesis of core amide analogue 29. Reagents and
conditions: (i) MsCl, Et3N, DMAP, DCM, 0 °C (74%); (ii) NaN3,
DMF, 140 °C (95%); (iii) PPh3, H2O, THF; (iv) 46, EDCI, HOBt,
DCM (75% over two steps); (v) DDQ, DCM, H2O; (vi) nC11H23CO2H,
EDCI, DMAP, DCM; (vii) TFA, Et3SiH, DCM then NaHCO3; (viii)
nC13H27COCl, NaHCO3, H2O, DCM, 0 °C (69% over four steps); (ix)
TBAF, THF; (x) 13, 1H-tetrazole, DCM, 0 °C then oxone, H2O (60%
over two steps); (xi) TFA, Et3SiH, DCM then NaHCO3 (98%); (xii) 6,
i
EDCI, HOBt, Pr2NEt, DCM (50%); (xiii) Pd(PPh3)4, Ph3SiH, PPh3,
THF (50%).

5498
M. R. Spyvee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5494–5498
5. Hek293 cells, stably transfected with human TLR2 and an
NF-kB reporter gene, were incubated in 10% fetal bovine
serum with test compounds for 30 min prior to stimulation
with Pam3CysSK4 (0.2 ng/ml) for 18 h. Prior to assaying
compounds for inhibitory activity, agonism dose–response
curves had been generated to establish the EC50 of 0.2 ng/
ml for Pam3CysSK4.
52 with 6, followed by palladium-catalyzed deallyla-
tion, afforded 29.
In conclusion, from an HTS campaign of ca. one hun-
dred thousand compounds we discovered a novel
phospholipid compound 1 as an antagonist of TLR2 sig-
naling. We engaged in preliminary SAR investigations
demonstrating that significant structural changes could
be made without loss of activity. The finding that amide
analogue 29 possessed optimal potency was particularly
beneficial because this would pave the way for subse-
quent analogues that would be more readily accessible.
Continuation of our SAR investigations will be reported
in due course.
6. Chow, J.; Gusovsky, F.; Hawkins, L.; Spyvee, M. PCT
Int. Appl. WO 2005039504, 2005.
7. Hek293 cells, stably transfected with human TLR4, MD-
2, and an NF-kB reporter gene, were incubated in 10%
fetal bovine serum with test compounds for 30 min prior
to stimulation with LPS (10 ng/ml) plus soluble CD14
(10 nM) for 18 h. Prior to assaying compounds for
inhibitory activity, agonism dose–response curves had
been generated to establish the EC50 of 10 ng/ml for LPS.
8. PBMCs were purified from heparinized blood of healthy
human donors as previously described.9 Cells were seeded
in 96-well plates at a density of 50,000 cells per well.
Pam3CysSK4 (at its EC50 of 50 ng/ml) and compound 1 (at
30, 10, 3, 1, 0.3, 0.1, and 0.03 lM) were added and
incubated for 6 h at 37 °C. Culture supernatants were
obtained by centrifuging for 5 min at 12,000 rpm and were
stored at À20 °C until assayed for TNFa by ELISA.
Compound 1 demonstrated an IC50 of 1.6 lM (mean value
from three donors).
Acknowledgments
We thank Donna Young, Minglun Wang, Kara Loiac-
ono, and Melinda Genest for developing and running
the biological assays.
References and notes
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