In vitro and in vivo inhibition of LPS-induced tumor necrosis factor-α production by dimeric gallotannin analogues

Article abstract of DOI:10.1016/S0968-0896(01)00251-6

Designed dimeric gallotannin analogues featuring two tetragalloylglucopyranose cores connected by various hydrocarbon linkers inhibit tumor necrosis factor-α secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells by up to 53% (5-24 μM concentration range) compared to control. Comparable suppression of tumor necrosis factor-α levels (～50% vs control) was observed in the plasma of rats co-treated with lipopolysaccharide and specific tannin analogues selected for their lack of interleukin 1-β stimulating activity. Copyright

Full text of DOI:10.1016/S0968-0896(01)00251-6

Bioorganic & Medicinal Chemistry 10 (2002) 47–55
In Vitro and In Vivo Inhibition of LPS-Induced Tumor Necrosis
Factor-ꢀ Production by Dimeric Gallotannin Analogues
Ken S. Feldman,a,* Sarah L. Wilson,a Michael D. Lawlor,a Charles H. Langb and
William J. Scheuchenzuberc
aDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA
bDepartment of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine,
Hershey, PA 17033, USA
cLife Sciences Consortium, The Pennsylvania State University, University Park, PA 16802, USA
Received 4 May 2001; accepted 29 June 2001
Abstract—Designed dimeric gallotannin analogues featuring two tetragalloylglucopyranose cores connected by various hydro-
carbon linkers inhibit tumor necrosis factor-a secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear
cells by up to 53% (5–24 mM concentration range) compared to control. Comparable suppression of tumor necrosis factor-a levels
(ꢀ50% vs control) was observed in the plasma of rats co-treated with lipopolysaccharide and specific tannin analogues selected for
their lack of interleukin 1-b stimulating activity. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
cleavage of the first-formed pro-TNF-a protein to its
soluble form, are under investigation.6 Promising results
have been reported upon application of some of these
strategies to the treatment of certain inflammatory dis-
eases, but attempts to ameliorate the devastating effects
of septic shock have met with little success.
The secretion of cytokines from activated macrophages
and related cells is an integral component of an effective
immune response to a viral or bacterial infection. How-
ever, many autoimmune and inflammatory diseases are
linked in part to chronic overproduction of the cytokine
tumor necrosis factor-a (TNF-a).1 In addition, a surge
of proinflammatory cytokines such as TNF-a2,3 and
interleukin 1-b (IL-1b) as a result of exposure to Gram-
negative bacterial lipopolysaccharide (LPS, active com-
ponent=lipid A) can lead to life-threatening septic
shock. Consequently, therapeutic strategies targeted
toward diminishing plasma TNF-a levels have received
much attention. Protocols designed to achieve this goal
have run the gamut from blocking the initial lipid A–
receptor(s) interaction4,5 as an anti-sepsis therapy to
sequestering nascent serum TNF-a with antibodies and
soluble receptors.6,7 In between these extreme points of
intervention, small molecules such as thalidomide deri-
vatives8,9 and nucleoside analogues10 have displayed
promise as inhibitors of enzymes or receptors in the
macrophage cytosolic signaling pathway, while peptide-
based metalloproteinase inhibitors, which may prevent
The naturally occurring gallotannin b-pentagalloylgluco-
pyranose [b-PGG, (1), Fig. 1], recently has been identi-
fied as an effective inhibitor of LPS-induced TNF-a
release both from human peripheral blood mononuclear
cells (h-PBMCs) and in live rats.11 The polyphenolic
tannins do not structurally or functionally resemble any
of the therapeutic agents discussed above, and so this
result draws attention to the possibility that prospecting
among members of the tannin class of secondary plant
metabolites12ꢁ14 may generate novel leads against septic
shock and other inflammatory diseases. b-PGG reduced
the output of TNF-a in vitro by as much as 90% versus
control. However, in vivo studies with rats exposed
severe limitations in using this simple monomeric tannin
as a treatment for septic shock. Despite lowering plasma
TNF-a levels in LPS-treated rats versus control, the
physiological symptoms of septic shock (increased heart
rate and blood glucose levels, decreased blood pressure)
were not measurably dampened. It is plausible that the
sustained septic shock symptoms can be traced to the
b-PGG-stimulated secretion of the companion cytokine
*Corresponding author. Tel.: +1-814-864-4654; fax: +1-814-863-
8403; e-mail: ksf@chem.psu.edu
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00251-6

48
K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
of molecules should be effective antagonists of LPS. In
addition, it is possible that a larger and more complex
tannin architecture might benefit from increased selec-
tivity in protein interactions19 and therefore require
smaller doses with potentially fewer unwanted side
effects than those observed with the monomer 1. This
model does not permit predictions about IL-1b stimu-
lating activity, and so this point will have to be deter-
mined through experiment.
Results and Discussion
Synthesis chemistry
The general strategy for preparation of the dimeric tan-
nins involved the coupling of selected diacid chlorides
with 2 equivof per- O-benzyl tetragalloylglucopyranose
(7).20 Stereochemically pure compounds were desired to
limit the number of structural variables in interpreting
the immunological assays, and this coupling procedure
is reported to provide isomerically pure b-anomeric
ester linkages using aromatic monoacid chlorides.20,21
Five compounds 3a–e were selected for initial evalua-
tion (Fig. 2). Dimer 3a, the most structurally con-
servative change, probes the effect of hydroxylation on
the presumably critical diaryl ether linker of the active
compounds. The alkyl linker in 3b provides more flex-
ibility and more hydrophobicity than the con-
formationally limited diaryl ether of 2. Compounds 3c–e
are all more rigid than the active diaryl ether-bearing
compounds, and the different aryl spacers enforce dif-
ferent core–core distances and orientations compared
with 2.
Figure 1. Immunomodulatory tannins, b-d-pentagalloylglucopyr-
anose (1), and a dimeric tannin analogue 2.
IL-1b,11 a known mediator of this response.15 Thus, the
design, synthesis, and evaluation of second-generation
tannin-based immunomodulators which did not them-
selves generate IL-1b became a high priority.
A design hypothesis emerged from the observation that
the dimeric gallotannin analogue 2, as well as the struc-
turally related naturally occurring dimeric ellagitannins
coriariin A and agrimoniin, stimulated secretion of
TNF-a from h-PBMCs, whereas the monomeric species
1 was largely inert at similar concentrations.16 Further-
more, the dose-response data for the dimeric species
were reminiscent of that recorded after exposure of h-
PBMCs to LPS. Taken together, these results might be
accommodated by a mechanism-of-action model which
features interaction of the dimeric tannins with the
obligate PBMC receptors, surface bound CD14 and
soluble LBP,17 that mediate the LPS-initiated septic
shock response. Although the details of the lipid A–
receptor interaction are far from secure, some data are
consistent with a model in which an appropriate three-
component interaction between the two receptors and
lipid A is necessary to trigger the signaling cascade that
begins by engaging another cell surface protein,
TLR4.18 It is not beyond the realm of speculation,
therefore, to posit that the active dimeric tannins might
present galloylated glucopyranose recognition elements
to each of the receptors, and that the digalloyl ether
linker common to all of these active compounds (cf. 2)
might be a critical element in orienting these putative
binding domains in a manner that facilitates productive
interaction between the bound receptors and TLR4.
Circumstantial evidence (vide infra) is suggestive of no
more than a secondary role for the diaryl ether linker in
the recognition event(s). In this scenario, the monomeric
species 1 may bind to one or both of the receptors in
competition with lipid A, but would provide no orga-
nizing framework to the receptor complex, and hence
fail to initiate signal transduction. This working
hypothesis can be tested by assembly of dimeric tannin
constructs that preserve the tetragalloylglucopyranose
endcaps but alter their relative orientation through
incorporation of linkers that differ from the digalloyl
ether moiety in 2. In the best case, members of this class
The requisite acid chlorides 6b–d are commercially
available, and biphenyl diacid chloride 6e22 was readily
prepared from the corresponding diacid. Diaryl ether
diacid chloride 6a was prepared in two steps from the
known Ullmann coupling product 4 (Scheme 1).23
Galloylated glucopyranose 7 was coupled with each of
Figure 2. Five dimeric tannin analogues designed as LPS antagonists
in a septic shock response.

K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
49
3).16 In each case, LPS was used as a positive control
while untreated cells were reserved as negative controls.
Two of the compounds, 3b and 3d, showed very low
levels of TNF-a production, and 3c and 3e produced no
measurable response from the h-PBMCs (data not
shown). Treatment of the h-PBMCs with the diaryl
ether-containing compound 3a, however, a species
which retains much the same overall shape as the active
dimeric gallotannin 2, led to significant cytokine release.
Despite lacking the active compounds’ diaryl ether
hydroxyls, 3a appears to act as an immunostimulator
and was therefore eliminated from further consideration
as a TNF-a inhibitor. This result also seems to lessen
the likelihood that the linker unit itself is a critical
recognition element for association with the receptors,
in accord with an implicit assumption of the design
hypothesis.
Scheme 1.
the diacid chlorides 6a–e using triethylamine as base in
CH2Cl2 to furnish dimers 8a–e in isolated yields of 17–
61% (Scheme 2). These successful bis acylations stand
in sharp contrast to previous attempts to prepare dimer
2 using a similar procedure, suggesting that the bis acid
chloride corresponding to the 2,3,4,4050-penta-O-benzyl
version of 6a is an uncharacteristically poor acylating
reagent.20 As anticipated, compounds 8a, 8c, and 8d
were formed with good diastereoselectivity for the b,b0-
anomer as determined by 1H NMR. Higher temperature
(refluxing THF) was necessary to prepare the biphenyl-
linked dimer 8e, which was obtained as a 4:1 ratio of
b,b0-to-a,b0 anomers. The coupling reaction to form 8b
resulted in a mixture of the three possible anomeric iso-
mers, but the b,b0 anomer was produced as the major
product, and a,a0 diester formation was completely
suppressed by the slow addition of diacid chloride 6b to
alcohol 7.21 Debenzylation of the phenolic hydroxyl
protecting groups in dimers 8a–e proceeded in excellent
yield to furnish the desired tannin analogues 3a–e.
Compounds 3b–e were then evaluated as mediators of
IL-1b production from h-PBMCs (Fig. 4). This cytokine
is also released as part of a septic shock response to
bacterial LPS exposure, and so dimers that caused
minimal IL-1b release would hold the greatest ther-
apeutic promise. The meta-phenyl dimer 3d led to sig-
nificant IL-1b production at higher concentrations (30
mM, 2110 pg/mL), while the para-phenyl isomer 3c dis-
played significantly less activity (30 mM, 690 pg/mL).
The other two substrates, 3b and 3e, ostensibly failed to
induce IL-1b secretion from the cells (<250 pg/mL at
30 mM), thus emerging as the top inhibitory candidates
for further in vitro and in vivo study.
TNF-a inhibition data for compounds 3b–e are shown
in Figures 5 and 6. h-PBMCs were incubated with a
fixed concentration of LPS (5 mg/mL for 3b–c, 10 mg/
mL for 3d–e) for 45 min, followed by addition of
various concentrations of each dimeric tannin. The cell
In vitro immunomodulatory activity
The five dimers were initially tested for their ability to
induce TNF-a secretion from h-PBMCs. The cells were
isolated and incubated with each of the dimeric gallo-
tannins 3a–e for 24 h, and the amount of TNF-a in the
culture supernatants was determined by ELISA (Fig.
Figure 3. TNF-a secretion from two subjects’ PBMCs upon exposure
to varying concentrations of dimeric tannins 3a,b, d and LPS (24 h).
Scheme 2.

50
K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
culture supernatants were harvested after 8 h24 and
assayed by ELISA for TNF-a release. To compensate
for the inherent variability of using cells from different
subjects, the cytokine secretion data were normalized by
calculating a percent of the control value, represented
by LPS treatment only.
values of 30% for both 3b and 3c at 5 mM at 23.5 mM
for 3b (Fig. 5). The most potent inhibitor of TNF-a
secretion was the meta-phenyl-linked dimer 3d, with
maximum inhibition of 53% at 11.8 mM (Fig. 6). The
biphenyl-linked compound 3e was slightly less potent at
27%. However, in a separate trial using a dose of 1 mg/
mL of LPS as an h-PBMC stimulant, a higher max-
imum inhibition value of 45% was observed with 3e at a
concentration of 5.6 mM. Eliminating LPS pre-treat-
ment by introducing the compounds and LPS at the
same time had little effect on the maximum inhibitory
values (data not shown).
All of the dimers inhibited TNF-a production in the
concentration range studied, 0–24 mM. The pentyl and
para-phenyl-linked dimers, 3b and 3c, respectively,
exhibited promising activity with maximum inhibition
In vivo immunomodulatory activity
Compounds 3b and 3e were chosen for study in a rat
model system since they appear incapable of indu-
cing secretion of significant quantities of IL-1b from
h-PBMCs and are therefore less likely to initiate or
sustain
a septic shock response. The experiment
involved nine animals: rats 1–4 were administered LPS
(1 mg/kg) and one of the two dimeric tannins (20 mg/rat
over 180 min), whereas rats 5–8 were controls and
received only the initial LPS treatment, and rat 9
received saline only (data not shown). Plasma samples
were collected from each animal at 90 and 180 min for
determination of cytokine levels by ELISA-based ana-
lysis. The TNF-a data (Fig. 7) indicated significant
reduction (ꢂ50%) of cytokine levels at 90 min in three
of the four tannin-treated rats, with an average decrease
of 58%. Encouragingly, the rats dosed with 3b and 3e
did not produce higher IL-1b amounts than the LPS
controls at 180 min (Fig. 8), and, in fact, the cytokine
levels are 17% lower on average in these rats. Unfortu-
nately, monitoring typical physiological symptoms of
Figure 4. IL-1b secretion from subject 30s PBMCs upon exposure to
varying concentrations of dimeric tannins 3b–e and LPS (24 h).
Figure 6. TNF-a secretion from subject 5’s and subject 6’s PBMCs
upon exposure to 1 or 10 mg/mL of LPS and varying concentrations of
3d or 3e (8 h).
Figure 5. TNF-a secretion from subject 40s PBMCs upon exposure to
5 mg/mL of LPS and varying concentrations of 3b or 3c (8 h).

K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
51
and in live rats, five dimeric gallotannin analogues were
prepared and analyzed for immunomodulatory activity.
Four of these compounds displayed significant inhibi-
tory properties in vitro, demonstrating that a great deal
of structural variability is tolerated in the linker span-
ning the two glucopyranose cores. While not as potent
in vitro as b-PGG, synthetic dimers 3b and 3e
showed similar activity in vivo without exacerbating
the LPS-initiated septic shock response as observed
with monomer 1. The mechanism by which tannins
operate as immunomodulators is a matter of specula-
tion at present and will be the subject of continuing
work in this area.
Experimental
General
All reactions were performed, with magnetic stirring, in
flame-dried glassware under a positive pressure of
argon. Commercial grade reagents and solvents were
used without further purification except as indicated
below. Dichloromethane, benzene, and triethylamine
were distilled from calcium hydride. THF was distilled
from sodium benzophenone ketyl or dianion. Methanol
was distilled from magnesium. N,N-Dimethylforma-
mide (DMF) was sequentially dried in three stages over
activated 3 A molecular sieves. Reaction product solu-
tions and chromatography fractions were concentrated
using a Buchi rotary evaporator at approximately 20
mmHg and then at ca. 0.1 mmHg (vacuum pump).
Column chromatography was performed using 32–63
mm silica gel and the indicated solvent. Infra-red (IR)
spectra were recorded on a Perkin–Elmer 1600 Series
FT infrared spectrophotometer. Magnetic resonance
spectra (1H NMR, 13C NMR) were recorded on either a
Bruker DPX-300, AMX-360, or DRX-400 spectro-
meter. Chemical shifts are reported in d units using tet-
ramethylsilane (TMS) or acetone as an internal
standard for 1H NMR and chloroform or acetone as the
internal standard for 13C NMR. High resolution fast
atom bombardment (FAB) mass spectra were run at the
University of Texas at Austin, TX, USA. Combustion
analyses were performed by Midwest Microlabs,
Indianapolis, IN, USA.
Figure 7. Plasma TNF-a levels in rats at 90 min after treatment with
LPS (1 mg/kg) and 3b (12.5 mg), or LPS and 3e (12.5 mg), or LPS only
(1 mg/kg).
Figure 8. Plasma IL-1b levels at 180 min from rats treated with LPS (1
mg/kg) and 3b (20 mg), or LPS and 3e (20 mg), or LPS only (1 mg/kg).
General procedure A: bisacylation of 2,3,4,6-tetra-
kis(3,4,5-tribenzyloxybenzoyl)-D-glucose (7). To a solu-
tion of 2,3,4,6-tetrakis(3,4,5-tribenzyloxybenzoyl)-d-
glucose (7)20 (300 mg, 0.160 mmol) in 1.5 mL of CH2Cl2
was added triethylamine (67 mL, 0.48 mmol) followed
by the appropriate diacid chloride (0.5 equiv). In some
cases (see text), slow addition of a solution of the diacid
chloride in CH2Cl2 dropwise via syringe pump over ca.
6 h afforded increased selectivity for the b-anomeric
linkage in the products. This solution was stirred at rt
for 18 h. The reaction mixture was treated with 1 N HCl
and extracted with EtOAc. The organic layer was
washed sequentially with H2O and brine, and then dried
over Na2SO4. After filtration and removal of the sol-
vents in vacuo, the product was purified by flash chro-
matography using 3:5:12 EtOAc/benzene/hexane.
septic shock in the rats so treated (arterial blood pres-
sure, heart rate, plasma glucose level) did not reveal any
broadly significant amelioration of the LPS-induced
effects. Heart rate, however, did return to pre-LPS levels
upon treatment with 3e (average beats/min at 180 min:
after LPS administration, 483ꢃ10; after LPS+3e,
385ꢃ33; saline control, 380ꢃ22).
Conclusions
Building upon earlier results in which the simple gallo-
tannin 1 was identified as a promising down-regulator
of LPS-mediated TNF-a secretion in both h-PBMCs

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K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
General procedure B: hydrogenation. To a solution of
the appropriate benzylated dimer in THF was added
10% Pd/C (0.5–1 equivPd). The reaction mixture was
purged four times with H2 and stirred at rt under one
atm of H2 for 18 h. The reaction mixture was filtered
through Celite which was rinsed with acetone. After
concentration, the resulting gray solid was triturated
with hexane and ether and dried under vacuum at 35 ꢄC.
(MH+, 7). Anal. calcd for C244H202O46: C, 75.74, H,
5.23. Found: C, 75.61, H, 5.23.
1,10-O-2,20,3,30,4,40,6,60-Tetrakis(3,4,5-trihydroxybenzoyl)-
ꢁ,ꢁ0-D-glucopyranosylisophthalate (3d). By use of gen-
eral procedure B, dimer 8d (150 mg, 0.039 mmol) was
hydrogenated using 32 mg (0.03 mmol) of Pd/C in 5 mL
of THF to afford 50 mg (76%) of meta-phenyl linked
dimer 3d: IR (KBr) 3405, 1702 cmꢁ1
;
1H NMR
1,10 -O-2,20,3,30,4,40,6,60 -Tetrakis(3,4,5-tribenzyloxyben-
((CH3)2CO), 300 MHz) d 8.58 (s, 1H), 8.45–7.85 (m
24H), 8.17 (dd, J=7.7, 1.7 Hz, 2H), 7.61 (t, J=7.9 Hz)
d 7.12 (s, 4H), 7.02 (s, 4H), 6.96 (s, 4H), 6.94 (s, 4H),
6.40 (d, J=7.9 Hz, 2H), 6.00 (appar t, J=9.6 Hz, 2H),
5.65 (appar t, J=9.6 Hz, 2H), 5.62 (appar t, J=9.6 Hz,
2H), 4.61–4.49 (m, 4H), 4.39 (dd, J=12.8, 4.5 Hz, 2H);
13C NMR (CDCl3, 75 MHz) d 166.0, 165.5, 165.4, 165.2,
163.8, 145.7, 145.6, 144.5, 139.1, 138.9, 138.7, 136.9,
135.3, 135.2, 130.3. 130.1, 121.1, 120.3, 120.9, 120.3,
120.2, 120.1, 110.0, 109.9, 109.8, 93.7, 73.8, 72.8, 71.5,
68.9, 62.4; MS (+FAB) 1706 (M+, 14); HRFABMS
calcd for C76H58O46: 1706.2199. Found: 1706.2221.
0
zoyl)-ꢁ,ꢁ0-D,D -glucopyranosylterephthalate (8c). By use
of general procedure A, terephthaloyl chloride (16 mg,
0.079 mmol) was coupled alcohol 7 and purified by flash
chromatography to afford 190 mg (61%) of benzylated
1
dimer 8c: IR (CDCl3) 1734 cmꢁ1; H NMR (CDCl3,
300 MHz) d 8.02 (s, 4H), 7.43–7.15 (m, 136H), 6.25 (d,
J=8.3 Hz, 2H), 6.05 (appar t, J=9.8 Hz, 2H), 5.82 (dd,
J=10.2, 8.3 Hz, 2H), 5.70 (appar t, J=9.8 Hz, 2H),
5.11–4.91 (m, 48H), 4.77 (d, J=9.4 Hz, 2H), 4.47–4.42
(m, 2H), 4.32 (dd, J=11.9, 6.2 Hz, 2H); 13C NMR
(CDCl3, 75 MHz) d 165.6, 165.5, 165.0, 164.8, 163.7,
152.6, 152.5, 152.4, 143.2, 143.1, 142.6, 137.4, 137.3,
137.2, 136.6, 136.3, 136.2, 132.9, 130.3, 128.5, 128.5,
128.4, 128.2, 128.2, 128.1, 128.0, 127.8, 127.5, 124.4,
123.6, 123.5, 109.3, 109.1, 109.0, 93.2, 75.1, 73.4, 73.3,
71.1, 71.1, 71.0, 69.8, 63.19; MS (+FAB) 3867 (MH+,
16). Anal. calcd for C244H202O46: C, 75.74, H, 5.23.
Found: C, 75.59, H, 5.29.
Biphenyl-4,40-dicarbonyl dichloride (6e).22 A solution of
biphenyl-4,40-dicarboxylic acid (200 mg, 0.83 mmol) in
15 mL of CH2Cl2 was stirred with oxalyl chloride (0.80
mL, 9.14 mmol) and one drop of DMF under Ar for
18 h. Removal of solvents in vacuo afforded 211 mg
(93%) of biphenyl-4,40-dicarbonyl dichloride (6e) as a
yellow solid: IR (CH2Cl2) 1781 cmꢁ1; 1H NMR (CDCl3,
360 MHz) d 8.25 (d, J=8.2 Hz, 4H), 7.78 (d, J=8.7 Hz,
4H); 13C NMR (CDCl3, 90 MHz) d 167.9, 145.8, 133.2,
132.1, 127.9.
1,10-O-2,20,3,30,4,40,6,60-Tetrakis(3,4,5-trihyroxybenzoyl)-
0
ꢁ,ꢁ0-D,D -glucopyranosylterephthalate (3c). By use of
general procedure B, dimer 8c (150 mg, 0.039 mmol)
was hydrogenated using 32 mg (0.03 mmol) of Pd/C in 5
mL of THF to afford 30 mg (99%) para-phenyl linked
1,10 -O-2,20,3,30,4,40,6,60 -Tetrakis(3,4,5-tribenzyloxyben-
0
dimer 3c: IR (KBr) 3422, 1702 cmꢁ1
;
1H NMR
zoyl)-ꢁ,ꢁ0-D,D -glucopyranosylbiphenyl-4,40-diester (8e).
((CD3)2CO, 300 MHz) d 8.5-7.4 (m, 24H), 8.05 (s, 4H),
7.13 (s, 4H), 7.02 (s, 4H), 6.97 (s, 4H), 6.94 (s, 4H), 6.37
(d, J=8.3 Hz, 2H), 6.02 (appar t, J=9.6 Hz, 2H), 5.65
(appar t, J=9.8 Hz, 2H), 5.64–5.58 (m, 2H), 4.61–4.54 (m,
4H) 4.38 (dd, J=12.6, 4.3 Hz, 2H); 13C NMR [(CD3)2CO,
75 MHz] d 165.5, 164.9, 164.9, 164.7, 163.3, 145.1, 145.1,
145.0, 138.5, 138.3, 138.2, 135.9, 133.3, 130.0, 128.4, 120.5,
119.7, 119.6, 119.4, 109.4, 109.2, 93.2, 73.2, 72.1, 70.9, 68.3,
61.8; MS (+FAB) 1706 (MH+ 7); HRFABMS calcd for
C76H58O46: 1706.2199. Found: 1706.2232.
To a solution alcohol 7 (440 mg, 0.24 mmol) in 3 mL
of dry THF was added triethylamine (100 mL, 0.72
mmol) followed by diacid chloride 6e (33 mg, 0.12
mmol). The resulting mixture was heated at reflux for
40 h. The reaction mixture was treated with 1 N HCl and
extracted with EtOAc. The organic layer was washed
sequentially with H2O and brine, and then dried over
Na2SO4. After removal of the solvents in vacuo, the
product was purified by flash chromatography using
3:5:12 EtOAc/benzene/hexane to afford 80 mg (17%) of
1
benzylated dimer 8e: IR (CDCl3) 1732 cmꢁ1; H NMR
1,10 -O-2,20,3,30,4,40,6,60 -Tetrakis(3,4,5-tribenzyloxyben-
zoyl)-ꢁ,ꢁ-D-glucopyranosylisophthalate (8d). By use of
general procedure A, isophthaloyl chloride (16 mg,
0.079 mmol) was coupled to alcohol 7 and purified by
flash chromatography to afford 150 mg (48%) of ben-
(CDCl3, 300 MHz, b,b0 isomer) d 8.08 (d, J=8.3 Hz,
4H), 7.57 (d, J=8.6 Hz, 4H), 7.47–7.18 (m, 136H), 6.35
(d, 2H, J=8.3 Hz, 2H), 6.07 (appar t, 2H, J=9.6 Hz),
5.87 (dd, J=9.4, 8.3 Hz, 2H), 5.76 (appar t, J=9.6 Hz,
2H), 5.15–4.80 (m, 50H), 4.49–4.43 (m, 2H), 4.36 (dd,
J=11.9, 6.2 Hz, 2H); 13C NMR (CDCl3 75 MHz, b, b0
isomer) d 168.6, 165.6, 165.5, 165.0, 164.2, 152.6, 152.5,
152.4, 144.6, 143.0, 142.5, 137.4, 137.3, 137.2, 136.6,
136.3, 136.2, 133.4, 132.6, 128.8, 128.7, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6,
127.5, 123.6, 123.5, 109.3, 109.1, 109.0, 75.1, 75.1, 71.2,
71.1, 71.0, 69.3, 63.1. Anal. calcd for C250H206O46: C,
76.10, H, 5.23. Found: C, 75.85, H, 5.31.
zylated dimer 8d: IR (CDCl3) 1730 cmꢁ1 1H NMR
;
(CDCl3, 300 MHz) d 8.76 (s, 1H), 8.19 (dd, J=7.9, 1.5
Hz, 2H) 7.42–7.15 (m, 137H), 6.26 (d, J=8.2 Hz, 2H),
6.01 (appar t, J=9.4 Hz, 2H), 5.81 (dd, J=9.8, 8.3 Hz,
2H), 5.74 (appar t, J=9.6 Hz, 2H), 5.09–4.90 (m, 48H),
4.76 (d, J=9.0 Hz, 2H), 4.41–4.30 (m, 4H); 13C NMR
(CDCl3, 75 MHz) d 165.7, 165.5, 165.0, 164.9, 163.6,
152.6, 152.6, 152.4, 143.2, 143.1, 142.6, 137.5, 137.3,
137.3, 136.6, 136.4, 136.4, 136.3, 129.3, 128.9, 128.9,
128.7, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7,
127.6, 124.4, 123.7, 123.6, 109.3, 109.1, 93.1, 75.2, 75.1,
73.3, 73.2, 71.2, 71.2, 71.0, 69.7, 63.1; MS (+FAB) 3867
1,10-O-2,020,3,30,4,40,6,60-Tetrakis(3,4,5-trihyroxybenzoyl)-
ꢁ,ꢁ0-D,D - glucopyranosylbiphenyl-4,40-diester (3e). By
use of general procedure B, dimer 8e (50 mg, 0.013

K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
53
mmol) was hydrogenated using 13 mg (0.012 mmol) of
Pd/C in 4 mL of THF to furnish 20 mg (87%) of
biphenyl linked dimer 3e: IR (KBr) 3448, 1702 cmꢁ1; 1H
NMR ((CD3)2CO, 300 MHz) d 8.28–8.08 (m, 24H), 8.05
(d, J=8.7 Hz, 4H), 7.79 (d, J=8.3 Hz, 4H), 7.14 (s,
4H), 7.03 (s, 4H), 7.00 (s, 4H), 6.95 (s, 4H), 6.40 (d,
J=8.3 Hz, 2H), 6.02 (appar t, J=9.6 Hz, 2H), 5.71–5.61
(m, 4H), 4.61–4.52 (m, 4H), 4.39 (dd, J=12.2, 4.7 Hz,
2H); 13C NMR ((CD3)2CO, 90 MHz) d 167.3, 166.8,
166.7, 166.5, 165.7, 146.9, 146.8, 146.5, 140.3, 140.1,
139.9, 138.9, 132.3, 132.3, 130.2, 129.4, 122.4, 121.6,
121.5, 121.4, 111.2, 111.1, 111.1, 94.7, 75.1, 74.0, 72.8,
70.2, 63.7; MS (+FAB) 1782 (M+); HRFABMS calcd
for C82H62O46: 1782.2512. Found: 1782.2507;
[a]2D0=ꢁ8.3 (CH3OH).
d, J=7.8, 1.2, 1H), 7.64 (ddd, J=8.2, 7.5, 1.8 Hz, 1H),
7.53–7.46 (m, 2H), 7.35 (appar d of t, J=7.6, 1.1 Hz,
1H), 7.21 (ddd, J=8.2, 2.6, 1.0 Hz, 1H), 7.14 (dd,
J=8.2, 1.0 Hz, 1H); 13C NMR ((CD3)2CO, 75 MHz) d
166.6, 166.0, 158.8, 155.6, 134.4, 132.6, 132.5, 130.3,
125.0, 124.7, 124.2, 122.5, 122.3, 118.4.
2,30-Oxy-di-benzoyl chloride (6a).25 Oxalyl chloride
(0.058 mL, 0.66 mmol) was added slowly dropwise to a
suspension of diacid 5 (0.057 g, 0.22 mmol) in 2 mL dry
CH2Cl2 and 1 drop DMF. The suspension gradually
turned to a clear yellow solution. The reaction mixture
stirred under Ar for 3 h. The solvent was removed in
vacuo to yield 0.063 g (97%) of diaryl ether diacid
1
chloride 6a as a yellow oil: IR (CDCl3) 1758 cmꢁ1; H
NMR (CDCl3, 300 MHz) d 8.19 (dd, J=8.3, 1.7 Hz, 1
Hz) 7.93–7.90 (m, 1H), 7.68 (appar t, J=2.1 Hz, 1H),
7.66–7.60 (m, 1H), 7.52 (appar t, J=8.1 Hz, 1H), 7.36–
7.31 (m, 2H), 7.00 (dd, J=8.3, 0.7 Hz, 1H); 13C NMR
(CDCl3, 75 MHz) d 167.6, 163.9, 156.9, 155.4, 136.1,
135.0, 134.3, 130.5, 126.8, 125.8, 125.4, 124.5, 120.5,
120.4.
1,10 -O-2,20,3,30,4,40,6,60 -Tetrakis(3,4,5-tribenzyloxyben-
0
zoyl)-ꢁ,ꢁ0-D,D -glucopyranosylpimelate (8b). By use of
general procedure A, pimeloyl chloride (13 mL, 0.079
mmol) was coupled to alcohol 7 (300 mg, 0.16 mmol)
and purified by flash chromatography to afford 140 mg
(45%) of benzylated dimer 8b (mixture of a,a0, b,b0, and
a,b0 anomers): IR (CDCl3) 1730 cmꢁ1
;
1H NMR
(CDCl3, 300 MHz, b,b0 isomer) d 7.43–7.18 (m, 136H),
6.09 (d, J=7.9 Hz, 2H), 5.94 (appar t, J=9.8 Hz, 2H),
5.69–5.60 (m, 4H), 5.13–4.72 (m, 50H), 4.32–4.27 (m,
4H); 13C NMR (CDCl3, 90MHz) d 171.5, 165.6, 165.5,
165.0, 165.0, 164.7, 152.6, 152.5, 152.4, 143.3, 143.1,
143.1, 143.0, 142.7, 142.6, 137.5, 137.3, 136.7, 136.4,
136.4, 136.2, 128.5, 125.5, 128.4, 128.3, 128.3, 128.2,
128.1, 128.1. 128.0, 127.8, 127.6, 124.5, 123.7, 123.6,
109.2, 109.1, 921, 75.1, 75.1, 71.2, 71.1, 71.0, 69.9, 63.1,
33.6, 28.1, 23.9; MS (+FAB) 3861 (MH+, 50).
1,10 -O-2,20,3,30,4,40,6,60 -Tetrakis(3,4,5-tribenzyloxyben-
0
zoyl)-ꢁ,ꢁ0-D,D -glucopyranosyl-(2,30-oxy-di-benzoate) (8a).
By use of general procedure A, diacid chloride 6a (31
mg, 0.11 mmol) was coupled to alcohol 7 (373 mg, 0.20
mmol) with triethylamine (88 mL, 0.63 mmol) in 2.5 mL
of CH2Cl2, and the crude product was purified by flash
chromatography to afford 147 mg (37%) of benzylated
1
dimer 8a: IR (CDCl3) 1731 cmꢁ1; H NMR (CDCl3,
300 MHz) d 7.97 (dd, J=7.9, 1.5 Hz, 1H), 7.76–7.73 (m,
1H), 7.66–7.65 (m, 1H), 7.41–7.15 (m, 138H), 7.13–7.00
(m, 2H), 6.72 (d, J=8.3 Hz, 1H), 6.29 (d, J=7.9 Hz,
1H), 6.26 (d, J=7.9 Hz, 1H), 6.04 (appar t, J=9.8 Hz,
1H), 5.97 (appar t, J=9.8 Hz, 1H), 5.82–5.68 (m, 4H),
5.10–4.9 (m, 48H) 4.93–4.72 (m, 2H), 4.37–4.27 (m, 4H);
13C NMR (CDCl3, 90 MHz) d 165.5, 165.4, 165.0,
164.9, 164.8, 164.8, 164.0, 162.8, 162.8, 162.4, 157.3,
156.7, 152.5, 152.5, 152.4, 152.4, 143.1, 143.1, 143.0,
143.0, 143.0, 142.5, 142.5, 137.5, 137.5, 137.4, 137.3,
137.3, 136.7, 136.6, 136.4, 136.4, 136.3, 136.3, 134.9,
132.6, 130.2, 130.2, 128.5, 128.4, 128.3, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.5, 124.8, 124.5, 124.5,
123.7, 123.7, 123.6, 120.6, 120.3, 119.5, 109.3, 109.1,
109.1, 92.9, 92.5, 77.2, 75.1, 75.1, 73.6, 73.4, 73.1, 71.2,
71.1, 71.1, 71.0, 69.8, 69.7, 67.5, 63.2, 62.9.
1,10-O-2,20,3,30,4,40,6,60-Tetrakis(3,4,5-trihyroxybenzoyl)-
0
ꢁ,ꢁ0-D,D -glucopyranosylpimelate (3b). By use of gen-
eral procedure B, dimer 8b (140 mg, 0.036 mmol) was
hydrogenated using 32 mg (0.03 mmol) of Pd/C in 5 mL
of THF to afford 60 mg (97%) of pentyl linked dimer
1
3b: IR (KBr) 3422, 1718 cmꢁ1; H NMR ((CD3)2CO,
300 MHz) d 8.17–7.45 (m, 24H), 7.06 (s, 4H), 6.91 (s,
4H), 6.90 (s, 4H), 6.82 (s, 4H), 6.05 (d, J=8.3 Hz, 2H),
5.79 (appar t, J=9.6 Hz, 2H), 5.48 (appar t, J=9.8 Hz,
2H), 5.32 (dd, J=9.8, 8.3 Hz, 2H), 4.28–4.23 (m, 2H);
13C NMR ((CD3)2CO, 75 MHz) d 171.3, 165.8, 165.2,
165.0, 145.5, 145.4, 145.4, 145.3, 138.9, 138.8, 138.6,
138.5, 120.8, 120.0, 119.9, 119.8, 109.7, 109.6, 109.5,
109.5, 92.2, 73.3, 72.6, 71.2, 68.6, 62.2, 33.6, 28.2, 24.4;
MS (+FAB) 1701 (MH+); HRFABMS calcd for
C75H64O46: 1700.2669. Found: 1700.2577; [a]2D0=+53.3
(CH3OH).
1,10-O-2,20,3,30,4,40,6,60-Tetrakis(3,4,5-trihyroxybenzoyl)-
0
ꢁ,ꢁ0-D,D -glucopyranosyl-(2,30-oxy-di-benzoate) (3a). By
use of general procedure B, dimer 8a (131 mg, 0.033
mmol) was hydrogenated using 17 mg (0.016 mmol) of
Pd/C in 2.5 mL of THF to afford 70 mg (99%) of diaryl
2,30-Oxy-di-benzoic acid (5).25 Carboxylic ester 423
.
(0.118 g, 0.412 mmol) was combined with LiOH H2O
1
ether linked dimer 3a: H NMR (CDCl3, 360 MHz) d
(0.105 g, 2.50 mmol) in 3 mL of MeOH and 1 mL of
H2O. The reaction mixture was heated to reflux under
Ar for 5 h. The solution was cooled to rt, acidified with
1 N HCl, and extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4. After
filtration and removal of solvents in vacuo, 0.099 g
(93%) of diaryl ether diacid 5 was collected as a white
8.18 (m, 24H), 7.91 (m, 1H), 7.71 (d, J=7.8 Hz, 1H),
7.60–7.53 (m, 2H), 7.40–7.34 (m, 2H), 7.28–7.24 (m,
2H), 7.19 (s, 4H), 7.06 (s, 2H), 7.04 (s, 2H), 7.02 (s, 2H),
6.98 (s, 2H), 6.97 (s, 2H), 6.94 (s, 2H), 6.39 (d, J=8.2
Hz, 1H), 6.35 (d, J=8.2 Hz, 1H), 6.04 (appar t, J=9.8
Hz, 1H), 5.97 (appar t, J=9.6 Hz, 1H), 5.71–5.56 (m,
4H), 4.61–4.40 (m, 6H); 13C NMR ((CD3)2CO, 90 MHz)
d 166.0, 165.4, 165.2, 165.1, 165.1, 164.1, 164.1, 162.6,
157.9, 156.6, 145.6, 145.5, 145.4, 138.9, 138.7, 138.6,
1
powder: H NMR ((CD3)2CO, 300 MHz) d 9.5 (br s,
2H), 8.00 (dd, J=7.9 Hz, 1.9 Hz, 1H), 7.75 (appar t of

54
K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
135.5, 132.3, 130.8, 130.7, 124.9, 124.6, 124.2, 121.8,
121.3, 121.3, 120.9, 119.6, 109.8, 109.8, 109.7, 93.4, 93.0,
73.6, 73.5, 72.8, 72.6, 71.3, 71.2, 68.8, 68.7, 62.3, 62.2;
MS (+MALDI) 1822 (MNa+); [a]D20=+ 43.8
(CH3OH).
only, and control rat 9 was given saline only. Blood
plasma samples (and hemodynamic measurements)
were collected prior to the start of the study and at 90
and 180 min and analyzed for TNF-a and IL-1b
by ELISA. The cytokine values reported are mean
ꢃSEM.
Immunological assays, general. LPS (Escherichia coli
055:b5 phenol extract), Ficoll-Histopaque-1077, Fetal
Bovine Serum (FCS), gentamicin (10 mg/mL), l-gluta-
mine (200 mM), Dextran B-512 (ave Mr 580,000), and
Trypan Blue stain (0.4%) were purchased from Sigma.
Hanks’ Balanced Salt Solution 1X, with phenol red
(HBSS) and RPMI 1640 1X (Mediatech) were pur-
chased from Fisher Scientific. A complete culture med-
ium (cRPMI) was prepared by addition of 10 mL of
gentamicin solution, 10 mL of l-glutamine solution,
and 100 mL of FCS to 1 L of RPMI 1640. Human and
rat TNF-a and IL-1b ELISA kits were purchased from
R&D Systems, Minneapolis, MN, USA and Biosource
International, Camarillo, CA, USA.
Acknowledgements
We thank the National Institutes of Health [GM 35727
(KSF) and GM 38032 (CHL)] for financial support of
this work.
References and Notes
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Isolation of h-PBMCs. Fresh heparinized blood was
obtained from healthy human subjects (ages 20–34
years). PBMCs were isolated from reported proce-
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viability was determined by Trypan Blue exclusion
(typically, viability exceeded 95%). Experiments were
conducted in a 5% CO2, 37 ꢄC humidified incubator for
the indicated time period.
h-PBMCs, Dimer dose response data. 0.5 mL Samples
of PBMC suspension (2ꢅ106 cells/mL in cRPMI) were
incubated for 24 h with the appropriate amounts of a
dimer (3a–e) or LPS stock solution in HBSS to furnish
the desired concentration range. Each concentration
value was run in triplicate, and blank runs ensured that
(bacterial) contamination did not complicate the
experiments. After 24 h, culture supernatants were har-
vested and analyzed for TNF-a by Enzyme Linked
Immunosorbent Assay (ELISA). The cytokine values
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h-PBMCs, Dimer inhibition data. 0.5 mL Samples of
PBMC suspension (2ꢅ106 cells/mL in RPMI 1640) were
incubated with 5 or 10 mg/mL of LPS in HBSS for 45
min. At this time, the non-control wells were treated
with the appropriate amount of a dimer (3b–e) stock
solution in HBSS to furnish the desired concentration
range. Each concentration value was run in triplicate,
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Rat inhibition data. Chronically catheterized, conscious,
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injection+5 mg/h as constant infusion). DMSO was
used to aid in solubility of the dimers. 10 min after
the initial injection, the animals were treated with a
1 mg/kg dose of LPS (E. coli, 026:B6, Difco, Detroit,
MI, USA). Rats 5–8 were administered LPS+DMSO

K. S. Feldman et al. / Bioorg. Med. Chem. 10 (2002) 47–55
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