Structure-activity relationships of lipopolysaccharide sequestration in guanylhydrazone-bearing lipopolyamines

Article abstract of DOI:10.1016/j.bmc.2008.11.051

The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of Gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C₁₆ alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C₁₆ chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C₁₆ analogue is significantly more active than the N-C₁₆-alkyl or N-C₁₆-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.

Full text of DOI:10.1016/j.bmc.2008.11.051

Bioorganic & Medicinal Chemistry 17 (2009) 709–715
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of lipopolysaccharide sequestration
in guanylhydrazone-bearing lipopolyamines
Wenyan Wu, Diptesh Sil, Michal L. Szostak, Subbalakshmi S. Malladi, Hemamali J. Warshakoon,
*
Matthew R. Kimbrell, Jens R. Cromer, Sunil A. David
Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally
highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules
that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or
adjunctive therapy of Gram-negative sepsis. We had previously identified in rapid-throughput screens
several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the
guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency.
In evaluating a congeneric set of guanylhydrazone analogues, we find that C16 alkyl substitution is optimal
in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated
with a C16 chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone
moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C16 analogue is
significantly more active, than the N-C16-alkyl or N-C16-acyl compounds that we had characterized earlier,
indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS seques-
tration activity.
Received 15 October 2008
Revised 15 November 2008
Accepted 18 November 2008
Available online 24 November 2008
Keywords:
Endotoxin
Lipopolysaccharide
Sepsis
Shock
Lipopolyamines
Guanylhydrazones
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
Endotoxins, or lipopolysaccharides (LPS), a structural compo-
a membrane-active peptide antibiotic known to sequester LPS
and abrogate its toxicity. The pronounced oto- and nephro-toxicity
of PMB precludes its systemic use and has led to the development
of an extracorporeal hemoperfusion cartridge based on PMB cova-
1
nent of the outer membrane of Gram-negative bacteria, play a
pivotal role in septic shock, a syndrome of systemic toxicity which
occurs frequently as a sequel to serious systemic Gram-negative
TM
lently immobilized on a polystyrene based fiber (Toraymyxin ,
8
,9
Toray Industries Inc., Tokyo). Approved for clinical use in Japan
in late 2000, Toraymyxin provides a clinically validated proof-of-
concept of the therapeutic potential of sequestering circulating
LPS. A major goal in our laboratory has been to develop small-mol-
ecule analogues of PMB that would sequester LPS with a potency
comparable to that of PMB and, importantly, be nontoxic and safe,
so that it can be used parenterally for the prophylaxis or therapy of
Gram-negative sepsis.
2
infections. The activation by LPS of the innate immune response,
mediated via toll-like receptor-4,3 leads to an uncontrolled
production of numerous inflammatory mediators, including tumor
necrosis factor-
a
(TNF-a), interleukin-1b (IL-1b), and interleukin-6
4
(
IL-6), precipitating a systemic inflammatory response. This cul-
minates in the frequently fatal syndrome of multiple system organ
failure. Despite continuing advances in antimicrobial chemother-
5
apy, the incidence of sepsis has risen almost threefold from 1979
through 2000, emphasizing an urgent, unmet need to develop
therapeutic options specifically targeting the pathophysiology of
sepsis.
The toxicity of LPS resides in its structurally highly conserved
glycolipid component called lipid A,7 which is composed of a
hydrophilic, bis-phosphorylated diglucosamine backbone, and a
hydrophobic domain of 6 (Escherichia coli) or 7 (Salmonella) acyl
chains in amide and ester linkages (Fig. 1). Polymyxin B (PMB) is
Our identification of the lipopolyamines as potential endotoxin-
sequestering molecules was based on two simple heuristics that
6
1
0–15
have been experimentally tested and validated
: (a) an optimal
distance of ꢀ14 Å is necessary between protonatable functions in
linear bis-cationic molecules for simultaneous ionic interactions
with the anionic phosphates on lipid A (Fig. 1), and (b) additional,
appropriately positioned hydrophobic group(s) are obligatory
for the interaction to manifest in neutralization of endotoxicity.
Leads obtained from high-throughput screening on focused
1
1,16,17
libraries
as well as from molecular modeling and in silico
1
8
docking studies suggested that a long-chain alkyl, rather than
an acyl group that we had previously characterized may allow
*
Corresponding author. Tel.: +1 785 864 1610; fax: +1 785 864 1920.
15
E-mail address: sdavid@ku.edu (S.A. David).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.051

7
10
W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
analogue with the terminal amine N-alkylated with a C16 chain
OH
O
with the other terminus of the molecule bearing an unsubstituted
guanylhydrazone moiety is marginally more active suggesting very
slight, if any, steric effects. Neither C16 analogue is significantly
O
O
O
HO
O
(
HO)2P
O
O
O
21
15
more active than the N-C16-alkyl or N-C16-acyl compounds that
we had characterized earlier, indicating that a primary amine
serves just as well as a guanylhydrazone in recognizing the lipid
A phosphate groups.
NH
NH O P(OH)2
O
O
O
O
O
O
HO
HO
2
2
. Results and discussion
O
O
.1. Synthesis of the guanylhydrazone compounds
The syntheses of the 8 series involved a straightforward con-
densation of either commercially available aminoguanidine 3a or
1
2
3
4
N-alkylhydrazinecarboximidamides 3b–g with the N ,N ,N ,N -tet-
raBoc-protected spermine-propanal precursor (6). N-Alkylhydra-
zinecarboximidamides were synthesized via S-methylation of
hydrazinecarbothiamide, followed by displacement with the corre-
1
2
3
sponding alkylamine (Scheme 1). N ,N ,N -triBoc-protected sperm-
1
5,20,21
ine 4, synthesized as reported earlier
was N-alkylated with
3
-bromopropan-1-ol, and the secondary amine was Boc-protected
in a one-pot procedure. The alcohol 5 was then oxidized to the cor-
responding aldehyde 6. The protected guanylhydrazone deriva-
tives 7a–g were obtained in good yields via condensation of 6
with 3a (aminoguanidine) or 3b–g. The Boc groups were finally
deprotected with TFA to afford the target compounds as orang-
ish-yellow solids (Scheme 2). The synthesis of 14 (Scheme 3), bear-
ing an N-alkyl substituent on one end and an unsubstituted
guanylhydrazone on the other terminus was carried out starting
Figure 1. The structure of lipid A, the toxic core of enterobacterial Gram-negative
lipopolysaccharide.
1
2
3
4
from N ,N ,N ,N -tetraBoc-protected spermine (9). N-alkylation
with n-C16 33I was carried out in the presence of NaH; the mono
H
for more favorable electrostatic interactions with the lipid A phos-
N-alkylated precursor 10 was isolated and subjected to N-alkyl-
ation on the other terminus with 3-bromopropoxy-tert-butyldi-
methylsilane. Sequential deprotection of the TBDMS group,
oxidation of the resultant alcohol to the aldehyde, condensation
with aminoguandine and finally, deprotection with TFA yielded
14 in 12% overall yield.
phate groups. Independent SAR leads obtained in iterative rounds
of screening focused libraries1
0,16,19,11,20,21,17
have all converged
on molecules bearing a homologated spermine (homospermine)
scaffold. However, we had identified in such screens several guan-
ylhydrazones as potent LPS binders.16 We were desirous of (i)
determining if the SAR pertaining to the hydrophobic substituent
derived for the acylpolyamines15 would also hold for molecules
with similar homospermine scaffolds grafted with guanylhydraz-
one functionalities, (ii) if the presence of the guanylhydrazone
functionality would afford greater LPS sequestration potency ow-
ing to reasons mentioned earlier, and (iii) if N-alkyl versus unsub-
stituted guanylhydrazone compounds would manifest in
differential endotoxin neutralization activities due to possible ste-
ric effects. In order to examine these questions, we have synthe-
sized a congeneric set of guanylhydrazone analogues. We find
that, as for the acylhomospermine compounds, the C16 alkyl chain
is optimal in the N-alkylguanylhydrazone series; a homospermine
2.2. LPS sequestration activities of the guanylhydrazone
compounds
We had, as mentioned earlier, identified in rapid-throughput
screens in focused libraries, several guanylhydrazones as potent
1
6,22
LPS binders.
It is to be noted that guanidines and, by extension,
guanylhydrazones, are known to interact with phosphate groups
via bidentate H-bonds by virtue of their planar geometry and mul-
tiple H-bond donor N-atoms (the ‘arginine fork’ principle23), which
is not possible in the case of simple primary amines that constitute
the cationic termini of the compounds that we have hitherto char-
H
NH
H
b
N
NH
2
a
N
NH • HI
R
NH2
2
H N
H
2
N
N
H
N
H
S
91%
23%-53%
S
H C
3
1
2
3b-g
R= H
3a (commercially available)
R= C
8
H
17
3b
3c
3d
3e
3f
R= C10
H
21
R= C
H
1
2
25
R= C14H29
R= C16H33
R= C18
H
37
3g
Scheme 1. Reagents and conditions: (a) MeI, EtOH, 60 °C, 30 min and (b) R-NH2, EtOH, reflux, 1.5 h.

W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
711
Boc
N
N
H
N
NH
Boc
N
N
H
N
N
Boc
N
N
H
N
N
Boc
Boc
Boc
c
a
b
2
42%
OH
70%
CHO 70%-100%
Boc
Boc
Boc
Boc
Boc
4
5
6
Boc
N
N
H
N
N
H
N
N
H
Boc
NH
d
NH
NH2
N
H
N
R
N
R
N
H
N
N
H
N
Boc
Boc
H
100%
3
• 5 CF COOH
H
7
a-g
8a-g
R= H
R= C H
7a
7b
7c
7d
7e
7f
7g
R= H
R= C H
8 17
8a
8b
8c
8d
8e
8f
8g
8
17
R= C10H21
R= C12H25
R= C10H21
R= C12H25
R= C14H29
R= C16H33
R= C18H37
R= C14
R= C16H33
R= C18
H
29
H
37
2 3
Scheme 2. Reagents and conditions: (a) i—3-bromopropan-1-ol, K CO , DMF, 60 °C, 24 h; ii—Boc2O (excess), MeOH, rt, 12 h; (b) PCC, DCM, rt, 4.5 h; (c) aminoguanidine (3a)
or 3b–3g, CH COOH (cat.), EtOH, 85 °C, 2 h; (d) CF3COOH, rt, 30 min. The pentatrifluoroacetate salt form is assumed.
3
Boc
N
N
H
N
N
H
Boc
N
N
Boc
Boc
Boc
a
N C16H33
N Boc
H
b
35%
6
3%
Boc
Boc
1
0
9
1
Boc
N
N
Boc
N C16H33
N
Boc
Boc
N
N
Boc
N C16H33
N
c
d
OTBDMS
H
N
Boc
Boc
7
3%
Boc
74%
O
1
12
Boc
N
N
Boc
N C16H33
N
Boc
H
N
N
H
H
NH
N
e
N C16H33
N
H
NH
NH2
3
• 5 CF COOH
N
H
NH2
100%
Boc
N
H
13
14
Scheme 3. Reagents and conditions: (a) C16
H33I, NaH, DMF, 0 °C, rt, 20 h; (b) 3-bromopropoxy-tert-butyl-dimethylsilane (excess), NaH, DMF, 0 °C, rt, 20 h; (c) i—TBAF (1 M in
THF), 2 h; ii—PCC, DCM, rt, 4.5 h; (d) 3a (aminoguanidine), CH
3
COOH (cat.), EtOH, 85 °C, 2 h; (e) CF COOH, rt, 30 min. The pentatrifluoroacetate salt form is assumed.
3
acterized.10,15,20,21,24 Since virtually all of our screening results
point to superior LPS recognition by homospermine scaffolds, we
wished to ‘graft’ guanylhydrazone functionalities on such a scaf-
fold. It was of interest to examine if substantial differences in activ-
ities would be apparent in compounds with an N-alkylated
guanylhydrazone functional group on one end and a free primary
amine on the other or, conversely, an unsubstituted guanylhydraz-
one on one terminus, and an N-alkylated secondary amine on the
other.
lipid A phosphates. Given the minor differences in potency be-
tween 8f and 14, we elected to characterize 8f in greater detail be-
cause of a higher toxicity for 14 in in vitro cellular assays (data not
shown). As expected, 8f inhibits, in a dose-dependent manner, LPS-
induced proinflammatory cytokine production in whole human
blood ex vivo (Fig. 3); the IC50 values in human blood are consider-
ably higher than in the NF-jB assay owing to the fact that these
compounds are bound to albumin strongly (manuscript submitted)
and albumin concentrations abound in the milieu of whole blood.
Compound 8f also affords dose-dependent protection against
lethal endotoxemic challenge in a murine model (Table 1), as we
In the NF-jB reporter gene assay performed in TLR4-expressing
HEK cells, we found a distinct enhancement in LPS-neutralizing po-
tency with increasing alkyl chain lengths in the 8 series of com-
pounds, with the optimal chain length being hexadecyl (8f); this
SAR is virtually identical to that observed with the acylhomosper-
mine compounds that we had examined earlier,15 which suggests
that a C16 hydrocarbon chain is optimal for maximal interactions
1
5
have observed with acylhomospermines.
It was of interest to examine if 8f with its considerably higher
than an amine, planar geometry and multiple H-bond donor
pK
atoms, would be more potent than the N-acyl - and N-alkyl-
a
15
21
homospermine leads that are currently in preclinical development.
Although much to our disappointment, we found no discernible
differences in potency between these compounds in a battery of
in vitro as well as animal experiments (data not shown), these re-
sults are, nonetheless, instructive in that they indicate that basicity
of the terminal cationic functionality, per se, is not a determinant
of LPS-sequestering activity, and that no increments in activity
25
with the polyacyl domain of lipid A. The activity of compound
4, also with a hexadecyl substituent on the terminal amine rather
1
than on the guanylhydrazone, was no different than that of 8f
within the limits of statistical significance (Fig. 2). This would ap-
pear to indicate that long-chain substituents on the cationic ter-
mini do not adversely affect Coulombic interactions with the

7
12
W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
are expected to be considerably less basic, and will be the subject
of a future publication.
NO Inhibition
NFκB Inhibition
1
000
8
a
3
3
. Experimental section
.1. Chemistry
8
b
8
c
8
d
8g
All of the solvents and reagents used were obtained commer-
1
00
8
f
8
e
cially and used as such unless noted otherwise. Moisture or air-
sensitive reactions were conducted under argon atmosphere in
oven-dried (120 °C) glass apparatus. Solvents were removed under
reduced pressure using standard rotary evaporators. Flash column
chromatography was carried out using silica gel 635 (60–
1
4
1
00 mesh), while thin-layer chromatography was carried out on
1
0
silica gel CCM pre-coated aluminum sheets. All yields reported re-
fer to isolated material judged to be homogenous by TLC, NMR
spectroscopy and mass spectrometry.
0
8
10
12
14
16
16*
18
Alkyl Carbon Chain Length
Figure 2. Relationship between alkyl chain length and potency of inhibition of LPS-
induced NF- B nuclear transactivation as measured in HEK cells expressing TLR4
and an NF- B-secreted alkaline phosphatase reporter gene construct, and of the
3
.2. General procedure for the synthesis of compounds 3b–g:26
j
j
induction of nitric oxide (NO) in murine macrophage J774.A1 cells. Compound 14 is
depicted in open symbols; its alkyl chain length is also C16, but is denoted with an
asterisk to differentiate it from 8f.
A solution of hydrazinecarbothioamide (1, 20.0 g, 219.4 mmol)
in anhydrous ethanol (200 mL) was brought to 60 °C and methyl-
iodide was added. After stirring for 30 min, the reaction suspen-
sion was filtered and white solid was yielded as compound 2.
The white solid was washed with ether (46.9 g, 91%). A solution
of compound
2 (1.0 equiv) and the corresponding amine
(
1.0 equiv) in ethanol was heated under reflux for 1.5 h. The solu-
1
0000
TNF-α
IL-6
tion was cooled and diluted with ether, giving a gummy pink pre-
cipitate. The crude product was extracted with hot ethanol and
concentrated under reduced pressure and the residue was recrys-
IL-8
IL-10
tallized from CH
3 2
NO to yield compound 3b–g (23–53%). Repre-
1
sentative data for the series are given below. 3b: H NMR
1
000
(
1
400 MHz, MeOD) d 0.86–0.97 (m, 3H), 1.2 6–1.45 (m, 10H),
.55–1.66 (br m, 2H), 3.17–3.25 (m, 2H); C NMR (126 MHz,
1
3
MeOD) d 14.43, 23.71, 27.72, 30.34, 32.96, 42.96, 42.18, 159.68;
MS (ESI) calculated for C m/z 186.18; found: 187.22
MH ). Compound 3g: H NMR (400 MHz, MeOD) d 0.80 (s,
J = 7.0 Hz, 3H), 1.04–1.36 (m, 30H), 1.44–1.61 (m, 2H), 2.76–2.85
100
9
22 4
H N
+
1
(
1
3
(
m, 1H), 3.03–3.14 (m, 1H), 4.51 (br s, 1H); C NMR (126 MHz,
MeOD) d 14.45, 23.75, 27.46, 27.72, 28.63, 30.13, 30.39, 30.52,
10
1
10
100
30.79, 33.09, 40.79, 42.17, 152.67; MS (ESI) calculated for
+
C H
19 42
N
4
m/z 326.34; found: 327.36 (MH ).
8
f Concentration (μM)
Figure 3. Inhibition of LPS-induced secretion of cytokines in human blood ex vivo
by 8f as determined by multiplexed cytokine bead array assays. A representative
experiment is depicted.
3.2.1. Synthesis of 5
To a solution of 4 (930 mg, 1.85 mmol) in anhydrous DMF
(
8 mL) was added K
2 3
CO (768 mg, 5.55 mmol), followed by 3-bro-
mopropanol (194 L, 2.22 mmol). The reaction mixture was stirred
l
at 60 °C for 18 h. The reaction mixture was diluted with dichloro-
Table 1
Dose-dependent protection by 8f in CF-1 mice challenged with a supralethal dose of
methane and the resulting solution was successively washed with
2
2
00 ng/mouse (LD100 = 100 ng) in cohorts of five animals. Lethality was recorded at
4 h post-challenge. Ratios denote dead/total. Asterixis indicate statistically signif-
water (2Â), brine (1Â) and dried over Na
2 4
SO . After removal of sol-
vent under vacuum, the residue was purified by flash column chro-
icant values, p < 0.05.
matography (hexane/EtOAc = 7:3) to afford the title compound 5 as
1
8
f Dose (
lg/mouse)
Lethality in CF-1 mice (alive/total)
a viscous oil (514 mg, 42%). H NMR (400 MHz, CDCl
3
) d 1.44 (br s,
*
2
1
00
00
0/5
36H), 1.57–1.77 (m, 8H), 3.09–3.40 (m, 11H), 3.55–3.79 (m, 7H),
*
13
1/5
4.89 (br s, 3H); C NMR (126 MHz, CDCl
3
) d 27.34, 27.42, 31.81,
5
0
0
2/5
5/5
3
5.86, 58.16, 78.60, 156.20; MS (ESI) calculated for C33H N O
64 4 9
+
m/z 660.46, found 683.49 (M+Na ).
3
.2.2. Synthesis of 6
To a solution of 5 (500 mg, 0.76 mmol) in anhydrous DCM
are to be gained by incorporating strongly basic functional groups.
Indeed, our preliminary acute toxicity studies in rodents indicate
that 8f is considerably more toxic than the aforementioned homo-
spermine leads. These data, collectively, have led us to consider the
exploration of analogues with multiple electron-donating groups
placed adjacent to the amines on the scaffold; such compounds
(20 mL) was added pyridinium chlorochromate (245 mg,
1.14 mmol) and the solution was stirred at ambient temperature
for 4.5 h. After removal of solvent under high vacuum, the residue
was purified by flash column chromatography (hexane/EtOAc =
3:1) to afford the title compound 6 as a viscous oil (349 mg,

W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
713
7
(
0%). 1H NMR (400 MHz, CDCl
m, 5H), 2.66–2.77 (m, 2H), 3.07–3.32 (m, 11H), 3.48–3.56 (m,
3
) d 1.40–1.55 (m, 36H), 1.61–1.82
Compound 7g: yield 70%; 1H NMR (400 MHz, CDCl
J = 6.6 Hz, 3H), 1.17–1.57 (m, 70H), 1.59–1.78 (br s, 8H), 2.49 (s,
1H), 2.97–3.54 (m, 18H), 6.72–7.23 (br m, 2H), 7.61 (br s, 1H);
3
C NMR (126 MHz, CDCl ) d 8.59, 13.96, 14.07, 14.12, 23.32,
25.65, 30.83, 32.19, 38.94, 40.61, 40.82, 42.33, 45.96, 62.23,
62.59, 62.76, 64.20, 64.98, 68.85, 74.42, 126.97, 131.75, 153.62,
3
) d 0.89 (t,
1
3
2
2
4
1
H), 9.81 (s, 1H); C NMR (126 MHz, CDCl
3
) d 24.55, 25.00,
1
3
7.26, 27.39, 27.42, 27.46, 27.87, 36.28, 40.05, 42.29, 42.64,
3.12, 43.61, 44.15, 44.76, 45.51, 45, 80, 78.37, 78.93, 154.42,
62 4 9
55.09, 199.95; MS (ESI) calculated for C33H N O m/z 658.45;
+
found: 681.37 (M+Na ).
102 8 8
169.20; MS (ESI) calculated for C52H N O m/z 966.78; found:
+
9
67.77 (MH ).
3
.3. General procedure for the synthesis of compounds 7a–g
3
.4. General procedure for the synthesis of compounds 8a–g
To a solution of 6 (100 mg, 0.15 mmol) in anhydrous ethanol
(
5 mL) was added compound 3a–g (1.3 equiv) followed by catalytic
The resulting 7a–g was dissolved in excess dry trifluoroacetic
amount of acetic acid. The solution was refluxed at 85 °C for 2 h.
After removal of solvent under high vacuum, the residue was puri-
fied by flash column chromatography (DCM/MeOH = 9:1) to afford
acid and stirred at room temperature for 30 min. Trifluoroacetic
acid was removed by purging nitrogen and the residue was thor-
oughly washed with diethyl ether to obtain the title compounds
as yellow flaky solid 8a–g (100%).
7
a–g as a yellow viscous oil (70–100%).
Compound 7a: yield 80%; H NMR (400 MHz, CDCl
1
Compound 8a: 1H NMR (500 MHz, MeOD) d 1.17–1.36 (m, 2H),
1.78–1.90 (br s, 4H), 2.06–2.23 (m, 4H), 2.75–2.84 (m, 2H), 3.05
3
) d 1.35–1.52
(
m, 36H), 1.56–1.91 (m, 5H), 2.39–2.61 (m, 2H), 2.97–3.33 (m,
1H), 3.34–3.62 (m, 3H), 7.32–7.63 (m, 3H); 13C NMR (126 MHz,
CDCl ) d 15.27, 25.52, 26.03, 27.08 27.63, 27.98, 28.05, 28.29,
13
1
(m, 12H), 3.40 (t, J = 7.1 Hz, 2H), 7.54 (t, J = 4.1 Hz, 1H); C NMR
3
(126 MHz, MeOD) d 36.38, 43.55, 44.39, 44.42, 44.59, 46.80,
2
4
1
C
8.46, 28.50, 37.38, 37.71, 43.07, 43.76, 44.21, 44.72, 46.52,
6.81, 53.45, 65.86, 78.98, 79.18, 79.50, 79.79, 80.00 80.24,
34 8
115.43, 117.75, 147.12, 155.70; MS (ESI) calculated for C14H N
+
+
m/z 314.29; found: 158.17 (MH +H [doubly charged species]),
+
55.50, 155.86, 156.10, 156.23; MS (ESI) calculated for
315.33 (MH ).
+
Compound 8b: 1H NMR (400 MHz, MeOD) d 0.85–0.97 (m, 3H),
1.27–1.48 (m, 10H), 1.76–1.94 (m, 4H), 2.03–2.28 (m, 4H), 2.74–
2.85 (m, 2H), 3.06–3.31 (m, 15H), 3.36–3.43 (m, 15H), 7.31–7.82
34
H
66
N
8
O
8
: m/z 714.50; found: 715.48 (MH ).
1
Compound 7b: yield 100%; H NMR (400 MHz, CDCl
3
) d0.80–0.90
(
m, 3H), 1.16–1.54 (m, 52H), 1.55–1.82 (m, 6H), 2.49 (br s, 2H),
1
3
13
2
.96–3.56 (m, 16H), 6.69–7.21 (br m, 1H), 7.63 (br s, 1H);
) d 14.1, 22.61, 25.55, 26.66, 28.45, 28.71,
1.75, 37.41, 37.70, 42.25, 44.25, 44.94, 46.40, 46.57, 53.50,
9.04, 79.24, 79.47, 79.55, 79.95, 80.14, 154.38, 154.46, 155.53,
56.12, 156.26; MS (ESI) calculated for C42 m/z 826.63;
C
(m, 1H); C NMR (126 MHz, MeOD) d 13.01, 22.29, 22.70, 22.81,
NMR (126 MHz, CDCl
3
23.97, 26.28, 28.53, 31.54, 36.41, 41.14, 43.71, 46.81, 115.55,
117.86, 161.59, 161.86; MS (ESI) calculated for C22H N m/z
50 8
3
7
1
+
+
+
426.42; found: 214.22 (MH +H ), 427.42 (MH ).
1
H
82
N
8
O
8
Compound 8c: H NMR (400 MHz, MeOD) d 0.83–1.00 (m, 3H),
1.23–1.47 (m, 14H), 1.59–1.70 (br m, 2H), 1.75–1.93 (m, 4H),
2.05–2.25 (m, 4H), 2.76–2.83 (m, 2H), 3.05–3.31 (m, 14H), 3.35–
3.43 (m, 4H), 7.37–7.67 (m, 1H); C NMR (126 MHz, MeOD) d
13.03, 22.32, 22.70, 22.81, 23.98, 26.28, 31.64, 36.40, 41.14,
43.71, 44.40, 44.43, 44.58, 146.44, 146.49, 146.54, 154.36; MS
+
found: 827.66 (MH ).
Compound 7c: yield 73%; 1H NMR (400 MHz, CDCl
J = 6.8 Hz, 3H), 1.14–1.56 (m, 56H), 1.55–1.80 (m, 6H), 2.49 (br s,
) d 0.87 (t,
3
1
3
2
H), 2.99–3.53 (m, 16H), 6.69–7.20 (br m, 1H), 7.63 (br s, 1H);
1
3
3
C NMR (126 MHz, CDCl ) d 13.10, 14.25, 21.64, 24.57, 27.43,
3
5
1
8
0.84, 36.40, 36.42, 36.69, 41.17, 43.26, 43.89, 45.46, 45.83,
2.45, 64.84, 78.52, 78.83, 78.85, 78.92, 78.96, 154.51, 154.57,
(ESI) calculated for
(MH +H ), 455.45 (MH ).
C
24
H
54
N
8
m/z 454.45; found: 228.23
+
+
+
55.13, 155.21, 155.25; MS (ESI) calculated for C44
H
86
N
8
O
8
m/z
Compound 8d: 1H NMR (400 MHz, MeOD) d 0.92 (t, J = 6.8 Hz,
3H), 1.24–1.46 (m, 18H), 1.58–1.70 (m, 2H), 1.77–1.90 (br m,
4H), 2.05–2.25 (m, 4H), 2.74–2.84 (m, 2H), 3.05–3.31 (m, 14H),
+
54.66; found: 855.70 (MH ).
Compound 7d: yield 80%; 1H NMR (400 MHz, CDCl
) d 0.87 (t,
J = 6.8 Hz, 3H), 1.20–1.54 (m, 60H), 1.59–1.81 (m, 6H), 2.50 (br s,
3
1
3
3.40 (t, J = 7.1 Hz, 2H), 7.40–7.67 (m, 1H); C NMR (126 MHz,
MeOD) d 13.03, 22.33, 22.70, 22.81, 23.97, 26.29, 28.40, 28.57,
29.07, 29.25, 29.34, 31.67, 36.40, 41.13, 43.71, 44.40, 44.43,
44.59, 115.43, 115.46, 117.47, 146.54, 146.57, 146.62, 154.37; MS
2
H), 3.03–3.53 (m, 16H), 6.70–7.22 (br m, 2H), 7.66 (br s, 1H);
1
3
3
C NMR (126 MHz, CDCl ) d 14.13, 15.27, 22.68, 26.69, 28.51,
2
4
7
1
8
8.69, 29.34, 29.59, 29.64, 37.42, 37.53, 37.69, 42.23, 43.79,
4.26, 44.69, 44.83, 44.91, 44.96, 45.00, 65.86, 79.02, 79.06,
9.37, 79.54, 79.89, 80.00, 80.11, 80.15, 155.27, 155.31, 155.50,
(ESI) calculated for
C
26
H
58
N
8
m/z 482.48; found: 242.25
+
+
+
(MH +H ), 483.49 (MH ).
56.06, 156.11, 156.24; MS (ESI) calculated for C46
90
H N
8
O
8
m/z
Compound 8e: 1H NMR (400 MHz, MeOD) d 0.92 (t, J = 6.8 Hz,
3H), 1.25–1.42 (m, 22H), 1.58–1.70 (m, 2H), 1.78–1.89 (br m,
4H), 2.04–2.25 (m, 4H), 2.73–2.84 (m, 2H), 3.04–3.30 (m, 14H),
+
82.69; found: 883.72 (MH ).
Compound 7e: yield 75%; 1H NMR (400 MHz, CDCl
) d 0.85 (t,
J = 6.8 Hz, 3H), 1.14–1.56 (m, 64H), 1.55–1.79 (m, 6H), 2.49 (br s,
3
1
3
3.40 (t, J = 7.1 Hz, 2H), 7.46–7.64 (m, 1H); C NMR (126 MHz,
MeOD) d 13.03, 22.33, 22.70, 22.81, 23.97, 26.29, 28.96, 31.67,
36.41, 41.15, 43.71, 44.40, 44.43, 44.59, 115.44, 117.74, 146.59,
2
H), 2.97–3.55 (m, 16H), 6.67–7.19 (br m, 2H), 7.61 (br s, 1H);
1
3
3
C NMR (126 MHz, CDCl ) d 14.13, 22.68, 26.68, 29.19, 29.69,
3
4
7
1
1.91, 37.42, 37.70, 42.29, 42.70, 42.73, 43.78, 44.24, 44.70,
4.94, 44.97, 45.02, 46.40, 46.85, 79.02, 79.37, 79.53, 79.83,
9.90, 80.07 80.13, 153.92, 153.98, 154.53, 155.51, 156.06,
62 8
146.60, 154.37; MS (ESI) calculated for C28H N m/z 510.51;
+
+
+
found: 256.31 (MH +H ), 511.60 (MH ).
Compound 8f: 1H NMR (400 MHz, MeOD) d 0.92 (t, J = 6.8 Hz,
3H), 1.26–1.46 (m, 26H), 1.58–1.69 (m, 2H), 1.79–1.91 (br m,
4H), 2.05–2.24 (m, 4H), 2.75–2.84 (m, 2H), 3.05–3.31 (m, 13H),
94 8 8
56.12, 156.25; MS (ESI) calculated for C48H N O m/z 910.72;
+
found: 911.75 (MH ).
Compound 7f: yield 71%; 1H NMR (400 MHz, CDCl
J = 6.8 Hz, 3H), 1.15–1.58 (m, 68H), 1.58–1.81 (m, 6H), 2.50 (br s,
13
3
) d 0.86 (t,
3.40 (t, J = 7.1 Hz, 2H), 7.51–7.56 (m, 1H); C NMR (126 MHz,
MeOD) d 13.05, 22.33, 22.70, 22.81, 23.96, 26.29, 28.57, 31.67,
2
H), 2.97–3.58 (m, 16H), 6.67–7.19 (br m, 2H), 7.66 (br s, 1H);
36.41, 41.15, 43.72, 44.41, 44.44, 44.60, 115.47, 117.81, 146.55,
1
3
3
C NMR (126 MHz, CDCl ) d 13.11, 21.66, 24.54, 25.68, 27.43,
66 8
146.59, 146.63, 154.36; MS (ESI) calculated for C30H N m/z
+
+
+
2
4
1
9
8.18, 28.68, 30.90, 36.42, 36.69, 41.20, 43.22, 43.90, 44.50,
5.53, 52.46, 78.03, 78.36, 78.53, 78.83, 79.12, 147.00, 148.09,
54.52, 155.10, 155.25; MS (ESI) calculated for C50
538.54; found: 270.28 (MH +H ), 539.56 (MH ).
1
Compound 8g: H NMR (400 MHz, MeOD) d 0.91 (t, J = 6.7 Hz,
3H), 1.21–1.47 (m, 29H), 1.58–1.69 (m, 2H), 1.77–1.88 (br m,
4H), 2.05–2.24 (m, 4H), 2.73–2.86 (m, 2H), 3.04–3.30 (m, 14H),
98
H N
8
O
8
m/z
+
38.75; found: 939.78 (MH ).

7
14
W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
3
1
4
.40 (t, J = 7.0 Hz, 2H), 7.51 (br s, 1H); 13C NMR (126 MHz, MeOD) d
13.47, 22.75, 23.12, 23.23, 24.38, 26.71, 28.85, 32.09, 36.83, 41.57,
4.14, 44.84, 44.86, 45.02, 147.03, 147.09, 154.78; MS (ESI) calcu-
79.25, 79.34, 79.93, 155.47, 200.99; MS (ESI) calculated for
C H N O m/z 882.70; found: 905.68 (M+Na ).
49 94 4 9
+
+
+
lated for C32
70
H N
8
m/z 566.574; found: 284.31 (MH +H ), 567.58
3.4.4. Synthesis of 13
+
(
MH ).
To a solution of 12 (100 mg, 0.11 mmol) in anhydrous ethanol
(
5 mL) was added compound 3a (1.3 equiv) followed by catalytic
3
.4.1. Synthesis of 10
Compound 9 (2.4 g, 3.98 mmol) was added to the suspension of
amount of acetic acid. The solution was refluxed at 85 °C for 2 h.
After removal of solvent under high vacuum, the residue was puri-
fied by flash column chromatography (DCM/MeOH = 95:5) to af-
ford the title compound 13 as yellow viscous oil (73%). 1H NMR
NaH (30 equiv) in DMF at 0 °C. After the reaction suspension was
stirred at 0 °C for 30 min, 1-iodohexadecane (1.68 g, 1.0 equiv)
which was dissolved in 5 mL DMF was added to the reaction. The
mixture was then stirred at room temperature for 24 h. The reac-
tion solution was quenched with 1 M HCl and extracted with
DCM (3Â). The extracted DCM layers were washed with brine
3
(500 MHz, CDCl ) d 0.88 (t, J = 6.9 Hz, 3H), 1.25 (br s, 27H), 1.45
(br s, 41H), 1.73 (br s, 4H), 2.43–2.56 (br m, 2H), 3.16 (br s, 14H),
1
3
3
3.05–3.32 (br m, 2H), 7.48 (br s, 3H); C NMR (126 MHz, CDCl )
d 14.13, 22.69, 25.55, 25.59, 25.86, 25.97, 26.87, 27.51, 27.59,
28.47.28.50, 29.36, 29.42, 29.61, 29.66, 29.69, 30.95, 31.10, 31.92,
42.98, 43.08, 44.71, 46.61, 46.73, 47.12, 53.44, 79.29, 79.45,
(
1Â) and dried over Na
2 4
SO . After removal of the solvent under re-
duced pressure, the desired compound 10 was isolated at 16%
1
EtOAc in hexane by flash column chromatography (35%). H NMR
79.97, 155.51, 155.96; MS (ESI) calculated for C50
938.75; found: 939.75 (MH ).
H
98
N
8
O
8
m/z
+
(
500 MHz, CDCl
.54 (m, 40H), 1.61–1.78 (m, 4H), 3.01–3.33 (br m, 14H);
NMR (126 MHz, CDCl ) d 14.13, 22.69, 25.55, 25.89, 25.95, 26.87,
8.45, 29.36, 29.70, 31.93, 37.29, 37.66 43.68, 44.18, 44.60, 44.66,
4.90, 46.52, 46.63, 46.80, 47.10, 79.12, 79.27, 155.48, 155.95,
3
) d 0.88 (t, J = 7.0 Hz, 3H), 1.25 (s, 27H), 1.37–
1
3
1
C
3
3.4.5. Synthesis of 14
2
4
1
8
The resulting compound 13 was dissolved in excess dry trifluo-
roacetic acid and stirred at room temperature for 30 min. Excess
solvent was removed by purging nitrogen and the residue was
56.11; MS (ESI) calculated for C46
H
90
N
4
O
8
m/z 826.68; found:
+
49.71 (M+Na ).
thoroughly washed with diethyl ether to obtain white solid 14
1
(
100%). H NMR (500 MHz, DMSO-d
6
) d 0.85 (t, J = 6.9 Hz, 3H),
3
.4.2. Synthesis of 11
1.18–1.34 (m, 24H), 1.51–1.69 (br m, 6H), 1.89–2.01 (br m, 4H),
2.62–2.69 (m, 2H), 2.83–3.06 (m, 13H), 3.19–3.27 (m, 2H), 7.49
(t, J = 4.0, 1H), 7.61–7.96 (br m, 3H), 8.64–8.95 (m, 6H), 12.01 (s,
To a solution of tert-butyl dimethylsilylchloride (1.2 equiv) and
imidazole (1.3 equiv) in anhydrous DMF was added 3-bromopro-
panol (1.0 equiv) at 0 °C. After 1 h, the reaction mixture was
brought to room temperature and stirred for 8 h. Then the reaction
solution was diluted with ether and the ethereal layer was washed
2
7
13
1H); C NMR (126 MHz, DMSO) d 13.92, 22.05, 22.32, 22.34,
22.64, 25.37, 25.83, 28.33, 29.00, 31.25, 42.87, 43.79, 43.86,
44.01, 46.10, 46.71, 115.80, 118.18, 147.44, 155.20; MS (ESI) calcu-
+
+
with saturated NaHCO
Na SO . Compound 10 (670 mg, 0.81 mmol) was added to a sus-
3
solution (1Â) and 10% HCl and dried over
lated for C30
(MH ).
H
66
N
8
m/z 538.54, found 270.30 (MH +H ), 539.54
+
2
4
pension of NaH (40 equiv) in DMF at 0 °C. After the reaction sus-
pension was stirred at 0 °C for 30 min, an excess of freshly
prepared 3-bromopropoxy-tert-butyl-dimethylsilane (8.1 mmol)
was added to the reaction and the solution stirred at room temper-
ature for 20 h. The reaction solution was quenched with 1 M HCl
and extracted with DCM (3Â). The extracted DCM layers were
3.5. Nitric oxide assay
Nitric oxide production was measured as total nitrite in murine
2
8
macrophage J774A.1 cells using the Griess assay as described
1
4
5
previously. J774A.1 cells were plated at ꢁ10 mL in a volume of
washed with brine (1Â) and dried over Na
the solvent under reduced pressure, the desired compound 10
510 mg, 63%) was isolated at 13% EtOAc in hexane by flash column
2
SO
4
. After removal of
40 L/well, in 384-well, flat-bottomed, cell culture-treated micro-
l
titer plates and subsequently stimulated with 10 ng/mL lipopoly-
saccharide (LPS). Concurrent to LPS stimulation, serially diluted
concentrations of test compounds were added to the cell medium
and left to incubate overnight for 16 h. Polymyxin B was used as
reference compound in each plate. Positive- (LPS stimulation only)
and negative-controls (J774A.1 medium only) were included in
each experiment. Nitrite concentrations were measured by adding
(
1
chromatography. H NMR (500 MHz, CDCl
3
) d 0.01 (s, 6H), 0.82–
0
.89 (m, 12H), 1.14–1.32 (m, 28H), 1.41 (s, 40H), 1.64–1.78 (br
13
m, 6H), 3.03–3.25 (br m, 16H), 3.52–3.64 (br m, 2H); C NMR
126 MHz, CDCl ) d 14.13, 18.27, 22.69, 25.93, 26.88, 28.49,
9.37, 29.70, 31.93, 44.34, 44.62, 44.89, 46.65, 46.83, 47.08,
(
3
2
6
9
0.78, 79.20, 155.44; MS (ESI) calculated for C55
98.80; found: 1021.94 (M+Na ).
110
H N
O
4 9
Si m/z
40
well; 0.1% NED solution in ddH
phoric acid solution in ddH O) and incubating for 15 min at room
l
L of supernatant to equal volumes of Griess reagents (50
lL/
+
2
O and 1% sulfanilamide, 5% phos-
2
3
.4.3. Synthesis of 12
temperature in the dark. Absorbance at 535 nm was measured
using a Molecular Devices Spectramax M2 multifunction plate
reader (Sunnyvale, CA). Nitrite concentrations were interpolated
from standard curves obtained from serially diluted sodium nitrite
standards.
To a solution of 11 (400 mg, 0.40 mmol) in anhydrous THF
20 mL) was added tetra-n-butyl-ammonium fluoride (1 M in
THF, 174 L, 0.17 mmol) and the solution was stirred at ambient
(
l
temperature for 2 h. After removal of solvent under high vacuum,
the residue was dissolved in 10 mL anhydrous DCM and pyridini-
um chlorochromate (130 mg, 0.60 mmol) was added. The reaction
mixture was stirred at room temperature for 4.5 h. After removal of
solvent under high vacuum, the residue was purified by flash col-
3.6. Inhibition of LPS-induced NF-
jB induction
The inhibition of induction of NF-
j
B, a key transcriptional acti-
umn chromatography (hexane/EtOAc = 3:1) to afford the title com-
vator of the innate immune system, was quantified using human
embryonic kidney 293 cells cotransfected with TLR4 (LPS receptor),
CD14 and MD2 (co-receptors), available from InvivoGen Inc. (HEK-
1
pound 12 as a viscous oil (260 mg, 74%). H NMR (500 MHz, CDCl
3
)
d 0.88 (t, J = 6.9 Hz, 3H), 1.25 (br s, 27H), 1.45 (br s, 41H), 1.73 (br s,
1
3
TM
32
5
H), 2.70 (br s, 2H), 3.16 (br s, 14H), 3.51 (br s, 2H), 9.79 (s, 1H);
C
Blue , San Diego, CA), as described elsewhere. Stable expression
of secreted alkaline phosphatase (seAP) under control of NF- B/
AP-1 promoters is inducible by LPS, and extracellular seAP in the
supernatant is proportional to NF- B induction. HEK-4 cells were
NMR (126 MHz, CDCl
3
) d 14.13, 22.69, 25.60, 25.95, 26.88, 27.52,
j
2
4
8.32, 29.37, 29.66, 30.95, 31.93, 41.09, 41.12, 43.32, 43.63,
4.61, 44.69, 45.80, 46.62, 46.78, 46.84, 46.86, 47.08, 79.11,
j

W. Wu et al. / Bioorg. Med. Chem. 17 (2009) 709–715
715
5
incubated at a density of ꢁ10 cells/mL in a volume of 80
lL/well,
References and notes
in 384-well, flat-bottomed, cell culture-treated microtiter plates
until confluency was achieved, and subsequently stimulated with
0 ng/mL lipopolysaccharide (LPS). Concurrent to LPS stimulation,
1
.
.
Rietschel, E. T.; Kirikae, T.; Schade, F. U.; Mamat, U.; Schmidt, G.; Loppnow, H.;
Ulmer, A. J.; Zähringer, U.; Seydel, U.; Di Padova, F., et al FASEB J. 1994, 8, 217.
Hurley, J. C. Drug Saf. 1995, 12, 183.
1
2
serially diluted concentrations of test compounds were added to
the cell medium using a rapid-throughput, automated protocol
employing a Bio-Tek P2000 liquid handler as described above,
and left to incubate overnight. Polymyxin B was used as reference
compound in each plate. Positive- (LPS stimulation only) and neg-
ative-controls (HEK-detection medium only) were included in each
experiment. seAP was assayed spectrophotometrically using an
alkaline phosphatase-specific chromogen (present in HEK-detec-
tion medium as supplied by the vendor) at 620 nm.
3. Ulevitch, R. J. Immunol. Res. 2000, 21, 49.
4.
5.
6.
Dinarello, C. A. Curr. Top. Microbiol. Immunol. 1996, 216, 133.
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biology of bacterial endotoxic lipopolysaccharides. In Immunotherapy of
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8. Vesentini, S.; Soncini, M.; Zaupa, A.; Silvestri, V.; Fiore, G. B.; Redaelli, A. Int. J.
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Vincent, J. L.; Laterre, P. F.; Cohen, J.; Burchardi, H.; Bruining, H.; Lerma, F. A.;
Wittebole, X.; De Backer, D.; Brett, S.; Marzo, D.; Nakamura, H.; John, S. Shock
2005, 23, 400.
3
.7. Multiplexed cytokine assay ex vivo in human blood
Aliquots (100 L) of fresh whole blood, anticoagulated with hep-
1
1
1
1
0. Burns, M. R.; Wood, S. J.; Miller, K. A.; Nguyen, T.; Cromer, J. R.; David, S. A.
Bioorg. Med. Chem. 2005, 13, 2523.
1. Burns, M. R.; Jenkins, S. A.; Wood, S. J.; Miller, K.; David, S. A. J. Comb. Chem.
2006, 8, 32.
2. David, S. A.; Bechtel, B.; Annaiah, C.; Mathan, V. I.; Balaram, P. Biochim. Biophys.
Acta 1994, 1212, 167.
3. David, S. A.; Mathan, V. I.; Balaram, P. J. Endotoxin Res. 1995, 2, 325.
l
arin, obtained by venipuncture from healthy human volunteers with
informed consent and as per guidelines approved by the Human
Subjects Experimentation Committee, was exposed to an equal vol-
ume of 20 ng/mLofEscherichia coli 0111:B4LPS, with graded concen-
trations of test compounds diluted in saline for 4 h in a 96-well
14. David, S. A.; Silverstein, R.; Amura, C. R.; Kielian, T.; Morrison, D. C. Antimicrob.
Agents Chemother. 1999, 43, 912.
15. Miller, K. A.; Suresh Kumar, E. V. K.; Wood, S. J.; Cromer, J. R.; Datta, A.; David, S.
A. J. Med. Chem. 2005, 48, 2589.
16. Wood, S. J.; Miller, K. A.; David, S. A. Comb. Chem. High Throughput Screen. 2004,
7, 733.
2
9,30
microtiter plate.
The effect of the compounds on modulating
cytokine production was examined using a FACSArray multiplexed
flow-cytometric bead array (CBA) system (Becton–Dickinson-
Pharmingen, San Jose, CA) as described elsewhere.10,20,21
17.
Wood, S. J.; Miller, K. A.; Lushington, G. H.; Burns, M. R.; David, S. A. Comb.
Chem. High Throughput Screen. 2006, 9, 27.
1
1
2
8. Guo, J. X.; Wood, S. J.; David, S. A.; Lushington, G. H. Bioorg. Med. Chem. Lett.
3
.8. Mouse lethality experiments
2006, 16, 714.
9. Burns, M. R.; Jenkins, S. A.; Vermeulen, N. M.; Balakrishna, R.; Nguyen, T. B.;
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1
0,14,20,21
where.
of LPS by
tality (LD100) dose for the batch of LPS used (E. coli 0111:B4; pro-
cured from Sigma) was first determined by administering
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administered as a separate ip injection into the other flank. Lethal-
ity was determined at 24 h post-LPS challenge.
The animals were sensitized to the lethal effects
2
1–33
D
-galactosamine.3
The lethal dose causing 100% mor-
2
2
2
2
D
-
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.051.