Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists

Article abstract of DOI:10.1021/acs.jmedchem.6b00177

Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.

Full text of DOI:10.1021/acs.jmedchem.6b00177

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Article
Discovery and Structure–Activity Relationships of the
Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists
Matthew D. Morin, Ying Wang, Brian Thomas Jones, Lijing Su, Murali M. R. P. Surakattula,
Michael Berger, Hua Huang, Elliot K. Beutler, Hong Zhang, Bruce Beutler, and Dale L. Boger
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00177 • Publication Date (Web): 06 Apr 2016
Downloaded from http://pubs.acs.org on April 7, 2016
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Discovery and Structure–Activity Relationships of the Neoseptins: A New Class
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of Toll-like Receptor-4 (TLR4) Agonists
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Matthew D. Morin, Ying Wang, Brian T. Jones, Lijing Su, Murali M. R. P. Surakattula, Michael
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‡
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Berger, Hua Huang, Elliot K. Beutler, Hong Zhang, * Bruce Beutler, * and Dale L. Boger *
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1Department of Chemistry and the Skaggs Institute of Chemical Biology, The Scripps Research
Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 USA
2Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas,
TX 75390 USA
3Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390 USA
4Department of Genetics, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA
9
2037 USA
Abstract. Herein, we report studies leading to the discovery of the neoseptins and a comprehensive
examination of the structure–activity relationships of this new class of small molecule mouse Toll-like
receptor 4 (mTLR4) agonists. The compound class, which emerged from screening a -helix mimetic
library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are
easy to produce and structurally manipulate, exhibit exquisite structure–activity relationships, are non-
toxic, and elicit improved and qualitatively different responses than lipopolysaccharide (LPS) even
though they share the same receptor.
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Introduction
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The human immune system is divided into innate and adaptive components. The innate
immune system represents the first line of defense in mammals, responding to pathogens and
abnormal cells through activation of multiple cell types including dendritic cells, macrophages,
neutrophils, and natural killer cells. The adaptive immune system entails antigen-specific T cell and B
cell responses mediated by antigen-presenting dendritic cells, and serves to protect the host in the
longer term by neutralizing the pathogens and abnormal cells through the action of T cell receptors
and antibodies.1,2 The Toll-like receptors (TLRs),3-6 the most widely recognized subset of the
pathogen-associated or damage-associated molecular pattern (PAMPs and DAMPs) receptors, act
as part of the initial innate immune response system, recognizing molecular signatures of infectious
pathogens or abnormal cells.3,5 Their activation not only initiates an intracellular signaling cascade
that leads to the activation of transcription factors that trigger the production of cytokines and
chemokines,3,5 but it also induces the adaptive immune response. In fact, the action of vaccines is
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due in part to the activation of the TLR system. TLR agonists are immune system enhancers and
could be useful in the treatment of not only infectious diseases but also cancer, representing a
complementary small molecule approach in the emerging area of immunotherapy.8-11 They would
also be attractive as new vaccine adjuvants for both infectious diseases and oncology that act by
well-defined mechanisms.8-11 However, a relatively small number of small molecule classes have
been found to behave as TLR agonists.8-11 Notable examples include the TLR7 agonists imiquimod,12
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isotoribine, and 8-oxo-9-benzyladenine as well as the TLR7/8 agonist resiquimod that today still
serve as the inspiration for nearly all such work on small molecule TLR agonists.16,17 Recent
additional work has described TLR2/TLR1 agonists.18,19 Relevant to the studies detailed herein and
among the TLRs, TLR4 selectively responds to LPS (lipopolysaccharide or endotoxin, Figure 1)5
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extracted from aggregated LPS by LPS binding protein (LBP), delivered by cluster of differentiation
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4 (CD14) to the deep hydrophobic pocket of myeloid differentiation protein 2 (MD-2) in complex
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with TLR4 that leads to receptor dimerization and adoption of the conformation that characterizes the
.23
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activated complex (LPS–MD2/TLR4)
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Figure 1. Top: Structure of Lipid A, a component of lipopolysaccharide (LPS, endotoxin), which is responsible for the
toxicity of Gram-negative bacteria. It is the innermost of three regions of LPS. While its toxic effects are damaging, the
sensing of Lipid A by the human immune system (TLR4/MD-2) is critical for the onset of immune responses to Gram-
negative bacteria, and the fight against the infection. Bottom: Key steps in the initial elaboration of a screening lead,
compound 1, to the compounds 5 and 6 examined herein.
Complementary to a genetic approach in whole organisms that induce random nucleic acid
mutations in search of phenotypic changes derived from altered genes impacting the immune system,
5
which was used to discover that LPS acts through activation of a Toll-like receptor (TLR4), we
explored the use of an alternative chemical genetics approach.24 The approach entailed screening
libraries of compounds in cell-based functional assays that serve as a read out of stimulated signaling
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25
of an immune response. In a screening campaign conducted with nearly 100,000 compounds using
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a functional assay that measures the stimulated release of TNF- from treated mouse macrophages,
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we identified a new class of activators of the immune response. The libraries screened in our efforts
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represent a unique compound collection populated by nontraditional compounds designed to target
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protein–protein27,28 or protein–DNA interactions as well as the major enzyme classes. The library
screening for mouse macrophage TNF- production resulted in the discovery of a single class of
active compounds found in an 8,000-membered -helix mimetic library, prepared as 400 mixtures of
27
2
0 compounds. It is worth noting that the functional activity measured in the screening assay is both
a rare activation event (activator/agonist vs inhibitor/antagonist) and was conducted with an assay
that is extraordinarily sensitive, such that even weak and rare activation for TNF- release proved
detectable and clear (Figure 2). The two active pools from the -helix mimetic library observed in the
screen (56G4 and 58G4) were retested as their individual compounds providing three closely related
active compounds 1–3 (Figure 2). From the initial screening results, it was also determined that even
small deviations from their structure resulted in no detection of activity, providing a first level
structure–activity relationship (SAR) directly from screen. Representative of this SAR data available
from the initial screen, activity was observed only with a central tyrosine side chain substituent and all
other closely related substituents in the library,27 including phenyl, 4-chlorophenyl, naphthyl, 4-
methoxyphenyl, and indolyl, were inactive. Similarly, the two active mixtures contained either a
homophenylalanine (HoPhe) or methionine (Met) side chain at the carboxylic acid terminus and all
other 18 residues in the library27 were inactive, including phenylalanine, 4-chlorophenylalanine, 4-
methoxyphenylalanine, naphthylalanine, tyrosine, tryptophan, leucine, valine, isoleucine, alanine,
glycine, asparagine, lysine, serine, threonine, aspartate, histidine, and aminobutyric acid (Abu).
Equipped with this information, approximately 220 compounds in a library exploring the C-terminus of
1
and 225 compounds varying the N-terminus of 1 were prepared. These libraries provided
analogues that approached the activity of 1, and others that exhibited an altered profile of TLR4
dependence. However, the removal of the entire N-terminus subunit along with capping the C-
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terminus carboxylic acid as its t-butyl ester, providing 6, resulted in both a significant structure
simplification as well as a further enhancement in efficacy. As these studies progressed, it was also
established that most initial changes to the central residue of 1 resulted in a loss in activity except the
removal of the linking oxygen atom in 1 to give 4, which provided an enhancement in mouse TLR4
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(mTLR4) agonist activity. These initial optimizations not only led to simplifying structural
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modifications, but they also provided two efficacious mTLR4 agonists 5 and 6 (neoseptin-326 and
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neoseptin-4 ), nearly matching the maximal agonist efficacy of LPS (Figure 1).
Primary screening of  -helix mimetic library
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wells (1-400)
N-terminal SAR
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Figure 2. Top: mTLR4 agonists emerged from screening an 8000-membered -helix mimetic library designed to
represent the i, i + 4, and i + 7 residues of all -helices. The library, consisting of 400 wells of 20 compounds each and
prepared as a 20 x 20 x 20-mix, was tested at 50 M for stimulated release of TNF- against mouse macrophages.
Bottom: mTLR4 agonists identified in the library. The individual compounds found in the two active wells (56G4 and 58G4)
were prepared and tested (50 M) for stimulated release of TNF- against mouse macrophages providing the lead
compounds 1–3.
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Compounds 5 and 6 exhibited well-defined dose-dependent activation and stimulated release
of TNF- (IC50 = 18.5 and 20.7 M, respectively) in the functional assay and both were selective for
mTLR4 activation among all mTLRs and other pattern recognition receptors. Although the target
identification for leads that emerge from an undirected functional assay such as the one used in this
work is usually challenging, especially if the lead compounds bear no structural resemblance to
known effector molecules, it proved straightforward for 5 and 6. By design, it was recognized that the
follow up assay of lead compounds against macrophages available from mice bearing germline
genetic defects or knockouts of the genes encoding each of the TLRs, other pattern recognition
receptors, or downstream signaling molecules could be used to establish whether the activity was
derived from known or presently unrecognized targets.31 By employing this approach, the TNF-
production induced by 5 was established to be solely dependent on both mTLR4 and mMD-2, and
independent of other mTLRs (Figure 3) and other pattern recognition receptors (not shown).
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Moreover, 5 proved nearly identical to LPS in its dependency on not only mTLR4 and mMD-2, but
also the downstream signaling molecules MyD88, TRIF, IRAK4, and IKBKG (Figure 3). Unlike LPS,32
the activity of 5 was independent of CD14, which serves to deliver LPS to mMD-2 bound to mTLR4.
Like LPS, 5 has been additionally shown to activate the NF-B, P38 MAPK, JNK, and ERK signaling
26
pathways (not shown). Compound 5 more effectively stimulates a type I interferon response than
LPS or MPLA, resulting in release of IFN- but not IFN- (5 > 6), activates IL-6 production, but fails to
induce pro-IL-1 expression and subsequent capase-1 mediated IL-1release thought to contribute
to the higher toxicity of LPS relative to MPLA.34 This IFN- production was also shown to be
26
dependent on mTLR4, mMD-2 and TRIF, whereas LPS but not 5 additionally required CD14. These
features represent potential advantages to the use of 5 as a stimulator of the immune system or as a
vaccine adjuvant. Like LPS and monophosphoryl Lipid A (MPLA), the binding of 5 with mMD-2 was
first inferred by inhibition of its mTLR4 agonist activity (stimulated TNF- release) by eritoran, a
competitive antagonist of the binding of LPS/MPLA with mMD-2,33 supported by a NMR detected
interaction, and unambiguously established in a X-ray co-crystal structure of 5 bound to mMD-2 in
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complex with the mTLR4 ectodomain.26 Like 5 and 6, the analogues disclosed herein are inactive
against human TLR4, but the results of their examination define structural regions, structural insights,
and approaches (e.g., dimerization) available for modifications to address this species dependent
activity.
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Figure 3. Assay for TNF- release upon treatment with 5 (50 M) using macrophages from mice containing disabling
germline mutations or knockouts of genes encoding mTLRs and downstream signaling proteins. All results are
representative of two independent experiments. Error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; ****
= P ≤ 0.0001.
When examined in vivo in mice as detailed herein, 5 was found to be an effective adjuvant
and, unlike LPS, is nontoxic.26 Unlike LPS or its active core Lipid A on which substantial work has
been conducted,35 5 and 6 are easy to prepare and structurally manipulate, are intrinsically much
more stable, and exhibit exquisite structure–activity relationships as detailed herein. Compound 5
also was found to elicit an improved and qualitatively different response than LPS even though they
share the same receptor, including an altered profile of downstream cytokine release. With a few
notable exceptions, some of which were disclosed as our own studies were underway, 5 and 6 are
among only a handful of small molecule agonists capable of TLR4 activation beyond those related to
LPS and Lipid A.36-38 Herein we report the synthesis and the results of a systematic study of the
structure–activity relationships of 5 and 6, and the ability of 5 to stimulate the immune response in
vivo in mice as established by its ability to serve as an efficacious adjuvant.
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Results and Discussion
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Compound Assessment. Mouse macrophages (1 x 10 cells) were stimulated with 5, 6 or the
candidate analogues (dissolved in DMSO at the concentrations indicated, <0.5% DMSO in assay)
and ultra-pure LPS (dissolved in H O, Enzo Life Sciences) for 4 h. Mouse TNF-α in the supernatants
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was measured by ELISA kits according to the manufacturer’s instructions (eBioscience and PBL
Assay Science). All data reported in Figures 4–12 is the mean of triplicates and SD is within +10% of
mean and all samples were assessed alongside LPS, 5, and DMSO controls. Mouse cells were from
wild type C57BL/6J mice.
Compound Synthesis and Results of Their Assessment. Synthesis of 5 and 6. Fischer
esterification of commercially available 3-hydroxy-4-nitrobenzoic acid provided methyl 3-hydroxy-4-
nitrobenzoate (cat. H
2
SO
4
, MeOH, reflux, 18 h, 99%; also commercially available) and was followed
o
by formation of the aryl triflate 7 (Tf
2
O, Et
3
N, CH
Cl
2 2
, 0 C, 18 h, 79%) (Scheme 1). Sonogashira
cross-coupling of 7 with [(4-triisopropylsilyloxy)phenyl]acetylene, silyl ether deprotection and
o
concurrent hydrolysis of the methyl ester 8 (LiOH, THF/MeOH/H
2
O 4:1:1, 25 C, 16 h, 81% for 2
steps), and coupling of the resulting carboxylic acid 9 with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine,
o
DMF, 25 C, 18 h, 98%) provided 10. Alkyne hydrogenation and concurrent nitro group reduction with
o
Pearlman’s catalyst (H
2
, Pd(OH)
2
/C, EtOAc, 25 C, 16 h, 98%) afforded 5.
Scheme 1
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Subsequent to the completion of much of the work detailed herein, an alternative synthesis of
5
was developed (Scheme 2). Wittig reaction of (4-benzyoxyphenylmethylene)triphenylphosphorane
o
with methyl 3-formyl-4-nitrobenzoate (NaN(SiMe
3 2
)
, THF, 25 C, 16 h, 55%) provided 13. Methyl ester
o
hydrolysis (LiOH, THF/MeOH/H
2
O 3:1:1, 25 C, 3 h, 91%), coupling of the resulting carboxylic acid
o
with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 18 h, 70%), and single-step nitro group
o
reduction, alkene hydrogenation, and benzyl ether hydrogenolysis (H
3%) provided 5.
Scheme 2
2
, Pd(OH)
2
, EtOAc, 25 C, 12 h,
9
As detailed in our work that provided the -helix mimetic library,27 aromatic nucleophilic
substitution of commercially available 3-fluoro-4-nitrobenzoic acid with the sodium alkoxide derived
o
from 2-[4-(triisopropylsilyloxy)phenyl]ethanol (NaH, THF, 25 C, 2 h, 85%) provided 14 in a reaction
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conducted at room temperature and on the free carboxylic acid (Scheme 3). Coupling of carboxylic
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
acid 14 with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 12 h, 90%) afforded 15. Silyl ether
o
deprotection (Bu
4
NF, THF, 25 C, 1 h, 91%) and subsequent nitro group reduction of 16 (Zn
o
nanopowder, saturated aqueous NH
4
Cl/acetone, 25 C, 1 h, 99%) provided 6.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3
Both 5 and 6, but not their corresponding carboxylic acids 11 and 12, exhibited well-defined,
dose-dependent stimulated TNF- release from treated mouse macrophages (Figure 4). As
31
summarized above, the TNF- production induced by 5 and 6 was established to be nearly identical
to LPS in its dependency in mice cell lines on TLR4, MD-2, MyD88, TRIF, TRAM, MAL, IRAK4, and
IKBKG (Figure 3), and in their independency of all other TLRs. Not surprisingly and unlike LPS, their
activity proved independent of CD14, which serves to deliver LPS to the cell surface receptor
complex. Finally, as detailed herein, 5 proved to be a robust, in vivo active adjuvant in a mouse model
of vaccination.
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Journal of Medicinal Chemistry
3
2
000
500
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
**
**
2000
1500
**
*
*
1
000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
00
0
LPS LPS
5
6
11
12
Figure 4. Activity (TNF- release) of 5 and 6 versus the corresponding carboxylic acids 11 and 12, assayed at 50 M
against mouse macrophages alongside LPS (5 ng/mL in red, and 1 ng/mL in blue). All data is the mean of triplicate assay
versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
Scan of the C-terminus residue. Although not the first of the modifications to be conducted on
5
or 6, we reestablished the unique activity of the L-HoPhe-OtBu C-terminus residue when coupled to
the core amine terminus found in 5, examining all C-terminus residues that were present in the
2
7
original -helix mimetic library (Figure 5). Thus, coupling of 9 with the L--amino acid t-butyl esters
o
(H
2
N-Xxx-OtBu, EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 12–24 h), bearing hydrogenolysis labile side
chain protecting groups as needed (H-Lys(Z)-OtBu and H-Asp(OBn)-OtBu), followed by concurrent
o
alkyne and nitro group reduction (H
2
, Pd(OH)
2
/C, EtOAc, 25 C, 16 h) provided 17–35. Included in
this group was L-HoPhe-OtBu, reconfirming its activity when tested alongside the companion
analogues. Most notable in the set of analogues was the inactivity of the compounds containing other
aryl residues (e.g., L-Phe-OtBu, 23), highlighting that the activity of 5 is not observed if the linker to
the aryl ring is shortened by even a single carbon regardless of the extended size of such aryl
residues (e.g., NapAla-OtBu, 25). Interestingly, 19–21, which progressively contain more elements of
this linking chain but lack the attached phenyl group, exhibited weak and progressively stronger
agonist activity, whereas Met-OtBu (26) was inactive.
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3
000
500
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
**
2
2000
1500
**
**
1000
*
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
500
** **
**
0
LPS 5 17 18 19 20 21 22 23 24 25
5
26 27 28 29 30 31 32 33 34 35
Figure 5. C-terminus scan, compounds prepared from 9 (overall % yields) and assayed (50 M) against mouse
macrophages alongside LPS (5 ng/mL) for stimulated release of TNF-. All data is the mean of triplicate assay versus
vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
Analogues defining the importance of the C-terminus t-butyl ester. Within the structure of 6,
five systematic variations on the t-butyl ester were examined including the free carboxylic acid 12.
These were prepared by a synthetic route analogous to that used for the preparation of 6 itself,
coupling intermediate 14 with the corresponding i-Pr, Et, or Me ester of L-HoPhe or 3-phenyl-1-
propylamine followed by subsequent tri-isopropylsilyl (TIPS) ether deprotection and nitro group
reduction with these latter two reactions carried out in an exchangeable order for the ethyl ester 41
(Scheme 4). Like the observations made with 5 below, replacement of the t-butyl ester with smaller
ester groups led to progressive losses in activity with only the i-Pr ester 40 exhibiting weak agonist
activity (Figure 6). Both the removal of the t-butyl ester altogether with 43 or its cleavage to provide
the carboxylic acid 12 led to a complete loss in activity.
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Journal of Medicinal Chemistry
Scheme 4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A more extensive series of C-terminus analogues of 5 was prepared and was conducted by
coupling intermediate 9 with a series of alternative ester or amide derivatives of L-HoPhe-XR (X = O
or NH) followed by concurrent alkyne and nitro group reduction by the protocol developed for 5 itself
(Figure 6). Like the observations made with 6, replacement of the t-butyl ester with smaller esters led
to progressive and complete loss in agonist activity with only the i-Pr ester 44 maintaining weak
activity (Figure 6). Similarly, its hydrolysis to the carboxylic acid 11 also resulted in a complete loss in
activity. Only the closely related ester 47 approached the activity of 5, whereas the related but slightly
larger ester 48 bearing an alkyne was inactive. Even extending the t-butyl group by a single carbon
with ester 49 resulted in a complete loss in agonist activity as did its replacement with the larger
adamantyl ester 50. Replacement of the t-butyl ester with an amide was not productive. Only a trace
of activity was observed with the corresponding t-butyl amide 51, representing a single heavy atom
change in 5 (amide NH for ester O), and perhaps a trace of activity was observed with the primary
carboxamide 59. Without knowledge at the time of the nature of the binding of 5 to MD-2 and inspired
by the lipid chains found in LPS, we also examined a series of long chain alkyl amides 54–58.
Although a hint of activity was seen with a selected member in the group (54), the very modest level
of activity observed and the lack of a SAR trend of increasing activity with the longer alkyl amides 55–
5
8 did not inspire continued work on this series.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2500
000
1500
000
**
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
**
**
**
1
**
5
00
0
**
**
**
*
**
Figure 6. Preparation of t-butyl ester analogues of 5 and the assessment of the ester analogues of 5 (above) and 6
Scheme 4) assayed (50 M) against mouse macrophages alongside LPS (1 ng/mL) for stimulated release of TNF-. All
data is the mean of triplicate assay versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤
.001.
(
0
Synthesis of analogues probing the HoPhe absolute stereochemistry and linker length.
Replacement of the C-terminus L-HoPhe-OtBu with its enantiomer D-HoPhe-OtBu as well as the
extension of the linker chain by one or two additional carbons was conducted by coupling 9 with the
corresponding amines following the protocols developed for 5 (Figure 7). The D-enantiomer 60
proved inactive confirming that the interaction of 5 with the mTLR4/mMD-2 complex is
diastereospecific (Figure 7). Remarkably and like the reduction in the length of the linker chain to the
phenyl group by one carbon with 23 that was inactive, its extension by either one or two carbons with
6
1 and 62 also led to essentially complete loss in agonist activity.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
500
000
*
*
**
1500
000
1
5
00
0
*
*
**
LPS
5
60
23
61
62
Figure 7. Preparation of the enantiomer (60) of 5 and analogues altering the HoPhe chain length and their assessment
assayed (50 M) against mouse macrophages alongside LPS (1 ng/mL) for stimulated release of TNF-. All data is the
mean of triplicate assay versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
Synthesis of analogues containing L-HoPhe aryl substituents and aryl ring replacements. A
small series of p-substituted L-HoPhe-OtBu derivatives were examined as well as a single analogue
in which the phenyl ring was replaced by a saturated cyclohexyl ring. These analogues were
prepared by coupling 9 with the corresponding amines followed by alkyne and nitro group reduction
(
Figure 8). Although the number of compounds in the series was not large, the results of their
examination were especially revealing. Incorporation of polar p-substituents (R) were not tolerated,
resulting in a complete loss in agonist activity with 66 and 67 (R = OH or NH ). Even benign but
2
electron dense halide substituents led to a substantial reduction (65, R = F) or loss (64, R = Cl) in
activity. However, nonpolar substituents represented by the analogue 63 (R = Me) exhibited the
robust activity of 5, surpassing its efficacy and indicating that there are likely substantial nonpolar
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Page 16 of 50
modifications to this region of the molecules that are possible. Consistent with such expectations, the
methyl ether derivative 68 (R = OMe) proved equally active with 5, recovering the full agonist activity
lost with the corresponding phenol 66 (R = OH). These latter results indicate that it is the nature of the
substituent (hydrophobic vs hydrophilic) and not its presence (steric) that influences activity, likely the
result of binding deep in the hydrophobic pocket of mMD-2. Especially revealing was the potent
agonist activity of 70, surpassing the activity of 5. Not only does this indicate that even the
unsaturation of the phenyl ring is not necessary and that hydrophobic saturated replacements are
well tolerated, but also that this is a region of the molecule that can be substantially modified in
continued optimization efforts. Dose-response studies established that 63, 68, and 70 exhibit IC50’s
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(21.6, 22.2, and 29.5 M respectively) comparable to those found for 5 and 6.
4
500
*
4000
*
**
**
3
3
2
500
000
500
***
**
2000
1
1
500
000
500
0
**
Figure 8. Preparation of analogues of 5 bearing HoPhe phenyl substituents or replacements and their assessment
assayed (50 M) against mouse macrophages alongside LPS (5 ng/mL) for stimulated release of TNF-. All data is the
mean of triplicate assay versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
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Journal of Medicinal Chemistry
Synthesis of analogues probing the amide. The importance of the linking amide was
1
2
established with a small set of key analogues. The N-methyl amide 71 of 5 was prepared by coupling
L-NMe-HoPhe-OtBu with 9 followed by nitro group and alkyne reduction (Figure 9). In addition, the
corresponding thioamide 72 was prepared from 5 itself by TIPS ether protection of the phenol,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
Lawesson’s reagent conversion to the thioamide (toluene, 60 C, 12 h) and subsequent silyl ether
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
deprotection (Bu
4
NF, THF, 25 C, 6 h, 76% for 3 steps). Reductive removal of the thiocarbonyl (H
2
,
Raney Ni, MeOH, 18 h) provided the amine 72, lacking the amide carbonyl. Each modification of the
linking amide resulted in a complete loss in activity (Figure 9). Not only does this highlight the
essential contribution of the secondary amide to the expression of the properties of 5, but it also
underscores how remarkable the behavior of 5 is where even the conservative single atom
replacement (thioamide S for amide O) in the amide completely disrupted the observed activity.
2500
**
2
000
1500
000
**
1
5
00
0
LPS
5
71
72
73
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Figure 9. Preparation of analogues of 5 bearing changes to the amide and their assessment assayed (50 M) against
mouse macrophages alongside LPS (1 ng/mL) for stimulated release of TNF-. All data is the mean of triplicate assay
versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of analogues probing the importance of the presence, position, and substitution of
the aniline amine. A similarly key series of analogues of 5 and 6 were prepared to establish the
importance of the presence and position of the aniline amine and the impact of its substitution. Those
prepared that are analogues of 5 include the aniline positional isomers 74 and 75, requiring their
synthesis from the commercially available 5-nitro- or 6-nitro-3-hydroxybenzoic acids by a synthetic
route modeled on that used for 5 itself (Scheme 5). Similarly, the analogue 76 of 5 lacking the aniline
amine was prepared from commercially available 3-hydroxybenzoic acid. Finally, the monomethyl and
dimethyl anilines 77 and 78 were prepared by nonselective methylation of 80, available in 2 steps
from 10, followed by silyl ether deprotection. The acetamide 79 was prepared from the same
intermediate by acylation with acetyl chloride followed by silyl ether deprotection.
Scheme 5
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The nitro precursor 16 (Scheme 3) to compound 6 was examined as an additional probe of the
importance of the presence of the aniline and the analogue 81 of 6 lacking the aniline amine was
prepared by phenol alkylation with 2-(4-triisopropylsilyloxyphenyl)ethyl bromide (K
2
CO , refluxing
3
o
acetone) followed by silyl ether deprotection (Bu
4
NF, THF 25 C) (Scheme 6).
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Scheme 6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Removal of the aniline amine (76 and 81), its mono- or di-methylation (77 and 78), its simple
acetylation (79), its replacement with a nitro group (16), and its movement to the 6-position on the
central ring (75) resulted in a complete loss in agonist activity (Figure 10). However and interestingly,
relocating the aniline amine in 5 to the adjacent 5-position on the central aromatic ring provided an
analogue 74 that retained much of the activity of 5. Finally, the corresponding carboxylic acids
derived from both series (74–79 or 16 and 81) were also prepared by acid-catalyzed t-butyl ester
o
deprotection (4 N HCl, dioxane, 25 C) and all were found to be inactive (not shown).
2
500
000
**
2
**
1500
000
**
1
500
0
Figure 10. Evaluation of analogues of 5 (Scheme 5) and 6 (Scheme 6) used to probe the importance of the presence,
position, and substitution of the aniline amine assayed (50 M) against mouse macrophages alongside LPS (1 ng/mL) for
stimulated release of TNF-. All data is the mean of triplicate assay versus vehicle control, error bars represent SEM. * =
P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
Synthesis of analogues probing the importance of the presence and position of the phenol
hydroxyl group. Positional isomers of the phenol substitution and the removal of the phenol hydroxyl
group altogether were examined with the analogues 82–84 of 5 prepared as detailed in Scheme 7.
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Sonogashira cross-coupling of the appropriately substituted alkynes with triflate 7, LiOH promoted
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrolysis of the methyl ester and simultaneous phenol silyl ether deprotection, and coupling of the
o
resulting carboxylic acids with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 12–24 h)
o
followed by alkyne and nitro group reduction (H
2
, Pd(OH)
2
/C, EtOAc, 25 C, 3 h) provided 82–84. The
corresponding methyl ether 85 of the phenol found in 5 was prepared by O-methylation of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
intermediate 10 (MeI, K
2
CO
3
, DMF, 25 C, 12 h) followed by alkyne and nitro group reduction.
Scheme 7
Analogues of 6 were prepared in which the phenol was moved from the p-position to the m-
position and in which it was removed altogether. Additionally, two analogues that contain a benign
substituent (Cl and F) adjacent to the p-phenol hydroxyl group were also prepared. Thus, aromatic
nucleophilic substitution of 3-fluoro-4-nitrobenzoic acid conducted at room temperature and on the
free carboxylic acid with a series of four sodium alkoxides provided the product carboxylic acids
o
(
Scheme 8). Their subsequent coupling with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25 C,
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Page 22 of 50
o
1
2 h), silyl ether deprotection (Bu
4
NF, THF, 25 C, 2 h if needed) and subsequent nitro group
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
reduction (Zn nanopowder, saturated aqueous NH
4
Cl/acetone, 25 C, 30 min) provided 86–89.
Scheme 8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Removal of the phenol hydroxyl group (84 and 89), its conversion to a methyl ether (85), and
its movement to the o-position (83) led to complete loss (84, 85, and 89) or near complete loss (83) in
agonist activity, indicating its importance to the expression of the properties of 5 and 6 (Figure 11).
Even the addition of rather benign substituents adjacent to the p-phenol (87 and 88, F and Cl) led to
the sharp reduction in the agonist activity of 6, suggesting that there may be constraints on further
substituting the aryl ring. Only the corresponding m-phenol positional isomers 82 and 86 retained the
activity of 5 and 6, exhibiting slightly reduced agonist activity and suggesting that the connecting
flexible linking chain allows adjustments in the aryl positioning to accommodate the relocated m-
phenol. The carboxylic acids derived from both series (82–85 and 86–89) were also prepared by acid-
o
catalyzed t-butyl ester deprotection (4 N HCl, dioxane, 25 C) and all were found to be inactive (not
shown).
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Journal of Medicinal Chemistry
2
500
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
**
2000
**
**
*
*
1
500
***
1000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
**
5
00
0
**
***
Figure 11. Evaluation of analogues of 5 (Scheme 7) and 6 (Scheme 8) used to probe the importance of the presence,
position, and substitution of the phenol hydroxyl group assayed (50 M) against mouse macrophages alongside LPS (1
ng/mL) for stimulated release of TNF-. All data is the mean of triplicate assay versus vehicle control, error bars represent
SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.
Synthesis of analogues probing the central residue linker to the phenol. Among the most
carefully explored regions of 5 and 6 was the linker extending off the central residue to the phenol.
This is the site that distinguishes 5 and 6, differing by the insertion of an oxygen atom in the linker for
6. Thus, both a two-atom and three-atom linker proved effective in early studies. A series of additional
linkers containing an oxygen atom were prepared as shown in Scheme 9. Two extend the linker
found in 6 by one or two additional carbons (90 and 91) and were prepared by O-alkylation of a
common intermediate with the respective iodides followed by nitro group reduction (Zn nanopowder,
o
saturated aq NH
4
Cl/acetone, 25 C, 20 min) and subsequent phenol silyl ether deprotection (Bu
4
NF,
o
25 C, 20 min). A third analogue 92 shortened the linker of 6 by one carbon, matching the linker
length of 5 but now containing an oxygen atom. Compound 92 was prepared by alkylation of the
benzylic bromide, methyl 3-bromomethyl-4-nitrobenzoate, with 4-triisopropyloxyphenol (68%).
Subsequent LiOH mediated hydrolysis of the methyl ester and concurrent silyl ether deprotection
followed by coupling of the resulting carboxylic acid with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine,
o
DMF, 25 C, 12 h) and a final nitro group reduction (Zn nanopowder, saturated aq NH
4
Cl/acetone, 25
oC, 20 min, 91% for 3 steps) provided 92.
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Scheme 9
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A series of saturated all carbon linkers were explored that cover the range of (CH
2
) where n =
n
0
-3, including 5 (n = 2). Extension of the linker found in 5 by one carbon, providing an all carbon
analogue 93 of 6, was prepared as shown in Scheme 10. Wittig reaction of methyl 3-formyl-4-
nitrobenzoate with 2-((4-triisopropylsilyloxyphenyl)ethylene)triphenylphosphorane provided the
o
substituted styrene (THF, 25 C, 1 h, 66%, 3:1 Z:E). Methyl ester hydrolysis and concurrent silyl ether
o
deprotection (LiOH, 25 C, 1 h, 99%), coupling of the resulting carboxylic acid with L-HoPhe-OtBu
o
(EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 12 h, 86%), and final nitro group and alkene reduction (H
2
,
o
Pd(OH)
2
, EtOAc, 25 C, 81%) provided 93. The analogue 94 of 5 containing one less carbon in the
linking chain was prepared by Suzuki coupling of 4-(triisopropylsilyloxy)phenyl boronic acid with
o
methyl 3-bromomethyl-4-nitrobenzoate (cat. PdCl
2
, K
2
CO
3
, acetone/H
2
O, 25 C, 1 h, 58%). LiOH
mediated methyl ester hydrolysis and silyl ether deprotection, coupling of the resulting carboxylic acid
o
with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25 C, 12 h, 51%), and subsequent nitro group
o
reduction (Zn nanopowder, saturated aqueous NH
4
Cl/acetone, 25 C, 1 h, 80%) provided 94. The
analogue 95 of 5, containing no linking chain and attached directly to the central residue (two carbons
shorter), was prepared by Suzuki coupling of triflate 7 with 4-(triisopropylsilyloxy)phenyl boronic acid
o
(cat. Pd(PPh
3 4
) , K
3
PO
4
, DME/H
2
O,85 C, 1.5 h, 98%). Methyl ester hydrolysis of the coupled product
conducted with LiOH under conditions that did not induce phenol silyl ether deprotection (1.1 equiv
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Journal of Medicinal Chemistry
o
LiOH, 1:1 t-BuOH/H
2
O, 25 C, 2 h, 84%), coupling of the carboxylic acid with L-HoPhe-OtBu (EDCI,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
HOAt, 2,6-lutidine, DMF, 25 C, 12 h, 93%), nitro group reduction by hydrogenation (H
2
, Pd/C, EtOAc,
o
o
25 C, 3 h, 98%), and a final silyl ether deprotection (Bu
4
NF, THF, 25 C, 2 h, 63%) provided 95. In
addition, the analogue 96 with a rigid alkyne two carbon linker was prepared by selective nitro group
reduction of intermediate 10 employed for the synthesis of 5 (Zn nanopowder, saturated aqueous
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
NH
4
Cl/acetone, 25 C, 15 min, 62%).
Scheme 10
Two additional analogues were prepared that contain a single atom heteroatom linker
Scheme 11). The first of these was the analogue 97 that contains a sulfur atom linker. This analogue
(
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is one that we expected to be an exceptional candidate since a sulfur atom is often regarded as an
excellent isosteric replacement for the two carbons of a double bond. Room temperature aromatic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nucleophilic substitution of 3-fluoro-4-nitrobenzoic acid with 4-(triisopropylsilyloxy)thiophenol (K
2
CO ,
3
o
DMF, 25 C, 12 h, 72%), followed by coupling of the carboxylic acid with L-HoPhe-OtBu (EDCI, HOAt,
o
2
,6-lutidine, DMF, 25 C, 12 h, 76%), and final nitro group reduction (Zn nanopowder, saturated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
aqueous NH
4
Cl/acetone, 25 C, 1 h, 97%) provided 97. An analogous approach was enlisted for the
synthesis of 98 that contains a single nitrogen atom linker. Nucleophilic substitution of methyl 3-
o
fluoro-4-nitrobenzoate with 4-benzyloxyaniline (DMSO, 110 C, 6 h, 91%), methyl ester hydrolysis
(LiOH, 93%), coupling of the carboxylic acid with L-HoPhe-OtBu (EDCI, HOAt, 2,6-lutidine, DMF, 25
oC, 12 h, 65%), and nitro group reduction and concurrent benzyl ether hydrogenolysis provided 98.
Scheme 11
All analogues that contained saturated linkers composed of two or three carbons or oxygen
were highly effective agonists and the results with analogues in this series exhibited well defined
trends (Figure 12). The effective agonists included not only 5 and 6, but also 92 that incorporates an
oxygen atom into the two atom linker of 5 and 93 that replaces the oxygen atom in the three atom
linker of 6 with a carbon methylene. Extending the linker of 6 by even one or two more carbon atoms
resulted in a near complete loss in agonist activity (analogues 90 and 91). Similarly shortening the
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Journal of Medicinal Chemistry
two carbon atom linker of 5 by one carbon with 94 led to a substantial loss in activity, and removing
the linker altogether with 95 led to essentially complete loss in activity. Additionally, replacement of
the flexible two carbon saturated linker of 5 with the rigid two carbon alkyne linker in 96 resulted in a
complete loss in activity. Similarly, replacement of the linker with a single nitrogen atom as found in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
9
8 resulted in an inactive compound. Surprisingly, replacement of the linker with a sulfur atom as in
7 resulted in a complete loss in activity despite the fact that it is isosteric with two carbon atoms. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
carboxylic acids derived from these series were also prepared by acid-catalyzed t-butyl ester
deprotection and all were found to be inactive (not shown).
2
500
**
2000
**
*
** **
1
1
500
000
*
*
5
00
0
** **
**
Figure 12. Evaluation of analogues of 5 and 6 (Schemes 9–11) used to probe the linker between the central aromatic ring
and the phenol assayed (50 M) against mouse macrophages alongside LPS (1 ng/mL) for stimulated release of TNF-.
All data is the mean of triplicate assay versus vehicle control, error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** =
P ≤ 0.001.
Adjuvant Activity of 5. Extensive in vitro characterization of 5 as a mTLR4 agonist and its
downstream signaling was disclosed elsewhere26 as summarized in the introduction, indicating it
effectively elicits the innate immune response. Since TLR4 signaling plays an essential role in the
generation of both innate and adaptive immune responses, we investigated whether 5 can also serve
as an adjuvant in vivo in mice, indicating stimulation of the adaptive immune response. We analyzed
IgG production against a protein antigen ovalbumin (OVA) in the absence or presence of escalating
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doses of 5. The data showed that 5 elicited a robust antibody response in a dose-dependent manner
measured 21 days after immunization (Figure 13a). In mice, immunoglobulin G1 (IgG1) is associated
with a Th2-like response, while a Th1 response is associated with the induction of IgG2a, IgG2b, and
IgG3 antibodies. We further assessed the effect of 5 on OVA-specific IgG subclasses and found that
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
induced significant increases in OVA-specific IgG1, IgG2a, IgG2b and IgG3 (Fig.13b), suggesting it
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
activated both Th1 and Th2-mediated immune responses. Taken together, these data indicate that 5
is an adjuvant in mouse and serves to stimulate the adaptive immune response. Just as significantly,
the study indicates that compounds in this new class are of a caliber that exhibit in vivo activity, being
active not only on its target proteins and in functional cellular assays, but in vivo in mice as well.
Figure 13. Adjuvant activity of 5. (a) Dose-dependent adjuvant activity of 5. C57BL/6 mice (4 per group) were immunized
i.m. by 100 g OVA with vehicle (propylene glycol) or 5 in vehicle (propylene glycol) at indicated doses. After 21 days,
serum titers of OVA-specific IgG were measured by ELISA. (b) Assessment of OVA-specific IgG subclasses. Mice (4 per
group) were immunized with 100 g of OVA mixed with 5 or vehicle alone. After 21 days, serum titers of OVA-specific IgG
subclasses indicated were measured by ELISA. Error bars represent SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001;
**** = P ≤ 0.0001.
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Journal of Medicinal Chemistry
Retrospective Interpretation of the Structure–Activity Relationships Based on an X-ray
1
2
Co-crystal Structure of Mouse TLR4/MD-2 with 5. The crystal structure of mTLR4/mMD-2/5
recently reported by us revealed that 5 binds as an asymmetrical dimer within the hydrophobic pocket
of mMD-2 (Figure 14).26 There it induces and stabilizes a dimerization interface between two
mTLR4/mMD-2 complexes, similar to the active complex induced by Lipid A. However, 5 and Lipid A
form overlapping but distinct molecular contacts with the receptor to achieve receptor dimerization
and activation. Two molecules of 5 bind tightly to each other, being stabilized by -stacking of the
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
central aniline rings (-stacking) and by two hydrogen bonds between the NH group of the amide
bond of each molecule of 5 and the t-butyl ester carbonyl group of the other (2.2 Å each, Figure 14,
panel B). Thus, it is not surprising that all modifications to the amide (71–73) resulted in complete loss
in activity. Similarly, removal of hydrogen bond acceptor, the carbonyl of the t-butyl ester (43), results
in complete loss of activity. What is unique is that these two hydrogen bonds represent key
interactions, stabilizing the ligand dimer formation and not the interaction of 5 with the protein target.
Additionally, a hydrogen bond between the side chain of mMD-2 residue Arg90 and one of the amide
carbonyl groups stabilizes binding to the target.
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