Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4)

Article abstract of DOI:10.1016/j.tetlet.2014.11.048

Abstract The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated solvolysis of anomeric oxazolines and an acid-mediated conversion of β-glucosamine and β-galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the pyranose ring. These compounds were evaluated for their influence on TLR4-mediated inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-acetamido-α-galactoside 17.

Full text of DOI:10.1016/j.tetlet.2014.11.048

Accepted Manuscript
Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as inhib-
itors of toll-like receptor 4 (TLR4)
Peter Wipf, Benjamin R. Eyer, Yukihiro Yamaguchi, Feng Zhang, Matthew D.
Neal, Chhinder P. Sodhi, Misty Good, Maria Branca, Thomas Prindle Jr., Peng
Lu, Jeffrey L. Brodsky, David J. Hackam
PII:
S0040-4039(14)01941-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.11.048
TETL 45432
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 November 2014
9 November 2014
12 November 2014
Please cite this article as: Wipf, P., Eyer, B.R., Yamaguchi, Y., Zhang, F., Neal, M.D., Sodhi, C.P., Good, M.,
Branca, M., Prindle, T. Jr., Lu, P., Brodsky, J.L., Hackam, D.J., Synthesis of anti-inflammatory α-and β-linked
acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4), Tetrahedron Letters (2014), doi: http://dx.doi.org/
10.1016/j.tetlet.2014.11.048
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Synthesis of anti-Inflammatory α- and β-
Linked Acetamidopyranosides as Inhibitors
of Toll-Like Receptor 4 (TLR4)
Wipf, P.; Eyer, B. R.; Yamaguchi, Y.; Zhang, F.; Neal, M. D.; Sodhi, C. P.; Good, M.; Branca, M.; Prindle, T.
Jr.; Lu, P.; Brodsky, J. L.; Hackam, D. J.

1
Tetrahedron Letters
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as
inhibitors of toll-like receptor 4 (TLR4)§
a
c
a
c
Peter Wipfa,b , Benjamin R. Eyer , Yukihiro Yamaguchi , Feng Zhang , Matthew D. Neal , Chhinder P.
Sodhic, Misty Goodc, Maria Brancac, Thomas Prindle Jr.c, Peng Luc, Jeffrey L. Brodskyd, and David J.
Hackamb,e
∗
aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
bDepartment of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260, USA
cDivision of Pediatric Surgery, Children’s Hospital of Pittsburgh of University of Pittsburgh and Department of Surgery, University of Pittsburgh School of
Medicine, Pittsburgh PA 15224, USA
dDepartment of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
eDivision of Pediatric Surgery, Bloomberg Children's Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like
receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation
in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated
solvolysis of anomeric oxazolines and an acid-mediated conversion of β-glucosamine and β-
galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the
pyranose ring. These compounds were evaluated for their influence on TLR4-mediated
inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed
Received in revised form
Accepted
Available online
Keywords:
Keyword_1: toll-like receptors (TLRs)
Keyword_2: anti-inflammatory
Keyword_3: cabohydrates
Keyword_4: eritoran
using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity
relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-
acetamido-α-galactoside 17.
2009 Elsevier Ltd. All rights reserved.
Keyword_5: lipid A mimetics
Carbohydrates are best known for their structural and energy
storage functions in biological systems, but they demonstrate
many other features, including the control of molecular and
Peptidoglycan fragments are recognized and induce signaling
by toll-like receptors (TLRs), activating a pathway in innate
immune cells that mediates an antimicrobial and pro-
cellular recognition events (1, 2).
Mono-, oligo- and
inflammatory response.
In addition to common
polysaccharides can be found as conjugates of peptides, lipids,
and secondary metabolites and modulate their physical and
biological properties (1, 3). 2-Amino-2-deoxyglycosides, mainly
glycosides of N-acetylglucosamine, are glycoconjugates present
in human milk, blood, bacterial lipopolysaccharide antigens and
plant root cells (4, 5). Glucosamine (Fig. 1) is a known treatment
for pain maintenance in osteoarthritis and has no adverse side
effects when compared to non-steroidal anti-inflammatory drugs
(NSAIDs), nor does it affect glucose metabolism (6). Post-
translational modification of nuclear and cytoplasmic proteins by
O-glycosidic attachment of β-N-acetylglucosamine is essential
for cell viability, and improves cell survival under stress (7).
immunodisorders, TLRs have also been linked to carcinogenesis,
and therefore they have evolved into major targets for drug
discovery (9).
Several toll-like receptor 4 (TLR4) antagonists are under
development as targeted therapeutics, including Eritoran from
Eisai Pharmaceuticals (suspended in human Phase III trials),
AV411 from Avigen (Phase II), and NI0101 from NovImmune
(Preclinical) (9). Lipid A mimetics similar to Eritoran have
become popular synthetic targets (10-12) and have sparked our
interest in the use of glucosamine as a central scaffold for SAR
elaboration of immunomodulating TLR ligands. As glucosamine
is a component of the disaccharide core of both Lipid A and
Eritoran (Fig. 1), we sought to mimic key structural features of
these large liposaccharides, while reducing molecular weight and
lipophilicity to generate more drug-like analogs. Using in silico
drug discovery followed by in vivo testing, we recently
discovered novel small molecule TLR4 inhibitory mono- and
Glucosamine-
conjugates have also shown promise as lead structures in drug
and
N-acetylglucosamine-derived
glyco-
development (8).
———
§ Dedicated to the memory of Prof. Harry Wasserman, in deep appreciation of his many insightful contributions to Organic Chemistry.
∗ Corresponding author. Tel.: +1-412-624-8606; fax: +1-412-624-0787; e-mail: pwipf@pitt.edu

2
Tetrahedron Letters
oligosaccharides (13).
In particular, isopropyl 2-
of GlcNAc glycosyl donors to form the oxazoline side product,
various alternatives to the N-acetyl group have been examined in
glycosylation reactions (15), including protection with phthaloyl
(16), tetrachlorophthaloyl (17, 18), 4,5-dichlorophthaloyl (19),
dithiasuccinoyl (20, 21), trichloro- (22, 23), and trifluoroacetyl
(22, 24), trichloroethoxycarbonyl (25-28), diacetyl (29),
dimethylmaleoyl (30), and thiodiglycoloyl (31) groups, or a 2-
azido group (5, 32-34).
acetamidopyranoside C34 was found to inhibit TLR4 in
enterocytes and macrophages in vitro and to reduce systemic
inflammation in mouse models of endotoxemia and necrotizing
enterocolitis (13). Subsequently, our strategy examined three
areas for further structure-activity relationship (SAR) studies of
this lead compound for TLR4-mediated inflammatory diseases:
an α- vs β-glycosidic linkage, the configuration at C-4 of the
pyranose (glucosamine vs galactosamine), and the length, size
and hydrophilicity of the glycosyl chain. The pyranose hydroxyl
and amino groups were O- and N-acetylated to mimic the longer
acyl chains in Lipid A and Eritoran (Fig. 1).
While these protective groups resolve the oxazoline issue,
each requires additional steps for protection and subsequent
substitution with the N-acetyl group in the desired analogs. Ring
opening of aziridines such as those generated from
iodosulfonamidation of glycals (35) or from the photolysis of
triazolines (36) present an additional alternative for the formation
of 2-amino-2-deoxyglycopyranosides, but these methods were
unsuccessful in our hands. In contrast, the opening of oxazoline
2 with alcohols under mild, copper(II)-catalyzed conditions
showed promise for formation of the β-glycosides 3,4,6-tri-
acetyl-N-acetylglucosamine
and
3,4,6-tri-acetyl-N-
acetylgalactosamine 1b in our preliminary studies (13, 14, 37,
Scheme 1). Furthermore, an acid catalyzed isomerization of the
β-anomers 1b was envisioned to lead to the thermodynamically
more stable α-anomers 1a, thus allowing a convergent approach
from readily available pentaacetates 3.
Scheme 1. Retrosynthetic analysis for α-and β-anomers 1 of
2-acetamido-2-deoxy-D-glucose and -galactose.
The fully protected galactosamine β-pentaacetate 5 (38) and
glucosamine β-pentaacetate 11 (39) were prepared from tetrols 4
and 10, respectively, using pertinent literature procedures
(Schemes 2 and 3). The corresponding oxazolines 6 and 12 were
subsequently obtained in high yields with TMSOTf in 1,2-
dichloroethane (14, 40-42). Other literature methods to access
glyco-oxazolines, including SnCl4 in dichloromethane, did not
lead to the desired products (43, 44). Oxazolines 6 and 12 were
then heated at reflux in CHCl3 in the presence of CuCl2 and
isopropanol, cyclohexanol, and geraniol as glycosyl acceptors to
give the corresponding C-2 glycosides in good yields as pure β-
anomers after recrystallization or chromatographic purification
(14). Six different derivatives were prepared with the goal to
vary lipophilicity, with 3 epimeric pairs differing only by their
Figure 1. Glycoconjugates.
The exploration of glycosylation methods for 2-amino-2-
deoxyglycopyranosides was based on traditional literature
protocols for carbohydrates, with some distinct differences due to
the presence of the C-2 nitrogen atom (4). Glycosylation of 2-
configuration at C-4.
Glycosylation of isopropanol gave
acetamido-2-deoxy-D-glucosamine
(N-acetylglucosamine,
pyranosides 7 and 13 with a clogP of -0.5, whereas the
cyclohexyl derivatives 8 and 14 were more lipophilic (clog P
0.6). The geraniol glycosides 9 and 15 (clogP 1.8) represented
the most lipophilic small molecule analogs of Lipid A and
Eritoran in this series (45).
GlcNAc) under standard Koenigs-Knorr conditions proceeded in
good diastereoselectivity for the 1,2-trans-glycoside, but resulted
in a large amount of the oxazoline side product (4). Furthermore,
these conditions were limited to reactive glycosyl acceptors with
a primary hydroxyl group which are often used in a large excess
(4). If the N-acetyl group cyclizes to the oxazolinium
intermediate and looses a proton to give the oxazoline, the
glycosylation frequently stalls (4, 14). Because of the tendency

3
Scheme 4. Acid-mediated tandem glycosylation and β- to α-
anomeric equilibration.
As discussed in the introduction, many chronic inflammatory
diseases and certain cancers trace their origin to increased
signaling from the innate immune receptor TLR4, and our
preliminary studies had identified 16 (C34) as a potent anti-
inflammatory agent in mouse models of endotoxemia and
necrotizing enterocolitis (13). In order to investigate the SAR of
16 and determine potential therapeutic benefits of analog
structures, we screened compounds 7-9, 13-15, and 17-18 for the
response on TLR4-mediated inflammatory signaling in cultured
enterocytes (non-transformed rat small intestinal IEC-6 cells) and
Scheme 2. Synthesis of three β-glycosides of 2-acetoamido-2-
deoxy-D-galactose (7-9).
monocytes (mouse RAW 264.7 cells).
Efficacy was
subsequently confirmed using the NF-kB-luciferase reporter
mouse, followed by assessment of pro-inflammatory cytokines
IL6, IL1β and TNFα via qRT-PCR (48).
As shown in Table 1, isopropyl-β-galactoside 7, isopropyl-α-
glucoside 16 and cyclohexyl-α-glucoside 18 produced the
strongest anti-inflammatory suppression of LPS-induced
cytokine mRNA levels in IEC6 cells, whereas geranyl-β-
galactoside 9 and β-glucosides 13-15 were comparatively
ineffective. These results, in combination with the intermediate
level of activity of cyclohexyl-β-galactoside 8 and isopropyl-α-
galactoside 17, indicate that a syn-configuration of acetate groups
at C-4 and anomeric substituents at C-1 are preferred with regard
to suppression of the pro-inflammatory IL6, IL1β and TNFα.
The larger, more lipophilic geranyl group at the anomeric carbon
in 9 and 15 also appears detrimental to bioactivity. The
analogous measurements in RAW cells are consistent with these
data (Table 1).
Scheme 3. Synthesis of three β-glycosides of 2-acetoamido-2-
deoxy-D-glucose (13-15).
The corresponding α-glycosides in the galactosamine and
glucosamine series proved more difficult to synthesize, and at
first all previously envisioned acid catalyzed isomerization
conditions starting with the corresponding β-glycosides were
unsuccessful.
Finally, heating the β-glucosamine and β-
galactosamine pentaacetates 11 and 5, respectively, in in situ
prepared 5% HCl in i-propanol and cyclohexanol, in analogy to
literature conditions used for the synthesis of methyl α-D-
glucosamine from D-glucosamine pentaacetate,(46) provided a
3:1 mixture of partly deacylated α:β anomers (Scheme 4). After
reacetylation with acetic anhydride in pyridine, the mixture of
anomers was separated by chromatography on SiO2 to give the
desired α-anomeric glycosides 16-18 in moderate yields.
Extensive NMR analyses were performed to assign the
configurations of all glycoside products (47).
Table 1. Biological evaluation of anti-inflammatory activities
Compound
Suppression of LPS-induced IL6,
IL1β, and TNFα mRNA levels in IEC6
cellsa
Suppression of LPS-induced IL6,
IL1β, and TNFα induction in RAW cellsb
Protection of IL6, IL1β, and TNFα
induction in micec
7
58.0% (p<0.01)
44% (p<0.01)
<20%
50% (p<0.01)
50% (p<0.05)
<20%
<64% (p<0.01)
<60% (p<0.01)
<20%
8
9
13
<20%
<20%
<20%
14
<20%
<20%
48%
15
30%
32%
<20%
16 (C34; ref. 13)
55% (p<0.01)
43% (p<0.01)
54% (p<0.05)
50% (p<0.01)
50% (p<0.01)
50% (p<0.01)
69% (p<0.01)
78% (p<0.01)
64% (p<0.01)
17
18
Treatment and dosages: aIEC6 cells (LPS 25 µg/mL, compounds 5 µg/mL, 30 min pretreatment). bRaw cells (LPS, 10 ng/mL, compounds 5 µg/mL, 30 min pre-
treatment). cMice (LPS 2.5 mg/kg, compounds 2.5 mg/kg, 30 min pre-pretreatment).

4
Tetrahedron
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A more complex pattern emerges in the in vivo biological
assessment. While acetamidopyranosides 9, 13, and 15 are still
inactive, protection of cytokine expression in mice is most
effective with 2.5 mg/kg of isopropyl-α-galactoside 17 (78%),
slightly surpassing isopropyl-β-galactoside 7 (64%), isopropyl-α-
glucoside 16 (69%) and cyclohexyl-α-glucoside 18 (64%).
Furthermore, cyclohexyl-β-glucoside 14 picks up moderate
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48. For a detailed description of assay conditions, see the
Supplementary Material.

5
Supplementary Material
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Synthetic procedures, spectroscopic data, and assay
conditions.