Glycolipids and benzylammonium lipids as novel antisepsis agents: Synthesis and biological characterization

Article abstract of DOI:10.1021/jm801333m

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagoni

Full text of DOI:10.1021/jm801333m

J. Med. Chem. 2009, 52, 1209–1213
1209
Glycolipids and Benzylammonium Lipids as Novel Antisepsis Agents: Synthesis and Biological
Characterization
Matteo Piazza, Clara Rossini, Silvia Della Fiorentina, Chiara Pozzi, Francesca Comelli, Isabella Bettoni, Paola Fusi,
Barbara Costa, and Francesco Peri*
Department of Biotechnology and Biosciences, UniVersity of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy
ReceiVed October 22, 2008
New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure
of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds,
their in vitro activity as lipid A antagonists on HEK cells, and the capacity to inhibit LPS-induced septic
shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the
panel as hits for antisepsis drug development.
Mammalian cells have developed the ability to respond to
minute (pM) quantities of endotoxin, unique, abundant, and
generally conserved surface glycolipids (the entire LPSa and
parts of its structure, lipid A) of Gram-negative bacteria.1,2 This
exceptional sensitivity is due to signal amplification mediated
by a cascade of sequential protein-endotoxin and protein-protein
interactions involving at least four extracellular and cell-surface
host proteins: LPS-binding protein (LBP), soluble (s) and
membrane-associated CD14, secreted and Toll-like receptor
(TLR) 4-associated forms of MD-2, and TLR4 itself.3-5 MD-2
plays an essential role in regulation of endotoxin mediated TLR4
activation, bridging recognition, normally initiated by CD14,
to receptor activation or antagonism.6-8 TLR4 trigger can be
remarkably sensitive and robust, stimulating prompt and power-
ful host defense responses to different species of invading
bacteria. However, an excessively potent host response generates
life-threatening syndromes such as acute sepsis and septic shock.
Gram-negative sepsis is the first cause of deaths in intensive
care units, and it is associated to a mortality of about 45%.9
Several pharmacologically active compounds have been devel-
oped and used in clinic to treat septic shock.10 However, because
Figure 1. Structure of Glycolipids 1-6 and of benzylammonium lipid
7.
of the serious side effects observed, there is still a major unmet
medical need for safer and more effective antisepsis agents. The
it. LPS can be complexated and neutralized by antibodies,14
majority of small molecules so far proposed as leads for drug
nonantibody proteins,15 and by small molecules such as the
development are synthetic mimics of the lipid A or naturally
antibiotic cyclic peptide polymixin B16 (PMB), a highly active
occurring underacylated forms of lipid A that act as antagonists
endotoxin-sequestring agents that has never been used in a clinic
on the TLR4.11 Among these compounds, worth noting is
because its high toxicity. Synthetic polycationic amphiphiles
were shown to protect animals against endotoxin-induced
lethality, and it was found that binding to LPS is a necessary
compound E5564,12 a synthetic lipid A analogue that is currently
in phase III clinical trials. The ethyl(6R)-6-[N-(2-chloro-4-
fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-
but not sufficient condition for the inhibition of endotoxic
242)13 is another small molecule that reached the clinical phase
activity.17 Among these compounds are notable linear or
III as antisepsis agent, and it was discovered by screening a
branched18 spermine derivatives and “Gemini” amphiphiles;19
large library of compounds. The antiendotoxic activity of this
this last class of molecules has been designed as surfactant and
compound has been related to selective inhibition of the
therefore presents high cytotoxicity and hemolytic activity.
intracellular signaling of the TLR4 pathway. Another class of
We recently published the synthesis and the biological activity
antisepsis agents directly target LPS by binding and sequestering
of compounds 1 and 2, derived from the natural monosaccharide
D-glucose20 (Figure 1). These glycolipids have some structural
* To whom correspondence should be addressed. Phone: +39.02.64483453.
Fax: +39 0.02.64483565. E-mail: francesco.peri@unimib.it.
elements in common with the branched spermine derivatives cited
above,18 namely two lipophilic (C14) appendages linked to a
positively charged amino group. We reported the activity of these
compounds in inhibiting the lipid A-stimulated cytokine production
in cells of the innate immune system such as macrophages and
dendritic cells. Conversely, TNF-R production inhibition was not
observed in macrophages and DC when these cells were treated
with agents that selectively stimulate other TLRs. Compound 2
a Abbreviations: AcOEt, ethyl acetate; ANOVA, analysis of variance;
sAP, secreted alkaline phosphatase; CD14, cluster differentiation antigen
14; CIP, calf intestinal alkaline phosphatase; DC, dendritic cell; DIPEA,
diisopropylethylamine; DMF, N,N-dimethylformamide; DMAP, N,N-dim-
ethylaminopyridine; Fmoc, fluorenylmethoxycarbonyl; HEK, human em-
brionic kidney; HOBt, N-hydroxybenzotriazole; LBP, lipid binding protein;
LPS, lipopolysaccharide; MD-2, protein MD-2; NF-kB, nuclear factor-kB;
SEM, standard error media; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TLR4, Toll-like receptor 4; TNF-R, tumor necrosis factor-R.
10.1021/jm801333m CCC: $40.75
2009 American Chemical Society
Published on Web 01/22/2009

1210 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Scheme 1. Synthetic Route to Glycolipids 1-6a
Brief Articles
a Reagents and conditions: (a) Dess-Martin periodinane, CH2Cl2, rt, 1 h; (b) cyclopentylamine, NaBH3CN, AcOH, CH2Cl2, MeOH, 24 °C, 12 h; (c)
TFA, CH2Cl2, 24 °C, 1 h; (d) CH3I, Na2CO3, DMF, 24 °C, 12 h; (e) CH3ONH2 ·HCl, pyridine, 24 °C, 12 h; (f) NaBH3CN, AcOH, CH2Cl2, 24 °C, 2 h; (g)
MsCl, pyridine, 24 °C, 5 h; (h) cyclopentanol, NaH, DMF, 24 °C, 12 h; (i) TsCl, DMAP, pyridine, 24 °C, 12 h; (l) NaN3, tetrabutylamonium iodide (TBAI),
DMF, 75 °C, 4 h; (m) PPh3, H2O, THF, 70 °C; (n) N-Fmoc-Pro, HOBt, DIC, DIPEA, DMF, 24 °C, 4 h; (o) piperidine/DMF 2:8, 24 °C, 1 h.
Scheme 2. Four-Step Synthesis of Compound 7a
inhibited the lipid A-induced NF-kB activation of HEK cells
selectively transfected with the human TLR4 while inactive on
HEK cells transfected with human TLR9 gene. Taken together,
these results suggest of a selective action of compound 2 on the
TLR4 signal pathway. Recent evidence indicates a pivotal role of
TLR4 in the development and maintenance of some other patholo-
gies such inflammation21 or painful neuropathies.22 We observed
an interesting activity of compound 2 in inhibiting both neuropathic
pain23 and carrageenan-induced acute local inflammation in animal
models.24
To gain information about the structure-activity relationship
for this new class of biologically active molecules, we designed
compounds 3-7 that share certain common key features with
compounds 1 and 2. Chemical modifications of the lead 2 were
focused on the amino group on C-6 of glucose and on its
substituents. We designed compound 3, in which the nitrogen
atom on glucose C-6 has been replaced by the oxygen of a
cyclopentyl ether, and compound 5, conserving a primary amino
group and lacking the cyclopentyl ring. In compound 4, we
inserted an O-methyl hydroxylamine function in C-6, while in
compound 6, the C-6 amine is condensed with a proline residue.
The unique structure of this amino acid provides a secondary
amine inserted into a five-membered cycle. In compound 7, the
cyclic core of the glucopyranose scaffold of 2 has been replaced
by an aromatic ring. The mutual disposition of the C-6 nitrogen
(or oxygen, in the case of 3) and of the two tetradecyl ethers
was conserved in all derivatives. The C-6, C-2, and C-3 positions
of glucose of compounds 1-6 correspond to C-1, C-3, and C-4
positions of the benzene ring in 7. The protonation state of the
nitrogen could greatly influence the biological activity and the
pharmacokinetic of these molecules, so all compounds but 3
are fully protonated and therefore positively charged at neutral
pH. Compounds 1-7 were prepared starting from commercially
available and inexpensive compounds: the methyl-ꢀ-D-glucopy-
a Reagents and conditions: (a) cyclopentylamine, HOBt, DIC, DIPEA,
DMF, 24 °C, 12 h; (b) tetradecyl bromide, NaH, DMF, 60 °C, 2 h; (c)
LiAlH4, tetrahydrofuran (THF), 60 °C, 12 h; (d) CH3I, Na2CO3, DMF, 24
°C, 3 h.
ranoside was transformed into glycolipids 1-6 according to the
synthetic way shown in Scheme 1, whereas 3,4-dihydroxy
benzoic acid was transformed in compound 7 through the four-
step synthesis depicted in the Scheme 2.
Methyl glucopyranoside 8, protected at C-4 as p-methoxy-
benzyl (PMB) ether, and with two tetradecyl ether groups in
C-2 and C-3, was prepared in gram quantities according to
published procedures.20 The oxidation of 8 to aldehyde 9 was
carried out by treating with Dess-Martin periodinane. The
reductive amination of 9 with cyclopentylamine and NaBH3CN
followed by acidic cleavage of the PMB group afforded
compound 1.20 The methylation of secondary amine group of
1 with iodomethane gave compound 220 that directly precipitated
in a pure form from the reaction crude after addition of light
petroleum. Alternatively, the aldehyde group of 8 was converted
to the corresponding O-methyl oxime derivative by reacting with
MeONH2 ·HCl in pyridine. The O-methyl oxime was then
reduced to hydroxylamine by treating with NaBH3CN and, after

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1211
removal of the PMB group with TFA, gave final compound
4.25 The C-6 hydroxyl of compound 8 was transformed into a
mesylate by reacting with mesyl chloride in pyridine, then
treatment with cyclopentanol in the presence of sodium hydride
afforded compound 3 with a cyclopentyl ether in C-6. The
reaction of 8 with p-toluensulfonyl(tosyl) chloride afforded the
C-6 tosylate that was converted into 10 by nucleophilic
displacement by using sodium azide. The conversion of the C-6
azide into a primary amine was accomplished in a clean and
efficient way by treating 10 with triphenylphosphine: the
hydrolysis of the aza-yilide intermediate in THF-water afforded
the corresponding amine. After cleavage of the PMB ether in
C-4, compound 5 was obtained, which was condensed with
N-Fmoc-L-proline in the presence of HOBt, DIC and the Hunig’s
base DIPEA. Finally, the cleavage of the Fmoc carbamate (20%
piperidine in DMF) from proline nitrogen afforded compound
6. This molecule showed an unexpected chemical instability
when dissolved in organic solvents at 24 °C, and decomposition
was observed in NMR samples. A half-life of about 48 h was
calculated based on the disappearance of compound 6 proton
signals in NMR spectra.
The activity of compounds 1-7 as inhibitors of the TLR4
signal pathway was tested in vitro using a HEK-Blue LPS
Detection Kit (InvivoGen). HEK-Blue-4 cells are HEK293 cells
stably transfected with TLR4, MD2, and CD14 genes. In
addition, these cells stably express an optimized alkaline
phosphatase gene engineered to be secreted (sAP), placed under
the control of a promoter inducible by several transcription
factors such as NF-kB and AP-1. This reporter gene allows
monitoring of the activation of TLR4 signal pathway by
endotoxin (LPS or lipid A). Moreover, using HEK-Blue
detection medium, phosphatase activity can be quantified
spectrophotometrically. Therefore, we exploited this simple, fast,
and reliable system to screen our compounds for their TLR4
antagonistic activity. HEK-Blue-4 cells were stimulated with
lipid A (10 nM) after pre-exposure to the molecules, at three
different concentrations (1, 5, and 10 µM) and phosphatase
activity was measured by spectrophotometric analysis (630 nm,
24 h later). As shown in Figure 2, compounds 3 and 4 did not
interfere with lipid A activity at least at the maximum
concentration employed and compound 1 showed a weak
inhibition (23%) only at the highest concentration (10 µM). On
the other hand, compounds 2, 5, 6, and 7 exhibited a significant
inhibition of the lipid A activity decreasing the phosphatase
activity in a concentration-dependent manner. It was possible
to calculate a linear regression relating the inhibitory activity
and the compound concentration (for compound 2 r2 ) 0.5624
p < 0.05; for compound 5 r2 ) 0.6810, p < 0.01; for compound
6 r2 ) 0.6389, p < 0.05; for compound 7 r2 ) 0.8553, p <
0.001). The IC50 comparison (Table 1) demonstrates that in this
experiment 7 is the most active compound in suppressing lipid
A activity: its potency is more than 3-fold greater than that of
compounds 2, 5, and 6.
Figure 2. In vitro TLR4 antagonistic activity of compounds 1-7.
HEK-Blue-4 cells stably transfected with TLR4 and an optimized
alkaline phosphatase gene engineered to be secreted (sAP) were treated
with lipid A (10 nM) alone or lipid A in the presence of one among
compounds 1-7 at three different concentrations (1, 5, 10 µM). NF-
kB activation was evaluated by measuring phosphatase activity,
expressed as absorbance (Abs) at 630 nm. The results are representative
for three independent experiments and data are expressed as mean (
SEM *p < 0.05; **p < 0.01 (ANOVA; Dunnett’s test).
Table 1. IC50 on Lipid A-Induced TLR4 Activation
compd
IC50 (µM)
confidence intervals
1
2
3
4
5
6
7
>10
5.013
>10
3.430-7.327
>10
5.487
5.871
1.675
3.102-9.705
3.531-9.726
0.910-3.084
Blue. Molecules were incubated with supernatant containing
HEK-Blue sAP produced with 24 h cells stimulation with lipid
A (10 nM) and no enzyme inhibition could be detected
(Supporting Information).
The cytotoxic potential of compounds 2, 5, 6, and 7 was
investigated. HEK-Blue-4 cells were grown in the presence of
molecules at highest concentration (10 µM). After 24 h growth,
the viability was evaluated using CellTiter Blue assay (Prome-
ga). As shown in Figure 3, for the HEK-Blue-4 cell line, no
inhibitory effect of compounds 2, 5, and 6 on cell viability was
detected. Compound 7 showed a diminution of the cell viability
of about 17%; however, there is no statistical difference between
the percentage of vitality cell in presence and absence of
compound 7, as demonstrated by Kruskal-Wallis ANOVA for
non parametric data.
It was important to verify that the observed inhibition in sAP
activity is not a consequence of direct interference of compounds
1-7 with the enzyme. Control experiments were done using a
similar enzyme, the recombinant calf intestinal alkaline phos-
phatase (CIP), by incubating molecules 2, 5, 6, and 7 (10 µM)
with different concentrations of CIP (0.4, 0.2, 0.02 mU/mL) in
the presence of HEK-Blue detection medium. Recombinant CIP
resulted active even at 0.02 mU/mL, demonstrating that
compounds tested were not able to inhibit phosphatase activity
(data not shown). An additional experiment was done to asses
if compounds 2, 5, and 7 interfere with sAP produced by HEK-
Finally, we tested active compounds 1, 2, 5, and 7 for their
capacity to protect animal against LPS-induced lethality. For
the lethal endotoxin shock model, C57BL/6J male mice were

1212 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Brief Articles
Figure 5. In vitro TNFR activation of HEK-Blue-4 cells. HEK-Blue-4
cells were treated with TNF-R (1 ng/mL) alone or in the presence of
compounds 2, 5, and 7 (10 µM). sAP production was evaluated by
measuring absorbance (Abs) at 630 nm.
Figure 3. Viability of HEK-Blue-4 cells in the presence of active
compounds 2, 5, 6, and 7. Cells were seeded and grown for 24 h in the
presence of the most effective molecules at maximum concentration
(10 µM). The cell viability was determined using CellTiter Blue cell
viability assay (Promega). As controls, we set up triplicate wells with
cells and 0.5% DMSO/EtOH and triplicate wells containing cells treated
with a compound known to be toxic to the cells (C+). Kruskal-Wallis
ANOVA for nonparametric data has been applied to analyze the
differences among groups.
its half-life of about 48 h is compatible with the in vitro tests duration
but significant degradation is observed after the time required for in
vivo tests (5 days). We are investigating the mechanism of degradation
of this compound and, once clarified, we will be able to design
chemically stable structural variants. Glycolipids 2, 5, and 6 are active
lipid A antagonists on HEK cells and protect mice from LPS-induced
lethality in a statistically significant way. Notably, compound 5 is the
most active in vivo and the 100% survival observed at 3 mg/kg dose
suggests a possible use of this compound at lower concentrations. The
potency of 5 is comparable to that of the best antisepsis agents so far
developed (compound TAK-242, in clinical phase III, showed
comparable activity in this test with an ED50 of 0.3 mg/kg).13 The
second most potent compound in vivo and the most potent in vitro is
molecule 7, with an aromatic ring instead of the pyranose ring of
glucose. Both compounds 5 and 7 have been selected for clinical
experimentation on the base of their in vivo activity and lack of toxicity
on cells. We are currently investigating the mechanism of action of
these compounds, and we are trying to identify their specific target(s)
in the TLR4 signaling pathway. Sepsis is a very complex disease
involving various cascades of events and many factors. For this reason,
further work is necessary to prove the potential clinical significance
of our compounds, especially their efficacy to improve survival even
when administered after the onset of the pathology.
Figure 4. Effect of the different compounds on LPS-induced lethality
in mice. Compounds were administered ip at 10 mg/kg, 30 min before
LPS challenge. Statistical analysis was performed using the log rank
test for nonparametric data. Each group consisted of eight mice.
intraperitoneally (ip) injected with 20 mg/kg LPS (from E. coli
055:B5, Sigma, Italy), and survival of mice was observed over
4 days. Compounds were administered ip 30 min before the
LPS. As shown in Figure 4, ip injection of 20 mg/kg LPS was
lethal to mice and all mice died within two days.
Experimental Section
Methyl 6-Deoxy-6-amino-2,3-di-O-tetradecyl-r-D-glucopyra-
noside (5). To a solution of methyl 6-deoxy- 6-amino-4-O-(4′-
methoxybenzyl)-2,3-di-O-tetradecyl-R-D-glucopyranoside (0.25 g, 0.35
mmol) in CH2Cl2 (5 mL), TFA (3.3 mL) was added and the solution
was stirred at rt for 2 h. Solvents were evaporated in vacuo, the residue
was purified by flash chromatography on silica gel (gradient starting
from AcOEt/MeOH/water 8: 2: 0.1), and pure product was obtained
as a colorless oil (0.18 g, 90% yield). Anal. (C35H71NO5) C, H, N.
Methyl 6-Deoxy-6-[(2-pyrrolidinyl)carbonylamino]-2,3-di-O-
tetradecyl-r-D-glucopyranoside (6). A solution of methyl 6-deoxy-
6-[[(1-fluorenylmethoxycarbonyl)-2-pyrrolidinyl]carbonylamino]-
2,3-di-O-tetradecyl-R-D-glucopyranoside (0.09 g, 0.100 mmol)
in piperidine/DMF 2:8 (4 mL) was stirred at rt for 1 h. After
this time, reaction was complete, as assessed by TLC analysis
(AcOEt, Rf product ) 0.12). Solvent was evaporated in vacuo,
the residue was purified by flash chromatography on silica gel
(gradient starting from petroleum AcOEt/MeOH 8:2), and pure
product was obtained as a colorless oil (0.06 g, 90% yield). Anal.
(C40H78N2O6) C, H, N.
N-(3,4-Bis-tetradecyloxy-benzyl)-N-cyclopentyl-N,N-dimethyl-
ammonium (7). To a solution of N-(3,4-bis-tetradecyloxy-benzyl)-
N-cyclopentylamine (0.3 g, 0.5 mmol) in anhydrous DMF (50 mL),
iodomethane (70 µL, 1 mmol) and Na2CO3 (160 mg, 1.5 mmol) were
added and the solution was heated at 60 °C for 1 h. After this time,
reaction was complete, as assessed by TLC analysis (CHCl3/MeOH
9:1, Rf product ) 0.47). Solvent was evaporated in vacuo, the residue
was dissolved in chloroform (40 mL) and washed with brine (2 × 40
mL). The organic layer was dried on sodium sulfate, filtered, and the
The administration of 10 mg/kg of the three molecules
significantly increased survival of mice. Particularly, the
compound 2 increased the survival rate from 0% to 25%,
compound 7 from 0% to 67%, and all mice that received
compound 5 survived. We further studied the protection from
LPS induced lethality exerted by compound 5, and we found
that also a smaller dose (3 mg/kg) evoked a 100% survival of
mice treated with LPS (data not shown). Further work is in
progress in order to establish the smallest effective dose of
compound 5 and to test whether the compound rescues mice
even when administered after the LPS challenge. Compound
1, which has the weakest TLR4 antagonistic activity in vitro,
was not effective either in the lethal endotoxin shock model.
All mice treated with this compound died within the second
day, exactly as mice treated with LPS alone.
Compounds 2, 5, and 7 were tested to assess their specificity
for TLR4 signal pathway. HEK-Blue-4 cells were grown with
TNF-R as a stimulus for TLR4 independent production of sAP
in presence of compounds. Results are depicted in Figure 5 and
clearly show no inhibitory effects on TNF-R induced activation.
By comparing the in vitro and in vivo activity of compounds 1-7,
it is possible to elucidate some aspects of structure-activity relationship
in this series. Compounds 3 and 4 lacking a protonatable amino group
were inactive in antagonizing the lipid A effect on HEK cells.
Compound 6 is relatively unstable when dissolved in solution at rt:

Brief Articles
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light petroleum (300 mg, 95% yield). Anal. (C40H78NO2) C, H, N.
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ethanol 1:1 and then diluted in PBS and added 20 µL per well of a
flat-bottom 96-well plate at three different concentrations (1, 5, 10 µM).
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Lethal Endotoxin Shock Model. For the lethal endotoxin shock
model, C57BL/6J male mice (9 weeks old, Harlan Italy) were
intraperitoneally (ip) injected with 20 mg/kg LPS (from E. coli 055:
B5, Sigma, Italy), and survival of mice was observed over 4 days. All
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ip 30 min before the LPS.
Supporting Information Available: General experimental meth-
ods, synthetic procedures, complete spectroscopic characterization of
target molecules (1H, 13C NMR, MS), full details for the experiments
on HEK cells and mice. This material is available free of charge via
the Internet at http://pubs.acs.org.
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